JP2006016348A - Remedy for metabolic osteopathia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a remedy for metabolic osteopathia, capable of improving condition of the metabolic osteopathia by an administration in a short period, without causing a side effect, to provide a health food, and to provide a method for producing the same. <P>SOLUTION: This remedy for the metabolic osteopathia is obtained by together mixing a young shoot of a plant belonging to Pinaceae family, water, and a glucide and then naturally fermenting the mixture. A plant belonging to Pinus genus is preferably used as the plant belonging to the Pinaceae family. Further, the remedy for the metabolic osteopathia, etc., is produced by using a fermented product which is obtained by using an yeast separated from the remedy for the metabolic osteopathia. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a therapeutic agent for metabolic bone diseases including osteoporosis and rheumatic bone diseases.

  Bone is composed of a bone matrix composed of about 80% inorganic substance such as calcium phosphate and about 20% organic substance such as collagen, and several types of bone cell groups. Bone is constantly undergoing metabolism, and bone metabolism is carried out by osteoclasts breaking a part of old bone (bone resorption) and osteoblasts repairing bone (bone formation).

  Examples of metabolic bone diseases include osteoporosis, rheumatic bone disease, osteomalacia, rickets, bone paget disease and osteoarthritis.

  Among these, for example, osteoporosis is a state in which bone resorption exceeds bone formation, resulting in decreased bone mass and increased bone vulnerability and fracture risk. Examples of symptoms of osteoporosis include low back pain, back pain, easy fracture, and spinal compression fracture. Although osteoporosis is not a fatal disease, there is a great deal of interest in this treatment because it greatly reduces the quality of life.

As a conventional therapeutic agent for osteoporosis, for example, as described in the prior art column of Patent Document 1, it is said that estrogen may induce cancer in the genitals. In calcitonin, the administration method is intramuscular. Because it is an injection, the burden on the patient is large. In addition, these therapeutic agents were not expected to have much therapeutic effect on osteoporosis.
Patent Publication 2003-95971

  An object of the present invention is to provide a therapeutic agent that can improve the pathology of metabolic bone disease.

  The therapeutic agent for metabolic bone disease of the present invention is obtained by mixing pine shoots, water, and carbohydrates and fermenting them naturally, and is an effective therapeutic agent for metabolic bone diseases. . The pine plant is preferably a pine plant, and the sugar is preferably sugar.

  The therapeutic agent for metabolic bone disease of the present invention is obtained by mixing pine shoots, water, and sugar and fermenting them naturally, and is an effective therapeutic agent for metabolic bone diseases.

  Furthermore, the present invention includes the above-described method for producing a therapeutic agent for metabolic bone disease. The method for producing a therapeutic agent for metabolic bone disease comprises (1) dissolving a saccharide in sterilized water and dissolving the saccharide. A step of preparing a solution; and (2) a step of adding a shoot of a pine family plant to the solution in which the carbohydrate is dissolved and allowing the solution to spontaneously ferment. The pine plant is preferably a pine plant, and the sugar is preferably sugar.

  The method for producing a metabolic bone disease therapeutic agent of the present invention includes (1) a step of dissolving sugar in sterilized water to prepare a solution in which sugar is dissolved, and (2) a pine in the solution in which the sugar is dissolved. And a step of natural fermentation.

  The natural fermentation is performed under anaerobic conditions at 10 to 70 ° C., preferably 20 to 60 ° C., for 2 to 8 months, preferably 5 to 8 months, more preferably 6 to 8 months.

  As a more specific method for producing a metabolic bone disease therapeutic agent, (1) a step of preparing an aqueous sugar solution in which sugar is dissolved in hot water and cooled to ambient temperature, and (2) the aqueous sugar solution is washed with water. A step of adding pine shoots, sealing them in a container, and allowing them to spontaneously ferment. This natural fermentation is preferably carried out in a place where the sealed container is exposed to direct sunlight until early winter.

  The present invention also intends a health food for improving metabolic bone disease symptoms obtained by mixing shoots of pineaceae plants, water, and carbohydrates and fermenting them naturally. The food (including health drinks) is preferably obtained by mixing pine shoots, water, and sugar, followed by natural fermentation.

  Further, the present invention provides a fermented product subjected to the natural fermentation described above, that is, a drug for treating metabolic bone disease or health food of any fermentation product using yeast isolated from the metabolic bone disease therapeutic agent of the present invention. Relates to the use in the manufacture of The yeast is a yeast disclosed in an international patent application (International Publication WO 01/95922) by the present applicant. On March 12, 2001, the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit. Center, 1st East, 1-chome, Tsukuba City, Ibaraki Pref. Center 6 (former name: National Institute of Bioscience and Human-Technology National Institute of Advanced Industrial Science and Technology), Yeast 1-1-3, Tsukuba City, Ibaraki Prefecture, whose name has been changed as of April 1, 2001) is a yeast deposited as Haruisan A-3 and identified by the accession number FERM BP-7499. In addition, the use of a series of yeasts having the same bacterial properties as the deposited yeast is also included in the present invention. Here, health foods (including health drinks) for improving metabolic bone disease symptoms are intended to improve the constitution and maintain health, particularly to improve the symptoms of metabolic bone diseases. It means foods and beverages such as supplements used.

  The therapeutic agent for metabolic bone disease of the present invention can improve the pathological condition of metabolic bone disease for a short period of time without side effects. Therefore, according to the present invention, it is a very useful metabolic bone disease. Health foods and health drinks for improving disease therapeutic agents and metabolic bone disease symptoms are obtained.

  The therapeutic agent for metabolic bone disease of the present invention is obtained by mixing pine shoots, water, and carbohydrates and fermenting them naturally.

  Examples of the pine family plant that can be used in the present invention include so-called fir (Abies firma Sieb. Zucc.), Vladivus fir (Abies homolepis Sieb. Zucc.), Abbies mariesii MT Mast., Abies sachalinensis (Friedr. MT Mast. Var marie, Abies sachalinensis (Friedr. Schmidt) MT Mast., Syrabe (Abies veitchii Lindl.), Cedar (Cedrus deodara (Roxb. Ex D. Don) G. Don), Guimatsu (Larix) gmelini (Rupr.) Kuzeneva), larch (Larix kaempferi (Lamb.) Carriere), German spruce (Picea abies (L.) Karst.), red spruce (Picea glehnii (Friedr. Schmidt) MT Masters), spruce (Picea jezoensis ( Siuc. Zucc.) Carriere var. Hondoensis, Picea jezoensis (Sieb. Zucc.) Carriere, Picea koyamae Shirasawa, Hara fir (Picea polita (Sieb. Zucc.) Carriere), Pinus x densi-thunbergii Uyeki) Japanese red pine (Pinus densiflora Sieb. Zucc.), Japanese cabbage (Pinus densiflora Sieb. Zucc. var. parviflora), Kitagoyou (Pinus parviflora Sieb. Zucc. Var. pentaphylla (Mayr) Henry), Pinus pumila (Pall.) Regel, Pinus rigida Mill., Strobe pine (Pinus strobus L.), Europe Pinus sylvestris L., Pinus teada L., Pinus thunbergii Parl., Pseudotsuga japonica (Shiras.) Beissn., Rice moth (Tsuga diversifolia (Maxim.) MT Mast. (Tsuga sieboldii Carriere) etc. are illustrated. Of these, the pine genus plants Aigromatsu, Japanese red pine, Taenshosho, Korean ginger, Japanese red ginger, Japanese Komatsu, Kitagoyo, Japanese pine, Rigid pine, Strobe pine, European red pine, Japanese pine, Japanese black pine are preferred. High pine, black pine, etc. are pines that are generally cultivated, and are preferable from the viewpoint of easy availability.

  Examples of the saccharide used in the present invention include sucrose, invert sugar, and maltose. Of these, sucrose is preferable in terms of easy availability. As sucrose, any sugar such as white sugar, brown sugar, tri-warm sugar, sugar beet sugar, and acne sugar can be used. preferable.

  The water to be used is preferably sterilized in advance, and it is preferable to prevent the propagation of various bacteria. As a sterilization method, a publicly known method can be used, and for example, it can be performed by a method such as boiling. .

  The therapeutic agent for metabolic bone disease of the present invention is obtained by adding a shoot of a pine family plant to an aqueous solution in which a saccharide is dissolved using the above-mentioned sterilized water, and spontaneously fermenting it. The sprout of a family plant may be a sprout collected from any kind of pine plant, but a sprout collected from a plant of the genus Pinus is particularly preferred. The time of collection is preferably when the flowers of the pine family plants bloom, and the shoots collected at this time are the most effective and preferable as a therapeutic agent for metabolic bone diseases. In the case of pine, depending on the climate of the land, in general, a reddish female flower blooms from the beginning of April to the end of June, and a yellow male flower blooms around the new branch. It is preferable to collect and use shoots at the tips of the branches after the flowers have finished blooming.

  Preparation of the undiluted | stock solution used for fermentation melt | dissolves saccharide | sugar in the ratio of about 0.5 kg with respect to about 1 liter of water, It adds at the ratio of about 25 shoots of a pine plant to the obtained solution. In this case, the carbohydrate does not need to be completely dissolved, and it may be present in the liquid without being dissolved. In addition, the stock solution to be fermented may contain shoots of pine family plants such as pine shoots at the above-mentioned ratio, and may contain leaves and flowers other than the shoots.

  Natural fermentation is performed under anaerobic conditions, and is allowed to stand at 10 to 70 ° C., preferably 20 to 60 ° C. for 2 to 8 months, preferably 5 to 8 months, more preferably 6 to 8 months. Do. As the anaerobic condition, for example, a method of filling a sealed light-shielding container is adopted, and this is achieved by placing the sealed container in a place exposed to direct sunlight until early winter. After the natural fermentation period has elapsed, the container is opened, and solid substances such as pine shoots are removed to obtain the therapeutic agent for metabolic bone disease of the present invention. In addition, the above-mentioned early winter time is a balance with the time for collecting shoots, that is, the preparation time. In the region where the shoots are produced, the flowering time is from the beginning of April to the beginning of May. If it is set from the middle to the beginning of June, the end of fermentation will be early winter (mid-November), but it is only a guideline and can be changed arbitrarily.

  The therapeutic agent for metabolic bone disease of the present invention uses a fermented product obtained by natural fermentation as it is, but a sweetener or a flavoring agent is added to the product to make it easy to drink, long-term For preservation and the like, various additives such as preservatives may be added.

  In order to apply the therapeutic agent for metabolic bone disease of the present invention, in general, in the case of an adult, about 30 to 50 milliliters is taken 2 to 3 times a day. In the case of a dwarf, half the adult dose is taken. Moreover, since the metabolic bone disease therapeutic agent of the present invention is safe without toxicity and mutagenicity, there is no problem even if an amount exceeding the above dose is used.

  The fermented product obtained by natural fermentation of the present invention can be used not only as a therapeutic agent for metabolic bone disease but also as a health food or a health drink. Health foods or health drinks are not intended for treatment, but are used to improve health and maintain health. Therefore, the health food for improving metabolic bone disease symptoms (including health drinks) of the present invention is also used to improve the symptoms of metabolic bone diseases, that is, to reduce or prevent those symptoms. Can do. In this case, in consideration of consumer preference, it is preferable to add the above-mentioned sweeteners, flavoring agents and the like to make it easy to eat, easy to drink, and suitable for preference.

  Furthermore, the present invention provides a metabolic bone therapeutic agent using a product fermented using yeast isolated from the fermentation product and an extract from the product, and health for improving metabolic bone disease symptoms. A method for producing food (including health drinks) is provided.

The separation of the yeast is also described in detail in International Publication WO 01/95922 pamphlet. Specifically, using the fermented product that is the therapeutic agent for metabolic bone disease of the present invention, the plate is cultured on a plate by the GPLP agar plate culture method, and the colony growing predominantly on the culture plate is caught and separated. Yeast was obtained. Morphological observations and characterization tests were performed on isolated yeast, and the literature (Kurtzman, CP et al., “The yeasts, A Taxonomic Study” 4th edition (1998), Elsevier Science BV; Barnett, JA et al., “Yeasts: Characeristics and Identification ", 3rd edition (2000), Cambridge University Press, these references are incorporated by reference). The number of yeast in the fermentation product was 1.4 × 10 ⁵ / g. Table 1 shows the results of the property test of the isolated yeast, and FIG. 1 shows an example of ascospores of the isolated yeast.

  Based on the above results, the isolated yeast is identified as Zygosaccharomyces bisporus morphologically and qualitatively. This Tigosaccharomyces bisporus is a genus of Ascomycetous yeast that joins between independent cells to form 1-4 ascospores of spherical to elliptical shape. Tigosaccharomyces bisporus is an osmotic-resistant yeast that is separated from fermented foods and soft drinks.

On the other hand, when the DNA homology test with the target strain was performed on the isolated yeast, the following results were obtained. That is, Takayuki Esaki et al., Journal of the Japanese Society for Bacteriology, 45, 851 (1990), and Masaaki Takahashi et al., Tokyo Agricultural University Isotope Center Research Report, No. 7, p. 69 (1993). Incorporated as part of the document), using the Tigosaccharomyces bisporus IFO 1131 and Tygosaccharomyces bailii IFO 1098 (Zygosaccharomyces bailii IFO 1098) as the target strain DNA-DNA hybridization test was performed by the photobiotin labeling method. The DNA was prepared according to Jahnke, K.-D. et al., Trans. Br. Mycol. Soc., Vol, 87, pp.175-191 (1986). Prepared as part). The results are shown in Table 2.

  According to the results of the homology test with the target strain described above, the yeast of the present invention is identified as T. saccharomyces bisporus morphologically and in nature, but the DNA sequence itself is not in T. saccharomyces bisporus. It is understood that this is a new strain that is close and different from any of the target strains.

The applicant named this new type of yeast presumed to belong to the genus Tigosaccharomyces as “Haruisan A-3”. It is not clear at this time whether this new yeast “Haruisan A-3” is not a new strain but a new species or genus. However, the applicant has identified a new species of yeast, isolated and named “Haruisan A-3”, on March 12, 2001, under the accession number FERM, in accordance with the Budapest Treaty on the International Approval of Deposits of Microorganisms in Patent Procedures. BP-7499, new name: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center, 1-1-1 Higashi 1-chome, Tsukuba City, Ibaraki, Japan Center 6 (former name: Ministry of Economy, Trade and Industry Deposited with the Institute (National Institute of Bioscience and Human-Technology National Institute of Advanced Industrial Science and Technology), 1-chome, East 1-chome, Tsukuba, Ibaraki, Japan 3). The name of the depositary institution was changed to an independent administrative institution on April 1, 2001, and changed to the National Institute of Advanced Industrial Science and Technology on April 1, 2001. .

  The isolated new species of yeast according to the present invention is considered to have greatly contributed to the effectiveness as a therapeutic agent for metabolic bone diseases together with shoots of pineaceae plants, and is an extremely useful yeast.

  Next, the metabolic bone disease therapeutic agent of the present invention will be described in detail with examples, but the present invention is not limited to the following examples.

(Example 1: Production example of metabolic bone therapeutic agent)
The therapeutic agent for metabolic bone disease of the present invention can be produced as follows.

  The pine shoots of black pine, red pine, and larch are collected when the pine has finished blooming (in mid-May to early June in Fukushima Prefecture), and the collected pine shoots are thoroughly washed. Add white sugar to hot water, dissolve it, cool it to room temperature, and then wash the pine shoots with water into a container, for example, a plastic container, and seal it in early winter (in Fukushima Prefecture, in November Manufactured by natural fermentation by placing it in a place exposed to direct sunlight until mid). When the container is opened in early winter and the pine is removed, the therapeutic agent for metabolic bone disease of the present invention can be obtained.

  The sugar water used was used at a ratio of about 1 kg of white sugar to about 2 liters of water, and about 50 pine trees were used.

As described above, about 1 liter of white sugar and about 50 pine shoots were produced in about 2 liters of water, and about 1.2 liters (about 60% of the prepared sugar water) had a white turbidity. A liquid metabolic bone disease therapeutic agent having When the contained components were analyzed for this therapeutic agent, the results shown in Table 3 were obtained.

  This analysis was mainly performed only for the above-mentioned test items relating to food in general, and the metabolic bone disease therapeutic agent of the present invention may contain other components. Therefore, it cannot be specified which component is particularly effective as a therapeutic agent for metabolic bone disease, but it has a medicinal effect as a therapeutic agent for metabolic bone disease.

Next, the number of molds, the number of yeasts, and the number of general bacteria (viable cells) contained in the therapeutic agent were examined for a plurality of metabolic bone disease therapeutic agents of the present invention produced as described above. For the number of molds and yeast, the GPLP agar plate culture method was used. For the number of general bacteria (viable cells), the pH of the medium was adjusted to 3.5 using tartaric acid by the SCDLP agar plate culture method with an antifungal agent. Two types of tests, one adjusted and one not adjusting the medium, were performed and measured. The results obtained are shown in Table 4.

According to the above results, it can be seen that no fungus is observed in the metabolic bone disease therapeutic agent of the present invention, and the number of general bacteria (viable bacteria) is extremely small. Also, yeast albeit slight variation by lot fermentation product, the fermentation product it can be seen that there is a yeast of the order of 10 ² to 10 ⁵ cells / g. This corresponds to a new type of yeast deposited by the yeast present in the fermentation product.

(Test Example 1: Oral toxicity of a therapeutic agent for metabolic bone disease)
Next, oral toxicity of the therapeutic agent for metabolic bone disease of the present invention was examined. Using the stock solution of the therapeutic agent for metabolic bone disease of the present invention and honey (10% by weight) added thereto, 2,000 mg / kg of each of these test substances is administered, and water for injection is administered as a control group. Group (dose 0 mg / kg) was administered by oral gavage using a disposable syringe (1 mL capacity) with an oral gastric tube attached to 5 SD male and female SD rats [Crj: CD (SD) IGS]. Thereafter, an observation period of 15 days (including the day of administration) was established, and signs of toxicity and approximate lethal dose were examined.

  No death was observed in males and females in the 2000 mg / kg administration group of the metabolic bone disease treatment agent of the present invention and honey added thereto, including the control group, throughout the test period. In addition, no changes due to test substance administration were observed in general conditions, body weight, and autopsy. From the above results, it was concluded that the approximate lethal dose of the therapeutic agent for metabolic bone disease of the present invention under the present test conditions was 2000 mg / kg or more for both males and females.

(Test Example 2: Mutagenicity of therapeutic agent for metabolic bone disease)
Next, the mutagenicity of the therapeutic agent for metabolic bone disease of the present invention was examined. Regarding the mutagenicity of the therapeutic agent for metabolic bone disease of the present invention, a modified method of Ames et al. (Maron, DM et al., “Revised methods for the Salmonella mutagenicity test”, Mutation Res., Vol. 113, pp.173- 215 (1983), which is incorporated by reference as a part of this application), and the histidine requirement of Salmonella typhimurium TA98, TA100, TA1535, TA1537, and tryptophan requirement of Escherichia coli. Each WP2 uvrA strain was treated and its mutagenicity was examined both with and without metabolic activation.

  The test doses were 312.5, 625, 1250, 2500 and 5000 μg / plate. As a result, the number of revertant colonies in the test substance group of each test strain did not depend on the dose dependency and the increase of more than double that of the negative control group regardless of the presence or absence of the metabolic activation system. Moreover, growth inhibition and precipitation of the test substance were not observed. The results are shown in FIGS. FIG. 2 shows the results when a base pair substitution strain (TA100: □, TA1535: ◯, WP2uvrA: Δ) is used. In the figure, A is a case where the metabolic activation is not performed, B is a case where the metabolic activation is performed, and the results when S9mix is added are shown. FIG. 3 shows the results when a frameshift strain (TA98: □, TA1537: ◯) was used. In the figure, A is a case where the metabolic activation is not performed, B is a case where the metabolic activation is performed, and the results when S9mix is added are shown. As a negative control substance, distilled water for injection used as a solvent at the time of preparation of the test substance was used. As positive control substances, 2- (2-Furyl) -3- (5-nitro-2-furyl) acrylamide (AF-2), 2-Aminoanthracene (2-AA), Sodium azide (SA), and , 9-Aminoacridine (9-AA). AF-2, 9AA, and 2-AA were dissolved in DMSO and SA was dissolved in distilled water for injection, and were used depending on the strain and metabolic activation, and depending on metabolic activation.

  From the above results, the mutagenicity of the therapeutic agent for metabolic bone disease of the present invention under this test condition was judged negative.

(Test Example 3: Osteoclast formation inhibitory effect of a therapeutic agent for metabolic bone disease)
The effect of inhibiting osteoclast differentiation was tested by the metabolic bone disease therapeutic agent of the present invention.

  Macrophage RAW264 cells were mixed with α-MEM medium (hereinafter referred to as “10% FBS-MEM”) supplemented with 10% fetal bovine serum (FBS) containing the osteoclast differentiation factor RANKL and the metabolic bone disease therapeutic agent of the present invention. ) And cultured for 3 days.

  As the therapeutic agent for metabolic bone disease of the present invention, those having a fermentation period of 2, 5 and 6 months were used, and these stock solutions were previously contained in the above medium at various concentrations (0.00003 to 1.0% v / v). It was. In general, Macrophage RAW264 cells differentiate into osteoclasts when cultured in a medium containing osteoclast differentiation factor RANKL for 3 days. If RANKL is not included, Macrophage RAW264 cells do not affect Macrophage RAW264 cells and become osteoclasts. Differentiation is not induced.

  The degree of osteoclast differentiation was assessed by measuring tartrate-resistant acid phosphatase (TRAP), a marker enzyme for osteoclasts.

  Specifically, the procedure of this test is to fix cells cultured on a 96-well plate with 90% ethanol, and then add TRAP enzyme reaction solution 100 ml (4-nitrophenyl phosphate disodium salt 6H2O (Wako Pure Chemical Industries, Ltd.) 1.36 50 mM Citrate Buffer (pH 4.6) containing mg / ml and 10 mM tartrate was added and reacted at 37 degrees for 20-30 minutes. The enzyme reaction was stopped by taking the total amount of the reaction solution and transferring it to a plate containing 100 ml of 0.1N-NaOH in advance, and the enzyme reaction product was measured at 405 nm absorbance.

  The results are shown in FIG. 4 (a) to (c) as bar graphs for each fermentation period of the therapeutic agent of the present invention. FIG. 4 shows the osteoclast formation inhibitory effect of the therapeutic agent of the present invention. The vertical axis shows the relative value (%) of the TRAP activity relative to the control group, that is, the medium containing the therapeutic agent of the present invention relative to the absorbance of the TRAP activity of the cells cultured in the medium not containing the therapeutic agent of the present invention (control). The absorbance of the TRAP activity of the cultured cells is shown as a percentage (%). The horizontal axis indicates the concentration (%, v / v) of the therapeutic agent stock solution of the present invention in the culture medium.

  In the medium containing the therapeutic agent of the present invention having a fermentation period of 6 months, the relative value of TRAP activity was remarkably reduced regardless of the concentration of the therapeutic agent, and osteoclast differentiation was greatly inhibited. In the medium containing the therapeutic agent for metabolic bone disease of the present invention having a fermentation period of 2 months and 5 months, the relative value of TRAP activity was significantly reduced in the region where the therapeutic agent concentration was high, and osteoclast differentiation was greatly inhibited.

(Test Example 4: Cytotoxicity of metabolic bone disease therapeutic agent)
In order to test the cytotoxicity of the therapeutic agent for metabolic bone disease of the present invention, the number of viable cells in each medium cultured in Test Example 3 was measured using an XTT assay. The XTT assay uses a tetrazolium salt as a chromogenic substrate and measures the number of living cells by measuring the absorbance.

  First, 50 ml of the XTT labeling mixture was added to the cell culture medium on the 96-well plate of Test Example 3 and cultured at 37 degrees for 18 hours, and the absorbance at 480 nm (control wavelength was 650 nm) was measured.

  The cell viability is shown as a line graph for each fermentation period of the therapeutic agent of the present invention in FIGS. The vertical axis shows the relative value (%) of the XTT activity, that is, the absorbance (%) of the XTT activity of the cells cultured in the medium containing the therapeutic agent of the present invention relative to the absorbance of the XTT activity of the cells cultured in the control medium. . The horizontal axis indicates the concentration (%, v / v) of the therapeutic agent stock solution of the present invention in the culture medium.

  4A to 4C, when the therapeutic agent concentration of the present invention is 0.3% or less, the XTT activity is almost 100% regardless of the fermentation period of the therapeutic agent. Thus, within the above range, it was determined that the cytotoxicity of the therapeutic agent of the present invention was almost the same as that of the control cells, that is, there was almost no cytotoxicity.

  When the therapeutic agent concentration was 1.0%, it decreased at the fermentation period of 6 months and 5 months, and showed a value close to that of the control group at the fermentation period of 2 months. This seems to be because no cytotoxic components were formed yet in the 2 months fermentation period.

  FIG. 5 shows micrographs of cells cultured in a control medium and a medium containing the metabolic bone disease therapeutic agent of the present invention (6-month fermentation, containing 0.03% v / v), respectively, and stained with TRAP.

  Compared to the control medium, the medium containing the therapeutic agent of the present invention has reduced red cells stained with TRAP. This result shows the possibility that the therapeutic agent for metabolic bone disease of the present invention inhibits the differentiation of osteoclast precursor cells to osteoclasts.

FIG. 1 is a micrograph (differential interference, × 2400) showing an example of ascospores of isolated yeast isolated from the therapeutic agent for metabolic bone disease (fermented product) of the present invention. As the medium, malto extract agar medium was used. FIG. 2 is a graph showing the mutagenicity screening test results of the therapeutic agent for metabolic bone disease of the present invention, using base pair substitution strains (TA100: □, TA1535: ◯, WP2uvrA: Δ). Results are shown. Further, in the figure, A shows the case where the metabolic activation is not performed (-S9), and B shows the case where the metabolic activation is performed (+ S9). FIG. 3 is a graph showing the mutagenicity screening test result of the therapeutic agent for metabolic bone disease of the present invention, and shows the results when using a frameshift strain (TA98: □, TA1537: ◯). . Further, in the figure, A shows the case where the metabolic activation is not performed (-S9), and B shows the case where the metabolic activation is performed (+ S9). FIG. 4 shows the relative value (%) based on the TRAP activity for each fermentation period (2, 5, 6 months) of the therapeutic agent for metabolic bone disease of the present invention (bar graph) and the relative value based on the XTT activity. (Line graph). FIG. 5 is a photomicrograph showing the state of cells in a medium containing the therapeutic agent for metabolic bone disease of the present invention.

Claims (1)

  A therapeutic agent for metabolic bone diseases obtained by mixing shoots of Pinaceae plants, water, and carbohydrates, followed by natural fermentation.