JP4717362B2 - Cell differentiation inducer - Google Patents

Description

  The present invention relates to a cell differentiation-inducing agent that exhibits differentiation-inducing activity against cancer cells such as leukemia and skin cancer, and is used as an anticancer agent or the like that suppresses the growth and proliferation of such cancer cells.

  In general, plant alkaloids are known as one of the active ingredients of anticancer agents. For example, vinca alkaloids, vincristine (trade name oncovin), and podophylline-type compounds are well known.

  The mechanism of action of anticancer drugs including these plant alkaloids includes cell division inhibition such as preventing mitosis, DNA damage, DNA synthesis inhibition, metabolic antagonism, cancer nutrition inhibition, enzyme action, and hormonal effects. In addition, not only tumor cells but also healthy normal cells have toxic effects, so serious side effects may occur, and the therapeutic effect is limited.

  An anticancer agent that does not cause such side effects is also known, and its active ingredient includes tocopherol (vitamin E) (see Patent Document 1).

  The mechanism of action of tocopherol (vitamin E) on cancer cells is not toxic to normal cells and is safe to administer for a long period of time due to its appropriate cell differentiation-inducing action, as well as various hematopoietic tumors and solid cells. Has a therapeutic or ameliorating effect on cancer.

  In addition, epidemiological studies suggest that a component comprising a water-soluble polymer fraction of green-yellow vegetables such as spinach is effective in preventing human cancer.

JP-A-6-256181

  However, the above-mentioned components showing cell differentiation-inducing action are obtained only from vitamin E and specific vegetables, and the drug efficacy has not been confirmed depending on the type of extraction material, and as many cell differentiation-inducing components as possible have been discovered. And establishment of its availability is required.

  In addition, the above-mentioned conventional plant-derived components were obtained from herbaceous foodstuffs such as green-yellow vegetables, and there was no knowledge investigating the cell differentiation-inducing properties of components extracted from non-food trees. .

  Accordingly, an object of the present invention is to provide a cell differentiation inducing agent containing a cell differentiation inducing component extracted from a conventionally unused plant, and particularly a cell differentiation inducing agent having a growth inhibitory effect on leukemia cells and skin cancer cells. It is to do.

  In order to solve the above problems, in the present invention, a hot water extract of a tree composed of cedar, pine, and oak and a hot water extract of psyllium are used in combination and are soluble in ethyl alcohol. Is used as an agent for inducing cell differentiation.

  The cell differentiation induction target cell is a cell differentiation inducer that is a cancer cell such as a leukemia cell or a skin cancer cell, that is, a leukemia cell differentiation inducer or a skin cancer cell differentiation inducer.

  As described above, the cell differentiation inducer of the present invention configured as described above has not been elucidated for its chemical components and its mechanism of action, but as is clear from the experimental results described later, it can be used for leukemia cells, skin cancer cells, etc. It has a remarkable differentiation-inducing effect on cancer cells. The cell differentiation inducer of the present invention is sufficiently reliable with respect to safety.

  Thus, the cell differentiation inducer of the present invention using the novel extract material becomes a new cell differentiation inducer of an active ingredient for cancer cells such as leukemia cells and skin cancer cells.

  The cell differentiation inducer of the present invention contains, as an active ingredient, an ethyl alcohol-soluble component in a water-soluble mixed extract in which a hot water extract of a tree containing cedar, pine and cocoons and a plantain hot water extract are used in combination. Because it is a cell differentiation inducer, it becomes a cell differentiation inducer with a novel water-soluble and ethyl alcohol-soluble extract component contained in each plant as an active ingredient, and particularly has an anti-proliferative effect on leukemia cells and skin cancer cells. It becomes a certain cell differentiation inducer.

  The hot water extract of a pulverized tree used in the present invention is a hot water extract of a tree made of cedar, pine and cocoons, and usually immerses the aforementioned predetermined tree in hot water to separate and remove the oil and fat component. The water-soluble component is fractionated.

  Cedar (scientific name: Cryptomeria japonica), which is one of the types of plants used in the present invention, is an evergreen tree of the cedar family and genus Cedar, and is a kind of genus.

  Similarly, the pine, which is one of the types of plants used in the present invention, may be any of the types belonging to the genus Pinus, which is representative of the Pinaceae family. For example, Japanese red pine, Japanese black pine, Japanese green pine, Japanese pine, etc.

  The cypress may be a species belonging to the cypress family (Chamaecyparis obtusa), for example, Thai cypress, Sawara, Benihi, North American main production areas such as Lawsonia, Note Caensis, Thiodes, and Chabohiba horticulture. Varieties).

  The part as the extraction raw material of these predetermined trees may be any part such as a leaf, a branch, a trunk, a root, and a bark.

  In the extraction, it is preferable to use a pulverized product of these trees or a cut product such as chips or sawdust, that is, a material having a surface area as large as possible with respect to the volume of the tree. In practice, it is preferable to use a material obtained by pulverizing a trunk or branch of a tree with a wood crusher, sawdust, by-product generated during sawing, sawdust, chips or the like because extraction can be performed at low cost.

  Psyllium (Plantago asiatica var.densiuscula) used in the present invention is a plant widely distributed as wild grass in Japan, and it has long been known as a edible or rabbit food, and a seed is also known as a herb with coughing and diuretic action. It is a plant.

  For such psyllium, the part used as an extraction raw material may be any of leaves, stems, roots, or the whole plant.

  Indispensable raw materials for extraction consisting of these three kinds of trees and one kind of herb are water-soluble mixed by mixing hot water extracts obtained by individually using each dry matter as an extraction raw material in a range of 10 to 90% by volume. An extract is prepared or a hot water extract may be produced from a mixture previously mixed in the predetermined range. Usually, the hot water extract is mixed in an equal volume or an extraction raw material is an equal volume ( A typical example is the case where trees are used as sawdust or sawdust.) A hot water extract is produced using each of them and a water-soluble mixed extract obtained by mixing the hot water extract produces favorable results.

  Also, in the hot water extraction process, it is convenient to extract the trees and psyllium separately from each other, in which case the trees are immersed in water and boiled under normal pressure (atmospheric pressure). It is boiled for 5 to 6 hours, and psyllium is immersed in water, boiled for about 1 hour or more and filtered, and each liquid is separated.

  And it leaves still as it is, and the oil-and-fat content which floated and isolate | separated is removed, and also a filter is passed, solid content is removed, and a water-soluble component is fractionated. A mixed extract is obtained by mixing water components extracted from trees and psyllium.

  The mixed extract obtained from the previous steps is commercially available as a nutrient solution for plants under the trade name “HB-101” from Flora Co., Ltd. The content of the hot water extract of the pulverized tree in this case Is 45% (% by weight) of cedar, 44% of cocoons, 0.5% of pine, and 0.5% of psyllium hot water extract.

  Regarding the safety of “HB-101”, the test result of acute toxicity (rat) is LD50 = 60 g / kg, and it has been found that there is no toxicity. As for long-term continuous toxicity, it has been found that there is no abnormality in livestock, poultry and fish.

  Furthermore, the volatile component of the water-soluble component of the above-mentioned mixed extract is evaporated, the obtained component (dried product) is dissolved in ethyl alcohol, and then the solid component obtained by evaporating ethyl alcohol is used as the active ingredient in the cell. Adjust differentiation inducer.

  Examples of the dosage form include oral preparations such as powders, fine granules, granules, tablets, coated tablets, capsules, injection preparations, and external preparations. At the time of formulation, it can be produced by a conventional method using an ordinary formulation carrier.

  That is, in order to produce an oral preparation, after adding an active ingredient and an excipient, and if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent, etc., the above dosage form is prepared by a conventional method. Formulate.

  Examples of the excipient include lactose, corn starch, glucose, sorbit, crystalline cellulose, and silicon dioxide. Examples of the binder include polyvinyl alcohol, methyl (ethyl) cellulose, gum arabic, tragacanth, gelatin, and polyvinylpyrrolidone. Examples of the disintegrant include starch, agar, gelatin, crystalline cellulose, calcium carbonate, dextrin, pectin, and the like. Tablets and granules may be coated with sugar, and other coatings as necessary.

  In order to produce an injectable preparation, a pH adjuster, a solubilizer, an isotonic agent and the like are added to an active ingredient, and if necessary, a solubilizing agent, a stabilizer and the like are added to prepare an ordinary preparation.

  The method for producing the external preparation is not limited and may be produced by a conventional method, that is, as a base material, for example, animal and vegetable oils, ester oils, waxes, higher alcohols, fatty acids, silicone oil, surfactants. , Phospholipids, clay minerals, purified water and the like, and pH adjusters, antioxidants, chelating agents and the like can be added as necessary.

  In addition, components such as blood flow promoters, bactericides, anti-inflammatory agents, cell activators, vitamins, amino acids, humectants, keratolytic agents, and the like can be blended as necessary. In addition, the addition amount of the base material is an amount that gives a concentration that is normally set in the manufacture of an external preparation.

  The clinical dose of the active ingredient in this invention varies depending on symptoms, severity, age, complications, etc. and is not limited, and also varies depending on the type of compound and administration route, but is usually 25 mg to 115 mg per adult day. The dose is preferably 50 mg to 250 mg, more preferably 100 mg to 500 mg, which is administered orally, intravenously or transdermally.

[Examples 1 to 3, Comparative Examples 1 and 2]
Plant for nutrient solution (Flora manufactured: HB-101) volatile components evaporated to dryness at room temperature, solid components thus obtained was dissolved in 95% ethyl alcohol, ethyl alcohol dissolution of this dry solid components The drug efficacy (proliferation inhibitory and differentiation-inducing activity against leukemia cells in humans) and safety when adjusted to a cell differentiation inducer by the following cell growth inhibition test with various concentrations of active ingredients were examined.

  The above-mentioned nutrient solution for plants (manufactured by Flora: HB-101) is obtained by adding 10 parts by weight of water to 1 part by weight of each pulverized tree made of cedar, straw or pine and boiled for 5 to 6 hours. The ripened product is filtered to remove the resin and solids, and 10 parts by weight of water is added to 1 part by weight of psyllium, and the simmered product is filtered through a filter for 10 hours. Remove, mix and concentrate each component (Brix25), add purified water, hot water extract content is 45% cedar, 44% pine, 0.5% pine, psyllium hot water extract It is adjusted to contain 0.5% and has a pH of 3.5.

<Cell growth inhibition test>
HL60 cells (human leukemia cells) are seeded on the following medium of a 12-well plate so as to be 100,000 cells / ml / well each, and the concentration of ethyl alcohol lysate in the above-mentioned dry formed component is 50 μg / ml (Example 1). ), 3 [mu] g / ml (example 2), 1 [mu] g / ml (example 3), 0.5 [mu] g / ml (Comparative example 1), 0 Pg / ml (Comparative example 2 and untreated silage), incubator 37 ° C. The culture was performed under the conditions of 3 days. In the meantime, the number of cells on the hemocytometer was counted every 24 hours using a microscope, the change in the increase / decrease was examined, the number of counted cells was recorded, and a graph was shown in FIG.

[Medium used]
RPMI-1640 Medium in 10% FCS
RPMI-1640 Medium SIGMA lot.22K2354
FCS HY Clone in USA

<Cell differentiation induction confirmation test>
(A) Specimen used: HL60 cells after cell growth suppression test (ethyl alcohol lysate concentration of 3 μg / ml added and non-added cells in HB101)
Test material: Color test Nitro Blue Tetrazolium SIGMA N6876
TPA (12-O-Tetradecanoyl-13-ace) stock solution
(B) Test method:
(1) Collecting 100 μl of HL60 cells cultured in a cell growth inhibition test (concentration of 3 μg / ml ethyl alcohol lysate (Example 2) and 0 μg / ml (Comparative Example 2: no addition)) of HB101 in dry formed components) And added to a 96-well plate.
(2) The same amount (100 μl) of NBT (nitroblue tetrazolium) in TPA (2 × 10 −6 M) solution was collected and added to each 96-well plate.
(3) After adding both solutions, the 96-well plate was incubated at 37 ° C. for 40 minutes.
(4) After completion of the culture, the color state of the cells was observed under a microscope.
(C) Evaluation method In this test, the cells induced to differentiate by the addition culture of HB101 are strongly colored in black compared to the non-addition group. After coloring, the differentiation-inducing activity in HB101-added culture was determined by comparing the degree of coloration in HB101-added section HL60 cells and non-added section HL60 cells.
(D) Test results From the above test results, the cell differentiation induction rate of HL60 cells in Comparative Example 2 (no addition group) was 0%, but the cell differentiation induction rate of HL60 cells in Example 2 was 4.2. %Met.

As is apparent from the results of the cell growth inhibition test and the cell differentiation induction confirmation test shown in FIG. 1, the concentration of the ethyl alcohol lysate in the dry formed component was set to 0.5 μg / ml of Comparative Example 1 or 0 μg / ml. In Comparative Example 2, almost no cell growth inhibitory effect on human leukemia cells was observed, but in the case of the active ingredient concentrations obtained in Examples 1, 2, and 3, stable cell growth inhibitory effects on human leukemia cells were obtained. Admitted. Further, as is clear from the above test result (d), cell differentiation induction was observed at a concentration of 1 to 50 μg / ml of the ethyl alcohol lysate in the dry component.

<Safety test>
(1) 2000 mg / kg of Example 2 was orally administered once to five Crj: CD (SD) IGS male rats, and the toxicity was examined. As a result, there were no deaths, and no abnormalities were observed in general condition, body weight, and necropsy. Therefore, the minimum lethal dose of Example 2 was determined to exceed 2000 mg / kg.

(2) A 500-fold water dilution of Example 2 was administered to rats (SD system, 3 animals) in the administration group using a gastric tube, and 5 mg of physiological saline was administered to the rats (SD system, 3 animals) in the control group. / Kg was administered. After breeding for 1 week, the blood was sacrificed after anesthesia. Total RNA was extracted from individual rat livers. Then, 1031 genes related to toxicity are selected, and a fluorescently labeled RNA extracted from liver cells is hybridized on a DNA chip to which a probe for detecting the gene is affixed. The amount was measured for each gene.
As for the genes that have more than 5 times increase / decrease in the genes related to toxins, there are changes in genes such as cytosolic epoxide hydrolase, stress activated protein kinase alpha II, N-heparan sulfate sulfotranferase, and connexin protein. Since there is a tendency to decrease and the amount of change is not large numerically, no toxic effect is seen, and the amount of change of other genes is also very small, so it is considered that there is almost no toxic reaction in Example 2. It was.

(3) A 500-fold water dilution of Example 2 was administered to rats (SD system, 3 animals) in the administration group with a gastric tube, and 5 mg of physiological saline was administered to the rats (SD system, 3 animals) in the control group. / Kg was administered. After breeding for 1 week, the blood was sacrificed after anesthesia. Serum was separated and collected from the blood collected at the time of blood removal of each rat by centrifugation (3000 rpm for 15 minutes), and this serum was used as a sample.
2 μl of sample serum was dropped onto the H4 protein chip, and after washing, immobilization was performed on the chip with 0.5 μl of EAMEAM. The H4 protein chip on which the protein was immobilized was set on a protein chip analyzer SELDI, and the molecular weight distribution was measured.
As a result, five main peaks were observed in the molecular weight of 5000 to 15000, but all of them were observed in both the control group and the administration group, and no clear tendency of the difference in the amount was observed.
In addition, 8 main peaks were observed at molecular weights of 15000 to 50000, all of which were observed in both the control group and the administration group, and there was a clear tendency for the difference in the amounts. There wasn't.
From these results, no difference in the amount of expressed protein in serum was observed between the administration group and the control group, and no toxic reaction was observed.

Chart showing results of cell growth inhibition test of Examples and Comparative Examples

Claims (1)

Each hot water in which 10 parts by weight of water is added to each 1 part by weight of a cedar, oak or pine tree pulverized product, and this is boiled for 5 to 6 hours to remove the resin and solids by filtration. 10 parts by weight of water is added to 1 part by weight of psyllium, and the hot water extract obtained by filtering the simmered product for 10 hours to remove solids is used for each hot water extract. Leukemia cells comprising an ethyl alcohol-soluble component as an active ingredient in a dry product of a water-soluble mixed extract whose content is adjusted to 45% cedar, 44% cocoon, 0.5% pine, and 0.5% psyllium Cell differentiation-inducing agent.

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