WO2018168879A1 - Composition having estrogen-like action, medicine, food and beverage containing same, method for producing composition having estrogen-like action and method for utilizing dna base sequence of sparassis crispa - Google Patents

Abstract

[Problem] To provide a composition having an estrogen-like action which comprises a hot water extract of Sparassis crispa mycelia.

Description

Estrogen-like composition, pharmaceutical, food and drink containing the same, method for producing estrogen-like composition, and method for using DNA sequence of Hanabiratake

The present invention relates to an estrogen-like action composition, a pharmaceutical, a food and drink containing the same, a method for producing the estrogen-like action composition, and a method for using the DNA sequence of Hanabiratake.

¡Hanaburatake (Sparassis 属 し crispa) is a kind of mushroom that belongs to the family Basidiomycota agaricidae and is classified into the genus Hanaburatake. Hanabiratake is called “Phantom Mushroom” because it is difficult to find mushrooms that grow naturally in Japan, and it is used as a Japanese food ingredient. It is called “Cauliflower Mushroom” overseas. It has been used as a luxury food in France and China.

In addition, natural Hanabiratake has long been used as a material for Kampo, but recently, cultivated products have come to the market in Japan and China, but quality control is difficult and is still fully utilized. It ’s hard to say. For example, since the appearance of a fruit body is relatively simple, it is not easy to distinguish at the species level, and there are cases where two or more taxonomic groups are confused.

Hanabiratake has been reported to have an anticancer effect, an antibacterial effect, an angiogenesis inhibitory effect, a hypoglycemic effect, an insulin secretion promoting effect, an immune activity, and the like (for example, Patent Document 1), and is against cancer, infectious diseases, diabetes, etc. Prevention / improvement effect is expected.

For this reason, Hanabiratake is eaten raw or dried fruit bodies or fruit body extract as health food. The effect is generally considered to be due to the action of β-D-glucan, which is a high molecular weight polysaccharide contained in the fruit body of Hanabiratake, and Hanabiratakeri, sesquiterpenoids, sparrole, etc., which are phthalide compounds. ing.

Japanese Unexamined Patent Publication No. 2006-117616

Chin, C.-S., Peluso, P., Sedlazeck, F. J., Nattestad, M., Concepcion, G. T., Clum, A., et al. (2016). Phased diploid genome assembly with single -molecule real-time sequencing. Nature Methods, 13 (12), 1050-1054. http://doi.org/10.1038/nmeth.4035 Kim, D., Langmead, B., & Salzberg, S. L. (2015). HISAT: a fast spliced aligner with low memory requirements. Nature Methods, 12 (4), 357-360. Http: // doi. org / 10.1016 / j.cell.2012.02.009 Pertea, M., Pertea, G. M., Antonescu, C. M., Chang, T.-C., Mendell, J. T., & Salzberg, S. L. (2015). StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology, 33 (3), 290-295. http://doi.org/10.1287/moor.16.2.351 Stanke M, Morgenstern B (2005). AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Research 33: W465-W467. Pmid: 15980513 Testa, A. C. (2015). CodingQuarry: highly accurate hidden Markov model gene prediction in fungal genomes using RNA-seq transcripts, 1-12. Http://doi.org/10.1186/s12864-015-1344-4 Haas, B. J., Salzberg, S. L., Zhu, W., Pertea, M., Allen, J. E., Orvis, J., et al. (2008). Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biology, 9 (1), R7. http://doi.org/10.1186/gb-2008-9-1-r7 Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. (2009). BLAST +: architecture and applications. BMC Bioinformatics 10: 421. pmid: 20003500 Bairoch A, Boeckmann B, Ferro S, Gasteiger E (2004) Swiss-Prot: juggling between evolution and stability. Briefings in Bioinformatics 5: 39-55. Pmid: 15153305 Chen L, Gong Y, Cai Y, Liu W, Zhou Y, Xiao Y, et al. (2016). Genome Sequence of the Edible Cultivated Mushroom Lentinula edodes (Shiitake) Reveals Insights Deto e0160336. doi: 10.1371 / journal.pone.0160336. Li L, Stoeckert CJ, Roos DS (2003). OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Research 13: 2178-2189. Pmid: 12952885 Katoh K, Standley DM (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772-780. Pmid: 23329690 Castresana J (2000). Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17: 540-552. Pmid: 10742046 Stamatakis A (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313. Pmid: 24451623 Floudas D, Binder M, Riley R, Barry K, Blanchette RA, Henrissat B, et al. (2012). The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Sanderson MJ (2003). R8s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock. Bioinformatics 19: 301-302. Pmid: 12538260 Martinez D, Challacombe J, Morgenstern I, Hibbett D, Schmoll M, Kubicek CP, et al. (2009). Genome, transcriptome, and secretome analysis of wood decay congus Postia placenta S A 106 (6): 1954-1959. Doi: 10.1073 / pnas.0809575106. Gaskell J, Kersten P, Larrondo LF, Canessa P, Martinez D, Hibbett D, et al. (2017). Draft genome sequence of a monokaryotic model brown-rot fungus Postia (Rhodonia) placenta SB12 14 . Doi: 10.1016 / j.gdata.2017.08.003. Rong C, Zhao S, Li D, Wang L, Wang S, Ma K, et al. (2015). Cloning of the A Mating-Type Locus from Lepista nuda and Characterization of Its Genetic Structure. Curr Microbiol. 71 : 669-677. Doi: 10.1007 / s00284-015-0902-y. Yin Y, Mao X, Yang J, Chen X, Mao F, Xu Y (2012). DbCAN: a web resource for automated carbohydrate-active enzyme annotation. Nucleic acids research 40: W445-W451. Pmid: 22645317 Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic D37: 391 Martinez D, Larrondo LF, Putnam N, Gelpke MD, Huang K, Chapman J, et al. (2004). Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nat Biotech. Okada H, Abe M, Asakawa-Minemura M, Hirata A, Qadota H, Morishita K, et al. (2010). Multiple functional domains of the yeast l, 3-beta-glucan synthase subunit Fks1p revitedoo -sensitive mutants. Genetics 184 (4): 1013-1024. doi: 10.1534 / genetics.109.109892.

On the other hand, there are few reports on the activity and active ingredients of the mycelium of Hanabiratake because of the difficulty in culturing it. In addition, the genome analysis data of Hanabiratake has not been acquired yet because of the difficulty of genome analysis. If whole genome analysis data can be obtained, it can be used not only for information on useful genes but also for markers and polymorphism information used for differentiation from related species and strains, systematic component analysis, and active component search It becomes possible to do.

The present invention has been made in view of the circumstances as described above, reveals the action of the mycelium of Hanabiratake, a composition containing a component derived from the mycelium of Hanabiratake, a drug containing less side effects, including food and drink The issue is to provide products and other products in a stable manner.

The estrogen-like action composition of the present invention is characterized in that it contains a hot water extract of the mycelium of the moss.

The medicament of the present invention is a medicament for treating or preventing a disorder or disease caused by estrogen deficiency, and is characterized by containing the estrogen-like action composition.

The food / beverage product of the present invention is a food / beverage product for treating or preventing a disorder or disease caused by estrogen deficiency, and is characterized by containing the estrogen-like action composition.

The method for producing an estrogen-like composition of the present invention is characterized in that it comprises a step of bringing a hot water extract into contact with hot water to obtain a hot water extract.

The quality control of the extract of the mycelium of the present invention, the establishment of the standard strain of the fungus, and the systematic component analysis method of the estrogen-like action composition are characterized by using the data of the whole genome of the algal bloom.

The utilization method for the effectiveness of the flower of the present invention is characterized by using information on the genomic DNA base sequence of the flower.

The utilization method relating to the effectiveness of the flower of the present invention is characterized by using the information of the DNA base sequence of the sugar-related enzyme gene of the flower.

The utilization method relating to the effectiveness of the flower of the present invention is characterized by the use of the information of the DNA base sequence of the gene of the beta-1,3-glucan synthase of the flower.

In the estrogen-like action composition of the present invention, the estrogen-like action is preferably an anti-arteriosclerosis action and visceral fat accumulation suppression by reducing the total cholesterol level, neutral fat level and free fatty acid level.

According to the estrogenic action composition, medicine and food and drink of the present invention, the estrogen-like action can treat or prevent disorders and diseases caused by estrogen deficiency.

According to the method for producing an estrogen-like action composition of the present invention, an estrogen-like action composition can be obtained with certainty.

According to the use of the data of the present genome of Hanabiratake and the effectiveness of the effect of Hanabiratake, quality control of the extract of mycelia, establishment of a standard strain of Hanabiratake, systematic component analysis of an estrogen-like composition Etc. are possible. This makes it possible to stably supply the active ingredient of the estrogen-like action composition, and to ensure quality control with respect to the effect and amount of the active ingredient.

It is a figure which shows the characteristics of the genome of a flower of a bamboo shoot (S. crispa). (A) is the contig, (b) is the GC content, (c) is the gene number, (d) is the gene expression level, and (e) is the segment overlap. The GC content was calculated as the percentage of G + C in a 20 kb non-overlapping window. Gene expression levels are shown in red (FPKM> = 100), orange (FPKM> = 10), green (FPKM> 0) and black (FPKM = 0). Large segment replicas with 90% sequence similarity or more are linked by orange (sequence length> 5 kb) and gray (sequence length> 2 kb) lines. It is a figure which shows the phylogenetic tree of other 25 types of fungi and Hanabira bamboo (S. crispa). MYA indicates 1 million years ago. It is a figure which shows the matA and matB gene loci of S. japonicus (S. crispa). The matA and matB loci are located in contigs 1 and 10, respectively. It is a figure which shows the variation | mutation of glycosyltransferase (GT). Three groups are shown: S. crispa (outermost ring), Phanerochaete chrysosporium (second ring from outside), Postia placenta (third ring), and Lentinula edodes (innermost ring). It is a figure which shows the diversity of a glycoside hydrolase. Three groups are shown: S. crispa (outermost ring), Phanerochaete chrysosporium (second ring from outside), Postia placenta (third ring), and Lentinula edodes (innermost ring). It is a figure which shows the structure of a beta-glucan synthase gene and protein. (A) shows the structure of the ScrFKS1 (on contig 5) and ScrFKS2 (on contig 4) genes in S. crispa. The intron-exon structure is shown at the top, and the transcriptome mapping results are shown at the bottom. The direction of transcription is indicated by an arrow. (B) shows the structures of the ScrFKS1 and ScrFKS2 proteins. The transmembrane domains predicted by TMHMM are shown shaded and their clusters are represented by TM1 and TM2. The regions homologous to the FKS1 domain (FKS1) and glucan synthase (GS) predicted by Pfam are shown in orange or green, respectively. The N-glycosylation sites predicted by ScanProsite are indicated by white triangles. The exon-intron junction is shown as a black triangle. It is a figure which shows the nucleotide sequence and deduced amino acid sequence of β-glucan synthase of S. ナ ビ crispa. The nucleotide sequence and deduced amino acid sequence of the β-glucan synthase gene ScrFKS1 is shown. The transmembrane domain predicted by TMHMM is shown shaded. Regions homologous to the FKS1 domain and glucan synthase predicted by Pfam are indicated by solid or diagonal lines, respectively. The N-glycosylation sites predicted by ScanProsite are boxed. Exon-intron junctions are indicated by triangles (a and b). It is a figure which shows the nucleotide sequence and deduced amino acid sequence of β-glucan synthase of S. ナ ビ crispa. The nucleotide sequence and deduced amino acid sequence of the β-glucan synthase gene ScrFKS2 is shown. The transmembrane domain predicted by TMHMM is shown shaded. Regions homologous to the FKS1 domain and glucan synthase predicted by Pfam are indicated by solid or diagonal lines, respectively. The N-glycosylation sites predicted by ScanProsite are boxed. Exon-intron junctions are indicated by triangles. It is the figure which showed the cell growth effect by the flower mycelia extract. FIG. 2 shows the expression profile of estrogen responsive genes when cells are stimulated between E ₂ and SCE and SCE-EtOAc samples using a DNA microarray assay. For _{E 2, SCE10, SCE100, SCE} -EtOAc, a diagram showing the results of a cluster analysis using estrogen responsive genes. It is a figure which shows the elution pattern obtained using the reverse phase column about the fraction extract of a mycelium of a bamboo shoot which eluted in the water | moisture content fraction. It is the figure which showed the result of the phosphorylation assay by the Western blotting of each fraction isolate | separated with the reverse phase column. It is the figure which showed transition of the body weight after a single oral administration to a mouse | mouth of the dried garlic mycelium mycelia extract and the hot water extract SCE (extracted dried material). It is the figure which showed transition of the body weight after a single oral administration to a mouse | mouth of the dried garlic mycelium mycelia extract and the hot water extract SCE (extracted dried material). It is a figure which shows the result of the blood biochemical test | inspection of the ApoE deficient mouse | mouth which orally administered for 6 weeks the dried garlic mushroom mycelium culture or the dried edible mycelium mycelia for 6 weeks. It is a figure which shows the accumulation amount of the visceral fat of the ApoE defect | deletion mouse | mouth which orally administrated for 6 weeks the dried wilt of mycelium mycelia or the dried mycelium mycelia extract for 6 weeks.

The present inventors have obtained a novel finding that the hot water extract of the fly agaric mycelium has an estrogen-like action while conducting research on the fly agaric mycelium. Therefore, by using an estrogen-like action composition containing a hot water extract of the leaf mycelia, pharmaceuticals and foods and drinks for the treatment and prevention of disorders and diseases caused by estrogen deficiency can be obtained.

Hereinafter, the estrogen-like action composition of the present invention, and medicines, foods and drinks containing the composition, and methods for producing the estrogen-like action composition will be described in more detail.

The estrogen-like action composition of the present invention contains a hot water extract of the leaflet of mycelium. The “mycelium” is an aggregate of mycelium, and here, it is distinguished from the “fruit body” of the so-called mushrooms.

Further, the mycelium of the bamboo shoot may be the mycelium itself, or may be a processed product such as a crushed material, a ground product, a tissue disrupted product, a dried product, or a dried crushed product. There is no restriction | limiting in particular about the origin, and any of the self-growth thing and an artificial culture may be sufficient as the mycelia bamboo shoot mycelium used by this invention, but if productivity is considered, an artificial culture is preferable.

There is no particular limitation on the harvesting time, growing year, culture method, culture period, etc. As for the mycelium of the fungus, different activities can be obtained by different production methods of the medium components, and any of them may be used in the present invention. These may be used alone or in combination of two or more. Higher activity can be expected by using a combination of the higher activity Hanabiratake mycelium.

In particular, in order to stabilize the quality of the estrogen-like composition, it is preferable to use a mycelium of Hanabiratake that has been cultured and standardized by Intertrade Corporation.

As the first quality control of the extract of the mycelium of the bamboo shoot, the present inventors established a standard strain of the mycelium of the bamboo shoot, and by using this strain, quality control regarding the stable supply of the mycelium of the mycelium and the effect and amount of the active ingredient We succeeded in acquiring whole genome analysis data to facilitate

Quality control is an important item for manufacturing, processing and sales of foods, health foods and pharmaceuticals in recent years, and it is required to use reliable scientific methods. The characteristics peculiar to the species / strain are often defined by the gene. By using the whole genome analysis data, it becomes possible to compare many items quickly and easily.

And, as a method for using the efficacy of Hanabiratake, information on the genomic DNA base sequence of Hanabiratake obtained by the present inventors for the first time, information on the DNA base sequence of the sugar-related enzyme gene of Hanabiratake, Beta-1,3- Information on the DNA base sequence of the glucan synthase gene can be used. By using such information, it is not only the advantage of establishing a standard strain, but it can also be used as a first step to search for and establish species and strains with high quality and high efficacy. it can. In fact, because there is a case where two or more taxonomic groups are confused in Hanabiratake because it is not easy to distinguish at the species level, for example, the whole genome of Hanabiratake is shown in the examples described later. It is useful to use the analysis data for quality control. As described above, the data for the whole genome analysis of Hanabiratake can be used for the quality control of the extract of mycelia, the establishment of a standard strain of Hanabiratake, and the systematic component analysis of the estrogenic action composition.

Then, the hot water extract can be obtained by contacting the mycelium of the moss with hot water. Specifically, the hot water extraction condition is preferably hot water at room temperature or higher, preferably 60 ° C. or higher, or water containing alcohol (hot water). For example, boiled garlic mycelium is boiled for about 5 to 20 minutes. The method of doing can be illustrated. In addition, since the active ingredient of the estrogen-like action composition of the present invention is water-soluble, it cannot be eluted by an extraction method using an organic solvent.

For example, a hot water extract can be obtained by appropriately drying and freezing the extract obtained under such hot water extraction conditions.

The estrogen-like action composition of the present invention has an estrogen-like action.

エ Estrogen deficiency is known to cause climacteric disorder and various diseases in women. Specifically, for example, women are known to have low LDL cholesterol levels before menopause but increase LDL cholesterol levels and increase arteriosclerosis after menopause, and estrogen deficiency is a major cause of this I know that. However, it is known that there are a carcinogenic risk, a thrombus risk, etc. as an adverse effect by the estrogen action. For this reason, side effects such as increased risk of endometrial cancer have been reported when estrogen treatment is used to improve postmenopausal arteriosclerosis in women, and it is rather dangerous to administer estrogen directly It is pointed out. Therefore, in recent years, instead of using estrogen directly for treatment, soy isoflavone has the same effect as estrogen, and has a relatively low risk of strong carcinogenesis (including proliferation of cancer cells) exhibited by estrogen. Atherosclerosis treatment methods using plant-derived estrogen-like substances such as these are drawing attention. However, soy isoflavone has cell growth activity and is not equivalent to silent estrogen.

For example, the Ministry of Health, Labor and Welfare's Food Safety Committee report “Basic concept of safety assessment of foods for specified health use containing soy isoflavones” (May 2006: see attached file) In the report on risk, etc., there is a report that “in sovitro study shows that intake of soy isoflavone has an anti-estrogenic effect on the development of breast cancer” (page 35). According to the Ministry of Health, Labor and Welfare's Food Safety Commission's newly developed food research committee, soy isoflavones are supposed to be "suppressed daily intake to about 30 milligrams" (January 31, 2006).

Here, “estrogen-like action” means the same action as estrogen, for example, anti-arteriosclerosis action. However, the present inventors have found that the estrogen-like action composition of the present invention has the good action of estrogen but does not have the disadvantages of estrogen.

Therefore, the medicament containing the estrogen-like composition of the present invention has no fear of side effects and is effective in treating or preventing disorders and diseases caused by estrogen deficiency.

Thus, a compound that has estrogenic activity but does not have negative activity such as proliferation activity of cancer cells is also called silent estrogen (Cell. Mol. Life Sci. 71: 2065 (2014) Etc.)

Here, the “disorder due to estrogen deficiency” includes, for example, climacteric disorder, and according to the medicament of the present invention, autonomic dysfunction due to climacteric disorder (facial flushing, sweating, coldness, sleep disorder, palpitation, Headache, etc.), psychiatric symptoms (depression, mental instability, decreased memory, etc.), other symptoms (stiff shoulders, joint pain, abdominal pain, loss of appetite, fatigue, etc.) (Shomoto Sakamoto et al .: Principal Obstetrics and Gynecology Revised version, Medical View, 2001) can be improved.

Also, “diseases caused by estrogen deficiency” include, for example, urogenital disorders such as atrophic vaginitis, prostate cancer, benign prostatic hyperplasia, osteoporosis, arteriosclerosis, memory impairment, Alzheimer's disease, thrombotic disease, and the like.

The medicament of the present invention is a medicament for treating or preventing a disorder or disease caused by estrogen deficiency, and comprises the aforementioned estrogen-like action composition of the present invention and a pharmaceutically acceptable ingredient. When the pharmaceutical of the present invention is prepared as an oral solid preparation, the estrogen-like active composition of the present invention includes excipients, if necessary, binders, disintegrants, lubricants, coloring agents, flavoring agents, flavoring agents, etc. After adding, tablets, coated tablets, granules, powders, capsules and the like can be produced by conventional methods.

As such additives, those commonly used in the field can be used.

Examples of excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and succinic acid.

Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, calcium phosphate, polyvinylpyrrolidone and the like.

Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose.

Examples of lubricants include purified talc, stearate, borax, and polyethylene glycol, and examples of flavoring agents include sucrose, orange peel, citric acid, and tartaric acid.

In addition, when preparing an oral liquid preparation, an oral solution, syrup, elixir and the like are added by a conventional method by adding a corrigent, a buffer, a stabilizer, a corrigent and the like to the estrogen-like action composition of the present invention. Can be manufactured. In this case, examples of the corrigent may include those listed above, examples of the buffer include sodium citrate, and examples of the stabilizer include tragacanth, gum arabic, and gelatin.

The dosage of the medicament of the present invention can be appropriately set according to the patient's symptoms, weight, age and the like.

The food or drink of the present invention is a food or drink for treating or preventing a disorder or disease caused by estrogen deficiency, and includes the aforementioned estrogen-like composition of the present invention and various additives.

Examples of the food and drink include various foods such as milk drinks, fruit juice processed drinks, energy drinks, cookies, and candy, health supplements, and the like.

Examples of additives to be added to food and drink include various sugars, emulsifiers, thickeners, acidulants, fruit juices, and the like.

Specifically, saccharides such as sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose, xylose, sorbitol, xylitol, erythritol, lactitol, palatinit, reduced starch syrup, reduced maltose starch syrup, etc. Examples include high-intensity sweeteners such as sugar alcohols, aspartame, stevia, acesulfame potassium, and sucralose.

Examples of the emulsifier include glycerin fatty acid ester and lecithin.

Examples of the thickening (stabilizing) agent include carrageenan, xanthan gum, guar gum, pectin, locust bean gum and the like.

In addition, examples of acidulants include citric acid, lactic acid, and malic acid, and examples of fruit juices include lemon juice, orange juice, and berry juice.

Besides these, various additives such as vitamins such as vitamin A, vitamin B, vitamin C, vitamin D and vitamin E and minerals such as calcium, iron, manganese and zinc can be added.

Moreover, the method for producing an estrogen-like composition of the present invention includes a step of bringing a hot water extract into contact with hot water to obtain a hot water extract.

In this step, for example, the dried mycelium mycelia can be dissolved in water and extracted with hot water at room temperature or higher, preferably 60 ° C. or higher, or water containing alcohol (hot water). More specifically, for example, a method of boiling blossom mycelia for about 5 to 20 minutes can be exemplified.

In addition, the method for producing an estrogen-like composition of the present invention can include a process of separation by centrifugation, a filtration process, a concentration / drying process, a freezing process, and the like.

The present invention is not limited to the above embodiment.

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

<Example 1> Genomic structure and general characteristics The genome sequence of Hanabiratake has not yet been determined. In order to standardize the Hanabiratake genome, the entire genome sequence of the Hanabiratake was determined.

Examples of the use of Hanabiratake Genome genome analysis data include evolutionary genome analysis by phylogenetic tree, DNA analysis or DNA analysis using rRNA / tRNA / ncRNA and its gene (DNA), aroma component synthases and β-glucan synthases Analysis of active ingredients using mutations and polymorphisms of synthases of useful ingredients, comprehensive and comprehensive gene analysis by RNA-Seq method, useful genes (effective ingredients by knockdown / knockout of genes by RNAi method or genome editing) ) And systematic classification of useful components. These examples of use and their use give an advantage to the production of pharmaceuticals, foods and drinks and estrogenic compositions with respect to the estrogenic compositions of the present invention.

(1) DNA sequence determination and genome assembly of Hanabiratake The liquid culture of the mycelium of the seed strain of S.crispa (Scrmy26 strain) was provided by Intertrade. Genomic DNA was extracted from this mycelium culture using a genome extraction kit (Macherey-Nagel: Nucleo Bond buffer set III). The genome of S. crispa mycelium (Scrmy26 strain) was sequenced using whole genome shotgun sequencing.

Specifically, using the P6-C4 reagent and 16 SMRT cells, the S. crispa genome was sequenced on the PacBio RSII platform (Pacific Biosciences) (21.3 Gbp (> 500 × coverage)). The average, N50, and longest inserted read lengths for the generated data were 9,616 bp, 13,871 bp, and 52,687 bp, respectively. The assembly of the obtained leads was performed by slightly changing a predetermined parameter set created by the developer using the Falcon pipeline (Non-patent Document 1).

Assembled contigs were obtained using the Arrow algorithm of the GenomicConsensus software package (https://github.com/PacificBiosciences/GenomicConsensus).

Genome sequence data is registered as Accession No. BFAD01000001-BFAD01000032 (PRJDB5582) in DDBJ BioProject Database of DNA Database Japan (DDBJ: http://www.ddbj.nig.ac.jp/).

(2) Prediction and functional annotation of genes encoding proteins RNA-Seq reads are first aligned with the reference genome of HISAT (Non-patent Document 2), and then StringTie (Non-patent Document 3) to construct a transcription sequence. Assembled by. AUGUSTUS (Non-patent Document 4) is used for parameters of the gene model of S. crispa using the obtained 15,184 transcripts, and exons and intron hints obtained from RNA-seq mapping data are used as input data did.

On the other hand, using CodingQuarry (Non-patent Document 5) developed for accurately predicting fungal genome genes, gene models were predicted using the above-mentioned transcription sequences derived from StringTie.遺 伝 子 13,156 consensus gene models were constructed using EvidenceModeler (Non-Patent Document 6) by combining the gene models predicted by AUGUSTUS, CodingQuarry, and StringTie. All predicted gene models were functionally annotated based on similarity to the annotated genes. Using BLASTP (Non-Patent Document 7), protein sequences were aligned to Swiss-Prot (Non-Patent Document 8) protein database Nr and fungal classification with e-value <1e-5.

(3) Results By assembling an approximately 21.3 Gbp read (> 500X coverage), a 39.0 Mb genomic sequence was obtained (Table 1).

This genome sequence assembly was composed of 32 contigs in total, with an L50 length of 3.18 Mb and 5 contigs representing N50 (FIG. 1, Table 2).

Based on the number of contigs and the number of chromosomes expected for mushrooms, the genome size was predicted to be close to the obtained size. In total, 13,157 protein-encoding genes were predicted, the average gene length was 1,669.3 bp, and the average number of exons was 5.7 (Table 3).

The number of genes in the genome of S. タ crispa was the same as the number of genes in the genome of other filamentous fungi (Non-patent Document 9). The predicted gene forms a transcript with an average length of 1.3 kb and a protein with an average length of 147 amino acids.

<Example 2> Comparison with other fungal genomes The predicted proteome of S. crispa was compared with 25 filamentous fungi that were sequenced.

(1) Preparation of phylogenetic tree and gene family expression analysis 26 species of fungi classified as Basidiomycota or Ascomycota with S. crispa were used for phylogenetic analysis. The protein sequences of these 26 fungi were compared by BLASTP with an e value <1e-5 and a hit count <500. 895 single copy orthologous genes were then determined by OrthoMCL (Non-Patent Document 10) using default parameters to obtain orthologous genes. Multi-sequence alignments of these 895 genes were calculated by MAFFT v7.309 (Non-Patent Document 11) software and combined into one long sequence for each species. Next, block regions stored in the aligned sequences were selected using Gblock 0.91b (Non-patent Document 12) using default parameters, and 221,266aa were obtained as final alignment lengths.

Alignment was input, and a phylogenetic tree was constructed by bootstrap analysis 1000 times using RAxML-8.2.9 (Non-Patent Document 13) software. Three fossil calibration points (Non-Patent Document 14) were fixed in the molecular clock analysis.

The most recent common ancestors (MRCA) Coprinopsis cinerea, Laccaria bicolor and Schizophyllum commune branched at 122.74 MYA. MSerpula lacrymans and Coniophora puteana MRCA diverged at 104.23 MYA. The MRCA of Pichia stipitis, Aspergillus niger, Cryphonectria parasitica, Stagonospora nodorum and Trichoderma reesei branched at 517.55 MYA.

Next, the branching time of other nodes was calculated by r8s v1.81 (Non-patent Document 15) software set to 1.8 by TN algorithm, PL method, and cross validation.

(2) Results The evolutionary history of S. crispa is a phylogenetic tree constructed using 895 single copy orthologous genes conserved in these 26 fungi obtained by OrthoMCL analysis (Fig. We investigated in 2).

According to molecular clock analysis, the brown rot fungus Postia placenta (Non-patent document 16, Non-patent document 17) is the fungus that is evolutionarily closest to S. crispa, and their branch was 94 million years ago (94 MYA).

<Example 3> Analysis of mating type loci Two mating type loci A and B in the genome sequence of S. crispa were identified on different contigs.

(1) Identification of matA and matB genes.

The matA gene was identified by mapping genomic protein sequences to the matA and MIP (mitochondrial intermediate peptidase) genes of Coprinopsis cinerea and Schizophyllum commune. The pheromone receptor gene was identified by Swiss-Prot annotation with the keyword “pheromone receptor”. The length of pheromone precursor is short, usually 50-60aa, so it cannot be predicted by normal genome annotation procedure. These sequences were searched in the approximately 20 kb flanking region of the pheromone receptor gene by Transdecoder (https://transdecoder.github.io/) software using Pfam search (https://pfam.xfam.org/) . The ORF (open reading frame) annotated in PF08015.9 was a pheromone precursor gene.

(2) Results Type A mating type loci were identified by homology search with HD1 and HD2 homeodomain transcription factors and MIP genes of Coprinopsis cinerea and Schizophyllum commune, and relatively high protein homology for MIP (66- 67% identity) was found, and low homology (28-34% identity) was found for HD1 and HD2 (Figure 3, Table 4).

However, all three genes were found to be located close together on the same contig (Contig 1). In addition, the arrangement of HD1 and HD2 genes (on different strands and showing characteristic outward transcription directions) is similar (Non-patent Document 18), suggesting that they are functional .

On the other hand, a total of 7 pheromone receptor genes and 3 pheromone precursor genes were found for the B-type mating loci mapped on Contig 10 (FIG. 3, Table 4).

Example 4 CAZymes and glycosyltransferases A total of 246 carbohydrate-related enzymes (CAZymes) were identified in the genome of S. crispa.

(1) Identification of CAZymes Carbohydrate-related enzyme (CAZymes) is HMM search by dbCAN HMM 5.0 (default cutoff threshold condition) and BLASTP search of CAZy database (Non-patent document 20) (e value ≤ 1e-6; fraction Ratio> = 0.2 and maximum hits = 500).

Next, the final CAZyme annotation was obtained by adding the BLASTP results screened using the threshold values used in the study of L. edodes (Non-Patent Document 9) to the common results. Therefore, the identification process used here is different from the identification process adopted by the CAZy system (Non-Patent Document 20), suggesting that it may occasionally conflict with previously published results.

(2) Results In the genome of S. crispa, 131 glycoside hydrolases (GH), 10 carbohydrate esterases (CE), 61 glycosyltransferases (GT), 4 polysaccharide lyases, 19 carbohydrate linkages A total of 246 carbohydrate-related enzyme gene (CAZymes) candidates, including modules (CBM) and 31 accessory active enzymes (AA), were identified.

The distribution of CAZymes in Hanabiratake (S. crispa) was compared with 25 other fungi.

Compared with the Agaricales genome, Polyporales generally has fewer CAZymes and S. crispa has the least number of GH (Tables 5 to 11).

Variety of Hanabiratake (S. scrispa) GT (Fig. 4) and GH (Fig. 5) from Phanerochaete sochrysosporium (Non-patent document 21), Postia placenta (Non-patent document 16) and Lentinula edodes (Non-patent document 9) Compared with. Compared to other fungi, S. crispa has the fewest number of genes in each category of CAZymes, and the group that includes Hanabiratake (S. crispa) or Hanabiratake (S. crispa) is GT1, GT5, GT17, GT69 and The number of GT family genes in GT90 is very small (Tables 5 to 11, FIG. 4).

On the other hand, S. crispa has a very small number of GH family genes (about half of Lentinula edodes), and most of the GH family was the lowest compared to the three other fungi (Tables 5-11). 5) Therefore, it has been confirmed that S. crispa is classified as a fungus with poor carbohydrate utilization.

<Example 5> Identification of β-glucan synthase The β-glucan synthase gene was identified by BLASTP search using the reported genes.

(1) Identification of β-glucan synthase gene Identification of β-glucan synthase gene is performed by BLAST search (Non-patent Document 19) using genes reported for type I and type II genes. Domain analysis of the transmembrane region was performed by Pfam or TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) database.
(2) Results Of the polysaccharides in the fungal cell wall, β-1,3-glucan (β-glucan) is the most abundant and widely used for various pharmacological actions. β-glucan is synthesized from uridine 5′-diphosphate (UDP) -glucose by β-glucan synthase. β-glucan synthase is a membrane protein consisting of a catalytic domain and a transmembrane domain. In Saccharomyces cerevisiae, there are two independent β-glucan synthase genes (FKS1 and FKS2). Identification of the β-glucan synthase gene was performed by BLASTP search using the reported gene. Here, two novel β-glucan synthase genes ScrFKS1 and ScrFKS2 were identified and found to be homologous to type I and type II genes, respectively (Table 12, FIG. 6).

The existence of these two different types is a common feature of Agaricomycetes mushroom (Non-patent Document 19). ScrFKS1 and ScrFKS2 are 77-90% or 77-92% homologous to previously reported type I and type II genes, respectively (Table 12), which revealed that they are new genes. A region homologous to the catalytic domain of FKS1 (Non-patent Document 22) and a region homologous to β-glucan synthase are identified by Pfam database search in both genes, and Pfam database search and transmembrane domain main are TMHMM database search (FIG. 6). , FIG. 7). The two transmembrane domains (TM1 and TM2) are in the same position, but there is a difference between them. The region between TM1 and TM2 is predicted to be outside the ScrFKS1 cell and localized inside the ScrFKS2. In addition, there are several N-glycosylation sites, some of which are well conserved among them (FIGS. 6, 7).

<Example 6> Preparation of an extract using the same strain In order to search for the active ingredient of the bamboo shoot, the extraction method of the fungus body was established and the extraction efficiency by the extraction method was evaluated.

(1) Materials and methods 1) Dry cells (mycelium) were dissolved in water and boiled for 10 minutes.
2) Separated with a centrifuge and filtered the supernatant.
3) Concentrated, dried and partially frozen.
4) The hot water extract of dried mycelium mycelia (hereinafter referred to as “SCE”) was adjusted to 10 mg / ml with sterile water.
5) The prepared SCE and ethyl acetate were mixed.
6) Centrifugation was performed to recover the ethyl acetate layer (supernatant).
7) The operations 5) to 6) were performed twice.
8) The ethyl acetate layer was concentrated to dryness to obtain an ethyl acetate extract of SCE (hereinafter referred to as “SCE-EtOAc”).
9) SCE-EtOAc was dissolved in methanol (10 mg / ml).
10) Phosphorylation assay and HPLC analysis (Documents 2 and 3).

(2) Results The extraction efficiency (recovery rate) of SCE was 30% and the extraction efficiency (recovery rate) of SCE-EtOAc was 2.5% with respect to 100% of the dried cells.

<Example 7> Evaluation of estrogen activity by cell proliferation assay The cell proliferation effect by HPLC fractionation of the mycelium extract of Hanabiratake was evaluated. The method followed (“Brefeldin A is an estrogenic, Erk1 / 2-activating component in the extract of Agaricusblazei mycelia.” (J. Agri. Food Chem., 2013).

In the cell proliferation assay, E ₂ (10 nM) and SCE (each adjusted to 1 μg / ml, 10 μg / ml, and 100 μg / ml) were added to the MCF-7 cell culture medium. After 72 hours of culture, sulforhodamine B (SRB ) Assay (Vichai V., Kirtikara K. Nat. Protoc. 2006) was used to calculate the cell growth rate relative to the control, and compared between each sample.

Specifically, the following materials and methods were used.

(1) Materials and methods 1) Cells and stimulating substances Cells: MCF-7 cells Stimulating substances: positive control (10 nM estrogen (E ₂ )), negative control (0.1% DMSO), SCE (1 μg / ml, 10 μg / ml) , 100 μg / ml).
2) Culture: MCF-7 cells were counted at 0.5 × 10 ⁴ cells per well and inoculated into a 24-well plate. As the medium, RPMI medium without phenol red supplemented with 10% charcoal dextran-treated FBS (CD-FBS) was used. The cells were cultured for 3 days at 37 ° C. in a CO ₂ incubator.
3) Stimulation: After confirming cell adhesion and proliferation, the medium was removed, a medium containing each stimulating substance was added to each well, and the cells were further cultured for 72 hours.
4) Fixation: The medium was removed and each well was washed with PBS. 10% trichloroacetic acid was added and the mixture was allowed to stand at 4 ° C. for 30 minutes, washed with distilled water, and dried.
5) Staining: Cells were stained for 20 minutes by adding 0.4% SRB / 1% acetic acid solution to each well.
6) Washing: Washed with 1% acetic acid to remove SRB and dried overnight.
7) Measurement: After adding 10 mM Tris-HCl buffer (pH 10.4) to each well and eluting the SRB taken up by the cells, the cells are transferred to a 96-well plate and microplate reader (SPECTRA MAX190 Molecular Devices) Was used to measure the absorbance (OD ₄₉₀ ).

(2) Results The results are shown in FIG. The cell proliferation assay was performed three times to evaluate reproducibility. It was confirmed that estrogen (E ₂ ) exhibited a cell growth promoting effect more than twice that of the control, whereas SCE had no proliferation activity. This result can be grounds for safe SCE intake.

<Example 8> Evaluation of estrogen activity using a DNA chip for estrogen activity evaluation Using an estrogen-responsive gene, an oligo DNA microarray (DNA chip) for evaluating estrogen activity of a chemical substance was prepared, and estrogen activity was evaluated. . Using an assay system (“DNA microarray-based gene expression profiling of estrogenic chemicals” (Cell. Mol. Life Sci., 2014)), cells between E ₂ and SCE samples (SCE10, SCE100, SCE-EtOAc) The expression profiles of estrogen-responsive genes during stimulation were compared, and the estrogenic activity of the extract of Hanabiratake was evaluated.

Specifically, the following materials and methods were used.

(1) Materials and Methods 1) Cells and stimulants Cells: Human-derived MCF-7 cells Stimulators: Positive control (10 nM estrogen (E ₂ )), negative control (0.1% DMSO), SCE10 (SCE 10 μg / ml) , SCE100 (SCE 100 μg / ml), SCE-EtOAc (extracted fraction of ethyl acetate, 10 μg / ml) were used.

Culture: MCF-7 cells were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS). The cells were cultured for 3 days at 37 ° C. using a CO ₂ incubator. Cells were subcultured using RPMI medium without phenol red supplemented with 10% charcoal dextran-treated FBS (CD-FBS). At this time, the number of cells was 1 × 10 ⁶ cells per petri dish and seeded on the petri dish. Cells After confirming that it has adhered to the Petri dish, cells were stimulated with each of _{E 2, DMSO, SCE10, SCE100} , SCE-EtOAc. Thereafter, the cells were further cultured at 37 ° C. for 3 days.

2) Cell recovery, 3) RNA extraction, 4) RNA amplification, 5) cDNA synthesis, and 6) cDNA labeling were performed using RNeasy® Plus® Micro® and Mini® Kits (QIAGEN). .

7) Measurement of fluorescence intensity Measurement was performed using a microarray scanner FLA8000 (FUJI).

8) Statistical analysis Statistical analysis of the obtained microarray data was performed by correlation analysis and cluster analysis. Correlation analysis was performed as follows. First, for each of estrogen-responsive genes (172 genes), the fluorescence intensity with compound without addition Sample of samples spiked with the compound (E ₂ + or SCE +) - the ratio of the fluorescence intensity of the (E ₂ or SCE-) determined It was. Next, after correcting the fluorescence intensity ratio using 28 internal control genes ((E ₂ + / E ₂ −) or (SCE + / SCE−)), each correction value was converted to a log ₂ value, An overall picture (gene expression profile) of 172 gene expression was obtained for each sample. The correlation between the two profiles was shown by a scatter plot, and the correlation coefficient (R value) was calculated based on linear regression.

Cluster analysis using estrogen-responsive genes is performed using the cluster analysis software Cluster 2.12 and Tree view (Eisen et al., 1998) using the correction values of each sample described in 8) to create a phylogenetic tree. And analyzed.

Note that cluster analysis is a method of statistically comparing each group by collecting clusters that have similar properties from groups that have different properties mixed together and creating clusters.

(2) Results DNA microarray assays are used between E ₂ and SCE samples a profile of expression of estrogen-responsive genes when stimulated cells were compared respectively, R value of SCE10 is 0.76, SCE100 is the R value 0.62 It was found to be high (FIG. 10).

On the other hand, the R value between SCE-EtOAc was as low as 0.04 mm. This is presumably because the active substance was extracted into the water fraction and hardly eluted in the ethyl acetate extract fraction (EtOAc fraction).

From the results of cluster analysis using estrogen responsive genes, SCE10 and SCE100 showed profiles similar both with E ₂ (Fig. 11).

From the above, SCE was found to have estrogenic activity similar to E _2, its activity is considered to elute in water fraction further.

Notice that the SCE results of evaluation DNA microarray assays estrogenic activity by <Example 9> signaling proteins have estrogenic activity similar to E _2, further its activity found to be eluted in water fraction It was. From this, the fraction of the extract of the mycelium extracted from the water fraction was separated using a reverse phase column, and the activity of each separated fraction was evaluated using a phosphorylation assay.

(1) Method 1) Separation by reverse phase column Separation by a reverse phase column was performed under the following conditions.

Elution condition: ₂ ml of SCE 10 mg / ml (dissolved in H ₂ O (distilled water)) was injected.
a: 100% H ₂ O → 20% AcN: 0 minutes → 60 minutes (60 minutes)
b: 20% AcN → 100% AcN: 60 minutes → 75 minutes (15 minutes)
c: 100% AcN → 100% H ₂ O: 75 minutes → 80 minutes (5 minutes)
Column: CAPCELL PAK C18 MG II S5 (20 mm x 250 mm)
Flow rate: 10 ml / min
The separated sample was concentrated and dried, weighed, adjusted to 10 mg / ml with sterilized H ₂ O, and then used for the phosphorylation assay by Western blotting.

2) Phosphorylation assay of SCE fraction by Western blotting Cell: MCF-7 cell Stimulant: Positive control (10 nM E ₂ ), SCE (10 μg / ml), 26 samples separated by reverse phase column (10 μg / ml) was used.

Stimulation time: 0 (Control), 5, 15, 30, and 60 minutes Antibodies that detect phosphorylated protein: Phospho-p44 / 42 MAPK (ERK), Phospho-Akt (Ser473) were used.

Antibody that detects total protein: p44 / 42MAPK (ERK), Akt antibody was used.

Method: Cells were subcultured as described above and seeded using a 6-well petri dish at 1 × 10 ⁵ cells per well. After culturing for 2 days, the medium was replaced with serum-free RPMI medium and further cultured for 24 hours. Each fraction of SCE was added to stimulate the cells. After the stimulation time, the cells were rapidly lysed and subjected to protein denaturation treatment, and then the variation in the amount of phosphorylated protein (P-ERK and P-Akt) was examined using Western blotting. The amount of phosphorylated protein was corrected using total protein (T-ERK and T-Akt).

(2) Results 1) An elution pattern shown in FIG. 12 was obtained using a reverse phase column under the above elution conditions. Fraction with the acetonitrile concentration shown in the figure was collected.
2) Separation and purification was carried out twice using a reverse phase column, and a total of 26 fractions could be obtained. After preparing them at 10 mg / ml, the activity was evaluated by a phosphorylation assay by Western blotting. As shown in FIG. 13, in P-ERK, stimulation such as E ₂ and SCE as positive controls was performed. After 5 to 15 minutes, an increase in activity was observed. Prominent examples are 6B3, 6B4, 6B6, 6B7, 6B10. On the other hand, fractions showing low activity such as 6B5 and 6B8 were also confirmed.

On the other hand, in P-Akt, high activity was observed in the 6B3, 6B4, and 6B7 fractions. Since only one result affects the state of cells at the time of stimulation and the amount ratio of active ingredients eluted in each fraction, the activity value is affected. The reproducibility was evaluated for the activity in each fraction.

<Example 10> Safety of extract As biological safety evaluation, a dried oriental mycelium mycelium culture extract and a hot water extract Shana (extracted dry matter) SCE (extracted dry product) were orally administered to a mouse once, and its acute toxicity Evaluated.

(1) Method The group composition shown in the following Table 7 was performed, and the dried cultivated mycelium and the dried mycelium mycelium were orally administered at a dose of 2000 mg / kg to 5 male / female ICR mice each. The animals were observed for 14 days after the administration, and after the observation on the 14th day, acute toxicity was evaluated by autopsy (visual observation).

(2) Results General condition after administration, changes in body weight (Figs. 14 and 15), and autopsy findings on day 14 after administration, caused by dried cultivated mycelium mycelia and hot water extract Shana (extracted dried product) No change was observed.

No effect was observed even after a single dose of garlic mushroom cultivated dried garlic mushroom mycelia hot water extract SCE (extracted garlic extract) up to 2,000 mg / kg, and the minimum lethal dose was 2,000 mg / kg or more for both males and females. Inferred.

<Example 11> Anti-arteriosclerotic action and visceral fat accumulation-reducing action BALB / c.KOR / Stm Slc-Apoeshl mice (hereinafter referred to as ApoE-deficient mice) fed with a high-fat diet were treated with 180 liters of dried mycelium mycelium. mg / kg / day, dried leaflet mycelium was orally administered at 1000 mg / kg / day for 6 weeks each day. At the end of the administration period, blood biochemical tests and visceral fat accumulation were measured.
(1) Method ApoE-deficient mice fed a high-fat diet were orally administered daily for 6 weeks each at 180 mg / kg / day for the dried mycelium mycelium and 1000 mg / kg / day for the mycelium extract. did. At the end of the administration period, blood was collected under anesthesia and then a blood biochemical test was performed. Visceral fat (peritesticular fat and perirenal fat) was removed, body fluid adhering to each organ was removed with physiological saline, and the weight was measured.

(2) Results Total cholesterol levels were statistically higher in the ApoE-deficient mice after 6 weeks of daily oral administration, compared to the non-administered group ApoE-deficient mice, respectively. Significantly lower trend. Neutral lipid levels were also statistically significantly lower in the ApoE-deficient mice treated with the dried agaric fungus mycelium and the dried extract of the mycelial mycelia compared to the non-administered group ApoE-deficient mice after 6 weeks of daily oral administration. The value trend was shown. The free fatty acid levels were also statistically significantly lower in the ApoE-deficient mice that were administered with the dried Candida mycelium culture extract and the dried Candida mycelium extract after 6 weeks of oral administration compared to the non-administered group ApoE-deficient mice. The value tendency was shown (FIG. 16). In addition, visceral fat accumulation was statistically significant in ApoE-deficient mice after administration of 6-day oral administration for 6 weeks, respectively, in the ApoE-deficient mice that received the dried clove mycelium mycelia extract and the dried Agaricus mycelium extract. Showed a low value tendency (FIG. 17).

Claims (9)

An estrogen-like action composition characterized by containing a hot water extract of Hanabiratake mycelium. A medicament for treating or preventing a disorder or disease caused by estrogen deficiency, comprising the estrogen-like composition of claim 1. A food or drink for treating or preventing a disorder or disease caused by estrogen deficiency, comprising the estrogen-like action composition according to claim 1. A method for producing an estrogen-like action composition comprising a step of bringing a hot water bamboo mycelium into contact with hot water to obtain a hot water extract. 品質 Quality control of the extract of the mycelium of the mycelium, the establishment of the standard strain of the fungus, and the systematic analysis of the estrogen-like composition. How to use Hanabiratake on the effectiveness of Hanabiratake based on genomic DNA base sequence information. The usage method for the effectiveness of the garlic bamboo based on the information of the DNA base sequence of the sugar-related enzyme gene of the garlic bamboo. The usage method for the efficacy of Hanabiratake, based on the DNA base sequence information of the beta-1,3-glucan synthase gene of Hanabiratake. 2. The estrogenic action composition according to claim 1, wherein the estrogenic action is an anti-arteriosclerosis action and visceral fat accumulation suppression by reducing total cholesterol level, neutral fat level and free fatty acid level.

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