JP5167462B2 - Novel compounds and osteoclast differentiation / proliferation inhibitors - Google Patents
Novel compounds and osteoclast differentiation / proliferation inhibitors Download PDFInfo
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Description
本発明は、例えば、飲食品、医薬品、化粧品の原料として用いることができる新規化合物、及び破骨細胞分化・増殖阻害剤に関する。 The present invention relates to, for example, a novel compound that can be used as a raw material for foods and drinks, pharmaceuticals, and cosmetics, and an osteoclast differentiation / proliferation inhibitor.
高齢化が年々進んでいる日本の現代社会においては、老化に伴う様々な疾患に悩む人々が増加している。例えば、老化に伴う疾患の一つである骨粗鬆症は、骨量が減少して骨が非常に脆くなる症状をいう。 In the modern Japanese society where aging is progressing year by year, an increasing number of people suffer from various diseases associated with aging. For example, osteoporosis, which is one of the diseases associated with aging, refers to a condition in which the bone mass decreases and the bone becomes very brittle.
正常な骨では、古くなった骨の部分を破骨細胞が分解する骨吸収と、骨吸収されたところに骨芽細胞が新しい骨を作り出す骨形成がバランスよく行われて、一定の骨量に保たれている。しかし、カルシウムの摂取不足、カルシウム吸収能力の低下、ホルモンバランスの乱れ等により、骨の代謝回転のバランスが崩れて骨吸収が骨形成を上回ることによって骨粗鬆症を招きやすくなる。特に、閉経後の女性は、ホルモンバランスの乱れにより、骨粗鬆症になりやすい。 In normal bone, bone resorption, in which osteoclasts break down old bone parts, and bone formation in which osteoblasts create new bone where bone resorption is performed, are balanced, resulting in a constant bone mass. It is kept. However, due to insufficient intake of calcium, a decrease in calcium absorption ability, disorder of hormone balance, etc., the balance of bone turnover is lost, and bone resorption exceeds bone formation, leading to osteoporosis. In particular, postmenopausal women are prone to osteoporosis due to hormonal balance disturbance.
したがって、骨粗鬆症に対しては、骨吸収を抑制するか、又は骨形成を促進することが必要であると考えられている。 Therefore, for osteoporosis, it is considered necessary to suppress bone resorption or promote bone formation.
そのため、従来の骨粗鬆症の治療薬としては、活性型ビタミンＤ３製剤、ビタミンＫ２製剤、エストロゲン製剤、カルシトシン製剤、カルシウム製剤、ビスホスホネート製剤等が用いられているが、これらの治療薬には以下のような副作用の問題があった。 Therefore, as active therapeutic agents for osteoporosis, active vitamin D3 preparation, vitamin K2 preparation, estrogen preparation, calcitocin preparation, calcium preparation, bisphosphonate preparation, etc. are used. There was a problem of side effects.
１）活性型ビタミンＤ３製剤：血中カルシウム量の増え過ぎによる食欲不振、倦怠感、腎不全等。
２）ビタミンＫ２製剤：心筋梗塞の場合などに服用するワーファリンという薬の効果低下。
３）エストロゲン製剤：心筋梗塞や脳卒中の危険性拡大、乳癌及び子宮体癌の発症率拡大等。
４）カルシトシン製剤：吐き気、顔面紅潮等。
５）カルシウム製剤：胃腸障害。
６）ビスホスホネート製剤：消化器症状、特に食道潰瘍。
７）活性型ビタミンＤ３製剤とカルシウム製剤の併用による副作用の相乗効果。
1) Active vitamin D3 preparation: Anorexia, malaise, renal failure, etc. due to excessive increase in blood calcium level.
2) Vitamin K2 preparation: Decreased effect of a drug called warfarin taken in the case of myocardial infarction.
3) Estrogen preparation: Increased risk of myocardial infarction and stroke, increased incidence of breast cancer and endometrial cancer.
4) Calcitocin preparation: nausea, hot flush, etc.
5) Calcium preparation: gastrointestinal disorders.
6) Bisphosphonate formulation: digestive symptoms, especially esophageal ulcers.
7) Synergistic effect of side effects by combined use of active vitamin D3 preparation and calcium preparation.
そのため、長期間摂取しても副作用の問題がなく、より安全性の高い骨粗鬆症治療剤の開発も進められており、例えば、下記特許文献１には、乳由来の塩基性タンパク質画分を有効成分とする骨強化剤が開示されている。 Therefore, development of a safer osteoporosis treatment agent that has no side-effect problems even when ingested for a long period of time has been promoted. For example, Patent Document 1 listed below discloses a basic protein fraction derived from milk as an active ingredient. A bone strengthening agent is disclosed.
下記特許文献２には、乳由来の塩基性タンパク質画分、特にラクトフェリンを有効成分とする破骨細胞分化抑制因子産生促進剤が開示されている。 Patent Document 2 listed below discloses an osteoclast differentiation inhibitor production promoter containing milk-derived basic protein fraction, particularly lactoferrin as an active ingredient.
下記特許文献３には、カゼインホスホペプチド及びゲニステインを有効成分とする骨強化剤が開示されている。 Patent Document 3 listed below discloses a bone strengthening agent containing casein phosphopeptide and genistein as active ingredients.
下記特許文献４には、コラーゲン又はコラーゲンの酵素分解物を有効成分とする骨粗鬆症予防・治療剤が開示されている。 Patent Document 4 listed below discloses an osteoporosis preventive / therapeutic agent containing collagen or an enzyme degradation product of collagen as an active ingredient.
下記特許文献５には、イソフラボンを主たる有効成分とする骨形成促進及び骨塩量減少防止用組成物が開示されている。
上記特許文献に記載されている食品由来の成分を有効成分とする様々な骨粗鬆症治療剤は、健康食品等として手軽に摂取でき、副作用の問題も少ないものの、いずれも十分満足できる効果が期待できるとは言えなかった。 Various osteoporosis therapeutic agents containing the ingredients derived from foods described in the above patent documents as active ingredients can be easily ingested as health foods, etc., and there are few side effects, but all can be expected to be sufficiently satisfactory. I could not say.
したがって、本発明は、例えば、破骨細胞の分化・増殖を阻害し、骨吸収を抑制することにより、十分な骨粗鬆症の予防・治療改善効果が期待でき、飲食品、医薬品、化粧品の原料として用いることができる新規化合物、及び破骨細胞分化・増殖阻害剤を提供することにある。 Therefore, the present invention can be expected to have a sufficient effect of preventing or treating osteoporosis by inhibiting osteoclast differentiation / proliferation and suppressing bone resorption, and is used as a raw material for foods, beverages, pharmaceuticals, and cosmetics. It is an object of the present invention to provide a novel compound and an osteoclast differentiation / proliferation inhibitor that can be used.
マコモタケは、漢方薬の原料として利用されており、口渇の抑制、解熱、整腸、消化、解毒など様々な生理活性効果が知られている。本発明者らは、豊富な食経験により安全性が確認されているマコモタケのさらなる生理活性を追及したところ、マコモタケの新たな生理活性効果として骨粗鬆症予防・治療効果を見出した。そこで、その生理活性成分について更なる研究を行った結果、本発明を達成するに至った。 Makomotake is used as a raw material for traditional Chinese medicine, and various physiologically active effects such as suppression of dry mouth, fever reduction, bowel regulation, digestion and detoxification are known. The present inventors have investigated the further physiological activity of Momotake, which has been confirmed to be safe by abundant dietary experiences, and found the effect of preventing and treating osteoporosis as a new physiological activity of Momotake. As a result of further research on the physiologically active component, the present invention has been achieved.
すなわち、本発明の新規化合物は、下記化学式（１）で示される。 That is, the novel compound of the present invention is represented by the following chemical formula (1).
本発明の新規化合物は、飲食品、医薬品又は化粧品の原料として用いられることが好ましい。 It is preferable that the novel compound of this invention is used as a raw material of food-drinks, a pharmaceutical, or cosmetics.
一方、本発明の破骨細胞分化・増殖阻害剤は、下記化学式（１）で示される化合物で示される化合物を有効成分として含有することを特徴とする。 On the other hand, the osteoclast differentiation / proliferation inhibitor of the present invention contains a compound represented by the following chemical formula (1) as an active ingredient.
また、本発明の破骨細胞分化・増殖阻害剤は、マコモタケの有機溶媒抽出物から調製された組成物を含有することが好ましい。 In addition, the osteoclast differentiation / proliferation inhibitor of the present invention preferably contains a composition prepared from an organic solvent extract of Makomotake.
また、本発明の破骨細胞分化・増殖阻害剤は、マコモタケの菌えい部の有機溶媒抽出物から調製された組成物を含有することが好ましい。 Moreover, it is preferable that the osteoclast differentiation / proliferation inhibitor of the present invention contains a composition prepared from an organic solvent extract of the cervix of the fungus of Makomotake.
本発明の新規化合物は、例えば、飲食品、医薬品、化粧品などの原料として用いることができ、骨粗鬆症の予防・治療改善効果が期待できる。 The novel compound of the present invention can be used, for example, as a raw material for foods and drinks, pharmaceuticals, cosmetics and the like, and can be expected to have an effect of preventing and / or improving osteoporosis.
また、本発明の破骨細胞分化・増殖阻害剤は、豊富な食経験により安全性が確認されているマコモタケ由来の成分を有効成分として含有するものであり、安全性が高く、更には十分な骨粗鬆症の予防・治療改善効果が期待できる。 Further, the osteoclast differentiation / proliferation inhibitor of the present invention contains a component derived from Makotake that has been confirmed to be safe by abundant dietary experience as an active ingredient, and is highly safe and further sufficient. Expected to be effective in preventing and treating osteoporosis.
本発明の新規化合物は、下記化学式（１）で表される。 The novel compound of the present invention is represented by the following chemical formula (1).
上記化学式（１）で表す化合物（以下、「化合物（１）」と記す）は、有機溶媒に可溶な化合物であり、例えば、マコモタケの溶媒抽出物を精製することで得られる。 The compound represented by the above chemical formula (1) (hereinafter referred to as “compound (1)”) is a compound that is soluble in an organic solvent, and can be obtained, for example, by purifying a solvent extract of Makomotake.
すなわち、まず、抽出原料となるマコモタケを、必要に応じて乾燥あるいは粉砕処理する。なかでも、マコモタケの菌えい部は、上記化合物（１）の含有量が高いことから、マコモタケの菌えい部を抽出原料として用いることで、上記化合物（１）の回収率を向上できる。ここで、マコモタケとは、イネ科マコモ属・多年草の水生植物であるマコモの若茎が黒穂菌によって肥大生育したものであり、中国、台湾を中心とした東アジアから東南アジアにかけて栽培されている中国野菜である。近年では日本でも栽培が行われており、これらの市販品を簡単に入手することができる。 That is, first, the mushroom that is the raw material for extraction is dried or pulverized as necessary. Especially, since the content of the above-mentioned compound (1) is high in the cervix of Momotake, the recovery rate of the above-mentioned compound (1) can be improved by using the cervix of Momotake as an extraction raw material. Here, Makomotake is a cultivated shoot of Macomo, an aquatic plant belonging to the genus Macomo and Perennial, that grows sproutingly with Aspergillus oryzae, and is cultivated from East Asia to Southeast Asia, mainly in China and Taiwan. Vegetables. In recent years, cultivation has been carried out in Japan, and these commercial products can be easily obtained.
次いで、この抽出原料のマコモタケを、抽出溶媒中に室温〜加温下で浸漬して、溶媒抽出物を得る。抽出溶媒としては、特に限定はなく、低級アルコール類（メタノール、エタノールなど）、アセトン、アセトニトリル、クロロホルム、ジクロロメタン、酢酸エチルなどが挙げられ、これらを、単独、もしくは２種類以上組み合わせて用いることができる。特に好ましい抽出溶媒としては、メタノール、エタノール、アセトン、ジクロロメタン及びこれらの混合溶媒が挙げられる。 Next, this extract raw material, Momotake, is immersed in an extraction solvent at room temperature to under heating to obtain a solvent extract. The extraction solvent is not particularly limited, and examples thereof include lower alcohols (methanol, ethanol, etc.), acetone, acetonitrile, chloroform, dichloromethane, ethyl acetate, and the like. These can be used alone or in combination of two or more. . Particularly preferred extraction solvents include methanol, ethanol, acetone, dichloromethane, and mixed solvents thereof.
この抽出溶媒には、夾雑物が多量に含まれている場合が多いことから、例えば、マコモタケを低級アルコール類あるいはアセトンなどの水溶性有機溶媒に、室温〜加温下で浸漬して、水溶性溶媒抽出画分を得た後、酢酸エチル、ヘキサン、クロロホルム、ジクロロメタンなどの非水溶性有機溶媒を用いて脂溶性成分と水溶性成分に分離して、非水溶性有機溶媒抽出画分を回収することが好ましい。こうすることで、夾雑物の極めて少ないマコモタケの溶媒抽出物（非水溶性有機溶媒抽出画分）を得ることができる。また、この溶媒抽出物は必要に応じて濃縮してもよく、更に乾燥してもよい。 Since this extraction solvent often contains a large amount of contaminants, for example, makomotake is soaked in a water-soluble organic solvent such as lower alcohols or acetone at room temperature to under heating to After obtaining the solvent-extracted fraction, separate the fat-soluble component and the water-soluble component using a water-insoluble organic solvent such as ethyl acetate, hexane, chloroform, or dichloromethane, and collect the water-insoluble organic solvent-extracted fraction. It is preferable. By carrying out like this, the solvent extract (non-water-soluble organic-solvent extraction fraction) of Makotake which has very few impurities can be obtained. Moreover, this solvent extract may be concentrated as needed, and may be further dried.
次いで、この溶媒抽出物を、カラム分離精製、再結晶等により精製することで、上記化合物（１）を得ることができる。 Next, the solvent extract is purified by column separation purification, recrystallization, or the like, whereby the compound (1) can be obtained.
溶媒抽出物の精製処理は、まずクロマトグラフィーを用いて一次精製して得られる粗精製物を、再結晶化、カラムクロマトグラフィー、薄層カラムクロマトグラフィーから選ばれた少なくとも１種以上の方法により二次精製することが好ましい。こうすることで、上記化合物（１）の回収率を向上できる。 For the purification of the solvent extract, first, a crude product obtained by primary purification using chromatography is subjected to at least one method selected from recrystallization, column chromatography, and thin layer column chromatography. Subsequent purification is preferred. By carrying out like this, the recovery rate of the said compound (1) can be improved.
上記一次精製に用いるクロマトグラフィーとしては、特に限定はなく、順相カラムクロマトグラフィー、逆相カラムクロマトグラフィー、サイズ排除カラムクロマトグラフィー、イオン交換カラムクロマトグラフィー等の従来一般的なクロマトグラフィーを用いることができる。また、一次精製方法としては、特に限定はなく、例えば、シリカゲルを担体としたクロマトグラフィーを用いた場合、クロロホルム、ジクロロメタン、ベンゼン、酢酸エチル、ヘキサンなどの非水溶性有機溶媒と、その混合溶媒、更にこれらにメタノール、エタノール、アセトンなどの水溶性有機溶媒を適当量加え、その比率を変え順次極性を上げながら溶出する方法が好ましく、ヘキサン／酢酸エチル、ヘキサン／ジクロロメタン、ジクロロメタン／アセトン、クロロホルム／アセトン、クロロホルム／メタノールの混合溶媒を用いる方法が特に好ましい。 The chromatography used for the primary purification is not particularly limited, and conventional general chromatography such as normal phase column chromatography, reverse phase column chromatography, size exclusion column chromatography, and ion exchange column chromatography may be used. it can. The primary purification method is not particularly limited. For example, when chromatography using silica gel as a carrier, a water-insoluble organic solvent such as chloroform, dichloromethane, benzene, ethyl acetate, hexane, and a mixed solvent thereof, Furthermore, it is preferable to add an appropriate amount of a water-soluble organic solvent such as methanol, ethanol or acetone, and change the ratio to elute while gradually increasing the polarity. Hexane / ethyl acetate, hexane / dichloromethane, dichloromethane / acetone, chloroform / acetone A method using a mixed solvent of chloroform / methanol is particularly preferable.
上記二次精製に用いるカラムクロマトグラフィーとしては、順相カラムクロマトグラフィー、ジオールカラムクロマトグラフィー、逆相カラムクロマトグラフィー、サイズ排除カラムクロマトグラフィーなどがあり、担体、溶出溶媒等の精製条件は各種クロマトグラフィーに対応して適宜選択することができる。これらカラムクロマトグラフィーを単独または組み合わせて採用することができ、オープンカラム、ＨＰＬＣカラムなどで適宜適用できる。 Column chromatography used for the secondary purification includes normal phase column chromatography, diol column chromatography, reverse phase column chromatography, size exclusion column chromatography and the like, and purification conditions such as carrier and elution solvent are various chromatography. Can be selected as appropriate. These column chromatography can be employed singly or in combination, and can be appropriately applied to open columns, HPLC columns and the like.
上記二次精製に用いる薄層クロマトグラフィーとしては、逆相担体または順相担体またはアミンや光学活性体で修飾した化学修飾担体を用いたものが挙げられ、担体、展開溶媒等の精製条件は各種クロマトグラフィーに対応して適宜選択することができる。 Examples of the thin layer chromatography used for the secondary purification include those using a reverse phase carrier or normal phase carrier or a chemically modified carrier modified with an amine or an optically active substance. It can be selected appropriately according to the chromatography.
本発明の新規化合物（化合物（１））は、豊富な食経験により安全性が確認されているマコモタケ由来の成分であり、安全性は既に立証されており、各種飲食品、医薬品、化粧品の原料として用いることができる。 The novel compound of the present invention (compound (1)) is a component derived from Makotake that has been confirmed to be safe by abundant dietary experience, and its safety has already been proven, and is a raw material for various foods, pharmaceuticals, and cosmetics. Can be used as
上記飲食品としては、例えば、（ａ）清涼飲料、炭酸飲料、果実飲料、野菜ジュース、乳酸菌飲料、乳飲料、豆乳、ミネラルウォーター、茶系飲料、コーヒー飲料、スポーツ飲料、アルコール飲料、ゼリー飲料等の飲料類、（ｂ）トマトピューレ、キノコ缶詰、乾燥野菜、漬物等の野菜加工品、（ｃ）乾燥果実、ジャム、フルーツピューレ、果実缶詰等の果実加工品、（ｄ）カレー粉、わさび、ショウガ、スパイスブレンド、シーズニング粉等の香辛料、（ｅ）パスタ、うどん、そば、ラーメン、マカロニ等の麺類（生麺、乾燥麺含む）、（ｆ）食パン、菓子パン、調理パン、ドーナツ等のパン類、（ｇ）アルファー化米、オートミール、麩、バッター粉等、（ｈ）焼菓子、ビスケット、米菓子、キャンデー、チョコレート、チューイングガム、スナック菓子、冷菓、砂糖漬け菓子、和生菓子、洋生菓子、半生菓子、プリン、アイスクリーム等の菓子類、（ｉ）小豆、豆腐、納豆、きな粉、湯葉、煮豆、ピーナッツ等の豆類製品、（ｊ）蜂蜜、ローヤルゼリー加工食品、（ｋ）ハム、ソーセージ、ベーコン等の肉製品、（ｌ）ヨーグルト、プリン、練乳、チーズ、発酵乳、バター、アイスクリーム等の酪農製品、（ｍ）加工卵製品、（ｎ）干物、蒲鉾、ちくわ、魚肉ソーセージ等の加工魚や、乾燥わかめ、昆布、佃煮等の加工海藻や、タラコ、数の子、イクラ、からすみ等の加工魚卵、（ｏ）だしの素、醤油、酢、みりん、コンソメベース、中華ベース、濃縮出汁、ドレッシング、マヨネーズ、ケチャップ、味噌等の調味料や、サラダ油、ゴマ油、リノール油、ジアシルグリセロール、べにばな油等の食用油脂、（ｐ）スープ（粉末、液体含む）等の調理、半調理食品や、惣菜、レトルト食品、チルド食品、半調理食品（例えば、炊き込みご飯の素、カニ玉の素）等が挙げられる。そして、飲食品への添加量としては、有効量が摂取できれば、特に限定されるものではなく、飲食品の味や品質安定性へ損なわないように適宜配合すればよいが、例えば５〜５００ｐｐｍ質量、好ましくは１０〜１００ｐｐｍ質量となるように配合すればよい。 Examples of the food and drink include (a) soft drink, carbonated drink, fruit drink, vegetable juice, lactic acid bacteria drink, milk drink, soy milk, mineral water, tea-based drink, coffee drink, sports drink, alcoholic drink, jelly drink, and the like. (B) Processed vegetable products such as canned tomato puree, mushrooms, dried vegetables, pickles, (c) Processed fruit products such as dried fruit, jam, fruit puree, canned fruit, (d) curry powder, wasabi, Spices such as ginger, spice blend, seasoning powder, (e) noodles (including raw noodles, dried noodles) such as pasta, udon, soba, ramen, macaroni, (f) breads such as bread, confectionery bread, cooking bread, donut , (G) Alpha-ized rice, oatmeal, rice cake, batter flour, etc. (h) Baked confectionery, biscuits, rice confectionery, candy, chocolate, chewing gum , Snacks, frozen confectionery, candied confectionery, Japanese confectionery, Western confectionery, half confectionery, pudding, ice cream confectionery, etc. ) Honey, royal jelly processed foods, (k) meat products such as ham, sausage, bacon, (l) dairy products such as yogurt, pudding, condensed milk, cheese, fermented milk, butter, ice cream, (m) processed egg products, (N) Processed fish such as dried fish, sea bream, chikuwa, fish sausage, processed seaweed such as dried seaweed, kelp, and boiled fish, processed fish eggs such as octopus, scallop, salmon roe, and sea bream, (o) dashi stock, soy sauce, Condiments such as vinegar, mirin, consomme base, Chinese base, concentrated soup stock, dressing, mayonnaise, ketchup, miso, salad oil, sesame oil, linoleic oil, diacyl glycero , Edible fats and oils such as Benibana oil, (p) Cooking, semi-cooked foods such as soups (including powder and liquid), side dishes, retort foods, chilled foods, semi-cooked foods (eg cooked rice, crab Tamanomoto) and the like. And as addition amount to food / beverage products, if an effective amount can be taken, it will not specifically limit, What is necessary is just to mix | blend suitably so that it may not impair the taste and quality stability of food / beverage products, For example, 5-500 ppm mass. Preferably, it may be blended so that the mass becomes 10 to 100 ppm.
上記化粧品としては、化粧水、乳液、ローション、ジェル、ファンデーション、クリーム、洗顔料、身体洗浄料等などが挙げられる。そして、化粧品への添加量としては、５〜５００ｐｐｍ質量が好ましく、１０〜１００ｐｐｍ質量がより好ましい。添加量が、上記範囲より少ないと、十分な生理活性効果が期待できず、上記範囲よりも多いと品質安定性に不具合が生じる場合がある。 Examples of the cosmetics include lotions, emulsions, lotions, gels, foundations, creams, facial cleansers, body washing agents, and the like. And as addition amount to cosmetics, 5-500 ppm mass is preferable, and 10-100 ppm mass is more preferable. If the addition amount is less than the above range, sufficient physiological activity effect cannot be expected, and if it is more than the above range, there may be a problem in quality stability.
上記医薬品としては、例えば、破骨細胞分化・増殖阻害剤、免疫賦活剤、メラニン生成抑制剤等が挙げられる。また、その形態としては、液剤、散剤、錠剤、丸剤、細粒剤、顆粒剤、カプセル剤、ゼリー、チュアブル、ペースト等の剤型が例示できる。 Examples of the pharmaceuticals include osteoclast differentiation / proliferation inhibitors, immunostimulators, melanin production inhibitors, and the like. Examples of the form include dosage forms such as liquids, powders, tablets, pills, fine granules, granules, capsules, jelly, chewable and paste.
次に、本発明の破骨細胞分化・増殖阻害剤について説明する。 Next, the osteoclast differentiation / proliferation inhibitor of the present invention will be described.
本発明の破骨細胞分化・増殖阻害剤は、下記化学式（１）で示される化合物（化合物（１）を有効成分として含有するものである。 The osteoclast differentiation / proliferation inhibitor of the present invention contains a compound represented by the following chemical formula (1) (compound (1) as an active ingredient.
そして、本発明の破骨細胞分化・増殖阻害剤は、マコモタケの有機溶媒抽出物から調製された組成物を含有することが好ましく、マコモタケの菌えい部の有機溶媒抽出物から調製された組成物を含有することがより好ましい。 And, the osteoclast differentiation / proliferation inhibitor of the present invention preferably contains a composition prepared from an organic solvent extract of Makomotake, a composition prepared from an organic solvent extract of the fungus colony of Makomotake It is more preferable to contain.
上記化合物（１）は、マコモタケに含まれている成分であり、また、有機溶媒に可溶な化合物である。このため、マコモタケの有機溶媒抽出液には、上記化合物（１）が含まれており、高い骨粗鬆症予防・治療効果が得られる。なかでも、マコモタケの菌えい部の有機溶媒抽出物には、特に上記化合物（１）が多く含まれていることから、マコモタケの菌えい部の有機溶媒抽出物から調製された組成物を用いることで、有効成分の含有量を向上させることができ、より優れた骨粗鬆症予防・治療効果が得られる。 The compound (1) is a component contained in Makotake and is a compound that is soluble in an organic solvent. For this reason, the organic solvent extract of Makomotake contains the compound (1), and a high osteoporosis prevention / treatment effect can be obtained. Especially, since the organic solvent extract of the fungus colony of Macomotake contains a large amount of the above compound (1), the composition prepared from the organic solvent extract of the fungus colony of Makomotake is used. Thus, the content of the active ingredient can be improved, and a more excellent osteoporosis prevention / treatment effect can be obtained.
なお、マコモタケの有機溶媒抽出液から得られる抽出物中には、通常、固形分あたり１ｐｐｍ〜１０００ｐｐｍ程度の上記化合物（１）が含まれている。 In addition, the extract obtained from the organic solvent extract of Makomotake usually contains about 1 ppm to 1000 ppm of the above compound (1) per solid content.
また、本発明の破骨細胞分化・増殖阻害剤は、更にイソフラボン、コラーゲンペプチド、ラクトフェリン、カゼインホスホペプチド、魚骨カルシウム、貝カルシウムから選ばれた１種以上を含むことが好ましい。これらの成分は、骨粗鬆症予防・治療効果を有することが知られており、相乗効果が期待できる。また、必要に応じて、無機塩類、有機酸類、糖質類、タンパク類、ペプチド類、アミノ酸類、脂質類を適宜配合することができる。 In addition, the osteoclast differentiation / proliferation inhibitor of the present invention preferably further contains one or more selected from isoflavones, collagen peptides, lactoferrin, casein phosphopeptides, fish bone calcium and shellfish calcium. These components are known to have osteoporosis prevention / treatment effects, and a synergistic effect can be expected. In addition, inorganic salts, organic acids, carbohydrates, proteins, peptides, amino acids, and lipids can be appropriately blended as necessary.
本発明の破骨細胞分化・増殖阻害剤の形態としては、液剤、散剤、錠剤、丸剤、細粒剤、顆粒剤、カプセル剤、ゼリー、チュアブル、ペースト等の剤型が例示できる。そして、上記化合物（１）の有効摂取量は、体重１ｋｇ当り、０．０１〜５ｍｇが好ましく、０．０５〜０．５ｍｇがより好ましい。 Examples of the form of the osteoclast differentiation / proliferation inhibitor of the present invention include dosage forms such as solution, powder, tablet, pill, fine granule, granule, capsule, jelly, chewable and paste. The effective intake of the compound (1) is preferably 0.01 to 5 mg, more preferably 0.05 to 0.5 mg per kg body weight.
以下、本発明の新規化合物及び破骨細胞分化・増殖阻害剤について具体的に説明する。 Hereinafter, the novel compound and osteoclast differentiation / proliferation inhibitor of the present invention will be described in detail.
なお、化合物の同定は、ESI/TOFMSスペクトル解析、赤外吸収（ＩＲ）スペクトル解析、核磁気共鳴（ＮＭＲ）スペクトル解析、ＤＥＰＴスペクトル解析、１Ｈ−１Ｈ ＣＯＳＹスペクトル解析、ＨＭＱＣスペクトル解析、ＨＭＢＣスペクトル解析にて行った。 For identification of the compounds, ESI / TOFMS spectrum analysis, infrared absorption (IR) spectrum analysis, nuclear magnetic resonance (NMR) spectral analysis, DEPT spectrum analysis, 1 H- 1 H COZY spectrum analysis, HMQC spectrum analysis, HMBC spectrum Analysis was performed.
ESI/TOFMSスペクトル解析は、ポジティブイオンモードで行った。 ESI / TOFMS spectral analysis was performed in positive ion mode.
赤外吸収（ＩＲ）スペクトル解析は、ＫＢｒ法で行った。 Infrared absorption (IR) spectrum analysis was performed by the KBr method.
核磁気共鳴（ＮＭＲ）スペクトル解析は、１Ｈ−ＮＭＲは５００ＭＨｚ、１３Ｃ−ＮＭＲは１２５ＭＨｚ、溶媒は重クロロホルムの条件で行った。 Nuclear magnetic resonance (NMR) spectrum analysis was performed under the conditions of 500 MHz for 1 H-NMR, 125 MHz for 13 C-NMR, and deuterated chloroform.
<実施例１>
［新規化合物］
マコモタケを葉部と菌えい部に切り分け、菌えい部(30.6 kg)を粉砕機で粉砕し、85%EtOH、アセトンで順次抽出を行った。このようにして得られた抽出物をCHCl3とEtOAcと水で溶媒分画し、CHCl3可溶部、EtOAc可溶部、水可溶部を得た。得られた画分の一部を破骨細胞形成阻害試験に供したところCHCl3可溶部、EtOAc可溶部に活性が確認された。そこでEtOAc可溶部7.2 gをフラッシュカラムクロマトグラフィー(silica gel 60 N (350 g),φ4 cm×60 cm)に供し、CH2Cl2:アセトン =10:0, 9:1, 6:4, 2:8, 0:10,100% MeOHの溶媒で順次段階的に溶出し、溶出画分MAK-E1〜MAK-E26を得た。これらを後述する破骨細胞形成阻害試験に供したところMAK-E3,4,9,11,13,24に活性が確認された。これらのうちMAK-E4 (135.8 mg)をフラッシュカラムクロマトグラフィー(silica gel 60 N, (200 g), φ2.5 cm×60 cm)に供し、ヘキサン:CH2Cl2=6:4,100%CH2Cl2,CH2Cl2:MeOH=1:1,100%MeOHの溶媒で順次段階的に溶出し、溶出画分MAK-E4-1〜MAK-E4-7を得た。そのMAK-E4-6(16.3 mg)を分取TLC(silica gel 60 F254,20×20 cm)に供し、ヘキサン:CH2Cl2=6:4で展開し、展開スポット画分MAK-E4-6-1〜MAK-E4-6-10を得た。また、MAK-E-4-7(76.7 mg)を分取TLC(silica gel 60 F254,20×20 cm)に供し、ヘキサン:CH2Cl2=6:4で展開し、展開スポット画分MAK-E4-7-1〜MAK-E4-7-10を得た。各画分をTLC及びNMRで比較したところ、MAK-E4-6-3,4、及びMAK-E4-7-3,4,5は類似した画分であったため、あわせてMAK-E4-7-1’とした。同様にMAK-E4-6-5,6、及びMAK-E4-7-6,7をあわせてMAK-E4-7-2’とし、MAK-E4-6-7,8,9、及びMAK-E4-7-8,9をあわせてMAK-E4-7-3’とした。MAK-E4-7-1'(10.2 mg)をSep pak(silica gel)に供し、得られた溶出部を順相カラムを用いたHPLC(Senshu pak AQUASIL SS-5251)に供し、ヘキサン:CHCl3=6:4で溶出させ、溶出画分MAK-E4-7-1’-1〜MAK-E4-7-1’-13を得た。その溶出画分MAK-E4-7-1’-2(5.3 mg)をsep pak(silica gel)に供し100%ヘキサンで溶出させ非吸着部と吸着部を得た。しかし得られた画分は量的に少なく化学構造同定のための分析が困難であったため、再度、マコモタケ菌えい部から抽出を行った。
<Example 1>
[New compound]
Makomotake was cut into leaves and fungal parts, and the fungal parts (30.6 kg) were pulverized with a pulverizer and extracted sequentially with 85% EtOH and acetone. The extract thus obtained was fractionated with CHCl 3 , EtOAc and water to obtain a CHCl 3 soluble part, an EtOAc soluble part and a water soluble part. When a part of the obtained fraction was subjected to an osteoclast formation inhibition test, activity was confirmed in the CHCl 3 soluble part and the EtOAc soluble part. Therefore, 7.2 g of the EtOAc soluble part was subjected to flash column chromatography (silica gel 60 N (350 g), φ4 cm × 60 cm), and CH 2 Cl 2 : acetone = 10: 0, 9: 1, 6: 4, Elution was performed stepwise with a solvent of 2: 8, 0:10, 100% MeOH to obtain elution fractions MAK-E1 to MAK-E26. When these were subjected to the osteoclast formation inhibition test described later, the activity was confirmed in MAK-E3,4,9,11,13,24. Of these, MAK-E4 (135.8 mg) was subjected to flash column chromatography (silica gel 60 N, (200 g), φ2.5 cm × 60 cm), hexane: CH 2 Cl 2 = 6: 4,100% CH 2 Elution was performed stepwise with a solvent of Cl 2 , CH 2 Cl 2 : MeOH = 1: 1, 100% MeOH to obtain elution fractions MAK-E4-1 to MAK-E4-7. The MAK-E4-6 (16.3 mg) was subjected to preparative TLC (silica gel 60 F 254 , 20 × 20 cm) and developed with hexane: CH 2 Cl 2 = 6: 4, and the developed spot fraction MAK-E4 -6-1 to MAK-E4-6-10 were obtained. In addition, MAK-E-4-7 (76.7 mg) was subjected to preparative TLC (silica gel 60 F 254 , 20 x 20 cm) and developed with hexane: CH 2 Cl 2 = 6: 4, MAK-E4-7-1 to MAK-E4-7-10 were obtained. When each fraction was compared by TLC and NMR, MAK-E4-6-3,4 and MAK-E4-7-3,4,5 were similar fractions. -1 '. Similarly, MAK-E4-6-5,6 and MAK-E4-7-6,7 are combined into MAK-E4-7-2 ', MAK-E4-6-7,8,9, and MAK- E4-7-8 and 9 were combined into MAK-E4-7-3 '. MAK-E4-7-1 '(10.2 mg) was subjected to Sep pak (silica gel), and the obtained elution part was subjected to HPLC (Senshu pak AQUASIL SS-5251) using a normal phase column, hexane: CHCl3 = Elution was performed at 6: 4 to obtain elution fractions MAK-E4-7-1′-1 to MAK-E4-7-1′-13. The eluted fraction MAK-E4-7-1'-2 (5.3 mg) was subjected to sep pak (silica gel) and eluted with 100% hexane to obtain a non-adsorbed part and an adsorbed part. However, since the obtained fraction was small in quantity and difficult to analyze for chemical structure identification, it was extracted again from the cervix of the genus Momotake.
すなわち、上記MAK-E4-7-1’-2画分を指標にして、以下のようにして破骨細胞形成阻害活性物質を単離した。 That is, the osteoclast formation inhibitory active substance was isolated as described below using the MAK-E4-7-1'-2 fraction as an index.
菌えい部(39.0 kg)を粉砕機で粉砕し、85%EtOH、アセトンで順次抽出を行った。得られた抽出物からヘキサン可溶部(40.0 g)を抽出し、上記と同様にしてフラッシュカラムクロマトグラフィー(silica gel 60 N, (350 g),φ4cm×60cm)に供し、溶出画分MAK-H1〜MAK-H26を得た。各画分について上記MAK-E4-7-1’-2を指標にしてTLC及びNMRで比較したところ、MAK-H6〜MAK-H10は類似した画分であったため、あわせてMAK-H6(107.0 mg)とした。これを分取TLC(silica gel 60 F254,20×20 cm)に供し、ヘキサン:CH2Cl2=6:4で展開し、展開スポット画分MAK-H6-1〜MAK-H6-12を得た。各画分をTLC及びNMRで比較したところ、MAK-H6-6,7,8,9,10は類似した画分であったため、あわせてMAK-H6-6-2’とした。MAK-H6-6-2’(25.9 mg)をSep pak(silica gel)に供し、得られた溶出部を順相カラムを用いたHPLC(Senshu pak AQUASIL SS-5251) に供し100%ヘキサンで溶出させ、溶出画分MAK-H6-6-2’-1〜MAK-H6-6-2’-40を得た。このうちTLC、NMRを指標にMAK-H6-6-2’-27,28,29,30,31,32,33をあわせてMAK-H6-6-2’-27’とし、MAK-H6-6-2’-34,35,36,37,38,39,40をあわせてMAK-H6-6-2’-34’とした。MAK-H6-6-2’-27’を先と同じ条件でHPLCに供し、溶出画分MAK-H6-6-27’-1〜MAK-H6-6-27’-20を得たが、この段階で量が少なくなったため、再度MAK-H6(106.0 mg)を上に示したものと同じ条件で分取TLCに供し、５つの展開スポット画分MAK-H6-1(2)〜MAK-H6-5(2)を得た。このうちMAK-H6-4(2)(58.0 mg)を先と同じ条件でSep pak(silica gel)、及び順相カラムHPLC(Senshu pak AQUASIL SS-5251) に供し、溶出画分MAK-H6-4(2)-1〜MAK-H6-4(2)-14を得た。その溶出画分MAK-H6-4(2)-10,11と、上記MAK-H6-6-2’-34’とをあわせたMAK-H6-6-2’-34”(24.0 mg)を疎水性フィルターに供し、２−プロパノールで溶出させた。このうち２−プロパノール不溶部から下記式（１）で表わされる新規化合物（化合物（１））(2.1 mg)が単離された。 The fungus cervix (39.0 kg) was pulverized with a pulverizer and extracted successively with 85% EtOH and acetone. A hexane soluble part (40.0 g) was extracted from the obtained extract and subjected to flash column chromatography (silica gel 60 N, (350 g), φ4 cm × 60 cm) in the same manner as above, and the eluted fraction MAK- H1 to MAK-H26 were obtained. When each fraction was compared by TLC and NMR using the above MAK-E4-7-1′-2 as an index, MAK-H6 to MAK-H10 were similar fractions, and therefore MAK-H6 (107.0 mg). This was subjected to preparative TLC (silica gel 60 F 254 , 20 × 20 cm), developed with hexane: CH 2 Cl 2 = 6: 4, and developed spot fractions MAK-H6-1 to MAK-H6-12 were obtained. Obtained. When each fraction was compared by TLC and NMR, MAK-H6-6,7,8,9,10 were similar fractions, so they were collectively referred to as MAK-H6-6-2 '. MAK-H6-6-2 '(25.9 mg) was applied to Sep pak (silica gel), and the resulting elution part was subjected to HPLC (Senshu pak AQUASIL SS-5251) using a normal phase column and eluted with 100% hexane. Elution fractions MAK-H6-6-2′-1 to MAK-H6-6-2′-40 were obtained. Of these, MAK-H6-6-2'-27,28,29,30,31,32,33 were combined into MAK-H6-6-2'-27 'using TLC and NMR as indicators, and MAK-H6- 6-2'-34,35,36,37,38,39,40 was combined into MAK-H6-6-2'-34 '. MAK-H6-6-2'-27 'was subjected to HPLC under the same conditions as above to obtain elution fractions MAK-H6-6-27'-1 to MAK-H6-6-27'-20. Since the amount decreased at this stage, MAK-H6 (106.0 mg) was again subjected to preparative TLC under the same conditions as shown above, and five developed spot fractions MAK-H6-1 (2) to MAK- H6-5 (2) was obtained. Of these, MAK-H6-4 (2) (58.0 mg) was subjected to Sep pak (silica gel) and normal phase column HPLC (Senshu pak AQUASIL SS-5251) under the same conditions as above, and the eluted fraction MAK-H6- 4 (2) -1 to MAK-H6-4 (2) -14 were obtained. MAK-H6-6-2'-34 ”(24.0 mg) of the elution fraction MAK-H6-4 (2) -10,11 and the above MAK-H6-6-2'-34 ' It was subjected to a hydrophobic filter and eluted with 2-propanol, from which a novel compound (compound (1)) (2.1 mg) represented by the following formula (1) was isolated from the 2-propanol insoluble part.
以下には、上記化合物（１）の同定の結果について示す。 Below, it shows about the result of identification of the said compound (1).
図１に示すように、化合物（１）はESI/TOFMSにおいてm/z687に[M+Na]+,の分子イオンピークを示したため、分子量664、分子式C46H80O2である化合物であることが明らかとなった。また、図８に示すように、IRスペクトルにおいて1740 cm-1に吸収が見られたため、カルボキシル基の存在が明らかとなった。1H-NMRスペクトルはステロイドに特徴的なピークを示していたが(図３)、ESI/TOFMSより決定した炭素原子の数は４６もあることからこの化合物はステロイドの脂肪酸エステルであることが示唆された。図４に示す13C-NMRスペクトルにはカルボキシル基由来のδ173.5のピークが観測された。また、図５に示すDEPTスペクトルより５個の四級炭素の存在が示唆された。図６に示すHMQCスペクトルの相関からCとそれに結合しているプロトンの帰属を明らかにすることができた。図７に示すCOSYスペクトルからH-20とH-21、H-22とH-23に相関が確認された。図８に示すHMBCスペクトルから、H-3とC-1’、H-3’とC-2’,3’、H-18’とC-16’,17’、H-1とC-3,5、H-2とC-3、H-4とC-3,5、H-6とC-8、H-7とC-9,14、H-11とC-9、H-12とC-9,13、H-14とC-12、H-15とC-14、H-16とC-13、H-17とC-13、H-18とC-12,13,14,17、H-19とC-1,5,9,10、H-20とC-17、H-21とC-17,20,22、H-22とC-17、H-26とC-23,24,25、H-27,28とC-24,25に相関が確認され、ステロイドの側鎖にあたる部分構造が確認された。また水酸基に結合するより低磁場シフトしたH-3とC-1’、H-3’とC-2’,3’、H-18’とC-16’,17’に相関が確認され、エステル結合した脂肪酸にあたる部分構造が確認された。 As shown in FIG. 1, compound (1) showed a molecular ion peak of [M + Na] +, at m / z 687 in ESI / TOFMS, and thus has a molecular weight of 664 and a molecular formula of C 46 H 80 O 2. It became clear. Further, as shown in FIG. 8, since absorption was observed at 1740 cm −1 in the IR spectrum, the presence of a carboxyl group was clarified. The 1 H-NMR spectrum showed a characteristic peak for steroids (Fig. 3), but the number of carbon atoms determined by ESI / TOFMS was 46, suggesting that this compound is a fatty acid ester of steroids. It was done. In the 13 C-NMR spectrum shown in FIG. 4, a peak of δ173.5 derived from a carboxyl group was observed. The presence of five quaternary carbons was suggested from the DEPT spectrum shown in FIG. From the correlation of the HMQC spectrum shown in FIG. 6, the assignment of C and the proton bonded thereto could be clarified. From the COZY spectrum shown in FIG. 7, correlations were confirmed between H-20 and H-21, and H-22 and H-23. From the HMBC spectrum shown in FIG. 8, H-3 and C-1 ′, H-3 ′ and C-2 ′, 3 ′, H-18 ′ and C-16 ′, 17 ′, H-1 and C-3 , 5, H-2 and C-3, H-4 and C-3, 5, H-6 and C-8, H-7 and C-9, 14, H-11 and C-9, H-12 And C-9,13, H-14 and C-12, H-15 and C-14, H-16 and C-13, H-17 and C-13, H-18 and C-12,13,14 , 17, H-19 and C-1,5,9,10, H-20 and C-17, H-21 and C-17,20,22, H-22 and C-17, H-26 and C Correlation between -23,24,25, H-27,28 and C-24,25 was confirmed, and a partial structure corresponding to the side chain of the steroid was confirmed. In addition, the correlation between H-3 and C-1 ′, H-3 ′ and C-2 ′, 3 ′, H-18 ′ and C-16 ′, 17 ′, which are shifted to a lower magnetic field than the hydroxyl group, was confirmed. A partial structure corresponding to an ester-linked fatty acid was confirmed.
以上より、マコモタケ菌えい部から得られた新規化合物を、上記式（１）で表わされるepisterol stearate（ステロイドのステアリン酸エステル）であると決定した。 Based on the above, it was determined that the novel compound obtained from the cervix of the mushroom was Episterol stearate (steroid stearate) represented by the above formula (1).
<試験例１>
［破骨細胞の分化・増殖阻害活性評価］
上記化合物（１）による破骨細胞の分化・増殖阻害活性を評価した。破骨細胞形成阻害活性試験はマウス由来の骨髄細胞と骨芽細胞様間質細胞の共存培養法を用いた。破骨細胞形成阻害活性は破骨細胞(TRAP陽性多核細胞)の形成した数をカウントして評価した。
<Test Example 1>
[Evaluation of osteoclast differentiation / proliferation inhibitory activity]
The osteoclast differentiation / proliferation inhibitory activity of the compound (1) was evaluated. The osteoclast formation inhibitory activity test used a co-culture method of mouse-derived bone marrow cells and osteoblast-like stromal cells. Osteoclast formation inhibitory activity was evaluated by counting the number of osteoclasts (TRAP positive multinucleated cells) formed.
・共存培養
マウス(雌 5〜7週齢)の脛骨，大腿骨から採取した骨髄細胞 1.3×108 cellと、 頭蓋骨から採取し培養した骨芽細胞様間質細胞 1×106 cellを培養液(10%FBS含有α-MEM)7.2 mlに懸濁し、48穴プレートに150μl /wellで分注した。
・ Cultivated culture Bone marrow cells collected from tibia and femur of mice (female 5-7 weeks old) 1.3 × 10 8 cells and osteoblast-like stromal cells 1 × 10 6 cells collected from the skull and cultured The suspension was suspended in 7.2 ml of (10% FBS-containing α-MEM), and dispensed into a 48-well plate at 150 μl / well.
上記化合物（１）のサンプル溶液としては、実施例１で得られたものを100 mg/mlとなるようにDMSO及びヘキサンに溶解し、さらに培養液で希釈し希釈系列を作製した。各濃度のサンプル溶液50 μlを1,25(OH)2VD3(20 ng/ml) 50 μlと共に培養液に添加し全量を250 μl/wellとした。CO2インキュベータを用いて培養 (37℃、CO2濃度5.0%)し、培養開始3日目にはサンプルを追加するため各wellの上澄み液100 μlを除去し、初日の添加濃度の２倍のサンプル溶液と1,25(OH)2D3(40 ng/ml) を各50 μl添加し、再び培養した。 As a sample solution of the above compound (1), the solution obtained in Example 1 was dissolved in DMSO and hexane so as to be 100 mg / ml, and further diluted with a culture solution to prepare a dilution series. 50 μl of sample solution of each concentration was added to the culture solution together with 50 μl of 1,25 (OH) 2 VD 3 (20 ng / ml) to make a total volume of 250 μl / well. Cultivate in a CO 2 incubator (37 ° C, CO 2 concentration 5.0%). On the third day of culture, remove 100 μl of the supernatant from each well to add the sample. The sample solution and 50 μl each of 1,25 (OH) 2 D 3 (40 ng / ml) were added and cultured again.
・TRAP陽性多核細胞のカウント
1週間程度培養した後、培養液を除去し、PBSで洗浄後10%ホルマリン含有PBS溶液を0.5 ml/well加え10分間固定した。次にEtOHを0.5 ml/well添加し、1分間再固定した。その後乾燥させ、TRAP反応液を0.3 ml/well加え、室温で30分間染色した。染色後は蒸留水（0.5 ml/well）で洗浄し、乾燥した。TRAP陽性であり、核を2個以上有する細胞をTRAP陽性多核細胞として、1 well当たりの個数をカウントした。なお、TRAP反応液は、Naphthol as-mx phosphate 1.5 mgを150 mlのN,N-dimethylformaldehydeに溶解し、これに、fast red violet lb salt 9 mgを15 mlのbuffer（50 mM 酒石酸Naを含む0.1 M酢酸Na緩衝液pH5.0）に添加した溶液を混合し、調製した。
・ Count of TRAP-positive multinucleated cells
After culturing for about 1 week, the culture solution was removed, washed with PBS, and then added with 10% formalin-containing PBS solution at 0.5 ml / well and fixed for 10 minutes. Next, EtOH was added at 0.5 ml / well and re-fixed for 1 minute. Thereafter, it was dried, 0.3 ml / well of TRAP reaction solution was added, and staining was performed at room temperature for 30 minutes. After staining, it was washed with distilled water (0.5 ml / well) and dried. Cells that were TRAP-positive and had 2 or more nuclei were counted as TRAP-positive multinucleated cells, and the number per well was counted. The TRAP reaction solution was prepared by dissolving 1.5 mg of Naphthol as-mx phosphate in 150 ml of N, N-dimethylformaldehyde, and adding 15 mg of fast red violet lb salt to 15 ml of buffer (0.1% containing 50 mM Na tartrate). The solution added to M Na acetate buffer (pH 5.0) was mixed and prepared.
・MTT assay
上記共存培養後の生細胞数をMTT assayで確認した。具体的には、共存培養後の培養液を除去せず、1 mg/mlのMTT試薬を125 μl/wellで添加した。次にCO2インキュベータを用いて37℃、CO2濃度5.0%の培養条件で2時間培養した後、培養液を除去し、dimethyl sulfoxide 100 μl/well加え吸光度 (λ=570 nm) を測定した。なお、MTT試薬は、3-(4,5-dimethyl-2-thiazoly)-2,5-diphenyl-2H-tertazolium bromideを1 mg/mlになるよう水に溶解して調製した。
・ MTT assay
The number of viable cells after the co-culture was confirmed by MTT assay. Specifically, without removing the culture solution after co-culture, 1 mg / ml MTT reagent was added at 125 μl / well. Next, after culturing at 37 ° C. under a CO 2 concentration of 5.0% for 2 hours using a CO 2 incubator, the culture solution was removed, 100 μl / well of dimethyl sulfoxide was added, and the absorbance (λ = 570 nm) was measured. The MTT reagent was prepared by dissolving 3- (4,5-dimethyl-2-thiazoly) -2,5-diphenyl-2H-tertazolium bromide in water to 1 mg / ml.
結果を図９にまとめて示す。図９に明らかなとおり、上記化合物（１）は、５０μｇ／ｍｌの濃度で細胞生存率に影響を与えず、破骨細胞の形成を有意に抑制した。 The results are summarized in FIG. As apparent from FIG. 9, the compound (1) did not affect the cell viability at a concentration of 50 μg / ml, and significantly suppressed the formation of osteoclasts.
<製造例１>
表１に示す配合で調合した組成物を常法にしたがってハードカプセルに２３０ｍｇ／カプセルで充填して、ハードカプセル食品を製造した。魚骨カルシウムは商品名「焼成ボニカル」（焼津水産化学工業株式会社製）を使用した。
<Production example 1>
According to a conventional method, the hard capsules were filled with 230 mg / capsule according to a conventional method to produce a hard capsule food. The fish bone calcium used the brand name "baking Bonical" (made by Yaizu Suisan Chemical Co., Ltd.).
<製造例２>
表２に示す配合で常法にしたがってゼリー飲料を製造した。このゼリー飲料はマコモタケの風味を感じることなく、また食感にも優れ、非常に飲み易かった。
<Production example 2>
A jelly beverage was produced according to a conventional method with the formulation shown in Table 2. This jelly beverage was very easy to drink without feeling the flavor of makomotake and excellent in texture.
<製造例３>
表３に示す配合で常法にしたがって乳飲料を製造した。この乳飲料は沈殿を生じることなく、またマコモタケの風味を感じることもなく、非常に飲み易かった。
<Production Example 3>
A milk beverage was produced according to a conventional method with the formulation shown in Table 3. This milk drink was very easy to drink without causing precipitation and without the taste of makomotake.
本発明の新規化合物は、飲食品、医薬品、化粧品等の原料として用いることができる。 The novel compound of the present invention can be used as a raw material for foods and drinks, pharmaceuticals, cosmetics and the like.
また、本発明の破骨細胞分化・増殖阻害剤は、そのまま、或いは飲食品、医薬品、化粧品等に配合して摂取することにより、骨粗鬆症の予防・治療改善に用いることができる。 In addition, the osteoclast differentiation / proliferation inhibitor of the present invention can be used for the prevention / treatment improvement of osteoporosis by ingesting it as it is or in combination with foods, beverages, pharmaceuticals, cosmetics and the like.
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