JP2018193345A - Testosterone secretion promoting composition - Google Patents
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本発明は、テストステロン分泌促進用組成物に関し、詳しくは男性更年期障害の予防及び／又は改善に有用なテストステロン分泌促進用組成物に関する。 The present invention relates to a composition for promoting testosterone secretion, and particularly relates to a composition for promoting testosterone secretion useful for preventing and / or improving male climacteric disorder.
テストステロンは、男性ホルモンの中で最も分泌量が多く、作用も強い。テストステロンは、男性だけでなく、女性においても量は少ないが分泌されており、性器及び骨格の男性化、ならびにタンパク質同化促進といった働きがあり、男性生殖器の発達、骨格や筋肉の発達、性欲・性衝動の亢進、及び脳や精神面の活力亢進に影響を及ぼすとされている。血中のテストステロン濃度は強いストレスを受けた場合や加齢に伴って低下し、男性更年期障害や思春期遅発症などの疾病を引き起こす（特許文献１及び２)。更に近年では、更年期障害が骨粗しょう症や鬱・認知症などの原因の要因として考えられている。 Testosterone is the most secreted and highly effective of male hormones. Testosterone is secreted in a small amount not only in men but also in women, and has functions such as genital and skeletal masculinization and protein assimilation, male genital development, skeletal and muscular development, libido / sexuality It is said that it has an effect on the enhancement of impulses and the vitality of the brain and mental aspects. The testosterone concentration in blood decreases with strong stress or with aging, and causes diseases such as male menopause and delayed onset of puberty (Patent Documents 1 and 2). In recent years, menopause has been considered as a cause of osteoporosis, depression / dementia and the like.
またテストステロンは、造血作用を有し、動脈硬化やメタボリックシンドロームの予防などに影響することが知られている（非特許文献１）。 Testosterone has a hematopoietic effect and is known to affect arteriosclerosis and prevention of metabolic syndrome (Non-patent Document 1).
このため、テストステロン剤として皮下注射させる方法で治療に使われてきているが、テストステロンの分泌促進作用を有する食品も知られ、マカ（特許文献３）、ツルニンジン属植物（特許文献４）、黒酢もろみ（特許文献５）、タマネギ加工品（特許文献６）等の摂取が提案されている。 For this reason, it has been used for treatment by the method of subcutaneous injection as a testosterone agent, but foods having a testosterone secretion-promoting action are also known, such as maca (patent document 3), plant of the genus Turrundin (patent document 4), black vinegar. Ingestion of moromi (Patent Document 5), processed onion (Patent Document 6) and the like has been proposed.
一方、特許文献７には、エノキタケ等の抽出物がテストステロン−５α−レダクターゼ阻害作用を有し、頭部に塗布することにより育毛効果を示すことが記載されている。
特許文献７の段落０００５に記載されているように、テストステロン−５α−レダクターゼ阻害剤が養毛・育毛剤として利用される理由は、代謝系における男性ホルモン（テストステロン）が、テストステロン−５α−レダクターゼの作用を受けて５α−ジヒドロテストステロンが生成・蓄積され、その結果毛根が萎縮し、抜毛・禿等を誘引すると考えられているからである。
On the other hand, Patent Document 7 describes that an extract such as enokitake has a testosterone-5α-reductase inhibitory action and exhibits a hair-growth effect when applied to the head.
As described in paragraph 0005 of Patent Document 7, a testosterone-5α-reductase inhibitor is used as a hair-restoring / hair-growth agent because the male hormone (testosterone) in the metabolic system is testosterone-5α-reductase. This is because it is considered that 5α-dihydrotestosterone is generated and accumulated under the action, and as a result, the hair roots atrophy and attracts hair removal, wrinkles and the like.
したがって、特許文献７はテストステロン−５α−レダクターゼ阻害作用による５α−ジヒドロテストステロンの生成・蓄積に関するものであり、本発明のテストステロンの分泌を促進させる組成物については開示も示唆もしていない。 Therefore, Patent Document 7 relates to the production and accumulation of 5α-dihydrotestosterone by the testosterone-5α-reductase inhibitory action, and does not disclose or suggest the composition for promoting the secretion of testosterone of the present invention.
本発明は、エノキタケ（Ｆｌａｍｍｕｌｉｎａ ｖｅｌｕｔｉｐｅｓ（Ｃｕｒｔ．：Ｆｒ．）Ｓｉｎｇ．）を原料とする、男性更年期障害の予防及び／又は改善に有用なテストステロンの分泌促進作用を有し、且つ、安全性が高い経口組成物を提供することを目的とする。 The present invention has a testosterone secretion-promoting action which is useful for the prevention and / or improvement of male climacteric disorders using Enokitake (Flamulina velutipes (Curt .: Fr.) Sing.) As a raw material, and has high safety. An object is to provide an oral composition.
前記目的を達成するため、本発明者らは鋭意研究の結果、エノキタケの乾燥粉末、抽出物又はそれらの処理物を有効成分とすることで、精巣細胞からのテストステロンの分泌を顕著に促進できることを見出し、本発明を完成した。 In order to achieve the above-mentioned object, the present inventors have found that, as a result of diligent research, the secretion of testosterone from testicular cells can be remarkably promoted by using dry powder of enokitake, extract or processed product thereof as an active ingredient. The headline and the present invention were completed.
すなわち、本発明の実施形態は以下の通りである。
（１）エノキタケの乾燥粉末、抽出物又はそれらの処理物を有効成分とするテストステロン分泌促進用経口組成物。
（２）エノキタケのエタノール抽出物又はその処理物を有効成分とする前記（１）に記載の組成物。
（３）男性更年期障害を予防及び／又は改善するために用いられる前記（１）又は（２）に記載の組成物。
（４）エタノール抽出物の抽出溶媒がエタノール水溶液である前記（２）又は（３）に記載の組成物。
（５）エノキタケの乾燥粉末、抽出物又はそれらの処理物が、セラミド、セレブロシド、グリコシルセラミド、スフィンゴイド塩基、スフィンゴ脂質、グリセロ脂質、ステリルグリコシド、ステロール配糖体、ステロール、脂肪酸、サポニン、ステロイド配糖体、ステロイド、レチノイド、レチノイン酸、レチノール、レチナール、カロテノイド及びテルペノイドから選ばれる少なくとも１種を含むものである前記（１）〜（４）のいずれかに記載の組成物。
（６）食品組成物又は医薬組成物である前記（１）〜（５）のいずれかに記載の組成物。
（７）食品組成物が健康食品、サプリメント、機能性表示食品、栄養補助食品、特定保健用食品、病者用食品又は高齢者用食品である前記（６）に記載の組成物。
（８）６０℃以上の条件で乾燥、又は凍結乾燥したエノキタケの重量に対し０．１倍量以上のエタノール水溶液を加え、０〜７８℃の温度で抽出し、固液分離した後の液層を乾燥することを含む、前記（２）〜（７）のいずれかに記載の組成物の製造方法。
（９）６０〜７０℃の条件で乾燥、又は凍結乾燥したエノキタケの重量に対し５倍量以上のエタノール水溶液を加え、１０〜３０℃の温度で抽出する前記（８）に記載の製造方法。
That is, the embodiment of the present invention is as follows.
(1) An oral composition for promoting testosterone secretion comprising a dried powder of enokitake, an extract or a processed product thereof as an active ingredient.
(2) The composition according to the above (1), which comprises an enokitake ethanol extract or a processed product thereof as an active ingredient.
(3) The composition according to (1) or (2), which is used for preventing and / or improving male climacteric disorder.
(4) The composition according to (2) or (3), wherein the extraction solvent for the ethanol extract is an aqueous ethanol solution.
(5) Enokitake mushroom dry powder, extract or processed product thereof is ceramide, cerebroside, glycosylceramide, sphingoid base, sphingolipid, glycerolipid, steryl glycoside, sterol glycoside, sterol, fatty acid, saponin, steroid The composition according to any one of (1) to (4) above, comprising at least one selected from glycosides, steroids, retinoids, retinoic acid, retinol, retinal, carotenoids and terpenoids.
(6) The composition according to any one of (1) to (5), which is a food composition or a pharmaceutical composition.
(7) The composition according to (6), wherein the food composition is a health food, a supplement, a functional labeling food, a dietary supplement, a food for specified health use, a food for the sick, or a food for the elderly.
(8) Liquid layer after adding 0.1 times or more amount of ethanol aqueous solution to the weight of enokitake mushroom dried or freeze-dried at 60 ° C. or higher, extracted at a temperature of 0-78 ° C., and solid-liquid separated The manufacturing method of the composition in any one of said (2)-(7) including drying.
(9) The production method according to (8), wherein an ethanol aqueous solution is added in an amount of 5 times or more the weight of enokitake mushroom dried or freeze-dried at 60 to 70 ° C, and extracted at a temperature of 10 to 30 ° C.
本発明のテストステロン分泌促進用経口組成物によれば、血中テストステロンを増加させることができる。したがって、本発明のテストステロン分泌促進用経口組成物は、男性更年期障害の予防及び／又は改善に有用である。また、本発明のテストステロン分泌促進用経口組成物は、健康食品、サプリメント、機能性表示食品、栄養補助食品、特定保健用食品、病者用食品又は高齢者用食品を含む種々の非加熱及び加熱加工食品に用いることができる。 According to the oral composition for promoting testosterone secretion of the present invention, blood testosterone can be increased. Therefore, the oral composition for promoting testosterone secretion of the present invention is useful for preventing and / or improving male climacteric disorder. The oral composition for promoting testosterone secretion according to the present invention includes various non-heated and heated foods including health foods, supplements, functional indication foods, dietary supplements, foods for specified health use, foods for the sick or foods for the elderly. Can be used for processed foods.
本発明においては、エノキタケの乾燥粉末、抽出物又はそれらの処理物をテストステロン分泌促進用経口組成物の有効成分として用いる。
エノキタケ乾燥粉末の調製法としては、好ましくは、エノキタケを、通常６０℃以上、好ましくは６０〜７０℃の条件で乾燥するか、あるいは、凍結乾燥し、フードプロセッサー等で細粉する方法が挙げられる。
In the present invention, a dried powder of enokitake, an extract or a processed product thereof is used as an active ingredient of an oral composition for promoting testosterone secretion.
The method for preparing enokitake mushroom dry powder is preferably a method in which enokitake mushroom is usually dried at 60 ° C. or higher, preferably 60 to 70 ° C., or freeze-dried and finely powdered with a food processor or the like. .
抽出物を得るために用いる溶媒としては、水；アルコール類、例えばメタノール、エタノール、プロパノール、ブタノール；ケトン類、例えばアセトン等が挙げられる。抽出物を一旦溶媒除去して乾燥物として用いる場合には、前述した任意の溶媒を単独で又は混合して用いることができる。一方、抽出物を溶媒に溶解した状態で用いる場合には、人体に対して有害な作用を示さない溶媒を用いる必要があり、この場合には、水又はエタノール水溶液を用いることが好ましく、エタノール水溶液を用いることが更に好ましい。抽出に際して、エノキタケは、そのまま用いることができ、また乾燥後に破砕又は粉砕して溶媒との接触を高めることもできる。
また、抽出物の形態は、液状、乾燥粉末状、顆粒状、ペースト状等、どのようなものでも用いることができ、特に限定されるものではない。
Solvents used to obtain the extract include water; alcohols such as methanol, ethanol, propanol, butanol; ketones such as acetone. When the extract is once solvent-removed and used as a dried product, any of the above-described solvents can be used alone or in combination. On the other hand, when the extract is used in a state dissolved in a solvent, it is necessary to use a solvent that does not have a harmful effect on the human body. In this case, it is preferable to use water or an aqueous ethanol solution. More preferably, is used. At the time of extraction, the enokitake mushroom can be used as it is, or can be crushed or pulverized after drying to enhance contact with the solvent.
The form of the extract can be any liquid, dry powder, granule, paste or the like, and is not particularly limited.
例えば、抽出溶媒としてエタノール水溶液を用いて抽出物を製造する方法としては、前記エノキタケ乾燥粉末を、該乾燥粉末の重量に対し通常０．１倍量以上、好ましくは５倍量以上（例えば５〜５０倍量、１０〜５０倍量などの範囲で）のエタノール水溶液（濃度：通常２０〜９０％、好ましくは３０〜５０％）と混合し、通常０〜７８℃、好ましくは１０〜６０℃、更に好ましくは１０〜３０℃（室温）の温度で抽出処理を行った後、抽出残渣を除去（遠心分離、又はフィルター等でのろ過）する方法等が例示される。 For example, as a method for producing an extract using an aqueous ethanol solution as an extraction solvent, the enokitake mushroom dry powder is usually 0.1 times or more, preferably 5 times or more (for example, 5 to 5 times) the weight of the dry powder. Mixed with an aqueous ethanol solution (concentration: usually 20 to 90%, preferably 30 to 50%) in a range of 50 times, 10 to 50 times, etc., usually 0 to 78 ° C, preferably 10 to 60 ° C, More preferably, after performing the extraction treatment at a temperature of 10 to 30 ° C. (room temperature), the extraction residue is removed (centrifugation or filtration with a filter or the like).
本発明において、乾燥粉末又は抽出物の処理物における「処理」とは、濃縮、粉砕、製粉、洗浄、加水分解、発酵、精製、圧搾、抽出、分画、ろ過、乾燥、粉末化、造粒、溶解、滅菌、ｐＨ調整、脱臭、脱色等を任意に選択、組み合わせた処理を示し、前記精製とは、イオン交換樹脂やシリカゲル等を用いたクロマトグラフィー・吸着脱離、イオン交換樹脂や電気透析膜を用いた脱塩、電気透析膜及び／又はＲＯ膜及び／又はＭＦ膜及び／又はＵＦ膜を用いた分離、濃縮及び／又は冷却を用いた結晶化・沈殿、遠心分離等を用いた固液分離等を任意に選択、組み合わせた処理を示す。 In the present invention, “treatment” in the processed product of dry powder or extract means concentration, pulverization, milling, washing, hydrolysis, fermentation, purification, pressing, extraction, fractionation, filtration, drying, powdering, granulation , Dissolution, sterilization, pH adjustment, deodorization, decolorization, etc. are arbitrarily selected and combined, and the purification refers to chromatography / adsorption / desorption, ion exchange resin or electrodialysis using ion exchange resin or silica gel. Desalination using membrane, separation using electrodialysis membrane and / or RO membrane and / or MF membrane and / or UF membrane, crystallization / precipitation using concentration and / or cooling, solidification using centrifugation, etc. The process which selected and combined liquid separation etc. arbitrarily is shown.
なお、本発明で用いるエノキタケの乾燥粉末、抽出物又はそれらの処理物には、セラミド、セレブロシド、グリコシルセラミド、スフィンゴイド塩基、スフィンゴ脂質、グリセロ脂質、ステリルグリコシド、ステロール配糖体、ステロール、脂肪酸、サポニン、ステロイド配糖体、ステロイド、レチノイド、レチノイン酸、レチノール、レチナール、カロテノイド及びテルペノイドから選ばれる１種又は２種以上を含むのがより好適である。また、これらの取得は、上述のような調製方法、製造方法等に限定されるものではなく、同様の品質のものが取得できるような様々な変形が可能である。 In addition, dry powder of enokitake used in the present invention, extract or processed product thereof includes ceramide, cerebroside, glycosylceramide, sphingoid base, sphingolipid, glycerolipid, steryl glycoside, sterol glycoside, sterol, fatty acid More preferably, it contains one or more selected from saponins, steroid glycosides, steroids, retinoids, retinoic acid, retinol, retinal, carotenoids and terpenoids. In addition, these acquisitions are not limited to the above-described preparation methods, production methods, and the like, and various modifications can be made so that the same quality can be acquired.
本発明の組成物の形態は、例えば錠剤、カプセル剤、顆粒剤、散剤、シロップ剤、液剤等を挙げることができる。これらの各種製剤は、エノキタケの乾燥粉末、抽出物又はそれらの処理物のみで製剤化してもよいし、常法にしたがって賦形剤、結合剤、崩壊剤、滑沢剤、矯味矯臭剤、溶解補助剤、懸濁剤、コーティング剤などの既知の補助剤を用いて製剤化することもできる。 Examples of the form of the composition of the present invention include tablets, capsules, granules, powders, syrups, and liquids. These various preparations may be formulated only with enokitake mushroom dry powder, extracts or processed products thereof, and according to conventional methods, excipients, binders, disintegrants, lubricants, flavoring agents, dissolution agents It can also be formulated using known adjuvants such as adjuvants, suspensions, and coating agents.
また、本発明においては、飲食品等と混合して健康食品、サプリメント、特定保健用食品、機能性表示食品（例えば、男性ホルモン増加用食品、男性更年期障害予防用食品）、栄養補助食品、病者用食品又は高齢者用食品として提供することも可能である。 In the present invention, health foods, supplements, foods for specified health use, functional indication foods (for example, foods for increasing male hormones, foods for preventing menopause), nutritional supplements, diseases, It is also possible to provide food for the elderly or food for the elderly.
本発明のテストステロン分泌促進用経口組成物の投与量は、患者の年令、体重、疾患の程度により異なるが、エノキタケの乾燥粉末、抽出物又はそれらの処理物では、乾燥粉末として、通常１日１５〜５００ｍｇであり、投与回数は、通常、経口投与では１日１〜３回である。
本発明のテストステロン分泌促進用経口組成物の製造原料であるエノキタケは、食用に供されており、安全性は確立されている。
The dose of the oral composition for promoting testosterone secretion according to the present invention varies depending on the age, body weight, and degree of disease of the patient. However, in the dried powder of enokitake, the extract or a processed product thereof, it is usually 1 day as a dry powder. It is 15 to 500 mg, and the frequency of administration is usually 1 to 3 times a day for oral administration.
Enokitake, which is a raw material for producing the oral composition for promoting testosterone secretion according to the present invention, is used for food and its safety has been established.
以下、本発明の実施例について述べるが、本発明はこれらの実施例のみに限定されるものではなく、本発明の技術的思想内においてこれらの様々な変形が可能である。 Examples of the present invention will be described below, but the present invention is not limited to these examples, and various modifications can be made within the technical idea of the present invention.
［実施例１］
（エノキタケのテストステロン分泌促進作用に関する動物試験）
（試料の調製）
１ｋｇのエノキタケを水洗した後、９０℃で２分間加熱処理し、凍結乾燥を行った。乾燥後、フードプロセッサーで処理し、エノキタケ粉末を得た。該粉末の水分含量は２．４％であった。
[Example 1]
(Animal test on the effect of enokitake on testosterone secretion)
(Sample preparation)
After washing 1 kg enokitake with water, it was heat-treated at 90 ° C. for 2 minutes and freeze-dried. After drying, it was processed with a food processor to obtain enokitake powder. The water content of the powder was 2.4%.
（動物試験の方法）
マウス（７〜８週令のｄｄＹ雄）１２匹を３群に分け、(i)標準飼料摂取群、(ii)シスプラチン投与＋標準飼料群、(iii)シスプラチン投与＋５％エノキタケ混餌群とした。(ii)及び(iii)群では、シスプラチン（２．５ｍｇ／ｋｇ体重）を腹腔投与した。(i)及び(ii)群は、マウス用標準飼料（ＭＦ、オリエンタル酵母工業株式会社）を、(iii)群は、エノキタケ粉末を５％になるようにマウス用標準飼料に混ぜたものを摂取させた。１週間これらの飼料でマウスを飼育した後、精巣を採取、初代培養し、テストステロン分泌量を測定した。
(Animal testing method)
Twelve mice (7-8 week old ddY males) were divided into three groups: (i) standard feed intake group, (ii) cisplatin administration + standard feed group, and (iii) cisplatin administration + 5% enokitake mixed group. In groups (ii) and (iii), cisplatin (2.5 mg / kg body weight) was administered intraperitoneally. Groups (i) and (ii) ingest standard feed for mice (MF, Oriental Yeast Co., Ltd.), and group (iii) ingests enokitake mushroom powder mixed with standard feed for mice at 5%. I let you. After breeding mice with these feeds for 1 week, testes were collected and cultured primarily, and the amount of testosterone secretion was measured.
（マウス精巣初代培養細胞の調製）
各群の精巣を細切し、０．０５％コラゲナーゼを含むMedium 199培地にて処理（３７℃、１５分）した後、ＤＭＥ／Ｆ−１２培地にて２回洗浄後、１０％ウシ胎児血清を含むＤＭＥ／Ｆ−１２培地にて懸濁し、ウェルあたり３×１０５cell／０．５ｍｌに調製し、２４ウェルプレートに播種し、一夜、培養した。
(Preparation of mouse testis primary culture cells)
The testis of each group was cut into small pieces, treated with Medium 199 medium containing 0.05% collagenase (37 ° C., 15 minutes), washed twice with DME / F-12 medium, and then with 10% fetal bovine serum. Was suspended in a DME / F-12 medium containing 2 × 10 5 cells / 0.5 ml per well, seeded in a 24-well plate, and cultured overnight.
（テストステロン分泌量の測定）
初代培養細胞のウェルをダルベッコＰＢＳ（−）で細胞を２回洗浄し、これに０．１％ウシ血清アルブミン（ＢＳＡ）を含むＤＭＥ／Ｆ−１２培地０．４５ｍｌと共に、試料に０．１％ウシ血清アルブミン（ＢＳＡ）を含むＤＭＥ／Ｆ−１２培地の１０倍希釈したものを、１／１０量添加した。２時間培養後、培地を採取し、培地中のテストステロン濃度をＥＩＡ法により測定した。具体的には、９６ウェルマイクロプレートにヤギ抗家兎ＩｇＧ抗体（２μｇ／well）を添加し、常温で１時間コーティングした。洗浄後、１％ＢＳＡを含む０．０１Ｍリン酸緩衝液（ｐＨ７．４）を添加した。約１５分後、ウェルを洗浄し、標準希釈液（０．５％ＢＳＡ，２５ｍＭ ＥＤＴＡ，０．１４Ｍ ＰＢＳ，ｐＨ７．４）に溶解したテストステロン又は前記培地試料、ビオチン化テストステロン及び抗テストステロン血清を順次添加し、撹拌し、２時間反応させた。洗浄後、ＨＲＰ標識アビジン（１万倍希釈）を添加し、３０分間反応させた。洗浄後、０．００２％オルトフェニレンジアミン溶液（０．００１２％過酸化水素を含むリン酸クエン酸緩衝液に溶解）を加えて１０分間呈色反応させ、２Ｍ硫酸にて反応を停止し、マイクロプレートリーダーで４９０ｎｍにおける吸光度を測定し、テストステロン分泌量を求めた。
(Measurement of testosterone secretion)
The wells of the primary culture cells were washed twice with Dulbecco's PBS (-), which was mixed with 0.45 ml of DME / F-12 medium containing 0.1% bovine serum albumin (BSA), and 0.1% of the sample was added. 1/10 amount of 10-fold diluted DME / F-12 medium containing bovine serum albumin (BSA) was added. After culturing for 2 hours, the medium was collected, and the testosterone concentration in the medium was measured by the EIA method. Specifically, goat anti-rabbit IgG antibody (2 μg / well) was added to a 96-well microplate and coated at room temperature for 1 hour. After washing, 0.01 M phosphate buffer (pH 7.4) containing 1% BSA was added. After about 15 minutes, the wells were washed, and testosterone dissolved in standard diluent (0.5% BSA, 25 mM EDTA, 0.14 M PBS, pH 7.4) or the medium sample, biotinylated testosterone and anti-testosterone serum were sequentially added. Added, stirred and allowed to react for 2 hours. After washing, HRP-labeled avidin (diluted 10,000 times) was added and allowed to react for 30 minutes. After washing, a 0.002% orthophenylenediamine solution (dissolved in a phosphate citrate buffer containing 0.0012% hydrogen peroxide) is added, and a color reaction is carried out for 10 minutes, and the reaction is stopped with 2M sulfuric acid. The absorbance at 490 nm was measured with a plate reader to determine the amount of testosterone secretion.
（結果）
各群から得た精巣初代培養細胞のテストステロン分泌量は、標準飼料摂取群に比し、シスプラチン投与＋標準飼料群で有意（＊、ｐ＜０．０５）に低下したのに対し、シスプラチン投与＋５％エノキタケ混餌群では、シスプラチン投与＋標準飼料群に比し有意（＊＊、ｐ＜０．０５）に増加していた。（図１）
(result)
Testosterone secretion of primary testicular cells obtained from each group was significantly decreased (*, p <0.05) in the cisplatin administration + standard diet group compared to the standard diet intake group, whereas cisplatin administration + 5 The% enokitake mushroom group increased significantly (**, p <0.05) compared to the cisplatin administration + standard feed group. (Figure 1)
［実施例２］
（マウス精巣由来の初代培養細胞を用いたエノキタケのテストステロン分泌促進活性試験）
（試料の調製）
実施例１で調製したエノキタケの凍結乾燥粉末の０．１ｇに２０倍量の３０％、５０％、あるいは８０％エタノール水溶液を添加し、室温で２時間撹拌した後に３０００ｒｐｍで１０分間遠心分離した上清を減圧乾固し、０．２ｍｌの３０％、５０％あるいは８０％エタノールに溶解した。これを１００００ｒｐｍで５分間遠心分離し、不溶物を除去した上清を試料として用いた。
[Example 2]
(Test of testosterone secretion promoting activity of enokitake using primary cultured cells derived from mouse testis)
(Sample preparation)
Add 20 times 30%, 50%, or 80% aqueous ethanol solution to 0.1 g of Enokitake lyophilized powder prepared in Example 1, stir at room temperature for 2 hours, and then centrifuge at 3000 rpm for 10 minutes. The supernatant was dried under reduced pressure and dissolved in 0.2 ml of 30%, 50% or 80% ethanol. This was centrifuged at 10,000 rpm for 5 minutes, and the supernatant from which insoluble matter was removed was used as a sample.
（マウス精巣初代培養細胞の調製）
マウス（ｄｄＹ系、雄、７〜８週令）から精巣を採取し初代培養を行った。具体的には、採取した精巣を細切し、０．０５％コラゲナーゼを含むMedium 199培地にて処理（３７℃、１５分）した後、ＤＭＥ／Ｆ−１２培地にて２回洗浄後、１０％ウシ胎児血清を含むＤＭＥ／Ｆ−１２培地にて懸濁し、ウェルあたり３×１０５cell／０．５ｍｌに調製し、２４ウェルプレートに播種し、一夜、培養した。
(Preparation of mouse testis primary culture cells)
Testes were collected from mice (ddY strain, male, 7-8 weeks old) and subjected to primary culture. Specifically, the collected testis was minced, treated with Medium 199 medium containing 0.05% collagenase (37 ° C., 15 minutes), washed twice with DME / F-12 medium, 10 The suspension was suspended in DME / F-12 medium containing 1% fetal bovine serum, adjusted to 3 × 10 5 cells / 0.5 ml per well, seeded in a 24-well plate, and cultured overnight.
（テストステロン分泌促進活性の測定法）
初代培養細胞のウェルをダルベッコＰＢＳ（−）で細胞を２回洗浄し、これに０．１％ウシ血清アルブミン（ＢＳＡ）を含むＤＭＥ／Ｆ−１２培地０．４５ｍｌと共に、前記の試料を０．１％ウシ血清アルブミン（ＢＳＡ）を含むＤＭＥ／Ｆ−１２培地にて１０倍希釈したものを、１／１０量添加した。無添加のものを陰性対照、ヒト絨毛性性腺刺激ホルモン（ｈＣＧ）０．０１ユニット／ｍｌを１／１０量添加したものを陽性対照とした。２時間培養後、培地を採取し、培地中のテストステロン濃度をＥＩＡ法によりテストステロン分泌量を測定した。具体的には、９６ウェルマイクロプレートにヤギ抗家兎ＩｇＧ抗体（２μｇ／well）を添加し、常温で１時間コーティングした。洗浄後、１％ＢＳＡを含む０．０１Ｍリン酸緩衝液（ｐＨ７．４）を添加した。約１５分後、ウェルを洗浄し、標準希釈液（０．５％ＢＳＡ，２５ｍＭ ＥＤＴＡ，０．１４Ｍ ＰＢＳ，ｐＨ７．４）に溶解したテストステロン又は前記培地試料、ビオチン化テストステロン及び抗テストステロン血清を順次添加し、撹拌し、２時間反応させた。洗浄後、ＨＲＰ標識アビジン（１万倍希釈）を添加し、３０分間反応させた。洗浄後、０．００２％オルトフェニレンジアミン溶液（０．００１２％過酸化水素を含むリン酸クエン酸緩衝液に溶解）を加えて１０分間呈色反応させ、２Ｍ硫酸にて反応を停止し、マイクロプレートリーダーで４９０ｎｍにおける吸光度を測定し、テストステロン分泌量を求めた。
(Measurement method of testosterone secretion promoting activity)
The wells of the primary culture cells were washed twice with Dulbecco's PBS (−), and 0.45 ml of the DME / F-12 medium containing 0.1% bovine serum albumin (BSA) was added to the above sample in the amount of 0. 1/10 amount of a 10-fold diluted solution in DME / F-12 medium containing 1% bovine serum albumin (BSA) was added. The additive without addition was used as a negative control, and the one with 0.01 unit / ml of human chorionic gonadotropin (hCG) added as a positive control. After culturing for 2 hours, the medium was collected, and the testosterone secretion amount in the medium was measured by the EIA method. Specifically, goat anti-rabbit IgG antibody (2 μg / well) was added to a 96-well microplate and coated at room temperature for 1 hour. After washing, 0.01 M phosphate buffer (pH 7.4) containing 1% BSA was added. After about 15 minutes, the wells were washed, and testosterone dissolved in standard diluent (0.5% BSA, 25 mM EDTA, 0.14 M PBS, pH 7.4) or the medium sample, biotinylated testosterone and anti-testosterone serum were sequentially added. Added, stirred and allowed to react for 2 hours. After washing, HRP-labeled avidin (diluted 10,000 times) was added and allowed to react for 30 minutes. After washing, a 0.002% orthophenylenediamine solution (dissolved in a phosphate citrate buffer containing 0.0012% hydrogen peroxide) is added, and a color reaction is carried out for 10 minutes, and the reaction is stopped with 2M sulfuric acid. The absorbance at 490 nm was measured with a plate reader to determine the amount of testosterone secretion.
（結果）
エノキタケ３０％エタノール抽出物、５０％エタノール抽出物、８０％エタノール抽出物添加により、無添加に比し２．４〜５．２倍のテストステロン分泌量の増加が認められた。（図２）
(result)
By adding Enokitake 30% ethanol extract, 50% ethanol extract, and 80% ethanol extract, an increase in the amount of testosterone secretion was observed by 2.4 to 5.2 times as compared with the case of no addition. (Figure 2)
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| JPH03117467A (en) * | 1989-09-29 | 1991-05-20 | Shimaya:Kk | Food and medicine for prevention and remedy of hypertension, hyperlipemia and obesity |
| JPH07278006A (en) * | 1994-04-11 | 1995-10-24 | Narisu Keshohin:Kk | Testosterone-5alpha-reductase inhibitor |
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| US20080299145A1 (en) * | 2007-06-01 | 2008-12-04 | Edward Stephen Morrissey | Formula for improving physical performance and related methods |
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| JPH03117467A (en) * | 1989-09-29 | 1991-05-20 | Shimaya:Kk | Food and medicine for prevention and remedy of hypertension, hyperlipemia and obesity |
| JPH07278006A (en) * | 1994-04-11 | 1995-10-24 | Narisu Keshohin:Kk | Testosterone-5alpha-reductase inhibitor |
| JP2004131482A (en) * | 2002-08-21 | 2004-04-30 | Chee Keung Chung | Anti-aging/climacteric symptom relief by using ganoderma lucidium spore |
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