JP7426059B2 - Hematopoietic stem cell differentiation promoter - Google Patents

Description

The present invention relates to an agent for promoting differentiation of hematopoietic stem cells into blood cells.

Blood is composed of blood cells and plasma, and has important functions in maintaining homeostasis of the living body, such as oxygen transport, waste removal, and immune function. However, in the elderly, aging causes a decrease in the number of blood cells in the blood and a decline in their function, which leads to a decline in immunity against infections, leading to various hematopoietic diseases such as anemia, neutrophilia, and thrombocytopenia. It has been reported that the patient is susceptible to the disease and causes a decline in QOL (Non-patent Documents 1-4, etc.). One of the causes of such a decrease in blood cells and functional decline is considered to be aging of hematopoietic stem cells that produce them (Non-Patent Document 5).

Hematopoietic stem cells exist in small numbers in the bone marrow, and have multipotency and self-renewal ability to differentiate into all blood cells, and continue to supply blood cells throughout an individual's life. Therefore, by transplanting hematopoietic stem cells, it is possible to reconstruct all blood cells from a state where there are no blood cells at all. Therefore, transplantation of autologous or allogeneic hematopoietic stem cells has been carried out as a means of treating blood diseases, immunodeficiency diseases, and the like. However, it has been reported that the proliferative ability and differentiation ability of hematopoietic stem cells decrease with age (Non-Patent Document 6), and there are cases where the blood cell system cannot be reconstituted even if hematopoietic stem cells are transplanted.

Therefore, functional recovery of hematopoietic stem cells is considered to be important for preventing and improving the decline in QOL caused by age-related failure of blood cell system reconstruction. So far, factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin (TPO) have been known as factors involved in promoting differentiation of hematopoietic stem cells (Non-Patent Documents 7, 8). In addition, an agent for inducing differentiation of pluripotent stem cells into hematopoietic stem cells and/or hematopoietic progenitor cells (Patent Document 1) consisting of a transforming growth factor-β (TGF-β) inhibitor, and cofilin as an active ingredient, Agents for inducing proliferation and/or differentiation of hematopoietic stem cells and/or hematopoietic progenitor cells (Patent Document 2) have also been reported. However, these have various problems such as being difficult to control in vivo and to use continuously on a daily basis, and it is desired to elucidate new factors to replace these.

Japanese Patent Application Publication No. 2012-70731 WO2003/057241 publication

Disordered hematopoiesis and myelodysplasia in the elderly. J. Am. Geriatr. Soc. 2003 Mar;51(3 Suppl):S22-6 Immunosenescence: emerging challenges for an aging population. Immunology. 2007 Apr;120(4):435-46. Epub 2007 Feb 15. Aging of the Innate Immune System: An Update.Curr Immunol. Rev. 2011 Feb 1;7(1):104-115. Unexplained anemia in the elderly. Semin Hematol. 2008 Oct;45(4):250-4. doi: 10.1053/j.seminhematol.2008.06.003. The aging haematopoietic stem cell compartment. Nat. Rev. Immunol. 2013 May;13(5):376-89. doi: 10.1038/nri3433. Epub 2013 Apr 15. Age-associated characteristics of murine hematopoietic stem cells. J. Exp. Med. 2000 Nov 6;192(9):1273-80. The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Nature. 1989 May 4;339(6219):27-30. The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells. Blood. 1996 Jun 15;87(12):4998-5005.

In view of the above circumstances, an object of the present invention is to discover a new substance that promotes differentiation of hematopoietic stem cells into blood cells, and to provide it as an agent for promoting differentiation of hematopoietic stem cells.

As a result of extensive research to solve the above problems, the present inventors have discovered that a supercritical extract of C. chinensis spores or a mixture of a supercritical extract of C. chinensis spores and an extract of other herbal medicines can be applied to blood cells of hematopoietic stem cells. The present inventors have discovered that the present invention has an excellent promoting effect on the differentiation into .

That is, the present invention includes the following inventions.
(1) An agent for promoting differentiation of hematopoietic stem cells into blood cells, which contains a supercritical extract of C. spores as an active ingredient.
(2) (A) A supercritical extract of Cinnamon spores, and (B) an extract of one or more crude drugs selected from cinnamon bark, carrot, plantain, licorice, chimpi, ginger, bellflower, and hawthorn. An agent for promoting differentiation of hematopoietic stem cells into blood cells, which contains as an active ingredient a mixture of
(3) An agent for promoting differentiation of hematopoietic stem cells into blood cells, which contains as an active ingredient a mixture of a supercritical extract of Cinnamon spores and extracts of one or two crude drugs selected from cinnamon bark and ginseng.
(4) A method for promoting differentiation of hematopoietic stem cells into blood cells, comprising the step of culturing hematopoietic stem cells in a medium containing the agent according to any one of (1) to (3).
(5) A method for producing blood cells, comprising the step of culturing hematopoietic stem cells in a medium containing the agent according to any one of (1) to (3).
(6) A composition for promoting differentiation of hematopoietic stem cells into blood cells, which contains the agent according to any one of (1) to (3).

The agent for promoting differentiation of hematopoietic stem cells into blood cells of the present invention can promote the differentiation of hematopoietic stem cells and efficiently induce blood cells, so it is possible to efficiently induce blood cells by promoting the differentiation of hematopoietic stem cells. It is effective in treating, improving, and preventing diseases or pathological conditions of organs.

The present invention will be explained in detail below.
1. Agent for promoting differentiation of hematopoietic stem cells into blood cells The agent for promoting differentiation of hematopoietic stem cells into blood cells according to the present invention (hereinafter sometimes referred to as "differentiation promoter for hematopoietic stem cells") is obtained by supercritical extraction of C. spores. or (A) a supercritical extract of Cinnamon spores, and (B) one selected from cinnamon bark, carrot, plantain, licorice, chimpi, ginger, bellflower, and hawthorn. Contains a mixture of extracts of two or more crude drugs as an active ingredient.

In the present invention, "hematopoietic stem cells" refer to myeloid cells [erythrocytes, platelets, granulocytes (eosinophils, basophils, neutrophils), monocytes, etc.] and lymphocytes via myeloid progenitor cells. Refers to cells that can differentiate into lymphoid cells (B cells, T cells, NK cells, etc.) via globular progenitor cells. Hematopoietic stem cells are characterized by being positive for CD34 antigen and negative for CD38 antigen (CD34+CD38-).

In the present invention, "differentiation of hematopoietic stem cells" refers to the division and proliferation of hematopoietic stem cells into hematopoietic progenitor cells (myeloid progenitor cells, lymphoid progenitor cells), and from hematopoietic progenitor cells into mature blood cells. . In the present invention, "promotion of differentiation of hematopoietic stem cells into blood cells" refers to activation of differentiation of hematopoietic stem cells into blood cells compared to before administering or ingesting the drug of the present invention, and specifically Specifically, the number of blood cells differentiated from hematopoietic stem cells (myeloid cells, lymphoid cells) or their progenitor cells, preferably the number of myeloid cells, more preferably the number of red blood cells. This means an increase in

Here, "blood cells" refer to all stages from hematopoietic stem cells to hematopoietic progenitor cells (including multipotent hematopoietic progenitor cells and unipotent hematopoietic progenitor cells), and finally to functional blood cells. Refers to cells. For example, red blood cells differentiated from hematopoietic stem cells, granulocytes (neutrophils, eosinophils, basophils), monocytes, lymphocytes (T cells, B cells, NK cells), platelets, macrophages, dendritic cells, etc. These include myeloid progenitor cells (erythroblasts, myeloblasts, promyelocytes, megakaryocytes, etc.) and lymphoid progenitor cells (monoblasts, lymphoblasts, etc.) that are in the pre-stage stage.

Ganoderma is a basidiomycete that belongs to the Ganoderma family (Ganodermataceae) and the genus Ganoderma, and is used in the herbal medicine "Reishi". According to the ancient Chinese medicinal books ``Bencao Gangme'' and ``Shennong Bencao Tejing,'' ganoderma is classified as red ganoderma (red turf), black ganoderma (black lucidum), purple lingzhi (purple zhi), and blue lingzhi (green zhi). ), yellow reishi (huangzhi), and white reishi (baikizhi) are described as being present. Kazuno reishi is also known as a type of red reishi. The "Stone Ganoderma spores" used in the present invention are not particularly limited as long as they are spores of Ganoderma genus Ganoderma in the family Ganodermaceae, for example, red Reishi, black Reishi, purple Reishi, blue Reishi, yellow Reishi. Examples include spores of grass and white reishi, but spores of red reishi (Ganoderma lucidum) and black reishi (Ganoderma sinense, Ganoderma japonicum, Ganoderma atrum) are more preferred.

Rock mushroom spores are brown powdery substances that appear on the fungal cap when the Reishi mushroom matures. In the present invention, the term "Moss spores" includes spores and sporangia containing a plurality of spores. For the extraction of C. chinensis spores, the recovered C. chinensis spores may be used as they are, but it is preferable to perform wall-breaking treatment to break down the cell walls of the spores by physical force. The method for treating the broken wall is not particularly limited, but can be carried out, for example, by atomization treatment, roll press treatment, grinding treatment, ultra-high pressure micro steam treatment, and other mechanical methods commonly used in industry. When using ruptured spores, they may be obtained by any of the methods described above, or commercially available products may be used.

In the present invention, the method for extracting C. chinensis spores is not particularly limited as long as it is a method in which the spores are brought into contact with a fluid in a supercritical state (supercritical fluid), but the method can safely and easily remove the solvent. Therefore, an extraction method using carbon dioxide in a supercritical state (supercritical carbon dioxide) is preferable. Supercritical carbon dioxide refers to carbon dioxide that has become a fluid under conditions of a temperature of 31° C. or higher and a pressure of 7 MPa or higher, and in the present invention, the supercritical state includes states in the vicinity thereof.

As extraction conditions using carbon dioxide in a supercritical state, the temperature is preferably 31 to 100°C, more preferably 31 to 80°C, even more preferably 31 to 60°C, and the pressure is preferably 7 to 100 MPa, and 7 to 50 MPa. is preferable, and 7 to 30 MPa is more preferable. Among these, it is particularly preferable that the temperature is 31 to 80°C and the pressure is 7 to 50 MPa, and most preferably the temperature is 31 to 60°C and the pressure is 7 to 30 MPa. The amount of supercritical carbon dioxide supplied during extraction is, for example, preferably 5 to 500 parts by weight, more preferably 10 to 100 parts by weight, per 1 part by weight of C. spores (in terms of dry matter). Further, the extraction time is preferably 30 minutes to 24 hours, more preferably 1 to 10 hours. Furthermore, organic solvents can also be used as co-solvents (entrainers). Examples of the co-solvent (entrainer) include ethanol, acetone, and the like. Among these, ethanol is preferred from the viewpoint of safety.

Extraction with carbon dioxide in a supercritical state can be performed, for example, by continuously blowing carbon dioxide under the above extraction conditions. The carbon dioxide fluid containing the extract of C. spores is then led to a separation tank and separated by conventional methods, such as reducing pressure, varying temperature, etc. At this time, the separation tank can be filled with an adsorbent that can adsorb the extracted solute or a medium (solvent, base) that can dissolve or disperse it, allowing for appropriate separation according to the extraction conditions. means can be adopted. The separated carbon dioxide can be transported to a liquefaction tank and reused.

"Keihi" (Japanese name: cinnamon, scientific name: CINNAMOMI CORTEX) is the bark of Cinnamomum cassia Blume (scientific name: Cinnamomum cassia Blume) of the Lauraceae family, or other plants of the same genus, with part of the bark or periderm removed. In herbal medicine (Japanese Pharmacopoeia), it is mainly used as a stomachic medicine. The extraction raw material used in the present invention is preferably the bark of the Japanese cinnamon tree, which is used as the herbal medicine "cinnamon bark."

"Carrot" (Japanese name: ginseng, scientific name: GINSENG RADIX) is the thin root of Panax ginseng (scientific name: Panax ginseng C. A. Meyer, also known as: Korean ginseng, Korean ginseng, medicinal ginseng), which is a perennial plant of the Araliaceae family. It is the root or a lightly blanched version of the root, and is mainly used in herbal medicine (Japanese Pharmacopoeia) as a health tonic and a stomachic medicine. Panax ginseng may be plants of the same genus or related species, such as Panax ginseng (scientific name: Panax japonicus C.A.Mey), Panax ginseng (scientific name: Panax notoginseng), Panax ginseng (scientific name: Panax quinquefolius), and Siberian ginseng (scientific name: Eleutherococcus senticosus). ) etc. The extraction raw material used in the present invention is preferably Panax ginseng root, which is used as the crude drug "ginseng."

``Plantago'' (Japanese name: Oobako, alias: ``Plantaginis Herba'', scientific name: PLANTAGINIS HERBA) is the entire plant of Plantago asiatica Linne (scientific name: Plantago asiatica Linne), a perennial plant of the Plantago family (Plantaginaceae). It is mainly used as an expectorant in herbal medicine (Japanese Pharmacopoeia). Plantain species that are native to Japan include Plantago asiatica, Plantago camtschatica, Plantago japonica, Plantago lanceolata, and its closely related naturalized species, Plantago major and Tubo plantain. (Plantago virginica), etc., and plants of the same genus or related species may also be used. The raw material for extraction used in the present invention is preferably the whole plant of Plantain, which is used as the herbal medicine "Chazenso".

"Glycyrrhiza" (Japanese name: Glycyrrhiza, scientific name: GLYCYRRHIZAE RADIX) is the root or shoot (stolon) of Glycyrrhiza uralensis (scientific name: Glycyrrhiza uralensis), a perennial plant of the Fabaceae family. In herbal medicine (Japanese Pharmacopoeia), it is mainly used as an analgesic, antispasmodic (gastrointestinal medicine), and an expectorant. Examples of daylily include Ural daylily (scientific name: Glycyrrhiza uralensis), Spanish daylily (scientific name: Glycyrrhiza glabra), Chinese daylily (scientific name: Glycyrrhiza echinata), and plants of the same genus or closely related thereof may be used. The extraction raw material used in the present invention is preferably licorice roots or stolons, which are used as the crude drug "licorice". Note that licorice is both a crude drug name (Japanese Pharmacopoeia) and a botanical name.

"Chinpi" (Japanese name: Chenpi, scientific name: AURANTII NOBILIS PERICARPIUM) is the mature pericarp of Citrus unshiu Marcowicz (scientific name: Citrus unshiu Marcowicz or Citrus reticulata Blanco), an evergreen shrub of the Rutaceae family, and is used as a herbal medicine in Japan. In the Pharmacopoeia), it is mainly used as a stomachic medicine. The raw material for extraction used in the present invention is preferably the peel of Mandarin orange, which is used as the herbal medicine "Chinpi".

"Ginger" (Japanese name: ginger, scientific name: ZINGIBERIS RHIZOMA) is the rhizome of ginger (scientific name: Zingiber officinale Roscoe), a perennial plant of the Zingiberaceae family, sometimes with the periderm removed, and is used as an herbal medicine. (Japanese Pharmacopoeia), it is mainly used as a stomachic medicine. ``Shokyo'' is made by drying raw ginger rhizomes, and ``kankyo'' is made by steaming and drying ginger rhizomes. The extraction raw material used in the present invention is preferably ginger rhizome, which is used as the herbal medicine "ginger".

"Platycodon grandiflorus" (Japanese name: Kikyo, scientific name: PLATYCODI RADIX) is the root of the bellflower (scientific name: Platycodon grandiflorus A. De Candolle), a perennial plant of the family Campanulaceae. ), and dried ones with the cork skin removed (bleached bellflowers). In herbal medicine (Japanese Pharmacopoeia), it is mainly used as an expectorant. The extraction raw material used in the present invention is preferably a bellflower root (raw dried bellflower) or a root with the cork skin removed (baked bellflower), which is used as the herbal medicine "bellflower". Incidentally, bellflower is both a crude drug name (Japanese Pharmacopoeia) and a botanical name.

"Hawthorn" (Japanese name: Yamaseko, scientific name: CRATAEGI FRUCTUS) is the false fruit of the hawthorn (Crataegus cuneata Siebold et Zuccarini) or great hawthorn (Crataegus pinnatifida Bunge var. major N. E. Brown) of the Rosaceae family, or It is cut vertically or horizontally, and is mainly used as a digestive absorption aid in herbal medicine (Japanese Pharmacopoeia). The extraction raw material used in the present invention is preferably hawthorn fruit (false fruit), which is used as the crude drug "hawthorn". Note that hawthorn is both a crude drug name (Japanese Pharmacopoeia) and a botanical name.

In the present invention, for the extraction of cinnamon bark, carrot, plantain, licorice, chimpi, ginger, bellflower, and hawthorn, the above-mentioned extraction raw materials may be used as they are, or they may be subjected to treatments such as drying, pulverization, and shredding. Good too.

The extraction method is not particularly limited, and may be, for example, a heating extraction method, a room temperature extraction method, or a cold extraction method. Examples of solvents used for extraction include water or hot water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3 -butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Among these solvents, water or hot water, lower alcohols, liquid polyhydric alcohols, and the like are preferred. These solvents may be used alone or in combination of two or more. Furthermore, a solvent whose pH has been adjusted by adding an acid or an alkali to the above extraction solvent can also be used.

There is no particular limitation on the amount of extraction solvent to be used; for example, it may be at least 10 times, preferably at least 20 times, the extraction raw material (dry weight), but in cases where concentration or isolation is performed after extraction. For convenience of operation, it is preferably 100 times or less. In addition, the extraction temperature and time depend on the target plant and the type of solvent used, but for example, 10 to 100°C, preferably 30 to 90°C, 30 minutes to 24 hours, preferably 1 to 10 hours. I can do it.

In addition, the extract may be used as it is as an extracted solution, but if necessary, concentration (concentration using an organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, It may be used after being subjected to treatments such as decolorization with activated carbon, deodorization, and ethanol precipitation. Furthermore, the extracted solution may be subjected to treatments such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

The differentiation promoter for hematopoietic stem cells according to the present invention may contain as an active ingredient the supercritical extract of Ganoderma spores obtained as described above;
It may contain as an active ingredient a mixture with extracts of one or more crude drugs selected from cinnamon bark, ginseng, plantain, licorice, chimpi, ginger, bellflower, and hawthorn. The extracts of herbal medicines used in combination with the supercritical extract of Cinnamon spores include cinnamon bark extract, carrot extract, plantain extract, licorice extract, chimpi extract, ginger extract, bellflower extract, and hawthorn extract. Any one type may be used, but two or more types are preferable, and three types are more preferable. Among these, cinnamon bark extract and carrot extract are preferred. When two or more types are used together, the combination and mixing ratio are not limited.

A supercritical extract of Ganoderma spores, or a supercritical extract of Ganoderma spores and an extract of one or more crude drugs selected from cinnamon bark, carrot, plantain, licorice, chimpi, ginger, bellflower, and hawthorn. (hereinafter referred to as "crude drug extract") has the effect of promoting differentiation of hematopoietic stem cells both at the biological level (in vivo) and at the culture level (in vitro). The agent can be administered to mammals including humans to promote hematopoietic stem cell differentiation, and can also be used as a stem cell culture medium additive to promote hematopoietic stem cell differentiation and produce blood cells. It can also be used as a research reagent or a medical reagent.

The agent for promoting differentiation of hematopoietic stem cells according to the present invention has the effect of promoting the differentiation of hematopoietic stem cells into blood cells, so that the hematopoietic stem cell differentiation function decreases or malfunctions. It is effective in treating, ameliorating, and preventing diseases or pathological conditions of the blood and hematopoietic organs caused by the lack of formation. Decreased differentiation function or insufficiency of hematopoietic stem cells is, for example, due to insufficient differentiation of hematopoietic stem cells, immature blood cells are produced, but due to qualitative abnormalities, they are destroyed in the bone marrow without fully maturing. This leads to ``hematopoiesis'' and ``dysplasia,'' in which the shape of the blood cells produced is abnormal. Therefore, diseases or pathological conditions of blood and hematopoietic organs associated with reduced or impaired differentiation function of hematopoietic stem cells include, for example, aplastic anemia (pancytopenia), myelodysplastic syndrome (MDS), thalassemia, and sideroblasts. sexual anemia, iron deficiency anemia, megaloblastic anemia, hemolytic anemia, erythroblastic aplasia, congenital anemia (e.g. sickle cellemia), paroxysmal nocturnal pigmenturia, secondary anemia (infectious disease) , malignant tumor, chronic disease, renal disease, liver disease, anemia associated with endocrine disease, etc.), malignant lymphoma, multiple myeloma, myeloproliferative diseases (polycythemia vera, essential thrombocythemia, myelofibrosis, etc.) ), idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), thrombocytopenia, autoimmune hemolytic anemia, as well as general anemic conditions (palpitations, shortness of breath, dizziness, difficulty standing up). dizziness, dyslexia, fatigue, fatigue, loss of appetite, nausea, headache, pale face, dark circles or dark circles on the skin, tinnitus, stiff shoulders, angular stomatitis, etc.), allergic diseases (hay fever, atopic dermatitis, etc.) Bronchial asthma, allergic rhinitis (runny nose, stuffy nose, sneezing), allergic conjunctivitis, allergic gastroenteritis, food allergies, urticaria, collagen disease, serum sickness, viral hepatitis, contact dermatitis, etc.), inflammatory bowel disease diseases (ulcerative colitis, Crohn's disease, etc.), autoimmune diseases (multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, pernicious anemia, etc.), infectious diseases (influenza virus, norovirus, herpes virus, AIDS) Viral infections, enterohemorrhagic Escherichia coli infections, etc.). Furthermore, "decreased differentiation function or insufficiency of hematopoietic stem cells" includes not only those caused by the above-mentioned diseases, but also those caused by administration of anticancer drugs and immunosuppressants, and radiotherapy for cancer.

The content of the crude drug extract in the agent for promoting differentiation of hematopoietic stem cells according to the present invention is not particularly limited, but depending on the nature of the extract (extract, concentrate, or dry product), for example, the content of the crude drug extract may be, for example, relative to the total amount of the drug. It is preferably 0.00001 to 10% by weight, more preferably 0.0001 to 1% by weight.

2. Method for promoting differentiation of hematopoietic stem cells, method for producing blood cells The present invention also provides a method for promoting differentiation of hematopoietic stem cells, which includes a step of culturing hematopoietic stem cells in a medium containing the above-described agent for promoting differentiation of hematopoietic stem cells, and a method for promoting differentiation of hematopoietic stem cells. The present invention relates to a method for producing blood cells, comprising the step of culturing them in a medium containing the above-mentioned hematopoietic stem cell differentiation promoter. Blood cells produced by inducing differentiation from hematopoietic stem cells in the method of the present invention can be delivered to patients as they are, or after separating the desired blood cells using separation means such as centrifugation or separation filters. It may also be prepared into an injectable formulation.

The medium used in the method of the present invention is not particularly limited, except for the addition of the above-mentioned hematopoietic stem cell differentiation promoter, and may include medium and additives commonly used for the culture and differentiation of hematopoietic stem cells. Just use it.

Specifically, the medium for culturing hematopoietic stem cells includes a basic medium containing components necessary for the survival and proliferation of stem cells (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids), such as Dulbecco's 's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle M edium: Nutrient Mixture F-12 (D-MEM/F-12), Glasgow Minimum Essential Medium (Glasgow MEM) and the like are used, but D-MEM/F-12 is preferred. In addition, the above medium may contain basic fibroblast growth factor (bFGF), leukocytes, etc., as necessary, in order to increase the proliferation rate of cells and/or to assist in inducing differentiation into blood cells. Migration factor (LIF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), transforming growth factor-α (TGF-α), granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin (EPO), stem cell factor (SCF), protease nexin I, protease nexin II, platelet derived growth factor (PDGF), tumor necrosis factor (TNF), vitamins, interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, etc.), insulin , transferrin, heparin, heparan sulfate, collagen, bovine serum albumin (BSA), fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement, etc. may be added, and antibiotics (penicillin, streptomycin, etc.) may be added. etc.) may be added. Each component of the medium is used after being sterilized by a suitable method. It is also possible to use a commercially available medium prepared by appropriately adding each of the above components to a basic medium.

In addition to the above, it is preferable that serum (for example, 10% FBS) be included at a content rate of 1 to 20%. However, since the components of serum differ depending on the lot, and the effectiveness varies, it is preferable to use the serum after performing a lot check.

According to the above-mentioned hematopoietic stem cell differentiation promoting agent according to the present invention or the method according to the present invention, the above-mentioned crude drug extract can be used alone, separately with a medium, or mixed with a medium to promote differentiation of hematopoietic stem cells. It can also be provided as a reagent kit. The kit can include an instruction manual, etc., if necessary. Alternatively, the above crude drug extract can be mixed with a medium and provided as a medium for promoting differentiation of hematopoietic stem cells.

The incubator used for culturing hematopoietic stem cells is not particularly limited as long as it is capable of culturing hematopoietic stem cells, but examples include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. Can be mentioned. The culture vessel may be either non-cell-adhesive or cell-adhesive, and is appropriately selected depending on the purpose. The cell-adhesive culture vessel may be one treated with a cell-supporting substrate such as an extracellular matrix for the purpose of improving adhesion with cells. Examples of cell-supporting substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin.

The concentration of the crude drug extract added to the medium used for stem cell culture can be appropriately determined according to the content of the crude drug extract in the above-mentioned hematopoietic stem cell differentiation promoter according to the present invention. In terms of dry matter, the concentration is, for example, 10 to 10,000 μg/mL, preferably 100 to 5,000 μg/mL. Moreover, these extracts may be added to the medium periodically during the culture period of stem cells.

The culture conditions for stem cells may follow the usual conditions used for culturing stem cells, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 36 to 37°C. The CO ₂ gas concentration is, for example, about 1-10%, preferably about 2-5%. Note that the medium is preferably replaced once every 2 to 3 days, and more preferably every day. The culture conditions can be appropriately varied and set within a range in which the stem cells can survive and proliferate.

In vitro determination of whether the differentiation of hematopoietic stem cells into blood cells has been promoted can be carried out by a method commonly used by those skilled in the art. Whether the expression level of a blood cell marker gene is significantly higher at the mRNA level or protein level in the stem cells cultured in the presence of the hematopoietic stem cell differentiation promoter according to the present invention, compared to the stem cells cultured in the presence of the agent. can be evaluated. Examples of blood cell marker genes include Hbb1, Bmaj, and Ter119 as red blood cell marker genes, Gr-1 and Fcgr3 as granulocyte marker genes, and CD11b/CD18 (Mac-1) and F4/ as monocyte/macrophage cell marker genes. Examples include, but are not limited to, 80, Id3, CD45R (Ptprc) as a B cell marker gene, and 2Ly1, CD2, and CD3e as T cell marker genes.

3. Composition for Promoting Differentiation of Hematopoietic Stem Cells into Blood Cells When administering the hematopoietic stem cell differentiation promoting agent of the present invention into a living body, it is possible to administer it as it is, but as long as the effects of the present invention are not impaired. It can be provided by being mixed with appropriate additives into various compositions such as pharmaceuticals (including quasi-drugs), food and drinks, etc.

When the hematopoietic stem cell differentiation promoter according to the present invention is incorporated into a pharmaceutical product, it is mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various preparations suitable for application to the affected area. can be converted into Pharmacologically and pharmaceutically acceptable additives include pharmaceutical bases, carriers, excipients, diluents, binders, lubricants, and coatings selected as appropriate depending on the dosage form and use. agent, disintegrant or disintegration aid, stabilizer, preservative, preservative, filler, dispersant, wetting agent, buffer, solubilizer or dissolution aid, tonicity agent, pH regulator, propellant , a coloring agent, a sweetener, a flavoring agent, a flavoring agent, etc. may be added as appropriate, and various formulations that can be administered orally or parenterally systemically or locally may be prepared by various known methods. When the pharmaceutical of the present invention is provided in each of the above forms, it can be manufactured by a manufacturing method commonly used by those skilled in the art, such as the manufacturing method shown in the Japanese Pharmacopoeia's General Preparations [2] Preparation Monograph.

The form of the pharmaceutical of the present invention is not particularly limited, but includes, for example, tablets, sugar-coated tablets, capsules, troches, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs. Oral preparations, injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, suppositories, ointments, lotions, sprays, transdermal absorption preparations , transmucosal absorption agents, and parenteral preparations such as patches. It may also be a dry product that is redissolved before use, or, in the case of injectable preparations, presented in unit-dose ampoules or multi-dose containers.

The pharmaceutical of the present invention can be administered orally or parenterally, but oral administration is preferred. When the pharmaceutical of the present invention is administered orally, it can be formulated into tablets (including sugar-coated tablets), capsules, granules, powders, troches, pills, oral solutions, emulsions, syrups, suspensions, elixirs, etc. It may be a dry product that is dissolved or redissolved before use. In addition, when administering the drug of the present invention parenterally, it is formulated into an injection (for example, a subcutaneous injection, an intravenous injection, an intramuscular injection, an intraperitoneal injection), an infusion, a suppository, etc., and then injected. Pharmaceutical formulations may be presented in unit-dose ampoules or multi-dose containers.

Formulations for oral administration include excipients such as, for example, starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; carboxymethylcellulose, calcium carboxymethylcellulose, Disintegrants or disintegration aids such as starch or hydroxypropylcellulose; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc. Coating agents such as hydroxypropyl methylcellulose, white sugar, polyethylene glycol, or titanium oxide; Bases such as vaseline, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat can be used; It is not limited to these.

Preparations for parenteral administration include solvents such as distilled water, saline, ethanol, glycerin, propylene glycol, macrogol, alum water, and vegetable oil; tonicity agents such as glucose, sodium chloride, and D-mannitol; and inorganic acids. , an organic acid, an inorganic base, an organic base, or the like can be used, but are not limited thereto.

Furthermore, in formulating a formulation, one or more active ingredients other than the crude drug extract, which is an active ingredient of the hematopoietic stem cell differentiation promoter of the present invention, may be used in combination. Active ingredients suitable for concomitant use include, for example, inorganic acid salts of iron such as iron chloride, iron sulfate, and iron phosphate; organic acid salts of iron such as iron citrate, iron gluconate, and iron lactate; heme iron; Examples include iron-protein binders such as ferritin, iron oxide, and the like. The iron compound is preferably one that can be used in pharmaceuticals or foods, such as iron citrate, sodium ferrous citrate, ferrous gluconate, iron lactate, ferrous fumarate, ferrous pyrophosphate, Examples include ferric pyrophosphate and ferrous sulfate. As the iron compound, commercially available products may be used, and one type or two or more types can be appropriately selected and used.

The pharmaceutical of the present invention functions as a prophylactic drug that suppresses the onset of the above-mentioned blood diseases and/or as a therapeutic drug that improves the blood disease to a normal state. Since the active ingredients of the pharmaceutical products of the present invention are derived from natural products, they are very safe and have no side effects. It can be administered orally or parenterally to mammals such as dogs, cats, etc. in a wide range of dosages.

The content of the hematopoietic stem cell differentiation promoter in the pharmaceutical of the present invention is not particularly limited, but is 0.001 to 30% by weight in terms of dry matter of the above crude drug extract based on the total weight of the preparation (composition). %, more preferably 0.01 to 10% by weight. The above-mentioned amounts are merely examples, and may be set and adjusted as appropriate in consideration of the type and form of the composition, the general usage amount, efficacy and effect, etc. Furthermore, the method of adding the active ingredient during formulation may be either added in advance or added during production, and may be appropriately selected in consideration of workability.

The dosage of the pharmaceutical of the present invention can be appropriately determined depending on the type of disease, the age, sex, body weight, and severity of symptoms of the subject to be administered. For example, when orally administered to adults, the daily dose is 0.1 to 1000 mg, preferably 1 to 500 mg, and more preferably 5 to 300 mg of the crude drug extract.

Furthermore, the hematopoietic stem cell differentiation promoter of the present invention can be incorporated into foods and drinks. In the present invention, foods and beverages include not only general foods and beverages, but also foods other than pharmaceuticals that can be taken for the purpose of maintaining or promoting health, such as health foods, functional foods, foods with health claims, or foods for special purposes. It is used in a meaning that includes. Health foods include foods provided under the names of nutritional supplements, health supplements, supplements, etc. Foods with health claims are defined by the Food Sanitation Law or the Food Promotion Law, and are foods for specified health uses and foods with nutritional function claims that can claim specific health effects, functions of nutritional ingredients, reduction of disease risk, etc., as well as foods with nutritional claims based on scientific evidence. This includes foods with functional claims that can be labeled with the functionality that has been reported to the Commissioner of the Consumer Affairs Agency. In addition, foods for special uses include foods for the sick, foods for the elderly, foods for infants, foods for pregnant women, etc. that are labeled as being suitable for specific subjects or patients with specific diseases. The agent for promoting differentiation of hematopoietic stem cells of the present invention is particularly useful for anemia, symptoms associated with anemia, and immunity in the elderly, pregnant women, menstruation, and diseases associated with bleeding tendency (gastric ulcer, duodenal ulcer, gastrointestinal polyps and cancer, hemorrhoids, etc.). When it is necessary to take it over a long period of time to improve or prevent muscle weakness, it can be suitably used in the above-mentioned health foods because it can be taken continuously on a daily basis. Here, indications such as specific health effects and functions of nutritional ingredients attached to food and beverages may be displayed on product containers, packaging, instructions, package inserts, etc., product flyers and pamphlets, newspapers and magazines, etc. It can be used as an advertisement for products, etc.

Furthermore, when the food and drink products of the present invention are used for mammals other than humans, they can be used to include pet food and feed.

The food or drink may be in any form suitable for eating, such as solid, liquid, granule, granule, powder, capsule, cream, or paste. In particular, the shapes of the above health foods include, for example, tablet, round, capsule, powder, granule, fine granule, troche, and liquid (including syrup, milk, and suspension). etc. are preferred.

Types of food and drink include breads, noodles, confectionery, dairy products, processed seafood/livestock foods, fats and oil processed foods, seasonings, and various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks) , milk drinks, etc.), concentrated stock solutions of the drinks, powders for preparation, etc., but are not limited thereto.

The food/beverage products of the present invention may appropriately contain commonly used additives depending on the type thereof. Any additive can be used as long as it is permissible under the Food Sanitation Act; for example, sweeteners such as glucose, sucrose, fructose, high-fructose corn syrup, aspartame, and stevia; citric acid, malic acid, etc. , acidulants such as tartaric acid; excipients such as dextrin and starch; binders, diluents, fragrances, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspensions. Examples include clouding agents and preservatives.

If the food or drink of the present invention is a general food or drink, it can be manufactured by including a step of adding a herbal medicine extract in the normal manufacturing process of the food or drink. In the case of health foods, it is sufficient to follow the manufacturing method for pharmaceuticals mentioned above. For example, for tablet-shaped supplements, additives such as excipients are added and mixed with crude drug extracts, and then processed using a tablet machine etc. It can be manufactured by applying pressure and molding. Capsule-shaped supplements are manufactured by filling liquid, suspension, paste, powder, or granular food compositions containing herbal medicine extracts into capsules, or by encapsulating and molding them with a capsule base. be able to. In addition, other materials (for example, minerals such as iron and potassium, vitamins such as vitamin C, vitamin B ₂ , vitamin B ₆ , vitamin B _12, folic acid, dietary fiber, etc.) may be added as necessary. can.

The amount of the crude drug extract in the food and drink products of the present invention may be any amount that can exert the effect of promoting differentiation of hematopoietic stem cells, but it is important to note that It may be set appropriately in consideration of taste, palatability, cost, etc.

When the food and drink of the present invention is taken for the purpose of preventing or improving the above-mentioned diseases or pathological conditions, the intake amount will vary depending on the condition of the subject, the form of intake, the intake amount, etc. 0.1 to 1000 mg, preferably 1 to 500 mg, more preferably 5 to 300 mg. The above amount may be ingested at one time, or may be divided into several times (2 to 4 times). It is preferable that the food/beverage products of the present invention are packaged or filled in a container such as a bag or a bottle, so that the amount of food/beverage products to be ingested at one time is used as a guideline for intake.

Hereinafter, the present invention will be explained in more detail with reference to Examples. However, the present invention is not limited to these.

[Example 1]
(Production Example 1) Supercritical extract of Cinnamon spores 1 kg of Cinnamon spores are placed in an extraction tank with an internal volume of 5 L, and supercritical carbon dioxide (temperature 60°C, pressure 25 MPa, carbon dioxide supply amount 15 m ³ ) is added to about 4 The mixture was fed for .5 hours, and then introduced into a separation tank (temperature: 40° C., pressure: 4 MPa) connected to the extraction tank to separate carbon dioxide gas and the extract, to obtain 15.9 g of a supercritical extract of C. spores.

(Production Example 2) Production of hot water extract of cinnamon bark 800 mL of purified water was added to 100 g of dried bark of cinnamon bark (Japanese cinnamon bark), extracted at 95 to 100°C for 2 hours, filtered, and the filtrate was concentrated, The mixture was freeze-dried to obtain 2.1 g of a hot water extract of cinnamon bark.

(Production Example 3) Production of hot water extract of carrots 800 mL of purified water was added to 100 g of dried Panax ginseng roots (carrots), extracted at 95 to 100°C for 2 hours, filtered, and the filtrate was concentrated, Freeze-drying yielded 6.7 g of a hot water carrot extract.

(Production Example 4) Production of hot water extract of plantain. 800 mL of purified water was added to 100 g of dried whole plantain plant (Chazenso), extracted at 95 to 100°C for 2 hours, filtered, and the filtrate was concentrated. , and freeze-dried to obtain 5.5 g of a hot water extract of plantain.

(Production Example 5) Production of hot water extract of licorice 800 mL of purified water was added to 100 g of dried licorice root, extracted at 95 to 100°C for 2 hours, filtered, and the filtrate was concentrated and freeze-dried. 5.1 g of hot water extract of licorice was obtained.

(Production Example 6) Production of hot water extract of Chimpi 800 mL of purified water was added to 100 g of dried peel of Mandarin orange (Chimpi), extracted at 95 to 100°C for 2 hours, filtered, and the filtrate was concentrated. , and lyophilized to obtain 6.1 g of a hot water extract of Chimpi.

(Production Example 7) Production of hot water extract of ginger. 800 mL of purified water was added to 100 g of dried ginger rhizome (Ginger ginger), extracted at 95 to 100°C for 2 hours, filtered, and the filtrate was concentrated. This was then freeze-dried to obtain 3.7 g of a hot water extract of Ginger.

(Production Example 8) Production of hot water extract of bellflower 800 mL of purified water was added to 100 g of dried bellflower root, extracted at 95 to 100°C for 2 hours, filtered, and the filtrate was concentrated and freeze-dried. 5.1 g of a hot water extract of Bellflower was obtained.

(Production Example 9) Production of hot water extract of hawthorn Add 800 mL of purified water to 100 g of dried hawthorn false fruit, extract at 95-100°C for 2 hours, filter, concentrate the filtrate, and freeze-dry. 2.6 g of a hot water extract of hawthorn was obtained.

[Example 2] Evaluation of the differentiation-promoting effect on hematopoietic stem cells Evaluation experiments were conducted as follows.

A6 cells (RIKEN BRC Cell Bank) were used as hematopoietic stem cells. The above hematopoietic stem cells maintained in 20% FBS DMEM/F12 (10 μg/mL bovine insulin, 10 μg/mL bovine Transferrin, 10 ng/mL bFGF) medium (manufactured by Gibco) were adjusted to a cell number of 7.5 × 10 ⁵ cells. The cells were seeded in a 12-well plate (manufactured by Falcon). Next, the above medium was mixed with a differentiation medium of 20% FBS DMEM/F12 (10 μg/mL bovine insulin, 10 μg/mL bovine transferrin, 10 ng/mL G-CSF, 10 ng/mL IL-3, 10 ng/mL IL-6, 15ng/mL EPO, 100ng/mL SCF) medium (manufactured by Gibco), test substances (each extract of Production Examples 1 to 18) were added to a final concentration of 100μg/mL, and cultured for 96 hours. . In the case of a mixture of extracts, each extract was added in the same ratio so that the total amount of extracts had the above concentration.

After 96 hours of culture, the cells were collected, washed twice with PBS(-), and RNA was extracted from the cells using Trizol Reagent (manufactured by Invitrogen). After reverse transcribing the extracted RNA into cDNA using a 2-STEP real-time PCR kit (manufactured by Applied Biosystems), real-time PCR (95°C: 15 60°C for 30 seconds, 40 cycles) to confirm the gene expression of Hbb1 (Hemoglobin subunit beta-1), a red blood cell marker. Other operations were performed according to established methods.

(Primer set for Hbb1)
5'-GAAAGGTGAACGCCGATGA-3' (SEQ ID NO: 1)
5'-CAAAGTACCGCTGGGTCCAA-3' (SEQ ID NO: 2)
(Primer set for 18srRNA (internal standard))
5'-CCGAGCCGCCTGGATAC-3' (SEQ ID NO: 3)
5'-CAGTTCCGAAAACCAACAAAATAGA-3' (SEQ ID NO: 4)

The effect of promoting differentiation of hematopoietic stem cells was determined by the relative expression level of Hbb1 gene (Hbb1 gene Expression level/18srRNA gene expression level) was set to 1, and relative Hbb1 gene expression level in cells cultured with the addition of the extract was calculated and evaluated. The results of these tests are shown in Table 1 below.

As shown in Table 1, supercritical extract of Cinnamon spores, cinnamon extract, carrot extract, plantain extract, licorice extract, chimpi extract, ginger extract, bellflower extract , and hawthorn extract were both found to have an excellent effect of promoting differentiation of hematopoietic stem cells (Production Examples 1 to 9). In addition, in the combination of the supercritical extract of C. chinensis spores and the extract of other crude drugs, a mixture of the supercritical extract of C. chinensis spores (Production example 1) and the cinnamon extract (Production example 2) (Production example 10) , A mixture of a supercritical extract of C. chinensis spores (Production example 1) and a carrot extract (Production example 3) (Production example 11), A supercritical extract of C. chinensis spores (Production example 1) and an extract of cinnamon bark (Production example 1) A remarkable synergistic effect was observed in the mixture (Production Example 18) of Example 2) and carrot extract (Production Example 3) in promoting differentiation of hematopoietic stem cells.

The agent for promoting differentiation of hematopoietic stem cells into blood cells of the present invention can efficiently induce differentiation of hematopoietic stem cells into blood cells in vivo or in vitro. Therefore, the present invention can be used in the field of manufacturing pharmaceuticals and supplements for treating, improving, and preventing diseases or pathological conditions of blood or hematopoietic organs associated with reduced or impaired differentiation function of hematopoietic stem cells, and the field of manufacturing transplant materials. can.

Claims (4)

An agent for promoting differentiation of hematopoietic stem cells into erythrocytes , which contains as an active ingredient a mixture of a supercritical extract of C. chinensis spores and an extract of one or two crude drugs selected from cinnamon bark and ginseng . A method for promoting differentiation of hematopoietic stem cells into red blood cells, comprising the step of culturing hematopoietic stem cells in a medium containing the agent according to claim 1 . A method for producing red blood cells, comprising the step of culturing hematopoietic stem cells in a medium containing the agent according to claim 1 . A composition for promoting differentiation of hematopoietic stem cells into red blood cells, comprising the agent according to claim 1 .