KR20090050856A - Whitening cosmetics containing fraction extract of horse riding extract and preparation method thereof - Google Patents
Whitening cosmetics containing fraction extract of horse riding extract and preparation method thereof Download PDFInfo
- Publication number
- KR20090050856A KR20090050856A KR1020070117510A KR20070117510A KR20090050856A KR 20090050856 A KR20090050856 A KR 20090050856A KR 1020070117510 A KR1020070117510 A KR 1020070117510A KR 20070117510 A KR20070117510 A KR 20070117510A KR 20090050856 A KR20090050856 A KR 20090050856A
- Authority
- KR
- South Korea
- Prior art keywords
- extract
- fraction
- methanol
- dichloromethane
- layer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
본 발명은 승마 추출물의 분획추출물을 함유하는 미백화장료와 이의 제조방법을 제공하는 것이다. The present invention is to provide a whitening cosmetics and a method for producing the same containing a fraction extract of horse riding extract.
본 발명에 따른 미백화장료는 승마의 물 또는 메탄올 추출물의 디클로로메탄분획추출물과 에틸아세테이트 분획추출물을 함유한다. 승마 추출물의 분획추출물은 기존의 미백물질로 개발된 알부틴보다 뛰어난 미백 기능성을 가지므로, 유연화장수(스킨), 영양화장수(로숀), 영양크림, 마사지크림, 엣센스, 팩, 유화형 화운데이션등 다양한 미백화장료로 사용될 수 있다.The whitening cosmetics according to the present invention contains dichloromethane fraction extract and ethyl acetate fraction extract of horse riding water or methanol extract. Since the fraction extract of horse riding extract has more whitening function than arbutin developed as a conventional whitening material, it has various whitening effects such as softening longevity (skin), nourishing longevity (losion), nourishing cream, massage cream, essence, pack, emulsified foundation, etc. Can be used as a cosmetic.
Description
본 발명은 미백화장료에 관한 것으로서, 더욱 상세하게는 승마 추출물의 분획추출물을 함유하는 미백화장료와 이의 제조방법에 관한 것이다.The present invention relates to a whitening cosmetics, and more particularly to a whitening cosmetics containing a fraction extract of a horse riding extract and a preparation method thereof.
산업화로 인한 환경오염은 일반생활의 장애를 초래하는데 특히 인체 보호막으로서 피부 질환이 증가할 뿐 아니라 삶의 질 향상이라는 인식변화로 인해 피부 관련 미용과 질환 완화 치료 소재에 대한 관심이 증가하고 있다. 또한 현대 가치관의 다양화와 개성화가 두드러지면서 상기와 같은 관심의 증가는 미백, 보습, 세포재생, 주름개선 등의 성분을 함유한 기능성 화장품의 개발로 이어지고 있으며 제품의 안전성을 지향하는 화장품 업계는 천연물 소재에 대한 연구개발을 활발하게 하고 있다.Environmental pollution caused by industrialization is causing obstacles in general life. Especially, as skin protection is increasing as a human protective film, interest in skin-related beauty and disease mitigation treatments is increasing due to a change in the perception that quality of life is improved. In addition, as the diversification and individualization of modern values stand out, the increase in interest has led to the development of functional cosmetics containing ingredients such as whitening, moisturizing, cell regeneration, and wrinkle improvement. We are actively researching and developing materials.
신체의 가장 바깥 부분에 위치하는 표피는 기저층, 유극층, 각질층의 순으로 배열되어 있다. 이를 구성하는 다양한 세포 종류 중 멜라닌세포(melanocyte)는 모발색과 피부색을 결정하는 페놀류의 생체고분자 물질로 검은 단백질의 복합체인 멜 라닌(melanin)을 합성하고 분비하여, 자외선에 의한 피부손상을 막아줄 뿐만 아니라 독성 약물의 흡수 등 여러 중요한 작용을 수행한다. 그러나 멜라닌의 과잉 생산은 기미, 주근깨와 같은 피부 질환을 초래하며, 심한 경우 피부암의 유발과도 관련이 있는 것으로 보고되고 있다. 따라서 최근에는 이러한 문제점을 예방 및 개선하기 위하여 멜라닌의 과잉생산을 억제하는 소재를 개발하려는 연구가 다양하게 진행되고 있다. The epidermis, located at the outermost part of the body, is arranged in order of the basal layer, the pole layer, and the stratum corneum. Among the various cell types, melanocytes are biopolymers of phenols that determine hair color and skin color. They synthesize and secrete melanin, a complex of black protein, to prevent skin damage caused by UV rays. In addition, it performs several important functions such as absorption of toxic drugs. However, excessive production of melanin causes skin diseases such as blemishes and freckles, and in severe cases, it has been reported to be associated with the induction of skin cancer. Therefore, in recent years, in order to prevent and improve such a problem, various researches are being conducted to develop a material that suppresses the overproduction of melanin.
미백 기능성 화장품의 개발은 멜라닌세포내 멜라닌의 생성을 억제하는 것, 멜라닌세포 자극물질을 조절하는 것, 멜라닌의 분해 및 배출을 촉진키는 것 등의 세 가지 방향으로 이루어지고 있다. 피부내 멜라닌합성증가와 기저층 멜란닌세포의 수적 증가는 기미, 주근깨와 같은 피부 이상을 일으키며 현대 피부 의학에서는 하이드퀴논(hydroquinone)과 레티노익산(retinoic acid)등의 피부 복합제재를 사용하거나 레이저 또는 박피술 등의 외과적인 치료방법을 이용하고 있으나 치료효과가 일시적이거나 여러 부작용을 유발하고 있어 이에 대한 새로운 접근이 필요하다.The development of whitening functional cosmetics consists of three directions: inhibiting melanin production in melanocytes, regulating melanocyte stimulants, and promoting melanin degradation and excretion. Increased melanin synthesis in the skin and an increase in the number of melanocytes in the basal layer cause skin abnormalities such as blemishes and freckles. In modern dermatology, skin complexes such as hydroquinone and retinoic acid are used or laser or dermabrasion is performed. Surgical treatment methods such as, but the treatment effect is a temporary or causing a number of side effects and a new approach is required.
멜라닌형성은 티로시네이즈(tyrosinase)라는 효소를 함유한 멜라닌세포내에서 멜라닌생합성이 이루어지는 생리학적인 반응 과정을 말하며 멜라닌은 L-티로신(L-tyrosine)이 다양한 반응을 거쳐 만들어지는 최종산물로서 티로시네이즈의 발현과 활성에 의해 조절된다. 티로시네이즈는 L-티로신의 수산기첨가 및 이로 인해 생성된 L-디하이드록시페닐알라닌(L-dihydroxyphenylalanine, L-DOPA)의 산화, 그 결과 O-도파퀴논(O-dopaquinone) 생성과 같은 일련의 반응 과정 중 초기 단계에 주로 영향을 미치는 효소로서 최근의 연구는 티로시네이즈의 활성을 제어, 억제하 는 세포내 신호전달계를 제어하는 방향으로 진행되고 있다. Melanin formation refers to a physiological reaction process in which melanin biosynthesis occurs in melanocytes containing an enzyme called tyrosinase. Melanin is the final product of L-tyrosine through various reactions. It is regulated by the expression and activity of naize. Tyrosinase is a series of reactions such as the hydroxylation of L-tyrosine and the oxidation of L-dihydroxyphenylalanine (L-DOPA), resulting in the production of O-dopaquinone. As an enzyme that mainly affects the early stages of the process, recent research is directed toward controlling intracellular signaling systems that control and inhibit tyrosinase activity.
티로시네이즈 이외에도 티로시네이즈-연관 단백질(tyrosinase-related protein)-1과 티로시네이즈-연관 단백질-2가 멜라닌형성에 작용하는 것으로 알려져 있으나, 그 기능은 아직 명확하게 밝혀지지 않고 있다. 자외선은 일산화질소(nitro oxide)를 생성하여 피부조직의 멜라닌세포내에서 구성효소인 티로시네이즈와 티로시네이즈-연관 단백질-1의 분비를 증가시켜 멜라닌 색소의 생성을 촉진하며, 생성된 멜라닌 색소들은 각질세포(keratinocyte)로 이동되어 기미와 주근깨를 비롯한 피부색소 침착을 유발한다. 따라서 멜라닌 합성에 영향을 주는 신호전달물질인 티로시네이즈-연관 단백질들의 활성을 저해시키는 기전을 가지며 세포독성이 낮고 안전성이 보장된 천연소재로부터 도출된 신소재 개발이 필요하다. 이러한 신소재는 생체내 멜라닌관련 대사의 기초 연구뿐만 아니라 멜라닌 생합성 저해제의 대체물질로 활용될 수 있을 것이다. In addition to tyrosinase, tyrosinase-related protein-1 and tyrosinase-related protein-2 are known to play a role in melanogenesis, but their function is not yet clear. UV light stimulates the production of melanin by producing nitro oxide and increasing the secretion of constituent enzymes tyrosinase and tyrosinase-associated protein-1 in melanocytes of skin tissues. They migrate to keratinocytes, causing skin pigmentation, including blemishes and freckles. Therefore, there is a need for the development of new materials derived from natural materials with low cytotoxicity and safety mechanisms that inhibit the activity of tyrosinase-associated proteins, signaling agents that affect melanin synthesis. This new material could be used as a substitute for melanin biosynthesis inhibitors as well as basic research on melanin-related metabolism in vivo.
미적 기능을 중시하는 관점에서 티로시네이즈 효소의 활성과 발현을 조절함으로서 미백을 목적으로 하는 다양한 소재에 대한 연구가 이루어지고 있으며 지금 까지 알려진 것으로 하이드로퀴논(hydroquinone), 아스코빅산(ascorbic acid), 코직산(kojic acid), 레티놀(retinole) 그리고 알부틴(arbutin) 등이 있다. 그러나 강력한 멜라닌 생합성 저해활성을 보이는 소재는 대부분 색소세포의 변성 또는 치사를 일으키고 세포 본래의 기능을 손상시키는 등의 부작용을 발생시킨다는 문제가 있다. Research on various materials aimed at whitening by controlling the activity and expression of tyrosinase enzymes from the point of view of aesthetic function is known, and hydroquinone, ascorbic acid and kojic acid are known so far. (kojic acid), retinol (retinole) and arbutin (arbutin). However, a material that exhibits strong melanin biosynthesis inhibitory activity has a problem of causing side effects such as causing denaturation or lethality of pigment cells and impairing the original function of cells.
또한, 한편으로는 독성이 상대적으로 적고 자연친화적인 미백화장료를 개발 하기 위하여 미백작용을 갖는 생약을 탐색하고 이를 이용하려는 연구도 시행되고 있다. 이러한 연구를 통하여 수종의 생약들이 도출되어 있으며 그 예로, 상지, 닥나무, 반하, 상황버섯 추출물 등이 있다.On the other hand, in order to develop whitening cosmetics with relatively low toxicity and nature-friendly, researches are being conducted to search for and use herbal medicines with whitening effects. Several kinds of herbal medicines have been derived from these studies, for example, upper limbs, mulberry, halveda, and situation mushroom extract.
그러나, 이러한 종래의 생약식물의 성분을 이용한 미백화장료들은 당해 약재가 가지는 장점뿐만 아니라 단점(또는 부작용을 일으키는 요소)도 함께 발현되는 관계로 다소의 부작용이 초래되거나, 또는 장기간 보관함에 있어서 변색 변취 등의 문제점이 있다. However, the whitening cosmetics using the components of the conventional herbal medicines not only have the advantages but also the disadvantages (or causing the side effects) of the medicinal herb, which causes some side effects, or discoloration and discoloration during long-term storage. There is a problem.
따라서, 본 발명이 해결하고자 하는 첫 번째 과제는, 미백효과가 뛰어난 승마 추출물의 분획추출물을 함유하는 미백화장료를 제공하는 것이다.Therefore, the first problem to be solved by the present invention is to provide a whitening cosmetics containing a fraction extract of the horse riding extract excellent in whitening effect.
본 발명이 해결하고자 하는 두 번째 과제는 상기의 승마 추출물의 분획추출물을 함유하는 미백화장료의 제조방법을 제공하는 것이다.The second problem to be solved by the present invention is to provide a method for producing a whitening cosmetics containing the fraction extract of the above-mentioned horse riding extract.
본 발명은 상기 첫 번째 과제를 달성하기 위해서,In order to achieve the first object of the present invention,
승마 추출물의 분획추출물을 함유하는 미백화장료를 제공한다.It provides a whitening cosmetic containing a fraction extract of a horse riding extract.
본 발명의 바람직한 일 실시예에 따르면, 상기 승마 추출물의 분획추출물을 함유하는 미백화장료는 승마의 물 또는 메탄올 추출물을 메탄올에 재용해한 후 헥산을 가하여 분획시킨 다음 헥산 분획층을 제거하고 남은 여액에 디클로로메탄을 가하여 재분획하여 얻은 디클로로메탄 분획추출물을 함유하는 미백화장료일 수 있다.According to an embodiment of the present invention, the whitening cosmetics containing the fraction extract of the horse riding extract is redissolved water or methanol extract of horse riding in methanol and fractionated by the addition of hexane and then removed the hexane fractionation layer in the remaining filtrate It may be a whitening cosmetic containing a dichloromethane fraction extract obtained by refraction by adding dichloromethane.
본 발명의 바람직한 다른 실시예에 따르면, 상기 승마 추출물의 분획추출물을 함유하는 미백화장료는 승마의 물 또는 메탄올 추출물을 메탄올에 재용해한 후 헥산을 가하여 분획시킨 다음 헥산 분획층을 제거하고 남은 여액에 디클로로메탄을 가하여 재분획시고, 다시 디클로로메탄 분획층을 제거하고 남은 여액에 에틸아세테이트를 가하여 재분획하여 얻은 에틸아세테이트 분획추출물을 함유하는 미백화장료일 수 있다.According to another preferred embodiment of the present invention, the whitening cosmetics containing the fraction extract of the horse riding extract is redissolved water or methanol extract of horse riding in methanol and fractionated by addition of hexane and then removed the hexane fractionation layer to the remaining filtrate It may be a whitening cosmetic containing ethyl acetate fraction extract obtained by re-fractionation by adding dichloromethane, removing the dichloromethane fractionation layer, and re-fractionation by adding ethyl acetate to the remaining filtrate.
본 발명의 바람직한 또 다른 실시예에 따르면, 상기 승마 추출물의 분획추출물을 함유하는 미백화장료는 승마의 메탄올 추출물을 메탄올에 재용해한 후 헥산을 가하여 분획시킨 다음 헥산 분획층을 제거하고 남은 메탄올층을 감압 농축하여 제조한 추출물을 다시 메탄올에 재용해한 후 디클로로메탄을 가하여 재분획하여 얻은 디클로로메탄 분획추출물을 함유하는 미백화장료일 수 있다.According to another preferred embodiment of the present invention, the whitening cosmetics containing the fraction extract of the horse riding extract is redissolved methanol extract of horse riding and fractionated by adding hexane and then removed the hexane fraction layer and the remaining methanol layer The extract prepared by concentrating under reduced pressure may be a whitening cosmetic containing a dichloromethane fraction extract obtained by re-dissolving again in methanol and re-fractionation by adding dichloromethane.
본 발명의 바람직한 또 다른 실시예에 따르면, 상기 승마 추출물의 분획추출물을 함유하는 미백화장료는 승마의 메탄올 추출물을 메탄올에 재용해한 후 헥산을 가하여 분획시킨 다음 헥산 분획층을 제거하고 남은 메탄올층을 감압 농축하여 제조한 추출물을 다시 메탄올에 재용해한 후 디클로로메탄을 가하여 재분획시킨 다음 디클로로메탄 분획층을 제거하고 남은 여액에 에틸아세테이트를 가하여 다시 재분획하여 얻은 에틸아세테이트 분획추출물을 함유한 미백화장료를 포함할 수 있다.According to another preferred embodiment of the present invention, the whitening cosmetics containing the fraction extract of the horse riding extract is redissolved methanol extract of horse riding and fractionated by adding hexane and then removed the hexane fraction layer and the remaining methanol layer The whitening cosmetics containing the ethyl acetate fraction extract obtained by re-dissolving the extract prepared by concentrating under reduced pressure again and re-fractionated with dichloromethane, removing the dichloromethane fraction layer and adding the ethyl acetate to the remaining filtrate. It may include.
본 발명의 바람직한 또 다른 실시예에 따르면, 상기 승마 추출물의 분획추출물을 함유하는 미백화장료는 상기 승마의 메탄올 추출물의 디클로로메탄 분획층을 칼럼 크로마토그래피에 적용하여 6 분획한 뒤 이 분획층 중 이동률이 큰 3 개 분획층의 분획추출물을 함유하는 미백화장료일 수 있다.According to another preferred embodiment of the present invention, the whitening cosmetics containing the fraction extract of the horse riding extract is divided into six fractions by applying the dichloromethane fraction layer of the methanol extract of horse riding to column chromatography and the mobility of the fraction layer is increased. It may be a whitening cosmetic containing a fraction extract of a large three fraction layer.
또한, 본 발명에 따른 조성물의 제형은 승마 추출물의 분획추출물을 함유한 화운데이션, 팩, 유연 마사지 크림, 영양 크림, 영양화장수(로션), 및 유연화장수(스킨)로 이루어진 군에서 선택된 어느 하나일 수 있다.In addition, the formulation of the composition according to the present invention may be any one selected from the group consisting of a foundation, a pack, a flexible massage cream, a nourishing cream, a nourishing cream (lotion), and a softening cream (skin) containing a fraction extract of a horse riding extract. have.
본 발명은 상기 두 번째 과제를 달성하기 위해서,In order to achieve the second object of the present invention,
상기의 승마 추출물의 분획추출물을 함유하는 미백화장료의 제조방법을 제공 한다.It provides a method of producing a whitening cosmetics containing the fraction extract of the above-mentioned riding extract.
본 발명의 바람직한 일 실시예를 따르면, 미백화장료의 제조방법은According to a preferred embodiment of the present invention, the method for producing a whitening cosmetic
승마를 물 또는 메탄올로 추출하여 용매 추출물을 제조하는 단계;Extracting equestrian with water or methanol to produce a solvent extract;
상기의 물 추출물 또는 메탄올 추출물에 메탄올을 가하여 재용해하는 단계;Adding redissolved methanol to the water extract or methanol extract;
상기 재용해하여 제조된 용액을 헥산으로 분획하는 단계;Fractionating the redissolved solution into hexane;
상기 헥산 분획층을 제거하고 남은 메탄올층을 디클로로메탄으로 분획하는 단계; 및Removing the hexane fractionation layer and fractionating the remaining methanol layer with dichloromethane; And
상기에서 얻어진 디클로로메탄 분획층을 감압 농축하여 분획추출물을 제조하는 단계Preparing a fraction extract by concentrating the obtained dichloromethane fraction layer under reduced pressure;
를 포함하는 미백화장료의 제조방법일 수 있다.It may be a method of manufacturing a whitening cosmetic comprising a.
본 발명의 바람직한 다른 실시예를 따르면, 미백화장료의 제조방법은According to another preferred embodiment of the present invention, the method for producing a whitening cosmetic
상기 디클로로메탄으로 분획하는 단계 이후에 After fractionating with dichloromethane
상기 디클로로메탄 분획층을 제거하고 남은 메탄올층을 에틸아세테이트로 분획 추출하는 단계; 및Removing the dichloromethane fractionation layer and fractionating the remaining methanol layer with ethyl acetate; And
상기에서 얻어진 에틸아세테이트 분획층을 감압 농축하여 분획추출물을 제조하는 단계Preparing a fraction extract by concentrating the ethyl acetate fraction layer obtained under reduced pressure;
를 포함하는 미백화장료의 제조방법일 수 있다.It may be a method of manufacturing a whitening cosmetic comprising a.
본 발명의 바람직한 또 다른 실시예에 따르면, 미백화장료의 제조방법은According to another preferred embodiment of the present invention, the method for producing a whitening cosmetic
상기 디클로로메탄으로 분획하는 단계 이후에 After fractionating with dichloromethane
상기에서 얻어진 메탄올 추출물의 디클로로메탄 분획층을 칼럼 크로마토그래 피를 실시하여 모두 6종류의 분획층을 얻은 후 이 분획층 중 이동률이 큰 3개 층을 분획하는 단계; 및 Distilling the dichloromethane fractionation layer of the methanol extract obtained above by column chromatography to obtain all six types of fractionation layers, and then fractionating three layers having high mobility in the fractionation layers; And
상기에서 얻어진 이동률이 큰 3개 분획층을 감압 농축하여 분획추출물을 제조하는 단계Preparing a fraction extract by concentrating the three fractional layers having a high mobility as described above under reduced pressure;
를 포함하는 미백화장료의 제조방법일 수 있다.It may be a method of manufacturing a whitening cosmetic comprising a.
본 발명의 바람직한 또 다른 실시예에 따르면, 미백화장료의 제조방법은According to another preferred embodiment of the present invention, the method for producing a whitening cosmetic
상기 헥산을 분획하는 단계 이후에After fractionating the hexane
상기 헥산 분획층을 제거하고 남은 메탄올층을 감압 농축하여 분획추출물을 제조하는 단계;Removing the hexane fractionation layer and concentrating the remaining methanol layer under reduced pressure to prepare a fraction extract;
상기 분획추출물을 메탄올에 재용해하는 단계;Re-dissolving the fraction extract in methanol;
상기 재용해하여 얻어진 용액을 디클로로메탄으로 분획하는 단계; 및Fractionating the solution obtained by redissolution into dichloromethane; And
상기에서 얻어진 디클로로메탄 분획층을 감압 농축하여 분획추출물을 제조하는 단계Preparing a fraction extract by concentrating the obtained dichloromethane fraction layer under reduced pressure;
를 포함하는 미백화장료의 제조방법일 수 있다.It may be a method of manufacturing a whitening cosmetic comprising a.
본 발명의 바람직한 또 다른 실시예에 따르면, 미백화장료의 제조방법은According to another preferred embodiment of the present invention, the method for producing a whitening cosmetic
상기 디클로로메탄으로 분획하는 단계 이후에 After fractionating with dichloromethane
상기 디클로로메탄 분획층을 제거하고 남은 메탄올층을 에틸아세테이트로 분획하는 단계; 및 Removing the dichloromethane fractionation layer and fractionating the remaining methanol layer with ethyl acetate; And
상기에서 얻어진 에틸아세테이트 분획층을 감압 농축하여 분획추출물을 제조하는 단계Preparing a fraction extract by concentrating the ethyl acetate fraction layer obtained under reduced pressure;
를 포함하는 미백화장료의 제조방법일 수 있다.It may be a method of manufacturing a whitening cosmetic comprising a.
본 발명의 바람직한 또 다른 실시예에 따르면, 미백화장료의 제조방법은According to another preferred embodiment of the present invention, the method for producing a whitening cosmetic
상기 디클로로메탄으로 분획하는 단계 이후에After fractionating with dichloromethane
상기에서 얻어진 메탄올 추출물의 디클로로메탄 분획층에 칼럼 크로마토그래피를 실시하여 모두 6 종류의 분획층을 얻은 후 이 분획층 중 이동률이 큰 3 개 층을 분획하는 단계; 및Performing column chromatography on the dichloromethane fractionation layer of the methanol extract obtained above to obtain all six kinds of fractionation layers, and then fractionating three layers having high mobility in the fractionation layers; And
상기에서 제조된 이동률이 큰 3 개 분획층을 감압 농축하여 분획추출물을 제조하는 단계Preparing a fraction extract by concentrating the three fractional layers having a high mobility as described above under reduced pressure;
를 포함하는 미백화장료의 제조방법일 수 있다.It may be a method of manufacturing a whitening cosmetic comprising a.
본 발명의 승마 추출물의 미백효과는 기존의 미백물질로 개발된 알부틴과 비교한 결과 본 발명의 승마 추출물의 분획추출물이 보다 강력한 미백 효과를 나타내었다. 따라서, 미백 효과가 뛰어난 화장품 조성물 및 이의 제조방법을 제공할 수 있다.The whitening effect of the equestrian extract of the present invention compared to the arbutin developed as a conventional whitening material, the fraction extract of the equestrian extract of the present invention showed a more powerful whitening effect. Therefore, it is possible to provide a cosmetic composition having excellent whitening effect and a preparation method thereof.
이하, 실시예 및 실험예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 그러나, 이들 실시예 및 실험예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예 및 실험예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited by these Examples and Experimental Examples according to the gist of the present invention is a common knowledge in the art. It will be self-evident for those who have.
본 발명자는 종래 알려져 있는 미백물질들이 갖는 문제점을 극복하고 보다 우수한 미백 원료를 찾기 위하여 천연 식물을 대상으로 미백 효능이 있는 유효 물질을 탐색하던 중, 승마 추출물이 매우 뛰어난 티로시네이즈 활성 저해 효과를 나타냄을 발견하고 본 발명을 완성하였다.The inventors of the present invention, while overcoming the problems of conventionally known whitening substances and searching for an effective substance having a whitening effect on natural plants in order to find a superior whitening raw material, the equestrian extract exhibits an excellent inhibitory effect of tyrosinase activity. And the present invention was completed.
승마(升麻)는 미나리아재비과에 속하는 Cimicifuga heracleifolia 또는 그 동속의 식물의 뿌리를 말하며 한의학에서 신농본초경에는 “약성이 능히 열을 식히며 독을 해한다”라고 하였다. 한방에서는 해열, 해독, 항염증약으로서 신열, 두통, 인후통 등에서 쓰이는데 메탄올, 에탄올, 부탄올, 등의 분획 중 부탄올 추출물에는 진통작용, 정상체온의 강하적용 등이 있어 주로 해열, 항염증에 작용이 있다고 알려져 있다. Horseback riding is Cimicifuga belonging to the buttercup family It refers to the root of heracleifolia or a plant of the same genus. In oriental medicine, it is used for fever, detoxification, and anti-inflammatory drugs such as fever, headache, and sore throat. Butanol extract in methanol, ethanol, butanol, etc. is known to have analgesic action and lowering of normal body temperature. have.
승마는 주로 C. acerina , C. dahurica , C. foetida , C. heracleifolia , C. racemosa, C. simplex 기원의 약재로 지난 45년간 생리활성물질에 대한 약리효과에 대한 연구가 광범위하게 이루어져 폐경기의 일과성 열감, 골다공증, 항염증, 면역결핍 바이러스를 포함한 항바이러스, 항말라리아, 항당뇨, 혈관확장 등에 대한 효능이 밝혀졌다. 이러한 약리적 효능과 더불어 승마는 피부미용에 관한 화장료로 사용되기도 한다. 예전부터 승마위풍탕 등이 한방 피부관리에 사용되었으며 피부발적, 안면홍조등에 대한 피부팩 재료로 사용되고 있다. Horseback riding is mainly derived from C. acerina , C. dahurica , C. foetida , C. heracleifolia , C. racemosa, and C. simplex , and has been extensively studied for pharmacological effects on bioactive substances over the past 45 years. Efficacy has been shown for antiviral, antimalarial, antidiabetic, vasodilation, including hot flashes, osteoporosis, anti-inflammatory, immunodeficiency virus. In addition to these pharmacological effects, horse riding is also used as a cosmetic for skin beauty. Horseback riding feng tang has been used for herbal skin care since ancient times and has been used as a skin pack material for skin redness and hot flashes.
본 발명자는 문헌 및 임상적 자료를 바탕으로 30여 종의 한방 약용작물을 대상으로 실험하여 비세포계(in vitro)에서의 버섯티로시네이즈(mushroom tyrosinase) 활성을 억제하는 천연물소재를 도출하였고 이것이 실제 세포계(in vivo)에서 작용하는 지를 B16F10세포를 이용하여 티로시네이즈 활성억제 및 멜라닌 생성 저해 효과를 스크린하여 연구조사하였다. 이중 승마는 현재 상업적으로 미백기능성 화장료로 널리 사용되고 있는 알부틴보다 우수한 미백관련 효과를 나타내어 승마추출물이 미백관련 기초 화장료로서 다양한 미백기능성 화장품 개발에 적용 가능하다는 것을 알게 되었다.Based on the literature and clinical data, the present inventors experimented with over 30 kinds of herbal medicinal crops and derived natural products that inhibited mushroom tyrosinase activity in the in vitro. To investigate the effect of tyrosinase activity inhibitory and melanin production screening using B16F10 cells in the actual cell system (in vivo). Among these, horseback riding showed superior whitening-related effects than arbutin, which is currently widely used as a whitening functional cosmetic, and found that horse riding extracts can be applied to various whitening functional cosmetics as a basic whitening-related cosmetic.
본 발명의 미백화장료에 함유되는 승마 추출물의 분획추출물의 보다 상세한 제조 방법은 다음과 같다.More detailed preparation method of the fraction extract of the horse riding extract contained in the whitening cosmetic of the present invention is as follows.
우선 승마를 세절하여 물 또는 80∼100% 메탄올로 가열 환류시키고 실온까지 냉각시킨 후 감압 여과하는 단계를 거친다. 상기 단계에서의 환류시 가열온도는 60∼95℃인데, 물의 끓는점은 100℃이고, 메탄올의 끓는점은 대략 75℃ 전후이기 때문이다. 60℃ 미만인 때에는 추출효율이 떨어지고 95℃를 초과하는 때에도 용매의 끓는점과 관련하여 열에 약한 물질이 분해될 우려가 있다. 따라서, 메탄올을 용매로 사용하는 경우에는 물을 사용하는 경우보다 추출온도를 낮게 조절하는 것이 바람직하며, 약 65℃ 정도가 가장 바람직하다. 한편, 상기 추출시간은 추출효율을 고려할 때에 2∼4시간인 것이 좋다.First, horse riding is chopped, heated to reflux with water or 80-100% methanol, cooled to room temperature and filtered under reduced pressure. The heating temperature at reflux in this step is 60 to 95 ℃, because the boiling point of water is 100 ℃, methanol boiling point is about 75 ℃. When the temperature is less than 60 ° C., the extraction efficiency is lowered, and even when the temperature is higher than 95 ° C., there is a concern that heat-sensitive substances are decomposed in relation to the boiling point of the solvent. Therefore, when methanol is used as a solvent, it is preferable to control the extraction temperature lower than when water is used, and about 65 ° C is most preferable. On the other hand, the extraction time is preferably 2 to 4 hours in consideration of the extraction efficiency.
다음으로, 상기에서 감압 여과하여 제조된 여과액을 30∼50℃에서 감압 농축하여 각 초기 용매 추출물을 제조하는 단계를 거친 후, 상기의 초기 용매 추출물에 메탄올을 가하여 재용해하는 단계를 수행하는데, 본 단계에서 사용되는 메탄올의 농도는 특별히 제한되지 않으며 예를 들어 50∼100% 메탄올일 수 있다. 다만, 최초 추출시 물 또는 80% 메탄올을 사용하여 추출한 추출물을 재용해시키는 때에는 50% 정도의 저농도 메탄올에 용해시키는 것이 바람직하고, 최초 추출시 100% 메탄올로 추출한 추출물의 재용해시에는 95% 정도의 고농도 메탄올에 용해시키는 것이 좋은데 그 이유는 최초 추출시 물의 함량이 많을수록 고농도의 메탄올에는 잘 용해되지 않기 때문이다. 이는 물의 극성이 메탄올보다 크기 때문이다. 추출물을 완전히 용해시키지 않은 상태에서 용매추출을 시도하면 추출물의 성질에 따라서 에멀젼 현상, 즉 두 용매의 분리가 일어나지 않는 현상이 일어난다. 본 발명에서는 이러한 에멀젼 현상을 되도록 피하기 위해서 물의 함량이 비교적 많은 물 또는 80% 메탄올 추출물은 50% 정도의 저농도 메탄올을 사용하고, 100% 메탄올 추출물은 95% 정도의 고농도 메탄올을 사용하여 완전히 재용해시켰다. 이후, 상기 재용해시 사용한 메탄올의 양과 동량의 헥산을 사용하여 2∼5회 분획하고 헥산 분획층을 제거한 후에 남은 메탄올층을 직접 동량의 디클로로메탄을 사용하여 2∼5회 분획한 다음, 상기 디클로로메탄 분획물을 20∼50℃에서 감압 농축하여 분획추출물을 제조할 수 있다. 상기 재용해시 95% 메탄올에 재용해한 경우에는 헥산 분획 이후에 남은 메탄올층을 직접 디클로로메탄으로 재분획하는 것이 아니라, 상기 메탄올층을 감압 농축하여 추출물을 얻은 다음 이를 다시 50% 정도의 저농도 메탄올에 재용해시켜 저농도 메탄올 용액을 얻은 이후에 디클로로메탄으로 재분획하는 것이 바람직한데 그 이유는 100% 메탄올과 디클로로메탄은 서로 섞이기 때문이다. Next, the filtrate prepared by filtration under reduced pressure is concentrated under reduced pressure at 30 to 50 ° C. to prepare each initial solvent extract, and then, methanol is added to the initial solvent extract to redissolve. The concentration of methanol used in this step is not particularly limited and may be, for example, 50 to 100% methanol. However, when re-dissolving the extract extracted with water or 80% methanol at the time of initial extraction, it is preferable to dissolve it in about 50% of low concentration methanol, and about 95% at the time of re-dissolution of the extract extracted with 100% methanol at the first extraction. It is better to dissolve in the high concentration of methanol because the higher the content of water at the time of initial extraction, the less it dissolves in the high concentration of methanol. This is because the polarity of water is greater than that of methanol. Attempting to extract the solvent without completely dissolving the extract results in an emulsion phenomenon, that is, no separation of the two solvents, depending on the nature of the extract. In the present invention, in order to avoid such an emulsion phenomenon, water with a relatively high content of water or 80% methanol extract is used as a low concentration methanol of about 50%, and 100% methanol extract is completely redissolved by using a high concentration methanol of about 95%. . Thereafter, two to five times the amount of methanol used for re-dissolution using the same amount of hexane and the hexane fractionation layer was removed, and then the remaining methanol layer was directly fractionated two to five times using the same amount of dichloromethane. The methane fractions may be concentrated under reduced pressure at 20-50 ° C. to produce a fraction extract. When redissolved in 95% methanol when re-dissolving, the methanol layer remaining after the hexane fraction is not directly re-fractionated with dichloromethane, but the methanol layer is concentrated under reduced pressure to obtain an extract, which is then reduced to about 50% methanol. It is desirable to re-dissolve in dichloromethane after redissolution to obtain a low methanol solution because 100% methanol and dichloromethane are mixed together.
그 다음으로, 상기에서 디클로로메탄으로 분획하고 남은 메탄올층을 재용해시 사용한 메탄올의 양과 동량의 에틸아세테이트를 사용하여 2∼5회 분획한 후 상기 에틸아세테이트 분획층을 20∼50℃에서 감압 농축하여 분획추출물을 제조할 수 있다.Then, fractionate with dichloromethane and fractionate 2-5 times using the same amount of ethyl acetate as the amount of methanol used to redissolve the remaining methanol layer, and then the ethyl acetate fraction layer was concentrated under reduced pressure at 20-50 占 폚. Fraction extracts can be prepared.
다른 한편으로는, 상기 메탄올 추출물의 디클로로메탄 분획층을 메탄올 수용액을 전개용액으로 칼럼 크로마토그래피를 실시하여 분획층을 얻은 후 상기 분획층을 20∼50℃에서 감압 농축하여 분획추출물을 제조할 수 있다. 본 단계에서 사용되는 메탄올 수용액 전개용액의 농도는 특별히 제한되지 않으며 예를 상기 100% 메탄올 추출물의 디클로로메탄의 경우에는 메탄올:물=9:1의 전개용액을 사용하였다. 그 이유는 전개용매의 극성을 조절하여 가장 많은 분획을 얻기 위해서이다. 또한, 칼럼 크로마토그래피의 종류는 특별히 제한되지 않으며 본 발명에서는 통상 사용하는 C18 칼럼 크로마토그래피를 사용하였다. On the other hand, the dichloromethane fractionation layer of the methanol extract may be subjected to column chromatography with an aqueous methanol solution to obtain a fractionation layer, and then the fractionation layer may be concentrated under reduced pressure at 20 to 50 ° C. to prepare a fraction extract. . The concentration of the methanol aqueous solution development solution used in this step is not particularly limited. For example, in the case of the dichloromethane of the 100% methanol extract, a developing solution of methanol: water = 9: 1 was used. The reason is to control the polarity of the developing solvent to obtain the largest fraction. In addition, the kind of column chromatography is not specifically limited, The C18 column chromatography used normally is used in this invention.
제조된 승마 추출물의 분획 추출물을 통상적인 기초 화장료, 다시 말하면 화운데이션, 팩, 유연 마사지 크림, 영양 크림, 영양 화장수(로션), 유연 화장수(스킨)등에 첨가하여 미백화장료를 제조하는 것이 가능하다.It is possible to prepare a whitening cosmetic by adding a fraction extract of the prepared horse riding extract to a conventional basic cosmetic, that is, a foundation, a pack, a flexible massage cream, a nourishing cream, a nourishing lotion (lotion), a flexible lotion (skin) and the like.
실시예Example 1 One
승마(건조중량, 30g)를 세절한 후 물(300 ㎖) 및 80% 메탄올(300 ㎖)로 각각 3시간 동안 환류시키고 실온까지 냉각시킨 후 감압 여과하였다. 환류시 온도는 물을 용매로 한 경우에는 95℃이었고, 80% 메탄올을 용매로 한 경우에는 65℃이었다. 상기와 같은 방법으로 3회 실시하여 얻어진 여과액(300 ㎖ × 3회)을 40℃에서 감압 농축하여 각각의 물추출물 및 80% 메탄올 추출물을 제조하였다. 상기에서 제조된 물추출물 및 80% 메탄올 추출물에 50% 메탄올(200 ㎖)을 가하여 각각 재용해하였다. 이 용액을 동량의 헥산(200 ㎖)으로 각각 3회 분획하였다. 이 분획하여 얻어 진 헥산 분획층을 제거하고 남은 각각의 50% 메탄올층을 동량의 디클로로메탄(200 ㎖)으로 다시 각각 3회 분획하였다. 상기에서 얻어진 디클로로메탄 분획층을 40℃에서 감압 농축하여 각각의 디클로로메탄 분획추출물을 제조하였다.Horse riding (dry weight, 30 g) was chopped and then refluxed with water (300 mL) and 80% methanol (300 mL) for 3 hours, cooled to room temperature and filtered under reduced pressure. The temperature at reflux was 95 ° C. with water as solvent and 65 ° C. with 80% methanol as solvent. The filtrate (300 mL × 3 times) obtained by performing the above-described method three times was concentrated under reduced pressure at 40 ° C. to prepare each water extract and 80% methanol extract. 50% methanol (200 mL) was added to the water extract and 80% methanol extract prepared above, and redissolved. The solution was partitioned three times with equal amounts of hexane (200 mL) each. The hexane fractionation layer obtained by this fractionation was removed, and each remaining 50% methanol layer was fractionated again three times with the same amount of dichloromethane (200 mL). The dichloromethane fractionation layer obtained above was concentrated under reduced pressure at 40 ° C. to prepare respective dichloromethane fraction extracts.
실시예Example 2. 2.
실시예 1에서 제조된 디클로로메탄 분획층을 제거하고 남은 각각의 50% 메탄올층을 다시 동량의 에틸아세테이트(200 ㎖)로 각각 3회 분획하였다. 상기에서 얻어진 에틸아세테이트 분획층을 40℃에서 감압 농축하여 각각의 에틸아세테이트 분획추출물을 제조하였다.The dichloromethane fractionation layer prepared in Example 1 was removed, and each remaining 50% methanol layer was again fractionated three times with the same amount of ethyl acetate (200 mL). The ethyl acetate fraction layer obtained above was concentrated under reduced pressure at 40 ° C. to prepare respective ethyl acetate fraction extracts.
실시예Example 3. 3.
승마(건조중량, 30g)를 세절한 후 100% 메탄올(300 ㎖)로 65℃에서 3시간 환류시키고 실온까지 냉각시킨 후 감압 여과하였다. 상기와 같은 방법으로 3회 실시하여 얻어진 여과액(300 ㎖ × 3회)을 40℃에서 감압 농축하여 용매 추출물을 제조하였다. 상기에서 얻어진 100% 메탄올 추출물을 95% 메탄올 200 ㎖를 가하여 재용해하였다. 이 용액을 동량의 헥산(200 ㎖)으로 3회 분획하였다. 분획하여 얻어진 헥산 분획층을 제거하고 남은 95% 메탄올층을 40℃에서 감압 농축하여 추출물을 얻은 후 다시 이 추출물에 50% 메탄올(200㎖)를 가하여 재용해하였다. 이 용액을 디클로로메탄(200 ㎖)으로 다시 3회 분획하였다. 상기에서 얻어진 디클로로메탄 분획층을 40℃에서 감압 농축하여 디클로로메탄 분획추출물을 제조하였다.Horse riding (dry weight, 30 g) was cut and then refluxed with 100% methanol (300 mL) at 65 ° C. for 3 hours, cooled to room temperature, and filtered under reduced pressure. The filtrate obtained by performing the above three times in the same manner (300 mL × 3 times) was concentrated under reduced pressure at 40 ℃ to prepare a solvent extract. The 100% methanol extract obtained above was redissolved by adding 200 ml of 95% methanol. This solution was partitioned three times with the same amount of hexane (200 mL). The resulting hexane fractionation layer was removed, and the remaining 95% methanol layer was concentrated under reduced pressure at 40 ° C. to obtain an extract, and then 50% methanol (200 mL) was added to the extract and redissolved. This solution was partitioned again three times with dichloromethane (200 mL). The dichloromethane fractionation layer obtained above was concentrated under reduced pressure at 40 ° C. to obtain a dichloromethane fraction extract.
실시예Example 4. 4.
실시예 3에서 얻어진 디클로로메탄 분획층을 제거하고 남은 50% 메탄올층을 다시 동량의 에틸아세테이트(200 ㎖)로 3회 분획하였다. 상기에서 얻어진 각 분획층을 40℃에서 감압 농축하여 에틸아세테이트 분획추출물을 제조하였다.The dichloromethane fractionation layer obtained in Example 3 was removed, and the remaining 50% methanol layer was fractionated three times with the same amount of ethyl acetate (200 mL). Each fraction layer obtained above was concentrated under reduced pressure at 40 ° C. to prepare an ethyl acetate fraction extract.
실시예Example 5. 5.
실시예 1 과 실시예 3에서 얻어진 승마의 80% 메탄올 추출물과 100% 메탄올 추출물의 디클로로메탄 분획층을 메탄올:물=9:1의 전개용액으로 칼럼 크로마토그래피(C18 column chromatography)를 실시하여 모두 6종류의 분획층을 각각 얻었다. 상기에서 얻어진 각 분획층을 40℃에서 감압 농축하여 에틸아세테이트 분획추출물을 제조하였다.Dichloromethane fractionation layers of 80% methanol extract and 100% methanol extract of horse riding obtained in Examples 1 and 3 were subjected to column chromatography (C18 column chromatography) with a methanol: water = 9: 1 solution. Different fractional layers were obtained, respectively. Each fraction layer obtained above was concentrated under reduced pressure at 40 ° C. to prepare an ethyl acetate fraction extract.
승마의 건조중량 30g 에서의 각 용매 및 추가 분획에 대한 수득율은 하기 표1 및 표2에 나타내었다. The yields for each solvent and additional fractions at 30 g dry weight of horse riding are shown in Tables 1 and 2 below.
표 1은 승마의 건조중량 30g 당 각 용매별 수득량 및 수득율을 나타낸다. (단위 : %) Table 1 shows the yield and yield for each solvent per 30 g dry weight of horse riding. (unit : %)
표 2는 각 용매 추출물의 추가 분획추출물의 수득량 및 수득율을 나타낸다. (단위 : %)Table 2 shows the yield and yield of additional fraction extract of each solvent extract. (unit : %)
실험예Experimental Example 1 : One : 비세포계Noncellular system 티로시네이즈Tyrosinase 활성 측정. Activity measurement.
50 mM 인산완충용액(pH 7.2) 120 ㎕에 100 ㎍/㎖ 농도의 티로신 40 ㎕와 60 unit 티로시네이즈 40 ㎕를 혼합하여 37℃에서 30분간 반응시킨 후 490 nm 파장에서 흡광도를 측정하여 티로시네이즈 활성을 측정하였으며(T, 총 티로시네이즈 활성), 이때 승마 추출액의 분획추출물의 시료액을 각각의 농도별로 상기의 반응액에 추가로 첨가하여 반응시켜 티로시네이즈 활성이 감소되는 (R, 감소된 티로시네이즈 활성) 추출시료액에 대하여 다음의 수학식 1을 적용하여 억제률(I, %)로 나타내었다. 40 μl of tyrosine at 100 μg / ml concentration and 40 μl of 60 unit tyrosinase were mixed in 120 μl of 50 mM phosphate buffer (pH 7.2) for 30 minutes at 37 ° C., and then absorbance was measured at 490 nm. Naze activity was measured (T, total tyrosinase activity), wherein the sample solution of the fraction extract of the horse riding extract was added to the reaction solution for each concentration to react to decrease tyrosinase activity (R, Reduced tyrosinase activity) was expressed as the inhibition rate (I,%) by applying the following equation (1) to the extracted sample solution.
각 추출용매 100 ㎍/㎖ 농도에서 티로시네이즈 활성 억제율을 조사한 결과는 표 3과 같았다. The results of investigating the inhibition rate of tyrosinase activity at the concentration of 100 μg / ml of each extraction solvent were shown in Table 3.
표 3은 용매 추출물 100㎍/㎖ 농도에서의 티로시네이즈 활성 억제율을 나타낸다. (단위 : %) Table 3 shows the inhibition rate of tyrosinase activity at the concentration of 100 μg / ml solvent extract. (unit : %)
이 중 티로시네이트 활성 억제율이 가장 미약한 50% 메탄올 수용액을 제외하고, 그 외 티로시네이즈 활성 억제율을 보이는 물, 80% 메탄올, 100% 메탄올추출물의 헥산, 에틸아세테이트 및 디클로로메탄 분획추출물의 티로시네이즈 활성 억제율을 농도별로 실험한 결과는 표4와 같다. Among these, except 50% methanol aqueous solution having the least inhibition of tyrosinate activity, water, 80% methanol, 100% methanol extract of hexane, ethyl acetate and dichloromethane fraction extract showed the tyrosinase activity inhibition rate. The results of experiments on the rate of inhibition of sinease activity are shown in Table 4.
표 4는 헥산, 에틸아세테이트 및 디클로로메탄 분획추출물의 농도별 티로시네이즈 활성 억제율을 나타낸다. (단위 : %)Table 4 shows the inhibition rate of tyrosinase activity by concentration of hexane, ethyl acetate and dichloromethane fraction extract. (unit : %)
비교예Comparative example 1 One
효능에 대한 대조 실험 대상으로 티로시네이즈 활성 저해제이며 미백기능성 화장품에 널리 사용되고 있는 알부틴을 사용하였다.Arbutin, an inhibitor of tyrosinase activity and widely used in whitening functional cosmetics, was used as a control subject for efficacy.
승마 추출물의 티로시네이즈 활성 억제율에 대한 객관적 비교 분석을 위해 현재 미백기능성 화장품에 널리 사용되고 있는 알부틴의 농도별 티로시네이즈 활성 억제율을 조사한 결과를 표 5에 나타내었다. Table 5 shows the results of examining the inhibition rate of tyrosinase activity by concentration of arbutin widely used in whitening functional cosmetics for the objective comparative analysis on the inhibition rate of tyrosinase activity of horse riding extract.
표 5는 알부틴의 농도별 티로시네이즈 활성 억제율을 나타낸다. (단위 : %)Table 5 shows the tyrosinase activity inhibition rate by the concentration of arbutin. (unit : %)
상기의 실험 결과로부터 승마 추출물의 에틸아세테이트 분획추출물은 동일 농도의 알부틴과 비교할 때 거의 동등한 수준의 티로시네이즈 활성 억제율을 보이고, 승마 추출물의 디클로로메탄 분획추출물은 동일 농도의 알부틴에 비해 저농도에서 현저히 높은 티로시네이즈 활성 억제율을 보임을 알 수 있다. 도 1 및 도 2는 표 4 및 표 5를 그래프로 나타낸 것으로 승마 추출물과 알부틴의 티로시네이즈 활성 억제율을 비교한 것이다.From the above experimental results, ethyl acetate fraction extract of equestrian extract showed almost the same level of inhibition of tyrosinase activity as compared to arbutin at the same concentration, and dichloromethane fraction extract of equestrian extract was significantly higher at low concentration than arbutin at the same concentration. It can be seen that the tyrosinase activity inhibition rate. 1 and 2 are the graphs of Table 4 and Table 5 to compare the inhibitory rate of tyrosinase activity of horse riding extract and arbutin.
이 중에서도 티로시네이즈 활성 억제율이 가장 높게 나타난 100% 메탄올의 디클로로메탄 분획추출물을 C18 칼럼 크로마토그래피(column chromatography)에 걸어서 분획 1-6 샘플을 얻은 후 동일 농도(100㎍/㎖)에서 이들의 티로시네이즈 활성 억제율을 조사하였다. 이를 표 6에 나타내었다.Among them, dichloromethane fraction extract of 100% methanol, which showed the highest inhibition of tyrosinase activity, was subjected to C18 column chromatography to obtain fraction 1-6 samples, and then their tyrosine at the same concentration (100 µg / ml). The inhibition of cinease activity was investigated. This is shown in Table 6.
이 분획물 들을 100㎍/㎖ 농도에서 실험한 결과, 활성이 높게 나온 상층 분획 1, 2, 3을 선택하여 농도별로 실험한 결과를 표 7에 나타내었다. As a result of experimenting these fractions at a concentration of 100 ㎍ / ㎖, the results of experiments by concentrations of the upper fractions 1, 2, 3 that showed high activity are shown in Table 7.
표 6은 100㎍/㎖ (1% DMSO)에서의 분획 1-6 샘플의 티로시네이즈 활성 억제율을 나타낸다. (단위 : %)Table 6 shows the tyrosinase activity inhibition rate of fraction 1-6 samples at 100 μg / ml (1% DMSO). (unit : %)
표 7은 분획 1-3의 농도별 티로시네이즈 활성 억제율을 나타낸다. (단위 : %)Table 7 shows the tyrosinase activity inhibition rate by concentration of fractions 1-3. (unit : %)
상기의 실험 결과로부터 승마의 100% 메탄올 추출액의 디클로로메탄 분획물을 C18 칼럼 크로마토그래피(전개용매; 메탄올:물=9:1)에 걸어서 얻어진 6개의 분획물 중 C18 칼럼 크로마토그래피에 있어서 이동률이 큰 3개 분획층에 티로시네이즈 활성을 억제하는 물질이 주로 분포되어 있음을 알 수 있다.According to the above experimental results, three dichloromethane fractions of 100% methanol extracts of horse riding were subjected to C18 column chromatography (developing solvent; methanol: water = 9: 1), and three of them were highly mobile in C18 column chromatography. It can be seen that a substance mainly inhibiting tyrosinase activity is distributed in the fractionation layer.
실험예Experimental Example 2 : 2 : 세포계Cell line 티로시네이즈Tyrosinase 활성 측정을 위한 B16F10세포 배양, 세포 성장 및 형태 관찰 B16F10 cell culture, cell growth and morphology observation for activity measurement
B16F10세포의 배양을 위해 90%의 Dulbecco's modified Eagle's medium (DMEM, Gibco BRL)에 10% fetal bovine serum과 1% penicillin-streptomycin이 포함된 성장배지를 사용하였으며, 5%의 CO2가 공급되는 37℃ 세포배양기에서 배양하였다. 실험을 위하여 배양 플라스크에 부착되어 있는 B16F10 세포를 trypsin-EDTA로 부유시켜 1×105개로 100 ㎜ 배양 용기에 나눈 다음, 24시간 동안 안정화시킨 후, 승마 추출물과 멜라닌세포 자극호르몬 (α-melanocyte stimulating hormone, α-MSH)을 처리하여 2일 또는 5일간 배양하였다. 이를 10% fetal bovine serum과 1% penicillin-streptomycin이 함유된 DMEM을 이용하여 100 mg/㎖의 농도를 가진 시료로 제작하여 사용하였다. For the cultivation of B16F10 cells, a growth medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in 90% Dulbecco's modified Eagle's medium (DMEM, Gibco BRL) was used, and 37 ° C with 5% CO 2. Cultured in a cell culture phase. For experiments, B16F10 cells attached to the culture flasks were suspended with trypsin-EDTA, divided into 1 × 10 5 cells in a 100 mm culture vessel, stabilized for 24 hours, and then equestrian extract and melanocyte stimulating hormone (α-melanocyte stimulating). hormone, α-MSH) was incubated for 2 or 5 days. This was prepared using a 100 mg / ㎖ sample using DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin.
B16F10 세포성장에 대한 승마 추출물의 영향을 MTT assay로 알아보기 위해 상기에서 제작돤 시료에 3-(4,5-Dimethylthiaxol-2-yl)-2,5-diphenyltetraxolium bromide thiazolyl blue(MTT)를 넣고, 2-4시간 정도 37℃에서 암상태로 두었다. 그 후 MTT 시약을 제거하고, 1 ㎖의 dimethyl sulfoxide (DMSO, Sigma)를 넣은 후, 540 ㎚에서 흡광도를 측정하였다. 승마 추출물로 처리한 군의 흡광도와 승마 추출물을 처리하지 않은 군의 흡광도를 각각 측정하여 다음의 수학식 2를 적용하여 세포 성장율로 나타내었다. In order to determine the effect of the equestrian extract on B16F10 cell growth by MTT assay, 3- (4,5-Dimethylthiaxol-2-yl) -2,5-diphenyltetraxolium bromide thiazolyl blue (MTT) was added to the sample prepared above. It was left dark at 37 degreeC for 2-4 hours. Thereafter, the MTT reagent was removed, 1 mL of dimethyl sulfoxide (DMSO, Sigma) was added thereto, and the absorbance was measured at 540 nm. The absorbance of the group treated with the equestrian extract and the absorbance of the group not treated with the equestrian extract, respectively, were measured and expressed as cell growth rates by applying the following equation (2).
표 8은 승마 추출물이 B16F10 세포 성장율에 미치는 영향을 나타낸다. (단위 : %)Table 8 shows the effect of the equestrian extract on B16F10 cell growth rate. (unit : %)
세포의 형태학적 변화는 200 배율로 위상차 현미경을 통해 관찰하였다. 도 3 과 같이 세포형태의 관찰에서도 α-MSH를 단독으로 처리한 세포의 경우에 멜라닌세포의 특징적인 형태인 수지상 세포돌기가 관찰되었으며, 이러한 현상은 승마 추출물로 처리한 군에서도 관찰되어 형태적 변화를 보여 주지 않았다.Morphological changes of the cells were observed through a phase contrast microscope at 200 magnification. In the observation of cell morphology as shown in FIG. 3, dendritic dendritic protrusion, which is a characteristic form of melanocytes, was observed in the cells treated with α-MSH alone, and this phenomenon was observed in the group treated with the equestrian extract. Did not show.
실험예Experimental Example 3 : 3: 세포계Cell line 티로시네이즈Tyrosinase 활성 측정 Active measurement
티로시네이즈활성은 B16F10 세포를 trypsin-EDTA로 부유시켜 1×105개로 100 ㎜ 배양 용기에 나눈 다음, 24시간 동안 안정화시킨 후, 세포성장에 영향을 주지 않는 10 ㎍/㎖ 농도의 승마추출물과 α-MSH를 처리하여 2일간 배양하고, 이와 같이 처리된 B16F10 세포를 phosphate-buffered saline(PBS)으로 세 번 씻어내고, 1,000 rpm에서 5분간 원심분리하여 상층액을 제거하였다. 상층액을 제거하고 남은 여액에 Lysis buffer(1% Triton X-100, 10 mM sodium phosphate, pH 7.0, 0.1 mM PMSF)를 첨가하여 30 분간 얼음에서 반응시켜 세포를 녹인 후, 원심분리하여 상층액을 취하여 티로시네이즈 활성 측정용액으로 사용하였다. 단백질 농도 50 ㎍의 용액을 취하여 0.1 M sodium phosphate buffer (pH 7.0)과 0.05% L-DOPA를 첨가하여 37℃에서 반응시키면서 10분 간격으로 405 nm에서 흡광도의 변화를 1시간 동안 관찰하였다.Tyrosinase activity was obtained by floating B16F10 cells with trypsin-EDTA, dividing 1 × 10 5 cells into 100 mm culture vessels, and stabilizing for 24 hours, and then riding riding at a concentration of 10 μg / ml, which did not affect cell growth. After treatment with α-MSH for 2 days, the B16F10 cells thus treated were washed three times with phosphate-buffered saline (PBS) and centrifuged at 1,000 rpm for 5 minutes to remove supernatant. After removing the supernatant, Lysis buffer (1% Triton X-100, 10 mM sodium phosphate, pH 7.0, 0.1 mM PMSF) was added to the remaining filtrate and allowed to react on ice for 30 minutes to dissolve the cells, followed by centrifugation. Was used as a measurement solution for tyrosinase activity. A 50 μg solution of protein concentration was taken, and 0.1 M sodium phosphate buffer (pH 7.0) and 0.05% L-DOPA were added and reacted at 37 ° C., whereupon the change of absorbance was observed at 405 nm for 10 hours.
상기 실험에 의하여 티로시네이즈 활성도를 측정한 결과 α-MSH에 의해 유도된 티로시네이즈 활성은 승마 추출물에 의해 표 9와 같은 억제율을 보이며 특히 승마의 100% 메탄올 추출물의 디클로로메탄 분획추출물의 티로시네이즈 활성 억제율이 가장 현저하였다.As a result of measuring the tyrosinase activity by the above experiments, the tyrosinase activity induced by α-MSH shows the inhibition rate as shown in Table 9 by the equestrian extract. Naze activity inhibition was the most remarkable.
표 9는 승마 추출물의 B16F10 세포내 티로시네이즈 활성 억제율을 나타낸다. (단위 : %)Table 9 shows the inhibition rate of B16F10 intracellular tyrosinase activity of the equestrian extract. (unit : %)
실험예Experimental Example 4 : 멜라닌농도 측정 4 melanin concentration measurement
B16F10세포내 멜라닌생성에 미치는 영향을 알아보기 위해 상기 실험예 2에서 배양한 승마 추출물을 처리하여 5일간 배양한 B16F10 세포를 phosphate-buffered saline(PBS)로 세 번 씻어내고 1,000 rpm에서 5분간 원심분리하여 상층액을 제거한 후 10%의 DMSO를 함유한 1 N NaOH를 가하여 80 ℃에서 1 시간 동안 반응시킨 후, 475 nm에서 흡광도를 측정하였다. 합성멜라닌을 이용하여 농도별 표준곡선을 작성한 후 멜라닌의 농도를 측정하였다. 승마 추출물을 처리하지 않은 세포의 멜라니 농도(T, 총 멜라닌 농도), 승마 추출액의 각각의 농도별로 처리했을 때 감소된 멜라닌 농도(R, 감소된 멜라닌 농도)를 추출시료액에 대하여 상기 수학식 1을 적용하여 억제율(I,%)로 나타내었다.In order to examine the effect on melanogenesis in B16F10 cells, B16F10 cells cultured for 5 days with horse riding extract cultured in Experiment 2 were washed three times with phosphate-buffered saline (PBS) and centrifuged at 1,000 rpm for 5 minutes. After removing the supernatant, 1 N NaOH containing 10% DMSO was added thereto, and the mixture was reacted at 80 ° C. for 1 hour, and then absorbance was measured at 475 nm. Synthetic melanin was used to prepare a standard curve for each concentration, and then the melanin concentration was measured. Melanin concentration (T, total melanin concentration) of the cells not treated with the equestrian extract, reduced melanin concentration (R, reduced melanin concentration) when treated for each concentration of the equestrian extract formula (1) It is expressed as the inhibition rate (I,%) by applying.
멜라닌 생성 억제율을 측정한 결과는 표 10과 같으며 모든 추출물 중 디클로로메탄 분획추출물의 멜라닌 생성 억제율이 가장 높았다.The results of measuring the melanin production inhibition rate are shown in Table 10. Among all the extracts, the melanin production inhibition rate of the dichloromethane fraction extract was the highest.
표 10은 세포내 멜라닌 생성에 미치는 영향을 나타낸다. (단위 : %)Table 10 shows the effect on intracellular melanin production. (unit : %)
세포외 멜라닌은 상기 실험예 2에서 배양한 B16F10 세포에 승마 추출물과 α-MSH를 처리한 다음 5일간 배양 후 배양 상층액을 취하여 404 nm에서 흡광도를 측정하고, 억제율은 상기 수학식 1을 적용하여 산출하였다. 세포외로 분비되는 멜라닌양을 조사한 결과는 표 11과 같으며 승마 추출물 중 100% 메탄올 추출물의 디클로로메탄 분획추출물의 멜라닌 생성 억제율이 가장 높았다. The extracellular melanin was treated with riding extract and α-MSH in the B16F10 cells cultured in Experimental Example 2, and then cultured for 5 days, the culture supernatant was measured, and the absorbance was measured at 404 nm. Calculated. The results of investigation of the amount of melanin secreted from the cells are shown in Table 11, and the inhibition rate of melanin production of the dichloromethane fraction extract of 100% methanol extract of horse riding extract was the highest.
표 11은 세포외 멜라닌 분비에 미치는 영향을 나타낸다. (단위 : %)Table 11 shows the effect on extracellular melanin secretion. (unit : %)
본 발명의 조성물은 승마 추출물의 분획추출물을 0.01 ∼ 10 중량%, 바람직하게는 0.1 중량%로 포함한다. 승마 추출물의 분획추출물의 함량이 0.01중량% 미만의 경우에는 미백효과가 떨어지고, 10 중량% 초과인 경우에는 제형 안정성이 떨어질 우려가 있다.The composition of the present invention comprises 0.01 to 10% by weight, preferably 0.1% by weight of the fraction extract of horse riding extract. When the content of the fraction extract of the horse riding extract is less than 0.01% by weight, the whitening effect is reduced, when the content of more than 10% by weight there is a fear that the formulation stability.
이상의 결과로 보아 추출물은 하기 처방예 1-6에서 보는 바와 같이 다양한 조성물인 유연화장수(스킨), 영양화장수(로숀), 영양크림, 마사지크림, 엣센스, 팩, 유화형 화운데이션 등에 첨가함으로서 미백기능성 화장품의 화장료료 사용될 수 있다.As a result of the above results, the extract is added to various compositions such as supple cosmetics (skin), nourishing cosmetics (lotion), nourishing cream, massage cream, essence, pack, emulsified foundation, and the like as shown in Formulations 1-6 below. Cosmetics can be used.
처방예에서 사용된 승마 추출물의 분획추출물은 100% 메탄올 추출물의 디클로로메탄 분획추출물을 사용하였다.As a fraction extract of the horse riding extract used in the formulation example, a dichloromethane fraction extract of 100% methanol extract was used.
처방예Prescription Example 1. 승마 추출물의 분획추출물을 함유한 1. Containing fraction extract of horse riding extract 화장료중In cosmetics 유연 화장수( Flexible lotion 스킨skin )의 )of 처방예는Prescription example 다음과 같다. As follows.
처방예Prescription Example 2. 승마 추출물의 분획추출물을 함유한 2. Contains fraction extract of horse riding extract 화장료중In cosmetics 영양화장수( Nutritional Cosmetics 로숀Lotion )의 처방예는 다음과 같다.Prescription example of) is as follows.
처방예Prescription Example 3. 승마 추출물의 분획추출물을 함유한 3. Containing Fraction Extract of Horse Riding Extract 화장료중In cosmetics 영양크림의 Nutrition cream 처방예는Prescription example 다음과 같다. As follows.
처방예Prescription Example 4. 승마 추출물의 분획추출물을 함유한 4. Contains fraction extract of horse riding extract 화장료중In cosmetics 유연 마사지 크림의 처방예는 다음과 같다. A prescription example of the flexible massage cream is as follows.
처방예Prescription Example 5. 승마 추출물의 분획추출물을 함유한 5. Containing fraction extract of horse riding extract 화장료중In cosmetics 팩의 Pack 처방예는Prescription example 다음과 같다. As follows.
처방예Prescription Example 6. 승마 추출물의 분획추출물을 함유한 6. Containing Fraction Extract of Horse Riding Extract 화장료중In cosmetics 유화형Oil painting 화운데이션의Foundation 처방예는Prescription example 다음과 같다. As follows.
도 1 은 각 용매의 에틸아세테이트 분획 및 알부틴의 활성억제율 비교한 것이다.1 is a comparison of the ethyl acetate fraction of each solvent and the inhibitory activity of arbutin.
도 2 는 각 용매의 디클로메탄 분획 및 알부틴의 활성억제율 비교한 것이다.Figure 2 compares the dichloromethane fraction and arbutin activity inhibition rate of each solvent.
도 3 은 승마 추출물처리에 대한 B16F10세포의 형태적 변화를 나타낸 도면이다. A는 정상군을 B는 대조군 (α-MSH처리군)을 C는 승마 추출물처리군을 나타낸다.Figure 3 is a diagram showing the morphological changes of B16F10 cells for the equestrian extract treatment. A represents a normal group, B represents a control group (α-MSH treatment group), C represents a horse riding extract treatment group.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019143197A1 (en) * | 2018-01-22 | 2019-07-25 | 원광대학교 산학협력단 | Skin whitening composition containing cimicifuga dahurica extract or fraction thereof |
| KR20190089748A (en) * | 2018-01-22 | 2019-07-31 | 원광대학교산학협력단 | A composition for skin whitening comprising Cimicifuga dahurica extract or a fraction thereof |
| KR20230147989A (en) | 2022-04-15 | 2023-10-24 | 주식회사 코씨드바이오팜 | A Cosmetic composition with the extract of plant belonging to the Cimicifuga genus for skin cooling |
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2007
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019143197A1 (en) * | 2018-01-22 | 2019-07-25 | 원광대학교 산학협력단 | Skin whitening composition containing cimicifuga dahurica extract or fraction thereof |
| KR20190089748A (en) * | 2018-01-22 | 2019-07-31 | 원광대학교산학협력단 | A composition for skin whitening comprising Cimicifuga dahurica extract or a fraction thereof |
| KR20230147989A (en) | 2022-04-15 | 2023-10-24 | 주식회사 코씨드바이오팜 | A Cosmetic composition with the extract of plant belonging to the Cimicifuga genus for skin cooling |
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