JP6093238B2 - Hematopoietic progenitor cell recovery agent for radiation damage - Google Patents

Description

  The present invention relates to a hematopoietic disorder-recovering agent and an immune function activator for a hematopoietic disorder and immune function reduction caused by radiation.

  Radiation has become a powerful weapon in chemotherapy and radiation therapy for malignant tumors. However, it is known that in a tumor-bearing state, the immune function of the living body is lowered through a lymphoid disorder and adversely affects the host. Clinically, suppression of hematopoietic cells such as anemia, leukopenia associated with lymphocyte depletion, and thrombocytopenia is observed (Non-patent Document 1). These symptoms are not desired for the original purpose of treatment. Therefore, there is a need for a useful method for preventing and improving hematopoietic / reticuloendothelial system disorders and immune function induced by radiation.

On the other hand, Ganoderma is known as a herbal medicine obtained from the fruit body of Amanentake mushroom, and is said to have tonicity, blood supplementation, mental stability, water use, and liver replacement in Chinese medicine. In recent years, Ganoderma can be supplied in large quantities due to the success of artificial cultivation, and various studies have been conducted on its effectiveness. For example, Ganoderma active steroids play a role in helping endonuclease function and show an apoptosis-inducing action that activates the function of genes that incorporate an apoptotic program. It has been reported that the uric acid level shows a dose-dependent inhibitory action (Non-patent Documents 2 and 3).
However, there has been no report on the relationship between ganoderma and hematopoietic disorders associated with radiation irradiation or immune function decline.

Experimental Hematology 18 (1): 18-22, 1990 Mie University Research Center for Social Collaboration 18: 129-134, 2010 Mie University Research Center for Social Collaboration 19: 77-83, 2011

  The present invention relates to providing a hematopoietic disorder recovering agent and an immune function activator for a hematopoietic disorder induced by radiation and a decrease in immune function.

  The present inventors have conducted extensive research on a substance having a radioprotective action from the viewpoint of analysis of hematopoietic progenitor cells and antibody-producing immune responses. As a result, Ganoderma is a hematopoietic / reticuloendothelial system induced by radiation. It has been found that it has an alleviating effect on injury, and Ganoderma promotes antibody immunity to sheep erythrocytes, and histological findings show recovery of hematopoietic foci and reduction of spleen / thymopathy. The present invention has been completed.

That is, this invention provides the hematopoietic disorder recovery agent by the radiation which uses ganoderma or its extract as an active ingredient.
Moreover, this invention provides the activator with respect to the immune function fall by the radiation which uses ganoderma or its extract as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the hematopoietic and reticuloendothelial system function impaired by irradiation of radiation and the antibody-producing ability of an immune lymphoid cell can be recovered. Ganoderma is very useful for the prevention and improvement of side effects in radiation therapy and the like because of its rich dietary experience and high safety.

It is a figure which shows the tissue slice of the thymus and spleen 10 days after X-ray irradiation.

Ganoderma used in the present invention is a fruit body of Ganoderma lucidum . Ganoderma is classified into red turf, black turf, green turf, white turf, yellow turf, purple turf, etc. depending on its color, but these are not different in the original plant, but due to differences in lineage, growth conditions, etc. . In the present invention, it is preferable to use Ganoderma belonging to the Gogaku Ganoderma GY (Minowa) system. Ganoderma belonging to the Gogaku Ganoderma GY series is classified and fixed by Yukio Naoi and is called Naoi Ganoderma.
Ganoderma may be used as it is or after it is dried, and may be further processed. Examples of the treatment include cutting, crushing, grinding, and crushing.

Ganoderma extract can be obtained by subjecting Ganoderma lucidum or drying or subjecting them to an extraction step. The reishi extract may be a commercially available one or various solvent extracts obtained by a conventional method.
The extraction means for obtaining the ganoderma extract is not particularly limited, and for example, solid-liquid extraction, immersion, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, stirring, and the like can be used.

  Examples of the solvent for extraction include water, lower alcohols, polyhydric alcohols, ketones, esters, ethers, polyethers, hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, Examples include supercritical carbon dioxide, oils, and mixtures thereof. Of these, water, lower alcohols, and alcohol-water mixtures are preferable, and water is more preferable.

  As a usage-amount of a solvent, 1-100 mL is preferable with respect to 1 g of ganoderma (dry weight conversion), As extraction time, 1 minute-10 days are preferable, and 30 minutes-1 day are more preferable. The extraction temperature at this time is 0 ° C to the boiling point of the solvent, more preferably 20 to 100 ° C.

  The extract of ganoderma obtained in this way may be used as it is as an extract or a fraction, may be used as a diluted solution diluted with an appropriate solvent, or may be a concentrated extract or a dry powder, or in a paste form. It may be prepared. It can also be lyophilized and diluted with a solvent used for extraction, such as water, at the time of use.

  The reishi extract may be a crude product as long as it conforms to food and pharmaceutical acceptable standards and exhibits the effects of the present invention. These purities may be increased by appropriately combining purification methods. Examples of purification means include precipitation using an organic solvent, centrifugation, ultrafiltration membrane, high performance liquid chromatograph, column chromatograph, and the like.

  Ganoderma extract preferably contains 2% by weight or more, preferably 3% by weight or more of polysaccharides from the viewpoint that the effects of the present invention are effectively exhibited. Examples of the polysaccharide include β-glucan, glucurono-β-glucan and the like. An example of the composition of the reishi extract is as described in the examples described later.

Ganoderma or its extract has a radiation-proofing effect, activates the hematopoietic / reticuloendothelial system function and immune function, which are damaged by irradiation, and promotes recovery, as shown in the Examples described later. Therefore, this invention provides the hematopoietic disorder recovery agent by the radiation which uses Ganoderma or its extract as an active ingredient, and the activator with respect to the immune function fall by radiation.
In the present specification, radiation means ionizing radiation, and ionizing radiation includes particle beams and electromagnetic waves.
Hematopoietic disorder recovery agent and activator for immune function reduction of the present invention is a pharmaceutical, food or other composition for preventing / suppressing / reducing / promoting / ameliorating hematopoietic disorder or immune function decrease caused by radiation Can be used for the manufacture of things.
These pharmaceuticals, foods and other compositions may contain pharmaceutically acceptable carriers, additives, other active ingredients, pharmacological ingredients, etc., as necessary, as long as the effects of the present invention are exhibited. .

  The content of the hematopoietic disorder recovering agent and the activator for reducing immune function in the above-mentioned pharmaceuticals, foods and other compositions is preferably 0.5% by weight or more, more preferably as a ganoderma extract (dry matter equivalent). 1% by weight or more, more preferably 10% by weight or more, on the other hand, preferably 100% by weight or less, more preferably 90% by weight or less, still more preferably 50% by weight or less.

The medicament can be administered in any dosage form. Examples of the dosage form include parenteral administration such as injections, suppositories, inhalants, transdermal absorption agents, external preparations, or oral administration such as tablets, capsules, granules, powders, syrups and the like.
In order to prepare pharmaceuticals of such various dosage forms, pharmaceutically acceptable excipients, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives , Flavoring agents, flavoring agents, coating agents, diluents and the like can be used in appropriate combinations.

Also, the form of the food or other composition can be solid, semi-solid or liquid. Examples of the food include various foods such as breads, cakes, noodles, confectionery, jelly, frozen foods, ice creams, dairy products, and beverages, and forms similar to the above-mentioned oral preparations (tablets, Capsules, syrups, etc.).
To prepare various forms of food or other compositions, other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, antioxidants, humectants, A thickener or the like can be used in appropriate combination.

The dose or intake of the hematopoietic disorder-restoring agent and activator for lowering immune function of the present invention may vary according to the condition, weight, sex, age or other factors of the subject, but in the case of oral administration or ingestion, adult 1 It is preferable that per day, the extract of ganoderma extract (in terms of dry matter) is 1 mg to 100 mg / kg per day.
The timing of administering or ingesting the hematopoietic disorder recovering agent and the activator for lowering immune function of the present invention may be any timing, but is preferably before or after irradiation, or simultaneously with irradiation. .

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

1. Ganoderma extract Naoi Ganoderma (Lot No. 0008) was used as the ganoderma. To 200 g of pulverized Naoi Reishi, 2000 mL of purified water was added, extracted by stirring at 100 ° C. for 2 hours, and centrifuged at 12,000 r / min for 10 minutes to obtain a hot water extract. Subsequently, the obtained hot water extract was freeze-dried in vacuum to obtain a ganoderma extract. The yield was 19.6%.
The chemical composition of Ganoderma lucidum extract was measured by a general analysis method. The crude ash content was 4.5% (% by weight, hereinafter the same), crude protein 12.4%, crude lipid 4.2%, crude fiber 30.2. %, Soluble carbohydrates 20.1%, water-soluble total sugars 26.8%, ergosterol 1.8%.
The polysaccharide content of Ganoderma extract was 14.2%, and its composition was as follows.

  In the next test, a certain amount of Ganoderma lucidum extract dissolved in physiological saline and sterilized at 120 ° C. for 20 minutes was used.

2. Experimental Animals and Breeding Conditions Six-week-old ICR / Slc male mice (Japan SLC Co., Ltd.) were preliminarily raised for 7 days, and then mice that showed no abnormality in general symptom observation and urinalysis were subjected to the test. The mice are 10 mice per group under the rearing conditions of a barrier system at a temperature of 23 ± 2 ° C. and a relative humidity of 55 ± 5%, and 5 mice are allowed to live together in a plastic cage, and a solid feed (CLEA CE-7) is used. And drinking tap water freely.

3. X-ray irradiation conditions Using an X-ray irradiation apparatus PHILIPS MG226 / 4.5 (Philips high-precision X-ray generator), one mouse at a time under irradiation conditions of a tube voltage of 200 KV, a tube current of 9 mA, and a dose rate of 0.365 Gy / min X-ray whole body irradiation with doses of 7.0 Gy and 5.0 Gy was performed while rotating horizontally in an acrylic capsule made of irradiation.

4). Test Example 1 Measurement of mature blood cell level and number of hematopoietic progenitor cells For measurement, three groups were set: a 5.0 Gy irradiated / ganoderma treated group, a non-irradiated group, and a 5.0 Gy irradiated group. The 5.0 Gy irradiation / ganoderma administration group and the 5.0 Gy irradiation group performed X-ray whole body irradiation with a dose of 5.0 Gy on the first day.
In the 5.0 Gy irradiation / ganoderma administration group, the dose of ganoderma extract was 500 mg / kg × 2 / day for 10 days from the next day of X-ray whole body irradiation, and the ratio of 0.2 mL / mouse body weight of 10 g to the mouse. Then, it was orally administered twice a day (morning and evening) every day. On day 11, heparinized peripheral blood was collected from each posterior aorta under ether anesthesia.
In the non-irradiated group and the 5.0 Gy irradiated group, the same amount of physiological saline was administered instead of the reishi extract, and the same treatment as in the 5.0 Gy irradiated / reishi administered group was performed.

  In the examination of the mature blood cell level, the red blood cell count, white blood cell count, and platelet count were measured with a fully automatic multi-item analyzer (JEOL). In addition, blood smears were prepared, stained with Giemsa, and the number of monocytes, lymphocytes and granulocytes were measured under a microscope.

Preparation of mouse spleen cell suspension and bone marrow cell suspension was carried out according to the method of Ninho (Yoshiyuki Nibo, in vitro colony formation method in mouse spleen cells, edited by the Japanese Society for Immunology) 1301-1304, 1978; Yoshiyuki Niho, in vitro colony formation method in mouse bone marrow cells, edited by Japanese Society for Immunology, Immunological Experiment Procedure B Japanese Immunology Society, Kanazawa: 1305-1310, 1978).
For the spleen cell suspension, the spleen was removed from the mouse, transferred to Eagles' MEM medium (Grand Island, NY, USA) that had been cooled to 4 ° C., and then two slide glasses. Was used to crush the spleen to obtain a spleen cell suspension. Further, it was filtered through a stainless steel mesh to obtain a single cell suspension, which was used for culture or detection of antibody-producing cells.
One femur bone marrow is suspended in DMEM medium (Nishisui Pharmaceutical Co. Tokyo, Japan) containing 20% fetal bovine serum (Sigma Chemical Co., St. Louis, Mo., USA). The number of bone marrow (nucleated) cells per bone was calculated.
In measurement of hematopoietic progenitor cells, BFU-E (Erythroid burst-forming cell), mouse spleen cells or bone marrow cells were 2 × 10 ⁶ cells / ml, CFU-E (Colony-forming unit- For measurement of erythropoetin dependent, late erythroblastic progenitor cells), mouse spleen cells or bone marrow cells at a concentration of 2 × 10 ⁵ cells / mL and a 35 mm diameter plastic Petri dish (Falcon, No. 1008, USA). ) In the presence of 2 U / mL EPO (human recombinant recombinant erythropoietin Sigma Chemical Co., St. Louis, Mo., USA).
For the measurement of CFU-GM (Granulocycles-macrophage colony-forming cell, granulocyte-macrophage colony-forming cell), the cells were 1% Pokeweed mitogen (GIBCO, Grand Island, N. Y., U.S.) at a concentration of 2 × 10 ⁵ cells / mL. S.A.) suspended in RPMI 1640 medium (Sigma Chemical Co., St. Louis, Mo., USA) containing 10% fetal calf serum, at 37 ° C., 5% CO ₂ . Cultured for one week.
The results are shown in Table 2.

From Table 2, in the 5.0 Gy irradiated group, compared to the non-irradiated group, peripheral red blood cell count, platelet count, peripheral white blood cell count, granulocyte count, lymphocyte count, monocyte count, early erythroid progenitor cell count, Both the number of late erythroid progenitor cells and the number of granulocyte macrophage colony forming cells were significantly decreased. As a result, it was confirmed that hematopoietic cells are damaged by irradiation.
On the other hand, in the 5.0 Gy irradiation / ganoderma administration group, a significant increase in the number of red blood cells and white blood cells centering on granulocytes was observed as compared to the 5.0 Gy irradiation group. In addition, a significant increase in the number of CFU-GM, which is a progenitor cell in the bone marrow and spleen, was observed, and it was revealed that administration of ganoderma stimulates the proliferation of hematopoietic cells. Furthermore, the number of undifferentiated erythroid hematopoietic progenitor cells, BFU-E, was significantly increased in the bone marrow and spleen.

5. Test Example 2 Measurement of Spleen / Thymus Index and Antibody Producing Cells In the measurement, three groups were set: a 5.0 Gy irradiated / ganoderma administration group, a non-irradiated group, and a 5.0 Gy irradiated group. Mouse spleen and thymus weight, spleen cell count, antibody-producing cell immune response test was performed according to a method improved by the method of Simpson et al. J Immunol Meth 21 (1-2): 159-165, 1978).
The 5.0 Gy irradiation / ganoderma administration group and the 5.0 Gy irradiation group performed X-ray whole body irradiation with a dose of 5.0 Gy on the first day. In the 5.0 Gy irradiation / ganoderma administration group, the dose of ganoderma extract was 500 mg / kg × 2 / day for 10 days from the next day of X-ray whole body irradiation, and the ratio of 0.2 mL / mouse body weight of 10 g to the mouse. Then, it was orally administered twice a day (morning and evening) every day. On day 11, all blood was collected under ether anesthesia and euthanized, and then the spleen and thymus were removed and weighed.
Furthermore, splenocytes 1 × 10 ⁷ cells / mL, mixed with sheep erythrocytes ^{1 × 10 6 cells / mL,} 10% guinea pig serum (complement serum) 1 mL were incubated for 1 hour at 37 ℃, 2,000r / min, 10 Centrifugation was performed for minutes. Absorbance at 413 nm of the obtained supernatant was measured to examine antibody-producing cells.
The results are shown in Table 3.

From Table 3, the spleen / thymus weight, the number of spleen cells, and the number of antibody-producing cells against sheep erythrocytes (QHS) in the 5.0 Gy irradiated group were 30.1%, 25.9%, and 22 respectively. .6%, 30.3% decrease.
On the other hand, in the 5.0 Gy irradiated / ganoderma administration group, the spleen / thymus weight, the number of spleen cells and QHS were close to the level of the non-irradiated group mice, and all the values were significantly increased compared to the 5.0 Gy irradiated group. Admitted. From these results, it was confirmed that Ganoderma was effective in activating the reticuloendothelial system function impaired by irradiation and the antibody-producing ability of immune lymphoid cells and promoting recovery.

6). Test Example 3 Histological Examination For measurement, two groups, a 7.0 Gy irradiation / ganoderma administration group and a 7.0 Gy irradiation group, were set, and X-ray whole body irradiation with a dose of 7.0 Gy was performed on the first day.
In the 7.0 Gy irradiation / ganoderma administration group, the dose of ganoderma extract is 500 mg / kg × 2 / day for 10 days from the next day of whole body X-ray irradiation. Then, it was orally administered twice a day (morning and evening) every day. In the 7.0 Gy irradiated group, the same amount of physiological saline was orally administered instead of Ganoderma extract.
On the 11th day after X-ray whole body irradiation, the spleen and thymus of 3 patients in each group were excised, fixed with 10% formalin solution at 4 ° C, and 4μ paraffin sections were prepared by a conventional method, followed by hematoxylin and eosin staining. And observed.
In addition, about the statistical process, the test was performed by Student's t-test. The results are shown in FIG.

From (a) of FIG. 1, in the thymus of the 7.0 Gy irradiated group, the destruction of lymphocytes was strong, and nuclear debris and cells showing nuclear enrichment were scattered, and large reticulum cells proliferated instead. . Lymphocytes decreased, the cortex / medullary boundary became unclear, and images replaced with reticulum cells were scattered.
From FIG. 1 (b), in the thymus of the 7.0 Gy irradiated / ganoderma administration group, the thymus weight after irradiation increased markedly, and histologically, lymphocytes decreased by irradiation with 7.0 Gy. The recovery tendency of the strong lymphocyte count was shown compared with the irradiation group.
Further, from FIG. 1 (c), in the spleen of the 7.0 Gy irradiated group, lymphocyte depletion of white pulp was observed and lymph follicles were reduced. Significant hemosiderin pigmentation, which is thought to be the destruction of red blood cells, was observed in the red spleen.
From (d) of FIG. 1, in the spleen of the 7.0 Gy irradiated / ganoderma administration group, the spleen weight after irradiation increased remarkably, and histologically, destruction of erythrocytes and hemosiderin pigmentation by irradiation were almost complete. No hematopoiesis recovery was observed. It was also confirmed that spleen damage was reduced.

  From these results, it was confirmed that the extract of Ganoderma is useful for promoting the recovery of radiation-induced hematopoietic damage and also activates the reduction of antibody-producing immune response.

Claims (4)

A hematopoietic progenitor cell recovery agent for radiation damage comprising a water extract of Reishi belonging to the Gotake Reishi GY (Minowa) system as an active ingredient. The hematopoietic progenitor cell recovery agent for radiation damage according to claim 1 , wherein the water extract of Ganoderma turf contains a polysaccharide having the composition shown in the following table .
A food for recovery of hematopoietic progenitor cells against radiation damage comprising a water extract of Ganoderma belonging to the Gogaku Ganoderma GY (Minowa) system as an active ingredient. The food for recovering hematopoietic progenitor cells against radiation damage according to claim 3, wherein the water extract of Ganoderma turf contains a polysaccharide having the composition shown in the following table.