KR102176018B1 - Streptomyces species strain and Composition for Skin Whitening Containing the same - Google Patents
Streptomyces species strain and Composition for Skin Whitening Containing the same Download PDFInfo
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- KR102176018B1 KR102176018B1 KR1020190035276A KR20190035276A KR102176018B1 KR 102176018 B1 KR102176018 B1 KR 102176018B1 KR 1020190035276 A KR1020190035276 A KR 1020190035276A KR 20190035276 A KR20190035276 A KR 20190035276A KR 102176018 B1 KR102176018 B1 KR 102176018B1
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Abstract
본 발명은 해양 퇴적토로부터 분리된 신규한 스트렙토마이세스 SCO736 균주, 이로부터 생성되는 신규 천연물, 및 이의 미백 용도에 관한 것이다.The present invention relates to a novel strain of Streptomyces SCO736 isolated from marine sediments, a new natural product produced therefrom, and a whitening use thereof.
Description
본 발명은 해양 퇴적토로부터 분리된 신규 균주, 및 이를 포함하는 미백용 조성물에 관한 것이다.The present invention relates to a new strain isolated from marine sediment, and a whitening composition comprising the same.
사람의 피부색을 결정하고 기미, 잡티들이 생성되는 현상에는 여러 요인들이 관여하는데, 멜라닌 색소를 만드는 멜라노사이트(melanocyte)의 활동성, 혈관의 분포, 피부의 두께, 카로티노이드, 빌리루빈 등 여러 인자들이 복합적으로 관여되어 있다. 이중에서도 가장 중요한 인자는 인체 내의 멜라노사이트에서 여러 효소들이 작용하여 생성되는 멜라닌 색소이다. 멜라닌 색소의 형성에는 유전적 요인, 호르몬 분비, 스트레스, 자외선 등의 생리적 요인 및 환경적 요인 등이 영향을 미친다고 알려져 있다.Several factors are involved in determining human skin color and the generation of spots and blemishes, including the activity of melanocytes that make melanocytes, distribution of blood vessels, skin thickness, carotenoids, and bilirubin. Has been. Among them, the most important factor is the melanin pigment produced by the action of various enzymes in melanocytes in the human body. It is known that physiological and environmental factors such as genetic factors, hormone secretion, stress, and UV rays affect the formation of melanin pigment.
사람의 피부색을 결정하는 멜라닌(melanin)은 멜라노사이트(melanocyte)에서 생성되는데, 멜라노사이트에는 티로시나제 등의 효소가 존재하며, 이들이 함께 작용하여 생체 내에 항상 존재하는 티로신(tyrosine)이라는 아미노산을 기질로 중합화 산화반응하여, 흑갈색의 색소인 멜라닌을 형성하게 된다. 이렇게 형성된 멜라닌은 멜라노사이트의 수지상 돌기를 통하여 케라티노사이트(keratinocyte)라는 표피 세포로 이동한다. 여기서 멜라닌은 핵 주변에 모자와 같은 구조를 형성하여 자외선으로부터 유전자를 보호하고, 자유 라디칼(free radical)을 제거하여 세포 내 단백질을 보호하는 등 중요한 역할을 하게 된다. 생체 내에는 멜라닌을 분해하는 효소가 없고, 다만 케라티노사이트가 노화되어 제 기능을 다하고 표피에서 떨어져나갈 때 케라티노사이트와 함께 피부로부터 제거된다. 하지만 멜라닌이 필요 이상으로 과다하게 생성되면 기미나 주근깨, 점 등과 같은 과색소침착증을 유발한다.Melanin, which determines human skin color, is produced in melanocytes, and enzymes such as tyrosinase exist in melanocytes, and they act together to polymerize an amino acid called tyrosine, which is always present in the living body, into a substrate. It reacts with oxidation to form melanin, a dark brown pigment. The melanin formed in this way moves to epidermal cells called keratinocytes through dendritic processes of melanocytes. Here, melanin plays an important role in protecting genes from ultraviolet rays by forming a cap-like structure around the nucleus and protecting proteins in cells by removing free radicals. There is no enzyme that breaks down melanin in the living body, but when keratinocytes are aged and functioned and fall off the epidermis, they are removed from the skin along with keratinocytes. However, when melanin is produced in excess than necessary, it causes hyperpigmentation such as spots, freckles, and spots.
이에 따라 멜라닌의 과도한 생성을 막아 피부에서 색소 침착을 억제하기 위한 미백제에 대한 연구가 이루어졌으며, 코직산(kojic acid), 알부틴(arbutin) 등과 같은 티로시나제 활성을 저해하는 억제제들, 하이드로퀴논 (hydroquinone), 비타민 A등을 비롯하여 대표적인 항산화제인 비타민 C 및 이들의 유도체 등이 보고되었다.Accordingly, studies have been conducted on whitening agents to inhibit pigmentation in the skin by preventing excessive production of melanin, and inhibitors that inhibit tyrosinase activity such as kojic acid and arbutin, hydroquinone, In addition to vitamin A, vitamin C, a representative antioxidant, and derivatives thereof have been reported.
그러나 기존의 미백 물질들은 그 효능이 약하거나 세포 독성 등을 동반하는 경우가 많아, 안전하고 미백 효능이 높은 천연물을 발굴하고자 하는 노력이 계속되고 있다.However, since the existing whitening substances have poor efficacy or are often accompanied by cytotoxicity, efforts are being made to discover natural products that are safe and have high whitening efficacy.
본 발명자들은 남극 해양퇴적토에서 분리된 미생물 스트렙토마이세스 SCO-736 균주를 연구하던 중, 이로부터 신규천연물인 안타로이드 (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo[c][1,5]oxazonine-2,7(1H,3H)-dione)를 발견하여 구조를 규명하였고, 안타로이드가 멜라닌 합성을 효과적으로 저해할 수 있음을 발견하여 본 발명을 완성하였다.The inventors of the present invention were studying the microorganism Streptomyces SCO-736 strain isolated from the Antarctic marine sediment, from which a novel natural product, antaroide (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4, 5-dihydrobenzo[c][1,5]oxazonine-2,7(1H,3H)-dione) was discovered and the structure was elucidated, and the present invention was completed by discovering that the antaroid can effectively inhibit melanin synthesis. I did.
남극 해양퇴적토에서 분리한 미생물인 스트렙토마이세스 SCO-736 균주는, 해양 방선균 (Marine Streptomyces) 의 일종으로, 이러한 방선균들은 아직까지 밝혀지지 않은 신규한 종들이 다수 존재하며, 이에 신규한 해양 방선균으로부터 아직까지 밝혀지지 않았던 새로운 천연물 또한 발견할 수 있을 것으로 기대되고 있다.Streptomyces SCO-736 strain, a microorganism isolated from the Antarctic marine sediment, is a type of Marine Streptomyces , and these actinomycetes have a number of novel species that have not been identified yet. It is expected to be able to discover new natural products that have not been revealed until now.
화학식 1로 표시되는 화합물인 안타로이드 (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihy- dro-benzo-[c][1,5]oxazonine-2,7(1H,3H)-dione)는, 해양 방선균 (Marine Streptomyces, SCO-736)에서 처음으로 발견 및 분리되었으며, 이의 활성에 대한 구체적인 연구결과는 아직까지 전혀 보고되지 않아, 신규천연물인 안타로이드의 새로운 활성에 대한 연구가 절실히 필요하다.Antaroide, a compound represented by Chemical Formula 1 (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihy- dro-benzo-[c][1,5]oxazonine-2, 7(1H,3H)-dione) was first discovered and isolated from marine actinomycetes (Marine Streptomyces, SCO-736), and no specific research results on its activity have yet been reported. Research on new activity is urgently needed.
본 발명의 일예는, 화학식 1을 가지는 화합물 또는 이의 약제학적으로 허용 가능한 염에 관한 것이다.An embodiment of the present invention relates to a compound having Formula 1 or a pharmaceutically acceptable salt thereof.
상기 화합물은 스트렙토마이세스 속 균주 또는 상기 균주의 배양물로부터 분리된 것을 특징으로 하는 것일 수 있다.The compound may be characterized in that it is isolated from a strain of the genus Streptomyces or a culture of the strain.
상기 화합물은 상기 균주의 배양물을 유기용매로 분획추출하고, 물 및 알코올로 이루어지는 군에서 선택된 1종 이상을 전개 용매로 사용하여 상기 분획추출물을 크로마토그래피 분리 및 정제한 분획물로부터 얻어지는 것을 특징으로 하는 것일 수 있다.The compound is characterized in that obtained from a fraction obtained by fractionating the culture of the strain with an organic solvent, and using at least one selected from the group consisting of water and alcohol as a developing solvent, and chromatographic separation and purification of the fractional extract. Can be.
상기 유기용매는 에틸아세테이트, 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올, 아세톤, 에테르, 클로로포름, 메틸렌클로라이드, 헥산, 시클로헥산, 및 디클로로메탄로 이루어진 군에서 선택된 1종 이상인 것일 수 있다.The organic solvent may be one or more selected from the group consisting of ethyl acetate, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, methylene chloride, hexane, cyclohexane, and dichloromethane.
상기 알코올은 메탄올일 수 있다.The alcohol may be methanol.
본 발명의 또 다른 일예는, 스트렙토마이세스 속 균주의 배양액을 유기용매로 분획 추출하는 단계; 및 분획추출물을 크로마토그래피로 분리 및 정제하는 단계를 포함하는, 화학식 1을 갖는 화합물 또는 이의 약제학적으로 허용 가능한 염의 제조방법을 제공하기 위한 것이다.Another example of the present invention is the step of fractionally extracting the culture solution of the strain of the genus Streptomyces with an organic solvent; And it is to provide a method for preparing a compound having Formula 1 or a pharmaceutically acceptable salt thereof, comprising the step of separating and purifying the fractional extract by chromatography.
상기 크로마토그래피는 농도 구배 크로마토그래피일 수 있다.The chromatography may be a concentration gradient chromatography.
상기 유기용매는 에틸아세테이트, 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올, 아세톤, 에테르, 클로로포름, 메틸렌클로라이드, 헥산, 시클로헥산, 및 디클로로메탄로 이루어진 군에서 선택된 1종 이상인 것일 수 있다.The organic solvent may be one or more selected from the group consisting of ethyl acetate, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, methylene chloride, hexane, cyclohexane, and dichloromethane.
상기 크로마토그래피는 물 및 알코올로 이루어지는 군에서 선택된 1종 이상을 전개 용매로 사용하여 상기 분획추출물을 분리 및 정제하는 것일 수 있다.The chromatography may be separating and purifying the fractionated extract using at least one selected from the group consisting of water and alcohol as a developing solvent.
상기 알코올은 메탄올인 것일 수 있다.The alcohol may be methanol.
본 발명의 또 다른 일예는, 화학식 1을 갖는 신규 화합물, 또는 이의 약리학적으로 허용 가능한 염을 유효성분으로 포함하는, 미백용 조성물에 관한 것이다.Another example of the present invention relates to a composition for whitening, comprising a novel compound having Formula 1, or a pharmacologically acceptable salt thereof as an active ingredient.
상기 조성물은 멜라노마 세포의 멜라닌 생성을 저해하는 것일 수 있다.The composition may be one that inhibits melanin production of melanoma cells.
상기 조성물은 용액, 연고, 외용연고, 크림, 에센스, 폼, 화장수, 영양화장수, 유연수, 유연화장수, 팩, 유액, 메이크업베이스, 비누, 세정료, 입욕제, 선크림, 선오일, 현탁액, 유탁액, 페이스트, 겔, 로션, 파우더, 비누, 계면활성제 함유 클린싱, 오일, 파운데이션, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션, 패치 및 스프레이로 이루어진 군에서 선택되는 제형을 가지는 것일 수 있다.The composition is a solution, ointment, external ointment, cream, essence, foam, lotion, nutrient lotion, softening water, softening lotion, pack, emulsion, makeup base, soap, cleaning agent, bathing agent, sun cream, sun oil, suspension, emulsion, It may have a formulation selected from the group consisting of paste, gel, lotion, powder, soap, surfactant containing cleansing, oil, foundation, powder foundation, emulsion foundation, wax foundation, patch, and spray.
상기 화장료 조성물은 피부 미백 또는 피부 색소 침착 개선용 화장료 조성물인 것일 수 있다.The cosmetic composition may be a cosmetic composition for skin whitening or skin pigmentation improvement.
본 발명의 또 다른 일예는, 화학식 1을 갖는 신규 화합물, 또는 이의 약리학적으로 허용 가능한 염을 유효성분으로 포함하는, 약학적 조성물, 화장료 조성물, 피부 외용제 조성물, 건강기능식품, 의약외품 조성물, 피부 색소 침착의 예방 또는 치료용 약학적 조성물, 또는 피부 색소 침착의 예방 또는 개선용 의약외품 조성물에 관한 것이다.Another example of the present invention is a pharmaceutical composition, a cosmetic composition, a composition for external application for skin, a health functional food, a quasi-drug composition, a skin pigment comprising a novel compound having Formula 1 or a pharmacologically acceptable salt thereof as an active ingredient It relates to a pharmaceutical composition for preventing or treating deposition, or a quasi-drug composition for preventing or improving skin pigmentation.
상기 약학적 조성물은 피부 색소 침착 질환의 예방 또는 치료용 약학적 조성물인 것일 수 있다. The pharmaceutical composition may be a pharmaceutical composition for preventing or treating skin pigmentation disorders.
본 발명의 또 다른 일예는, 미백 활성을 가지는 스트렙토마이세스 속 균주에관한 것이다.Another example of the present invention relates to a strain of the genus Streptomyces having whitening activity.
본 발명의 또 다른 일예는, 스트렙토마이세스 디아스타티쿠스 아종 아데스시아쿠스 SCO-736 균주의 균체, 상기 균주의 배양물, 상기 균주의 파쇄물, 및 상기 균주의 추출물로 이루어지는 군에서 선택된 1종 이상을 포함하는, 미백용 조성물에 관한 것이다.Another example of the present invention is one or more selected from the group consisting of the bacterial body of the Streptomyces diastaticus subspecies Adesciacus SCO-736 strain, the culture of the strain, the lysate of the strain, and the extract of the strain. It relates to a composition for whitening, including.
본 발명의 또 다른 일예는, 스트렙토마이세스 디아스타티쿠스 아종 아데스시아쿠스 SCO-736 균주의 균체, 상기 균주의 배양물, 상기 균주의 파쇄물, 및 상기 균주의 추출물로 이루어지는 군에서 선택된 1종 이상을 포함하는, 피부 외용제 조성물, 건강기능식품, 피부 색소 침착의 예방 또는 치료용 약학적 조성물, 또는 피부 색소 침착의 예방 또는 개선용 의약외품 조성물에 관한 것이다.Another example of the present invention is one or more selected from the group consisting of the bacterial body of the Streptomyces diastaticus subspecies Adesciacus SCO-736 strain, the culture of the strain, the lysate of the strain, and the extract of the strain. It relates to a composition for external application for skin, a health functional food, a pharmaceutical composition for preventing or treating skin pigmentation, or a quasi-drug composition for preventing or improving skin pigmentation.
본 발명의 또 다른 일예는, 스트렙토마이세스 속 균주 또는 상기 균주의 배양액을 유기용매로 분획추출하는 단계; 및 분획추출물을 크로마토그래피로 분리 및 정제하는 단계를 포함하는, 미백용 조성물의 제조방법에 관한 것이다.Another example of the present invention is the step of fractionally extracting a strain of the genus Streptomyces or a culture solution of the strain with an organic solvent; And it relates to a method for preparing a whitening composition comprising the step of separating and purifying the fractional extract by chromatography.
상기 유기용매는 에틸아세테이트, 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올, 아세톤, 에테르, 클로로포름, 메틸렌클로라이드, 헥산, 시클로헥산, 및 디클로로메탄로 이루어진 군에서 선택된 1종 이상일 수 있다.The organic solvent may be at least one selected from the group consisting of ethyl acetate, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, methylene chloride, hexane, cyclohexane, and dichloromethane.
상기 크로마토그래피는 물 및 알코올로 이루어지는 군에서 선택된 1종 이상을 전개 용매로 사용하여 상기 분획추출물을 분리 및 정제하는 것일 수 있다.The chromatography may be separating and purifying the fractionated extract using at least one selected from the group consisting of water and alcohol as a developing solvent.
상기 알코올은 메탄올일 수 있다.The alcohol may be methanol.
이하, 본 발명을 더욱 자세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명자들은 남극의 해양퇴적토를 채취한 뒤, 클린벤치에서 건조 후 상기 건조물을 멸균된 해수에 현탁하여 1/20으로 희석한 후 마린 브로스 (Marine broth) 고체배지에 접종하고, 27℃에서 배양하면서 생성된 단일 균주를 마린 브로스 (Marine broth) 고체배지에 다시 접종하여 순수한 균주를 분리 및 선별하였다. 이와 같이 분리된 SCO-736 균주는 전형적인 스트렙토마이세스 속에 속하는 균주의 형태적 양상이 관찰되었다 (도 1 참조).The present inventors collected the sea sediment of Antarctica, dried in a clean bench, suspended the dried product in sterilized seawater, diluted 1/20, inoculated in a marine broth solid medium, and cultured at 27°C. The resulting single strain was re-inoculated in Marine broth solid medium to isolate and select pure strains. The SCO-736 strain isolated as described above was observed in the form of a strain belonging to the typical Streptomyces genus (see FIG. 1).
본 발명의 일예에 따른 스트렙토마이세스 속 균주는, 서열번호 3의 16S rRNA 유전자 염기서열을 가지며, 이차대사산물로서 하기의 화학식 1로 표시되는 화합물을 분비하는 것을 특징으로 한다. 상기 균주 SCO-736는 생물자원센터(Korean Collection for Type Culture)에 2019년 1월 30일자로 기탁하여, 수탁번호 KCTC 13805BP 를 부여받았다. 상기 스트렙토마이세스 속 SCO-736 균주의 16S rRNA genes 염기서열은 서열번호 3과 같다.The strain of the genus Streptomyces according to an embodiment of the present invention has a 16S rRNA gene nucleotide sequence of SEQ ID NO: 3, and secretes a compound represented by the following formula (1) as a secondary metabolite. The strain SCO-736 was deposited on January 30, 2019 with the Korean Collection for Type Culture, and was given the accession number KCTC 13805BP. The base sequence of the 16S rRNA genes of the SCO-736 strain of the genus Streptomyces is shown in SEQ ID NO: 3.
상기 스트렙토마이세스 속 균주는, 하기 (1) 내지 (4) 중 1 이상의 특징을 가지는 것일 수 있다:The strain of the genus Streptomyces may have one or more of the following characteristics (1) to (4):
(1) 미백 활성을 가짐,(1) has whitening activity,
(2) 멜라노마 세포의 멜라닌 생성을 저해함,(2) inhibits the production of melanin in melanoma cells,
(3) 멜라닌 세포 자극 호르몬 (Melanocyte-Stimulating Hormone, αMSH)의 처리 조건에서 멜라닌 합성을 억제함,(3) inhibiting melanin synthesis under the conditions of melanocyte-stimulating hormone (αMSH) treatment,
(4) 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용 가능한 염을 분비함.(4) Secreting the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.
상기 스트렙토마이세스 속 균주의 상기 (3) 멜라닌 세포 자극 호르몬 (Melanocyte-Stimulating Hormone, αMSH)의 처리 조건에서 멜라닌 합성을 억제하는 억제율은, 알부틴(arbutine) 대비 1배 내지 20배, 1배 초과 내지 20배, 2배 내지 20배, 3배 내지 20배, 4배 내지 20배, 5배 내지 20배, 6배 내지 20배, 7배 내지 20배, 8배 내지 20배, 9배 내지 20배, 10배 내지 20배, 11배 내지 20배, 12배 내지 20배, 13배 내지 20배, 14배 내지 20배, 또는 15배 내지 20배일 수 있다.The inhibition rate of inhibiting melanin synthesis under the treatment conditions of the (3) melanocyte-stimulating hormone (αMSH) of the strain of the genus Streptomyces is 1 to 20 times, more than 1 times to arbutine. 20 times, 2 times to 20 times, 3 times to 20 times, 4 times to 20 times, 5 times to 20 times, 6 times to 20 times, 7 times to 20 times, 8 times to 20 times, 9 times to 20 times , 10 times to 20 times, 11 times to 20 times, 12 times to 20 times, 13 times to 20 times, 14 times to 20 times, or 15 times to 20 times.
본 발명자들은 해양퇴적토로부터 분리된 스트렙토마이세스 디아스타티쿠스 아종 아데스시아쿠스 SCO-736 균주를 액체 배양한 후, 에틸아세테이트를 이용하여 추출물을 조제하였고, 이로부터 화학식 1로 표시되는 안타로이드 (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo[c][1,5]oxazonine-2,7(1H,3H)-dione) 화합물을 분리하여 화합물의 구조를 동정하였고, 또한 상기 화합물들의 미백 효능을 평가함으로써 본 발명을 완성하였다.The present inventors liquid cultured strain of Streptomyces diastaticus subspecies Adesciacus SCO-736 isolated from marine sediment, and then prepared an extract using ethyl acetate, from which an anthoid represented by Chemical Formula 1 (Antaroide, Isolation of 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo[c][1,5]oxazonine-2,7(1H,3H)-dione) compound And also completed the present invention by evaluating the whitening efficacy of the compounds.
본 발명의 또 다른 일 예는, 상기 스트렙토마이세스 디아스타티쿠스 아종 아데스시아쿠스 SCO-736 균주를 고체 배지에서 1차 배양한 이후, 이로부터 생성된 콜로니를 액체 배지에 접종하여 대량 액체 배양하는 방법에 관한 것이다.Another example of the present invention is a method for mass liquid culture by inoculating the resulting colonies in a liquid medium after primary culturing the Streptomyces diastaticus subspecies Adesciacus SCO-736 strain in a solid medium It is about.
본 발명의 또 다른 일 예는, 스트렙토마이세스(Streptomyces, SCO-736)로부터 화학식 1로 표시되는 화합물을 수득하는 방법에 관한 것이다.Another example of the present invention relates to a method for obtaining a compound represented by Formula 1 from Streptomyces ( SCO-736).
본 발명의 또 다른 일예는, 상기 스트렙토마이세스 디아스타티쿠스 아종 아데스시아쿠스 SCO-736 균주, 상기 균주의 배양물, 상기 배양물의 건조물, 상기 배양물의 농축물, 상기 배양물의 유기용매 추출물로 이루어진 군에서 선택된 1종 이상을 유효성분으로 포함하는 미백제 또는 피부 색소 침착 질환의 치료용 조성물에 관한 것이다.Another example of the present invention is the group consisting of the Streptomyces diastaticus subspecies Adesciacus SCO-736 strain, a culture of the strain, a dried product of the culture, a concentrate of the culture, and an organic solvent extract of the culture It relates to a composition for the treatment of a whitening agent or skin pigmentation disorders comprising at least one selected from as an active ingredient.
상기 건조물과 농축물은, 상기 배양물을 통상적인 방법으로 건조 또는 농축하여 얻어진 것을 의미한다. 상기 유기용매 추출물은, SCO-736 균주의 배양물을 에틸아세테이트, 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올, 아세톤, 에테르, 클로로포름, 메틸렌클로라이드, 헥산, 시클로헥산, 및 디클로로메탄로 이루어진 군에서 선택된 1종 이상으로 추출하여 얻어진 추출물일 수 있다.The dried product and the concentrate mean that the culture is dried or concentrated by a conventional method. The organic solvent extract is 1 selected from the group consisting of ethyl acetate, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, methylene chloride, hexane, cyclohexane, and dichloromethane from the culture of the SCO-736 strain. It may be an extract obtained by extracting more than one species.
상기 추출은 초임계추출, 고압추출 또는 초음파추출법 등의 추출장치를 이용한 방법, 또는 셀라이트 또는 폴리스틸렌 또는 폴리아마드 흡착수지를 이용하는 방법으로 대체 가능하나, 이에 한정되지 않는다. 추출 시 용매를 배양액 분량의 1 내지 3 부피 배 첨가하여 추출하는 것이 바람직하며, 상온에서 추출하는 것이 바람직하다. 추출 회수는 1 내지 3회인 것이 바람직하며, 감압건조는 회전진공농축기 (Rotary Vacuum Evaporator)를 사용하는 것이 바람직하나 이에 한정되지 않는다. 상기 감압건조 시 온도는 20 내지 40 ℃가 바람직하고, 더욱 바람직하게는 25 내지 35℃, 예를 들어 30℃인 것이 더욱 바람직하나, 이에 한정되지 않는다.The extraction may be replaced by a method using an extraction device such as supercritical extraction, high pressure extraction, or ultrasonic extraction, or a method using celite, polystyrene, or polyamide adsorption resin, but is not limited thereto. During extraction, it is preferable to extract the solvent by adding 1 to 3 times the volume of the culture solution, and it is preferable to extract at room temperature. The number of extractions is preferably 1 to 3 times, and drying under reduced pressure is preferably performed using a rotary vacuum evaporator, but is not limited thereto. The temperature for drying under reduced pressure is preferably 20 to 40°C, more preferably 25 to 35°C, more preferably 30°C, but is not limited thereto.
본 발명의 또 다른 일예에 의하면, 상기 추출물을 분리 및 정제하여 소획물을 얻을 수 있으며, 예를 들어 상기 소획물은 농도 구배를 가지는 크로마토그래피를 사용하여 상기 추출물을 분리 및 정제하여 얻어질 수 있다. 상기 크로마토그래피는 역상 크로마토그래피, 컬럼 크로마토그래피, 액체 크로마토그래피, 친화성 크로마토그래피, 흡착 크로마토그래피, 이온교환 크로마토그래피, HPLC, 및 UPLC로 이루어지는 군에서 선택된 1종 이상의 크로마토그래피 방법일 수 있다. 일예로, 상기 소획물은 전개용매로 메탄올 수용액을 사용하고 농도 구배를 가지는 역상 칼럼 크로마토그래피를 사용하여 상기 추출물을 분리하여, 메탄올 농도 60%(v/v)의 분획에서 얻어진 크로마토그램 분획물일 수 있다. 예를 들어, 상기 추출물의 물/메탄올 분획물은 상기 유기용매 추출물을 메탄올 20, 40, 50, 60, 70, 80, 100%(v/v) 용액으로 추가로 분획하여 얻어진 소획물일 수 있으며, 일예로 네 번째 크로마토그램인 메탄올 60%(v/v) 분획물일 수 있다.According to another embodiment of the present invention, the extract may be separated and purified to obtain a small fraction, for example, the small fraction may be obtained by separating and purifying the extract using chromatography having a concentration gradient. . The chromatography may be one or more chromatographic methods selected from the group consisting of reverse phase chromatography, column chromatography, liquid chromatography, affinity chromatography, adsorption chromatography, ion exchange chromatography, HPLC, and UPLC. As an example, the small fraction may be a chromatogram fraction obtained from a fraction having a methanol concentration of 60% (v/v) by separating the extract using an aqueous methanol solution as a developing solvent and using reverse phase column chromatography having a concentration gradient. have. For example, the water/methanol fraction of the extract may be a small fraction obtained by further fractionating the organic solvent extract into a
본 발명의 또 다른 일예에 의하면, 상기 소획물을 분리 및 정제하여 화학식 1로 표시되는 화합물을 얻을 수 있으며, 예를 들어 상기 소획물을 크로마토그래피로 분리하여 얻을 수 있다. 상기 크로마토그래피는 역상 크로마토그래피, 컬럼 크로마토그래피, 액체 크로마토그래피, 친화성 크로마토그래피, 흡착 크로마토그래피, 이온교환 크로마토그래피, HPLC, 및 UPLC로 이루어지는 군에서 선택된 1종 이상의 크로마토그래피 방법일 수 있다. 일예로, 상기 소획물을 액체 크로마토그래피를 사용하여 정제하여, 머무름 시간 10 내지 50분, 10 내지 45분, 10 내지 40분, 10 내지 30분, 15 내지 50분, 15 내지 45분, 15 내지 40분, 15 내지 35분, 20 내지 50분, 20 내지 45분, 20 내지 40분, 20 내지 35분, 25 내지 50분, 25 내지 45분, 25 내지 40분, 25 내지 35분, 30 내지 50분, 30 내지 45분, 30 내지 40분, 30 내지 35분, 31 내지 50분, 31 내지 45분, 31 내지 40분, 31 내지 35분, 31 내지 34분, 31 내지 33분, 31 내지 32분, 또는 31분에서 화학식 1의 화합물을 얻을 수 있다. 상기 액체 크로마토그래피는 역상 액체 크로마토그래피일 수 있다.According to another embodiment of the present invention, the small fraction may be separated and purified to obtain a compound represented by Formula 1, for example, the small fraction may be obtained by separating the fraction by chromatography. The chromatography may be one or more chromatographic methods selected from the group consisting of reverse phase chromatography, column chromatography, liquid chromatography, affinity chromatography, adsorption chromatography, ion exchange chromatography, HPLC, and UPLC. As an example, the fraction was purified using liquid chromatography, and retention times of 10 to 50 minutes, 10 to 45 minutes, 10 to 40 minutes, 10 to 30 minutes, 15 to 50 minutes, 15 to 45 minutes, 15 to 40 minutes, 15 to 35 minutes, 20 to 50 minutes, 20 to 45 minutes, 20 to 40 minutes, 20 to 35 minutes, 25 to 50 minutes, 25 to 45 minutes, 25 to 40 minutes, 25 to 35 minutes, 30 to 50 minutes, 30 to 45 minutes, 30 to 40 minutes, 30 to 35 minutes, 31 to 50 minutes, 31 to 45 minutes, 31 to 40 minutes, 31 to 35 minutes, 31 to 34 minutes, 31 to 33 minutes, 31 to The compound of formula 1 can be obtained in 32 minutes or 31 minutes. The liquid chromatography may be reverse phase liquid chromatography.
상기 분획물 및/또는 소분획물은 화학식 1로 표시되는 화합물을 비롯하여 유용한 이차대사산물을 함유하여 우수한 미백 효과를 나타낼 수 있다.The fraction and/or the small fraction may contain useful secondary metabolites, including the compound represented by Formula 1, to exhibit excellent whitening effect.
본 발명의 또 다른 일예에 의하면, 스트렙토마이세스 디아스타티쿠스 아종 아데스시아쿠스 SCO-736 균주의 이차대사산물 중 미백 효능을 갖는 물질이 포함되어 있음을 확인하였다 (실시예 2 및 4). 이러한 유용한 이차대사 산물은 상기 균주의 배양 후 약 6일 째부터 생산되는 것으로 나타났다. 따라서, 본 발명의 일예에 의한 미백제 또는 피부 색소 침착 질환의 치료용 조성물의 유효 성분으로, 스트렙토마이세스 SCO-736 균주 뿐만 아니라 스트렙토마이세스 SCO-736 균주의 배양물도 사용할 수 있다. 상기 목적을 달성하기 위하여, 본 발명의 일예는 화학식 1로 표시되는 화합물인 안타로이드 (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydro- benzo-[c][1,5]oxazonine-2,7(1H,3H)-dione)를 유효성분으로 포함하는 피부 미백용 화장료 조성물을 제공한다.According to another embodiment of the present invention, it was confirmed that a substance having a whitening effect was included among the secondary metabolites of the Streptomyces diastaticus subspecies Adesciacus SCO-736 strain (Examples 2 and 4). These useful secondary metabolism products were found to be produced from about 6 days after cultivation of the strain. Therefore, as an active ingredient of the composition for the treatment of whitening agents or skin pigmentation disorders according to an example of the present invention, not only the Streptomyces SCO-736 strain but also the culture of the Streptomyces SCO-736 strain may be used. In order to achieve the above object, an example of the present invention is an antaroid (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydro- benzo-[c), a compound represented by Formula 1 It provides a cosmetic composition for skin whitening comprising ][1,5]oxazonine-2,7(1H,3H)-dione) as an active ingredient.
상기 "안타로이드"는 다음과 같은 화학식 1로 표시되는 물질이다.The above "Antaroid" is a material represented by the following formula (1).
상기 안타로이드 (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5- dihydrobenzo-[c][1,5]oxazonine-2,7(1H,3H)-dione)는 멜라닌 생성을 저해하여 피부의 색소 침착을 예방, 치료, 개선할 수 있고, 미백효능을 나타낼 수 있다.The antaroide (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5- dihydrobenzo-[c][1,5]oxazonine-2,7(1H,3H)-dione) is By inhibiting melanin production, it can prevent, treat, and improve skin pigmentation, and can exhibit whitening effects.
본 발명의 일예에 따른 화학식 1로 표시되는 화합물인 "안타로이드"는 멜라닌 생성을 저해할 수 있다. 상기 "멜라닌 생성 저해"는 피부에서 멜라닌 합성을 억제하는 것, 생성된 멜라닌의 분해를 촉진하는 것과 같이 피부에서 멜라닌의 함량을 낮추고, 색소의 침착을 억제하는 기전을 모두 포함할 수 있다. 예를 들어, 멜라노마 세포의 멜라닌 생성을 저해하거나, 멜라닌 세포 자극 호르몬 (Melanocyte-Stimulating Hormone, αMSH)로 인해 증가된 멜라닌 합성을 억제하는 기전일 수 있다."Antaroid", which is a compound represented by Chemical Formula 1 according to an embodiment of the present invention, may inhibit melanin production. The "melanogenesis inhibition" may include all mechanisms of inhibiting melanin synthesis in the skin, reducing the content of melanin in the skin, and inhibiting the deposition of pigments, such as promoting the decomposition of the produced melanin. For example, it may be a mechanism that inhibits melanin production of melanoma cells, or inhibits increased melanin synthesis due to melanocyte-Stimulating Hormone (αMSH).
상기 SCO-736 균주 및/또는 이로부터 분리된 상기 화학식 1로 표시되는 화합물은, 적은 농도로 강한 미백 효능을 가져 폭넓은 미백제로 사용될 수 있다는 이점을 갖는다.The SCO-736 strain and/or the compound represented by Formula 1 isolated therefrom has the advantage that it can be used as a wide range of whitening agents with strong whitening efficacy at a small concentration.
본 발명의 일 예에 따른 미백용 조성물은, 화학식 1로 표시되는 물질 및 이와 유사한 것으로 당 분야에서 인지될 수 있는 유도체, 이성질체 등을 제한 없이 포함할 수 있다.The composition for whitening according to an embodiment of the present invention may include, without limitation, derivatives, isomers, etc. that are recognized in the art as substances represented by Formula 1 and similar.
본 발명에서 "피부 미백"은 예컨대 기미, 주근깨, 검버섯 및 잡티 등의 예방 또는 개선이 있으나 이에 제한되는 것은 아니며, 피부에서의 색소침착을 예방 또는 개선, 치료하는 모든 활성을 포함한다.In the present invention, "skin whitening" includes, for example, prevention or improvement of spots, freckles, age spots, and blemishes, but is not limited thereto, and includes all activities of preventing, improving, and treating pigmentation in the skin.
상기 유효성분으로 사용되는 화학식 1로 표시되는 화합물인 안타로이드 (Antaroide, 5-hydroxy- 5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo[c][1,5]oxa- zonine-2,7(1H,3H)-dione)는 그 자체, 또는 염의 형태로 사용될 수 있다. 상기 염으로는 약학적으로 허용 가능한 염인 유리산에 의하여 형성된 산 부가염이 바람직하다.Antaroide, a compound represented by Formula 1, used as the active ingredient (Antaroide, 5-hydroxy- 5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo[c][1,5]oxa-zonine -2,7(1H,3H)-dione) can be used by itself or in the form of a salt. The salt is preferably an acid addition salt formed by a free acid, which is a pharmaceutically acceptable salt.
본 발명의 또 다른 예에서, SCO-736 균주로부터 상기 화학식 1의 화합물을 분리 정제하여 제조하는 방법이 제공된다. 상기 방법은 다음의 단계를 포함할 수 있다:In another example of the present invention, a method for preparing the compound of Formula 1 by separating and purifying it from the SCO-736 strain is provided. The method may include the following steps:
1) 상기 SCO-736 균주를 배양한 배양액에 초산에틸 유기용매를 가하여 추출하는 단계; 1) extracting the SCO-736 strain by adding an organic solvent of ethyl acetate to the culture solution;
2) 상기 얻어진 추출물을 20 내지 100%의 메탄올/물 혼합 용액을 이용하여 C-18 역상 오픈 컬럼 크로마토그래피하여 분획물을 얻는 단계; 및2) obtaining a fraction by performing C-18 reverse phase open column chromatography on the obtained extract using a methanol/water mixture solution of 20 to 100%; And
3) 상기 분획물들 중 첫 번째와 네 번째 분획물을 각각 Acetonitrile : H2O = 48 : 52, 용액으로 고성능 액체 컬럼 크로마토그래피로 분리 정제 하는 단계.3) Separating and purifying the first and fourth fractions of the above fractions by high performance liquid column chromatography as a solution of Acetonitrile: H2O = 48: 52, , respectively.
상기 균주의 배양기간은, 유효한 이차대사 산물의 생산이 활발한 약 4일 이상, 바람직하게는 4 내지 20일, 보다 바람직하게는 6 내지 15일로 설정될 수 있다.The culture period of the strain may be set to about 4 days or more, preferably 4 to 20 days, more preferably 6 to 15 days, in which the production of an effective secondary metabolite product is active.
상기 화학식 1로 표시되는 유용한 이차대사산물의 생산에 최적 조건을 제공하기 위하여, 균주의 배양 배지는 Marine broth (BD) 등일 수 있다. 상기 Marine broth 배지 형태는 고체배지와 액체배지 모두 가능하며, 바람직하게는 액체배지일 수 있다. 예를 들어, 상기 제조방법에 있어서 단계 1)의 액체 배양은 Marine broth 액체배지를 사용하여 균주 콜로니 2-3개당 액체배지 20 내지 30 ml의 비율로 접종하여 수행하는 것이 바람직하다.In order to provide optimal conditions for the production of useful secondary metabolites represented by Formula 1, the culture medium of the strain may be Marine broth (BD) or the like. The marine broth medium form may be both a solid medium and a liquid medium, preferably a liquid medium. For example, in the preparation method, the liquid culture of step 1) is preferably performed by inoculating the liquid medium in a ratio of 20 to 30 ml of the liquid medium per 2-3 colonies of the strain using Marine broth liquid medium.
상기 배양액의 유기용매 추출 시, 용매를 배양액 분량의 1 내지 3 부피 배 첨가하여 추출하는 것이 바람직하며, 예를 들어 약 2 부피 배 첨가하여 추출하는 것이 더욱 바람직하다. 감압건조는 회전진공농축기 (Rotary Vacuum Evaporator)를 사용하는 것이 바람직하나 이에 한정되지 않는다. 상기 감압건조 시 온도는 20 ~ 40℃인 것이 바람직하고 30℃인 것이 더욱 바람직하나 이에 한정되지 않는다. When extracting the organic solvent of the culture solution, it is preferable to extract the solvent by adding 1 to 3 times the volume of the culture solution, for example, it is more preferable to extract by adding about 2 times the volume. For drying under reduced pressure, it is preferable to use a rotary vacuum evaporator, but is not limited thereto. The temperature during drying under reduced pressure is preferably 20 to 40°C, and more preferably 30°C, but is not limited thereto.
상기 단계 2) 또는 단계 3)의 컬럼 크로마토그래피는 실리카겔, 세파덱스, RP-18, RP-8, RP-4, 폴리아미드, 도요펄 (Toyopearl) 및 XAD 수지로 이루어진 그룹으로부터 선택된 충진제를 이용한 컬럼 크로마토그래피일 수 있다. 컬럼 크로마토그래피는 필요에 따라 적절한 충진제를 선택하여 수차례 실시할 수 있으며, 용매로 클로로포름 (CHCl3)-메탄올, 초산에틸 (ethyl acetate)-메탄올, 염화메틸렌 (dichloromethane)-메탄올, 메탄올-물 또는 아세토나이트릴-물을 이용할 수 있으나 이에 한정되지 않는다. The column chromatography of step 2) or step 3) is a column using a filler selected from the group consisting of silica gel, Sephadex, RP-18, RP-8, RP-4, polyamide, Toyopearl, and XAD resin. It can be chromatography. Column chromatography can be performed several times by selecting an appropriate filler as necessary, and as a solvent, chloroform (CHCl 3 )-methanol, ethyl acetate-methanol, methylene chloride-methanol, methanol-water or Acetonitrile-water may be used, but is not limited thereto.
본 발명의 구체 예에서, 용리 조건에 물과 메탄올을 사용하였으며, 20% 메탄올/80% 물로 시작하여 20, 40, 50, 60, 70, 80, 100%까지 메탄올의 함량을 증가시켰다. 이로부터 얻어진 분획물을 C-18을 이용한 컬럼 크로마토그래피법을 이용하여 주성분을 분석한 결과, 60% 메탄올/40% 물 분획물에서 1종의 화합물이 확인되었다. 상기 분획물에 추가적으로 48% 아세토나이트릴/52% 물 조건 및 분당 2 ml의 유속의 용리조건으로, 10 mm × 250 mm의 RP-18 컬럼을 이용한 역상 액체 크로마토그래피법을 통해 정제하였으며 머무름 시간 31분에 상기 화학식 1로 표시되는 화합물인 안타로이드 (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo[c][1,5]oxazonine- 2,7(1H,3H)-dione)를 수득하였다.In an embodiment of the present invention, water and methanol were used for the elution conditions, and the content of methanol was increased to 20, 40, 50, 60, 70, 80, and 100% starting with 20% methanol/80% water. As a result of analyzing the main component of the fraction obtained therefrom using a column chromatography method using C-18, one compound was identified in the 60% methanol/40% water fraction. In addition to the fraction, the fraction was purified through reverse phase liquid chromatography using a 10 mm × 250 mm RP-18 column under conditions of 48% acetonitrile/52% water and an elution condition at a flow rate of 2 ml per minute, and retention time of 31 minutes In the compound represented by Formula 1, the antaroide (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo[c][1,5]oxazonine-2,7(1H) ,3H)-dione) was obtained.
상기 분리한 화합물들을 MS 및 핵분광기를 이용하여 화합물의 구조를 분석하여 화학구조를 동정하였으며, 화합물 안타로이드 (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo[c][1,5]oxazonine-2,7(1H,3H)-dione)는 분자량 361이며, 분자식이 C20H27NO5인 연갈색 오일상의 물질인 것을 확인하였다.The isolated compounds were analyzed by analyzing the structure of the compound using MS and nuclear spectroscopy to identify the chemical structure, and the compound antaroid (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5- It was confirmed that dihydrobenzo[c][1,5]oxazonine-2,7(1H,3H)-dione) has a molecular weight of 361 and a light brown oily substance having a molecular formula of C 20 H 27 NO 5 .
본 발명의 또 다른 일예는, 스트렙토마이세스 속 균주 또는 상기 균주의 배양액을 유기용매로 분획추출하는 단계; 및 분획추출물을 크로마토그래피로 분리 및 정제하는 단계를 포함하는, 미백용 조성물의 제조방법에 관한 것이다.Another example of the present invention is the step of fractionally extracting a strain of the genus Streptomyces or a culture solution of the strain with an organic solvent; And it relates to a method for preparing a whitening composition comprising the step of separating and purifying the fractional extract by chromatography.
본 발명의 또 다른 일예는, 상기 분획추출물을 크로마토그래피로 분리 및 정제하는 단계 이후에, 상기 크로마토그래피로 얻어진 크로마토그램 분획물을 얻는 단계를 추가로 포함할 수 있다. 일예로, 상기 크로마토그래피는 역상 칼럼 크로마토그래피일 수 있다. 예를 들어, 상기 크로마토그래피는 전개용매로 메탄올 수용액을 사용한 역상 칼럼 크로마토그래피일 수 있다. 상기 크로마토그래피는 농도 구배를 가지는 역상 칼럼 크로마토그래피일 수 있으며, 예를 들어 메탄올 함량이 점차 증가되는 메탄올 수용액을 전개용매로 사용한 농도 구배 역상 칼럼 크로마토그래피일 수 있다. 일예로, 상기 크로마토그래피는 전개용매로 20, 40, 50, 60, 70, 80, 100%(v/v)까지 메탄올의 함량을 증가시킨 메탄올 수용액을 사용한 역상 칼럼 크로마토그래피일 수 있다. 상기 분획물은, 상기 역상 칼럼 크로마토그래피로 얻어진 크로마토그램 분획물일 수 있다. 일예로, 상기 분획물은 전개 용매로 메탄올 수용액을 사용한 역상 칼럼 크로마토그래피로 얻어진 크로마토그램 분획물일 수 있으며, 예를 들어 메탄올 농도 50 내지 70%(v/v), 50 내지 65%(v/v), 50 내지 64%(v/v), 50 내지 63%(v/v), 50 내지 62%(v/v), 50 내지 61%(v/v), 50 내지 60%(v/v), 55 내지 70%(v/v), 55 내지 65%(v/v), 55 내지 64%(v/v), 55 내지 63%(v/v), 55 내지 62%(v/v), 55 내지 61%(v/v), 55 내지 60%(v/v), 56 내지 70%(v/v), 56 내지 65%(v/v), 56 내지 64%(v/v), 56 내지 63%(v/v), 56 내지 62%(v/v), 56 내지 61%(v/v), 56 내지 60%(v/v), 57 내지 70%(v/v), 57 내지 65%(v/v), 57 내지 64%(v/v), 57 내지 63%(v/v), 57 내지 62%(v/v), 57 내지 61%(v/v), 57 내지 60%(v/v), 58 내지 70%(v/v), 58 내지 65%(v/v), 58 내지 64%(v/v), 58 내지 63%(v/v), 58 내지 62%(v/v), 58 내지 61%(v/v), 58 내지 60%(v/v), 59 내지 70%(v/v), 59 내지 65%(v/v), 59 내지 64%(v/v), 59 내지 63%(v/v), 59 내지 62%(v/v), 59 내지 61%(v/v), 59 내지 60%(v/v), 또는 60%(v/v) 메탄올 농도에서 분리된 분획물일 수 있다.Another example of the present invention may further include a step of obtaining a chromatogram fraction obtained by chromatography after separating and purifying the fraction extract by chromatography. For example, the chromatography may be reverse-phase column chromatography. For example, the chromatography may be reverse-phase column chromatography using an aqueous methanol solution as a developing solvent. The chromatography may be reverse-phase column chromatography having a concentration gradient, for example, a concentration gradient reverse-phase column chromatography using an aqueous methanol solution whose methanol content is gradually increased as a developing solvent. As an example, the chromatography may be reverse-phase column chromatography using an aqueous methanol solution in which the content of methanol is increased to 20, 40, 50, 60, 70, 80, 100% (v/v) as a developing solvent. The fraction may be a chromatogram fraction obtained by the reverse phase column chromatography. As an example, the fraction may be a chromatogram fraction obtained by reverse phase column chromatography using an aqueous methanol solution as a developing solvent, for example, a methanol concentration of 50 to 70% (v/v), 50 to 65% (v/v) , 50 to 64% (v/v), 50 to 63% (v/v), 50 to 62% (v/v), 50 to 61% (v/v), 50 to 60% (v/v) , 55-70% (v/v), 55-65% (v/v), 55-64% (v/v), 55-63% (v/v), 55-62% (v/v) , 55 to 61% (v/v), 55 to 60% (v/v), 56 to 70% (v/v), 56 to 65% (v/v), 56 to 64% (v/v) , 56 to 63% (v/v), 56 to 62% (v/v), 56 to 61% (v/v), 56 to 60% (v/v), 57 to 70% (v/v) , 57-65% (v/v), 57-64% (v/v), 57-63% (v/v), 57-62% (v/v), 57-61% (v/v) , 57 to 60% (v/v), 58 to 70% (v/v), 58 to 65% (v/v), 58 to 64% (v/v), 58 to 63% (v/v) , 58 to 62% (v/v), 58 to 61% (v/v), 58 to 60% (v/v), 59 to 70% (v/v), 59 to 65% (v/v) , 59 to 64% (v/v), 59 to 63% (v/v), 59 to 62% (v/v), 59 to 61% (v/v), 59 to 60% (v/v) , Or may be a fraction separated at a concentration of 60% (v/v) methanol.
본 발명의 또 다른 일예는, 상기 크로마토그래피로 얻어진 크로마토그램 분획물을 얻는 단계 이후에, 상기 크로마토그램 분획물을 분리 및 정제하여 화학식 1로 표시되는 화합물을 얻는 단계를 추가로 포함할 수 있다. 일예로, 상기 크로마토그램 분획물은 크로마토그래피, 역상 크로마토그래피, 컬럼 크로마토그래피, 액체 크로마토그래피, 친화성 크로마토그래피, 흡착 크로마토그래피, 이온교환 크로마토그래피, HPLC, 및 UPLC로 이루어지는 군에서 선택된 1종 이상의 방법으로 분리 및 정제될 수 있으며, 일예로 역상 액체 크로마토그래피로 분리 및 정제될 수 있다. 예를 들어, 상기 크로마토그램 분획물은 크로마토그래피의 머무름 시간 10 내지 50분, 10 내지 45분, 10 내지 40분, 10 내지 30분, 15 내지 50분, 15 내지 45분, 15 내지 40분, 15 내지 35분, 20 내지 50분, 20 내지 45분, 20 내지 40분, 20 내지 35분, 25 내지 50분, 25 내지 45분, 25 내지 40분, 25 내지 35분, 30 내지 50분, 30 내지 45분, 30 내지 40분, 30 내지 35분, 31 내지 50분, 31 내지 45분, 31 내지 40분, 31 내지 35분, 31 내지 34분, 31 내지 33분, 31 내지 32분, 또는 31분에서 화학식 1의 화합물을 얻을 수 있다. Another example of the present invention may further include the step of obtaining a compound represented by Formula 1 by separating and purifying the chromatogram fraction after the step of obtaining the chromatogram fraction obtained by the chromatography. As an example, the chromatogram fraction is one or more methods selected from the group consisting of chromatography, reverse phase chromatography, column chromatography, liquid chromatography, affinity chromatography, adsorption chromatography, ion exchange chromatography, HPLC, and UPLC. It can be separated and purified by, for example, can be separated and purified by reverse phase liquid chromatography. For example, the chromatogram fraction has a retention time of 10 to 50 minutes, 10 to 45 minutes, 10 to 40 minutes, 10 to 30 minutes, 15 to 50 minutes, 15 to 45 minutes, 15 to 40 minutes, 15 To 35 minutes, 20 to 50 minutes, 20 to 45 minutes, 20 to 40 minutes, 20 to 35 minutes, 25 to 50 minutes, 25 to 45 minutes, 25 to 40 minutes, 25 to 35 minutes, 30 to 50 minutes, 30 To 45 minutes, 30 to 40 minutes, 30 to 35 minutes, 31 to 50 minutes, 31 to 45 minutes, 31 to 40 minutes, 31 to 35 minutes, 31 to 34 minutes, 31 to 33 minutes, 31 to 32 minutes, or The compound of formula 1 can be obtained in 31 minutes.
이상 본 명세서에 기재된 수치값은 달리 명시되어 있지 않은 한 균등범위까지 포함하는 것으로 해석되어야 한다.The numerical values described in the present specification should be construed as including an equivalent range unless otherwise specified.
상기한 바와 같이, 본 발명은 스트렙토마이세스 디아스타티쿠스 아종 아데스시아쿠스 SCO736 균주의 액체 배양법을 개발하여, 화학식 1로 표시되는 화합물인 안타로이드 (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo-[c]- [1,5]oxazonine-2,7(1H,3H)-dione)를 분리, 정제하였다. 이 화합물은 미백 효능이 뛰어나며, 따라서 스트렙토마이세스 디아스타티쿠스 아종 아데스시아쿠스 SCO-736 균주의 액체 배양액 추출물, 혹은 화학식 1로 표시되는 화합물을 포함하는 분획물, 또는 그 화학식 1의 화합물 혹은 이들의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 약학적 조성물은, 미백제 개발을 위한 후보물질로서 유용하게 이용될 수 있다.As described above, the present invention develops a liquid culture method of the strain of Streptomyces diastaticus subspecies Adesciacus SCO736, and is an antaroid, a compound represented by Chemical Formula 1 (Antaroide, 5-hydroxy-5-(6-methyl- 7-oxooctyl)-4,5-dihydrobenzo-[c]-[1,5]oxazonine-2,7(1H,3H)-dione) was isolated and purified. This compound is excellent in whitening effect, and therefore, a liquid culture extract of the strain of Streptomyces diastaticus subspecies Adesciacus SCO-736, a fraction containing a compound represented by Formula 1, or a compound represented by Formula 1 or a pharmaceutical thereof. A pharmaceutical composition containing an acceptable salt as an active ingredient can be usefully used as a candidate material for the development of a whitening agent.
또한, 상기 SCO-736 균주로부터 분리된 화학식 1의 화합물인 안타로이드(Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo[c][1,5]oxazonine-2,7(1H,3H)-dione)가 우수한 미백 효능이 있는 것으로 나타났다. 따라서, 화학식 1로 표시되는 화합물의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 미백제 또는 치료용 조성물이 제공될 수 있다.In addition, the antaroid (Antaroide, 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo[c][1,5]oxazonine, which is a compound of Formula 1 isolated from the SCO-736 strain. -2,7(1H,3H)-dione) was found to have excellent whitening effect. Accordingly, a whitening agent or a therapeutic composition containing a pharmaceutically acceptable salt of the compound represented by Formula 1 as an active ingredient may be provided.
본 발명의 일예에 따른 화학식 1의 화합물은 천연물 유래이므로 안전한 물질이며, 멜라닌 생성을 효과적으로 억제함으로써 피부의 색소침착을 예방, 치료, 개선할 수 있어, 미백제 개발을 위한 조성물의 제조에 매우 유용하게 이용될 수 있다. The compound of formula 1 according to an example of the present invention is a safe substance because it is derived from a natural product, and it can prevent, treat, and improve pigmentation of the skin by effectively inhibiting the production of melanin. Can be.
도 1은 해양퇴적토로부터 분리한 스트렙토마이세스 디아스타티쿠스 아종 아데스시아쿠스 SCO-736 균주의 Marine 고체배지에서의 형태를 나타내는 사진이다.
도 2는 배양한 SCO-736 균주의 전체 추출물 분획 후, 그 중 미백효과가 가장 뛰어난 Fraction 4번 째의 크로마토그램 중 UV 210, 254 nm에서 화학식 1로 표시되는 화합물이 분리된 피크를 화살표로 나타낸 도면이다.
도 3a는 실시예 3에서 분리 정제된 화합물의 구조를 동정하기 위해 핵자기공명(NMR) 분석을 수행한 결과를 나타낸 도면이다.
도 3b는 실시예 3에서 분리 정제된 화합물의 구조를 동정하기 위한 질량분석 결과를 나타낸 도면이다.
도 4는 화학식 1로 표시되는 화합물의 농도 의존적 멜라닌 억제 효과를 확인한 결과를 나타낸 도면이다.1 is a photograph showing the morphology of the strain of Streptomyces diastaticus Adesciacus SCO-736 isolated from marine sediment in Marine solid medium.
FIG. 2 is an arrow showing the peak of the compound represented by Formula 1 at
3A is a diagram showing the results of nuclear magnetic resonance (NMR) analysis in order to identify the structure of a compound separated and purified in Example 3. FIG.
3B is a diagram showing the results of mass spectrometry for identifying the structure of a compound separated and purified in Example 3. FIG.
4 is a diagram showing the results of confirming the concentration-dependent melanin inhibitory effect of the compound represented by Chemical Formula 1.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.
실시예 1. 균주 선별 및 동정Example 1. Strain selection and identification
1-1: 퇴적토 내 균주 분리1-1: Isolation of strains in sediment
2016년 5월 2일 남극 인근 36°00′42.7″N 126°39′41.8″E 에서 퇴적토를 채집하였다. 채집된 퇴적토는 클린벤치에서 건조 후, 건조물을 멸균된 해수에 현탁하여 1/20으로 희석한 뒤 마린 브로스 (Marine broth) 고체배지에 접종하고, 27℃에서 배양하면서 생성된 단일 균주를 마린 브로스 (Marine broth) 고체배지에 다시 접종하여 순수한 균주를 분리 및 선별하였다. On May 2, 2016, sedimentary soil was collected at 36°00′42.7″N 126°39′41.8″E near Antarctica. The collected sediment is dried in a clean bench, and the dried product is suspended in sterilized seawater, diluted 1/20, inoculated in a marine broth solid medium, and cultured at 27°C. Marine broth) solid medium was inoculated again to isolate and select pure strains.
순수 분리된 SCO-736 균주는 도 1 과 같이 스트렙토마이세스 속에 속하는 균주들과 같은 형태적 양상이 관찰되었다.The purely isolated SCO-736 strain was observed in the same morphology as the strains belonging to the genus Streptomyces as shown in FIG. 1.
1-2: 균주의 동정1-2: Identification of strain
실시예 1-1에서 분리된 균주의 종 동정을 위해 마린브로스 (Marine broth) 액체배지에서 4일 동안 27℃에서 배양된 균주 1ml을 취하여 “Tissue Genomic DNA Isolation kit” (Cosmogenetech co, Ltd.. Seoul, South Korea)를 이용하여 제조회사 프로토콜에 의하여 genomic DNA를 추출하였다. 종 분석을 위해 16S rRNA 유전자 증폭을 27F 및 1492R 프라이머를 사용하여 PCR을 수행하였다. PCR에 사용한 프라이머 서열은 표 1에 나타내었다. In order to identify the strain isolated in Example 1-1, 1 ml of the strain cultured at 27°C for 4 days in a marine broth liquid medium was taken and the “Tissue Genomic DNA Isolation kit” (Cosmogenetech co, Ltd.. Seoul) , South Korea) was used to extract genomic DNA according to the manufacturer's protocol. PCR was performed using 27F and 1492R primers to amplify the 16S rRNA gene for species analysis. The primer sequences used for PCR are shown in Table 1.
PCR 산물은 “PCR purification kit” (Cosmogenetech co, Ltd.. Seoul, South Korea)을 이용하여 정제 후, capillary electrophoresis (Applied Biosystems 3730XL)를 이용하여 염기서열을 분석하였다. The PCR product was purified using a “PCR purification kit” (Cosmogenetech co, Ltd.. Seoul, South Korea), and then the base sequence was analyzed using capillary electrophoresis (Applied Biosystems 3730XL).
SCO-736 균주로부터 얻어진 16S rRNA 유전자 염기서열을 GenBank/EMBL/DDBJ 데이터베이스의 BLAST 서치를 활용하여 이전에 보고된 균주들의 정보와 비교하였다. 그 결과 SCO-736 균주의 16S rRNA 유전자 염기서열은 Streptomyces diastaticus, Streptomyces diastaticus subsp. ardesiacus, 및 Streptomyces fradiae strain SMS_SU23 균주들과 99.9% 상동성을 나타내었으며, 본 발명의 스트렙토마이세스 속 SCO-736 균주는 Streptomyces 속에 속하는 균주로 판단되었다. Streptomyces diastataticus는 strepomyces속의 알칼리성(Alkaliphile) 및 고온성(thermophilic)세균으로 oligomycin A, oligomycin C, rimocidin 및 leukotriene-A4 hydrolase-inhibitor 8 (S) -amino-2 (R) -methyl-7-oxononanoic acid를 생산하는 특징을 가진다.The 16S rRNA gene sequence obtained from the SCO-736 strain was compared with information of previously reported strains using BLAST search of the GenBank/EMBL/DDBJ database. As a result, the 16S rRNA gene sequence of the SCO-736 strain was Streptomyces diastaticus, Streptomyces diastaticus subsp. ardesiacus , and Streptomyces fradiae strain SMS_SU23 showed 99.9% homology with the strains, and the strain SCO-736 of the genus Streptomyces of the present invention was determined to be a strain belonging to the genus Streptomyces. Streptomyces diastataticus is an alkaline (Alkaliphile) and thermophilic bacteria of the genus strepomyces. It has a characteristic to produce.
상기 스트렙토마이세스 속 SCO-736 균주의 16S rRNA genes 염기서열을 표 2에 나타내었다 (서열번호 3):The base sequence of the 16S rRNA genes of the SCO-736 strain of the genus Streptomyces is shown in Table 2 (SEQ ID NO: 3):
실시예 2. 균주 배양물 및 추출물 제조Example 2. Preparation of strain culture and extract
실시예 1에서 동정된 스트렙토마이세스 속 균주를 2.5L 배양 용기에 해수가 포함된 1리터 배양액 (10 g/L 전분, 2 g/L 효모 추출물, 4 g/L 펩톤)에 7일 동안 27℃, 130 rpm 조건에서 배양 후, 스트렙토마이세스 속 SCO-736 균주의 배양물을 얻었다.The strain of the genus Streptomyces identified in Example 1 was placed in a 1 liter culture solution containing seawater (10 g/L starch, 2 g/L yeast extract, 4 g/L peptone) in a 2.5 L culture vessel at 27°C for 7 days. After culturing under conditions of, 130 rpm, a culture of SCO-736 strain of Streptomyces sp. was obtained.
스트렙토마이세스 속 SCO-736 균주의 배양물에 에틸아세테이트 1L를 넣어 두 층을 분리하고 이로부터 에틸아세테이트 층을 얻었다. 이후 에틸아세테이트 층으로부터 진공회전 농축기를 이용하여 유기 용매를 제거하고 이의 추출물을 얻었다. 상기 에텔아세테이트 추출물 (3.4 g)은 H2O와 MeOH의 20, 40, 50, 60, 70, 80, 100%까지 MeOH의 단계적 구배에 따르는 C-18 실리카를 이용한 역상 칼럼 크로마토그래피로 분획하였다. 그 중 60% 메탄올/ 40% 물로 분획한 네 번째 분획에서 유기 용매를 제거하였으며, 그 분획물을 3ml 메탄올에 녹이고, 그 용액에 대한 고성능 액체 크로마토그래피를 수행하여 최종적으로 순수한 화합물 (화학식 1)을 획득하였으며, 스트렙토마이세스 속 SCO-736 균주의 배양물은 하기 화학식 1로 표시되는 화합물을 포함하는 것을 확인하였다.The two layers were separated by adding 1 L of ethyl acetate to the culture of SCO-736 strain of the genus Streptomyces, and an ethyl acetate layer was obtained therefrom. Thereafter, the organic solvent was removed from the ethyl acetate layer using a vacuum rotary concentrator, and an extract thereof was obtained. The ether acetate extract (3.4 g) was fractionated by reverse-phase column chromatography using C-18 silica according to a stepwise gradient of MeOH to 20, 40, 50, 60, 70, 80, 100% of H2O and MeOH. Among them, the organic solvent was removed from the fourth fraction fractionated with 60% methanol/40% water, and the fraction was dissolved in 3 ml methanol, and the solution was subjected to high performance liquid chromatography to finally obtain a pure compound (Formula 1). And, it was confirmed that the culture of the strain SCO-736 of the genus Streptomyces contained a compound represented by the following formula (1).
[화학식 1][Formula 1]
실시예 3. 화합물의 분리 및 정제Example 3. Isolation and purification of the compound
실시예 2에서 얻은 스트렙토마이세스(Streptomyces, SCO-736) 배양액에 에틸아세테이트 1L를 넣어 두 층을 분리하고 이로부터 에틸아세테이트 층을 얻었다. 이후 에틸아세테이트 층으로부터 진공회전 농축기를 이용하여 유기 용매를 제거하고 이의 추출물을 얻었다. 상기 에텔아세테이트 추출물 (3.4 g)은 H2O와 MeOH의 20, 40, 50, 60, 70, 80, 100%까지 MeOH의 단계적 구배에 따르는 C-18 실리카를 이용한 역상 칼럼 크로마토그래피로 분획하였다.The two layers were separated by adding 1 L of ethyl acetate to the culture solution of S treptomyces ( SCO-736) obtained in Example 2, and an ethyl acetate layer was obtained therefrom. Thereafter, the organic solvent was removed from the ethyl acetate layer using a vacuum rotary concentrator, and an extract thereof was obtained. The ether acetate extract (3.4 g) was fractionated by reverse phase column chromatography using C-18 silica according to a stepwise gradient of MeOH up to 20, 40, 50, 60, 70, 80, 100% of H 2 O and MeOH.
그 중 LC-MS 분석에서 특이한 분자량과 자외선 흡광을 보이는 피크인 60% 메탄올/ 40% 물로 분획한 네 번째 분획 (도 2)의 유기 용매를 제거하였으며, 그 분획물을 3ml 메탄올에 녹이고, 그 용액에 대한 고성능 액체 크로마토그래피를 수행하여 최종적으로 순수한 화합물을 획득하였으며, 이를 "안타로이드 (Antaroide)"로 명명하였다. 사용한 고성능 액체 크로마토그래피의 조건은 다음과 같다:Among them, the organic solvent of the fourth fraction (Fig. 2) fractionated with 60% methanol / 40% water, which is a peak showing a specific molecular weight and ultraviolet absorption in LC-MS analysis, was removed, and the fraction was dissolved in 3 ml methanol, and the solution was High-performance liquid chromatography was performed for the final pure compound, which was designated as "Antaroide". The conditions of the high performance liquid chromatography used are as follows:
Phenomenex Luna C-18 (2), 250×10.0 mmPhenomenex Luna C-18 (2), 250×10.0 mm
2.0 mL/분, 5 mm, 100 Å, UV = 210 nm, 아세토나이트릴 (CH3CN) : 물 (H2O) = 48 : 522.0 mL/min, 5 mm, 100 Å, UV = 210 nm, acetonitrile (CH 3 CN): water (H 2 O) = 48: 52
상기와 같은 방법에 따라 안타로이드 (13 mg)를 스트렙토마이세스(streptomyces, SCO-736)로부터 머무름 시간 31분에 분리하였다. Antaroid (13 mg) was isolated from Streptomyces (SCO-736) at a retention time of 31 minutes according to the above method.
실시예 4. 화합물의 동정Example 4. Identification of compounds
실시예 3에서 분리 정제된 화합물의 구조를 동정하기 위하여 핵자기공명(NMR)과 질량분석(MS)을 수행하였다. Nuclear magnetic resonance (NMR) and mass spectrometry (MS) were performed to identify the structure of the compound separated and purified in Example 3.
도 3a 및 표 3에 도시된 NMR 분석 결과와, 도 3b에 도시된 질량분석 결과를 근거로, 분리 정제된 화합물을 분자량 361, 화학식 1의 구조를 가진 5-hydroxy-5-(6-methyl-7-oxooctyl)-4,5-dihydrobenzo[c][1, 5]oxazonine-2,7(1H,3H)-dione으로 동정하였다. 화학식 1의 구조를 가지는 화합물은 아직까지 보고된 바 없는 천연에서 처음으로 분리된 신규한 화합물임을 확인하였다. 하기 화학식 1의 화합물을 "안타로이드(Antaroide)"로 명명하였다.Based on the NMR analysis results shown in Figs. 3a and 3 and the mass spectrometry results shown in Fig. 3b, the separated and purified compound has a molecular weight of 361 and 5-hydroxy-5-(6-methyl-) having a structure of Formula 1 It was identified as 7-oxooctyl)-4,5-dihydrobenzo[c][1, 5]oxazonine-2,7(1H,3H)-dione. It was confirmed that the compound having the structure of Formula 1 is a novel compound isolated for the first time in nature, which has not yet been reported. The compound of the following formula 1 was named "Antaroide".
a 800 MHz for 1H NMR and 200 MHz for 13C NMR. a 800 MHz for 1 H NMR and 200 MHz for 13 C NMR.
b Numbers of attached protons were determined by analysis of 2D spectra. b Numbers of attached protons were determined by analysis of 2D spectra.
[화학식 1][Formula 1]
실시예 5. 멜라닌 생성 저해능 측정Example 5. Measurement of melanin production inhibitory ability
실시예 3에서 분리된 화학식 1로 표시되는 화합물의 멜라닌생성 저해 활성을 이용하여 측정하였다.It was measured using the melanogenesis inhibitory activity of the compound represented by Formula 1 isolated in Example 3.
구체적으로, B16F10 멜라노마 세포 (ATCC, USA)를 1 x 104 cells/well로 48 웰 플레이트에 300 μL씩 분주한 후에 인큐베이터에서 하룻밤 동안 배양하였으며, 배지로는 DMEM(Dulbecco's Modified Eagle's Medium)high glucose 배지(ATCC, USA)를 사용하였다. 멜라닌 생성 촉진 자극원인 Alpha-MSH (0.1 uM, Sigma-Aldrich, USA)와 DMSO에 용해시켜 10, 25, 50ppm 농도로 제조한 안타로이드를 동시에 섞은 배지를 제조하였다. 양성대조군으로는 알부틴 50ppm을 사용하였다. 분주된 웰 내의 배지를 제거한 후에 제조된 배지를 웰 당 450μL씩 넣어주었다. 72 시간 후 배지를 완전히 제거하고, 1N NaOH 를 웰 당 200μL씩 넣어준 후 60℃에서 1시간 동안 배양하여 세포로부터 멜라닌을 용해시켰다. 용해된 멜라닌 시료 100μL를 96 웰 플레이트에 옮겨 담고 마이크로플레이트 리더(Molecular Devices, Sunnyvale, CA, USA) 405nm에서 흡광도를 측정하였다. 흡광도의 차이로부터 멜라닌 생성 저해도는 다음 수학식 1과 같이 산출하였다. 멜라닌 생성 저해도 측정 결과를 도 4에 나타내었다. Specifically, B16F10 melanoma cells (ATCC, USA) were dispensed at 1 x 10 4 cells/well into a 48-well plate at 300 μL, and cultured overnight in an incubator, and DMEM (Dulbecco's Modified Eagle's Medium) high glucose Medium (ATCC, USA) was used. A medium was prepared by simultaneously mixing Alpha-MSH (0.1 uM, Sigma-Aldrich, USA), which is a stimulator for promoting melanogenesis, and anthroids prepared at 10, 25, and 50 ppm concentrations by dissolving in DMSO. 50ppm of arbutin was used as a positive control. After removing the medium in the dispensed well, the prepared medium was added at 450 μL per well. After 72 hours, the medium was completely removed, 200 μL of 1N NaOH was added per well, and then cultured at 60° C. for 1 hour to dissolve melanin from the cells. 100 μL of the dissolved melanin sample was transferred to a 96-well plate and absorbance was measured at 405 nm in a microplate reader (Molecular Devices, Sunnyvale, CA, USA). From the difference in absorbance, the degree of inhibition of melanin production was calculated as shown in Equation 1 below. The measurement results of melanin production inhibition are shown in FIG. 4.
[수학식 1][Equation 1]
멜라닌생성 저해도(%) = 1 - (ODS-ODc)/(ODm-ODc)×100Inhibition of melanogenesis (%) = 1-(ODS-ODc)/(ODm-ODc)×100
ODS: 시험물질(안타로이드)과 alpha-MSH를 처리한 시료의 흡광도ODS: Absorbance of the sample treated with the test substance (anthroid) and alpha-MSH
ODm: alpha-MSH 만 처리한 시료의 흡광도ODm: absorbance of a sample treated with only alpha-MSH
ODc: 시험물질(안타로이드) 및 alpha-MSH 미처리 음성대조군(1N NaOH 100 μL)의 흡광도ODc: Absorbance of test substance (anthroid) and alpha-MSH untreated negative control (
그 결과, 도 3에서 확인되는 바와 같이, 화학식 1로 표시되는 화합물 (안타로이드, Antaroide)는 10ppm, 25ppm, 50ppm 으로 농도가 증가할수록 멜라닌 생성 저해 효과가 더욱 증가하였다. 특히 동일 농도인 50ppm 에서는 양성대조군인 알부틴과 비교하여 약 42% 높은 저해 활성도를 보였으며, 특히 저농도인 10ppm에서도 약 43%의 높은 멜라닌 생성 저해 활성을 나타내었다. As a result, as shown in FIG. 3, the inhibitory effect of melanin production was further increased as the concentration of the compound represented by Formula 1 (Antaroid, Antaroide) increased to 10ppm, 25ppm, and 50ppm. In particular, at the same concentration of 50 ppm, the inhibitory activity was about 42% higher than that of the positive control arbutin, and in particular, even at the low concentration of 10 ppm, the inhibitory activity of melanin production was about 43%.
상기와 같은 결과에 따라, 본 발명의 안타로이드(Antaroide)는 멜라닌 생성 저해 활성이 매우 우수하며, 멜라닌 생성 저해를 통해 피부 색소 침착과 관련된 다양한 질병 및 상태에 적용 가능함을 확인하였다. According to the results as described above, it was confirmed that the antaroide of the present invention has excellent melanin production inhibitory activity, and can be applied to various diseases and conditions related to skin pigmentation through melanin production inhibition.
본 명세서는 본 발명의 기술 분야에서 통상의 지식을 가진 자이면 충분히 인식하고 유추할 수 있는 내용은 그 상세한 기재를 생략하였으며, 본 명세서에 기재된 구체적인 예시들 이외에 본 발명의 기술적 사상이나 필수적 구성을 변경하지 않는 범위 내에서 보다 다양한 변형이 가능하다. 따라서 본 발명은 본 명세서에서 구체적으로 설명하고 예시한 것과 다른 방식으로도 실시될 수 있으며, 이는 본 발명의 기술 분야에 통상의 지식을 가진 자이면 이해할 수 있는 사항이다.In the present specification, details that can be sufficiently recognized and inferred by those of ordinary skill in the technical field of the present invention have been omitted, and the technical spirit or essential configuration of the present invention other than the specific examples described in the present specification is changed. More various modifications are possible within the range that does not. Accordingly, the present invention may be implemented in a manner different from that specifically described and illustrated in the present specification, which is a matter that can be understood by those of ordinary skill in the technical field of the present invention.
<110> Ewha University - Industry Collaboration Foundation <120> Streptomyces species strain and Composition for Skin Whitening Containing the same <130> DPP20190386KR <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R primer <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 1095 <212> DNA <213> Unknown <220> <223> Streptomyces sp. <400> 3 agctccggcg gtgcaggatg agcccgcggc ctatcagctt gttggtgagg taatggctca 60 ccaaggcgac gacgggtagc cggcctgaga gggcgaccgg ccacactggg actgagacac 120 ggcccagact cctacgggag gcagcagtgg ggaatattgc acaatgggcg aaagcctgat 180 gcagcgacgc cgcgtgaggg atgacggcct tcgggttgta aacctctttc agcagggaag 240 aagcgaaagt gacggtacct gcagaagaag cgccggctaa ctacgtgcca gcagccgcgg 300 taatacgtag ggcgcaagcg ttgtccggaa ttattgggcg taaagagctc gtaggcggct 360 tgtcgcgtcg gttgtgaaag cccggggctt aaccccgggt ctgcagtcga tacgggcagg 420 ctagagttcg gtaggggaga tcggaattcc tggtgtagcg gtgaaatgcg cagatatcag 480 gaggaacacc ggtggcgaag gcggatctct gggccgatac tgacgctgag gagcgaaagc 540 gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacggt gggcactagg 600 tgtgggcaac attccacgtt gtccgtgccg cagctaacgc attaagtgcc ccgcctgggg 660 agtacggccg caaggctaaa actcaaagga attgacgggg gcccgcacaa gcggcggagc 720 atgtggctta attcgacgca acgcgaagaa ccttaccaag gcttgacata caccggaaag 780 catcagagat ggtgcccccc ttgtggtcgg tgtacaggtg gtgcatggct gtcgtcagct 840 cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtcc cgtgttgcca 900 gcaactcttc ggaggttggg gactcacggg agaccgccgg ggtcaactcg gaggaaggtg 960 gggacgacgt caagtcatca tgccccttat gtcttgggct gcacacgtgc tacaatggcc 1020 cggtacaatg agctgcgata ccgcaaggtg gagcgaatct caaaaagccg gtctcagttc 1080 ggattggggt ctgca 1095 <110> Ewha University-Industry Collaboration Foundation <120> Streptomyces species strain and Composition for Skin Whitening Containing the same <130> DPP20190386EN <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R primer <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 1095 <212> DNA <213> Unknown <220> <223> Streptomyces sp. <400> 3 agctccggcg gtgcaggatg agcccgcggc ctatcagctt gttggtgagg taatggctca 60 ccaaggcgac gacgggtagc cggcctgaga gggcgaccgg ccacactggg actgagacac 120 ggcccagact cctacgggag gcagcagtgg ggaatattgc acaatgggcg aaagcctgat 180 gcagcgacgc cgcgtgaggg atgacggcct tcgggttgta aacctctttc agcagggaag 240 aagcgaaagt gacggtacct gcagaagaag cgccggctaa ctacgtgcca gcagccgcgg 300 taatacgtag ggcgcaagcg ttgtccggaa ttattgggcg taaagagctc gtaggcggct 360 tgtcgcgtcg gttgtgaaag cccggggctt aaccccgggt ctgcagtcga tacgggcagg 420 ctagagttcg gtaggggaga tcggaattcc tggtgtagcg gtgaaatgcg cagatatcag 480 gaggaacacc ggtggcgaag gcggatctct gggccgatac tgacgctgag gagcgaaagc 540 gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacggt gggcactagg 600 tgtgggcaac attccacgtt gtccgtgccg cagctaacgc attaagtgcc ccgcctgggg 660 agtacggccg caaggctaaa actcaaagga attgacgggg gcccgcacaa gcggcggagc 720 atgtggctta attcgacgca acgcgaagaa ccttaccaag gcttgacata caccggaaag 780 catcagagat ggtgcccccc ttgtggtcgg tgtacaggtg gtgcatggct gtcgtcagct 840 cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtcc cgtgttgcca 900 gcaactcttc ggaggttggg gactcacggg agaccgccgg ggtcaactcg gaggaaggtg 960 gggacgacgt caagtcatca tgccccttat gtcttgggct gcacacgtgc tacaatggcc 1020 cggtacaatg agctgcgata ccgcaaggtg gagcgaatct caaaaagccg gtctcagttc 1080 ggattggggt ctgca 1095
Claims (16)
The cells of the Streptomyces SCO736 strain deposited with the accession number KCTC 13805BP having the ability to inhibit melanogenesis of melanoma cells, the culture of the strain, the lysate of the strain, the extract of the cells, the extract of the culture, and the extract of the lysate A composition for whitening comprising at least one selected from the group consisting of.
The whitening composition of claim 1, wherein the whitening composition inhibits skin pigmentation or melanin production.
The method of claim 1, wherein the composition is a solution, ointment, external ointment, cream, essence, foam, lotion, nutrient lotion, softening water, softening lotion, pack, emulsion, makeup base, soap, detergent, bath agent, sun cream, sun oil , Suspension, emulsion, paste, gel, lotion, powder, soap, cleansing with surfactants, oil, foundation, powder foundation, emulsion foundation, wax foundation, patch and spray, having a formulation selected from the group consisting of, for whitening Composition.
The whitening composition according to claim 1, wherein the whitening composition is a pharmaceutical composition, a cosmetic composition, an external composition for skin, a health functional food, or a quasi-drug composition.
The whitening composition of claim 4, wherein the pharmaceutical composition is a pharmaceutical composition for preventing or treating skin pigmentation disorders.
The whitening composition of claim 4, wherein the cosmetic composition is a cosmetic composition for whitening or improving skin pigmentation.
[화학식 1]
The whitening composition of claim 1, wherein the whitening composition comprises a compound having the following formula 1, or a pharmacologically acceptable salt thereof:
[Formula 1]
The whitening composition of claim 1, wherein the extract is obtained by fractionally extracting the cells, the culture of the strain, or the lysate of the strain with an organic solvent.
The method of claim 1, wherein the extract is fractionally extracted from the cells of the strain, the culture of the strain, or the lysate of the strain with an organic solvent, and at least one selected from the group consisting of water and alcohol is used as a developing solvent. A composition for whitening, characterized in that obtained from a fraction obtained by chromatographic separation and purification of the fractional extract.
The method of claim 9, wherein the organic solvent is at least one selected from the group consisting of ethyl acetate, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, methylene chloride, hexane, cyclohexane, and dichloromethane. Dragon composition.
The whitening composition according to claim 9 or 10, wherein the alcohol is methanol.
Streptomyces SCO736 strain deposited with accession number KCTC 13805BP having the ability to inhibit melanin production of melanoma cells.
[화학식 1]
The strain of claim 13, wherein the strain produces a compound having Formula 1 below:
[Formula 1]
The strain according to claim 13, wherein the strain inhibits melanin synthesis under conditions of treatment with melanocyte-stimulating hormone (αMSH).
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| KR1020190035276A KR102176018B1 (en) | 2019-03-27 | 2019-03-27 | Streptomyces species strain and Composition for Skin Whitening Containing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20200114109A (en) * | 2019-03-27 | 2020-10-07 | 이화여자대학교 산학협력단 | Novel compounds and Composition comprising the same for Skin Whitening |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101879852B1 (en) * | 2017-04-28 | 2018-07-19 | 주식회사 만방바이오 | Novel Streptomyces sp. strain and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101879852B1 (en) * | 2017-04-28 | 2018-07-19 | 주식회사 만방바이오 | Novel Streptomyces sp. strain and use thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200114109A (en) * | 2019-03-27 | 2020-10-07 | 이화여자대학교 산학협력단 | Novel compounds and Composition comprising the same for Skin Whitening |
| KR102288356B1 (en) * | 2019-03-27 | 2021-08-09 | 이화여자대학교 산학협력단 | Oxazonine-based compound and Composition comprising the same for whitening |
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