JP5768230B2 - Estrogenic agent - Google Patents

Description

  The present invention relates to a drug for treating or preventing disorders and diseases associated with estrogen deficiency, and a health supplement for supplementing estrogen deficiency.

Himematsutake is a mushroom that grows naturally in Brazil. Its scientific name is "Agaricus blazei Murrill" and its name is "Kagariharatake". Himematsutake has long been called the “God's Mushroom” in the field, and it has been known that Himematsutake has a preventive / improving effect on cancer, infectious diseases, diabetes, etc. Extracts of fruiting bodies and fungal bodies are eaten as health foods. By the way, in general, the effect is such as β-D-glucan, which is a high molecular polysaccharide contained in the fruit body of Himematsutake, glucomannan protein polysaccharide (ATOM) contained in mycelium, mannan protein complex It is thought to be due to the action of the body (AB-FP). However, it is suggested that various active ingredients such as lectin and polyphenol oxidase are effective, and that various active ingredients such as steroid apoptosis-inducing action are acting in combination. (For example, Patent Documents 1-6)
In addition, it has been reported that the water-soluble complex component extracted from the fruit body and the fruit body of Himematsutake has an anti-arteriosclerotic action, and this action is due to the protein polysaccharide complex component of the extract. It has been suggested. (Patent Document 7)
As described above, the extract of Himematsutake is known to have various pharmacological actions and has great potential for use in the treatment of diseases such as cancer and adult diseases. The body of is not revealed. In order to use Himematsutake as a medicine, not as a health food, it is necessary to elucidate its pharmacological action at the substance level.

  By the way, it has been conventionally known that estrogen deficiency causes women's menopause and various diseases. Specific examples include genitourinary disorders such as atrophic vaginitis, prostate cancer, benign prostatic hyperplasia, osteoporosis, arteriosclerosis, memory impairment, Alzheimer's disease, thrombotic disease and the like.

  In particular, arteriosclerosis is one of the major causes of heart disease and cerebrovascular disease, and progression of arteriosclerosis involves various factors such as individual constitution and lifestyle habits, including dietary habits. It is necessary to elucidate the functional mechanism at the gene level involved. Women also have low LDL (low density lipoprotein) cholesterol levels before menopause, but it is known that LDL cholesterol levels increase and arteriosclerosis progresses after menopause, and estrogen deficiency is the main cause. I know it is the cause.

  However, for example, estrogen treatment for improving postmenopausal arteriosclerosis in women has known side effects such as an increased risk of endometrial cancer, and it is rather dangerous to administer estrogen directly. It is pointed out that it is. Against this background, in recent years, attention has been paid to a method for treating arteriosclerosis using a plant-derived estrogen-like substance such as soybean isoflavone instead of directly using estrogen for treatment. In this way, interest in useful substances in foods is increasing, and health foods that can be taken safely without any side effects are drawing attention. In particular, for elderly people, pregnant women, infants, children, etc., there is a demand for a method of treatment and prevention using food-derived ingredients that have no side effects.

Japanese Laid-Open Patent Publication No. 1-6 7 1 94 Japanese Laid-Open Patent Publication No. 1-6 7 1 95 Japanese Patent Laid-Open No. 2-786 30 Japanese Patent Laid-Open No. 2-1 24 8 29 Japanese Patent Application Laid-Open No. 6-12 864 Japanese Patent Application Laid-Open No. 7-258081 JP 2004-359645 A

  The present invention provides a therapeutic agent for a disorder or disease caused by deficiency of estrogen, using as an active ingredient a substance extracted from the mycelium of Japanese matsutake mushroom or a processed product thereof, and health for supplementing deficiency of estrogen The challenge is to provide supplements.

  The inventors of the present invention examined the action of the extract of the mycelium of the matsutake mushroom in research on the matsutake mushroom, and in detail, as described later in the examples, in vivo, the inhibitory effect on the oxidized LDL level in serum, It has been found that it has an effect of improving atherosclerosis.

In addition, when the gene expression level was examined using a DNA microarray, it was found that the extract of mycelium of E. matsutake has an estrogen-like action. As a result of examining the relationship between the above effect and the estrogen-like action, Estrogen-like effects induced by administration of mycelium extract are caused by activation of Erk1 / 2 and Akt, which are known to play an important role in suppressing oxidized LDL levels and improving atherosclerosis I found out that it was. Thereby, it was clarified that the above-mentioned effect exhibited by the extract of the mycelium of Himematsutake was due to an estrogen-like action.
Further, as a result of examining the estrogen-like action at the substance level, the composite component fraction containing a large amount of brefeldin A showed a particularly strong estrogen-like action. The present invention relating to drugs and the like has been completed.

Based on the above findings, the present invention provides the following extract of the mycelium of matsutake mushroom, the drug and the production method thereof, the health supplement, and the production method of Brefeldin A.
<1> Solvent extract from Himematsutake fungus bodies and / or processed products thereof, which has an estrogenic action.
<2> The first extract of Mycobacterium matsutake, whose estrogenic action is an anti-atherosclerotic action.
<3> The first or second Himematsutake fungus body extract, wherein the solvent is water, hot water or an organic solvent.
<4> The extract of Himematsutake mycelia according to any one of 1 to 3 above, wherein the extract contains Brefeldin A represented by the following formula (I).

<5> The above-mentioned extract of mycelia bryoderdin A, wherein Brefeldin A is Brefeldin A represented by the following formula (II).

<6> A drug for treating or preventing a disorder or disease associated with estrogen deficiency, comprising the extract according to any one of the first to fifth as an active ingredient.
<7> A drug for treating or preventing arteriosclerosis or hyperlipidemia, the drug containing any one of the extracts from 1 to 5 as an active ingredient.
<8> A health supplement for supplementing deficiency of estrogen, comprising the extract according to any one of the first to fifth as an active ingredient.
<9> A health supplement for lowering the oxidized LDL value, comprising any one of the extracts from 1 to 5 as an active ingredient.
<10> A drug for treating or preventing a disorder or disease associated with estrogen deficiency, which comprises brefeldin A represented by the above formula (I).
<11> A drug for treating or preventing arteriosclerosis or hyperlipidemia, which comprises brefeldin A represented by the above formula (I).
<12> The tenth or eleventh drug, wherein Brefeldin A is Brefeldin A represented by the above formula (II).
<13> The tenth or eleventh drug, wherein the Brefeldin A is obtained by solvent extraction from a mycelium of Himematsutake and / or a processed product thereof.
<14> A health supplement for supplementing deficiency of estrogen, which contains brefeldin A represented by the above formula (I).
<15> A health supplement for lowering the oxidized LDL value, comprising brefeldin A represented by the above formula (I).
<16> The fourteenth or fifteenth health supplement, wherein the Brefeldin A is Brefeldin A represented by the above formula (II).
<17> The fourteenth or fifteenth health supplement, wherein Brefeldin A is obtained by solvent extraction from a mycelium of Himematsutake and / or a processed product thereof.
<18> The method for producing the tenth or eleventh drug, wherein at least the brefeldin A represented by the above formula (I) is solvent-extracted from at least mycelia of himematsutake and / or a processed product thereof. The manufacturing method of the chemical | medical agent containing this.
<19> A method for producing Brefeldin A represented by the above formula (I), wherein the solvent is extracted from the mycelium and / or the treated product thereof.
<20> The method for producing the nineteenth Brefeldin A, wherein the Brefeldin A is Brefeldin A represented by the above formula (II).

  The mycelium extract of the pine agaric of the present invention has an estrogen-like action. For example, it is an estrogen-like action agent in an anti-arteriosclerosis agent, a blood oxidation LDL inhibitor, hormone replacement therapy, etc., and supplements estrogen. It can be used as a health supplement or supplement. In addition, Himematsutake is an edible mushroom, so there is no fear of side effects when used as described above.

  Further, by using Brefeldin A contained in the mycelium extract of Himematsutake as an active ingredient, more effective utilization is possible.

  Furthermore, the extract of Himematsutake mycelium is easy to formulate because it is in a state where the active ingredient Brefeldin A is included by the protein polysaccharide complex component contained in the extract.

  In addition, because of the novel finding that brefeldin A can be extracted from the mycelium of Japanese matsutake mushrooms, a safer drug than when extracted from actinomycetes when using brefeldin A as an antibiotic or anticancer agent, etc. Can be provided.

It is a figure which shows the effect with respect to myocardial ischemia by a Japanese apricot powder. (A) and (B) represent the accumulation of technetium before and after administration of Japanese apricot powder, respectively. It is a figure which shows the influence with respect to a serum oxidation LDL level by ingestion of a Japanese apricot powder. The data is calculated as the weight ratio of oxidized LDL to total LDL (ng oxidized LDL / μgLDL) and shows the mean and sub-deviation. * P <0.05, ** P <0.01. It is a figure which shows the in-vitro peroxidation assay of LDL. Each of the fractions was prepared from a Japanese matsutake extract, and shows an ethanol extraction fraction (Fraction I), a boiling water extraction fraction (Fraction II), and a salt-soluble fraction (Fraction III). The immunological analysis result of the coronary artery is shown. Rabbits (Group I: AC) not given himematsutake powder were examined for arteriosclerotic disorders in the septal coronary artery, and rabbits (group II: DF) given himematsutake powder and control group (group III: figure GI) The lesion of the left coronary artery was examined. Each figure shows the result of Masson trichrome staining (A, D, G), immunostaining with RAM11 (B, E, H), and staining with an antibody against smooth muscle α-actin (C, F, I) . Each figure shows a magnified image of × 100. It is the figure which carried out the immunohistochemical dyeing | staining of the aorta. A to J are group I rabbits, and K to T are group II rabbit aorta 100 times magnified images. Specifically, RAM11 (A, K), smooth muscle α-actin (B, L), type I collagen (C, M), type III collagen (D, N), type IV collagen (E, O), Ki67 Analysis was performed using (F, P), BAX (G, Q), LOX-1 (H, R) and CD36 (I, S) antibodies. Apoptosis was stained by the TUNEL method (J, T). The MCF-7 cell proliferation activity by a himematsutake extract or a himematsutake mycelium culture solution is shown. The concentration of each matsutake mushroom extract (ABE), or the results when using pure water and 10 nM E2 are shown. The experiment was performed at least three times, and the ratio (Proliferation Index;%) of the obtained 490 nm absorbance to the 490 nm absorbance obtained in the control experiment was calculated, and the average and standard deviation were shown. It is a figure which shows the comparison of the expression profile of 203 estrogen response genes when a MCF-7 cell is stimulated with a Japanese matsutake extract or E2. The gene expression profile obtained using the 2 μg / mL Japanese matsutake extract solution and the gene expression profile obtained using E2 (average values of three experimental results) were compared and shown as a scatter diagram. It is a figure which shows the result confirmed by real-time RT-PCR about the gene (Table 1) which showed the different gene expression by a Himematsutake extract and E2 stimulation by DNA microarray analysis. The graph shows the average and standard deviation of the values obtained by log ₂ conversion of the results of E2 (gray graph) or Japanese matsutake extract (white graph). * P <0.05, ** P <0.01. It is a figure which shows the rapid activation of Erk1 / 2 and Akt by the Japanese apricot extract (ABE) in HUVEC and TIB-67 cell. It is a figure which shows the inhibition of the activation of Erk1 / 2, Akt, and p38MAP kinase by stimulation with oxidized LDL (OxLDL) by the extract of Himematsutake in HUVEC and TIB-67 cells. Proteins are extracted using SDS sample buffer and analyzed by Western blotting. It is a figure which shows the analysis result of mRNA of GPX3, LOX-1, and eNOS in HUVEC cell by real-time RT-PCR. The amount of each mRNA was normalized by the amount of β-actin. Data represent the average of the results of three experiments. ** P <0.01. It is a figure which shows the result about the Western blotting analysis of GPX3 in a HUVEC cell, and eNOS protein. It is a schematic diagram which shows the influence which the Japanese apricot extract extracts in a vascular endothelial cell. (A) Chromatography by HPLC of the ethyl acetate fraction. Every 10 fractions were mixed with the eluate and examined for Erk1 / 2 phosphorylation activity. (B) Erk1 / 2 phosphorylation activity of the fraction 51-60 in which Erk1 / 2 phosphorylation activity was observed was re-chromatographed. It is a figure which shows the cell growth activity evaluation of Brefeldin A by a SRB test. It is a figure which shows activation of Erk1 / 2 protein by Brefeldin A. It is a figure which shows the estrogen activity evaluation (1) of Brefeldin A by a reporter gene test. It is a figure which shows the estrogen activity evaluation of a Japanese apricot mycelium extract by a reporter gene test. It is a figure which shows the estrogen activity evaluation (2) of Brefeldin A by a reporter gene test. It is a figure which shows the comparison of the activity of a mycelium and a fruiting body. It is a figure which shows extraction of Brefeldin A by various solvent.

  As described above, the present invention is based on the novel finding that the extract of the mycelium of matsutake mushroom has an estrogen-like action.

  Here, “estrogen-like action” means an action similar to that of estrogen, such as an anti-arteriosclerosis action. That is, the present invention has a good action of estrogen. In addition, although it is known that there are a carcinogenic risk, a thrombus risk, etc. as a bad influence by an estrogen action, in this invention, the fault which such an estrogen has is improved.

  The “estrogenic action” will be described in more detail. As a similar example, the estrogenic action of soybean isoflavone is known. One of the reasons why soy isoflavones are recognized as an active ingredient in health foods is that they not only have the same effect as estrogen, but also do not have the risk of strong carcinogenesis (including cancer cell growth) that estrogen exhibits . For example, the Ministry of Health, Labor and Welfare's Food Safety Commission report “Fundamental Concepts for Safety Evaluation of Foods for Specified Health Use Including Soy Isoflavone” (May 2006: See attached file) In the report on risks, etc., there is a report that “in vitro studies have shown that intake of soy isoflavone has an anti-estrogenic effect on the development of breast cancer” (page 35).

  Thus, the drug of the present invention has an estrogen-like action as in the case of soy isoflavone and does not have the disadvantages of estrogen, so there is no risk of side effects, and disorders associated with estrogen deficiency, It is effective for treatment or prevention of diseases.

  Here, the “disorder associated with estrogen deficiency” includes, for example, climacteric disorder, and according to the agent of the present invention, autonomic dysfunction due to climacteric disorder (facial flushing, sweating, coldness, sleep disorder, palpitations) , Headache, etc.), psychiatric symptoms (depression, mental instability, decreased memory, etc.), other symptoms (stiff shoulders, joint pain, abdominal pain, loss of appetite, fatigue, etc.) (supervised by Masakazu Sakamoto et al .: Principal Obstetrics and Gynecology 1 Revised version, Medical View, 2001). “Diseases associated with estrogen deficiency” include, for example, genitourinary disorders such as atrophic vaginitis, prostate cancer, benign prostatic hyperplasia, osteoporosis, arteriosclerosis, memory impairment, Alzheimer's disease, thrombotic disease, etc. .

  Moreover, the health supplement of the present invention is useful for the improvement of skin spots, wrinkles and the like and for cosmetic purposes such as breast augmentation.

  Specifically, the drug and health supplement of the present invention contain Brefeldin A represented by the following formula (I) as an active ingredient.

  Brefeldin A has 6 asymmetric carbons (carbon atoms at 1, 4, 5, 7, 9, and 15), and several isomers exist. Yes. For example, Brefeldin A having a configuration represented by the following formula (II) and its epimer 7-epi-brefeldin A, 4,7-epi-brefeldin A, etc. are known ( J. Chem. Soc., 1, 2387 (1982); Angew. Chem. Int. Ed. Engl., 23, 145 (1984)). Among these stereoisomers, brefeldin A represented by the following formula (II) is most preferable.

  Brefeldin A is extracted from actinomycetes (Eupenicillium brefeldianum) and the like, as an antibiotic (for example, JP-A-46-19542), and an anticancer agent (for example, DE 4305249, AU 9346338) and the like. However, the present inventors found for the first time that brefeldin A is contained in the mycelium of the basidiomycete himematsutake, and further, brefeldin A is anti-arteriosclerosis. It has been found that estrogen-like action such as action is exhibited.

  Brefeldin A contained in the present invention may be extracted from actinomycetes and purified as described above, but commercially available products (for example, manufactured by MOL and sold by Funakoshi Co., Ltd.) are also available. This may be used. In addition, Brefeldin A can be used after it has been subjected to solvent extraction from the mycelium of Himematsutake and / or its processed product.

  Hereinafter, a method for obtaining Brefeldin A from the mycelium of Himematsutake and / or a processed product thereof will be described. As described above, conventionally, it was not known at all that the mycelium of Himematsutake contained brefeldin A, so the method described below is also a novel method for producing brefeldin A. .

  Brefeldin A, which is an active ingredient in the present invention, can be extracted and isolated from the mycelium of Agaricus blazei and / or its processed product.

  The “mycelium” is an aggregate of mycelia, and here refers to a vegetative body of Himematsutake, which is distinguished from so-called “fruit bodies” called mushrooms. The processed mycelium includes a crushed product, a ground product, a tissue disrupted product, a dried product, a dried crushed product, and the like of Himematsutake. There are no particular restrictions on the origin of the mycelium of the matsutake mushroom used in the present invention, and it may be either a self-generated one or an artificial culture. However, considering productivity, an artificial culture is preferable.

  In addition, mutant strains (naturally occurring or artificially induced) can also be used in the present invention, and further, the ability to produce brefeldin A even if it is a zygote or genetically recombinant strain of Himematsutake. Anything having can be used in the method of the present invention.

  There are no particular restrictions on the harvest time, growth year, culture method, culture period, and the like of Japanese matsutake. Himematsutake mycelium can be obtained with different activities by different production methods of 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 high activity himematsutake.

  For example, the mycelium of Himematsutake can be cultured by a solid culture method or a liquid culture method usually used for culture of basidiomycetes. In solid culture, since the culture time is long and the extraction process is complicated, it is preferable to use a liquid culture method in consideration of the convenience of operation. Examples of the liquid medium include glycol, saccharose, and maltose. , Carbon sources such as water candy, dextrin, sucrose, starch, sugar, nitrogen sources such as ammonium sulfate, ammonium nitrate, sodium nitrate, natural complex nutrient sources such as malt extract, yeast extract, corn staple, phosphate, agnesium A normal sterilized liquid medium adjusted to around pH 6.0 comprising an inorganic salt such as a salt or potassium salt and other trace elements can be used. Organic and inorganic substances that help the growth of bacteria and promote the production of Brefeldin A can also be added to the medium. When inoculated with the seeds of Japanese matsutake mushrooms in the medium and cultured for about 15 to 25 days at a temperature of about 30 to 25 days under aerobic conditions while preventing contamination of bacteria, mycelium grows. The mycelium of matsutake mushroom can be obtained by centrifuging or filtering the culture system in stages. Production of brefeldin A by the mycelium of Himematsutake varies depending on the medium and culture conditions, but in the agar medium surface culture method, which is a solid culture method in the flask, the accumulation usually reaches its maximum within 7 to 21 days. . Therefore, it is preferable to stop the culture when the accumulation amount of brefeldin A in the mycelium reaches the maximum. And the obtained mycelium can also be made into a powder form by freeze-drying, for example.

  A solvent extract is obtained from the mycelium and / or processed product of Japanese matsutake mushrooms thus obtained. This solvent extract contains Brefeldin A. The solvent can be used with water, hot water or a water-containing organic solvent. In this case, for example, hot water is 40 ° C. or higher, preferably 60 ° C. or higher, and the water-containing organic solvent is an aqueous solution containing about 20 to 60% by weight of lower alcohol, preferably ethyl alcohol. Is mentioned.

  Since Brefeldin A is a fat-soluble substance, isolation and purification from the mycelium can be performed using this characteristic. That is, for example, solvent extraction with ethyl acetate, chloroform, ethyl acetate, ether, ethyl alcohol, etc .; synthetic adsorbents such as silica gel, alumina, ODS, Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation), and HW40 (Tosoh) Column chromatography using a gel filtration agent such as a high-performance liquid chromatography; preparative thin layer chromatography using silica gel or the like as a carrier is effective.

  When Brefeldin A is used as a drug, it can be made into a preparation according to the administration route together with a normal production carrier. The brefeldin A used in the present invention is, for example, formulated in the form of tablets, capsules, granules, powders, liquids and the like for oral administration. In preparing a solid preparation for oral administration, conventional excipients, binders, lubricants, other colorants, disintegrants, and the like can be used.

  Examples of the excipient include lactose, starch, talc, magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate, gum arabic and the like, and binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, Examples include gum arabic, shellac and sucrose, and examples of lubricants include magnesium stearate and talc. In addition, colorants and disintegrants that are generally known can be used.

  The tablet may be coated by a known method. The liquid preparation may be an aqueous or oily suspension, solution, syrup, elixir, or the like, and is prepared by a commonly used method. When preparing an injection, pH adjusters, buffers, stabilizers, isotonic agents, local anesthetics, etc. may be added to didepside to produce injections for subcutaneous, intramuscular and intravenous injections by conventional methods. it can. In addition, as a base for producing a suppository, for example, an oleaginous base such as cacao butter, polyethylene glycol, lanolin, fatty acid triglyceride, witepsol (registered trademark of Dynamit Nobel) can be used.

  In addition, without isolating and purifying Brefeldin A from the Japanese matsutake mycelium, for example, a hot water extract from the Japanese matsutake mycelium can be used as a medicine as it is. Although there may be problems in terms of safety, etc., by cultivating actinomycetes, etc., heat extracting the cells, and using the extract containing Brefeldin A as it is for oral administration, Is a kind of edible mushroom, and it has been customary for a long time to eat a mycelium or fruit body extracted with hot water as a supplement. Therefore, in terms of safety, there is no problem in using the hot water extract as it is as a medicine from Himematsutake mycelium, and in this case also, brefeldin A contained in the extract is effectively and safely used. Can be taken orally.

  Even if the extract of himematsutake mycelium is taken orally, the dosage form is not particularly limited, but it is a tablet, pill, powder, troche, sachet packaging, oblate preparation that is usually used for oral intake. , Elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or liquid media), and sterile packaging powders.

  In addition, the extract of Himematsutake mycelium can be mixed with an additive, diluted with an additive, or encapsulated in a container such as a capsule, packaging, or paper. When the additive serves as a diluent, it may be a solid, semi-solid, or liquid material that serves as a base, extender, or medium for the bioactive ingredient. Examples of additives include lactose, dextrose, sucrose, sorbitol, mannitol starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. Etc. can be illustrated. In addition, lubricants such as talc, magnesium stearate, mineral oil; wetting agents; emulsifiers or dispersants; preservatives such as methyl- and propylhydroxybenzoic acid; sweeteners; Methods well known in the art can also be employed to formulate the release of the active ingredient to be fast, sustained or slow after administration to a patient. Furthermore, an estrogen-like agent other than Brefeldin A used in the present invention, an anti-atherosclerotic agent, and the like can be used in combination.

  Here, Brefeldin A itself has a property that is hardly soluble in water, but α-glucan, β-glucan, β-galactoglucan, xyloglucan, and protein polysaccharide complex contained in the mycelium extract. Depending on the component, Brefeldin A is in an included state. Therefore, in the case where an extract is used as the medicine from the mycelium of Himematsutake, the use of the solid preparation for oral administration, excipients, tablets and the like exemplified above can be omitted.

  The dose varies depending on the patient's symptoms, weight, age, etc. and cannot be taken uniformly, but the amount of brefeldin A is preferably in the range of about 10 to 3000 mg per day for normal ingredients. It is desirable to administer this usually in 1 to 4 divided doses per day.

  As described above, the agent of the present invention comprising an extract of the mycelium of matsutake mushroom or Brefeldin A contained therein as an active ingredient exerts an estrogen-like action. For example, improvement of climacteric disorder, atrophic vaginitis It exerts a remarkable effect on the prevention and treatment of diseases such as urogenital disorders such as prostate cancer, benign prostatic hyperplasia, osteoporosis, arteriosclerosis, memory impairment, Alzheimer's disease, and thrombotic disease.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated still in detail, this invention is not limited to an Example.

<Example 1> Cultivation and extraction method of Himematsutake mycelium An inoculum of Himematsutake was obtained from Mona Corporation. Then, the sterilized liquid medium for basidiomycetes was inoculated with inoculum of Himematsutake, and the mycelium was grown by shaking culture at 30 ° C. for 14 days under aerobic conditions while preventing contamination with various bacteria. The mycelium obtained by centrifuging the culture system was lyophilized after treatment with glucanase. A powder obtained by processing this powder (hereinafter, referred to as “himematsutake powder”) was used in the administration experiment. In addition, Japanese matsutake powder was extracted with 10 times the weight of hot water of 90 ° C. or higher for 10 minutes, and the hot water extract was centrifuged to obtain a supernatant. This supernatant liquid (hereinafter referred to as “Himamatsutake extract” and indicated as “ABE” in the drawing) was subjected to the following activity evaluation and the like.

<Example 2> Blood Sample Preparation Method Peripheral blood samples were collected from 8 hyperlipidemic patients before and after daily administration of Japanese pine bamboo powder (250 mg / day). Four months after discontinuing the administration, the administration was resumed and, for 3 months, Japanese red pine powder was administered, and blood samples were collected regularly. Moreover, the blood sample used as a control was one voluntarily provided by 6 healthy people. All blood samples were approved by the ethics committee of Tokyo Women's Medical University and received individual consent.

<Example 3> Suppressive effect of oxidized LDL level in serum by administration of Japanese apricot powder (1) Measurement of oxidized LDL level First, familial hyperlipidemia, angina pectoris, myocardial ischemia and mild non-insulin dependence A 56-year-old male patient with diabetes mellitus was given himematsutake powder (250 mg / day). Before administration, the patient's myocardial blood flow had a low accumulation of technetium in the anterior part of the left ventricle (FIG. 1A), but normal levels of technetium accumulation appear 4 months after oral ingestion (FIG. 1B) and the amount of oxidized LDL decreased from 1.5 ng to 0.1 ng per 5 μg LDL protein after 11 months.

  From this finding, we hypothesized that Japanese red pine powder contains chemicals that reduce oxidized LDL levels. It was thought that this would promote recovery of vascular damage and prevent and improve arteriosclerosis.

  Next, we examined the effects of daily administration of Japanese apricot powder on serum oxidized LDL levels in hyperlipidemic patients. Blood was collected from hyperlipidemic patients (n = 8) and healthy individuals (n = 6) who had been ingested for 1 year, and the serum oxidized LDL level was measured. Thereafter, the hyperlipidemic patient stopped taking 4 months and then resumed taking 3 months again. Blood samples were collected before and after daily administration of Japanese red pine powder (250 mg / day) for 1 year, and the oxidized LDL level in the serum was measured by sandwich ELISA. The oxidized LDL fraction in serum was separated from various blood components by ultracentrifugation, and the protein concentration was determined using BCA reagent (BioRad; Hercules, Calif., USA). After adding 100 μl of diluted LDL (50 μg / ml) fraction to microtiter wells pre-coated with anti-oxidized phosphatidylcholine monoclonal antibody (DLH3; 0.5 μg / well), wells were washed thoroughly The oxidized LDL bound to the wells was detected using an anti-apo B protein polyclonal antibody as a primary antibody and a secondary antibody bound to alkaline phosphatase. Finally, the wells were incubated with paranitrophenyl phosphate (1 mg / ml, dissolved in 1 M triethanolamine buffer (pH 9.8)) for 10-30 minutes at 37 ° C. The amount of oxidized LDL was measured as absorbance at 405 nm using a microplate reader, and quantified using oxidized LDL prepared using copper as a standard substance.

(2) Results As shown in FIG. 2, in the control group (healthy person group), the oxidized LDL level in the serum remained unchanged throughout the year. In contrast, the patient group found a remarkable improvement in oxidized LDL levels. These results suggest that Himematsutake powder reduced the level of oxidized LDL in the serum of hyperlipidemic patients.

  In addition, we used human-derived LDL (0.2 mg / mL), Japanese matsutake extract (2, 20, 200 μg / mL), acetylcysteine (AcCys: 20 μg / mL) or butylhydroxytoluene (BHT: 2 μg / mL). Then, an oxidation reaction was carried out with 5 μM copper sulfate at 37 ° C. for 3 hours, and after the reaction, the amount of TBARS (thiobarbituric acid-reactive substance) was measured. As a result, as shown in FIG. 3, it was clarified that Japanese red pine powder does not promote LDL lipid peroxidation in vitro.

  Therefore, it was suggested that the inhibitory effect of the oxidized LDL level in the serum by administration of the Japanese pine agar powder is due to the removal of oxidized LDL from the serum by specific cells.

<Example 4> Immunohistochemical analysis of the effect of Japanese red pine powder on coronary arteries (1) Test method Using two groups of inbred Japanese white rabbits (group I and II) with hyperlipidemia, arteriosclerosis The effects of Japanese pine bamboo powder were investigated.

Sixteen inbred white rabbits of about 2.5 kg were randomly divided into the following three groups.
・ Group I: Japanese pine mushroom powder not administered (n = 8)
・ Group II: Himematsutake powder administered (n = 6)
・ Control: Untreated group (n = 2)
Initially Group A and II rabbit aorta were injured using a balloon tip catheter. Thereafter, all rabbits were raised separately, and each rabbit had free access to food (100 g / day) and water. Group I and II rabbit groups were fed a high cholesterol-containing diet [0.5% cholesterol added to CLEA Rabbit Diet CR-3 (Clear)] for 12 weeks. For the next 23 weeks, Group II rabbits were given a CR-3 diet containing 0.01% Japanese matsutake powder (3.3 mg / kg, equivalent to human consumption). The rabbit group was fed with normal CR-3 diet. The control rabbit group received only CR-3 diet for 35 weeks. At the end of the experiment, the Group I and Group II rabbits weighed approximately 3.5 kg. At the end of the 12th and 35th weeks, the thickness of the rabbit intima and the thickness of the intima of the descending abdominal aorta were measured using ultrasound echo. After the end of the administration period (35 weeks later), the rabbits were anesthetized with Nembutal (50 mg / kg), dissected and used for the subsequent experiments.

  In addition, all rabbit tissue sections and descending aorta were stained using conventional methods with hematoxylin eosin (HE), mason trichrome and Victoria Blue. Immunochemical analysis of all rabbits includes anti-rabbit macrophage 11 (RAM11) monoclonal antibody, anti-human oxidized LDL receptor (LOX-1) monoclonal antibody, mouse antibody to Ki67 (MIB-1) protein, smooth muscle An antibody against sex actin (αSMA), a mouse anti-BAX protein monoclonal antibody for detecting Bcl2 protein, an anti-CD36 protein antibody for detecting oxidized LDL receptor and mouse antibodies against human collagen types I, III and VI . Apoptosis was evaluated by the TUNEL method.

(2) Results While feeding the high cholesterol diet for 23 weeks, four Group I rabbits (50%) died of myocardial infarction, but Group II rabbits were not premature. Between group I and II rabbits, the differences are the brain, pituitary, skeletal muscle, kidney, ureter, aorta, pancreas, testis, rectum, lung, liver, spleen, thymus, stomach, small intestine and adrenal gland. It was not found. However, histological differences were found only in the coronary arteries.

  Intimal proliferation due to infiltration of foamed macrophages was observed in the coronary arteries and both ventricles of the atrium in group I rabbits, particularly in the ventricular septum, and the presence of intimal atherosclerotic disorder (Fig. 4A). Indicated. Prominent uplifted pigment growth with smooth muscle cells immunostained with foamed macrophages and smooth muscle α-actin (FIG. 4C), evident by RAM 11 immunostaining (FIG. 4B), was observed in coronary lesions of Group I rabbits It was done. The group II rabbit group (FIGS. 4D-F) showed no clear differences from the control rabbit group (FIGS. 4G-I) and showed little damage to the coronary arteries.

  These results revealed that atherosclerosis was observed in the coronary arteries of Group I rabbits but not in Group II rabbits or the control group. It has been shown that atherosclerosis is improved.

  In addition, the following findings were obtained by measuring the intima thickness of the descending aorta by echocardiography.

  Similar increases in intimal thickness (I: 0.63 ± 0.17 μm; II: 0.67 ± 0.10 μm) were observed for all rabbits in Groups I and II during the 12 weeks on the high-fat diet. However, it returned to normal levels (I: 0.38 ± 0.04 μm; II: 0.41 ± 0.08 μm) 23 weeks after stopping the high-fat diet. The control group consistently showed the same intimal thickness (0.40 ± 0 μm) throughout the dosing period. This finding suggests that Japanese red pine mushroom powder does not change the thickness of the descending aortic intima as a whole but acts directly on the arteriosclerotic site. This was also revealed by histological data as a significant difference from untreated animals (FIG. 5).

  Histopathology of the aorta has shown that the intima thickens due to fiber growth and macrophage infiltration. Immunohistochemical staining with RAM 11 showed significant macrophage proliferation in Group I rabbits (FIG. 5A) compared to Group II rabbits (FIG. 5K). For α-smooth muscle actin, type I, III, IV collagen, Ki67 (MIB-1), BAX, LOX-1 and CD36, immunostaining for the intima was good, but group I (FIG. 5B-I ) And Group II rabbits (Fig. 5L-S) were not found. TUNEL analysis of the intima of the aorta revealed that there were more cells showing apoptosis in group I rabbits (FIG. 5J) than in group II rabbits (FIG. 5T). This indicates that the damage to cells associated with arteriosclerosis was more prominent in Group I rabbits.

<Example 5> Estrogen-like action of the extract of Himematsutake mycelium We considered that the mechanism of improving the vascular damage caused by the extract of Himematsutake mycelium may be due to the estrogen-like action. Proliferation of MCF-7 cells derived from breast cancer The effect of the extract of Himematsutake was investigated.
(1) Sulforhodamine B (SRB) colorimetric evaluation Human breast cancer-derived MCF-7 cells were obtained from JCRB Cell Bank (National Institute of Pharmaceutical and Food Health, Tokyo) and RPMI 1640 medium (Invitrogen; Carlsbad, CA, USA). And supplemented with 10% fetal bovine serum (FBS) in the presence of 5% carbon dioxide and subcultured. The medium was replaced with 10% dextran coated charcoal-treated FBS (DCC-FBS) and phenol red-free RPMI 1640 medium for treatment of cells with 17β estradiol (hereinafter referred to as E2) or Himematsutake extract . The SRB analysis was evaluated according to a report by Powells et al. (2003).
(2) Analysis results As shown in FIG. 6, the cell growth inhibition rate reached 50% at an extract concentration of 4 μg / ml, and reached 80% at 6 and 8 μg / ml. It became clear that there was a quantitative dependence on inhibition.

  Thus, although the extract of Himematsutake mycelia has the effect of improving the vascular damage confirmed in Example 4, it was confirmed that it does not have the cancer cell proliferating activity of estrogen. The result of the present example shows the safety of administering the extract of the mycelium of Himematsutake.

<Example 6> Consideration 1 at gene expression level
It was investigated by using a DNA microarray whether or not an estrogen-like action was exhibited at the gene expression level using a himematsutake extract.

(1) cDNA microarray assay
The cDNA microarray is a mechanically spotted cDNA fragment (500 bp to 1.5 kb) of 203 genes including 172 genes that respond to estrogen on a glass substrate, manufactured by InfoJeans (Tsukuba). The product (trade name EstrArray) was used. For EstrArray analysis evaluation, MCF-7 cells were cultured in RPMI 1640 medium containing 10% DCC-FBS and no phenol red for 3 days, 10 nM E2, 2 μg / ml Japanese apricot extract, or solvent Only (DMSO or sterile Milli-Q water as a control) was treated for 72 hours. Total RNA was isolated using ISOGEN (Wako Pure Chemicals; Tokyo) according to the product instructions. Antisense RNA (aRNA) was purified from 5 mg total RNA using the RiboAmp RNA Amplification kit (Takara Bio; Japan). EstrArray analysis evaluation was performed based on the results of three independent experiments using different cultured cells.

Processing of the obtained data is performed only when the signal value of Cy3 and Cy5 is 30 or more for each gene or the corresponding signal area is 50 or more, and the expression ratio is calculated using Microsoft Excel. Used to do. The average of the Cy3 or Cy5 signal values obtained from the two spots was determined, and the ratio of the Cy3 value to the Cy5 value was calculated for each gene. In order to standardize the data obtained, for 28 control genes, first remove genes with a Cy3 and Cy5 signal area of less than 100, and then average the ratio of the Cy3 and Cy5 signal intensities for the remaining genes. Asked. Finally, standard values were calculated for all genes using the average Cy3 / Cy5 values determined for 28 control genes. The standardized Cy3 / Cy5 values were further converted to log ₂ and used for further statistical analysis.

  Correlation analysis and t-test were performed using SPSS 12.0J software (SPSS Japan; Tokyo). As the UniGene names of 120 genes, the registered names of Entrez Gene database (www.ncbi.nlm.nih.gov) were used. These 120 genes were selected as genes that respond to estrogen with extremely high reproducibility.

(2) Results As shown in FIG. 7, similar gene expression profiles were obtained between cells treated with 10 nM E2 and 2 μg / ml extract (correlation coefficient or R = 0.63). It is possible that a substance exhibiting an estrogen-like action exists.

<Example 7> Consideration 2 at gene expression level
To find differences in gene expression profiles between the extract and E2, ANOVA and t tests were performed on a total of 172 estrogen responsive genes. Table 1 shows the statistically significant change between 10 nM E2 and the degree of gene expression change due to the treatment of Japanese apricot extract (average ratio of log ₂ converted values obtained by three EstrArray analyzes). The indicated genes are indicated. The accession number indicates the number of each gene registered in the Entrez Gene database. Most genes showed similar responses, but the six genes (CEBPB, RBBP8, IER3, DHCR24, ARGGDIA and IGFBP5) responded significantly differently to this treatment (P <0.05).

(1) Real-time quantitative RT-PCR
To confirm these differences, real-time quantitative RT-PCR was performed.
Real-time quantitative reverse transcription (RT) -PCR was performed with the SYBR Green Kit (Invitrogen; Carlsbad, Calif., USA) using a two-step qRT-PCR method. PCR was performed 3 times for each gene using the primers listed in Table 2. The PCR cycle was first denatured at 95 ° C. for 3 minutes using a LightCycler (Roche, Mannheim, Germany) and then subjected to 50 cycles of 95 ° C. for 5 seconds and 60 ° C. for 30 seconds. β-actin was used as an internal standard.

(2) Results As shown in Table 1 and FIG. 8, among the selected genes, all the genes other than DHCR24 showed statistically significant differences in gene expression. Therefore, when comparing the effects of E2 and Japanese apricot extract on cells, it is clear that some genes show distinct differences but many genes have similar effects that cannot be distinguished at the gene expression level. became.

<Example 8> Activation of Erk1 / 2, Akt and p38 induced by himematsutake extract (1) Test method In order to examine intracellular signal transduction induced rapidly by himematsutake extract, human umbilical vein endothelium was examined. Cells (HUVEC cells) and TIB-67 cells derived from macrophage cells were used.

  HUVEC cells (Cambrex; East Rutherford, NJ, USA) are Dulbecco without phenol red supplemented and supplemented with calf serum (DCC-FBS) treated with 10% (v / v) dextran-coated charcoal. The modified eagle medium (DMEM) (Invitrogen; Carlsbad, Calif., USA) was used in a humid environment of 95% air and 5% carbon dioxide.

  Macrophage-derived TIB-67 cells were obtained from ATCC (Manassas, VA, USA) and subcultured at 37 ° C. in the presence of 5% carbon dioxide using RPMI 1640 medium containing 10% FBS.

  Serum-depleted cells were stimulated with S. matsutake extract (100 μg / mL), E2 (10 nM) or solvent alone, and after the indicated time, cells were lysed to purify the protein, and SDS buffer solution Dissolved in. Proteins were separated by electrophoresis using a 5-20% gel gradient SDS polyacrylamide gel, transferred to a nitrocellulose membrane, and then detected using each phosphorylated antibody. The total protein amount was detected using an antibody that detects the total protein. Cells were treated with 1 μM ICI 182,780 (ICI) for 15 minutes for an estrogen response inhibition experiment.

(2) Results
When HUVEC cells were treated with Japanese apricot extract, it was found that the estrogenic action induced abruptly was due to the activation of Erk1 / 2 and Akt, but that was not the case with TIB-67 cells. Activation was not seen (FIG. 9). The rapid activation of Erk1 / 2 and Akt by Himematsutake extract was observed not to be suppressed by treatment with the estrogen antagonist ICI 182,780 (ICI), so the signal pathway distinct from ERα and ERβ is a response by the extract It was presumed to exist in the route. It is noteworthy that the activation states of Erk1 / 2 and Akt returned to their original levels within 30 minutes.

In this example, the estrogen antagonist ICI 182,780, unlike estrogen (E ₂ ), did not inhibit phosphorylation of Erk1 / 2 and Akt, but this result indicates that the Japanese pine extract has no estrogenic effect Rather, it is because the estrogen-like action exhibited by the extract of Himematsutake has a signal transduction pathway different from that of estrogen. In fact, bisphenol A, which is known as a representative example of a chemical substance having an estrogenic action, is a GPR30 receptor or an estrogen-related receptor. Examples have been reported that act via γ (ERRγ) (Filardo et al., Mol. Endocrinol., 16, 70-84, 2002; Okada et al., Envirn. Health Perspect., 116, 32-38 pages, 2008). In addition, the fact that chemical substances with estrogen-like action exhibit different signal transduction pathways than estrogen is a basis for improving the shortcomings of estrogen, such as the risk of carcinogenesis / thrombosis risk of estrogen and low specificity to tissues. Is.

<Example 9> Inhibitory effect on signal by oxidized LDL (1) Test method The effect of Japanese apricot extract on activation of Erk1 / 2, Akt and p38MAP kinase by stimulation with oxidized LDL (OxLDL) was examined.

  In order to examine HUVEC and TIB-67 cells for 6 hours with Japanese apricot extract (100μg / mL), LDL (50μg / mL), oxidized LDL (50μg / mL) or solvent alone, to examine the inhibitory effect of oxidized LDL signal, Japanese apricot extract (100 μg / mL) or anti-LOX-1 antibody (Ab: 5 μg / mL) was added to the cells 1 hour before the addition of oxidized LDL. Proteins were extracted using SDS sample buffer and analyzed by Western blotting.

Total protein was extracted with SDS buffer and sonicated on ice for 30 seconds. After adding sample preparation buffer for SDS-PAGE and incubating at 95 ° C for 5 minutes, the protein sample (20 mg) was separated by SDS-PAGE using a 5-20% gradient gel method, and a semi-dry transfer cell (BIO-RAD) And then transferred to a nitrocellulose membrane (Millipore; Billerica, Mass., USA) at 1 mA / cm ² for 1 hour.

  Phosphorylated Erk1 / 2 (P-Erk1 / 2), phosphorylated Akt (P-Akt), and phosphorylated p38 (P-p38) contain Tris buffer containing 0.1% Tween-20 and 5% BSA Used for membranes previously blocked in solution (TBST-BSA). Membrane has enough antibodies against Erk1 / 2, P-Erk1 / 2, Akt, P-Akt, p38, and P-p38 (Santa Cruz Biotechnology; Santa Cruz, CA) (1: 200 to 1: 1000) ) And then reacted in TBST-BSA overnight at 4 ° C. After the reaction, a rabbit or mouse antigen-antibody complex was detected using a goat antibody diluted to 3000 fold with TBST-BSA for a rabbit or mouse IgG (Cell Signaling Technology) conjugated with horseradish peroxidase. Each protein band was visualized using ECL-Plus Western blot detection (Amersham Pharmacia Biotech; Arlington Heights, Illinois, USA).

(2) Results The rapid activation reaction of Erk1 / 2, Akt or p38 induced by oxidized LDL was suppressed by Japanese matsutake extract as well as anti-LOX-1 (oxidized LDL receptor) antibody (Ab) (Fig. 10). In other words, it is suggested that the extract of Himematsutake can be used in the same way as atherosclerosis treatment targeting the oxidized LDL response.

<Example 10> Analysis at the level of gene expression Next, the effect of the extract of Himematsutake on vascular endothelial cells was analyzed at the level of gene expression. Using real time RT-PCR, GPX3 in HUVEC cells, LOX-1, and were analyzed for mRNA levels of accumulated eNOS. Total RNA was prepared after treating HUVEC cells for 24 hours with Japanese matsutake extract (100 μg / mL), oxidized LDL (50 μg / mL) or solvent alone.

  The result is shown in FIG. Himematsutake extract increased the expression of two genes involved in the reduction of reactive oxygen species, glutathione peroxidase 3 (GPX3) and α-synuclein (SNCA). Estrogens have been shown to increase the expression of GPX3 and SNCA genes separately from the estrogen receptor pathway, indicating that the antioxidant effect induced by the extract of Japanese matsutake reduces serum oxidized LDL levels (Figure 1). It is thought that it shows that it contributes. In addition, himematsutake extract increases the expression of nitric oxide synthase gene (eNOS). Oxidized LDL induces a decrease in eNOS gene expression, but the addition of himematsutake extract increased the eNOS gene expression (FIG. 11). In cells treated with the extract of Himematsutake, the expression of MCP-1 (monocyte chemotactic protein-1) gene is decreased, and the gene expression of MCP-1 in response to oxidized LDL is inhibited. Therefore, these results can well explain the results of the eNOS gene.

  Furthermore, Western blotting analysis of GPX3 and eNOS proteins in HUVEC cells was performed in order to confirm the influence of the extract of Himematsutake on GPX3 and eNOS genes at the protein level (FIG. 12).

  The results are shown in FIG. As shown in FIG. 12, it can be seen that the extract of Himematsutake increases the expression of eNOS and GPX3 genes.

  Therefore, from the results of Examples 9 and 10, as shown in the schematic diagram of FIG. 13, it was found that the extract of Himematsutake increases the expression of eNOS, GPX3, and SNCA genes through activation of Akt and Erk proteins. . GPX3 and SNCA genes reduce the generation of reactive oxygen species (ROS) in mitochondria. On the other hand, eNOS protein was found to play a role in maintaining balanced vascular function by decreasing MCP-1 gene expression and increasing nitric oxide (NO) concentration.

<Example 11> Isolation of Brefeldin A To 100 g of the dried body of cultured mycelium obtained in Example 1, 2 liters of ethyl acetate is added and stirred for 2 hours. The solution after stirring was filtered, and the supernatant was dried under reduced pressure at 40 ° C or lower. The obtained concentrate was dissolved in 80% methanol, HPLC (condition: 5 mg / 50 ml was adsorbed on the reverse phase column 5C18-200, the column was washed with water, and 1 ml / min with 0-60% acetonitrile gradient. Fractions were obtained by detection at 210 nm.

As a result of measuring Erk1 / 2 phosphorylation activity (Erk1 / 2 phosphorylation activity obtained by quantification of phosphorylated Erk1 / 2 and total Erk1 / 2 shown in Example 9) for each of the obtained fractions, FIG. As shown in 14A, strong Erk1 / 2 phosphate activity was shown only in fraction No. 51-60, which is a fraction eluted with 50-60% acetonitrile.
Therefore, the same fraction was further purified by HPLC under the same conditions, fraction No. 58 (FIG. 14B) showing phosphorylation activity was rechromatographed, and 100 g of dried mycelium as a single component was cultured. 148 mg of Erk phosphate active ingredient was obtained.

  The active ingredient thus obtained was considered to have a macrolactone type chemical structure and exhibited the following physical properties. The following values all agree with the literature values of (+)-Brefeldin A (Tetrahedron Letters, Volume 47, Issue 37, 11, 6527-6530) and are identified as (+)-Brefeldin A It was. In the comparative experiment, a standard product manufactured by Wako Pure Chemical Industries, Ltd. was used.

(Data obtained by measurement)
1) Specific rotation (in methanol): [a] ²⁵ _D = +92.09. Method: (19.6 mg / 5 mL methanol).

2) NMR data: ¹ H NMR (CD ₃ OD, 75 MHz) δ 7.46 (dd, 1H), 5.82 (ddd, 1H), 5.76 (ddd, 1H), 5.27 (dd, 1H), 4.84 (qdd, 1H ), 4.22 (m, 1H), 4.05 (td, 1H), 2.44-0.87 (m, 12H), 1.25 (d, 3H); ¹³ C NMR (CD ₃ OD, 75 MHz) δ 167.2, 153.9, 136.9, 130.2, 116.5, 75.4, 72.0, 71.8, 52.0, 44.2, 42.9, 40.6, 33.8, 31.8, 26.8, 19.9.

3) FT-IR data: 3366, 2927, 1712, 1644, 1449, 1257 cm ⁻¹ . Method: 1.06 mg of a sample was weighed and mixed uniformly with 200.8 mg of KBr, and then measured 32 times at 2 cm ⁻¹ with an FTS-135 apparatus (Bio-Rad Laboratories).

  As shown in the above-mentioned experiment, Brefeldin A is contained in an amount of about 0.15% in terms of dry weight with respect to the mycelium of Himematsutake. This content is quite high as the content of secondary metabolites, excluding the basic constituents of fungi, and Brefeldin A is one of the main chemical components quantitatively produced by mycelium. It is believed that there is.

    In view of the fact that only the fraction containing brefeldin A in the above experiment shows Erk1 / 2 phosphate activity, the mycelium extract has a therapeutic effect on the estrogenic action and arteriosclerosis. It is shown that Ferdin A plays an important role.

<Example 12> Evaluation of cell growth activity of Brefeldin A by SRB test (1) Analysis method Human breast cancer-derived MCF-7 cells were cultured in the presence of 5% carbon dioxide and 10 ⁴ in a 24-well culture plate. The cells were increased to cells / well, and 10 nM E ₂ or 10 pM to 1 μM Brefeldin A (BFA) was added and cultured for 3 days. Thereafter, the cells were fixed with 10% trichloroacetic acid at 4 ° C., and the cell growth activity was evaluated by the SRB test. The results are shown with the value of the control experiment as 100%. Each test was performed 6 times and the standard deviation (SD) was calculated.
(2) Results The results are shown in FIG. Brefeldin A did not show cell proliferation activity at any concentration. On the other hand, 1 μM showed cell growth inhibitory action. This result is in agreement with the result obtained in “<Example 5> Estrogen-like action of Himematsutake mycelium extract”.

<Example 13> Activation of Erk1 / 2 protein by Brefeldin A (1) Analytical method [Example 8] Method used in the activation of Erk1 / 2, Akt and p38 induced by Japanese matsutake extract Was used to evaluate the activation of the Erk1 / 2 protein by Brefeldin A.
(2) Results The results are shown in FIG. Brefeldin A was found to promote phosphorylation of Erk1 / 2 protein. This response was not inhibited by the estrogen antagonist ICI 182,780 (ICI). This result is in agreement with the result of the mycelium extract of matsutake mushroom shown in “<Example 8> Activation of Erk1 / 2, Akt and p38 induced by the extract of matsutake mushroom”.

<Example 14> Evaluation of estrogen activity of Brefeldin A by reporter gene test 1
(1) Analysis method Evaluation of estrogen activity using the plasmid T3 (A + B) -pTK-Luc for reporter gene testing in which the estrogen response element (ERE) of human estrogen receptor α gene is introduced upstream of the luciferase gene. went. Test methods and plasmids were according to literature (Li et al., 2002: Gene 294, 279-290). Briefly described below. 2.0 μg of reporter gene DNA per 10 ⁵ cells was mixed with β-galactosidase gene DNA, and MCF-7 cells were transfected with Lipofectin (Gibco-BRL). After culturing for 24 hours, E ₂ (10 nM), Brefeldin A (BFA: 0.01 nM, 1 nM, 100 nM), or each concentration of Aedes mycelium (ABE) was added, and then 24 more Incubate for hours. Luciferase activity was measured using a kit, and the result was corrected by the expression level of β-galactosidase gene, and the value of the control experiment was expressed as 100%. Each test was performed 4 times and the standard deviation (SD) was calculated.
(2) Results The results are shown in FIG. Significant estrogen responsive element-dependent luciferase activity was observed in both Brefeldin A and Japanese apricot mycelium extract. This means that Brefeldin A activates the promoter in an estrogen responsive element-dependent manner, that is, Brefeldin A has estrogenic activity.

<Example 15> Evaluation of estrogen activity of a mycelia extract of Japanese matsutake mushroom by reporter gene test (1) Analysis method Promoters A and B of human estrogen receptor α gene having an estrogen response element (ERE) were introduced upstream of the luciferase gene. Estrogen activity was evaluated using plasmid pG-ERα-AB for reporter gene testing. Test methods and plasmids were according to the literature (Inoue et al., 2002: J. Pharmacol. Toxicol. Methods 47, 129-135.). Briefly described below. 2.0 μg of reporter gene DNA per 10 ⁵ cells was transfected into MCF-7 cells using Lipofectin (Gibco-BRL). After culturing for 24 hours, E ₂ (10 nM), Himematsutake mycelium extract (ABE: 0.01-8 μg / ml), or 1 μM ICI 182,780 (ICI) was added to each extract of Himematsutake mycelium After adding the sample, the cells were further cultured for 24 hours. Luciferase activity was measured using a kit, and the value of the control experiment was expressed as 100%. Each test was performed 4 times and the standard deviation (SD) was calculated. ** indicates that the risk factor (p value) is less than 0.01.
(2) Results The results are shown in FIG. A significant estrogen responsive element-dependent luciferase activity was observed in the mycelium extract. This indicates that the extract of mycelium is an estrogenic activity. This activity, like estrogen, was inhibited by the antagonist ICI 182,780.

<Example 16> Evaluation of estrogen activity of Brefeldin A by reporter gene test 2
(1) Analytical Method The test was carried out by the method shown in “Evaluation of estrogen activity of Brefeldin A by reporter gene test (Part 1)”. E ₂ (10 nM) or Brefeldin A (0.01 nM or 1 nM BFA) is added and divided into 2 groups, 1 μM ICI 182,780 (ICI) is added to 1 group, and the other group Not added.
(2) Results The results are shown in FIG. The estrogen response element-dependent luciferase activity exhibited by Brefeldin A was inhibited by ICI 182,780. Further, although this result is different from the result of ICI 182,780-independent Erk1 / 2 activation obtained in “<Example 13> Activation of Erk1 / 2 protein by Brefeldin A”, <Example 15> This agrees with the results of the mycelium extract of It is considered that this phenomenon is characteristic of Brefeldin A in combination with a phenomenon that does not show the proliferation activity of MCF-7 cells.

<Comparative Example 1> Comparison of activity with fruit body The following comparative example shows that the effect of Brefeldin A obtained from the dried mycelium used in this example cannot be expected when the fruit body is used. Became.

(1) Origin of fruit body sample and extraction method The following three commercially available fruit body samples were used. One is a dried fruit body (Company A), and the remaining two are processed fruit body extracts (extracts).

Company I: Daiai Co., Ltd. (Product name) Dried “Shinsenen”: Fruiting body A (Preparation method) Finely grind 5 g of dried Agaricus koji and extract with 40 ml of boiling water for 20 minutes.

Company II: S ・ S ・ I SFB: Fruit body B (Product name) Kyowa's Agaricus 茸 Sengyo dew granule SSG-PLUS35
(Preparation method) Dissolve 2 bags (4.0 g) in 40 ml of water.

Company III: Yoshida Kogay Co., Ltd. YN: Fruit body C (Product name) Super Gold Agaricus Extract Granules 2800 (contains an extract granule equivalent to 2800 mg of dried Agaricus 中 in one package. This is in 28 g of raw Agaricus 茸. Equivalent to).

  (Preparation method) Dissolve 2 packs (5.6 g) in 40 ml of water.

(2) Treatment after extraction After extraction with water (or boiling water), the mixture was filtered through a MILEX 0.45 μm filter, dried 3 ml and weighed. Further, the concentration was adjusted to 5.0 mg / ml, and HPLC analysis (elution with methanol using Wako Pure Chemical Industries, reverse phase column 5C18-200, detection at 210 nm absorption wavelength) was performed. The pattern of blefeldin A elution fraction for all fractions was compared. The dry weight of the water extract was about 1 to 5 g including the mycelium extract, and the fruit body extract did not exhibit particularly high extraction efficiency.

(3) Results The results are shown in FIG. The agaricus (Himematsutake) mycelium preparation (FIG. 20A) used in this Example was compared with three agaricus fruit body preparations (FIGS. 20B, C, and D). None of the fruit body preparations showed a significant peak in the HPLC retention time at which brefeldin A was eluted, but a significant peak was found in the mycelium specimen used in this example. It became clear that the quantitative ratio was at least 150 times in terms of the integral value. This indicates that Brefeldin A hardly exists in the fruit body, and even if the fruit body is used for treatment, the effect derived from Brefeldin A is hardly expected. From this fact, it was found that the effect seen in the mycelium of the matsutake mushroom was peculiar to the mycelium of the matsutake mushroom and was different from the effect in the fruiting body.

<Comparative example 2> Extraction efficiency of brefeldin A with various solvents (1) Analysis method A: water extraction, B: ethanol extraction, C: methanol extraction, D: ethyl acetate Extraction was performed, and after filtration through a MILEX 0.45 μm filter, a portion was dried and the weight was measured. Furthermore, the concentration was adjusted to 5.0 mg / ml, HPLC analysis (elution with methanol using Wako Pure Chemical Industries, reverse phase column 5C18-200, detection at 210 nm absorption wavelength) was performed, and Brefeldin A elution pattern was obtained. Comparison was made (peak positions are shown in the figure).

(2) Results Extracts of the mycelium of himematsutake were extracted using various solvents, and the weight of brefeldin A in the obtained fraction was quantified. The results (weight% of BFA fraction) are 0.015% (water extraction), 0.036% (ethanol extraction), 0.029% (methanol extraction) and 0.012% (ethyl acetate extraction) as shown in FIG. It was found that Brefeldin A can also be extracted using the above solvent.

Claims (2)

(1) A drug for treating or preventing a disorder or disease associated with estrogen deficiency, which contains purified brefeldin A represented by the following formula (II) :
Or
(2) A drug for treating or preventing arteriosclerosis or hyperlipidemia, which contains purified brefeldin A represented by the following formula (II)
A manufacturing method of
A process of obtaining a solvent extract by solvent extraction of the mycelium of himematsutake and / or a processed product thereof; and
Purifying the Brefeldin A from the solvent extract using chromatography,
The manufacturing method of the chemical | medical agent characterized by including.   The following steps,
A process of obtaining a solvent extract by solvent extraction of the mycelium of himematsutake and / or a processed product thereof; and
A step of purifying Brefeldin A represented by the following formula (II) from the solvent extract using chromatography.
The manufacturing method of Brefeldin A characterized by including these.