JP2020055795A - Adipocyte differentiation inhibitor and cosmetics containing the same, and adipocyte differentiation inhibiting method - Google Patents
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Abstract
Description
本発明は、脂肪細胞分化抑制剤及びこれを含有する化粧料、並びに脂肪細胞分化抑制方法に関する。 The present invention relates to a fat cell differentiation inhibitor, a cosmetic containing the same, and a method for suppressing fat cell differentiation.
近年、３−ヒドロキシ酪酸（以下、「３ＨＢ」ともいう）は、美白効果や、細胞賦活効果、保湿効果などがあることから、化粧水、乳液、クリーム、パック、分散液、洗浄料等の化粧品や、軟膏剤、クリーム剤、外用液剤等の外用医薬品（以下、「化粧料」と総称する）として利用されるようになっている（例えば、特許文献１記載の美白化粧料、細胞賦活化粧料及び保湿化粧料）。 BACKGROUND ART In recent years, 3-hydroxybutyric acid (hereinafter also referred to as “3HB”) has a whitening effect, a cell activating effect, a moisturizing effect, and the like, and therefore cosmetics such as a lotion, an emulsion, a cream, a pack, a dispersion, and a cleaning agent. And external medicines such as ointments, creams and liquids for external use (hereinafter collectively referred to as “cosmetics”) (for example, whitening cosmetics and cell-activating cosmetics described in Patent Document 1) And moisturizing cosmetics).
また、３ＨＢに関する研究が進むにつれて、当該３ＨＢには、様々な遺伝子の発現やタンパク質の活性に影響するシグナル伝達物質としての作用があることが判明し、特許文献２には、３ＨＢが脂肪細胞からのアディポネクチンの分泌を抑制するという新たな特徴に着目し、体重調節による抗肥満作用のためにアディポネクチンの分泌を調節（抑制）する、３ＨＢを有効成分としたアディポネクチン分泌調節剤が提案されている（特許文献２）。 Further, as research on 3HB progresses, it has been found that the 3HB has an action as a signal transmitter that affects the expression of various genes and the activities of proteins. Focusing on a new feature of inhibiting adiponectin secretion, an adiponectin secretion regulator containing 3HB as an active ingredient, which regulates (suppresses) adiponectin secretion for an anti-obesity effect by controlling body weight, has been proposed ( Patent Document 2).
特許文献２においては、アディポネクチンの産生を抑制することによって痩身効果が発揮されるとの知見を基に、アディポネクチンの分泌を抑制して血中のアディポネクチン濃度を適正な濃度に調節するために、体重調節による抗肥満作用を有する用途剤としてアディポネクチン分泌調節剤を用いている。 In Patent Document 2, based on the finding that a slimming effect is exerted by suppressing the production of adiponectin, the body weight is adjusted to suppress the adiponectin secretion and adjust the blood adiponectin concentration to an appropriate concentration. An adiponectin secretion regulator is used as an agent having an anti-obesity effect by regulation.
アディポネクチンの産生と肥満との関係については、特許文献２に記載されているように、アディポネクチンの産生を抑制することで痩身効果を示す（別の言い方をすれば、肥満の抑制に繋がる）という説も唱えられているが、アディポネクチンの産生と肥満との関係についてはこの他にも多数の説が唱えられている。 Regarding the relationship between adiponectin production and obesity, the theory that, as described in Patent Document 2, suppressing adiponectin production has a slimming effect (in other words, leads to suppression of obesity). There are many other theories about the relationship between adiponectin production and obesity.
例えば、アディポネクチンは、脂肪前駆細胞が分化した脂肪細胞から産生されるだけでなく、この脂肪細胞が成熟した脂肪細胞（成熟脂肪細胞）からも産生され、興味深いことに、両者から産生されるアディポネクチンの量は、脂肪細胞より成熟脂肪細胞の方が少ないという説も唱えられている。この説からすれば、より脂肪の蓄積が進んだ成熟脂肪細胞が増えることでアディポネクチンの産生量が減少するのであるから、アディポネクチンの産生が減っている場合の方が肥満が進行していることになる。 For example, adiponectin is produced not only from adipocytes from which preadipocytes have been differentiated, but also from mature adipocytes (mature adipocytes). Interestingly, adiponectin is produced from both. It has also been suggested that the amount is less in mature adipocytes than in adipocytes. According to this theory, the amount of adiponectin production decreases as the number of mature adipocytes with more accumulated fat increases, so obesity progresses when adiponectin production decreases. Become.
このように、アディポネクチンの産生を抑制することが肥満の抑制に繋がるとは必ずしも言えないという実情がある以上、アディポネクチンの産生と肥満の抑制とを関連付けてアディポネクチンの産生に着目することにさほど意義はない。 Thus, since there is a fact that suppressing adiponectin production does not necessarily lead to suppression of obesity, it is not so significant to focus on adiponectin production by linking adiponectin production with suppression of obesity. Absent.
特許文献２は、アディポネクチンの産生と肥満の抑制とを関連付けてアディポネクチンの産生に着目しているが、特許文献２においては、脂肪前駆細胞を脂肪細胞に分化させた後、この分化後の脂肪細胞を３ＨＢを含む無血清培地で培養することで、アディポネクチンの産生量を指標として、３ＨＢに関する、脂肪細胞からのアディポネクチンの分泌抑制能を評価している。しかしながら、アディポネクチンの産生量の変化は、脂肪細胞が成熟脂肪細胞に成熟したことに起因するものであるのか、或いは、３ＨＢの作用に起因するものであるのかを明確に区別することができない以上、３−ヒドロキシ酪酸に関するアディポネクチンの分泌抑制能が実験的に示されていると言えず、アディポネクチンの分泌の抑制と肥満の抑制との関係についても具体的な実験データが示されているわけでない。したがって、特許文献２の内容を鑑みても、アディポネクチンと肥満の抑制とを関連付けてアディポネクチンの産生に着目することにさほど意義があるとは考え難い。 Patent Document 2 focuses on adiponectin production by associating adiponectin production with suppression of obesity. In Patent Document 2, after differentiation of preadipocytes into adipocytes, the adipocytes after this differentiation Is cultured in a serum-free medium containing 3HB to evaluate the ability of 3HB to suppress the secretion of adiponectin from fat cells using the amount of adiponectin produced as an index. However, since the change in the amount of adiponectin produced is due to the maturation of adipocytes into mature adipocytes or due to the action of 3HB, it cannot be clearly distinguished, The ability to inhibit the secretion of adiponectin with respect to 3-hydroxybutyric acid cannot be said to be experimental, and no specific experimental data has been shown regarding the relationship between the suppression of adiponectin secretion and the suppression of obesity. Therefore, even in view of the contents of Patent Document 2, it is unlikely that there is much significance in focusing on adiponectin production by associating adiponectin with suppression of obesity.
これらの事情に鑑みれば、抗肥満作用を有する化粧料を開発する上では、アディポネクチンの産生とは無関係に、実際の体内での脂肪の蓄積を抑制して肥満を抑えられる物質や、当該物質の使用方法を見出したり、そのような物質がどのようにして脂肪の蓄積を抑制するのかを解明したりすることが極めて重要である。 In view of these circumstances, in developing a cosmetic having an anti-obesity effect, regardless of the production of adiponectin, a substance capable of suppressing fat accumulation in the actual body and suppressing obesity, It is extremely important to find out how to use it and to clarify how such substances suppress fat accumulation.
本発明は以上の実情に鑑みなされたものであり、体内での脂肪の蓄積の抑制を可能にする脂肪細胞分化抑制剤及びこれを用いた化粧料、並びに脂肪細胞分化抑制方法の提供を、その目的とする。 The present invention has been made in view of the above circumstances, and provides an adipocyte differentiation inhibitor capable of suppressing the accumulation of fat in the body, a cosmetic using the same, and a method for inhibiting adipocyte differentiation. Aim.
本発明者らは、体内での脂肪の蓄積を抑制する手段について鋭意研究を重ねた結果、脂肪前駆細胞を脂肪細胞へ分化誘導させる際の培地に３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が含まれていることで、この３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が分化過程において脂肪前駆細胞に作用して脂肪細胞への分化が抑制され、更に、脂肪細胞中の脂肪酸合成が抑制されることで、結果的に体内での脂肪の蓄積が抑えられること、即ち、３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が脂肪蓄積抑制効果を有することを見出した。更に、本発明者らは、脂肪細胞を培養する際に培地に３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が含まれていることで、これらが脂肪蓄積抑制効果を発揮し、脂肪細胞内での脂肪滴の蓄積が抑えられることも見出した。尚、分化とは細胞が特定の役割を持つことであるため、本願において、「脂肪細胞への分化」とは、脂肪前駆細胞から脂肪細胞へ分化すること、及び細胞が脂肪酸を合成して細胞内に脂肪滴を蓄積することを含む概念である。 The present inventors have conducted intensive studies on means for suppressing fat accumulation in the body. As a result, the medium for inducing differentiation of preadipocytes into adipocytes contains 3-hydroxybutyric acid or 3-hydroxybutyrate. The fact that 3-hydroxybutyric acid or 3-hydroxybutyrate acts on preadipocytes during the differentiation process to suppress differentiation into adipocytes, and further suppresses fatty acid synthesis in adipocytes As a result, they found that fat accumulation in the body was suppressed, that is, 3-hydroxybutyric acid or 3-hydroxybutyrate had a fat accumulation inhibitory effect. Furthermore, the present inventors, when culturing adipocytes, the medium contains 3-hydroxybutyric acid or 3-hydroxybutyrate, thereby exhibiting the effect of suppressing fat accumulation, and They also found that the accumulation of fat droplets was suppressed. In the present application, "differentiation into fat cells" refers to the differentiation of preadipocytes into fat cells, and the cell synthesizes fatty acids to cause cell differentiation. This is a concept that involves accumulating fat droplets in the interior.
即ち、上記目的を達成するための本発明に係る脂肪細胞分化抑制剤の特徴構成は、３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩を有効成分とし、脂肪前駆細胞、前記脂肪前駆細胞から脂肪細胞への分化過程にある細胞、又は前記脂肪細胞に作用して、前記脂肪細胞への分化を抑制する点にある。 That is, the characteristic configuration of the adipocyte differentiation inhibitor according to the present invention for achieving the above-described object includes, as an active ingredient, 3-hydroxybutyric acid or 3-hydroxybutyrate, from a preadipocyte to the adipocyte to the adipocyte. On the cells in the process of differentiation or the adipocytes to suppress the differentiation into the adipocytes.
上記特徴構成によれば、脂肪前駆細胞が脂肪細胞へと分化する過程や脂肪細胞の培養過程において、３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が脂肪前駆細胞や脂肪細胞に作用することで脂肪細胞への分化（脂肪細胞内での脂肪滴の蓄積を含む）が抑制される。このように、上記特徴構成では、アディポネクチンの産生とは無関係に、脂肪細胞への分化を抑制することで、体内での脂肪の蓄積を抑えることができる。 According to the above feature configuration, in the process of preadipocytes differentiating into adipocytes or the process of culturing adipocytes, 3-hydroxybutyric acid or 3-hydroxybutyrate acts on preadipocytes or adipocytes, thereby adipocytes (Including the accumulation of lipid droplets in fat cells) is suppressed. As described above, in the above-described characteristic configuration, accumulation of fat in the body can be suppressed by suppressing differentiation into fat cells regardless of production of adiponectin.
また、本発明に係る脂肪細胞分化抑制剤の更なる特徴構成は、前記脂肪前駆細胞又は前記脂肪細胞がヒト由来である点にある。 A further characteristic configuration of the adipocyte differentiation inhibitor according to the present invention is that the preadipocyte or the adipocyte is derived from a human.
上記特徴構成によれば、ヒト由来の脂肪前駆細胞が脂肪細胞へ分化するのを抑制することができ、また、ヒト由来の脂肪細胞での脂肪滴の蓄積を抑制することができるため、人に対して好適に用いることができる。 According to the above feature configuration, it is possible to suppress human-derived fat precursor cells from differentiating into fat cells, and also to suppress accumulation of fat droplets in human-derived fat cells. It can be suitably used.
尚、上記のように、３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩を有効成分として含有していることで、脂肪細胞中の脂肪酸合成が抑制され、体内での脂肪の蓄積が抑えられることから、３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩は、脂肪細胞において脂肪酸合成を抑制する働きを持っている。 As described above, by containing 3-hydroxybutyric acid or 3-hydroxybutyrate as an active ingredient, fatty acid synthesis in fat cells is suppressed, and accumulation of fat in the body is suppressed. 3-hydroxybutyric acid or 3-hydroxybutyrate has a function of suppressing fatty acid synthesis in fat cells.
即ち、本発明に係る脂肪細胞分化抑制剤の更なる特徴構成は、前記３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が、前記脂肪細胞において脂肪酸合成を抑制する働きを持つ点にある。 That is, a further characteristic configuration of the adipocyte differentiation inhibitor according to the present invention is that the 3-hydroxybutyric acid or 3-hydroxybutyrate has a function of suppressing fatty acid synthesis in the adipocyte.
上記脂肪細胞分化抑制剤は、一般的に用いられ得る添加剤（界面活性剤や希釈剤、安定剤など）とともに、化粧料の原料として好適に使用することができる。 The adipocyte differentiation inhibitor can be suitably used as a raw material for cosmetics together with commonly used additives (surfactants, diluents, stabilizers, etc.).
即ち、本発明に係る化粧料の特徴構成は、上記脂肪細胞分化抑制剤を含有する点にある。 That is, the characteristic configuration of the cosmetic according to the present invention is that the cosmetic contains the above-mentioned adipocyte differentiation inhibitor.
また、本発明に係る化粧料の更なる特徴構成は、前記３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩を０．１ｍＭ以上９５ｍＭ未満含有している点にある。 Further, a further feature of the cosmetic according to the present invention is that the cosmetic contains the above-mentioned 3-hydroxybutyric acid or 3-hydroxybutyrate in an amount of 0.1 mM or more and less than 95 mM.
化粧料に３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が０．１ｍＭ以上９５ｍＭ未満含有していることで、当該化粧料を人に対して使用した場合に、脂肪前駆細胞や脂肪細胞に作用して脂肪細胞への分化（脂肪細胞内での脂肪滴の蓄積を含む）を抑制でき、優れた脂肪蓄積抑制効果を発揮する。尚、０．１ｍＭ未満であると、３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩による脂肪蓄積抑制効果が発揮され難くなる。 When 3-hydroxybutyric acid or 3-hydroxybutyrate is contained in the cosmetic at 0.1 mM or more and less than 95 mM, when the cosmetic is used for humans, it acts on pre-adipocytes and fat cells. Differentiation into fat cells (including accumulation of fat droplets in fat cells) can be suppressed, and an excellent fat accumulation inhibitory effect is exhibited. In addition, when it is less than 0.1 mM, the effect of suppressing the accumulation of fat by 3-hydroxybutyric acid or 3-hydroxybutyrate becomes difficult to be exhibited.
また、上記目的を達成するための脂肪細胞分化抑制方法の特徴構成は、脂肪前駆細胞、前記脂肪前駆細胞から脂肪細胞への分化過程にある細胞、又は前記脂肪細胞に、３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩を作用させ、前記脂肪細胞への分化を抑制する分化抑制工程を含む点にある。 In addition, the characteristic configuration of the method for suppressing adipocyte differentiation for achieving the above object is as follows: a preadipocyte, a cell in the process of differentiation from the preadipocyte to an adipocyte, or the adipocyte contains 3-hydroxybutyric acid or 3 The method further comprises a step of inhibiting differentiation into adipocytes by allowing hydroxybutyrate to act.
上述したように、本発明者らは、鋭意研究を重ねた結果、脂肪前駆細胞を脂肪細胞へ分化誘導させる際の培地に３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が含まれていることで、この３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が分化過程において脂肪前駆細胞に作用して脂肪細胞への分化が抑制されること（即ち、前駆脂肪細胞から脂肪細胞への分化が抑制されたり、脂肪細胞中の脂肪酸合成が抑制されたりすること）を発見し、更に、脂肪細胞を培養する際の培地に３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が含まれていることで、これらが脂肪蓄積抑制効果を発揮し、脂肪細胞内での脂肪滴の蓄積が抑えられることも発見した。上記特徴構成によれば、脂肪前駆細胞、脂肪前駆細胞から脂肪細胞への分化過程にある細胞、又は脂肪細胞に対して３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が作用して脂肪細胞への分化（脂肪細胞内での脂肪滴の蓄積を含む）を抑制することができる。 As described above, the present inventors have conducted intensive studies and found that 3-hydroxybutyric acid or 3-hydroxybutyrate is contained in a medium for inducing differentiation of preadipocytes into adipocytes, The action of 3-hydroxybutyric acid or 3-hydroxybutyrate on preadipocytes during the differentiation process to suppress the differentiation into adipocytes (that is, the differentiation of preadipocytes into adipocytes, That fatty acid synthesis in the cells is suppressed), and that the medium used for culturing the adipocytes contains 3-hydroxybutyric acid or 3-hydroxybutyrate to suppress fat accumulation. It has also been found that it exerts an effect and suppresses the accumulation of fat droplets in fat cells. According to the above-mentioned characteristic configuration, 3-hydroxybutyric acid or 3-hydroxybutyrate acts on preadipocytes, cells undergoing differentiation from preadipocytes to adipocytes, or adipocytes to differentiate into adipocytes. (Including the accumulation of lipid droplets in fat cells).
また、本発明に係る脂肪細胞分化抑制方法の更なる特徴構成は、前記分化抑制工程は、前記脂肪前駆細胞、前記脂肪前駆細胞から前記脂肪細胞への分化過程にある細胞、又は前記脂肪細胞を、前記３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩を含む培地で培養する工程である点にある。 Further, a further characteristic configuration of the method for inhibiting adipocyte differentiation according to the present invention, wherein the differentiation inhibitory step is a step in which the preadipocyte, the cell in the process of differentiation from the preadipocyte to the adipocyte, or the adipocyte is performed. Culturing in a medium containing the above-mentioned 3-hydroxybutyric acid or 3-hydroxybutyrate.
上記特徴構成によれば、３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩を含む培地で、脂肪前駆細胞、脂肪前駆細胞から脂肪細胞への分化過程にある細胞、又は脂肪細胞を培養するため、３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が脂肪前駆細胞、脂肪前駆細胞から脂肪細胞への分化過程にある細胞、又は脂肪細胞に作用し易く、脂肪細胞への分化を効果的に抑制できる。 According to the above feature configuration, in order to culture preadipocytes, cells in the process of differentiation from preadipocytes to adipocytes, or adipocytes in a medium containing 3-hydroxybutyric acid or 3-hydroxybutyrate, Hydroxybutyric acid or 3-hydroxybutyrate easily acts on preadipocytes, cells undergoing differentiation from preadipocytes to adipocytes, or adipocytes, and can effectively suppress differentiation into adipocytes.
更に、本発明に係る脂肪細胞分化抑制方法の更なる特徴構成は、前記培地は、前記３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩のモル濃度が０．１ｍＭ以上９５ｍＭ未満である点にある。 Further, a further characteristic configuration of the method for inhibiting adipocyte differentiation according to the present invention is that the medium has a molar concentration of the 3-hydroxybutyric acid or 3-hydroxybutyrate of 0.1 mM or more and less than 95 mM.
上記特徴構成によれば、細胞に対する毒性を抑えた上で、脂肪細胞への分化を効果的に抑制することができる。尚、０．１ｍＭ未満であると、脂肪細胞への分化が抑制され難くなる。 According to the above-mentioned characteristic configuration, differentiation into fat cells can be effectively suppressed while suppressing toxicity to cells. In addition, when it is less than 0.1 mM, it becomes difficult to suppress differentiation into fat cells.
また、本発明に係る脂肪細胞分化抑制方法の更なる特徴構成は、前記脂肪前駆細胞又は前記脂肪細胞がヒト由来である点にある。 Further, a further characteristic configuration of the method for suppressing adipocyte differentiation according to the present invention is that the preadipocytes or the adipocytes are derived from humans.
上記特徴構成によれば、ヒト由来の脂肪前駆細胞が脂肪細胞へ分化するのを抑制することができ、また、ヒト由来の脂肪細胞での脂肪滴の蓄積を抑制することができる。 According to the above configuration, it is possible to suppress the differentiation of human preadipocytes into adipocytes, and to suppress the accumulation of lipid droplets in human adipocytes.
尚、上記のように、３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩を作用させることで、脂肪細胞中の脂肪酸合成が抑制されることから、３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩は、脂肪細胞において脂肪酸合成を抑制する働きを持っている。 As described above, the action of 3-hydroxybutyric acid or 3-hydroxybutyrate suppresses the synthesis of fatty acids in adipocytes. Has a function of suppressing fatty acid synthesis.
即ち、本発明に係る脂肪細胞分化抑制方法の更なる特徴構成は、前記３−ヒドロキシ酪酸又は３−ヒドロキシ酪酸塩が、前記脂肪細胞において脂肪酸合成を抑制する働きを持つ点にある。 That is, a further characteristic configuration of the method for suppressing adipocyte differentiation according to the present invention is that the 3-hydroxybutyric acid or 3-hydroxybutyrate has a function of suppressing fatty acid synthesis in the adipocyte.
以下、本発明の実施形態に係る脂肪細胞分化抑制剤、化粧料、及び脂肪細胞分化抑制方法について説明する。 Hereinafter, an adipocyte differentiation inhibitor, a cosmetic, and an adipocyte differentiation inhibitor method according to an embodiment of the present invention will be described.
〔３ＨＢ〕
３ＨＢ（（Ｒ）−３−ヒドロキシ酪酸）は、３ＨＢ生産性のハロモナス菌を添加した発酵プロセスを行い、得られた発酵液からハロモナス菌を分離除去し、精製することにより得られる。発酵プロセスは、果汁等の糖質栄養源を含有する原料液に、３ＨＢ生産性のハロモナス菌をそのまま添加し、好気発酵、嫌気発酵を順に行うプロセス（特開２０１３−０８１４０３号公報等参照）として実施することができる。これにより、糖質が３ＨＢに変換され、発酵液中に生産されることになる。生産された３ＨＢは、常法にて、膜分離、分離精製を経た後、純粋な３ＨＢとして用いられる。
[3HB]
3HB ((R) -3-hydroxybutyric acid) is obtained by performing a fermentation process to which 3HB-producing halomonas bacteria are added, separating and removing halomonas bacteria from the obtained fermented solution, and purifying the resultant. The fermentation process is a process in which 3HB-producing halomonas bacteria are directly added to a raw material liquid containing a carbohydrate nutrient such as fruit juice, and aerobic fermentation and anaerobic fermentation are sequentially performed (see Japanese Patent Application Laid-Open No. 2013-084033). It can be implemented as As a result, the saccharide is converted into 3HB and is produced in the fermentation liquor. The produced 3HB is used as pure 3HB after membrane separation and separation / purification by a conventional method.
〔脂肪細胞分化抑制剤〕
脂肪細胞分化抑制剤は、上記３ＨＢを有効成分として含有するものであり、当該３ＨＢは、それ自体の他、遊離酸や種々の塩（３−ヒドロキシ酪酸塩）として用いることができ、塩には金属塩（無機塩）及び有機塩のいずれも含む。また、脂肪細胞分化抑制剤は、３ＨＢを主たる機能性成分の一つとして含んでいれば、必要に応じて種々の添加剤を含んでいても良く、３ＨＢのもつ特性が現れる態様であれば、配合割合に特に制限はない。更に、３ＨＢは、必ずしも脂肪細胞分化抑制剤中で最も多い成分である必要はない。尚、脂肪細胞分化抑制剤には、精製された３ＨＢに代えて、発酵プロセスにより得られた発酵液をそのまま用いても良いし、その発酵液をベースに種々の添加剤を添加したものを用いても良い。
(Adipocyte differentiation inhibitor)
The adipocyte differentiation inhibitor contains the above 3HB as an active ingredient. The 3HB itself can be used as a free acid or various salts (3-hydroxybutyrate) in addition to itself. Includes both metal salts (inorganic salts) and organic salts. In addition, the adipocyte differentiation inhibitor may contain various additives as needed as long as it contains 3HB as one of the main functional components, provided that the properties of 3HB appear. There is no particular limitation on the mixing ratio. Furthermore, 3HB does not necessarily have to be the most abundant component in adipocyte differentiation inhibitors. In addition, instead of the purified 3HB, the fermented solution obtained by the fermentation process may be used as it is as the fat cell differentiation inhibitor, or a product obtained by adding various additives based on the fermented solution may be used. May be.
この脂肪細胞分化抑制剤によれば、有効成分である３ＨＢが脂肪前駆細胞や脂肪前駆細胞から脂肪細胞への分化過程にある細胞、脂肪細胞に対して作用することで、脂肪細胞への分化（脂肪細胞内での脂肪滴の蓄積を含む）が抑制され、脂肪細胞中の脂肪酸合成が抑制される。特に、脂肪前駆細胞、脂肪前駆細胞から脂肪細胞への分化過程にある細胞、又は脂肪細胞がヒト由来である場合、３ＨＢの高い分化抑制効果が発揮されることを実験的に確認している。 According to this adipocyte differentiation inhibitor, 3HB as an active ingredient acts on preadipocytes, cells in the process of differentiation from preadipocytes to adipocytes, and adipocytes, whereby differentiation into adipocytes ( (Including the accumulation of lipid droplets in fat cells), and the synthesis of fatty acids in fat cells is suppressed. In particular, it has been experimentally confirmed that when a preadipocyte, a cell undergoing differentiation from a preadipocyte to an adipocyte, or an adipocyte is of human origin, a high 3HB differentiation inhibitory effect is exhibited.
〔化粧料〕
本発明の脂肪細胞分化抑制剤は、化粧料の原料として用いることができる。この化粧料には、必要に応じて水や一般的な化粧料に配合される種々の公知の添加成分を本発明の効果を損なわない程度に任意の処方で含有することができるが、３ＨＢによって、脂肪前駆細胞や脂肪細胞に作用して脂肪細胞への分化（脂肪細胞内での脂肪滴の蓄積を含む）が抑制され、優れた脂肪蓄積抑制効果が有効に発揮されるようにするという観点からすると、３ＨＢを０．１ｍＭ以上９５ｍＭ未満含有していることが好ましく、０．１ｍＭ以上１９ｍＭ以下含有していることがより好ましい。尚、公知の添加成分としては、例えば、水性基剤や油性基剤、界面活性剤、保湿剤、増粘剤、ゲル化剤、酸化防止剤、防腐剤、殺菌剤、キレート剤、ｐＨ調整剤、紫外線吸収剤、還元剤、酸化剤、高分子粉体、無機粉体、ビタミン類及びその誘導体類、糖類及びその誘導体類、有機酸類、酵素類、アルコール類、香料、色素などを例示することができる。
(Cosmetics)
The adipocyte differentiation inhibitor of the present invention can be used as a raw material for cosmetics. This cosmetic can contain water and various known additives to be added to general cosmetics, if necessary, in an arbitrary formulation so as not to impair the effects of the present invention. , Which acts on pre-adipocytes and adipocytes to suppress differentiation into adipocytes (including the accumulation of lipid droplets in adipocytes), and to exert an excellent effect of suppressing fat accumulation effectively In view of this, it is preferable that 3HB be contained in a range of 0.1 mM to less than 95 mM, and more preferably 0.1 mM to 19 mM. Known additives include, for example, aqueous bases and oil bases, surfactants, humectants, thickeners, gelling agents, antioxidants, preservatives, bactericides, chelating agents, pH adjusters , UV absorbers, reducing agents, oxidizing agents, polymer powders, inorganic powders, vitamins and their derivatives, sugars and their derivatives, organic acids, enzymes, alcohols, flavors, pigments, etc. Can be.
具体的には下記表１に示す処方にて、化粧水を構成することができる。 Specifically, a lotion can be constituted by the formulation shown in Table 1 below.
具体的には下記表２に示す処方にて、乳液を構成することができる。 Specifically, an emulsion can be formed according to the formulation shown in Table 2 below.
具体的には下記表３に示す処方にて、クリームを構成することができる。 Specifically, a cream can be formed according to the formulation shown in Table 3 below.
このような化粧水や乳液、クリームなどの化粧料によれば、この化粧料を人の肌に塗布することで、有効成分として含まれている３ＨＢが人の脂肪前駆細胞や脂肪前駆細胞から脂肪細胞への分化過程にある細胞、脂肪細胞に対して作用して、脂肪細胞への分化が抑制されるため、結果的に、体内での脂肪の蓄積を抑えることができる。 According to such cosmetics as lotion, milky lotion, cream, etc., by applying this cosmetic to human skin, 3HB contained as an active ingredient is converted from human fat precursor cells or fat precursor cells into fat. It acts on cells and adipocytes in the process of cell differentiation, and suppresses adipocyte differentiation. As a result, accumulation of fat in the body can be suppressed.
〔脂肪細胞分化抑制方法〕
本発明の脂肪細胞分化抑制方法は、脂肪前駆細胞、脂肪前駆細胞から脂肪細胞への分化過程にある細胞、又は脂肪細胞に、３ＨＢを作用させ、脂肪細胞への分化を抑制する分化抑制工程を含み、具体的に、分化抑制工程において、脂肪前駆細胞、脂肪前駆細胞から脂肪細胞への分化過程にある細胞、又は脂肪細胞を、３ＨＢを含む培地で培養する。この脂肪細胞分化抑制方法によれば、脂肪前駆細胞、脂肪前駆細胞から脂肪細胞への分化過程にある細胞、又は脂肪細胞に対して３ＨＢを作用させ、脂肪細胞への分化（脂肪細胞内での脂肪滴の蓄積を含む）を抑制し、脂肪細胞中の脂肪酸合成を抑制することができる。
(Adipocyte differentiation suppression method)
The method for inhibiting adipocyte differentiation of the present invention comprises a step of suppressing differentiation into adipocytes by causing 3HB to act on preadipocytes, cells undergoing differentiation from preadipocytes to adipocytes, or adipocytes. Specifically, in the differentiation inhibiting step, preadipocytes, cells undergoing differentiation from preadipocytes to adipocytes, or adipocytes are cultured in a medium containing 3HB. According to this method of inhibiting adipocyte differentiation, 3HB acts on preadipocytes, cells undergoing differentiation from preadipocytes to adipocytes, or adipocytes to differentiate into adipocytes (intraadipocytes). (Including the accumulation of fat droplets) and the synthesis of fatty acids in adipocytes.
尚、３ＨＢを含む培地は、その３ＨＢのモル濃度が０．１ｍＭ以上９５ｍＭ未満（より望ましくは０．１ｍＭ以上１９ｍＭ以下）である場合、細胞に対する毒性を抑えた上で、脂肪細胞への分化を効果的に抑制することができることを実験的に確認している。また、特に、脂肪前駆細胞、脂肪前駆細胞から脂肪細胞への分化過程にある細胞、又は脂肪細胞がヒト由来である場合、３ＨＢの高い分化抑制効果が発揮されることを実験的に確認している。 When the molar concentration of 3HB is 0.1 mM or more and less than 95 mM (more preferably, 0.1 mM or more and 19 mM or less), the medium containing 3HB suppresses cell toxicity, and then differentiates into adipocytes. It has been experimentally confirmed that it can be effectively suppressed. In addition, it was experimentally confirmed that particularly when preadipocytes, cells in the process of differentiation from preadipocytes to adipocytes, or adipocytes were derived from humans, the high differentiation inhibitory effect of 3HB was exhibited. I have.
〔３ＨＢの分化抑制能の確認試験１〕
以下、ヒト白色脂肪前駆細胞を分化誘導下・被験物質（３ＨＢＮａ）添加下で培養し、被験物質による分化抑制能を検証した方法及び結果について説明する。尚、図１には、本確認試験１において行ったヒト白色脂肪前駆細胞に関する培養過程及びオイルレッドＯ染色による測定過程を模式的に図示した。
[Confirmation test 1 of the ability of 3HB to suppress differentiation]
Hereinafter, a method and results of verifying the ability of a test substance to inhibit differentiation by culturing human white adipose precursor cells under differentiation induction and addition of a test substance (3HBNa) will be described. FIG. 1 schematically shows the culture process of human white adipose precursor cells and the measurement process by oil red O staining performed in this confirmation test 1.
尚、検証のための各種試験に使用した被験物質、細胞、試薬、試験用キットは以下の通りである。
（被験物質）
・（Ｒ）−３−ヒドロキシ酪酸ナトリウム（発酵プロセス由来）
（細胞）
・ヒト白色脂肪前駆細胞（皮下脂肪）（タカラバイオ株式会社製）
（試薬及び試験用キット）
・脂肪前駆細胞増殖培地キット（タカラバイオ株式会社製）
・脂肪分化誘導培地（株式会社バイオ未来工房製）
・脂肪維持培地（株式会社バイオ未来工房製）
・ホルムアルデヒド溶液（富士フイルム和光純薬株式会社製）
・２−プロパノール（富士フイルム和光純薬株式会社製）
・オイルレッドＯ（Ｓｉｇｍａ Ａｌｄｒｉｃｈ製）
・生細胞数測定試薬ＳＦ（ナカライテスク株式会社製）
・ＲＮｅａｓｙ Ｍｉｃｒｏ Ｋｉｔ（ＱＩＡＧＥＮ製）
・ダルベッコＰＢＳ（日水製薬株式会社製）
Test substances, cells, reagents, and test kits used for various tests for verification are as follows.
(Test substance)
-Sodium (R) -3-hydroxybutyrate (from fermentation process)
(cell)
・ Human white fat precursor cells (subcutaneous fat) (Takara Bio Inc.)
(Reagent and test kit)
-Preadipocyte growth medium kit (Takara Bio Inc.)
・ Fat differentiation induction medium (manufactured by Bio Mirai Kobo Co., Ltd.)
・ Fat maintenance medium (Bio Mirai Kobo Co., Ltd.)
・ Formaldehyde solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
・ 2-propanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
・ Oil Red O (manufactured by Sigma Aldrich)
・ Viable cell count reagent SF (Nacalai Tesque, Inc.)
・ RNeasy Micro Kit (QIAGEN)
・ Dulbecco PBS (Nissui Pharmaceutical Co., Ltd.)
各種試験は、以下のように行った。
１．被験物質の調製
１５ｇの３ＨＢＮａを精製水に溶解させ、４９４ｇ／Ｌ（ｐＨ６．７）の被験物質溶液を調製した。この被験物質溶液は、所定モル濃度の３ＨＢを含む培地となるように適宜希釈した上で、１０％（ｖ／ｖ）となるように各培地に溶解後、ろ過滅菌（０．２２μｍ）し、ストックとして使用した。
Various tests were performed as follows.
1. Preparation of Test Substance 15 g of 3HBNa was dissolved in purified water to prepare a test substance solution of 494 g / L (pH 6.7). The test substance solution was appropriately diluted to a medium containing 3HB at a predetermined molar concentration, dissolved in each medium to 10% (v / v), and sterilized by filtration (0.22 μm). Used as stock.
２．オイルレッドＯ染色試験
２−１．細胞培養
脂肪前駆細胞を、増殖培地を用いて６×１０４ｃｅｌｌｓ／１ｍＬ／ウェルで、２４ウェルプレートに播種し、ＣＯ２インキュベータ内（５％ＣＯ２、３７℃）で１日間培養した（７０％コンフルエント）。その後、０．７６ｍＭ、３．８ｍＭ、１９ｍＭの３ＨＢを含む分化誘導培地に置換して３日間培養した（分化誘導処理）。しかる後、０．７６ｍＭ、３．８ｍＭ、１９ｍＭの３ＨＢを含む分化維持培地に交換して更に３日間培養した。
２−２．オイルレッドＯ染色
分化維持培地で３日間培養した細胞について、十分な脂肪滴の蓄積を確認した後、オイルレッドＯ染色を実施した。具体的には、以下の手順で行った。
（１）プレート各ウェルの細胞をＰＢＳで２回洗浄する。
（２）４％ＰＦＡで１０分間、室温にて固定する。
（３）ＰＢＳで２回洗浄する。
（４）固定された細胞を６０％イソプロパノールに１分間置換する。
（５）オイルレッドＯ染色液に置換し、２０分間、室温にて染色を行う。
（６）染色液を除去後、６０％イソプロパノールで１回洗浄する。
（７）ＰＢＳで２回洗浄する。
（８）ＰＢＳ中にて顕微鏡で染色細胞を観察、撮影する。
（９）ＰＢＳを除去後、５００μＬのイソプロパノールを添加し、染色しているオイルレッドを抽出する。
（１０）抽出液を１．５ｍＬチューブに移し、遠心分離により細胞片を除去した後、上清について４９２ｎｍの光学濃度にて吸光度測定を行う。尚、測定した値からブランク（プレートウェルのみでオイルレッドＯ染色を実施した区画）値を引いた値を測定値とした。
2. Oil red O dyeing test 2-1. Cell culture Preadipocytes were seeded in 24-well plates at 6 × 10 4 cells / 1 mL / well using growth medium and cultured in a CO 2 incubator (5% CO 2 , 37 ° C.) for 1 day (70 % Confluent). Thereafter, the medium was replaced with a differentiation-inducing medium containing 0.76 mM, 3.8 mM, and 19 mM of 3HB and cultured for 3 days (differentiation induction treatment). Thereafter, the medium was replaced with a differentiation maintaining medium containing 0.76 mM, 3.8 mM, and 19 mM of 3HB, and the cells were cultured for further 3 days.
2-2. Oil Red O Staining After confirming sufficient accumulation of lipid droplets in cells cultured in a differentiation maintenance medium for 3 days, oil red O staining was performed. Specifically, the procedure was as follows.
(1) The cells in each well of the plate are washed twice with PBS.
(2) Fix with 4% PFA for 10 minutes at room temperature.
(3) Wash twice with PBS.
(4) Replace fixed cells with 60% isopropanol for 1 minute.
(5) Replace with oil red O staining solution and perform staining for 20 minutes at room temperature.
(6) After removing the staining solution, wash once with 60% isopropanol.
(7) Wash twice with PBS.
(8) Observe and photograph the stained cells with a microscope in PBS.
(9) After removing the PBS, 500 μL of isopropanol is added to extract the stained oil red.
(10) The extract is transferred to a 1.5 mL tube, and cell debris is removed by centrifugation. Then, the absorbance of the supernatant is measured at an optical density of 492 nm. In addition, a value obtained by subtracting a blank value (a section in which oil red O staining was performed only in the plate well) from the measured value was defined as a measured value.
３．生細胞数測定試験
３−１．細胞培養
上記２−１と同手法にて細胞培養を実施した。
３−２．生細胞数の計測
培養終了後に各ウェルから培養上清を除去し、終濃度１０％の生細胞数測定試薬（ＷＳＴ−８）を含む維持培地を１ｍＬ／ウェルで添加し、３７℃にて反応させる。添加後３０分及び９０分後の吸光度（測定波長４５０ｎｍ、参照波長６２０ｎｍ）をプレートリーダーにて測定し、両値から６０分間の吸光度の変化量を算出し、生細胞数として計測した。
3. Viable cell count measurement test 3-1. Cell culture Cell culture was performed in the same manner as in 2-1.
3-2. Measurement of the number of viable cells After completion of the culture, the culture supernatant was removed from each well, and a maintenance medium containing a viable cell count reagent (WST-8) having a final concentration of 10% was added at 1 mL / well, and reacted at 37 ° C. Let it. Absorbance 30 minutes and 90 minutes after the addition (measurement wavelength 450 nm, reference wavelength 620 nm) was measured with a plate reader, and the change in the absorbance for 60 minutes was calculated from both values, and counted as the number of living cells.
４．遺伝子発現試験
４−１．細胞培養
脂肪前駆細胞を、増殖培地を用いて３×１０５ｃｅｌｌｓ／２ｍＬ／ウェルで、６ウェルプレートに播種し、ＣＯ２インキュベータ内（５％ＣＯ２、３７℃）で１日間培養した（７０％コンフルエント）。その後、０．７６ｍＭ、３．８ｍＭ、１９ｍＭの３ＨＢを含む分化誘導培地に置換して３日間培養した。しかる後、０．７６ｍＭ、３．８ｍＭ、１９ｍＭの３ＨＢを含む分化維持培地に交換して更に３日間培養した。
４−２．トータルＲＮＡの回収
培養終了後に、１９ｍＭの３ＨＢを含む処理区のみについて、トータルＲＮＡを回収した。具体的には、各ウェルの培養上清を除去した後、１×ＰＢＳを０．３ｍＭ／ウェルで添加して細胞を洗浄し、ＰＢＳを除去した後に５０μＬのトリプシンＥＤＴＡ溶液を添加して３７℃で反応させた。その後、各ウェルに４５０μＬの１０％ＦＢＳを含むＰＢＳを添加し、トリプシン反応を停止させた。しかる後、細胞剥離液を回収し、遠心力５００×ｇで１０分間、室温にて遠心分離を行い、上清を除去して沈殿した細胞を回収した。回収した細胞からのＲＮＡ抽出は以下の手順で行った。
（１）回収した細胞に３５０μＬのＲＬＴ ｂｕｆｆｅｒを添加し、細胞塊を緩やかに懸濁する。
（２）細胞懸濁液を１分間、ボルテックス（攪拌）処理する。
（３）細胞懸濁液に３５０μＬの７０％エタノールを添加し、ピペットにて緩やかに懸濁する。
（４）反応液をＲＮｅａｓｙ ＭｉｎＥｌｕｔｅ Ｓｐｉｎ Ｃｏｌｕｍｎに移し、遠心力８０００×ｇで１５秒間遠心分離を行い、ろ液を破棄する。
（５）カラムに３５０μＬのＢｕｆｆｅｒ ＲＷ１を添加し、遠心力８０００×ｇで１５秒間遠心分離を行い、ろ液を破棄する。
（６）１０μＬのＤＮａｓｅ Ｉ ｓｔｏｃｋ溶液を７０μＬのＢｕｆｆｅｒ ＲＤＤに添加し混和する。
（７）カラムに上記（６）のＤＮａｓｅ溶液８０μＬを添加して室温で１５分間静置して反応させる。
（８）カラムに３５０μＬのＢｕｆｆｅｒ ＲＷ１を添加し、遠心力８０００×ｇで１５秒間遠心分離を行い、ろ液を破棄する。
（９）カラムを新たな２ｍＬ Ｃｏｌｌｅｃｔｉｏｎ Ｔｕｂｅに移し、５００μＬのＢｕｆｆｅｒ ＲＰＥを添加し、遠心力８０００×ｇで１５秒間遠心分離を行い、ろ液を破棄する。
（１０）カラムに５００μＬの８０％エタノールを添加し、遠心力８０００×ｇで１５秒間遠心分離を行い、ろ液を破棄する。
（１１）カラムを新たな２ｍＬ Ｃｏｌｌｅｃｔｉｏｎ Ｔｕｂｅに移し、遠心力１２０００×ｇで５分間遠心分離を行い、ろ液を破棄する。
（１２）カラムを新たなチューブに移し、１４μＬのＲＮａｓｅフリー水を添加し、１２０００×ｇで５分間遠心分離を行い、ろ液をＲＮＡサンプルとした。
５−３．ＤＮＡマイクロアレイ解析
単離した各トータルＲＮＡをクラボウ株式会社において、Ａｆｆｉｍｅｔｒｉｘ社製のＧｅｎｅＣｈｉｐ Ｈｕｍａｎ Ｇｅｎｏｍｅ Ｕ１３３ Ｐｌｕｓ ２．０ Ａｒｒａｙにて遺伝子発現解析を実施し、遺伝子発現に差異があった遺伝子についてＧＯ機能解析とｐａｔｙｗａｙ解析を実施した。
4. Gene expression test 4-1. Cell culture Preadipocytes were seeded in 6-well plates at 3 × 10 5 cells / 2 mL / well using growth medium and cultured for 1 day in a CO 2 incubator (5% CO 2 , 37 ° C.) (70 % Confluent). Thereafter, the cells were replaced with a differentiation-inducing medium containing 0.76 mM, 3.8 mM, and 19 mM of 3HB and cultured for 3 days. Thereafter, the medium was replaced with a differentiation maintaining medium containing 0.76 mM, 3.8 mM, and 19 mM of 3HB, and the cells were cultured for further 3 days.
4-2. Recovery of Total RNA After the completion of the culture, total RNA was recovered only from the treated section containing 19 mM 3HB. Specifically, after removing the culture supernatant of each well, the cells were washed by adding 1 × PBS at 0.3 mM / well, and after removing the PBS, 50 μL of trypsin EDTA solution was added thereto, and the mixture was added at 37 ° C. Was reacted. Thereafter, 450 μL of PBS containing 10% FBS was added to each well to stop the trypsin reaction. Thereafter, the cell detachment solution was collected, centrifuged at 500 × g for 10 minutes at room temperature at room temperature, and the supernatant was removed to collect the precipitated cells. RNA extraction from the collected cells was performed according to the following procedure.
(1) 350 μL of RLT buffer is added to the collected cells, and the cell mass is gently suspended.
(2) Vortex (stir) the cell suspension for 1 minute.
(3) Add 350 μL of 70% ethanol to the cell suspension and gently suspend with a pipette.
(4) Transfer the reaction solution to RNeasy MinElute Spin Column, perform centrifugation at 8000 × g for 15 seconds, and discard the filtrate.
(5) Add 350 μL of Buffer RW1 to the column, perform centrifugation at 8000 × g for 15 seconds, and discard the filtrate.
(6) Add 10 μL of DNase I stock solution to 70 μL of Buffer RDD and mix.
(7) Add 80 μL of the DNase solution of the above (6) to the column and allow it to stand at room temperature for 15 minutes to react.
(8) 350 μL of Buffer RW1 is added to the column, centrifuged at 8000 × g for 15 seconds, and the filtrate is discarded.
(9) Transfer the column to a new 2 mL Collection Tube, add 500 μL of Buffer RPE, centrifuge at 8000 × g for 15 seconds, and discard the filtrate.
(10) Add 500 μL of 80% ethanol to the column, perform centrifugation at 8000 × g for 15 seconds, and discard the filtrate.
(11) Transfer the column to a new 2 mL Collection Tube, perform centrifugation at 12,000 × g for 5 minutes, and discard the filtrate.
(12) The column was transferred to a new tube, 14 μL of RNase-free water was added, centrifuged at 12,000 × g for 5 minutes, and the filtrate was used as an RNA sample.
5-3. DNA microarray analysis The isolated total RNAs were subjected to gene expression analysis at Kurabo Industries, Ltd. using GeneChip Human Genome U133 Plus 2.0 Array manufactured by Affimetrix, and GO function analysis was performed for the genes having different gene expression. Pataway analysis was performed.
以下、各種試験結果について説明する。 Hereinafter, various test results will be described.
１．オイルレッドＯ染色試験の結果
表４は、分化誘導処理を行わずに培養した細胞、即ち脂肪前駆細胞（コントロール１）、３ＨＢを含まない培地を用いて分化誘導処理を行って培養した細胞、即ち脂肪細胞（コントロール２）、０．７６ｍＭ、３．８ｍＭ、１９ｍＭの３ＨＢを含む培地を用いて分化誘導処理を行って培養した細胞に関するオイルレッドＯ染色試験の結果をまとめた表であり、また、図２は、コントロール１の吸光度に対する割合を縦軸としたグラフである。また、図３〜図７は、オイルレッドＯ染色試験の結果を示す顕微鏡写真であり、図３はコントロール１、図４はコントロール２、図５は３ＨＢのモル濃度が０．７６ｍＭ、図６は３ＨＢのモル濃度が３．８ｍＭ、図７は３ＨＢのモル濃度が１９ｍＭの場合に関するものであり、図中の赤色部分が染色された脂肪滴である。
表４から明らかなように、分化誘導処理の有無によって、吸光度に大きな差が出ており、分化誘導処理を行って培養することで培養後の細胞に脂肪が蓄積されることを確認することができ、図２と図３〜図７とを比較しても、分化誘導処理を行って培養することで、赤色部分が増加、即ち、細胞内に脂肪が蓄積されていることが確認できる。また、分化誘導処理を行って培養した場合、培地中に含まれる３ＨＢのモル濃度が高くなるほど吸光度が減少、即ち、脂肪の蓄積量が少なくなっていることが確認でき、特に、３ＨＢのモル濃度が３．８ｍＭ及び１９ｍＭである場合において、濃度依存的な有意な脂肪の蓄積の抑制を確認することができた。また、図３〜図７を比較しても、３ＨＢのモル濃度が高くなるほど赤色部分が減少、即ち、細胞内に蓄積された細胞が少なくなっていることが確認できる。このことから、３ＨＢが、脂肪前駆細胞から脂肪細胞への分化を抑制する分化抑制能を有することが分かり、特に、分化過程において３ＨＢがモル濃度３．８ｍＭ及び１９ｍＭで存在していることで、３ＨＢの分化抑制能が十分に発揮されることが明らかになった。
1. Results of Oil Red O Staining Test Table 4 shows cells cultured without performing differentiation induction treatment, that is, cells cultured by performing differentiation induction treatment using a medium containing no preadipocytes (control 1) and 3HB, FIG. 9 is a table summarizing the results of an oil red O staining test on cells cultured by performing a differentiation induction treatment using a medium containing 3HB at 0.76 mM, 3.8 mM, and 19 mM in adipocytes (Control 2); FIG. 2 is a graph in which the ratio to the absorbance of Control 1 is plotted on the vertical axis. 3 to 7 are micrographs showing the results of the oil red O staining test. FIG. 3 shows control 1, FIG. 4 shows control 2, FIG. 5 shows a molar concentration of 3HB of 0.76 mM, and FIG. FIG. 7 shows the case where the molar concentration of 3HB is 3.8 mM, and FIG. 7 shows the case where the molar concentration of 3HB is 19 mM.
As is clear from Table 4, there is a large difference in the absorbance depending on the presence or absence of the differentiation inducing treatment, and it is confirmed that fat is accumulated in the cultured cells by performing the differentiation inducing treatment and culturing. 2 and FIG. 3 to FIG. 7, it can be confirmed that, by performing the differentiation induction treatment and culturing, the red portion increases, that is, fat is accumulated in the cells. In addition, when the cells were cultured after the differentiation induction treatment, the absorbance was reduced as the molar concentration of 3HB contained in the medium was increased, that is, the amount of accumulated fat was reduced. In particular, the molar concentration of 3HB was confirmed. Was 3.8 mM and 19 mM, concentration-dependent significant suppression of fat accumulation could be confirmed. Also, comparing FIG. 3 to FIG. 7, it can be confirmed that as the molar concentration of 3HB increases, the red portion decreases, that is, the number of cells accumulated in the cells decreases. This indicates that 3HB has the ability to suppress differentiation from preadipocytes to adipocytes, and in particular that 3HB is present at a molar concentration of 3.8 mM and 19 mM during the differentiation process. It was revealed that the ability of 3HB to suppress differentiation was sufficiently exerted.
２．生細胞数測定試験の結果
表５は、分化誘導処理を行わずに培養した細胞（コントロール１）、３ＨＢを含まない培地を用いて分化誘導処理を行って培養した細胞（コントロール２）、０．７６ｍＭ、３．８ｍＭ、１９ｍＭの３ＨＢを含む培地を用いて分化誘導処理を行って培養した細胞に関する生細胞数測定試験の結果をまとめた表であり、図８は、細胞生存度を縦軸としたグラフである。
表５及び図８から明らかなように、分化誘導処理の有無、培地中の３ＨＢの有無、培地中の３ＨＢのモル濃度にかかわらず、生細胞数に大きな変化は見られない。このことから、オイルレッドＯ染色試験によって確認された脂肪の蓄積量の減少が、細胞数が減少したことによるものでないことが明らかになった。
2. Results of viable cell count measurement test Table 5 shows cells cultured without performing the differentiation inducing treatment (Control 1), cells cultured by performing the differentiation inducing treatment using a medium not containing 3HB (Control 2), 0. FIG. 8 is a table summarizing the results of a viable cell count measurement test on cells cultured by performing differentiation induction treatment using a medium containing 76 mM, 3.8 mM, and 19 mM 3HB. It is the graph which did.
As is clear from Table 5 and FIG. 8, there is no significant change in the number of viable cells regardless of the presence or absence of the differentiation inducing treatment, the presence or absence of 3HB in the medium, and the molar concentration of 3HB in the medium. This revealed that the decrease in the amount of accumulated fat, which was confirmed by the oil red O staining test, was not due to a decrease in the number of cells.
３．遺伝子発現試験の結果
表６は、脂肪前駆細胞から脂肪細胞への分化に関する遺伝子についての遺伝子発現解析の結果をまとめた表であり、表７は、脂肪酸の合成に関する遺伝子についての遺伝子発現解析の結果をまとめた表である。
表６に示すように、ペルオキシソーム増殖剤応答性受容体δ（ｐｅｒｏｘｉｓｏｍｅ ｐｒｏｌｉｆｅｒａｔｏｒ−ａｃｔｉｖａｔｅｄ ｒｅｃｅｐｔｏｒ ｄｅｌｔａ）、リポタンパク質リパーゼ（ｌｉｐｏｐｒｏｔｅｉｎ ｌｉｐａｓｅ）、ペリリピン１（ｐｅｒｉｌｉｐｉｎ １）、脂肪酸結合タンパク質３（ｆａｔｔｙ ａｃｉｄ ｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ ３， ｍｕｓｃｌｅ ａｎｄ ｈｅａｒｔ（ｍａｍｍａｒｙ−ｄｅｒｉｖｅｄ ｇｒｏｗｔｈ ｉｎｈｉｂｉｔｏｒ））、マトリックスメタロペプチダーゼ１（間質コラゲナーゼ）（ｍａｔｒｉｘ ｍｅｔａｌｌｏｐｅｐｔｉｄａｓｅ １（ｉｎｔｅｒｓｔｉｔｉａｌ ｃｏｌｌａｇｅｎａｓe））、ステアロイルＣｏＡデサチュラーゼ（Δ９デサチュラーゼ）（ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ （ｄｅｌｔａ−９−ｄｅｓａｔｕｒａｓｅ））の遺伝子発現が抑制されているため、脂肪前駆細胞のまま未分化の細胞があり、分化後の脂肪を蓄える脂肪細胞の数が相対的に少なくなる結果として、上記オイルレッドＯ染色試験において、培地中に３ＨＢが存在する場合に脂肪の蓄積が減少したと考えられる。このことから、３ＨＢに、脂肪前駆細胞や脂肪前駆細胞から脂肪細胞への分化過程にある細胞に作用して分化を抑制する作用があることが遺伝子レベルで明らかになった。
また、表７に示すように、ヒドロキシアシルＣｏＡデヒドロゲナーゼ／３−ケトアシルＣｏＡチオラーゼ／エノイルＣｏＡヒドラターゼ（三機能タンパク質）αサブユニット（ｈｙｄｒｏｘｙａｃｙｌ−ＣｏＡ ｄｅｈｙｄｒｏｇｅｎａｓｅ／３−ｋｅｔｏａｃｙｌ−ＣｏＡ ｔｈｉｏｌａｓｅ／ｅｎｏｙｌ−ＣｏＡ ｈｙｄｒａｔａｓｅ （ｔｒｉｆｕｎｃｔｉｏｎａｌ ｐｒｏｔｅｉｎ）， ａｌｐｈａ ｓｕｂｕｎｉｔ）、ヒドロキシアシルＣｏＡデヒドロゲナーゼ（ｈｙｄｒｏｘｙａｃｙｌ−ＣｏＡ ｄｅｈｙｄｒｏｇｅｎａｓｅ）、アセチルＣｏＡアセチルトランスフェラーゼ２（ａｃｅｔｙｌ−ＣｏＡ ａｃｅｔｙｌｔｒａｎｓｆｅｒａｓｅ ２）、ＥＬＯＶＬ脂肪酸エロンガーゼ５（ＥＬＯＶＬ
ｆａｔｔｙ ａｃｉｄ ｅｌｏｎｇａｓｅ ５）、ステアロイルＣｏＡデサチュラーゼ（Δ９デサチュラーゼ）（ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ（ｄｅｌｔａ−９−ｄｅｓａｔｕｒａｓｅ））、脂肪酸シンターゼ（ｆａｔｔｙ ａｃｉｄ ｓｙｎｔｈａｓｅ）の遺伝子発現が抑制されているため、脂肪細胞中の脂肪の蓄積量が少ないと考えられる。このことから、３ＨＢには、脂肪酸の合成自体を抑制する作用があることも明らかになった。
3. Results of gene expression test Table 6 is a table summarizing the results of gene expression analysis of genes relating to differentiation of preadipocytes into adipocytes, and Table 7 shows the results of gene expression analysis of genes relating to fatty acid synthesis. Is a table summarizing.
As shown in Table 6, peroxisome proliferator-activated receptor δ (peroxisome proliferator-activated receptor delta), lipoprotein lipase, lipoprotein lipase 1, perilipin 1 and fatty acid binding protein 3 (dipate indipate) muscle and heart (mammary-derived growth inhibitor)), matrix metallopeptidase 1 (interstitial collagenase) (matrix metallopeptidase 1 (interstitial collagenase )), stearoyl-CoA desaturase (delta 9-desaturase) (stearoyl-CoA d Since the gene expression of Saturase (delta-9-desaturase) is suppressed, there are undifferentiated cells as preadipocytes, and as a result of a relatively small number of adipocytes that store fat after differentiation, In the oil red O staining test, it is considered that fat accumulation was reduced when 3HB was present in the medium. From this, it was revealed at the gene level that 3HB acts on pre-adipocytes and cells undergoing differentiation from pre-adipocytes to adipocytes to suppress differentiation.
In addition, as shown in Table 7, hydroxyacyl-CoA dehydrogenase / 3-ketoacyl-CoA thiolase / enoyl-CoA hydratase (trifunctional protein) α-subunit (hydroxyacyl-CoA dehydrogenase / 3-ketoacyl-CoA thiolase / enoyl-Cohydrylase) protein, alpha subunit), hydroxyacyl-CoA dehydrogenase, acetyl-CoA acetyltransferase 2 (ELOVL fatty acid elongase 5 (ELOV), and acetyl-CoA acetyltransferase 2 (ELOVL fatty acid elongase 5).
fatty acid elongase 5), stearoyl-CoA desaturase (delta 9-desaturase) (stearoyl-CoA desaturase (delta -9-desaturase)), since the gene expression of fatty acid synthase (fatty acid synthase) is suppressed, the fat in adipocytes Is considered to be low. This also revealed that 3HB has an effect of suppressing the synthesis of fatty acids itself.
〔３ＨＢの分化抑制能の確認試験２〕
以下、ヒト白色脂肪前駆細胞を分化誘導段階から被験物質（３ＨＢＮａ）を添加した場合（条件Ａ）、及び分化維持段階の途中から被験物質（３ＨＢＮａ）を添加した場合（条件Ｂ）における被験物質による分化抑制能を検証した方法及び結果について説明する。尚、図９には、確認試験２におけるオイルレッドＯ染色試験の流れを模式的に示した。尚、図９中において、オイルレッドＯ染色による測定過程は確認試験１と同様であるため、図示を省略し、条件Ａ及び条件Ｂの場合についての細胞培養過程のみを図示した。
[Confirmation test 2 of 3HB's ability to inhibit differentiation]
Hereinafter, depending on the test substance when the test substance (3HBNa) is added to the human white adipose precursor cells from the differentiation induction stage (condition A) and when the test substance (3HBNa) is added during the differentiation maintenance stage (condition B). The method and results of verifying the ability to inhibit differentiation will be described. FIG. 9 schematically shows a flow of the oil red O staining test in the confirmation test 2. In FIG. 9, the measurement process by oil red O staining is the same as the confirmation test 1, so that the illustration is omitted, and only the cell culture process for the conditions A and B is illustrated.
尚、検証のための各種試験では、確認試験１と同様の被験物質、細胞、試薬、試験用キットを使用した。 In various tests for verification, the same test substances, cells, reagents, and test kits as those used in confirmation test 1 were used.
各種試験は、以下のように行った。
１．被験物質の調製
４５．９８％の３ＨＢＮａ溶液を、所定モル濃度（０．７６ｍＭ、３．８ｍＭ、１９ｍＭ）の３ＨＢを含む培地となるように適宜希釈した上で試験培地に添加し、ろ過滅菌（０．２２μｍ）後に使用した。
Various tests were performed as follows.
1. Preparation of Test Substance A 45.98% 3HBNa solution is appropriately diluted to a medium containing 3HB at a predetermined molar concentration (0.76 mM, 3.8 mM, 19 mM), and then added to the test medium. 0.22 μm).
２．オイルレッドＯ染色試験
２−１．細胞培養
確認試験１と同様に、増殖培地上での培養、分化誘導培地上での培養及び分化維持培地上での培養を行った。
具体的に、条件Ａでは、増殖培地上での培養を１日間、０．７６ｍＭ、３．８ｍＭ、１９ｍＭの３ＨＢを含む分化誘導培地上での培養を３日間、０．７６ｍＭ、３．８ｍＭ、１９ｍＭの３ＨＢを含む分化維持培地上での培養を１８日間行い、分化維持培地上での培養時は、２〜３日おきに培地を交換した。
また、条件Ｂでは、増殖培地上での培養を１日間、３ＨＢを含まない分化誘導培地上での培養を３日間、３ＨＢを含まない分化維持培地上での培養を１１日間、０．７６ｍＭ、３．８ｍＭ、１９ｍＭの３ＨＢを含む分化維持培地上での培養を７日間行い、分化維持培地上での培養時は、２〜３日おきに培地を交換した。
２−２．オイルレッドＯ染色試験
条件Ａ及び条件Ｂで培養した細胞について、確認試験１と同様の手順でオイルレッドＯ染色を実施した。
2. Oil red O dyeing test 2-1. Cell culture In the same manner as in Confirmation test 1, culture on a growth medium, culture on a differentiation induction medium, and culture on a differentiation maintenance medium were performed.
Specifically, under the condition A, culture on a growth medium for 1 day, culture on a differentiation-inducing medium containing 0.76 mM, 3.8 mM, and 19 mM 3HB for 3 days, 0.76 mM, 3.8 mM, Culture on a differentiation maintenance medium containing 19 mM 3HB was performed for 18 days, and during culture on the differentiation maintenance medium, the medium was replaced every two to three days.
In condition B, culture on a growth medium for 1 day, culture on a differentiation induction medium not containing 3HB for 3 days, culture on a differentiation maintenance medium without 3HB for 11 days, 0.76 mM, Culture on a differentiation maintenance medium containing 3.8 mM and 19 mM 3HB was performed for 7 days, and during culture on the differentiation maintenance medium, the medium was replaced every 2 to 3 days.
2-2. Oil Red O Staining Test Oil red O staining was performed on the cells cultured under the conditions A and B in the same procedure as the confirmation test 1.
３．生細胞数測定試験
３−１．細胞培養
上記２−１と同手法により条件Ａ及び条件Ｂにて細胞培養を実施した。
３−２．生細胞数の計測
確認試験１と同様の手順で行った。
3. Viable cell count measurement test 3-1. Cell culture Cell culture was performed under the conditions A and B in the same manner as in 2-1.
3-2. Measurement of the number of viable cells The same procedure as in confirmation test 1 was performed.
以下、各種試験結果について説明する。 Hereinafter, various test results will be described.
１．オイルレッドＯ染色試験の結果
表８は、条件Ａにて３ＨＢのモル濃度を０．７６ｍＭ、３．８ｍＭ、１９ｍＭとした培地で培養した細胞、条件Ｂにて３ＨＢのモル濃度を０．７６ｍＭ、３．８ｍＭ、１９ｍＭとした培地で培養した細胞、３ＨＢを添加していない培地で培養した細胞（コントロールＡ，Ｂ）に関するオイルレッドＯ染色試験の結果をまとめた表である。図１０は、オイルレッドＯ染色試験の結果を示すグラフであり、同図において、縦軸はコントロールＡ又はコントロールＢの吸光度に対する割合である。
表８及び図１０から分かるように、分化誘導段階から培地中に３ＨＢが存在している場合（条件Ａ）と、分化維持段階の途中から培地中に３ＨＢが存在している場合（条件Ｂ）とを比較すると、条件Ａの方が若干吸光度の減少割合が大きくなっているが、両者の間に顕著な差は見られず、いずれの条件でも、培地中に含まれる３ＨＢのモル濃度が高くなるほど吸光度が減少、即ち、脂肪の蓄積量が少なくなっている。特に、条件Ａについては３ＨＢのモル濃度が３．８ｍＭ、１９ｍＭの場合に有意な脂肪蓄積の抑制を確認でき、条件Ｂについては３ＨＢのモル濃度が１９ｍＭの場合に有意な脂肪蓄積の抑制を確認できた。尚、条件Ｂについては、３ＨＢを含まない培地で細胞を培養している間に脂肪の蓄積量が経時的に増加していくことを確認している。以上のことから、３ＨＢは、脂肪前駆細胞から脂肪細胞へと既に変化している細胞に対しても作用することが分かり、脂肪細胞での脂肪滴の蓄積を抑制するという分化抑制能を３ＨＢが有することが明らかとなった。
1. Results of Oil Red O Staining Test Table 8 shows cells cultured in a medium in which the molar concentration of 3HB was 0.76 mM, 3.8 mM, and 19 mM under condition A, and the molar concentration of 3HB was 0.76 mM under condition B. It is the table | surface which summarized the result of the oil red O staining test about the cell (control A, B) which culture | cultivated in the culture medium which adjusted to 3.8 mM and 19 mM, and the culture medium which did not add 3HB. FIG. 10 is a graph showing the results of the oil red O staining test. In FIG. 10, the vertical axis indicates the ratio of the control A or the control B to the absorbance.
As can be seen from Table 8 and FIG. 10, when 3HB is present in the medium from the differentiation induction stage (condition A), and when 3HB is present in the medium during the differentiation maintenance stage (condition B). When compared with the above, the decrease rate of the absorbance was slightly larger in the condition A, but no remarkable difference was observed between the two, and in any condition, the molar concentration of 3HB contained in the medium was high. The absorbance decreases, that is, the amount of accumulated fat decreases. In particular, significant suppression of fat accumulation was confirmed when the molar concentration of 3HB was 3.8 mM and 19 mM for condition A, and significant suppression of fat accumulation was confirmed when the molar concentration of 3HB was 19 mM for condition B. did it. In addition, about the condition B, while culturing the cell in the medium which does not contain 3HB, it has confirmed that the accumulation amount of fat increases with time. From the above, it was found that 3HB also acts on cells that have already changed from preadipocytes to adipocytes, and that 3HB has a differentiation inhibitory ability of suppressing the accumulation of lipid droplets in adipocytes. It became clear to have.
２．生細胞数測定試験の結果
表９は、条件Ａにて３ＨＢのモル濃度を０．７６ｍＭ、３．８ｍＭ、１９ｍＭとした培地で培養した細胞、条件Ｂにて３ＨＢのモル濃度を０．７６ｍＭ、３．８ｍＭ、１９ｍＭとした培地で培養した細胞、上記コントロールＡ，Ｂに関する生細胞数測定試験の結果をまとめた表である。また、図１１は、生細胞数測定試験の結果を示すグラフであり、同図において、縦軸は細胞生存度である。
表９及び図１１から明らかなように、条件Ａ及び条件Ｂのいずれの条件であっても、培地中の３ＨＢのモル濃度にかかわらず、生細胞数に大きな変化は見られない。したがって、上記条件Ａ及び条件Ｂのいずれの条件においても確認できた脂肪の蓄積量の減少は、細胞数が減少したことによるものでないことが明らかとなった。
2. Table 9 shows that cells cultured in a medium having a molar concentration of 3HB of 0.76 mM, 3.8 mM, and 19 mM under condition A, a molar concentration of 3HB of 0.76 mM under condition B, It is the table | surface which summarized the result of the viable cell number measurement test regarding the cell cultured by the culture medium which made 3.8 mM and 19 mM, and said control A and B. FIG. 11 is a graph showing the results of the viable cell count measurement test. In FIG. 11, the vertical axis represents the cell viability.
As is clear from Table 9 and FIG. 11, no significant change is observed in the number of viable cells regardless of the molar concentration of 3HB in the medium under any of the conditions A and B. Therefore, it was clarified that the decrease in the amount of accumulated fat, which could be confirmed under any of the conditions A and B, was not due to a decrease in the number of cells.
体内での脂肪の蓄積の抑制を可能にする脂肪細胞分化抑制剤及びこれを用いた化粧料、並びに脂肪細胞分化抑制方法に利用することができる。 The present invention can be used for a fat cell differentiation inhibitor capable of suppressing the accumulation of fat in the body, a cosmetic using the same, and a method for suppressing fat cell differentiation.
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