KR20220099773A - composition for senotherapy - Google Patents
composition for senotherapy Download PDFInfo
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- KR20220099773A KR20220099773A KR1020210002058A KR20210002058A KR20220099773A KR 20220099773 A KR20220099773 A KR 20220099773A KR 1020210002058 A KR1020210002058 A KR 1020210002058A KR 20210002058 A KR20210002058 A KR 20210002058A KR 20220099773 A KR20220099773 A KR 20220099773A
- Authority
- KR
- South Korea
- Prior art keywords
- piperine
- senescent cells
- cells
- senescent
- acid
- Prior art date
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- 238000012080 senotherapy Methods 0.000 title description 4
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- A61P39/00—General protective or antinoxious agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
Abstract
Description
본 발명은 세노테라피용 조성물에 관한 것이다.The present invention relates to a composition for senotherapy.
세포노화는 조직의 노화에 기여할 뿐 아니라 조직기능 저하와 노화 관련된 질환의 발병에도 중요한 역할을 하는 것으로 알려져 있다. 노화가 진행될수록 기능이 감소되어 있는 노화세포가 증가하고 증가된 노화세포는 노화관련분비인자(senescence-associated secretory phenotype, SASP)라고 하는 염증성 인자들을 분비하여 조직의 염증과 질환을 유발하게 된다. 최근 노화세포를 선택적으로 제거할 수 있는 세놀리틱스(senolytics)제제나 노화세포의 기능을 정상으로 회복시켜 SASP의 분비를 억제할 수 있는 세노몰픽스(senomorphics)와 같은 세노테라퓨틱스(senotherapeutics)를 처리하여 조직의 노화를 억제함으로써 수명을 연장시키거나 노인성 질환을 치료할 수 있다는 가능성이 보고되면서 보다 효과적이고 안전한 신 세노테라퓨틱스이 발굴에 관심이 집중되고 있다. 세노테라퓨틱스 제제는 노화억제, 노화관련 기능저하 예방, 노인성 질환 치료 등 광범위하게 적용이 가능하기 때문에 최근 해외 우수연구기관이나 기업의 가장 주요 타깃으로 인식되고 있다.Cellular aging is known not only to contribute to tissue aging, but also to play an important role in the deterioration of tissue function and the onset of aging-related diseases. As aging progresses, the number of senescent cells with reduced function increases, and the increased senescent cells secrete inflammatory factors called senescence-associated secretory phenotype (SASP) to induce tissue inflammation and disease. Recently, senolytics that can selectively remove senescent cells or senotherapeutics such as senomorphics that can inhibit the secretion of SASP by restoring the function of senescent cells to normal As the possibility of prolonging lifespan or treating geriatric diseases by inhibiting tissue aging by treating Cenotherapeutics is recognized as the main target of excellent overseas research institutes and companies because it can be widely applied to inhibit aging, prevent aging-related functional decline, and treat geriatric diseases.
2015년에 세놀리틱스가 노화조직의 기능을 개선할 수 있음이 개발된 후, 다양한 화합물을 이용하여 노화조직의 개선 및 일부 질환을 치료할 수 있음이 보고되면서, 세놀리틱스를 포함하는 세노테라퓨틱스에 관심이 폭발적으로 증가하고 있다. 그러나, 현재까지 보고된 물질들은 기존 화합물 라이브러리를 이용하여 스크리닝을 통해 발굴된 약제로써, 이미 항암제로 알려져 있거나 기존의 다른 약물로 사용이 되고 있어, 인체에 장기간 투여할 경우 부작용이 나타날 수 있는 한계가 있다. 그럼에도 불구하고 세노테라퓨틱스의 동물실험 결과, 기존 질환 치료제와는 다른 기전인 노화세포 억제를 통한 조직 기능회복, 질환 치료 등의 효과가 나타나기 때문에 세노테라퓨틱스의 개발에 대한 관심은 더욱 증가되고 있다.After it was developed that senolytics can improve the function of aging tissues in 2015, it was reported that various compounds can be used to improve aging tissues and treat some diseases. Interest in Ticks is exploding. However, the substances reported so far are drugs discovered through screening using the existing compound library, and are already known as anticancer drugs or used as other drugs. have. Nevertheless, as a result of animal experiments of cenotherapy, interest in the development of cenotherapeutics is increasing, as it shows effects such as tissue function recovery and disease treatment through suppression of senescent cells, a mechanism different from that of existing disease treatments. have.
본 발명의 목적은 피페린(piperine)을 투여하여 노화세포의 형태가 정상세포와 유사하게 회복되는 것을 확인하여, 이를 포함하는 세노테라퓨틱스 제제를 제공함에 있다.An object of the present invention is to provide a senotherapeutic preparation comprising the same by confirming that the morphology of senescent cells is restored similarly to that of normal cells by administering piperine.
1. 피페린 또는 이의 약학적으로 허용되는 염을 포함하는 노화세포를 정상세포로 회복하는 세노몰픽(Senomorphic)용 시약 조성물. 1. A reagent composition for senomorphic that restores senescent cells to normal cells containing piperine or a pharmaceutically acceptable salt thereof.
2. 피페린 또는 이의 약학적으로 허용되는 염을 포함하는 노화세포를 정상세포로 회복하는 세노몰픽(Senomorphic)용 약학 조성물. 2. A pharmaceutical composition for senomorphic that restores senescent cells to normal cells containing piperine or a pharmaceutically acceptable salt thereof.
3. 피페린 또는 이의 약학적으로 허용되는 염을 포함하는 노화세포를 정상세포로 회복하는 세노몰픽(Senomorphic)용 화장료 조성물. 3. A cosmetic composition for senomorphic that restores senescent cells to normal cells containing piperine or a pharmaceutically acceptable salt thereof.
4. 피페린 또는 이의 약학적으로 허용되는 염을 포함하는, 세포 노화로 유발되는 질환의 예방 또는 치료용 약학 조성물로서, 상기 질환은 암, 동맥경화, 당뇨병, 관절염 또는 조직 섬유증인 약학 조성물.4. A pharmaceutical composition for preventing or treating a disease caused by cellular aging, comprising piperine or a pharmaceutically acceptable salt thereof, wherein the disease is cancer, arteriosclerosis, diabetes, arthritis, or tissue fibrosis.
본 발명의 목적은 후추 추출물의 노화세포를 정상세포로 회복하도록 하는 효과를 확인하여, 이를 포함하는 세노몰픽용 시약 조성물을 제공하는 것이다. An object of the present invention is to provide a reagent composition for senomorphic containing the effect of the pepper extract to restore senescent cells to normal cells.
본 발명의 목적은 후추 추출물의 노화세포를 정상세포로 회복하도록 하는 효과를 확인하여, 이를 포함하는 세노몰픽용 약학 조성물을 제공하는 것이다. It is an object of the present invention to provide a pharmaceutical composition for senomorphic comprising the effect of the pepper extract to restore senescent cells to normal cells.
또한, 본 발명의 목적은 후추 추출물의 노화세포를 정상세포로 회복하도록 하는 효과를 확인하여, 이를 포함하는 세노몰픽용 화장료 조성물을 제공하는 것이다. In addition, an object of the present invention is to provide a cosmetic composition for senomorphic containing the effect of the pepper extract to restore senescent cells to normal cells.
도 1은 노화세포에 피페린을 투여한 횟수 및 간격을 간략하게 표시한 것이다.
도 2는 세포 독성 반응 실험을 통해 세포 독성을 확인한 것으로, 피페린을 도 1과 같이 이틀 간격으로 3회 처리하여도 세포 독성이 발생되지 않는 것을 확인한 것이다.
도 3은 피페린 처리에 의한 노화 세포 성장을 비교하여 확인한 것으로, A는 cell proliferation assay를, B는 viable cell count로 노화세포의 성장을 확인한 것이다.
도 4는 노화세포의 노화 유도 단백질의 발현을 확인한 결과이다.
도 5는 노화세포 특이적 베타-갈락토시데이즈(SA-Beta-Gal) 염색 결과를 통해 노화세포가 감소한 것을 나타낸 것이다.
도 6은 노화세포의 미토콘드리아 기능 및 세포 내 활성산소 생성을 비교한 것이다.
도 7은 피페린에 의한 노화세포 특이적 분비인자인 SASP (senescence-associated secretory phenotype)를 비교하여 확인한 것이다.1 shows briefly the number and interval of administration of piperine to senescent cells.
FIG. 2 shows that cytotoxicity was confirmed through a cytotoxic reaction experiment, and it was confirmed that cytotoxicity did not occur even when piperine was treated three times at two-day intervals as shown in FIG. 1 .
Figure 3 is confirmed by comparing the growth of senescent cells by piperine treatment, A is cell proliferation assay, B is the growth of senescent cells confirmed by viable cell count.
4 is a result confirming the expression of the senescence-inducing protein in senescent cells.
5 is a senescent cell-specific beta- through the galactosidase (SA-Beta-Gal) staining results show that the senescent cells are reduced.
6 is a comparison of mitochondrial function and intracellular free radical production of senescent cells.
7 is a comparison of senescence-associated secretory phenotype (SASP), which is a senescent cell-specific secretion factor by piperine, and confirmed.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 피페린 또는 이의 약학적으로 허용되는 염을 포함하는 노화세포를 정상세포로 회복하는 세노몰픽용 시약 조성물에 관한 것이다.The present invention relates to a reagent composition for senomorphic that restores senescent cells to normal cells containing piperine or a pharmaceutically acceptable salt thereof.
본 발명의 피페린은 하기 화학식 1로 표시되는 화합물일 수 있다:The piperine of the present invention may be a compound represented by the following formula (1):
[화학식 1][Formula 1]
본 발명에 있어서, 피페린은 후추에서 추출한 것일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, piperine may be extracted from pepper, but is not limited thereto.
본 명세서에서 사용된 "약학적으로 허용되는"이라는 문구는 건강한 의학적 판단의 범위 내에서 과도한 독성, 자극, 알레르기 반응 또는 다른 문제나 합병증이 없으며, 합리적인 이득/위험 비율에 상응하는 인간의 조직과의 접촉에 사용하기 적합한 화합물, 물질, 조성물 및/또는 투여형을 지칭하기 위한 것이다.As used herein, the phrase "pharmaceutically acceptable" means that there is no undue toxicity, irritation, allergic reaction, or other problems or complications within the scope of healthy medical judgment, It is intended to refer to a compound, substance, composition and/or dosage form suitable for use in contact.
또한, 본 명세서에서 사용된 "약학적으로 허용되는 염"은 모 화합물이 그의 산 또는 염기 염을 제조함으로써 변형된, 개시된 화합물의 유도체를 의미한다. 약학적으로 허용되는 염의 예는 아민과 같은 염기성 잔기의 무기 또는 유기산 염; 카복실산과 같은 산성 잔기의 알칼리 또는 유기 염; 등을 포함하나 이에 제한되는 것은 아니다. 약학적으로 허용되는 염은 예를 들어 무독성 무기 또는 유기산으로부터 형성된 모 화합물의 통상적인 무독성 염 또는 4 차 암모늄염을 포함한다. 예를 들어, 그러한 통상적인 무독성 염은 염산, 브롬산, 황산, 설팜산, 인산, 질산 등과 같은 무기산으로부터 유도된 염; 아세트산, 프로피오닉산, 석시닉산, 글리코릭산, 스테아릭산, 락틱산, 말산, 타르타르산, 시트릭산, 아스코빅산, 파모익산, 말레익산, 하이드록시말레익산, 페닐아세틱산, 글루타믹산, 벤조익산, 살리실산, 설파닐릭산, 2-아세톡시벤조익산, 퓨마릭산, 톨루엔설포닉산, 메탄설포닉산, 에탄 디설포닉산, 옥살릭산, 이세티오닉산, 등과 같은 유기산으로부터 얻어진 염을 포함한다.Also, as used herein, "pharmaceutically acceptable salt" refers to a derivative of a disclosed compound wherein the parent compound has been modified by preparing an acid or base salt thereof. Examples of pharmaceutically acceptable salts include inorganic or organic acid salts of basic moieties such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like, but are not limited thereto. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid and the like; Acetic acid, propionic acid, succinic acid, glycoric acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salts obtained from organic acids such as salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethane disulfonic acid, oxalic acid, isethionic acid, and the like.
본 명세서에 기술된 화합물의 약학적으로 허용되는 염은 통상적인 화학적 방법에 의해 염기성 또는 산성 잔기를 함유하는 모 화합물로부터 합성될 수 있다. 일반적으로, 이러한 염은 화합물의 유리산 또는 염기 형태를 물 또는 유기 용매 중 또는 이들의 혼합물 중 화학량론적 양의 적절한 염기 또는 산과 반응시킴으로써 제조될 수 있다: 일반적으로 에틸 아세테이트, 에탄올, 이소프로판올 또는 아세토니트릴과 같은 비수성 매질일 수 있다. Pharmaceutically acceptable salts of the compounds described herein can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base form of a compound with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or mixtures thereof: usually ethyl acetate, ethanol, isopropanol or acetonitrile. It may be a non-aqueous medium such as
본 발명에 있어서, 세노테라피(Senotherapy)란 세포의 노화를 특이적인 타겟으로 하는 치료제 및 전략의 개발을 위한 초기 단계의 기초 연구분야로서, 노화 및 노화 관련 질환과 관련된 세포 상태의 변화에 관련된 요법을 의미한다.In the present invention, senotherapy is a basic research field at the initial stage for the development of therapeutic agents and strategies specifically targeting cellular aging, and is a therapy related to aging and changes in cellular status related to aging-related diseases. it means.
상기 세노테라피 용도의 전략 또는 치료제로서, ① DNA 손상, 산화 스트레스, 단백질 독성 스트레스, 텔로미어 단축과 같은 세포 노화의 유발을 방지함으로써 노화 상태를 예방하거나 역전시키는 제제 또는 전략인 지로프로텍터(Geroprotector), ② 전염증성 SASP(senescence-associated secretory phenotype) 생산을 방해하는 약제로서, 글루코코르티코이드(Glucocorticoid), 스타틴(Statin), 룩소리티닙(Ruxolitinib)과 같은 JAK 1/2 억제제, NF-kB 및 p38 억제제, IL-1α 블로킹 제제, 손상된 미토파지(mitophagy) 경우의 미토콘드리아 고갈제(Mitochondrial deplete) 등을 포함하는 SASP 억제제, ③ 생존 경로 및 항-세포자멸(anti-apoptosis) 메커니즘을 타겟팅하여 노화세포의 사멸을 특이적으로 유도하는 작은 분자(small molecule), 항체 및 항체-매개 약물 전달 제제를 포함하는 세놀리틱(Senolytic) 제제, ④ 세포의 사멸 없이 노화세포만을 선택적으로 노화 표현형을 억제하는 작은 분자를 포함하는 세노몰픽(Senomorphic) 제제, ⑤ 노화 관련 질환에 대한 저항성을 높이고, 개체의 수명을 연장시키기 위한 세포 내 유전자 편집 전략을 포함하는 유전자 테라피(Gene therapy) 등이 포함될 수 있다.As a strategy or therapeutic agent for the senotherapy use, ① DNA damage, oxidative stress, proteotoxic stress, by preventing the induction of cellular aging, such as telomere shortening, an agent or strategy that prevents or reverses the aging state Geroprotector (Geroprotector), ② A drug that interferes with the production of pro-inflammatory SASP (senescence-associated secretory phenotype),
상기 SASP 인자들로는, IL-6, IL-7, IL-1a, IL-1b, IL-13, IL-15, IL-8, GRO-a, GRO-b, GRO-g, MCP-2, MCP-4, MIP-1a, MIP-3a, HCC-4, Eotaxin, Eotaxin-3, TECK, ENA-78, I-309, I-TAC, GM-CSE, G-CSE, IFN-γ, BLC, MIF, Amphiregulin, Epiregulin, Heregulin, EGF, bFGF, HGF, KGF(FGF7), VEGF, Angiogenin, SCF, SDF-1, PIGF, NGF, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGFBP-7, MMP-1, MMP-3, MMP-10, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, PAI-1, PAI-2, tPA, uPA, Cathepsin B, ICAM-1, ICAM-3, OPG, sTNFRI, TRAIL-R3, Fas, sTNFRⅡ, uPAR, SGP130, EGF-R, PGE2, TGF-β1, Nitric oxide(NO), Reactive oxygen species(ROS), Fibronectin, Collagen 및 Laminin의 인자들을 포함하는 것일 수 있다.The SASP factors include, IL-6, IL-7, IL-1a, IL-1b, IL-13, IL-15, IL-8, GRO-a, GRO-b, GRO-g, MCP-2, MCP -4, MIP-1a, MIP-3a, HCC-4, Eotaxin, Eotaxin-3, TECK, ENA-78, I-309, I-TAC, GM-CSE, G-CSE, IFN-γ, BLC, MIF , Amphiregulin, Epiregulin, Heregulin, EGF, bFGF, HGF, KGF(FGF7), VEGF, Angiogenin, SCF, SDF-1, PIGF, NGF, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGFBP- 7, MMP-1, MMP-3, MMP-10, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, PAI-1, PAI-2, tPA, uPA, Cathepsin B, ICAM -1, ICAM-3, OPG, sTNFRI, TRAIL-R3, Fas, sTNFRII, uPAR, SGP130, EGF-R, PGE2, TGF-β1, Nitric oxide (NO), Reactive oxygen species (ROS), Fibronectin, Collagen and It may include factors of laminin.
본 발명에서 세노몰픽(Senomorphic) 용도는 노화 및 노화 관련 질환과 관련된 세포 상태의 변화로서, 세포 노화 상태의 정상 상태로의 전환 요법을 위한 용도를 의미할 수 있고, 이는 세포의 사멸 없이 노화세포만을 선택적으로 노화 표현형을 억제하는 물질을 의미하는 것일 수 있다. 또한, 세노몰픽 용도는 노화세포의 기능을 정상으로 회복시키는 특이적인 용도를 의미하는 것일 수 있고, 이러한 경우 p16 또는 p21과 같은 노화 유도 단백질의 발현 수준이 정상세포의 수준으로 되돌아가도록 하거나, SASP(senescence-associated secretory phenotype) 인자의 분비를 억제하는 능력을 의미하는 것일 수 있다. In the present invention, the use of senomorphic (Senomorphic) is a change in cellular status associated with aging and senescence-related diseases, and may refer to use for a therapy for converting the cellular senescence state to a normal state, which is only senescent cells without apoptosis. It may refer to a substance that selectively inhibits the aging phenotype. In addition, the senomorphic use may refer to a specific use to restore the function of senescent cells to normal, and in this case, the expression level of a senescence-inducing protein such as p16 or p21 returns to the level of normal cells, or SASP ( It may mean the ability to inhibit the secretion of senescence-associated secretory phenotype) factors.
또한, 상기 세노몰픽 용도는 항노화 용도와는 완전히 구별되는 것으로서, 굳이 '노화(ageing)'라는 단어를 사용하여 표현하자면 '역노화' 용도와 유사한 개념으로서, 항노화는 항산화능(anti-oxidant activity) 등에 기반하여 세포의 노화 진행을 느리게 하거나 멈추는 반면, 상기 세노몰픽 효능은 노화 세포를 정상 세포의 수준으로 회복시키는 것으로서, 단순 항산화능으로 입증되는 것이 아니고, SASP 인자들의 억제를 포함한 특이적 인자들의 분비를 억제하여 정상 세포 수준으로 되돌리는 것으로, 그 약리기전과 효과 등에 현저한 차이가 존재한다.In addition, the cenomorphic use is completely distinct from the anti-aging use, and to express it using the word 'ageing', it is a concept similar to the 'reverse aging' use, and anti-aging is an anti-oxidant. activity), while slowing or stopping the aging process of cells, the senomorphic effect is to restore senescent cells to the level of normal cells, and is not demonstrated by simple antioxidant activity, but is a specific factor including inhibition of SASP factors By suppressing their secretion and returning them to normal cellular levels, there is a marked difference in their pharmacological mechanisms and effects.
본 발명의 시약 조성물은 in vitro에서 노화 세포에 처리되는 시약 조성물일 수 있다.The reagent composition of the present invention may be a reagent composition that is treated with senescent cells in vitro.
본 발명의 시약 조성물은 화합물의 안정성을 확보하기 위하여 적절한 보존제를 더 포함할 수 있으며, 상기 보존제의 종류 및 혼합량 등은 통상의 기술자에게 자명한 사항이다.The reagent composition of the present invention may further include an appropriate preservative in order to secure the stability of the compound, and the type and mixing amount of the preservative are obvious to those skilled in the art.
본 발명의 시약 조성물은 연구 목적, 사용되는 세포의 종류 및 세포의 수에 따라 다양한 농도로 처리될 수 있으나, 세포 독성 등을 고려하여 실험에 맞는 적절한 농도로 처리할 수 있다.The reagent composition of the present invention may be treated at various concentrations depending on the purpose of research, the type of cell used and the number of cells, but may be treated at an appropriate concentration for the experiment in consideration of cytotoxicity and the like.
본 발명은 피페린 또는 이의 약학적으로 허용되는 염을 포함하는 노화세포를 정상세포로 회복하는 세노몰픽용 약학 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for senomorphic that restores senescent cells to normal cells containing piperine or a pharmaceutically acceptable salt thereof.
상기 "피페린" 또는 "이의 약학적으로 허용되는 염"은 전술한 바와 같다. The "piperine" or "a pharmaceutically acceptable salt thereof" is as described above.
상기 세노몰픽 용도는 전술한 바와 같다.The cenomorphic use is the same as described above.
본 발명에서 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the pharmaceutical composition may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. , but is not limited thereto.
본 발명의 약학적 조성물에 함유될 수 있는 담체, 부형제 및 희석제로는 락토오즈, 덱스트로즈, 수크로스, 덱스트린, 말토덱스트린, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있으나, 이에 제한되는 것은 아니다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있으나, 이에 제한되는 것은 아니다.Carriers, excipients and diluents that may be contained in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not limited. In the case of formulation, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며 이에 제한되지는 않으나, 이러한 고형제제는 상기 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있으나, 이에 제한되는 것은 아니다.Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, etc., but these solid preparations include at least one or more excipients in the compound, for example, starch, calcium carbonate , sucrose or lactose, gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used, but the present invention is not limited thereto.
경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있으나, 이에 제한되는 것은 아니다.Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. may be used, but is not limited thereto.
또한, 본 발명은 피페린 또는 이의 약학적으로 허용되는 염을 포함하는 노화세포를 정상세포로 회복하는 세노몰픽용 화장료 조성물에 관한 것이다. In addition, the present invention relates to a cosmetic composition for senomorphic that restores senescent cells to normal cells containing piperine or a pharmaceutically acceptable salt thereof.
상기 "피페린" 또는 "이의 약학적으로 허용되는 염"은 전술한 바와 같다.The "piperine" or "a pharmaceutically acceptable salt thereof" is as described above.
상기 세노몰픽 용도는 전술한 바와 같다.The cenomorphic use is the same as described above.
본 발명에서 화장료 조성물은 상기 피페린 뿐만 아니라, 화장료 조성물에 통상적으로 이용되는 성분들을 포함할 수 있으며, 예를 들어, 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the cosmetic composition may include not only the piperine, but also components commonly used in the cosmetic composition, for example, conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances; And it may include a carrier, but is not limited thereto.
본 발명의 화장료 조성물을 첨가할 수 있는 제품으로는, 예를 들어, 수렴화장수, 유연화장수, 영양화장수, 각종크림, 에센스, 팩, 파운데이션 등과 같은 화장품류와 클렌징, 세안제, 비누, 트리트먼트, 미용액 등이 있으나, 이에 제한되는 것은 아니다.Products to which the cosmetic composition of the present invention can be added include, for example, cosmetics such as astringent lotion, softening lotion, nutrient lotion, various creams, essence, pack, foundation, and cleansing, face wash, soap, treatment, and serum. and the like, but is not limited thereto.
본 발명의 화장료 조성물의 구체적인 제형으로서는 스킨로션, 스킨 소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스처 로션, 영양로션, 맛사지크림, 영양크림, 모이스처 크림, 핸드크림, 에센스, 영양에센스, 팩, 비누, 샴푸, 클렌징폼, 클렌징로션, 클렌징크림, 바디로션, 바디클렌저, 유액, 립스틱, 메이컵 베이스, 파운데이션, 프레스파우더, 루스파우더, 아이섀도 등의 제형을 포함할 수 있으나, 이에 제한되는 것은 아니다.Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, essence, nourishing essence, pack, It may include formulations such as soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, lipstick, make-up base, foundation, press powder, loose powder, eye shadow, etc., but is limited thereto not.
본 발명은 피페린 또는 이의 약학적으로 허용되는 염을 포함하는 세포 노화로 유발되는 질환의 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating a disease induced by cellular aging, comprising piperine or a pharmaceutically acceptable salt thereof.
전술한 바와 같이, 본 발명의 조성물은 노화 세포를 정상 세포르 되돌릴 수 있으므로, 세포 노화로 의해 유발되는 다양한 질환에 대한 치료 효능을 나타낼 수 있다.As described above, since the composition of the present invention can return senescent cells to normal cells, it can exhibit therapeutic efficacy for various diseases caused by cellular senescence.
세포 노화로 유발되는 질환은 예를 들면 암, 동맥경화, 당뇨병, 관절염 또는 조직 섬유증 등일 수 있으나, 이에 제한되는 것은 아니다.The disease caused by cellular aging may be, for example, cancer, arteriosclerosis, diabetes, arthritis, or tissue fibrosis, but is not limited thereto.
이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다. Hereinafter, examples will be given to describe the present invention in detail.
실시예Example
1. 노화세포의 제작1. Preparation of senescent cells
노화 인간 이배체 섬유 아세포(senescent-human diploid fibroblast cell; S-HDF)의 지속적인 배양(subculture, 비율 1:4)을 통해 노화 세포주를 제작하였다. 이후, 노화세포 특이적 염색법인 노화관련-β-갈락토시데이즈(senescentassociated-β-galactosidase, SA-β-Gal) 염색을 통해 노화 여부를 확인하였다.A senescent cell line was prepared through continuous subculture (ratio 1:4) of senescent-human diploid fibroblast cells (S-HDF). Thereafter, aging was confirmed through senescent-associated-β-galactosidase (SA-β-Gal) staining, a senescent cell-specific staining method.
SA-β-Gal 염색은 Senescenceβ-Galactosidase Staining Kit (Cell Signaling Technology, 카탈로그 넘버: 9860)를 이용하여 프로토콜에 따라 염색하였고, 다음날, 광학현미경을 통해 세포에 염색이 되어있는지 확인하였다. 대조군으로는 비노화 인간 이배체 섬유 아세포(non-senescent human diploid fibroblast; NS-HDF)를 이용하였다.SA-β-Gal staining was performed using the Senescenceβ-Galactosidase Staining Kit (Cell Signaling Technology, catalog number: 9860) according to the protocol, and the next day, it was confirmed whether the cells were stained through an optical microscope. As a control, non-senescent human diploid fibroblasts (NS-HDF) were used.
2. 노화세포에 피페린의 처리2. Treatment of piperine in senescent cells
피페린은 서울대 오원근 교수님 연구실로부터 제공 받았다. 구체적으로, 피페린은 필발 (Piper Longum)식물에서 분리하여 사용하였고 분리 방법은 다음과 같다. Piperine was provided by Professor Won-Geun Oh's laboratory at Seoul National University. Specifically, piperine was isolated and used from a Piper Longum plant, and the separation method is as follows.
필발의 말린 과일 (1 kg)을 100% 에탈올 (EtOH, 2 Liter x 3회)로 실온에서 1 주동안 추출하였다. 에탄올 추출물을 농축하여 건조된 조 추출물 (60 g)을 수득하고, 이를 물레 현탁 시킨 후에 동량의 에틸아세테이트 (EtOAc, 3 x 500 mL)로 분획하였다. 상기 에틸 아세테이트 분획물을 컬럼 크로마토그래피 (Column Chromatography)를 실시하기 위하여 실리카 겔에 흡착시키고 n-핵산 (n-Hexane, 용매 A) 및 에틸아세테이트 (용매 B)의 용매 구배 (10:0, 8:2, 6:4, 4:6, 2:8, 및 0:10)로 용리하여 20개의 소 분획물(1-20)로 획득하였다. 얻어진 소 분획물 5에서 재 결정화 과정을 통하여 피페린 (30 mg)을 획득하였다. The dried fruits (1 kg) were extracted with 100% ethanol (EtOH, 2 Liter x 3 times) at room temperature for 1 week. The ethanol extract was concentrated to obtain a dried crude extract (60 g), which was suspended on a water wheel and fractionated with the same amount of ethyl acetate (EtOAc, 3 x 500 mL). The ethyl acetate fraction was adsorbed on silica gel to perform column chromatography, and a solvent gradient (10:0, 8:2) of n-nucleic acid ( n -Hexane, solvent A) and ethyl acetate (solvent B) was performed. , 6:4, 4:6, 2:8, and 0:10) obtained in 20 small fractions (1-20). Piperine (30 mg) was obtained through recrystallization from the obtained
또한, 피페린을 Sigma사(카탈로그 넘버: P49007)에서 구입하여 사용하기도 하였다. In addition, piperine was purchased from Sigma (catalog number: P49007) and used.
6웰 플레이트(6-well plate)에 피페린을 처리하기 전, 셀 컨플루언스(cell confluence)가 70%가 되도록 노화세포를 접종하였다. 노화세포에 피페린을 20 ㎍/ml의 농도로 2일 간격, 3회 투여한 후에 노화 세포의 형태, 노화마커, 노화세포의 기능 등을 비교 검증하였다. 구체적인 투여 일수 및 간격은 도 1과 같다.Before treatment with piperine in a 6-well plate, senescent cells were inoculated so that the cell confluence was 70%. After administration of piperine to senescent cells at a concentration of 20 μg/ml for 2 days, 3 times, the morphology of senescent cells, senescence markers, and functions of senescent cells were compared and verified. Specific administration days and intervals are shown in FIG. 1 .
3. 세포 독성 여부 확인3. Check for cytotoxicity
(1) 실험 방법(1) Experimental method
세포 독성 실험은 LDH 정량 키트 (LDH assay kit) (Dogen, 카탈로그 넘버: DG-LDH500)를 이용하여 제안된 프로토콜에 따라 시행하였다.The cytotoxicity test was performed according to the proposed protocol using an LDH assay kit (Dogen, catalog number: DG-LDH500).
구체적으로, 먼저 상기 2의 방법으로 피페린을 노화 세포에 3회 처리한 이틀 후에 세포배양 상층액을 취득하였다. 취득된 상층액 (10 ㎕)을 96웰 플레이트에 분주한 다음에 LDH 반응 용액 (100 ㎕)을 각 웰에 첨가하여 빛이 차단된 상온에서 30분간 반응을 시켰다. 반응시간이 끝나면 마이크로플레이트 리더기 (microplates reader)을 이용하여 파장대 (wavelength) 450 nm에서 각 웰의 흡광도를 측정하여 세포 독성을 확인하였다. 정확한 세포 독성을 계산하기 위해서 노화 세포에 용해 용액 (lysis solution)을 처리한 군 (high control), 세포 배양액에 용해 용액을 처리한 군 (volume control), 실험 과정에 자연적으로 죽거나 손상된 세포에서 방출되는 LDH 양을 측정하기 위한 군(low control), 그리고 세포배양액의 FBS에 포함되어 있는 LDH를 측정하기 위한 군 (Background control)을 같이 준비하여 각 군에서 측정된 흡광도 수치를 가지고 세포 독성을 계산하였다. 세포독성 계산식은 다음과 같다.Specifically, the cell culture supernatant was obtained two days after first treating senescent cells with piperine three times by the method of 2 above. The obtained supernatant (10 μl) was aliquoted into a 96-well plate, and then an LDH reaction solution (100 μl) was added to each well and reacted for 30 minutes at room temperature where light was blocked. When the reaction time was over, the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader to confirm cytotoxicity. In order to calculate the exact cytotoxicity, the group treated with the lysis solution on senescent cells (high control), the group treated with the lysis solution in the cell culture medium (volume control), A group for measuring the amount of LDH (low control), In addition, a group (background control) for measuring LDH contained in FBS of the cell culture medium was prepared, and cytotoxicity was calculated using the absorbance values measured in each group. The cytotoxicity calculation formula is as follows.
[수학식 1][Equation 1]
세포독성(%)=[(실험군의 흡광도-background control의 흡광도)-(low control의 흡광도-background control의 흡광도)]/[(high control의 흡광도-volume control의 흡광도)-(low control의 흡광도-background control의 흡광도)] X 100. Cytotoxicity (%) = [(absorbance of experimental group-absorbance of background control)-(absorbance of low control-background absorbance of control)]/[(absorbance of high control - absorbance of volume control)-(absorbance of low control - absorbance of background control)]
(2) 실험 결과(2) Experimental results
노화세포에서 상기 2의 방법으로 피페린을 처리하여 세포 영향을 확인하기 위해 세포 독성 반응을 확인한 결과, 피페린을 노화세포에 이틀 간격으로 3회 동안 처리하여도 0.5% 정도의 독성 비율이 나타났고, 이는 세포 독성이 발생되지 않았다는 것을 의미한다(도 2).As a result of confirming the cytotoxic reaction in senescent cells by treating piperine in the method of 2 above to confirm the cellular effect, the toxicity rate was about 0.5% even when piperine was treated with
4. 노화세포 성장 비교4. Comparison of senescent cell growth
노화세포는 일반적으로 세포 분열이 잘 되지 않아 세포 성장이 느리다. 피페린에 의한 노화 세포 성장 효과를 확인하기 위해, 상기 2의 방법으로 피페린을 노화세포에 이틀 간격으로 두 차례 정도 처리하여 노화 세포의 성장이 증가되는 것을 확인하였고, 그 세포 성장이 피페린을 3회 처리한 경우 더욱 증가하는 것을 볼 수 있었다(도 3). 또한, 노화세포의 성장을 유도한다고 알려진 니코틴아마이드(nicotinamide, NA) 보다 피페린 처리에 따른 노화세포 성장이 더 증가된 것을 확인할 수 있었다(도 3). 이를 통해, 피페린이 노화세포의 성장을 유도함을 알 수 있었다. Senescent cells generally do not divide well, so cell growth is slow. In order to confirm the effect of piperine on the growth of senescent cells, it was confirmed that the growth of senescent cells was increased by treating senescent cells with piperine twice at an interval of two days in the
5. 노화세포의 노화 유도 단백질 발현 확인5. Confirmation of senescence-inducing protein expression in senescent cells
(1) 실험 방법(1) Experimental method
피페린을 상기 2의 방법으로 노화 세포에 3회 처리한 후 이틀 후 세포를 획득하였고, 획득한 세포에 RIPA 버퍼(프로테아제 억제제 포함)를 첨가하여, 초음파 분쇄기로 세포를 분쇄하였다. 13,000rpm 에서 10분간 원심분리(4℃)하여 상층액을 획득한 다음, BCA 프로테인 정량법을 통해서 단백질을 정량하였다. 동량의 단백질을 5X sample buffer에 첨가하여 잘 섞은 다음, 10분 동안 100℃ 열 블록(heat block)에서 가열하였고, 상온에서 식힌 후, SDS-PAGE로 단백질은 크기별로 분리한 다음, PVDF 막으로 이동시킨 후 5% BSA TBS-T 용액을 이용하여 상온에서 한 시간 동안 쉐이커를 사용하여 블로킹하였다. 일차 항체(1st antibody)는 p16INK4a (Invitrogen, 카탈로그 넘버: MA5-17142)와 p21 (Santa Cruz, 카탈로그 넘버: sc-397)를 1:1000으로 냉각 환경(cold room)에서 16시간 동안 쉐이커에서 반응시켰다. 다음 날, TBS-T 용액을 사용하여 10분씩 3번 막(membrane)을 씻어준 후, 이차 항체(2nd antibody)를 반응시켰는데, 이차 항체는 Goat anti-mouse 또는 anti-rabbit HRP 접합된 항체를 1:3000을 사용하여 상온에서 한 시간 동안 쉐이커를 이용하여 반응시켰다. 다음 날 TBS-T 용액을 사용하여 10분씩 3번 막을 씻어준 후, ECL 용액을 사용하여 발광을 확인하였고, 각 발광이 확인된 단백질은 ImageJ 소프트웨어(NIH)를 통해서 밴드 강도(band intensity)를 정량화하였다. 대조군으로는 NS-HDF에서의 단백질 발현량을, 단백질 동량 확인은 β-actin 단백질 발현량을 이용하였다.After treating the senescent cells with piperine three times in the method of 2 above, cells were obtained two days later, RIPA buffer (including protease inhibitors) was added to the obtained cells, and the cells were crushed with a sonication machine. The supernatant was obtained by centrifugation (4° C.) at 13,000 rpm for 10 minutes, and then the protein was quantified by BCA protein quantification. The same amount of protein was added to 5X sample buffer, mixed well, heated in a heat block at 100 ° C for 10 minutes, cooled at room temperature, and protein was separated by size by SDS-PAGE, and then transferred to PVDF membrane. After drying, blocking was performed using a shaker for 1 hour at room temperature using 5% BSA TBS-T solution. The primary antibody was reacted with p16 INK4a (Invitrogen, catalog number: MA5-17142) and p21 (Santa Cruz, catalog number: sc-397) at 1:1000 in a shaker for 16 hours in a cold room. made it The next day, after washing the
(2) 실험 결과(2) Experimental results
노화세포에 상기 2의 방법으로 피페린을 처리하여 노화세포에서 증가된 노화 유도 단백질 p16INK4a 및 p21의 발현을 확인한 결과, 노화세포(S-HDF)에 증가되어 있는 노화 유도 단백질(p16INK4a 및 p21)의 발현이 정상세포(NS-HDF)에서 보이는 발현과 유사하게 피페린을 처리한 노화세포(S-HDF + 피페린)에서 노화유도 단백질의 발현이 감소한 것을 알 수 있었다(도 4). As a result of confirming the increased expression of the senescence-inducing proteins p16 INK4a and p21 in the senescent cells by treating the senescent cells with piperine in the method of 2 above, the senescence-inducing proteins (p16 INK4a and p21) increased in the senescent cells (S-HDF) ), it was found that the expression of the senescence-inducing protein was reduced in the senescent cells (S-HDF + piperine) treated with piperine similar to the expression seen in the normal cells (NS-HDF) (FIG. 4).
도 4 상부는 이를 웨스턴 블롯으로 확인한 것이고, 도 4 하부의 막대 그래프는 단백질의 발현을 정량화한 것으로 노화세포에 피페린을 처리한 결과가 정상세포에 가깝게 나타난 것을 알 수 있다.The upper part of FIG. 4 is confirmed by Western blot, and the bar graph in the lower part of FIG. 4 quantifies the protein expression, and it can be seen that the result of treating senescent cells with piperine is close to normal cells.
6. 베타-갈락토시데이즈(SA-Beta-Gal) 염색에 의한 노화세포 확인6. Confirmation of senescent cells by beta-galactosidase (SA-Beta-Gal) staining
(1) 실험 방법(1) Experimental method
상기 2의 방법으로 피페린을 노화 세포에 3회 처리한 다음, 이틀 후에 세포배양액을 제거하였고, SA-β-Gal 염색 키트를 이용하여 프로토콜에 따라서 염색하였다. 다음 날, 광학현미경을 통해서 세포의 염색 여부를 확인하였고, 염색된 세포를 각기 다른 위치에서(n=7) 이미지를 획득한 후에 SA-β-Gal 염색 정도를 Matlab 소프트웨어(MathWorks Inc.)를 이용하여 이미지 분석하여 정량화 하였다.The senescent cells were treated with piperine three times by the method of 2 above, and then the cell culture medium was removed two days later, and stained according to the protocol using the SA-β-Gal staining kit. The next day, it was confirmed whether the cells were stained through an optical microscope, and after acquiring images of the stained cells at different locations (n=7), the degree of SA-β-Gal staining was measured using Matlab software (MathWorks Inc.). and quantified by image analysis.
(2) 실험 결과 (2) Experimental results
노화세포 특이적 베타-갈락토시데이즈(SA-Beta-Gal)는 노화세포 마커로, 이를 노화세포가 얼마나 있는지 확인하는 염색법으로 사용된다. 염색이 많이 되면 노화세포가 많이 있는 것을, 염색이 적게 되면 노화세포가 적게 있는 것을 의미한다. Senescent cell-specific beta-galactosidase (SA-Beta-Gal) is a senescent cell marker and is used as a staining method to determine how many senescent cells are present. When the staining is high, it means that there are many senescent cells, and when the staining is low, it means that there are few senescent cells.
피페린을 노화세포에 처리하여 노화세포 특이적 염색법으로 확인한 결과, 피페린을 처리한 노화세포에서 SA-Beta-Gal 염색이 유의적으로 감소한 것을 확인하였다(도 5).As a result of treating senescent cells with piperine and confirming by a senescent cell-specific staining method, it was confirmed that SA-Beta-Gal staining was significantly reduced in the senescent cells treated with piperine ( FIG. 5 ).
7. 노화 세포 미토콘드리아의 기능 및 세포 내 활성 산소의 생성 비교7. Comparison of senescent cell mitochondrial function and intracellular free radical production
(1) 실험 방법(1) Experimental method
상기 2의 방법으로 피페린을 노화 세포에 3회 처리한 다음, 이틀 후에 반응성 산소종(reactive oxygen species; ROS)의 생성량을 비교하기 위해 디하이드로에티듐(dihydroethidium; DHE)(Molecular Probes, 카탈로그 넘버:D23107) 염색 시료를 5 μM로 세포에 처리하여 37℃ 세포배양기에서 반응시켰다. 30분 후에 세포를 획득한 다음, 1X PBS로 한 차례 씻어주었고, FACS Staining Buffer (Invitrogen, 카탈로그 넘버: 00-4222-57)로 잘 섞어준 후에 유세포분석기를 통해서 DHE 발현을 분석하였다. 실험군 당 1x104개 세포 수에서의 형광 발현을 확인하였고, 대조군(negative control)은 피페린을 처리하지 않은 노화세포로 하였으며, 양성 대조군은(positive control)으로 니코틴아마이드(nicotinamide; NA) 5 μM을 처리한 노화세포로 하였다.After treating senescent cells with piperine three times in the method of 2 above, two days later, to compare the amount of reactive oxygen species (ROS) produced, dihydroethidium (DHE) (Molecular Probes, catalog number) :D23107) The cells were treated with 5 μM of the staining sample and reacted in a cell incubator at 37°C. After 30 minutes, the cells were harvested, washed once with 1X PBS, mixed well with FACS Staining Buffer (Invitrogen, catalog number: 00-4222-57), and then DHE expression was analyzed by flow cytometry. Fluorescence expression was confirmed at the number of cells of 1x10 4 cells per experimental group, the control group (negative control) was senescent cells not treated with piperine, and nicotinamide (NA) 5 μM was used as the positive control group (positive control). Treated senescent cells were used.
(2) 실험 결과(2) Experimental results
노화세포는 미토콘드리아 기능이 감소되어 있어, 산화적 손상을 유도하는 활성산소(reactive oxygen species, ROS)가 세포 내에 증가되어 있다. 도 6A는 노화세포에 피페린을 처리하여 미토콘드리아 기능을 확인한 것이고, 도 6B는 노화세포에 피페린을 처리하여 세포 내 ROS 생성을 확인한 것이다. 그 결과, 피페린 처리 시 미토콘드리아의 기능은 개선되고, 세포 내 ROS 생성은 감소되는 것을 알 수 있었다. 또한, 피페린 처리에 따른 노화세포에서의 미토콘드리아 막 전위 값과 ROS 생성 결과가 항산화제로 알려져 있는 니코틴 아마이드(nicotinamide, NA)를 노화세포에 처리한 결과와 유사했다. In senescent cells, mitochondrial function is reduced, and reactive oxygen species (ROS) that induces oxidative damage are increased in the cell. Figure 6A shows that the senescent cells are treated with piperine to confirm the mitochondrial function, and Figure 6B shows that the senescent cells are treated with piperine to confirm the intracellular ROS generation. As a result, it was found that the mitochondrial function was improved upon treatment with piperine, and intracellular ROS production was reduced. In addition, the results of mitochondrial membrane potential and ROS generation in senescent cells following piperine treatment were similar to those of senescent cells treated with nicotinamide (NA), known as an antioxidant.
8. 피페린에 의한 노화세포 특이적 분비인자의 비교8. Comparison of senescent cell-specific secretion factors by piperine
(1) 실험 방법(1) Experimental method
피페린에 의한 노화세포에서의 노화 세포 특이적 분비 인자인 SASP(senescence-associated secretory phenotype) 인자의 발현을 확인하고자 효소결합면역침강분석법(enzyme-linked immunosorbent assay, ELISA)을 통해 확인하였다.In order to confirm the expression of SASP (senescence-associated secretory phenotype) factor, which is a senescent cell-specific secretory factor, in senescent cells by piperine, it was confirmed through enzyme-linked immunosorbent assay (ELISA).
구체적으로, 노화세포와 비노화세포를 각각 6웰 플레이트(3 x 104 cells/6 well plates)에 접종하고, 다음 날, 상기 2의 방법으로 피페린을 각 세포에 이틀 간격으로 3차례 처리한 후에 세포 배양액을 획득하였다. 획득된 배양액을 가지고 노화 세포 특이적 분비 인자인 SASP 인자 중, IL-8 (R&D System, 카탈로그 넘버: DY208-05), IL-6 (R&D System, 카탈로그 넘버: DY206-05), 그리고 TGF-β1 (R&D System, 카탈로그 넘버: DY240-05) 사이토카인을 ELISA 실험방법을 통해서 분석하였다.Specifically, senescent cells and non-senescent cells were inoculated into 6-well plates (3 x 10 4 cells/6 well plates), respectively, and the next day, piperine was treated with each cell three times at two-day intervals in the method of 2 above. Afterwards, a cell culture medium was obtained. Among the SASP factors that are senescent cell-specific secreted factors with the obtained culture medium, IL-8 (R&D System, catalog number: DY208-05), IL-6 (R&D System, catalog number: DY206-05), and TGF-β1 (R&D System, catalog number: DY240-05) Cytokines were analyzed through an ELISA test method.
(2) 실험 결과(2) Experimental results
노화세포가 조직에 축적되어 분비하는 노화세포 특이적 분비인자인 SASP에 의해 노화 조직의 염증이 증가되고 질환 유발이 유도되는 것으로 알려져 있어, SASP 분비 억제 기능이 매우 중요하다. 본 실험에서 피페린을 노화세포에 처리한 결과, SASP로 알려진 3가지 종류의 사이토카인인 IL-6, IL-8 및 TGF-β1의 분비가 피페린을 처리하지 않은 노화세포에 비해 감소한 것을 확인하였다(도 7).It is known that inflammation of senescent tissues is increased and disease induction is induced by SASP, which is a senescent cell-specific secretion factor that senescent cells accumulate and secrete in tissues, so the function of inhibiting SASP secretion is very important. As a result of treating senescent cells with piperine in this experiment, it was confirmed that the secretion of three types of cytokines known as SASP, IL-6, IL-8 and TGF-β1, was decreased compared to senescent cells not treated with piperine. (Fig. 7).
Claims (4)
A reagent composition for senomorphic that restores senescent cells to normal cells containing piperine or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition for senomorphic that restores senescent cells to normal cells containing piperine or a pharmaceutically acceptable salt thereof.
A cosmetic composition for senomorphic that restores senescent cells to normal cells containing piperine or a pharmaceutically acceptable salt thereof.
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| KR100492309B1 (en) | 2002-03-20 | 2005-06-03 | 대한민국 | Pesticidal and Fungicidal Composition Containing Black Pepper Extracts |
| KR100846125B1 (en) * | 2007-03-30 | 2008-07-15 | 바이오스펙트럼 주식회사 | Compositions for improving skin wrinkle comprising piperine as an active ingredient |
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| KR100492309B1 (en) | 2002-03-20 | 2005-06-03 | 대한민국 | Pesticidal and Fungicidal Composition Containing Black Pepper Extracts |
| KR100846125B1 (en) * | 2007-03-30 | 2008-07-15 | 바이오스펙트럼 주식회사 | Compositions for improving skin wrinkle comprising piperine as an active ingredient |
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| David Banji 외, "Piperine and curcumin exhibit synergism in attenuating D-galactose induced senescence in rats", European Journal of Pharmacology, 제703권, 제91-99면 (2012. 11. 29. 온라인 공개) * |
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