JP6420645B2 - PGC1α expression promoter - Google Patents

PGC1α expression promoter Download PDF

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JP6420645B2
JP6420645B2 JP2014243670A JP2014243670A JP6420645B2 JP 6420645 B2 JP6420645 B2 JP 6420645B2 JP 2014243670 A JP2014243670 A JP 2014243670A JP 2014243670 A JP2014243670 A JP 2014243670A JP 6420645 B2 JP6420645 B2 JP 6420645B2
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pgc1α
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pgc1α expression
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JP2016108244A (en
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博 吉野
博 吉野
重則 奈良
重則 奈良
真逸 大島
真逸 大島
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Shiratori Pharmaceutical Co Ltd
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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.

Nature, 464, 1313-1319(2010)Nature, 464, 1313-1319 (2010) Biochimica et Biophysica Acta, General Subjects, 496(2), 458-74 (1977)Biochimica et Biophysica Acta, General Subjects, 496 (2), 458-74 (1977) 日本薬理学雑誌, 74, 459-466 (1978)Japanese Journal of Pharmacology, 74, 459-466 (1978) J Appl Physiol 115, 260-267 (2013)J Appl Physiol 115, 260-267 (2013) Toxicology In Vitro, 27(6), 1830-1837 (2013)Toxicology In Vitro, 27 (6), 1830-1837 (2013)

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.

内臓脂肪におけるPGC1α発現促進作用を示す図である。***<0.001。It is a figure which shows the PGC1 (alpha) expression promotion effect in visceral fat. *** <0.001. 筋肉細胞におけるPGC1α発現促進作用を示す図である。It is a figure which shows the PGC1 (alpha) expression promotion effect | action in a muscle cell.

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2%HS(Horse Serum)/DMEM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、COインキュベーター(5%CO、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、CO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、COインキュベーター(5% CO、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、CO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、CO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 2 % HS (horse serum) / DMEM 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 was replaced with a sample-containing differentiation medium and cultured in a CO 2 incubator to process the sample. 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及びクリルオイル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. As a negative control, a test medium without addition of caffeine and krill oil was used.

(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、有意水準を記載する場合は*<0.05、**<0.01、***<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 an average value ± standard error. When describing a significance level, * <0.05, ** <0.01, and *** <0.001.

(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.

Claims (3)

(a)カフェインと、(b)クリルオイルとを含有する筋肉細胞におけるPGC1α発現促進剤。 A PGC1α expression promoter in muscle cells containing (a) caffeine and (b) krill oil . (a)カフェインと(b)クリルオイルの含有質量比(a/b)が、1/5〜5/1である請求項1記載の筋肉細胞におけるPGC1α発現促進剤。 The PGC1α expression promoter in muscle cells according to claim 1 , wherein the mass ratio (a / b) of (a) caffeine and (b) krill oil is 1/5 to 5/1. 筋肉細胞におけるPGC1α発現促進剤が、筋肉細胞におけるPGC1α発現促進用の医薬品、医薬部外品、化粧品又は機能性食品組成物である請求項1又は2記載の筋肉細胞におけるPGC1α発現促進剤。The PGC1α expression promoter in muscle cells according to claim 1 or 2, wherein the PGC1α expression promoter in muscle cells is a pharmaceutical, quasi-drug, cosmetic or functional food composition for promoting PGC1α expression in muscle cells.
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