JP4912720B2 - 茸 -derived osteoclast formation inhibitor - Google Patents

Description

  The present invention finds that Anninkou native to Chile and Argentina in South America has a unique fragrance and is locally appreciated and has an effect of inhibiting the formation of osteoclasts. An object of the present invention is to provide an osteoclast formation inhibitor effective for metabolic bone diseases such as osteoporosis, rheumatoid arthritis, periodontal disease, etc. by obtaining an aqueous extract from the substance or mycelium as a raw material.

An'ninkou of the present invention is referred to as a scientific name Gurifora-Garugaru (Grifola gargal) or Gurifora-Sorudurenta (Grifola sordulenta), is an edible mushroom with a strong aroma native to South America Chile Patagonia Toka Argentina, in Japan not yet known It was. The present applicant collects various strains of Griphora Gargal and Griphora Soldurenta locally and holds more than 10 kinds as inoculum. In the present invention, these moths belonging to Griphora Gargal or Griphora Soldurenta are called Anninkou.
Typical examples of Grifola spp. Are Grifola frondosa , Japanese name, and Maiko. Maiko has succeeded in artificial cultivation, and has been cultivated in a polypropylene bag with a medium containing nutrients in hardwood sawdust and can be easily purchased throughout Japan throughout the year. However, maiko has no osteoclast formation inhibitory effect.

  Since bone is a hard tissue and difficult to handle as research material, elucidation of its metabolism was delayed. Recently, methods for isolating osteoblasts, bone cells, and osteoclasts that constitute bone have been established and their metabolism is being investigated. Bone chemically consists of about 80% minerals such as calcium phosphate and about 20% organics such as collagen. Osteoblasts are present on the inner surface of bone tissue and actively secrete bone matrix proteins such as collagen. Crystals of calcium phosphate and hydroxyapatite are deposited on this matrix protein, resulting in a hard bone tissue.

  In contrast to such highly active osteoblasts, there are flat and inactive osteoblasts on the surface of bone tissue. Although these cells have poor matrix synthesis ability, they communicate with bone cells buried in the bone matrix through processes, and may have some signal transduction. Bone cells extend many elongated cell processes, and the cell processes contact each other and other cells in the bone through gap junctions to form a highly developed intercellular network. By this network, bone cells are thought to control each other and carry out bone metabolism. In addition, osteolysis by osteoclasts seems to play an important role in physiological calcium homeostasis.

Osteoclasts are multinucleated giant cells having a diameter of 20 to 100 μm having 2 to several tens of nuclei, and are cells that lyse bone. Organizationally, it is a tartrate-resistant acid phosphatase positive multinucleated cell.
Osteoclasts arise from hematopoietic stem cells. The generated precursor osteoclasts differentiate into osteoclasts, and finally some osteoclasts fuse to form multinucleated mature osteoclasts.
When osteoclasts dissolve bone matrix (bone resorption), while osteoblasts synthesize bone matrix, bone formation, growth, and metabolism are carried out smoothly and bone strength is maintained. Conceivable.

In the present invention, the formation of osteoclasts is inhibited by the water-soluble components in the fruit bodies and mycelium of the ginseng. Furthermore, the present inventors have succeeded in an artificial cultivation method of Anninkou which is not yet known in Japan.
There exist patent document 1 and patent document 2 as an osteoclast formation inhibitor. Patent Document 1 is an osteoclast formation inhibitor containing sulfated glycosaminoglycan or a salt thereof as an active ingredient, and Patent Document 2 is an osteoclast formation inhibitor containing interleukin 18 or a functional derivative thereof. It is. The present invention has been completed by finding an osteoclast formation inhibitory effect on a specific moss.
Japanese Patent Laid-Open No. 2004-210715 JP-A-10-236974

In general, Anninkou has a strong fragrance. In addition, a large amount of aroma components are released especially during mycelium culture. Benzaldehyde and cinnamaldehyde were specified as fragrance components. Although the mycelium can be cultured easily, it was difficult to generate fruit bodies. Even if the same operation is performed, a child entity may or may not occur, and the amount of the occurrence varies. Moreover, even if the primordial was formed, it took a long time for its growth, the yield was low, or the fruiting body was small, and it was difficult to artificially cultivate.
As a result of completing a method for cultivating fruit bodies in large quantities, the intake of anninkou of the experimental personnel and their families increased. As a result, it has been recognized among concerned parties that Anninkou ameliorates metabolic bone disease. Therefore, it was necessary to confirm this fact at least in cell units.

The object of the present invention is to solve the above-mentioned problems, and the composition thereof is an osteoclast containing an aqueous extract of the fruit body of the scientific name Grifola gargal and / or Grifola sordulenta as an active ingredient. It is a cell formation inhibitor or an osteoclast formation inhibitor containing an aqueous extract of mycelium of the scientific name Grifola gargal and / or Grifola sordulenta as an active ingredient.

  The present inventors succeeded in artificial cultivation of the fruit body of Anninkou, and distributed a part of the fruit body obtained to the research personnel. As a result, we obtained several information that the pain of osteoporosis was relieved. In order to confirm this information, the osteoclast formation inhibitory effect was confirmed at the cellular level for various fruit body extracts. As a result, the water extract and the ethyl acetate extract were remarkably effective. At the same time, toxicity tests were conducted to confirm that the water extract and ethyl acetate extract of Anninkou were not cytotoxic.

  According to the present invention, it is possible to prevent, ameliorate, and cure diseases caused by abnormal bone metabolism, including osteoporosis, simply by eating anninko as a delicious candy. Furthermore, it becomes possible to ingest more active ingredients by extracting the active ingredients from the carrot and providing them as so-called health foods. Furthermore, it was confirmed that the mycelium that can be easily cultivated has the same effect without affecting the fruit body.

The Anninkou according to the present invention can be collected by actually visiting Chile and Argentina in South America, entering forests such as Antarctic beech, and searching for Anninkou. Spores or mycelia can be separated from the collected ginseng, and the ginseng can be artificially cultivated by the cultivation method of the present invention. Alternatively, the spore or mycelium can be obtained by purchasing fruit bodies of cultivated anninkou sold by the present applicant.
The fruit body of anninkou is similar to maiko in shape, but it is slightly small, wide in leaf width, pale in color, ocher or light brown maiko, has fragrance, and excellent taste .

  In cultivating anninkou, the medium may be a medium usually used for the cultivation of maiko, and a medium combined with sawdust from Antarctic beech growing in South America is also preferred. For example, Antarctic beech and other hardwood sawdust are used as the medium base material, and rice bran, bran, beer lees, etc. are used as the nutrient material, and the medium base material / nutrient material = 4/1 to 11/1, preferably 5/1 Mix at a ratio of 9/1 (volume ratio) and adjust the water content to 60-70% to make a medium. In use, these media are put in a culture bottle or a culture bag, sterilized under high pressure, and used after cooling.

  In the first step, inoculation and culture of the inoculum may be the same as in ordinary maiko, but the temperature is 18-30 ° C, preferably 18-25 ° C, relative humidity 50-80%, preferably 60-70. % Substantially dark conditions are desirable. In the first step, the mycelium spreads throughout the medium, and a strong aroma mainly composed of benzaldehyde spreads throughout the culture chamber. This intense fragrance is characteristic of Anninkou. The content of benzaldehyde in the mycelium on the surface of the medium at this time is several hundred to 700 μg / g, and most is 200 to 500 μg / g.

The second step is a step of stimulating sufficiently spread mycelium. Stimulation uses light irradiation, low temperature, or both light irradiation and low temperature stimulation. The light stimulus has an illuminance of about 100 to 3000 Lx. Low temperature stimulation can also be used, and the temperature is lowered by 7 to 17 ° C., preferably 10 to 15 ° C. from the temperature in the first step.
The timing of stimulating is extremely important, and a stable primordium cannot be reliably formed if it is too late or too early. As described above, a strong fragrance mainly composed of benzaldehyde is diffused during mycelial culture, but this fragrance may suddenly drastically decrease. The benzaldehyde content of the mycelium on the medium surface at this time is 100 μg / g or less, for example, 30 to 80 μg / g. Give a stimulus without missing this time.

The third step of growing the primordium formed in the second step is almost the same as ordinary maiko, and is cultured at a relative humidity of 90 to 100%. When culturing continues, the surface changes from white to gray and then to black. After the surface of the primordium has turned black, the culture bottles are removed by opening the culture bottle lid or opening the culture bag, etc., and the culture is promoted by reducing the concentration of carbon dioxide in the culture medium. be able to.
According to the method of the present invention, it is possible to stably harvest 80 to 120 g of fruit bodies of anninkou from 1 kg of the medium in about 80 to 120 days after inoculation with the inoculum.

In the third step, the fourth step can be performed just before the fruiting body is sufficiently developed. That is, in a state where the fruiting body has grown 80 to 95%, the carbon dioxide concentration and humidity are increased and the cultivation is continued for 20 to 60 hours, whereby an anninkou having a strong aroma can be obtained. Increasing the carbon dioxide concentration and humidity can be achieved by surrounding the culture shelf with a transparent plate, individually enclosing the culture medium, or covering the culture shelf.
The cultivated anninkou obtained through the fourth step has a strong fragrance, and the main component of the fragrance is benzaldehyde.
Anninkou cultivated by the method of the present invention can be distinguished by a light-colored maiko-like appearance and a strong aroma mainly composed of benzaldehyde.

For the culture of mycelium, any of the solid culture method and liquid culture method usually used for culture of basidiomycetes can be used.
Any carbon source that can be assimilated, such as glucose, sucrose, maltose, starch, etc. can be used as the carbon source. As the nitrogen source, ammonium sulfate, ammonium nitrate, sodium nitrate, urea and the like, and natural complex nutrient sources such as potato extract, carrot extract, malt extract, onion extract, corn steep liquor, yeast extract, peptone and the like can be used. The pH is slightly acidic, preferably 4-5. The culture temperature is 15 to 27 ° C, preferably 19 to 25 ° C.
The culture is usually performed under aerobic conditions. For example, a shaking culture method or an aeration and agitation culture method is used.

The degree of osteoclast formation inhibitory effect and the degree of cytotoxicity can be tested by the following methods.
The fruit body of anninkou, its crushed product and / or dried product are extracted with water, warm water or hot water, or boiled and extracted to obtain an extract with a constant concentration. Alternatively, the mycelium culture is centrifuged. The obtained centrifuged product is directly or after drying, extracted with water, warm water or hot water, or boiled to obtain an extract with a constant concentration as a sample solution.

A certain amount of sample solution, UAMS-32 cells, which are osteoblast-like cell lines, and mouse bone marrow cells contain 10 ⁻⁸ M active vitamin D ₃ and 10 ⁻⁶ M prostaglandin E ₂ . Osteoclasts are formed by culturing in the culture for 5-6 days. The medium is changed once every 2-3 days. After confirmation of osteoclast formation, the osteoclast formation inhibitory effect of the sample solution can be measured by staining the osteoclast with TRAP and measuring under a microscope. At the same time, the MTT activity can also be measured to determine the degree of cytotoxicity of the sample.

A control value is obtained by performing the above-described experiment without adding a sample solution, and the osteoclast formation inhibitory effect and cytotoxicity can be objectively measured by comparison with this control value.
As a result of the experiment, the fruiting body of Anninkou was confirmed to have a remarkable inhibitory effect on osteoclast formation and was not cytotoxic at all. As for the mycelium of Anninkou, the effect of inhibiting osteoclast formation was confirmed without reaching the fruit body.

Osteoclast formation inhibition test A mouse coculture method of bone marrow cells and osteoblast-like stromal cells was used. Osteoclast formation inhibitory activity was evaluated by counting the number of osteoclasts (multinucleated, TRAP positive) formed.
Bone marrow cells 1.3 × 10 ⁸ collected from the tibia and femur of mice (♀, 5-7 weeks old) and 1.0 × 10 ⁶ osteoblast-like stromal cells collected from the skull and cultured Suspended in 7.2 ml. The culture solution was α-MEM (Minimum Essential Medium Alpha Medium) containing 10% fetal calf serum. This was dispensed in a 48-well plate at 150 μl / well.

The samples were obtained by extracting the fruit bodies of anninkou obtained by the inventors' artificial cultivation method at room temperature using chloroform, ethyl acetate and purified water. The sample was dissolved in methanol so as to be 100 mg / ml, and further diluted with a culture solution to prepare a dilution series of each solvent. However, since the water extraction sample did not dissolve in methanol, it was dissolved in distilled water. 50 μl of each concentration of the sample solution was added to the culture solution along with 50 μl of 20 ng / μl of 1α, 25 dihydroxyvitamin D ₃ (hereinafter referred to as 1,25 (OH) ₂ VD ₃ ) to make a total volume of 250 μl / well. The cells were cultured at 37 ° C. and a carbon dioxide gas concentration of 5.0% using a carbon dioxide incubator.

To add a sample on the third day of the culture, 100 μl of the supernatant of each well was removed, and each of the sample solution twice as much as the addition concentration on the first day and 1,25 (OH) ₂ VD ₃ (40 ng / ml) was added. 50 μl was added to each well and cultured again under the same conditions. After culturing for about one week, the culture solution was removed, washed with PBS, and then added with 10% formalin-containing PBS solution at 0.5 ml / well and fixed for 10 minutes. The cells adhered to the well bottom. Next, 0.5 ml / well of ethanol was added and refixed for 1 minute. Thereafter, it was dried and subjected to TRAP reaction to stain osteoclasts.

TRAP is an abbreviation for tartrate-resistant phosphatase, which is a marker for osteoclasts. The TRAP reaction solution is obtained by dissolving naphthol AS-MX phosphate in 150 ml of N, N-dimethylformaldehyde and adding 15 ml of buffer (50 mM sodium tartrate). Prepared by adding 9 mg of fast red violet 1b salt to 0.1 m sodium tartrate buffer solution (pH = 5.0).
Osteoclasts with two or more nuclei stained by the TRAP reaction are counted under a microscope, the number of osteoclasts per well is counted, and the control is the same amount of 10% fetal bovine instead of the test substance. A serum-containing α-MEM medium is added. The relative number of osteoclasts in relation to solvent and concentration with 100 as the control is shown in FIG.

After co-culture with the cytotoxicity test , 1 mg / ml MTT reagent was added at 125 ml / well without removing the culture medium. Subsequently, it culture | cultivated on the conditions of 37 degreeC and a carbon dioxide gas concentration 5.0% for 2 hours using the carbon dioxide gas incubator. After completion of the culture, the culture solution was removed, 100 μl / well of dimethyl sulfoxide was added, and the absorbance at a wavelength of λ = 570 nm was measured and also shown in FIG. Also in this case, the relative osteoclast number of each experimental result was shown with the control cultured without adding the sample solution as 100.
The MTT reagent was prepared by dissolving 3- (4,5-dimethyl-2-thiazoly) -2,5-diphenyl-2H-tertazolium bromide in water to 1 mg / ml.

  As is clear from FIG. 1, significant osteoclast formation inhibitory effects were observed at concentrations of 12.5, 25, and 50 μg / ml in both the ethyl acetate extraction part and the water extraction part, and the cytotoxicity was less than that of the control. Therefore, Anninkou has been demonstrated to be effective for osteoporosis and other diseases of abnormal bone metabolism.

It is a graph which shows the effect by making the extract by a solvent coexist, and showed the relative osteoclast formation number and cytotoxicity which made control 100.

Claims (2)

An osteoclast formation inhibitor containing, as an active ingredient, an aqueous extract of the fruiting body of Grifola gargal and / or Grifola sordulenta . An osteoclast formation inhibitor containing, as an active ingredient, an aqueous extract of the mycelium of Grifola gargal and / or Grifola sordulenta .