JP2020125293A - Osteoclast differentiation inhibitor and postmenopausal osteoporosis preventive/improver - Google Patents
Osteoclast differentiation inhibitor and postmenopausal osteoporosis preventive/improver Download PDFInfo
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- JP2020125293A JP2020125293A JP2020016793A JP2020016793A JP2020125293A JP 2020125293 A JP2020125293 A JP 2020125293A JP 2020016793 A JP2020016793 A JP 2020016793A JP 2020016793 A JP2020016793 A JP 2020016793A JP 2020125293 A JP2020125293 A JP 2020125293A
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- decenoic acid
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Abstract
Description
本発明は１０−ヒドロキシ−２−デセン酸を有効成分とする破骨細胞分化抑制剤および閉経後骨粗鬆症予防・改善剤に関する。 The present invention relates to an osteoclast differentiation inhibitor and a postmenopausal osteoporosis-preventing/ameliorating agent containing 10-hydroxy-2-decenoic acid as an active ingredient.
骨は体を支える重要な器官であり、生命維持に必要なカルシウムなどの代謝器官でもある。骨は破骨細胞による骨の破壊と骨芽細胞による骨の形成のバランスにより恒常性が保たれており、そのバランスの崩壊が様々な骨の疾患の原因となる。骨の疾患に骨粗鬆症がある。骨粗鬆症は特発性骨粗鬆症、閉経後骨粗鬆症、退行性または老人性骨粗鬆症、続発性骨粗鬆症などに分類される。特発性骨粗鬆症は体内のホルモンやビタミンの量は正常で明らかな原因はないにもかかわらず、小児期や青年期に発症する。閉経後骨粗鬆症は、エストロゲンが減少し破骨細胞前駆体の動員および反応亢進をまねき、結果として骨吸収が増大する。退行性または老人性骨粗鬆症は、骨芽細胞の数の減少や活性低下と関連し、破骨細胞の活性亢進とは一次的に関連していない。カルシウムの吸収に必要なビタミンＤ合成の減少などが原因と考えられる。続発性骨粗鬆症は骨粗鬆症全体の５％未満とされており、その原因としては内分泌性疾患、薬物誘導などが挙げられる。 Bone is an important organ that supports the body and is also a metabolic organ such as calcium that is necessary for life support. Bone maintains homeostasis by the balance between bone destruction by osteoclasts and bone formation by osteoblasts, and disruption of that balance causes various bone diseases. Osteoporosis is a bone disease. Osteoporosis is classified into idiopathic osteoporosis, postmenopausal osteoporosis, degenerative or senile osteoporosis, secondary osteoporosis and the like. Idiopathic osteoporosis occurs in childhood and adolescence, although the amount of hormones and vitamins in the body is normal and has no apparent cause. Postmenopausal osteoporosis is a decrease in estrogen, leading to the recruitment and hyperactivity of osteoclast precursors, resulting in increased bone resorption. Degenerative or senile osteoporosis is associated with a decreased number of osteoblasts and decreased activity, and not primarily with increased osteoclast activity. It is considered that the cause is a decrease in vitamin D synthesis necessary for calcium absorption. Secondary osteoporosis accounts for less than 5% of all osteoporosis, and its causes include endocrine diseases and drug induction.
特に閉経後骨粗鬆症は高齢の女性に多い。破骨細胞前駆体の動員および反応亢進を招き、結果として骨吸収が増大する。 Postmenopausal osteoporosis is particularly common in older women. This leads to the recruitment and hyperreactivity of osteoclast precursors, resulting in increased bone resorption.
骨粗鬆症の予防、治療法としてはカルシウム、活性型ビタミンDの摂取、選択的エストロゲン受容体調整薬、ビスホスホネート、抗ＲＡＮＫＬ（Receptor activator of NF-κB ligand）抗体などの投与などが行われている。 As a method for preventing and treating osteoporosis, ingestion of calcium, activated vitamin D, selective estrogen receptor modulator, bisphosphonate, anti-RANKL (Receptor activator of NF-κB ligand) antibody, etc. are administered.
さらに、ローヤルゼリーが骨芽細胞の分化マーカーであるプロコラーゲン１α１遺伝子の発現を高め、骨形成を促進させる作用も報告されている（特許文献１）。
また、ローヤルゼリーに含まれる１０−ヒドロキシ−２−デセン酸にエストロゲン作用を有することが知られている（特許文献２）。エストロゲン欠乏による骨粗鬆症の原因として、骨細胞などに由来するRANKLやOPG（オステオプロテゲリン）、Sema3Aがエストロゲンによって発現調節され、それらの適切な発現は骨量維持に関与するものと考えられるのに対し（非特許文献１）、本発明は１０−ヒドロキシ−２−デセン酸が破骨細胞のFFAR4に直接的に結合することで、NF-κBシグナルの活性化が抑制されることによるもので作用機序はエストロゲン作用とは異なっている。
Furthermore, it has been reported that royal jelly enhances the expression of procollagen 1α1 gene, which is a differentiation marker of osteoblasts, and promotes bone formation (Patent Document 1).
Further, it is known that 10-hydroxy-2-decenoic acid contained in royal jelly has an estrogenic effect (Patent Document 2). As a cause of osteoporosis due to estrogen deficiency, RANKL, OPG (osteoprotegerin) and Sema3A derived from bone cells are regulated by estrogen, and their proper expression is considered to be involved in bone mass maintenance. (Non-patent document 1), the present invention is based on the fact that 10-hydroxy-2-decenoic acid directly binds to FFAR4 of osteoclasts to suppress the activation of NF-κB signal. The order is different from estrogen action.
本発明は、１０−ヒドロキシ−２−デセン酸を有効成分とする、破骨細胞分化の抑制剤、閉経後骨粗鬆症予防・改善剤を提供することにある。 The present invention is to provide an inhibitor of osteoclast differentiation and a preventive/ameliorating agent for postmenopausal osteoporosis, which comprises 10-hydroxy-2-decenoic acid as an active ingredient.
本発明者らは、鋭意研究の結果、１０−ヒドロキシ−２−デセン酸に破骨細胞分化を抑制する作用を見出し、本発明を完成するのに至った。
すなわち、本発明は以下からなる：
１． １０−ヒドロキシ−２−デセン酸を有効成分とする破骨細胞前駆体から破骨細胞への分化抑制剤。
２． １０−ヒドロキシ−２−デセン酸を有効成分とする閉経後骨粗鬆症の予防・改善剤。
３． １０−ヒドロキシ−２−デセン酸を有効成分とし、破骨細胞前駆体から破骨細胞への分化抑制作用を有するものであることを特徴とする、破骨細胞前駆体から破骨細胞への分化抑制用食品組成物。
４． １０−ヒドロキシ−２−デセン酸を有効成分とし、閉経後骨粗鬆症の予防・改善作用を有するものであることを特徴とする、閉経後骨粗鬆症の予防・改善用食品組成物。
５． １０−ヒドロキシ−２−デセン酸を有効成分とし、１０−ヒドロキシ−２−デセン酸が破骨細胞のFFAR4に直接的に結合することで、NF-κBシグナルの活性化が抑制されることを特徴とする、破骨細胞前駆体から破骨細胞への分化抑制剤。
６． １０−ヒドロキシ−２−デセン酸を有効成分とし、１０−ヒドロキシ−２−デセン酸が破骨細胞のFFAR4に直接的に結合することで、NF-κBシグナルの活性化が抑制されることを特徴とする、閉経後骨粗鬆症の予防・改善剤。
７． １０−ヒドロキシ−２−デセン酸を有効成分とし、１０−ヒドロキシ−２−デセン酸が破骨細胞のFFAR4に直接的に結合することで、NF-κBシグナルの活性化が抑制され、破骨細胞前駆体から破骨細胞への分化抑制作用を有するものであることを特徴とする、破骨細胞前駆体から破骨細胞への分化抑制用食品組成物。
８． １０−ヒドロキシ−２−デセン酸を有効成分とし、１０−ヒドロキシ−２−デセン酸が破骨細胞のFFAR4に直接的に結合することで、NF-κBシグナルの活性化が抑制され、閉経後骨粗鬆症の予防・改善作用を有するものであることを特徴とする、閉経後骨粗鬆症の予防・改善用食品組成物。
As a result of intensive research, the present inventors have found that 10-hydroxy-2-decenoic acid has an action of suppressing osteoclast differentiation, and completed the present invention.
That is, the present invention consists of the following:
1. An agent for suppressing differentiation of an osteoclast precursor into an osteoclast, which comprises 10-hydroxy-2-decenoic acid as an active ingredient.
2. A preventive/ameliorating agent for postmenopausal osteoporosis, which comprises 10-hydroxy-2-decenoic acid as an active ingredient.
3. Differentiation of osteoclast precursors to osteoclasts, which comprises 10-hydroxy-2-decenoic acid as an active ingredient and has an action of suppressing differentiation of osteoclast precursors to osteoclasts. Suppressive food composition.
4. A food composition for preventing/ameliorating postmenopausal osteoporosis, which comprises 10-hydroxy-2-decenoic acid as an active ingredient and has a preventive/ameliorating action on postmenopausal osteoporosis.
5. 10-Hydroxy-2-decenoic acid as an active ingredient, 10-hydroxy-2-decenoic acid is directly bound to FFAR4 of osteoclasts, and the activation of NF-κB signal is suppressed. An agent for suppressing differentiation of an osteoclast precursor into an osteoclast.
6. 10-Hydroxy-2-decenoic acid as an active ingredient, 10-hydroxy-2-decenoic acid is directly bound to FFAR4 of osteoclasts, and the activation of NF-κB signal is suppressed. A preventive/ameliorating agent for postmenopausal osteoporosis.
7. By using 10-hydroxy-2-decenoic acid as an active ingredient and directly binding 10-hydroxy-2-decenoic acid to FFAR4 of osteoclasts, activation of NF-κB signal is suppressed, and osteoclasts are suppressed. A food composition for suppressing the differentiation of osteoclast precursors into osteoclasts, which has an effect of suppressing the differentiation of precursors into osteoclasts.
8. By using 10-hydroxy-2-decenoic acid as an active ingredient and directly binding 10-hydroxy-2-decenoic acid to FFAR4 of osteoclasts, activation of NF-κB signal is suppressed and postmenopausal osteoporosis A food composition for preventing/ameliorating postmenopausal osteoporosis, which has a preventive/ameliorating effect on
本発明によると、破骨細胞分化を抑制することにより、破骨細胞分化抑制剤、さらには閉経後骨粗鬆症予防・改善剤を提供することができる。 According to the present invention, it is possible to provide an osteoclast differentiation inhibitor, and further a postmenopausal osteoporosis preventive/ameliorating agent by suppressing osteoclast differentiation.
本発明において、破骨細胞分化を抑制する関与成分として１０−ヒドロキシ−２−デセン酸を見出した。さらに、閉経後骨粗鬆症は、破骨細胞前駆体の動員および反応亢進をまねき、結果として骨吸収が増大することで引き起こされる。そこで、１０−ヒドロキシ−２−デセン酸が破骨細胞前駆体から破骨細胞への分化を抑制することで、閉経後骨粗鬆症の予防・改善剤としても用いることができる。 In the present invention, 10-hydroxy-2-decenoic acid was found as a component involved in suppressing osteoclast differentiation. In addition, postmenopausal osteoporosis is caused by increased recruitment and hyperreactivity of osteoclast precursors, resulting in increased bone resorption. Therefore, 10-hydroxy-2-decenoic acid can be used as a prophylactic/ameliorating agent for postmenopausal osteoporosis by suppressing the differentiation of osteoclast precursors into osteoclasts.
本発明の一態様は、１０−ヒドロキシ−２−デセン酸を有効成分とする破骨細胞前駆体から破骨細胞への分化抑制剤に関する。本明細書において、破骨細胞前駆体から破骨細胞への分化抑制を単に破骨細胞分化抑制ともいう。
本発明の別の態様は、１０−ヒドロキシ−２−デセン酸を有効成分とする閉経後骨粗鬆症の予防・改善剤に関する。
本発明の一態様は、食品組成物に関し、１０−ヒドロキシ−２−デセン酸を有効成分とし、破骨細胞前駆体から破骨細胞への分化抑制作用を有するものであることを特徴とする、破骨細胞前駆体から破骨細胞への分化抑制用食品組成物に関する。また、１０−ヒドロキシ−２−デセン酸を有効成分とし、閉経後骨粗鬆症の予防・改善作用を有するものであることを特徴とする、閉経後骨粗鬆症の予防・改善用食品組成物に関する。
One aspect of the present invention relates to an agent for suppressing differentiation of an osteoclast precursor into an osteoclast, which contains 10-hydroxy-2-decenoic acid as an active ingredient. In the present specification, suppression of osteoclast differentiation from osteoclast precursors is also referred to simply as osteoclast differentiation suppression.
Another aspect of the present invention relates to a prophylactic/ameliorating agent for postmenopausal osteoporosis, which comprises 10-hydroxy-2-decenoic acid as an active ingredient.
One aspect of the present invention relates to a food composition, which is characterized by containing 10-hydroxy-2-decenoic acid as an active ingredient and having an action of suppressing differentiation from an osteoclast precursor to an osteoclast. The present invention relates to a food composition for suppressing the differentiation of osteoclast precursors into osteoclasts. Further, the present invention relates to a food composition for preventing/ameliorating postmenopausal osteoporosis, which comprises 10-hydroxy-2-decenoic acid as an active ingredient and has a preventive/ameliorating action on postmenopausal osteoporosis.
本発明の剤は、有効成分である１０−ヒドロキシ−２−デセン酸を薬理学的に許容される一種もしくはそれ以上の担体と一緒に混合し、製剤学の技術分野においてよく知られている任意の方法により製造することができる。 The agent of the present invention is prepared by mixing 10-hydroxy-2-decenoic acid, which is an active ingredient, with one or more pharmaceutically acceptable carriers, and is well known in the technical field of pharmacy. It can be manufactured by the method of.
薬理学的に許容される担体としては、製剤素材として慣用の各種有機あるいは無機担体物質が用いられる。具体例としては、固形製剤における賦形剤、滑沢剤、結合剤、崩壊剤、液状製剤における溶剤、溶解補助剤、懸濁化剤、等張化剤、緩衝剤、無痛化剤などが挙げられる。製剤化の際には、必要に応じて、防腐剤、抗酸化剤、着色剤、甘味剤などの製剤添加剤を用いてもよい。 As the pharmacologically acceptable carrier, various organic or inorganic carrier substances that are commonly used as pharmaceutical materials can be used. Specific examples include excipients in solid preparations, lubricants, binders, disintegrants, solvents in liquid preparations, solubilizing agents, suspending agents, isotonic agents, buffering agents, soothing agents and the like. To be Upon formulation, formulation additives such as preservatives, antioxidants, coloring agents and sweeteners may be used if necessary.
薬理学的に許容される添加剤としては、例えば、ショ糖、デンプン、マンニトール、ソルビトール、乳糖、グルコース、セルロース、タルク、リン酸カルシウム、炭酸カルシウム等の賦形剤、セルロース、メチルセルロース、ヒドロキシプロピルセルロース、ポリプロピルピロリドン、ゼラチン、アラビアゴム、ポリエチレングリコール、ショ糖、デンプン等の結合剤、デンプン、カルメロース、ヒドロキシプロピルスターチ、ナトリウム-グリコール-スターチ、炭酸水素ナトリウム、リン酸カルシウム、クエン酸カルシウム等の崩壊剤、ステアリン酸マグネシウム、タルク、ラウリル硫酸ナトリウム等の滑沢剤、クエン酸、メントール、グリチルリチン酸二アンモニウム、グリシン、オレンジ油等の矯味剤、安息香酸ナトリウム、亜硫酸水素ナトリウム、メチルパラベン、プロピルパラベン等の保存剤、クエン酸、クエン酸ナトリウム、酢酸等の安定化剤、メチルセルロース、ポビドン、ステアリン酸アルミニウム等の懸濁剤、界面活性剤等の分散剤、水、生理食塩水、オレンジジュース等の希釈剤、カカオ脂、ポリエチレングリコール、白灯油等のベースワックスなどが挙げられるが、それらに限定されるものではない。 Examples of pharmacologically acceptable additives include sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, excipients such as calcium carbonate, cellulose, methyl cellulose, hydroxypropyl cellulose, poly Binders such as propylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, starch, carmellose, hydroxypropyl starch, sodium-glycol-starch, disintegrating agents such as sodium hydrogen carbonate, calcium phosphate, calcium citrate, stearic acid Lubricants such as magnesium, talc, sodium lauryl sulfate, citric acid, menthol, diammonium glycyrrhizinate, flavoring agents such as glycine and orange oil, preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben, and citric acid. Acids, sodium citrate, stabilizers such as acetic acid, methyl cellulose, povidone, suspension agents such as aluminum stearate, dispersants such as surfactants, water, physiological saline, diluents such as orange juice, cocoa butter, Examples thereof include base waxes such as polyethylene glycol and white kerosene, but are not limited thereto.
本発明の剤の投与対象は、通常哺乳動物である。哺乳動物としては、例えば、マウス、ラット、ハムスター、モルモット等のげっ歯類やウサギ等の実験動物、ブタ、ウシ、ヤギ、ウマ、ヒツジ、ミンク等の家畜、イヌ、ネコ等のペット、ヒト、サル、カニクイザル、アカゲザル、マーモセット、オランウータン、チンパンジーなどの霊長類等を挙げることが出来るが、これらに限定されるものではない。哺乳動物は、好ましくは霊長類（ヒト等）又はげっ歯類（マウス等）である。 The administration target of the agent of the present invention is usually a mammal. Examples of mammals include, for example, mice, rats, hamsters, experimental animals such as rodents and rabbits such as guinea pigs, pigs, cows, goats, horses, sheep, minced domestic animals, dogs, pets such as cats, humans, and the like. Examples include primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees, but are not limited thereto. The mammal is preferably a primate (human etc.) or a rodent (mouse etc.).
１０−ヒドロキシ−２−デセン酸はローヤルゼリー特有の成分であり、他の食品には存在しないことが知られている。ローヤルゼリーは若い働き蜂の咽頭腺により分泌される乳白色のペースト状物質であり、その成分は必須アミノ酸をはじめとするアミノ酸が豊富に含まれ良質なタンパク質を構成している。さらに、ビタミン類、ミネラル類、炭水化物などの微量成分を含んでいる。例えばビタミン類ではビタミンＢ１、ビタミンＢ２、ビタミンＢ６、ナイアシン、パントテン酸、ビタミンＡ、ビタミンＣ、ビタミンＥなどが挙げられる。ミネラル類ではカリウム、マグネシウム、カルシウム、銅、鉄、リンなどがあげられる。炭水化物ではブドウ糖、果糖などが挙げられる。さらに、アセチルコリン様物質、有機酸、脂肪酸などが含まれている。 It is known that 10-hydroxy-2-decenoic acid is a component unique to royal jelly and does not exist in other foods. Royal jelly is a milky white paste-like substance secreted by the pharyngeal glands of young bees, and its components are rich in amino acids including essential amino acids and constitute high-quality proteins. In addition, it contains trace components such as vitamins, minerals and carbohydrates. Examples of vitamins include vitamin B1, vitamin B2, vitamin B6, niacin, pantothenic acid, vitamin A, vitamin C and vitamin E. Minerals include potassium, magnesium, calcium, copper, iron and phosphorus. Examples of carbohydrates include glucose and fructose. In addition, it contains acetylcholine-like substances, organic acids, fatty acids and the like.
１０−ヒドロキシ−２−デセン酸は、その由来や製法に制限はなく、天然物であってもよく合成品であってもよい。さらに、分画などにより部分精製したものや高度に精製したものを使用してもよい。天然物としてはローヤルゼリーを用いることができ、生ローヤルゼリー、乾燥ローヤルゼリー、調整ローヤルゼリーなどを挙げることができる。また、ローヤルゼリーの原産国は例えば日本、中国、台湾、タイ、ブラジル、ヨーロッパ諸国、オセアニア諸国、アメリカなどを挙げることができ、いずれの原産国のローヤルゼリーを用いてもよい。また、複数の原産国のローヤルゼリーを適宜混合して用いてもよい。 The origin and manufacturing method of 10-hydroxy-2-decenoic acid are not limited, and may be a natural product or a synthetic product. Furthermore, those partially purified by fractionation or highly purified may be used. Royal jelly can be used as a natural product, and examples thereof include raw royal jelly, dried royal jelly, and conditioned royal jelly. The country of origin of royal jelly can be, for example, Japan, China, Taiwan, Thailand, Brazil, European countries, Oceania countries, the United States, etc. Royal jelly of any country of origin may be used. Moreover, you may mix and use the royal jelly of several countries of origin suitably.
さらに、ローヤルゼリーは加熱、遠心分離、アルコール抽出、ろ過などの加工を行ったものであってもよい。 Further, the royal jelly may be processed by heating, centrifugation, alcohol extraction, filtration and the like.
さらに、各種クロマトグラフィーを用いて１０−ヒドロキシ−２−デセン酸を精製してもよい。精製方法としてはゲルろ過クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー、疎水クロマトグラフィー、逆相クロマトグラフィー、順相クロマトグラフィー、限外ろ過などを挙げることができ、これら単独若しくは組み合わせることにより精製を行うことも可能である。 Furthermore, 10-hydroxy-2-decenoic acid may be purified using various chromatographies. Examples of the purification method include gel filtration chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, reverse phase chromatography, normal phase chromatography, ultrafiltration and the like. It is also possible to do so.
ゲルろ過クロマトグラフィーは、種々な分子量のタンパク質を分離できるゲルろ過クロマトグラフィー用の担体があり、分子量が約１万以下のタンパク質を分離できるゲルろ過クロマトグラフィー用の担体が好ましい。イオン交換クロマトグラフィーに用いられているイオン交換基としては、陰イオン交換体、陽イオン交換体などがあり、陰イオン交換体としては、ジエチルアミノエチル基（ＤＥＡＥ基）、四級アミノエチル基（ＱＡＥ）などを例示することができる。また、陽イオン交換体としては、カルボキシメチル（ＣＭ）基、スルホプロピル（ＳＰ）基を例示することができる。疎水クロマトグラフィーに用いられる担体としてはブチル基（Ｂｕｔｙｌ基）、エチル基（Ｅｔｈｙｌ基）、フェニル基（Ｐｈｅｎｙｌ基）が結合した担体を例示することができる。逆相クロマトグラフィーに用いられる担体としとはオクタデシル基（Ｃ１８）、オクタデシル基とはアルキル基の長さが異なるＣ３０、Ｃ８、Ｃ４などが結合した担体が例示される。順相クロマトグラフィーに用いられる担体としてはシリカゲルのほか、シアノプロピル基、ジオール構造を有する官能基、アミノプロピル基、ポリアミンなどが結合した担体が例示される。 The gel filtration chromatography has a carrier for gel filtration chromatography capable of separating proteins having various molecular weights, and a carrier for gel filtration chromatography capable of separating proteins having a molecular weight of about 10,000 or less is preferable. Ion exchange groups used in ion exchange chromatography include anion exchangers and cation exchangers, and examples of anion exchangers include diethylaminoethyl group (DEAE group) and quaternary aminoethyl group (QAE). ) Etc. can be illustrated. Examples of the cation exchanger include a carboxymethyl (CM) group and a sulfopropyl (SP) group. Examples of the carrier used in the hydrophobic chromatography include carriers to which a butyl group (Butyl group), an ethyl group (Ethyl group), and a phenyl group (Phenyl group) are bound. Examples of the carrier used for the reverse phase chromatography include octadecyl group (C18) and carriers in which C30, C8, C4 and the like having different alkyl groups from the octadecyl group have different lengths. Examples of the carrier used for normal phase chromatography include silica gel and carriers to which a cyanopropyl group, a functional group having a diol structure, an aminopropyl group, a polyamine and the like are bound.
１０−ヒドロキシ−２−デセン酸は塩であってもよく、その塩として、例えばナトリウム塩、カリウム塩及びマグネシウム塩等の金属塩並びにモノメチルアミン、トリメチルアミン等の有機アミン塩などを挙げることができる。 The 10-hydroxy-2-decenoic acid may be a salt, and examples of the salt include metal salts such as sodium salt, potassium salt and magnesium salt, and organic amine salts such as monomethylamine and trimethylamine.
１０−ヒドロキシ−２−デセン酸に各種成分を添加することも可能である。各種成分としては、例えば糖、脂質、乳化剤、増粘剤、調味料、香料、酸味調整剤、保存料、果汁、香料、各種栄養成分などが挙げられ、本発明の効果を損なわない範囲で使用することができる。また、各種成分は単独で用いても良いし二種以上を混合して用いてもよい。例えば糖としては、蔗糖、異性化糖、グルコース、フラクトース、パラチノース、トレハロース、ラクトース、キシロース等を例示することができる。乳化剤としては、蔗糖脂肪酸エステル、グリセリン脂肪酸エステル、レシチン等を例示することができる。増粘剤としてはカラギーナン、アラビアガム、キサンタンガム、グァーガム、ペクチン、ローカストビーンガム増粘剤澱粉、ジェランガム等を例示することができる。酸味調整剤としては、クエン酸、乳酸、リンゴ酸、フマル酸、グルコン酸、酒石酸等を例示することができる。保存料としては、安息香酸及びその塩、ソルビン酸及びその塩、パラベン、亜硫酸ナトリウム、ペクチン分解物、グリシン等を例示することができる。果汁としては、トマト果汁、梅果汁、リンゴ果汁、レモン果汁、オレンジ果汁、ベリー系果汁等を例示することができる。香料としては、ハーブ、スパイスなどの香辛料、フルーツ系香料、バニラなどの香料等を例示することができる。この他、好ましい他の栄養成分として、ビタミンＤ等のビタミン類やカルシウム、マグネシウム、鉄、マンガン、亜鉛等のミネラル類等が挙げられる。 It is also possible to add various components to 10-hydroxy-2-decenoic acid. Examples of the various components include sugars, lipids, emulsifiers, thickeners, seasonings, flavors, acidity regulators, preservatives, fruit juices, flavors, various nutritional components, etc. can do. The various components may be used alone or in combination of two or more. Examples of sugars include sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose, xylose and the like. Examples of the emulsifier include sucrose fatty acid ester, glycerin fatty acid ester, lecithin and the like. Examples of the thickener include carrageenan, gum arabic, xanthan gum, guar gum, pectin, locust bean gum thickener starch, gellan gum and the like. Examples of the sourness adjusting agent include citric acid, lactic acid, malic acid, fumaric acid, gluconic acid, tartaric acid and the like. Examples of preservatives include benzoic acid and its salts, sorbic acid and its salts, parabens, sodium sulfite, pectin degradation products, glycine and the like. Examples of the fruit juice include tomato juice, plum juice, apple juice, lemon juice, orange juice, berry juice and the like. Examples of the flavor include spices such as herbs and spices, fruit flavors, flavors such as vanilla, and the like. In addition to these, preferable other nutritional components include vitamins such as vitamin D and minerals such as calcium, magnesium, iron, manganese, and zinc.
本発明の破骨細胞分化抑制剤または閉経後骨粗鬆症の予防・改善剤は食品、医薬品として供することもできる。食品の具体的形態としては、例えば、飲料類、菓子、キャンディ、ガム、パン、畜肉製品、乳製品、レトルト食品、即席食品、冷凍食品、ゼリー状食品、養蜂産品、漬物、調味料等を挙げることができる。これらの食品は、いわゆる健康食品、機能性食品、特定保健用食品、栄養機能食品、栄養補助食品、サプリメントなどとしても有用である。また、それらの食品としての形状としては、顆粒、粉末、タブレット、カプセル、チュアブル、ドリンク、ゼリー、ペースト、粒などを挙げることができる。 The agent for suppressing osteoclast differentiation or the agent for preventing/ameliorating postmenopausal osteoporosis of the present invention can be provided as a food or a medicine. Specific forms of foods include, for example, beverages, confectionery, candy, gum, bread, meat products, dairy products, retort foods, instant foods, frozen foods, jelly-like foods, beekeeping products, pickles, seasonings and the like. be able to. These foods are also useful as so-called health foods, functional foods, foods for specified health uses, nutritionally functional foods, dietary supplements, supplements and the like. Further, examples of the shape of these foods include granules, powders, tablets, capsules, chewables, drinks, jellies, pastes and granules.
医薬品の具体的形態としては、粉末、錠剤、顆粒剤、丸剤、散剤、カプセル、液状、シロップ、ペースト、乳液などを挙げることができる。 Specific forms of the medicine include powders, tablets, granules, pills, powders, capsules, liquids, syrups, pastes, emulsions and the like.
また、本発明の破骨細胞分化抑制剤または閉経後骨粗鬆症の予防・改善剤は、カルシウム、ビタミンD、エストロゲン等と一緒に摂ってもよい。 Further, the agent for suppressing osteoclast differentiation or the agent for preventing/ameliorating postmenopausal osteoporosis of the present invention may be taken together with calcium, vitamin D, estrogen and the like.
本発明の実施例を以下に示すが、本発明は下記の実施例に制限されるものではない。 Examples of the present invention are shown below, but the present invention is not limited to the following examples.
(破骨細胞分化抑制活性の測定法)
破骨細胞前駆体から破骨細胞への分化抑制活性測定は以下に示す方法で行った。
(Method for measuring osteoclast differentiation inhibitory activity)
The activity of inhibiting the differentiation of osteoclast precursors into osteoclasts was measured by the method described below.
メスのC57BL/6Jの大腿骨と脛骨より骨髄細胞を採取し、10 ng/mlのM-CSF（Macrophage-colonystimulating factor）及び10% ウシ胎児血清を加えたaMEM(mMinimumessential medium, a modification)培地で2日培養し、骨髄由来単球マクロファージ前駆細胞(BMM;Bone mallow-derived monocyte/macrophage precursor cells)を作成した。これらの細胞にRANKLを12.5~50ng/mlになるように加えた培地にて３日間培養し、破骨細胞分化を行なった（破骨細胞分化培養）。なお、添加するRANKLは骨髄由来単球マクロファージ前駆細胞の分化の状態により12.5~50ng/mlの間で適宜調整した。被験物質はRANKLと同時に添加した。得られた細胞はPBSで洗浄後4%パラホルムアルデヒドにて15分間室温にて固定を行った。その後アセトン(50%)エタノール(50%)溶液を加え30秒静置した。TRAP（酒石酸抵抗性酸性ホスファターゼ）染色溶液(NaphatolAS-MX phosphate 0.1 mg/ml、N,N-Dimetylformamide 10 ml/ml、Fast redviolet LB salt 0.6 mg/ml、 TRAP Buffer(酢酸Na 5.44 g/l、酒石酸Na 10.5 g/l))で５分間静置し、TRAP染色を行なった。３核以上の細胞を破骨細細胞とし、一視野当たり３核以上の破骨細細胞数をカウントし破骨細胞分化抑制活性の評価を行なった。 Bone marrow cells were collected from female C57BL/6J femurs and tibias, and agar (mMinimumessential medium, a modification) medium containing 10 ng/ml M-CSF (Macrophage-colony stimulating factor) and 10% fetal calf serum was used. After culturing for 2 days, bone marrow-derived monocyte-macrophage precursor cells (BMM) were prepared. These cells were cultured for 3 days in a medium containing RANKL at 12.5 to 50 ng/ml for osteoclast differentiation (osteoclast differentiation culture). The RANKL to be added was appropriately adjusted within the range of 12.5 to 50 ng/ml depending on the state of differentiation of bone marrow-derived monocyte macrophage progenitor cells. The test substance was added at the same time as RANKL. The obtained cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Thereafter, a solution of acetone (50%) and ethanol (50%) was added and the mixture was allowed to stand for 30 seconds. TRAP (tartrate-resistant acid phosphatase) staining solution (Naphatol AS-MX phosphate 0.1 mg/ml, N,N-Dimetylformamide 10 ml/ml, Fast redviolet LB salt 0.6 mg/ml, TRAP Buffer (Na acetate 4.44 g/l, tartaric acid Na 10.5 g/l)), the mixture was allowed to stand for 5 minutes, and TRAP staining was performed. The cells having 3 or more nuclei were used as osteoclasts, and the number of osteoclasts having 3 or more nuclei was counted per visual field to evaluate the osteoclast differentiation inhibiting activity.
（ローヤルゼリーの破骨細胞分化抑制活性の測定法）
生ローヤルゼリーについて以下に示す方法により、被験物質を作成し、破骨細胞分化抑制活性の測定を行った。DMSOジメチルスルホキシドに生ローヤルゼリーを溶解し、濃度200 mg/mlの溶液を得た。さらに遠心分離を行い固形成分を沈殿させ、上清を採取した。上清をAcrodisc DMSO safe filter(PALL)にて濾過滅菌を行い被験物質として、破骨細胞分化抑制活性の測定を行った。その結果、ローヤルゼリーについて破骨細胞分化抑制活性が確認された。
(Method for measuring the osteoclast differentiation inhibitory activity of royal jelly)
A test substance was prepared from raw royal jelly by the method described below, and the osteoclast differentiation inhibitory activity was measured. Raw royal jelly was dissolved in DMSO dimethyl sulfoxide to obtain a solution having a concentration of 200 mg/ml. Further centrifugation was performed to precipitate solid components, and the supernatant was collected. The supernatant was sterilized by filtration with an Acrodisc DMSO safe filter (PALL), and the osteoclast differentiation inhibitory activity was measured as a test substance. As a result, osteoclast differentiation inhibitory activity was confirmed for royal jelly.
（動物試験）
骨におけるローヤルゼリーの作用を調べるために、卵巣摘出閉経後骨粗鬆症モデルマウスを用いて実験を行なった。具体的には、9週齢のC57BL/6Jマウスのメスに対して卵巣摘出（OVX）またはSham手術を行い、コントロール群には生理食塩水、テスト群には1.0 g/kg body weight体重の生ローヤルゼリーを経口ゾンデにて毎日投与した。４週間後にマウスを安楽殺し、大腿骨はμCT解析、脛骨は骨形態計測解析を行なった。μCT解析の結果より、OVX群よりも生ローヤルゼリー投与OVX群で骨量の減少が抑制された画像が得られた。骨量（Bone volume/Ttissuevolume (BV/TV)）はOVX群に対し生ローヤルゼリー投与OVX群では減少の抑制が認められた（図１）。また、海綿骨の指標である骨梁数TrabecularNumber(Tb. N)はOVXにより減少したが生ローヤルゼリー投与OVX群では減少の抑制が認められた。また、TrabecularSeparation(Tb. Sep)骨梁間隔とTrabecularSpacing (Tb. Spa)骨梁中心距離はOVX群ではSham群と比較して増加したが生ローヤルゼリー投与OVX群では増加の抑制が認められた。また、骨形態計測解析より、OsteoclastNumber/Bonesurface (N.Oc/BS) 破骨細胞数は生ローヤルゼリー投与OVX群、OVX群と比較して有意に減少しており、OsteoclastSurface/BoneSurface (Oc.S/BS),破骨細胞面、浸食面Eroded Surface/Bone Surface (ES/BS)も同様の傾向が認められた。また、骨形成速度や骨芽細胞面BoneFormation Rate(BFR),Osteoblast Surface/Bone Surface (Ob.S/BS)などの骨芽細胞関連パラメータについては有意な変化は見られなかった。
(Animal test)
In order to examine the effect of royal jelly on bone, an experiment was performed using ovariectomized postmenopausal osteoporosis model mice. Specifically, ovariectomy (OVX) or Sham surgery was performed on 9-week-old C57BL/6J mouse females, saline was used for the control group, and 1.0 g/kg body weight of the test group was used. Royal jelly was administered daily by oral sonde. Four weeks later, the mouse was euthanized, and the femur was subjected to μCT analysis and the tibia was subjected to bone morphometric analysis. From the results of μCT analysis, the images in which the decrease in bone mass was suppressed in the raw royal jelly-administered OVX group were obtained compared to the OVX group. A decrease in bone volume (Bone volume/Ttissue volume (BV/TV)) was observed in the OVX group in the raw royal jelly-administered OVX group (Fig. 1). The trabecular number (Tb. N), which is an index of cancellous bone, decreased by OVX, but was suppressed in the OVX group treated with raw royal jelly. Trabecular Separation (Tb. Sep) trabecular spacing and Trabecular Spacing (Tb. Spa) trabecular center distance increased in the OVX group compared to the Sham group, but were suppressed in the OVX group treated with raw royal jelly. In addition, from bone morphometric analysis, OsteoclastNumber/Bonesurface (N.Oc/BS) osteoclast number was significantly reduced compared to the live royal jelly-administered OVX group, OVX group, and OsteoclastSurface/BoneSurface (Oc.S/ BS), osteoclast surface, and erosion surface Ereded Surface/Bone Surface (ES/BS) also showed the same tendency. No significant changes were observed in osteoblast-related parameters such as bone formation rate, bone formation rate (BFR), and osteoblast surface/bone surface (Ob.S/BS).
以上のことから、ローヤルゼリーは破骨細胞分化もしくは機能を抑制し、OVXによる骨量の減少を抑制する効果を持つことが示唆された。 From the above, it was suggested that royal jelly suppresses osteoclast differentiation or function, and has an effect of suppressing the decrease in bone mass caused by OVX.
（ローヤルゼリーからの関与成分の分離・同定）
ローヤルゼリーに破骨細胞前駆体から破骨細胞への分化抑制活性及び卵巣摘出マウスによる骨量の減少を抑制する効果が確認されたので、ローヤルゼリーから破骨細胞分化抑制活性を示す関与成分の分離、同定を行った。
(Separation and identification of components involved in royal jelly)
Since it was confirmed that royal jelly has an inhibitory activity on osteoclast differentiation from osteoclast precursors and an effect of suppressing the loss of bone mass by ovariectomized mice, separation of components involved in osteoclast differentiation inhibitory activity from royal jelly, Identification was performed.
生ローヤルゼリーに４倍量のメタノールを加え撹拌後、−２０℃で一晩放置した。一晩放置後、９，２００×ｇで１０分間遠心分離を行い、上清（Ａ）と沈殿物を得た。沈殿物にさらに同量のメタノールを加え、撹拌後、−２０℃で２時間放置した。２時間放置後、９，２００×ｇで１０分間遠心分離を行い、上清（Ｂ）と沈殿物を得た。上清（Ａ）と上清（Ｂ）を混合し、孔径０．４５μｍのフィルター（50CP045AS アドバンテック東洋）でろ過を行い、脱タンパク質画分とした。脱タンパク質画分をロータリーエバポレータ―により溶媒及び水を飛ばした後、残留物を酢酸エチル及び精製水で溶解し、該酢酸エチル及び該精製水で分液を行った。酢酸エチル層に破骨細胞分化抑制活性が確認されたので、酢酸エチル層をプールした。酢酸エチル層をロータリーエバポレータ―により溶媒を飛ばした後、残留物をクロロホルムで溶解し、クロロホルムで平衡化したシリカゲルカラム（ID20×200mm シリカゲル60 63−200μｍ メルク）にアプライした。クロロホルムで洗浄後、１０％メタノールを含有するクロロホルムで分画し、破骨細胞分化抑制活性が確認された画分をプールし、シリカゲル画分とした。シリカゲル画分をロータリーエバポレータ―により溶媒を飛ばした後、残留物をメタノールで溶解し、精製水でメタノール濃度が５０％になるように調整し、５０％メタノールで平衡化したBiotage SNAPS 30g Column Ultra C18（Biotage）にアプライした。５０％メタノールで分画し、破骨細胞分化抑制活性が確認された画分をプールし、SNAPS画分とした。SNAPS画分をロータリーエバポレータ―により溶媒及び水を飛ばした後、残留物をメタノールで溶解し、精製水でメタノール濃度が４０％になるように調整し、４０％メタノールで平衡化したCOSMOSIL Packed Column 5C18−AR−II（10ID×250mm ナカライテスク）にアプライした。４０％メタノールで分画し、破骨細胞分化抑制活性が確認された画分をプールし、ＡＲＩＩ画分とした。ＡＲＩＩ画分をエバポレータ及び吹付式試験管濃縮装置で蒸発乾固させたものを精製画分とした。 Four times the amount of methanol was added to the raw royal jelly, and the mixture was stirred and then left overnight at -20°C. After standing overnight, centrifugation was performed at 9,200×g for 10 minutes to obtain a supernatant (A) and a precipitate. The same amount of methanol was further added to the precipitate, and after stirring, the mixture was left at -20°C for 2 hours. After standing for 2 hours, centrifugation was performed at 9,200×g for 10 minutes to obtain a supernatant (B) and a precipitate. The supernatant (A) and the supernatant (B) were mixed and filtered with a filter (50CP045AS Advantech Toyo) having a pore size of 0.45 μm to obtain a deproteinized fraction. After removing the solvent and water from the deproteinized fraction using a rotary evaporator, the residue was dissolved in ethyl acetate and purified water, and the ethyl acetate and purified water were used for liquid separation. Since osteoclast differentiation suppression activity was confirmed in the ethyl acetate layer, the ethyl acetate layer was pooled. After the solvent was removed from the ethyl acetate layer by a rotary evaporator, the residue was dissolved in chloroform and applied to a silica gel column (ID20×200 mm silica gel 60 63-200 μm Merck) equilibrated with chloroform. After washing with chloroform, the mixture was fractionated with chloroform containing 10% methanol, and the fractions confirmed to have osteoclast differentiation inhibitory activity were pooled to obtain a silica gel fraction. After removing the solvent from the silica gel fraction by using a rotary evaporator, the residue was dissolved in methanol, adjusted to 50% methanol concentration with purified water, and equilibrated with 50% methanol Biotage SNAPS 30g Column Ultra C18 (Biotage) was applied. Fractions were fractionated with 50% methanol, and the fractions in which osteoclast differentiation suppression activity was confirmed were pooled to obtain SNAPS fractions. After removing the solvent and water from the SNAPS fraction with a rotary evaporator, the residue was dissolved in methanol and adjusted to a methanol concentration of 40% with purified water, and equilibrated with 40% methanol. COSMOSIL Packed Column 5C18 -Applied to AR-II (10 ID x 250 mm Nakarai Tesque). Fractions were fractionated with 40% methanol, and the fractions in which osteoclast differentiation suppression activity was confirmed were pooled and used as the ARII fraction. The ARII fraction was evaporated to dryness using an evaporator and a spray-type test tube concentrator, and the purified fraction was obtained.
HPLCによる精製画分の確認
HPLC (High Performance liquid chromatography)による分析
精製画分及び１０−ヒドロキシ−２−デセン酸をそれぞれメタノールで溶解し、Chromasterシステム（日立ハイテクサイエンス）で制御するCOSMOSILPacked Column5C18-AR-II(4.6ID×150mm)で分析を行った。溶出溶媒は5.9mmo/L H3PO4及び5.0mmol/L NaH2PO4を含む50%メタノールを用いてイソクラティックにより分析を行った。カラム温度は40℃に維持した。カラム流出物は、210nmで検出した。その結果、精製画分及び１０−ヒドロキシ−２−デセン酸の保持時間が6.5分とほぼ一致し、精製画分は１０−ヒドロキシ−２−デセン酸と推定された（図２、３）。
Confirmation of purified fractions by HPLC
Analysis by HPLC (High Performance liquid chromatography) The purified fraction and 10-hydroxy-2-decenoic acid were each dissolved in methanol and controlled by Chromaster system (Hitachi High-Tech Science) COSMOSIL Packed Column 5C18-AR-II (4.6ID×150mm) The analysis was carried out. The elution solvent was isocratically analyzed using 50% methanol containing 5.9 mmo/LH 3 PO 4 and 5.0 mmol/L NaH 2 PO 4 . The column temperature was maintained at 40°C. Column effluent was detected at 210 nm. As a result, the retention times of the purified fraction and 10-hydroxy-2-decenoic acid almost coincided with 6.5 minutes, and the purified fraction was estimated to be 10-hydroxy-2-decenoic acid (Figs. 2 and 3).
LC/MS分析
精製画分は１０−ヒドロキシ−２−デセン酸と推定されたので、精製画分及び１０−ヒドロキシ−２−デセン酸をそれぞれメタノールで溶解し、LC/MSにより比較を行った。LC/MS分析はAcquity UPLCシステム（Waters） 及びXevo QTof MSシステム（Waters）を用いて行った。Acquity UPLC HSST3カラム（2.1ID×100mm 1.8μm,Waters）をAcquity UPLCシステムで制御した。溶出溶媒は（Ａ）0.1%酢酸、（Ｂ）メタノールとした。分離はA99.5%・B0.5%からA0%・B100%の20分間の直線濃度勾配、次に、B100%を5分間維持することにより行った。流速は0.4mL/minに制御し、カラム温度は40℃に維持した。
LC/MS analysis Since the purified fraction was estimated to be 10-hydroxy-2-decenoic acid, the purified fraction and 10-hydroxy-2-decenoic acid were each dissolved in methanol, and comparison was performed by LC/MS. LC/MS analysis was performed using Acquity UPLC system (Waters) and Xevo QTof MS system (Waters). An Acquity UPLC HSST3 column (2.1 ID×100 mm 1.8 μm, Waters) was controlled by the Acquity UPLC system. The eluting solvent was (A) 0.1% acetic acid and (B) methanol. Separation was performed by maintaining a linear concentration gradient of A99.5%/B0.5% to A0%/B100% for 20 minutes, and then maintaining B100% for 5 minutes. The flow rate was controlled at 0.4 mL/min and the column temperature was maintained at 40°C.
Xevo QTof MSシステムによる質量分析は以下の条件で行った;
モード: ネガィブモード
イオン化法: ESI, Capillary voltage: 3.0KV
Cone voltage: 15V
質量範囲: m/z 50〜1000
Mass spectrometry on the Xevo QTof MS system was performed under the following conditions;
Mode: Negative mode Ionization method: ESI, Capillary voltage: 3.0KV
Cone voltage: 15V
Mass range: m/z 50-1000
LC/MS分析の結果、精製画分及び１０−ヒドロキシ−２−デセン酸について保持時間が10.7分にそれぞれ単一のピークが確認された（図４）。さらに、保持時間10.7分のMSスペクトルを確認したところ、m/z185.11と質量も一致していることが確認された（図５）。さらに、m/z185.11についてコリジョンエネルギーを10〜25eVに変化させ、生成したフラグメントを積算して比較を行ったところフラクメンテーションパターンが精製画分と１０−ヒドロキシ−２−デセン酸で一致し（図６）、精製画分は１０−ヒドロキシ−２−デセン酸と推定された。 As a result of LC/MS analysis, a single peak was confirmed at a retention time of 10.7 minutes for the purified fraction and 10-hydroxy-2-decenoic acid (Fig. 4). Furthermore, when the MS spectrum with a retention time of 10.7 minutes was confirmed, it was confirmed that the mass also corresponded to m/z 185.11 (FIG. 5). Furthermore, when the collision energy was changed from 10 to 25 eV for m/z 185.11 and the generated fragments were integrated and compared, the fractionation pattern was consistent between the purified fraction and 10-hydroxy-2-decenoic acid. (FIG. 6), the purified fraction was estimated to be 10-hydroxy-2-decenoic acid.
（ローヤルゼリーと１０−ヒドロキシ−２−デセン酸の破骨細胞分化抑制活性の比較）
生ローヤルゼリーをメタノールで0.25mg/mL、0.5mg/mLになるように調製し、遠心分離を行い上清を得た。その時の上清の１０−ヒドロキシ−２−デセン酸濃度は、それぞれ25μM、50μMであったので１０−ヒドロキシ−２−デセン酸をメタノールで溶解し、25μM、50μMになるように調製し、破骨細胞分化抑制活性を比較した。その結果、１０−ヒドロキシ−２−デセン酸と生ローヤルゼリーの破骨細胞分化抑制活性はほぼ同じであり、生ローヤルゼリーに含まれる関与成分の主成分は１０−ヒドロキシ−２−デセン酸と推定された（図７）。
(Comparison of osteoclast differentiation inhibiting activity of royal jelly and 10-hydroxy-2-decenoic acid)
Raw royal jelly was prepared with methanol to 0.25 mg/mL and 0.5 mg/mL, and centrifuged to obtain a supernatant. The 10-hydroxy-2-decenoic acid concentrations of the supernatant at that time were 25 μM and 50 μM, respectively, so 10-hydroxy-2-decenoic acid was dissolved in methanol to prepare 25 μM and 50 μM, and osteoclast was prepared. The cell differentiation inhibiting activity was compared. As a result, 10-hydroxy-2-decenoic acid and raw royal jelly have almost the same osteoclast differentiation inhibitory activity, and the main component of the components involved in raw royal jelly was presumed to be 10-hydroxy-2-decenoic acid. (Fig. 7).
（１０−ヒドロキシ−２−デセン酸のレセプターの検討）
本実施例に示す(破骨細胞分化抑制活性の測定方法)に従い、骨髄由来単球マクロファージ前駆細胞(BMM)及び破骨細胞(Osteoclast)を得た。
(Study of 10-hydroxy-2-decenoic acid receptor)
Bone marrow-derived monocyte macrophage progenitor cells (BMM) and osteoclasts (Osteoclast) were obtained according to the (method for measuring osteoclast differentiation inhibitory activity) shown in this example.
BMM、Osteoclastについて以下に示す方法により、RT-PCR(リアルタイム-PCR)を行ったところ、図８に示すようにFfar1、Ffar2、Ffar3、Gpr84のmRNAの発現はBMMと比較して減少していたが、Ffar4のmRNAの発現は増加しており、FFAR4は10-ヒドロキシ-2-デセン酸のレセプターとして機能する可能性が導かれた。 When RT-PCR (real-time-PCR) was performed on BMM and Osteoclast by the method described below, the expression of mRNA for Ffar1, Ffar2, Ffar3, and Gpr84 was decreased as compared with BMM, as shown in FIG. However, the expression of Ffar4 mRNA was increased, suggesting that FFAR4 may function as a receptor for 10-hydroxy-2-decenoic acid.
なお、RT-PCRは以下の方法により測定を行った。
BMM、OsteoclastについてNIPPON GENE社のISOGENを用いてRNAを抽出し、抽出された0.5 μgのtotalRNAをThermo Fisher Scientific社のSuperscriptIII reverse transcriptaseを用いてcDNAの合成を行った。次にcDNAについてリアルタイムPCR解析システムとしてBio-Rad社のCFX384 Touch、リアルタイムPCR用試薬としてTOYOBO社のSYBR Green Realtime PCR Master Mixを用いてRT-PCRを行った。なお、プライマーは表１に示すものを用い、Gapdhを用いて補正を行った。
In addition, RT-PCR was measured by the following method.
For BMM and Osteoclast, RNA was extracted using ISOGEN from NIPPON GENE, and 0.5 μg of the extracted total RNA was subjected to cDNA synthesis using Superscript III reverse transcriptase from Thermo Fisher Scientific. Next, RT-PCR was performed on the cDNA using CFX384 Touch from Bio-Rad as a real-time PCR analysis system and SYBR Green Realtime PCR Master Mix from TOYOBO as a real-time PCR reagent. The primers shown in Table 1 were used, and correction was performed using Gapdh.
１０−ヒドロキシ−２−デセン酸のFFAR4を介した作用を調べるために、FFAR4 (GPR120) Reporter Assay Kit (Cayman Chemical)を用いて、１０−ヒドロキシ−２−デセン酸とFFAR4との親和性を検討した。その結果、図９に示すように１０−ヒドロキシ−２−デセン酸との親和性が確認され、EC50は1.025mMであった。なお、FFAR4 (GPR120) Reporter Assay Kitは添付のマニュアルに従い操作を行った。 To investigate the action of 10-hydroxy-2-decenoic acid via FFAR4, the affinity between 10-hydroxy-2-decenoic acid and FFAR4 was examined using FFAR4 (GPR120) Reporter Assay Kit (Cayman Chemical). did. As a result, as shown in FIG. 9, the affinity with 10-hydroxy-2-decenoic acid was confirmed, and the EC50 was 1.025 mM. The FFAR4 (GPR120) Reporter Assay Kit was operated according to the attached manual.
製品プロトコールに従ってレンチウイルスベクターを作成し、BMMsに24時間感染させた後、RANKLと１０−ヒドロキシ−２−デセン酸で刺激を行い、Ffar4をターゲットとしたshRNA(短鎖ヘアピンRNA)レンチウイルスベクターによりノックダウンを行った(Kim Y, Hayashi M, Ono T, Yoda T, Takayanagi H, Nakashima T., Mod Rheumatol. 2020 Jan;30(1):85-92.)。その際のノックダウン効率は9割程度であった。
図１０に示すようにFfar4をノックダウンしていないControlでは１０−ヒドロキシ−２−デセン酸により、破骨細胞分化は抑制されているのに対し、Ffrar4をノックダウンしたshFfar4では１０−ヒドロキシ−２−デセン酸により、破骨細胞分化は抑制されなかった。これらの結果より、１０−ヒドロキシ−２−デセン酸のレセプターはFFAR4と推定された。
Lentivirus vector was prepared according to the product protocol, infected with BMMs for 24 hours, stimulated with RANKL and 10-hydroxy-2-decenoic acid, and then shRNA (short hairpin RNA) lentivirus vector targeting Ffar4 was used. Knockdown was performed (Kim Y, Hayashi M, Ono T, Yoda T, Takayanagi H, Nakashima T., Mod Rheumatol. 2020 Jan;30(1):85-92.). The knockdown efficiency at that time was about 90%.
As shown in FIG. 10, 10-hydroxy-2-decenoic acid suppresses osteoclast differentiation in Control that does not knockdown Ffar4, whereas 10-hydroxy-2 in shFfar4 that knocks down Ffrar4. -Decenoic acid did not suppress osteoclast differentiation. From these results, the receptor for 10-hydroxy-2-decenoic acid was estimated to be FFAR4.
１０−ヒドロキシ−２−デセン酸の存在・非存在下で破骨細胞分化培養を行い、血清スタベーションを6時間行った後、RANKL・１０−ヒドロキシ−２−デセン酸刺激を行った。その際、0、5、20分で細胞を採取し、Western Blot分析を行った。その結果、図１１に示すようにControl（１０−ヒドロキシ−２−デセン酸非存在下）ではIκBαのバンドはしだいに薄くなりIκBαは分解され、１０Ｈ２ＤＡ（１０−ヒドロキシ−２−デセン酸存在下）ではIκBαのバンドの変化はなく、IκBαは分解されていないものと考えられた。 Osteoclast differentiation culture was performed in the presence/absence of 10-hydroxy-2-decenoic acid, serum starvation was performed for 6 hours, and then RANKL·10-hydroxy-2-decenoic acid stimulation was performed. At that time, cells were collected at 0, 5, and 20 minutes, and Western Blot analysis was performed. As a result, as shown in FIG. 11, in Control (in the absence of 10-hydroxy-2-decenoic acid), the band of IκBα gradually became thinner and IκBα was decomposed, and 10H2DA (in the presence of 10-hydroxy-2-decenoic acid). Therefore, there was no change in the IκBα band, suggesting that IκBα was not degraded.
なお、Western Blot分析は以下の方法により行った。
得られた細胞は冷PBSで洗浄し、Roche Bioscience社のComplete protease inhibitor cocktailを含むリシスバッファー及びRoche Bioscience社のPhosSTOPを用いて氷上で10分反応させ細胞の溶解を行った。溶解物は15,000rpm・10分遠心分離を行い清浄化した。抽出されたタンパク質はSDS-PAGEを行い、PVDF膜(Millipore)に転写した。転写されたPVDF膜はBlocking One P またはBullet Blocking One(ナカライテスク)でブロッキングを行い室温で2時間一次抗体と反応させ続いて2次抗体と反応させた。バンドはChemi-Lumi One Ultra(ナカライテスク)で可視化を行った。なお、一次抗体についてphospho-IκBα(p-IκBα)、IκBαに対する抗体はCell Signaling Technology、β-actinに対する抗体はSigma-Aldrichより購入した。
コントロール(TurboGFP positive control vector)、ノックダウンベクター(shFfar4)をBMMsにそれぞれ24時間感染させた後、破骨細胞分化培養を行った。血清スタベーションを6時間行った後、RANKL・１０−ヒドロキシ−２−デセン酸刺激を行った。その際、0、5、20分で細胞を採取し、Western Blot分析を行った。その結果、コントロールではIκBαのバンドの変化はなく、IκBαは分解されていないものと考えられた。一方、ノックダウンベクター(shFfar4)はIκBαのバンドは薄くなりIκBαは分解されているもの考えられた。なお、Western Blot分析は本段落に示す方法と同様に行った。
The Western Blot analysis was performed by the following method.
The obtained cells were washed with cold PBS, and lysed by reacting for 10 minutes on ice using a lysis buffer containing Complete protease inhibitor cocktail from Roche Bioscience and PhosSTOP from Roche Bioscience. The lysate was clarified by centrifugation at 15,000 rpm for 10 minutes. The extracted protein was subjected to SDS-PAGE and transferred to a PVDF membrane (Millipore). The transferred PVDF membrane was blocked with Blocking One P or Bullet Blocking One (Nacalai Tesque), reacted with the primary antibody for 2 hours at room temperature, and then reacted with the secondary antibody. The band was visualized with Chemi-Lumi One Ultra (Nacalai Tesque). Regarding the primary antibody, phospho-IκBα (p-IκBα), an antibody against IκBα were purchased from Cell Signaling Technology, and an antibody against β-actin was purchased from Sigma-Aldrich.
BMMs were infected with a control (Turbo GFP positive control vector) and a knockdown vector (shFfar4) for 24 hours, and then osteoclast differentiation culture was performed. After performing serum starvation for 6 hours, RANKL 10-hydroxy-2-decenoic acid stimulation was performed. At that time, cells were collected at 0, 5, and 20 minutes, and Western Blot analysis was performed. As a result, there was no change in the IκBα band in the control, and it was considered that IκBα was not degraded. On the other hand, in the knockdown vector (shFfar4), the band of IκBα became thin and it was considered that IκBα was degraded. The Western Blot analysis was performed in the same manner as the method described in this paragraph.
（作用機序の考察）
RANKL による破骨細胞分化の調節はRANKにRANKLが結合するとIKK複合体が活性化され、活性化したIKK複合体により、IκBαがリン酸化・分解されることでNF-κBは核内に移行し、破骨細胞分化が誘導される。一方、１０−ヒドロキシ−２−デセン酸がFFAR4に結合するとIKK複合体の活性化が抑制され、IκBαのリン酸化・分解が抑えられた。また、Ffar4のノックダウンを行なったshFfar4群では１０−ヒドロキシ−２−デセン酸によるIκBαのリン酸化・分解の抑制が減弱した。このことから１０−ヒドロキシ−２−デセン酸はNF-κB経路を抑制することにより、破骨細胞分化抑制がなされると考えられる。
(Discussion of mechanism of action)
The regulation of osteoclast differentiation by RANKL is the activation of the IKK complex when RANKL binds to RANK, and the activated IKK complex phosphorylates and degrades IκBα to translocate NF-κB into the nucleus. , Osteoclast differentiation is induced. On the other hand, when 10-hydroxy-2-decenoic acid bound to FFAR4, activation of the IKK complex was suppressed, and phosphorylation/degradation of IκBα was suppressed. In the shFfar4 group in which Ffar4 was knocked down, the inhibition of phosphorylation/degradation of IκBα by 10-hydroxy-2-decenoic acid was diminished. From this, it is considered that 10-hydroxy-2-decenoic acid suppresses osteoclast differentiation by suppressing the NF-κB pathway.
本発明により、破骨細胞前駆体から破骨細胞への分化抑制剤、閉経後骨粗鬆症の予防・改善剤を提供することが可能となる。 According to the present invention, it becomes possible to provide an agent for suppressing the differentiation of osteoclast precursors into osteoclasts and a preventive/ameliorating agent for postmenopausal osteoporosis.
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