WO2018168879A1 - Composition ayant une action similaire à celle de l'œstrogène, médicament, aliment et boisson la contenant, procédé de production d'une composition ayant une action similaire à celle de l'œstrogène et méthode d'utilisation d'une séquence de base d'adn de sparassis crispa - Google Patents

Composition ayant une action similaire à celle de l'œstrogène, médicament, aliment et boisson la contenant, procédé de production d'une composition ayant une action similaire à celle de l'œstrogène et méthode d'utilisation d'une séquence de base d'adn de sparassis crispa Download PDF

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WO2018168879A1
WO2018168879A1 PCT/JP2018/009817 JP2018009817W WO2018168879A1 WO 2018168879 A1 WO2018168879 A1 WO 2018168879A1 JP 2018009817 W JP2018009817 W JP 2018009817W WO 2018168879 A1 WO2018168879 A1 WO 2018168879A1
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estrogen
action
mycelium
composition
hanabiratake
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PCT/JP2018/009817
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English (en)
Japanese (ja)
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敏雄 中西
喜幸 古谷
真弘 田中
亮一 木山
佳代子 川口
廣瀬　玉紀
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学校法人東京女子医科大学
株式会社インタートレード
国立研究開発法人産業技術総合研究所
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Priority to JP2019506060A priority Critical patent/JP6861422B2/ja
Publication of WO2018168879A1 publication Critical patent/WO2018168879A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/30—Oestrogens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

Definitions

  • 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 ⁇ ⁇ 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.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the estrogen-like action can treat or prevent disorders and diseases caused by estrogen deficiency.
  • an estrogen-like action composition can be obtained with certainty.
  • FIG. 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 ⁇ -glucan synthase of S. ⁇ ⁇ crispa.
  • the nucleotide sequence and deduced amino acid sequence of the ⁇ -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 ⁇ -glucan synthase of S. ⁇ ⁇ crispa.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • a processed product such as a crushed material, a ground product, a tissue disrupted product, a dried product, or a dried crushed product.
  • 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.
  • 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.
  • 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.
  • 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.
  • the hot water extract can be obtained by contacting the mycelium of the moss with hot water.
  • 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).
  • hot water preferably boiled garlic mycelium is boiled for about 5 to 20 minutes. The method of doing can be illustrated.
  • 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.
  • 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.
  • 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.
  • soy isoflavone 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.
  • soy isoflavone has cell growth activity and is not equivalent to silent estrogen.
  • estrogen-like action means the same action as estrogen, for example, anti-arteriosclerosis action.
  • 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.
  • 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.
  • 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.)
  • 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.
  • 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.
  • 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.
  • excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and succinic acid.
  • binder examples include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, calcium phosphate, polyvinylpyrrolidone and the like.
  • disintegrant examples include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose.
  • lubricants examples include purified talc, stearate, borax, and polyethylene glycol
  • flavoring agents include sucrose, orange peel, citric acid, and tartaric acid.
  • 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.
  • a corrigent may include those listed above
  • examples of the buffer include sodium citrate
  • 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.
  • additives to be added to food and drink include various sugars, emulsifiers, thickeners, acidulants, fruit juices, and the like.
  • 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.
  • high-intensity sweeteners such as sugar alcohols, aspartame, stevia, acesulfame potassium, and sucralose.
  • emulsifier examples include glycerin fatty acid ester and lecithin.
  • thickening (stabilizing) agent examples include carrageenan, xanthan gum, guar gum, pectin, locust bean gum and the like.
  • examples of acidulants include citric acid, lactic acid, and malic acid
  • examples of fruit juices include lemon juice, orange juice, and berry juice.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Genome genome analysis data examples include evolutionary genome analysis by phylogenetic tree, DNA analysis or DNA analysis using rRNA / tRNA / ncRNA and its gene (DNA), aroma component synthases and ⁇ -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.
  • RNA-Seq method useful genes (effective ingredients by knockdown / knockout of genes by RNAi method or genome editing) )
  • 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).
  • 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/).
  • 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.
  • Non-patent Document 5 CodingQuarry
  • Gene models were predicted using the above-mentioned transcription sequences derived from StringTie. ⁇ ⁇ ⁇ 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.
  • 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.
  • 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).
  • the number of genes in the genome of S. ⁇ 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.
  • Non-Patent Document 13 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 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).
  • 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.
  • Example 4 CAZymes and glycosyltransferases A total of 246 carbohydrate-related enzymes (CAZymes) were identified in the genome of S. crispa.
  • 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).
  • 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.
  • ⁇ -glucan synthase gene was identified by BLASTP search using the reported genes.
  • ⁇ -glucan synthase is a membrane protein consisting of a catalytic domain and a transmembrane domain.
  • Saccharomyces cerevisiae there are two independent ⁇ -glucan synthase genes (FKS1 and FKS2). Identification of the ⁇ -glucan synthase gene was performed by BLASTP search using the reported gene.
  • FKS1 and FKS2 Two novel ⁇ -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).
  • Non-patent Document 19 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 ⁇ -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.
  • there are several N-glycosylation sites some of which are well conserve
  • Example 6 Preparation of an extract using the same strain
  • the extraction method of the fungus body was established and the extraction efficiency by the extraction method was evaluated.
  • SCE-EtOAc ethyl acetate extract of SCE
  • 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 (“Greeneldin A is an estrogenic, Erk1 / 2-activating component in the extract of Agaricusblazei mycelia.” (J. Agri. Food Chem., 2013).
  • E 2 (10 nM) and SCE were added to the MCF-7 cell culture medium.
  • SRB sulforhodamine B Assay
  • Cells and stimulating substances Cells: MCF-7 cells Stimulating substances: positive control (10 nM estrogen (E 2 )), negative control (0.1% DMSO), SCE (1 ⁇ g / ml, 10 ⁇ g / ml) , 100 ⁇ g / 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.
  • CD-FBS charcoal dextran-treated FBS
  • Example 8 Evaluation of estrogen activity using a DNA chip for estrogen activity evaluation
  • an oligo DNA microarray (DNA chip) for evaluating estrogen activity of a chemical substance was prepared, and estrogen activity was evaluated.
  • 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
  • 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.
  • 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 ⁇ g / ml) , SCE100 (SCE 100 ⁇ g / ml), SCE-EtOAc (extracted fraction of ethyl acetate, 10 ⁇ g / ml) were used.
  • 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.
  • FBS fetal bovine serum
  • 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.
  • 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.
  • 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).
  • 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.
  • mice 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.
  • 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.
  • 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.
  • 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).
  • 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).

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Abstract

Le problème décrit par la présente invention consiste à fournir une composition ayant une action similaire à celle de l'œstrogène qui comprend un extrait dans l'eau chaude de mycélia de Sparassis crispa .
PCT/JP2018/009817 2017-03-13 2018-03-13 Composition ayant une action similaire à celle de l'œstrogène, médicament, aliment et boisson la contenant, procédé de production d'une composition ayant une action similaire à celle de l'œstrogène et méthode d'utilisation d'une séquence de base d'adn de sparassis crispa WO2018168879A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024006723A1 (fr) 2022-06-30 2024-01-04 The Procter & Gamble Company Articles absorbants et procédés et appareils pour fabriquer des articles absorbants comprenant des parties frangibles
JP7440838B1 (ja) 2023-07-06 2024-02-29 株式会社インタートレードヘルスケア 卵巣機能欠落生体の内臓脂肪蓄積抑制飲食品、及び卵巣機能欠落生体の内臓脂肪蓄積抑制薬

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Publication number Priority date Publication date Assignee Title
WO2006126488A1 (fr) * 2005-05-25 2006-11-30 Unitika Ltd. Extrait de sparassis crepu

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WO2006126488A1 (fr) * 2005-05-25 2006-11-30 Unitika Ltd. Extrait de sparassis crepu

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HONG KI BAE ET AL.: "Hypocholesterolemic Effects of the Cauliflower Culinary-Medicinal Mushroom,Sparassis crispa (Higher Basidiomycetes), in Diet-Induced Hypercholesterolemic Rats", INTERNATIONAL JOURNAL OF MEDICINAL MUSHROOMS, vol. 17, no. 10, 2015, pages 965 - 975, ISSN: 1521-9437 *
NISHIBORI, KOZO ET AL.,: "Seibutsu-kogaku kaishi /Maitake mushroom/", vol. 92, no. 10, 2014, pages 572 - 575, ISSN: 0919-3758 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024006723A1 (fr) 2022-06-30 2024-01-04 The Procter & Gamble Company Articles absorbants et procédés et appareils pour fabriquer des articles absorbants comprenant des parties frangibles
JP7440838B1 (ja) 2023-07-06 2024-02-29 株式会社インタートレードヘルスケア 卵巣機能欠落生体の内臓脂肪蓄積抑制飲食品、及び卵巣機能欠落生体の内臓脂肪蓄積抑制薬

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