JP2016108245A - Body fat reduction promoter - Google Patents
Body fat reduction promoter Download PDFInfo
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- JP2016108245A JP2016108245A JP2014243671A JP2014243671A JP2016108245A JP 2016108245 A JP2016108245 A JP 2016108245A JP 2014243671 A JP2014243671 A JP 2014243671A JP 2014243671 A JP2014243671 A JP 2014243671A JP 2016108245 A JP2016108245 A JP 2016108245A
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Abstract
Description
本発明は、体脂肪減少促進剤に関する。 The present invention relates to a body fat reduction promoter.
世界的に肥満者数は増加傾向にあり、肥満は心臓への負担増加、膝・腰等の関節に対する負担増加、生活習慣病の原因等、様々な悪影響を及ぼす。また、肥満の原因は中性脂肪であり、中性脂肪が過剰に蓄積し、脂肪細胞が増加・肥大することで理想体型を超えて体が大きくなり、外見的にも問題となる。その肥満を改善させるためには、中性脂肪を分解する必要がある。 The number of obese people is increasing worldwide, and obesity has various adverse effects such as increased burden on the heart, increased burden on joints such as knees and hips, and causes of lifestyle-related diseases. In addition, the cause of obesity is neutral fat. Neutral fat accumulates excessively, and fat cells increase and enlarge, resulting in a body that exceeds the ideal body size, which is also a problem in appearance. In order to improve the obesity, it is necessary to break down neutral fat.
中性脂肪、特に体脂肪の蓄積を抑制したり、分解する作用を有する成分、例えばカフェインが報告されており（非特許文献１〜４）、分岐アミノ酸等のアミノ酸類、各種の植物抽出物を配合したサプリメントや飲食品が販売されている。 Ingredients, such as caffeine, have been reported that have an action of inhibiting or degrading accumulation of neutral fat, especially body fat (Non-Patent Documents 1 to 4), amino acids such as branched amino acids, and various plant extracts. Supplements and foods and drinks are sold.
しかしながら、従来の体脂肪燃焼促進剤等は、その効果が十分でない等の問題がある。
従って、本発明は、体脂肪、特に皮下脂肪と内臓脂肪の両者を減少させる効果を有する体脂肪減少促進剤を提供することにある。
However, conventional body fat combustion promoters have problems such as insufficient effects.
Therefore, this invention is providing the body fat reduction promoter which has the effect of reducing both body fat, especially subcutaneous fat and visceral fat.
そこで本発明者は、種々の動物由来の成分の皮下脂肪及び内臓脂肪に対する作用を検討してきたところ、カフェインと構成脂肪酸としてω３不飽和脂肪酸を含むリン脂質との組み合わせが、同様に構成脂肪酸としてω３不飽和脂肪酸を含むトリグリセリドである魚油に比べて格別顕著に優れた皮下脂肪や内臓脂肪の分解を促進し、皮下脂肪細胞や内臓脂肪細胞への分化を抑制し、体脂肪減少促進剤として有用であることを見出し、本発明を完成した。 Therefore, the present inventor has studied the action of various animal-derived components on subcutaneous fat and visceral fat. Compared with fish oil, which is a triglyceride containing ω3 unsaturated fatty acid, it promotes the degradation of subcutaneous fat and visceral fat, which is outstandingly superior, suppresses differentiation into subcutaneous fat cells and visceral fat cells, and is useful as a body fat reduction promoter As a result, the present invention was completed.
すなわち、本発明は、次の〔１〕〜〔４〕を提供するものである。
〔１〕（ａ）カフェインと、（ｂ）構成脂肪酸としてω３不飽和脂肪酸を含むリン脂質とを含有する体脂肪減少促進剤。
〔２〕体脂肪が、皮下脂肪及び／又は内臓脂肪である〔１〕記載の体脂肪減少促進剤。
〔３〕体脂肪減少促進が、皮下脂肪及び／又は内臓脂肪の分解促進、あるいは皮下脂肪細胞及び／又は内臓脂肪細胞の分化抑制である〔１〕又は〔２〕記載の体脂肪減少促進剤。
〔４〕（ｂ）構成脂肪酸としてω３不飽和脂肪酸を含むリン脂質が、クリルオイル由来である〔１〕〜〔３〕のいずれかに記載の体脂肪減少促進剤。
That is, the present invention provides the following [1] to [4].
[1] A body fat reduction promoter containing (a) caffeine and (b) a phospholipid containing a ω3 unsaturated fatty acid as a constituent fatty acid.
[2] The body fat reduction promoter according to [1], wherein the body fat is subcutaneous fat and / or visceral fat.
[3] The body fat reduction promoter according to [1] or [2], wherein the promotion of body fat reduction is the promotion of degradation of subcutaneous fat and / or visceral fat or the suppression of differentiation of subcutaneous fat cells and / or visceral fat cells.
[4] (b) The body fat reduction promoter according to any one of [1] to [3], wherein the phospholipid containing ω3 unsaturated fatty acid as a constituent fatty acid is derived from krill oil.
本発明の体脂肪減少促進剤によれば、皮下脂肪及び／又は内臓脂肪が減少し、また皮下脂肪細胞及び／又は内臓脂肪細胞への分化が抑制されるため、皮下及び内臓の脂肪の蓄積が抑制され、肥満が改善される。 According to the body fat reduction-promoting agent of the present invention, subcutaneous fat and / or visceral fat is reduced, and differentiation into subcutaneous fat cells and / or visceral fat cells is suppressed. It is suppressed and obesity is improved.
本発明の体脂肪減少促進剤の有効成分は、（ａ）カフェインと、（ｂ）構成脂肪酸としてω３不飽和脂肪酸を含むリン脂質との２成分である。 The active ingredients of the body fat reduction promoter of the present invention are two components: (a) caffeine and (b) phospholipids containing ω3 unsaturated fatty acids as constituent fatty acids.
（ａ）カフェインは、化学名１，３，７−トリメチルキサンチンであり、コーヒー等に含まれる成分である。コーヒー豆から抽出することもできるが、公知の合成法により製造することもできる。カフェインは、中枢神経興奮作用、強心作用などを有し、脂肪燃焼効果を有することが知られているが、（ｂ）のリン脂質との併用により作用がどのように変化するのかは知られていない。 (A) Caffeine has the chemical name 1,3,7-trimethylxanthine and is a component contained in coffee and the like. Although it can be extracted from coffee beans, it can also be produced by a known synthesis method. Caffeine has a central nervous excitatory action, a cardiotonic action, etc., and is known to have a fat burning effect, but it is known how its action changes when used in combination with phospholipid (b). Not.
（ｂ）構成脂肪酸としてω３不飽和脂肪酸を含むリン脂質としては、ω３高度不飽和脂肪酸であるドコサヘキサエン酸、エイコサペンタエン酸、リノレン酸、α−リノレン酸を構成脂肪酸とするリン脂質が挙げられる。
当該（ｂ）リン脂質は、純粋なものを使用してもよいし、当該（ｂ）リン脂質を含有する天然脂質を用いてもよい。（ｂ）リン脂質を含有する天然脂質としては、例えばオキアミ由来油、特に南極オキアミ由来油（クリルオイル）が好ましい。オキアミ由来油には、ω３高度不飽和脂肪酸含有リン脂質が３５〜７０質量％含まれるので、これを用いるのが好ましい。これらのオキアミ由来油は市販品を使用することができる。
(B) Examples of phospholipids containing ω3 unsaturated fatty acids as constituent fatty acids include phospholipids containing ω3 highly unsaturated fatty acids such as docosahexaenoic acid, eicosapentaenoic acid, linolenic acid, and α-linolenic acid.
The (b) phospholipid may be pure, or a natural lipid containing the (b) phospholipid may be used. (B) As a natural lipid containing a phospholipid, for example, a krill-derived oil, particularly an Antarctic krill-derived oil (krill oil) is preferable. The krill-derived oil contains 35 to 70% by mass of ω3 polyunsaturated fatty acid-containing phospholipid, and is preferably used. A commercial item can be used for these krill origin oils.
本発明の体脂肪減少促進剤は、体脂肪減少促進効果の点から、（ａ）成分と（ｂ）成分の含有質量比（ａ／ｂ）が１／１００〜１００／１であるのが好ましく、１／５０〜５０／１であるのがより好ましく、１／２０〜２０／１であるのがさらに好ましく、１／１０〜１０／１であるのがさらに好ましく、１／５〜５／１であるのがさらに好ましい。 In the body fat reduction promoter of the present invention, the mass ratio (a / b) of the component (a) to the component (b) is preferably 1/100 to 100/1 from the viewpoint of the effect of promoting body fat reduction. 1/50 to 50/1, more preferably 1/20 to 20/1, further preferably 1/10 to 10/1, and 1/5 to 5/1. More preferably.
（ａ）カフェインと（ｂ）リン脂質とを含有する組成物は、後記実施例に示すように、皮下脂肪分解促進作用、内臓脂肪分解促進作用、皮下脂肪細胞への分化抑制作用、内臓脂肪細胞への分化抑制作用を有することから、皮下脂肪及び内臓脂肪を含む体脂肪減少促進剤として有用である。本発明の体脂肪減少促進剤を摂取すれば、皮下脂肪及び内臓脂肪の分解が促進され、皮下脂肪細胞及び内臓脂肪細胞への分化成熟が抑制されるので、肥満が予防又は改善され、肥満に伴う種々の生活習慣病が予防又は改善される。 The composition containing (a) caffeine and (b) phospholipid, as shown in Examples below, has a subcutaneous lipolysis promoting action, a visceral lipolysis promoting action, a differentiation inhibiting action into subcutaneous fat cells, a visceral fat Since it has an action of suppressing differentiation into cells, it is useful as a body fat reduction promoter containing subcutaneous fat and visceral fat. By ingesting the body fat loss-promoting agent of the present invention, degradation of subcutaneous fat and visceral fat is promoted and differentiation and maturation into subcutaneous fat cells and visceral fat cells is suppressed. Various lifestyle-related diseases that accompany it are prevented or ameliorated.
本発明の体脂肪減少促進剤は、医薬品、医薬部外品、化粧品及び機能性食品に配合して使用することができる。また、その投与形態は、経口投与又は経皮投与が好ましい。 The body fat reduction promoter of the present invention can be used by blending it with pharmaceuticals, quasi drugs, cosmetics and functional foods. The administration form is preferably oral administration or transdermal administration.
本発明の体脂肪減少促進剤を、医薬品、医薬部外品、化粧品、機能性食品として使用する場合には、（ａ）成分及び（ｂ）成分の混合物をそのまま使用することができるが、効果を損なわない範囲において、例えば、セルロース及びその誘導体、デンプン及びその誘導体、天然及び合成高分子等の乳糖、賦形剤、ステアリン酸及びその塩類、天然及び合成ワックス等の滑沢剤、糖類、酸味剤、香料等の種々の担体を配合することができる。また、本発明の医薬品、医薬部外品、化粧品又は機能性食品は、投与方法、投与経路に応じて散剤、顆粒剤、錠剤、丸剤、硬カプセル剤、軟カプセル剤、シロップ剤等の剤型とすることができる。 When the body fat reduction promoter of the present invention is used as a pharmaceutical product, quasi-drug, cosmetic product, or functional food, the mixture of the component (a) and the component (b) can be used as it is. For example, cellulose and derivatives thereof, starch and derivatives thereof, lactose such as natural and synthetic polymers, excipients, stearic acid and salts thereof, lubricants such as natural and synthetic wax, saccharides, acidity Various carriers such as agents and fragrances can be blended. The pharmaceutical, quasi-drug, cosmetic or functional food of the present invention is a powder, granule, tablet, pill, hard capsule, soft capsule, syrup, etc., depending on the administration method and administration route. Can be a mold.
また、本発明の体脂肪減少促進剤には、体脂肪減少に寄与する他の成分、例えばガルシニアエキス、ギムネマ、カプサイシン、コロハ種子エキス、難消化性デキストリン、その他食物繊維等を配合することができる。 In addition, the body fat reduction promoter of the present invention may contain other components that contribute to body fat reduction, such as garcinia extract, gymnema, capsaicin, fenugreek seed extract, indigestible dextrin, and other dietary fibers. .
本発明の体脂肪減少促進剤中には（ａ）成分及び（ｂ）成分を合計で１〜１００質量％、さらに５〜８０質量％、さらに１０〜７０質量％含有するのが好ましい。また、本発明の体脂肪減少促進剤の摂取量は、（ａ）成分及び（ｂ）成分合計で１日当たり、１０〜５０，０００mgが好ましく、さらに１００〜５，０００mgが好ましい。 The body fat reduction promoter of the present invention preferably contains 1 to 100% by mass, further 5 to 80% by mass, and further 10 to 70% by mass of component (a) and component (b). In addition, the intake of the body fat reduction promoter of the present invention is preferably 10 to 50,000 mg, more preferably 100 to 5,000 mg per day in total for the component (a) and component (b).
次に実施例を挙げてさらに本発明を詳細に説明する。 Next, the present invention will be described in more detail with reference to examples.
実施例１（脂肪分解促進作用）
（１）培地調製
増殖培地はPGM-2 bullet kitに付属のPreadipocyte Basal Medium-2（５００mL）に、PGM-2 bullet kit に付属のFBS, L-glutamine, GA-1000 SingleQuatsを全量添加し、１０％ FBS, ２mM L-glutamine,５０μg/mL Gentamicin, ３７ng/mL Amphotericin-Bの終濃度となるように作成した。
分化培地は２００mLの増殖培地に、PGM-2 bullet kit に付属のinsulin, dexamethasone, indomethacin, isobutyl-methylxanthineを全量添加して作成した。
Example 1 (facilitating lipolysis)
(1) Medium preparation For growth medium, add the whole amount of FBS, L-glutamine, GA-1000 SingleQuats attached to PGM-2 bullet kit to Preadipocyte Basal Medium-2 (500 mL) attached to PGM-2 bullet kit. % FBS, 2 mM L-glutamine, 50 μg / mL Gentamicin, 37 ng / mL Amphotericin-B.
The differentiation medium was prepared by adding all of insulin, dexamethasone, indomethacin, and isobutyl-methylxanthine included in the PGM-2 bullet kit to a 200 mL growth medium.
（２）サンプル調製
分化培地を用いて、それぞれ目的のサンプル終濃度となるよう調製した。カフェイン、フィッシュオイル含有培地は０．４５μｍのフィルターで滅菌して試験に用いた。クリルオイル含有培地はフィルターを透過しなかったため、クリーンベンチで無菌的に調製し、フィルター滅菌せずに試験に用いた。クリルオイルおよびフィッシュオイルは溶媒を用いず培地に直接添加して懸濁し、エマルジョンの状態で試験を行った。
陰性対照としてサンプル無添加分化培地を用いた。陽性対照として、１０μM Isoprotetenol含有培地を用いた（下記注）。１０μM Isoprotetenol含有培地は、Adipolysis Assay Kit付属のIsoprotetenol Solutionを分化培地に1/1000添加してフィルター滅菌して調製した。
（注）Isoproterenolはアドレナリンβレセプターのアゴニストとして機能する。アドレナリンが脂肪細胞のβレセプターに結合すると、プロテインキナーゼＡが活性化され、このプロテインキナーゼＡが、ホルモン感受性リパーゼを活性化する。このホルモン感受性リパーゼは、中性脂肪を遊離脂肪酸と遊離グリセロールに変換する。このようなメカニズムによって、Isoproterenolは中性脂肪の分解を促進する。
(2) Sample preparation Each sample was prepared using a differentiation medium so as to have a target final concentration. The caffeine and fish oil-containing medium was sterilized with a 0.45 μm filter and used for the test. Since the krill oil-containing medium did not permeate the filter, it was prepared aseptically on a clean bench and used for testing without filter sterilization. Kuryl oil and fish oil were added directly to the medium without using a solvent and suspended, and the test was conducted in the form of an emulsion.
A sample-free differentiation medium was used as a negative control. As a positive control, a medium containing 10 μM Isoprotetenol was used (note below). The 10 μM Isoprotetenol-containing medium was prepared by adding 1/1000 of Isoprotetenol Solution attached to the Adipolysis Assay Kit to the differentiation medium and filter sterilizing.
(Note) Isoproterenol functions as an adrenergic β receptor agonist. When adrenaline binds to the adipocyte β receptor, protein kinase A is activated, and this protein kinase A activates hormone-sensitive lipase. This hormone sensitive lipase converts neutral fat into free fatty acids and free glycerol. By this mechanism, Isoproterenol promotes the breakdown of neutral fat.
（３）ヒト前駆脂肪細胞の前培養
ヒト前駆脂肪細胞（皮下脂肪および内臓脂肪）は、増殖培地を用いてＴ−７５フラスコに起眠し、８０％ コンフルエントになるまで培養した。培地交換は２日おきに行い０．２５％Trypsin/EDTAを用いて細胞を剥離し、細胞を回収して本試験に用いた。試験には購入後継代数２の細胞を用いた。
(3) Preculture of human preadipocytes Human preadipocytes (subcutaneous fat and visceral fat) were put to sleep in a T-75 flask using a growth medium and cultured until they became 80% confluent. The medium was changed every two days, and the cells were detached using 0.25% Trypsin / EDTA, and the cells were collected and used for this test. For the test, cells with passage number 2 were used after purchase.
（３）脂肪細胞の分化誘導およびサンプル処理
前駆脂肪細胞を、増殖培地を用いて１０，０００cells/１００μL/wellで９６ウェルプレートに播種し、ＣＯ2インキュベーター（５％ ＣＯ2、３７℃）内で２４時間培養した。培養後、１００％コンフルエントになっていることを確認し、２００μLの分化培地と交換し、ＣＯ2インキュベーターで１０日間培養して脂肪細胞へ分化誘導した。分化誘導後１０日目にサンプル含有分化培地に交換し、ＣＯ2インキュベーターで培養してサンプル処理した。培養終了時に位相差顕微鏡で細胞観察を行った後、培地を回収して−２０℃保存し、後日脂肪分解量を測定した。
(3) Adipocyte differentiation induction and sample treatment Preadipocytes are seeded in a 96-well plate at 10,000 cells / 100 μL / well using a growth medium, and in a CO 2 incubator (5% CO 2 , 37 ° C.). Cultured for 24 hours. After culturing, the cells were confirmed to be 100% confluent, replaced with 200 μL of differentiation medium, and cultured in a CO 2 incubator for 10 days to induce differentiation into adipocytes. On day 10 after differentiation induction, the sample was replaced with a sample-containing differentiation medium, cultured in a CO 2 incubator, and processed. At the end of the culture, the cells were observed with a phase-contrast microscope, and the medium was collected and stored at −20 ° C., and the amount of lipolysis was measured at a later date.
（４）遊離グリセロール量定量（吸光法）
中性脂肪（トリグリセリド）は、加水分解により脂肪酸とグリセロールを生ずる。グリセロール濃度の測定により，生体内における脂質分解を評価することができる。グリセロールの定量は、基本的にAdipolysis Assay Kit（下記注）を用いた吸光法により行った。なお、培地やサンプルに含まれるグリセロールや、サンプルによる培地の着色の影響を補正するために、サンプル含有培地（ｎ＝３）についても測定を行い、その平均値をレファレンスとして、細胞処理を行った測定値から差し引いた。
（注）グリセロールを各種酵素で処理し、５４０nmの吸光度測定で検出可能なQuinoneimine dye を生ずる方法。
(4) Quantitative determination of free glycerol (absorption method)
Neutral fats (triglycerides) produce fatty acids and glycerol by hydrolysis. Lipid degradation in vivo can be evaluated by measuring glycerol concentration. Glycerol was quantitatively determined by an absorption method using an Adipolysis Assay Kit (note below). In addition, in order to correct the influence of glycerol contained in the medium and the sample and the coloring of the medium by the sample, the sample-containing medium (n = 3) was also measured, and the cell treatment was performed using the average value as a reference. Subtracted from measured value.
(Note) A method that produces quinoneimine dye that can be detected by measuring absorbance at 540 nm by treating glycerol with various enzymes.
（５）統計解析
比較試験区間（無添加区vsサンプル添加区）では有意差検定を行った。検定はStudent’s t-testで両側検定を行いＰ＜０．０５（帰無仮説が５％未満）のものを図中に示した。グラフデータは平均値±標準誤差として示した。有意水準を記載する場合、*Ｐ＜０．０５、**Ｐ＜０．０１、***Ｐ＜０．００１とした。
(5) Statistical analysis Significant difference test was performed in the comparative test section (no additive group vs. sample added group). The test was Student's t-test and two-sided test was performed, and P <0.05 (null hypothesis is less than 5%) is shown in the figure. The graph data is shown as mean ± standard error. When describing a significance level, it was set as * P <0.05, ** P <0.01, *** P <0.001.
（６）試薬
被験物質
・カフェイン（抽出物）（白鳥製薬株式会社）
・クリルオイル（清光薬品工業株式会社、ω３高度不飽和脂肪酸リン脂質41質量％含有）
・フィッシュオイル（日油株式会社、サンオメガDHA27）
（７）細胞
・ヒト前駆脂肪細胞（皮下脂肪） (Lonza, Cat. No. PT-5020, Lot. No. 0000279746, 38歳、アジア人種、女性、BMI=26)
・ヒト前駆脂肪細胞（内臓脂肪） (Lonza, Cat. No. PT-5005, Lot. No. 0000310955, 92歳、白色人種、女性、BMI=19)
・ヒト前駆脂肪細胞（内臓脂肪） (Lonza, Cat. No. PT-5005, Lot. No.8F3482, 46歳、白色人種、男性、BMI=26)
(6) Reagent Test substance ・ Caffeine (extract) (Shirotori Pharmaceutical Co., Ltd.)
・ Krill oil (Seiko Pharmaceutical Co., Ltd., containing ω3 highly unsaturated fatty acid phospholipid 41% by mass)
・ Fish oil (NOF Corporation, San Omega DHA27)
(7) Cells ・ Human preadipocytes (subcutaneous fat) (Lonza, Cat. No. PT-5020, Lot. No. 0000279746, 38 years old, Asian, female, BMI = 26)
・ Human preadipocytes (visceral fat) (Lonza, Cat. No. PT-5005, Lot. No. 0000310955, 92 years old, Caucasian, female, BMI = 19)
・ Human preadipocytes (visceral fat) (Lonza, Cat. No. PT-5005, Lot. No.8F3482, 46 years old, white, male, BMI = 26)
（８）結果
ｉ）皮下脂肪分解促進作用を図１に示す。
図１より、処理３時間後で、カフェイン及びクリルオイルを併用した場合、カフェイン及びクリルオイル単独で作用させた場合に比べて、顕著な皮下脂肪分解促進作用を示した。
(8) Results i) The subcutaneous lipolysis promoting action is shown in FIG.
As shown in FIG. 1, when caffeine and krill oil were used in combination 3 hours after the treatment, the effect of promoting subcutaneous lipolysis was significantly higher than when caffeine and krill oil were used alone.
ii）内臓脂肪分解促進作用を図２に示す。
図２より、処理２４時間及び４８時間後で、カフェイン及びクリルオイルを併用した場合、カフェイン単独で作用させた場合に比べて、顕著な内臓脂肪分解促進作用を示した。
なお、Isoproterenolは、陽性対照サンプルである。
ii) Visceral lipolysis promoting action is shown in FIG.
From FIG. 2, when caffeine and krill oil were used together 24 hours and 48 hours after the treatment, the visceral lipolysis was significantly promoted compared with the case where caffeine alone was used.
Isoproterenol is a positive control sample.
実施例２（脂肪細胞への分化抑制作用）
（１）培地調製、サンプル調製、ヒト前駆細胞の前培養、統計解析、試薬は実施例１と同様に行った。
Example 2 (Inhibition of differentiation into adipocytes)
(1) Medium preparation, sample preparation, preculture of human progenitor cells, statistical analysis, and reagents were performed in the same manner as in Example 1.
（２）脂肪細胞の分化誘導およびサンプル処理
前駆脂肪細胞を、増殖培地を用いて１０，０００cells/１００μL／wellで９６ウェルプレートに播種し、ＣＯ2インキュベーター（５％ＣＯ2、３７℃）内で２４時間培養した。培養後、１００％コンフルエントになっていることを確認し、２００μＬのサンプル含有分化培地と交換し、ＣＯ2インキュベーターで培養して脂肪細胞への分化誘導を開始した。分化誘導後４日目と７日目に新しいサンプル含有分化培地に交換した。各種サンプル解析は分化誘導開始後４日目、７日目、１０日目にオイルレッド染色を行い、脂肪細胞分化率を経時的に解析した。各ｎ＝５で試験した。カフェインおよびクリルオイルの濃度効果解析は、１０日目にオイルレッド染色を行い、脂肪細胞分化率を解析した。各ｎ＝３で試験した。
(2) Adipocyte differentiation induction and sample treatment Preadipocytes are seeded in a 96-well plate at 10,000 cells / 100 μL / well using a growth medium, and in a CO 2 incubator (5% CO 2 , 37 ° C.). Cultured for 24 hours. After culturing, the cells were confirmed to be 100% confluent, replaced with 200 μL of sample-containing differentiation medium, and cultured in a CO 2 incubator to initiate differentiation into adipocytes. On day 4 and day 7 after differentiation induction, the sample was replaced with a new sample-containing differentiation medium. In various sample analyzes, oil red staining was performed on the 4th, 7th, and 10th days after the start of differentiation induction, and the adipocyte differentiation rate was analyzed over time. Each n = 5 was tested. Concentration effect analysis of caffeine and krill oil was performed by oil red staining on the 10th day to analyze adipocyte differentiation rate. Each n = 3 was tested.
（３）オイルレッド染色
オイルレッド溶液は、１５０mgのオイルレッドＯを５０mLの１００％ ２−プロパノールに溶解した後、脱イオン水を３３mL加え、ろ紙で濾過して調製した。オイルレッド染色は、以下のプロトコールで行い、染色後に位相差顕微鏡観察および定量化を行った。
(3) Oil Red Staining The oil red solution was prepared by dissolving 150 mg of oil red O in 50 mL of 100% 2-propanol, adding 33 mL of deionized water, and filtering with filter paper. Oil red staining was performed according to the following protocol. After staining, phase contrast microscopy and quantification were performed.
（４）結果
（ｉ）皮下脂肪細胞への分化抑制作用を図３に示す。図３より、カフェイン及びクリルオイルを併用した場合、カフェイン及びクリルオイル単独で作用させた場合に比べて顕著な皮下脂肪細胞への分化抑制作用を示した。
(4) Results (i) The action of inhibiting differentiation into subcutaneous fat cells is shown in FIG. FIG. 3 shows that when caffeine and krill oil are used in combination, the effect of inhibiting differentiation into subcutaneous adipocytes is more marked than when caffeine and krill oil are used alone.
（ii）内臓脂肪細胞への分化抑制作用を図４に示す。図４よりカフェイン及びクリルオイルを併用した場合、カフェイン及びクリルオイル単独で作用させた場合に比べて顕著な内臓脂肪細胞への分化抑制作用を示した。
なお、LiClは陽性対照である。
(Ii) The action of inhibiting differentiation into visceral fat cells is shown in FIG. As shown in FIG. 4, when caffeine and krill oil were used in combination, the effect of inhibiting differentiation into visceral adipocytes was marked compared to when caffeine and krill oil were used alone.
LiCl is a positive control.
Claims (4)
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