JP2016108244A - PGC1α EXPRESSION PROMOTER - Google Patents
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Abstract
Description
本発明は、ＰＧＣ１α発現促進剤に関する。 The present invention relates to a PGC1α expression 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、中性脂肪を分解し、分解して生じた遊離脂肪酸をエネルギーとして消費(燃焼)させる必要がある。しかしながら、効果的に遊離脂肪酸を消費(燃焼)させるためには、ある程度継続的に有酸素運動を実施する必要があり(例えばウォーキングであれば１０分以上等)、遊離脂肪酸を消費(燃焼)させられなければ、遊離脂肪酸が中性脂肪に再合成され、再び脂肪細胞に蓄積するため、痩せないこととなる。一般的に痩せるためには運動が必要である。
しかし、遊離脂肪酸や糖を燃焼してエネルギーを産生する役割を持つミトコンドリアの増加・活性化させることにより、運動を必要とせず、肥満を改善させられる可能性がある。そのミトコンドリアの増加・活性化に影響を与えるのが遺伝子転写を制御するタンパク質であるＰＧＣ−１α(Peroxisome proliferator-activated receptor γ coactivator-１α)であり、ＰＧＣ−１αの発現を促進することにより、ミトコンドリアが増加することが知られている（非特許文献１）。
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 and consume (burn) free fatty acids generated by the decomposition as energy. However, in order to consume (burn) free fatty acids effectively, it is necessary to carry out aerobic exercise to some extent continuously (for example, 10 minutes or more if walking), and consume (burn) free fatty acids. Otherwise, free fatty acids will be re-synthesized into neutral fat and will again accumulate in the adipocytes, which will not fade. Generally, exercise is necessary to lose weight.
However, by increasing and activating mitochondria that play a role in producing energy by burning free fatty acids and sugars, there is a possibility that obesity can be improved without requiring exercise. PGC-1α (Peroxisome proliferator-activated receptor γ coactivator-1α), a protein that regulates gene transcription, affects the increase and activation of mitochondria. By promoting the expression of PGC-1α, mitochondria Is known to increase (Non-Patent Document 1).
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）が種々報告されており（非特許文献２〜５）、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。 Various components (for example, caffeine) having an action of suppressing or decomposing neutral fat, particularly body fat, have been reported (Non-patent Documents 2 to 5), amino acids such as branched amino acids, Supplements and foods and drinks containing plant extracts 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、ミトコンドリアの増加・活性化に着目した新たな体脂肪燃焼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 new body fat combustion promoter which paid its attention to the increase and activation of a mitochondria.
そこで本発明者は、種々の動物由来の成分のＰＧＣ１α発現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、これらの成分をそれぞれ単独で作用させた場合に比べて相乗的に優れた内臓脂肪細胞及び筋肉細胞におけるＰＧＣ１α発現促進作用を有し、ミトコンドリアの増加・活性化を介してエネルギー消費を促進させる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 examined the action of various animal-derived components on the expression of PGC1α, and the combination of caffeine and a phospholipid containing a ω3 unsaturated fatty acid as a constituent fatty acid acts on each of these components alone. It has been found that it has a synergistically superior PGC1α expression promoting action in visceral adipocytes and muscle cells compared to the case where it is applied, and is useful as an agent for promoting energy consumption through the increase and activation of mitochondria. Completed the invention.
すなわち、本発明は、次の〔１〕〜〔３〕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ＰＧＣ１α発現促進剤。
〔２〕内臓脂肪細胞及び／又は筋肉細胞におけるＰＧＣ１αの発現促進剤である〔１〕記載のＰＧＣ１α発現促進剤。
〔３〕（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〕又は〔２〕記載のＰＧＣ１α発現促進剤。
That is, the present invention provides the following [1] to [3].
[1] A PGC1α expression promoter containing (a) caffeine and (b) a phospholipid containing a ω3 unsaturated fatty acid as a constituent fatty acid.
[2] The PGC1α expression promoter according to [1], which is a PGC1α expression promoter in visceral fat cells and / or muscle cells.
[3] (b) The PGC1α expression promoter according to [1] or [2], wherein the phospholipid containing a ω3 unsaturated fatty acid as a constituent fatty acid is derived from krill oil.
本発明のＰＧＣ１α発現促進剤によれば、内臓細胞及び／又は筋肉細胞におけるＰＧＣ１αの発現が顕著に促進されるため、ミトコンドリアの増加・活性化を介してエネルギー消費が促進される。 According to the PGC1α expression promoting agent of the present invention, expression of PGC1α in visceral cells and / or muscle cells is remarkably promoted, so that energy consumption is promoted through increase / activation of mitochondria.
本発明のＰＧＣ１α発現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 PGC1α expression promoter of the present invention are two components: (a) caffeine and (b) a phospholipid containing a ω3 unsaturated fatty acid as a constituent fatty acid.
（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、ＰＧＣ１α発現に対する作用については知られていない。 (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, and the like, and is known to have a fat burning effect, but is not known for its action on PGC1α expression.
（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ＰＧＣ１α発現促進剤は、ＰＧＣ１α発現促進効果の点から、（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。 The PGC1α expression promoter of the present invention preferably has a mass ratio (a / b) of (a) component to (b) component of 1/100 to 100/1 from the viewpoint of the PGC1α expression promoting effect. / 50 to 50/1 is more preferable, 1/20 to 20/1 is further preferable, 1/10 to 10/1 is further preferable, and 1/5 to 5/1 is preferable. Is more preferable.
（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、優れた内臓脂肪細胞及び筋肉細胞におけるＰＧＣ１α発現促進作用を有することから、エネルギー消費促進剤として有用である。本発明のＰＧＣ１α発現促進剤を摂取すれば、内臓脂肪細胞及び筋肉細胞におけるミトコンドリアの増加・活性化を介し、エネルギー消費が促進され、肥満が予防又は改善され、痩身効果が得られる。 The composition containing (a) caffeine and (b) phospholipid has an excellent PGC1α expression promoting action in visceral fat cells and muscle cells, as shown in Examples below. Useful. When the PGC1α expression promoter of the present invention is ingested, energy consumption is promoted through the increase and activation of mitochondria in visceral fat cells and muscle cells, obesity is prevented or improved, and a slimming effect is obtained.
本発明のＰＧＣ１α発現促進剤は、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 PGC1α expression promoter of the present invention can be used by being blended with pharmaceuticals, quasi drugs, cosmetics and functional foods. The administration form is preferably oral administration or transdermal administration.
本発明のＰＧＣ１α発現促進剤を、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 PGC1α expression promoter of the present invention is used as a pharmaceutical, quasi-drug, cosmetic, or functional food, the mixture of the component (a) and the component (b) can be used as it is, but the effect 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, acidulants Various carriers such as 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ＰＧＣ１α発現促進剤には、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 PGC1α expression promoter of the present invention may contain other components that contribute to reducing body fat, such as garcinia extract, gymnema, capsaicin, fenugreek seed extract, indigestible dextrin, and other dietary fibers.
本発明のＰＧＣ１α発現促進剤中には（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ＰＧＣ１α発現促進剤の摂取量は、（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 PGC1α expression 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 amount of the PGC1α expression promoter of the present invention is preferably 10 to 50,000 mg, more preferably 100 to 5,000 mg per day in terms of the total of 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
（１）ヒト筋芽細胞の培養
ヒト筋芽細胞は、凍結細胞を融解して１０mLのＤＭＥＭ／２％ＨＳ（Ｈｏｒｓｅ Ｓｅｒｕｍ）培地（以下試験培地と称する）に添加し、１８０ｇで５分間遠心した後上清を除去し、６．２５mLの試験培地に細胞を再懸濁した。これを９６ｗｅｌｌプレートの各ｗｅｌｌに２００μＬずつ播種し、ＣＯ２インキュベーター（５％ＣＯ２、３７℃）で７２時間培養して筋管細胞に分化させた。培養後、各サンプルを添加した試験培地に交換し、ＣＯ２インキュベーターで培養してＲＮＡ抽出に用いた。
（２）内臓脂肪細胞の培養
ヒト前駆脂肪細胞（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。
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。サンプル処理時間３時間の細胞を、ＲＮＡ抽出に用いた。
Example 1
(1) Culture of human myoblasts For human myoblasts, frozen cells were thawed and added to 10 mL of DMEM / 2% HS (horse serum) medium (hereinafter referred to as test medium) and centrifuged at 180 g for 5 minutes. The post-supernatant was removed and the cells were resuspended in 6.25 mL test medium. 200 μL of this was seeded on each well of a 96-well plate and cultured in a CO 2 incubator (5% CO 2 , 37 ° C.) for 72 hours to differentiate into myotube cells. After culturing, the medium was replaced with a test medium to which each sample was added, and the cells were cultured in a CO 2 incubator and used for RNA extraction.
(2) Culture of visceral adipocytes Human preadipocytes (visceral fat) were asleep in a T-75 flask using a growth medium and cultured until 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.
Preadipocytes were seeded in a 96-well plate at 10,000 cells / 100 μL / well using growth medium and cultured in a CO 2 incubator (5% CO 2 , 37 ° C.) 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 the 10th day after differentiation induction, the sample-containing differentiation medium was replaced, and the cells were cultured in a CO 2 incubator and processed. Cells with a sample treatment time of 3 hours were used for RNA extraction.
（２）サンプル調製
試験培地を用いて、カフェイン１０００μｇ／mL、カフェイン２０００μｇ／mL、クリルオイル１０００μｇ／mL、カフェイン＋クリルオイル各１０００μｇ／mLとなるよう調製した。カフェイン添加試験培地は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。陰性対照にはカフェイン無添加の試験培地を用いた。
(2) Sample preparation Using a test medium, caffeine was prepared at 1000 μg / mL, caffeine at 2000 μg / mL, krill oil at 1000 μg / mL, and caffeine + krill oil at 1000 μg / mL. The caffeine-added test 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. A test medium without caffeine was used as a negative control.
（３）トータルＲＮＡ抽出およびｃＤＮＡ化
細胞からのトータルＲＮＡの回収およびｃＤＮＡ化はＦａｓｔＬａｎｅ Ｃｅｌｌ ＲＴ−ＰＣＲキットの手順に従い行った。
(3) Total RNA extraction and cDNA conversion Total RNA from cells and cDNA conversion were performed according to the procedure of FastLane Cell RT-PCR kit.
（４）リアルタイムＰＣＲ
リアルタイムＰＣＲはＳＹＢＲ Ｇｒｅｅｎ Ｉを用いたインターカレーター法で行った。具体的にはＲｅａｌ−ｔｉｍｅ ＰＣＲ専用プレートに表１のように反応液を調製し、ＰＣＲ反応を行った。
(4) Real-time PCR
Real-time PCR was performed by an intercalator method using SYBR Green I. Specifically, a reaction solution was prepared as shown in Table 1 on a Real-time PCR-dedicated plate, and a PCR reaction was performed.
ＰＣＲ反応は９５℃・３０ｓｅｃ−（９５℃・１０ｓｅｃ−６０℃・３０ｓｅｃ）×４５ｃｙｃｌｅｓ−９５℃・１５ｓｅｃ−５５℃・１５ｓｅｃ−９５℃・１５ｓｅｃの条件で行い、ＰＣＲ反応終了後にＰＣＲ産物の解離曲線を求めた。
相対発現量はΔΔＣｔ法により、コントロールの発現量を１．０となるよう算出した。内部標準遺伝子としてＡＣＴＢ（β−Ａｃｔｉｎ）遺伝子を用いた。試験に使用したプライマー配列を表２に示した。
PCR reaction is carried out under the conditions of 95 ° C., 30 sec- (95 ° C., 10 sec-60 ° C., 30 sec) × 45 cycles-95 ° C., 15 sec-55 ° C., 15 sec-95 ° C., 15 sec, and the PCR product dissociation curve after completion of the PCR reaction Asked.
The relative expression level was calculated by the ΔΔCt method so that the control expression level was 1.0. The ACTB (β-Actin) gene was used as an internal standard gene. Table 2 shows the primer sequences used in the test.
（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。
(5) Statistical analysis Significant difference test was performed in the comparative test section (non-added section vs. sample-added section). 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.
（６）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）
(6) Test substance / caffeine (extract) (Shirotori Pharmaceutical Co., Ltd.)
・ Krill oil (Seiko Pharmaceutical Co., Ltd., containing 41 mass% of ω3 highly unsaturated fatty acid phospholipid)
（７）細胞
・ヒト骨格筋筋芽細胞（Ｇｉｂｃｏ，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．８Ｆ３４８２，４６歳、白色人種、男性、ＢＭＩ＝２６）
(7) Cells / human skeletal muscle myoblasts (Gibco, Cat. No. PT-5020, Lot. No. 0000279746, 38 years old, Asian race, female, BMI = 26)
-Human preadipocytes (visceral fat) (Lonza, Cat. No. PT-5005, Lot. No. 8F3482, 46 years old, white race, male, BMI = 26)
（８）結果
ｉ）内臓脂肪細胞におけるＰＧＣ１αの発現促進作用を図１に示す。
図１より、カフェイン及びクリルオイルの併用により、内臓脂肪細胞におけるＰＧＣ１α発現促進作用は、相乗的に増強された。
ii）筋肉細胞におけるＰＧＣ１α発現促進作用を図２に示す。
図２より、カフェイン及びクリルオイルの併用により、筋肉細胞におけるＰＧＣ１α発現促進作用は、相乗的に増強された。
(8) Results i) The action of promoting the expression of PGC1α in visceral fat cells is shown in FIG.
From FIG. 1, the combined use of caffeine and krill oil synergistically enhanced the PGC1α expression promoting action in visceral fat cells.
ii) PGC1α expression promoting action in muscle cells is shown in FIG.
From FIG. 2, the combined use of caffeine and krill oil synergistically enhanced the PGC1α expression promoting action in muscle cells.
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| SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY, vol. 47, no. 1, JPN6018014486, 30 November 2011 (2011-11-30), pages 49 - 58 * |
| THE FEBS JOURNAL, vol. 281, no. 3, JPN6018014487, 15 November 2013 (2013-11-15), pages 739 - 749 * |
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