JP2019043928A - プロテクチンdxを有効成分として含有する高脂血症又は脂肪肝疾患の予防又は治療用組成物 - Google Patents
プロテクチンdxを有効成分として含有する高脂血症又は脂肪肝疾患の予防又は治療用組成物 Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/191—Carboxylic acids, e.g. valproic acid having two or more hydroxy groups, e.g. gluconic acid
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P3/04—Anorexiants; Antiobesity agents
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/3262—Foods, ingredients or supplements having a functional effect on health having an effect on blood cholesterol
Abstract
Description
マルジョン、水性組成物、リポソーム、マイクロビーズ及びミクロソームが含まれる。
以下、本発明を詳細に説明する。
ただし、下記実施例は本発明を例示するのみであり、本発明の内容が下記実施例に限定されるものではない。
細胞培養、試薬及び抗体
ヒトの肝臓癌細胞HepG2細胞(ATCC、Manassas、VA、USA)は、10%牛胎児血清(FBS、fetal bovine serum、Invitrogen)に100units/mLペニシリン及び100μg/mlストレプトマイシン(Invitrogen)を含む、高グルコースダルベッコ変形イーグル培地(DMEM、Dulbecco’s modified Eagle medium、Invitrogen、Carisbad、CA、USA)で培養した。細胞は37℃、5%CO2条件で培養した。
この研究は機関動物検討委員会(高麗大学校機関動物保護及び利用委員会、ソウル、大韓民国)の承認を得た。動物実験は実験室動物の管理と使用のためのガイド(NIH publication、8th edition、2011)により行われた。対照群と8週の雄C57BL/6J(B6)マウスに対する2グループに正常食餌(ND、Brogaarden、Gentofte、Denmark)とHFD(高脂肪食餌、High Fat Diet、Research Diets、New Brunswick、NJ、USA)を8週間それぞれ投与した。HFD(High Fat Diet)食餌群に対してPDXを8週間腹腔内に投与した(1μg/mice/day)。エタノール注入群を対照群に用いた。
HepG2を収得して、タンパク質を4℃で60分間溶解緩衝液(PRO-PREP;Intron Biotechnology、Seoul Korea)で抽出した。タンパク質試料(35μg)を12%SDS-PAGEに適用してニトロセルロース膜(Amersham Bioscience、Westborough、MA、USA)に移して1次抗体で検出した後、2次抗体と西洋ワサビペルオキシダーゼ(Santa Cruz Biotechnology)を接合させた2次抗体で調査した。サンプルはECLキットで検出した。
HepG2細胞の総タンパク質を、免疫沈殿緩衝液(IPバッファー:50mM Tris-HCL、pH7.8、150mM NaCl、1% IGEPAL CA630)で抽出し、1mg/mLの濃度で稀釈した。FOXO1(Santa Cruz Biotechnology)に対するポリクロナール抗体を1:150の稀釈率で混合物に添加して、サンプルを4℃で一晩培養した。培養後、Protein A/G-Sepharoseビーズ懸濁液(Santa Cruz Biotechnology)50μLを各試料に添加して、4℃で1時間柔らかに混合した。試料を12,000rpmで30秒間遠心分離して;ビーズをIP緩衝液で3回洗浄した。分離したビーズを1×SDS-PAGEローディング緩衝液に再懸濁して、95℃で5分間加熱し、ボルテックスミキサーで撹拌してフラッシュ遠心分離した。上澄液を電気泳動分離及びウエスタンブロット分析のため、12%SDSポリアクリルアミドゲル上にローディングした。
70%コンフルエントで、ORP150に対する0-20nmol/L低干渉(si)RNAオリゴヌクレオチドをSanta Cruz Biotechnologyから購入して、トランスフェクションして遺伝子発現を抑制した。スクランブルsiRNAを対照群に使用した。2又は4μgのpCMV3-0RP150(Sino Biological、Beijing、China)を一過的にトランスフェクションさせてORP150発現を過発現させた。pCMV3空ベクターを対照群に使用した。製造社の支持に従い、Lipofectamine2000(Invitrogen)を使用してトランスフェクションを行った。
HepG2細胞及びマウス肝臓の切片をオイルレッドO(Oil-red O)法により染色して、細胞中性脂肪濃度(TG)蓄積を測定した。40分間10%ホルマリンで固定後、肝細胞をOil-red O溶液で、37℃で1時間染色した。Oil-red O染色されたTG含量を、各サンプルにイソプロパノールを添加して定量化した。混合物を8分間25℃で柔らかに撹拌した。最後に、100μlのイソプロパノール抽出サンプルを510nmで、分光光度計で分析した。
総脂質をクロロホルム:メタノール(2:1、v/v)混合物を使用して抽出した。有機層を乾燥させた後、即時60%エタノールに溶解させた。抽出したTGは製造社の指針(Biovision、Milpitas、CA、USA)により比色分析キットを用いて測定した。
全ての分析はSPSS/PC統計プログラム(Windowsバージョン12.0;SPSS、Chicago、IL、USA)を用いて行った。結果は最も高い値の倍数で表示した(means±全てのin vitro実験は少なくとも3回行った。統計分析にはStudent'st test又はtwo-way ANOVAを用いた。
肝細胞におけるPDXに起因するERストレスに対するパルミチン酸誘導TG蓄積抑制効果
200μMパルミチン酸とPDX(0-2μm)の存在下で、HepG2細胞を24時間Oil-red O染色し、TG蓄積はイソプロピルアルコールで抽出して定量した。
本発明者らは、ORP150が、ERストレス及びTG蓄積に対するPDXの抑制効果に関与するか否かを調査した。
本発明者らは、24時間2μmのPDXの存在下でスクランブルsiRNA又はsiFOXO1で形質転換されたHepG2細胞でORP150に対するウエスタンブロット分析を行った。
本発明者らは、24時間200μMパルミチン酸で処理及び/又はORP150 0-4μgを処理したHepG2細胞において、SREBP1発現に対するウエスタンブロット分析を行った。
本発明者らは、マウスから脂肪蓄積に対するPDXの影響を評価した。このため、マウス肝臓疾患に対してH&E染色及びOil-red 0染色により組織学的分析及びウエスタンブロット分析を行い、TG蓄積はTG分析キットを用いて測定した。
本発明者らは、8週間の前記実験方法により、PDXを投与したマウスの体重、日々のエネルギー摂取量、肝臓重量及び副睾丸脂肪量を各グループ当たり5匹ずつ測定した。
Claims (6)
- 下記化学式1で表示されるプロテクチンDX又はこれの薬学的に許容可能な塩を有効成分として含む、高脂血症又は脂肪肝疾患の予防又は治療用薬学的組成物。
- 前記プロテクチンDXは肝臓組織の中性脂肪含量を減少させる効果を有することを特徴とする、請求項1記載の組成物。
- 化学式1で表示されるプロテクチンDX又はこれの塩を有効成分として含む、高脂血症又は脂肪肝疾患の予防又は改善用食品組成物。
- 前記脂肪肝臓疾患は、肝炎、肝硬変、肝細胞癌、アルコール性脂肪肝、非アルコール性脂肪肝、栄養性脂肪肝、飢餓性脂肪肝及び脂肪肝からなる群より選ばれることを特徴とする、請求項1又は請求項3記載の組成物。
- 化学式1で表示されるプロテクチンDX又はこれの薬学的に許容可能な塩を有効成分として含む、肝臓保護用薬学的組成物。
- 化学式1で表示されるプロテクチンDX又はこれの塩を有効成分として含む、肝臓保護用食品組成物。
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KR1020170112118A KR101981534B1 (ko) | 2017-09-01 | 2017-09-01 | 프로텍틴 dx를 유효성분으로 함유하는 고지혈증 또는 지방간 질환 예방 또는 치료용 조성물 |
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EP4358949A1 (en) * | 2021-06-25 | 2024-05-01 | Université Laval | Derivatives of the protectin 10s,17s-dihda (pdx) and use thereof as antiviral, anti-inflammatory, and anti-diabetic agents |
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JP2009120607A (ja) * | 2007-10-25 | 2009-06-04 | Kanazawa Univ | 新規な肝疾患の予防又は治療薬並びにインスリン抵抗性改善剤 |
JP2014129323A (ja) * | 2012-05-29 | 2014-07-10 | Ueno Fine Chem Ind Ltd | 肝臓への脂肪蓄積抑制剤 |
JP2015522535A (ja) * | 2012-05-10 | 2015-08-06 | ソルテックス エヌエー エルエルシー | 天然の特異的炎症収束性メディエータおよびその前駆物質を含有する、抗炎症活性を有する油 |
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EP2948163B1 (en) | 2013-01-25 | 2020-03-11 | Université Laval | Use of protectin dx for the stimulation of muscular il-6 secretion |
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- 2017-09-21 JP JP2017180866A patent/JP2019043928A/ja active Pending
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JP2009120607A (ja) * | 2007-10-25 | 2009-06-04 | Kanazawa Univ | 新規な肝疾患の予防又は治療薬並びにインスリン抵抗性改善剤 |
JP2015522535A (ja) * | 2012-05-10 | 2015-08-06 | ソルテックス エヌエー エルエルシー | 天然の特異的炎症収束性メディエータおよびその前駆物質を含有する、抗炎症活性を有する油 |
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