KR100465977B1 - Nanoemulsion having Compound K by nano-emulsion technology, and skin care and pharmaceutical compositions for external applications utilizing thereof - Google Patents
Nanoemulsion having Compound K by nano-emulsion technology, and skin care and pharmaceutical compositions for external applications utilizing thereof Download PDFInfo
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Abstract
본 발명은 생전환(bioconversion)법에 의하여 제조된 인삼의 주요대사산물인 화합물 K(20-O-β-D-글루코피라노실-20(S)-프로토파낙사다이올)을 주요 유효성분으로 하고, 나노유화기술을 사용하여 미세한 유화입자 및 리포좀 내부에 상기 화합물 K를 함유시킴으로써 피부흡수를 극대화한 피부외용제 조성물에 관한 것으로, 더욱 상세하게는 고압유화기술 및 용매추출법 등의 나노유화기술을 사용하여, 레시틴 등의 피부친화성이 우수한 유화제와 함께 미세한 유화입자 및 리포좀 내에 상기 화합물 K를 함유시키는 방법 및 이를 제형화함으로써 경피흡수율을 크게 향상시켜 우수한 피부세포 증식 및 콜라겐 생합성 촉진 효과를 갖는 피부 노화방지용 화장료 및 의약료 조성물에 관한 것이다.The present invention is a compound K (20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol) which is a major metabolite of ginseng prepared by the bioconversion method as the main active ingredient In addition, the present invention relates to an external skin composition for maximizing skin absorption by containing the compound K in the fine emulsified particles and liposomes using nanoemulsification technology, and more specifically, using nanoemulsification technology such as high pressure emulsion technology and solvent extraction method. By containing the compound K in the fine emulsified particles and liposomes together with an emulsifier such as lecithin and excellent skin affinity, and formulating the same, it greatly improves the transdermal absorption rate and has an effect of promoting skin cell proliferation and collagen biosynthesis. It relates to a cosmetic for preventing and a pharmaceutical composition.
Description
본 발명은 생전환(bioconversion)법에 의하여 제조된 인삼의 주요대사산물인 20-O-β-D-글루코피라노실-20(S)-프로토파낙사다이올(이하 '화합물 K(Compound K)'라 한다)을 나노유화기술을 사용하여 미세한 유화입자 및 리포좀 내에 함유시킴으로써 피부흡수를 극대화한 피부 외용제 조성물에 관한 것으로, 더욱 상세하게는 고압유화 및 용매추출법 등의 나노유화기술을 이용하여 레시틴 등의 피부친화성이 우수한 유화제와 함께 미세한 유화입자 및 리포좀 내에 화합물 K를 함유시키는 방법및 이를 제형화함으로써 경피흡수율을 크게 향상시켜 우수한 피부세포 증식 및 콜라겐 생합성 촉진 효과를 갖는 피부 노화방지용 화장료 및 의약료 조성물에 관한 것이다. 또한, 화합물 K를 제형화함에 있어서 미세한 유화입자 및 리포좀 외에 500nm이상 1000㎛ 이하의 일반유화입자에 포집하여 사용하거나, 오일에 용해하여 그대로 사용하거나, 캡슐화하여 사용할 수도 있다. 제형화 기술의 일반적인 방법을 포함한 미세유화기술을 적용하여 화합물 K의 효능을 화장료 및 의약료로서 응용하기 위한 모든 방법을 의미한다.The present invention is a major metabolite of ginseng prepared by the bioconversion method 20-O-β-D- glucopyranosyl-20 (S) -protopanaxadiol (hereinafter 'Compound K) The present invention relates to an external composition for skin that maximizes skin absorption by incorporating into the fine emulsified particles and liposomes using nanoemulsification technology, and more specifically, lecithin and the like using nanoemulsification technology such as high pressure emulsion and solvent extraction method. A method of containing compound K in fine emulsified particles and liposomes together with an emulsifier with excellent skin affinity, and formulating the same, greatly improves transdermal absorption, thereby improving skin aging and collagen biosynthesis. It relates to a composition. In addition, in formulating the compound K, in addition to the fine emulsified particles and liposomes, it may be collected and used in general emulsified particles of 500 nm or more and 1000 μm or less, dissolved in oil or used as is, or encapsulated. Means all methods for applying the efficacy of compound K as a cosmetic and a medicament by applying microemulsification techniques including general methods of formulation techniques.
피부는 인체의 일차 방어막으로서 체내의 제기관을 온도 및 습도 변화와 자외선, 공해물질 등 외부환경의 자극으로부터 보호해 주며, 체온조절 등의 생체 항상성 유지에 중요한 역할을 하고 있다. 그러나, 외부로부터 받는 과도한 물리적·화학적 자극, 스트레스 및 영양결핍 등은 피부의 정상기능을 저하시키고 탄력손실, 각질화, 주름생성 등의 피부 노화현상을 촉진시키게 되는데, 이러한 현상을 방지하고 보다 건강하고 탄력있는 피부를 유지하기 위하여 종래 각종 동물, 식물, 미생물 등으로부터 얻은 생리활성물질들이 강화된 화장품을 사용함으로써 피부의 고유기능을 유지시키고 피부세포를 활성화시켜 피부노화를 효과적으로 억제하기 위한 노력이 있어 왔다.Skin is the body's primary protective film that protects internal organs from changes in temperature and humidity, and from stimuli from the external environment such as ultraviolet rays and pollutants, and plays an important role in maintaining homeostasis such as body temperature control. However, excessive physical and chemical stimuli from the outside, stress and nutritional deficiency decrease the normal function of the skin and promote skin aging phenomenon such as loss of elasticity, keratinization and wrinkle formation. In order to maintain the skin, there have been efforts to effectively inhibit skin aging by maintaining skin's intrinsic function and activating skin cells by using cosmetics that have been enhanced with bioactive substances obtained from various animals, plants, and microorganisms.
그러나, 기존의 화장품 원료들은 효능이 미진하거나 피부 부작용을 유발하는 등 여러 가지 문제점을 가지고 있었다.However, conventional cosmetic raw materials had various problems such as insufficient efficacy or cause skin side effects.
따라서, 피부 부작용을 유발하지 않으면서 피부 노화방지 효과를 갖는 원료에 대한 연구가 활발히 진행되고 있으며, 그 중 인삼 추출물에 대한 관심은 매우높아 꾸준한 연구가 진행되고 있다. 그간의 인삼추출물에 대한 연구방향을 살펴보면, 인삼추출물(ginseng extract)에서 인삼 사포닌(ginseng saponin)의 추출, 인삼 어글리콘(aglycon)의 제조 및 인삼 사포닌의 정제를 통해 인체내부에서의 주요 대사산물인 화합물 K를 제조, 분리 정제하는 방향으로 전개되었다.Therefore, studies on raw materials having an anti-aging effect without causing skin side effects are being actively conducted, and among them, the interest in ginseng extract is very high, and steady research is being conducted. Looking at the research direction of ginseng extract in the meantime, ginseng extract (ginseng saponin) from ginseng extract (ginseng saponin), ginseng aglycon (aglycon) and ginseng saponin through the purification of ginseng saponin It developed in the direction to prepare and isolate compound K.
인삼 사포닌은 담마란(dammarane) 타입의 트리테르펜(triterpene)인 비당부의 R1, R2 및 R3 위치의 알코올성 OH기에 글루코스(glucose), 람노스(rhamnose), 자일로스(xylose) 및 아라비노스(arabinose)와 같은 당류가 에테르결합된 구조를 가지며, 현재까지 인삼에서 총 29종이 밝혀졌다. 상기 인삼 사포닌의 각 성분은 1964년 Shibata가 인삼에 함유된 배당체란 뜻으로 진세노사이드(ginsenoside)라 명명하였으며, 박층크로마토그래피(TLC)에서 분리된 이동거리 순으로 올레아닌(oleanine)계 사포닌인 진세노사이드-Ro와 진세노사이드-Ra, -Rb1, -Rb2, -Rc, -Rd, -Re, -Rf, -Rg1, -Rg2, -Rg3 및 -Rh등으로 명명하였다.Ginseng saponins are glucose, rhamnose, xylose and arabinose in the alcoholic OH groups at the R1, R2 and R3 positions of the non-sugar moiety, a triterpene of the dammarane type. Sugars such as) has an ether-bonded structure, a total of 29 species have been identified in ginseng to date. Each component of the ginseng saponin was named ginsenoside as a glycoside contained in Shibata ginseng in 1964, and it was an oleanine-based saponin in order of the distance separated from thin layer chromatography (TLC). Ginsenoside-Ro and ginsenoside-Ra, -Rb1, -Rb2, -Rc, -Rd, -Re, -Rf, -Rg1, -Rg2, -Rg3 and -Rh, etc. were named.
이러한 인삼 사포닌은 750여종의 식물에 함유된 다른 식물의 사포닌과는 화학구조가 상이할 뿐 아니라 약리효능도 다른 것으로 밝혀졌다. 특히, 인삼사포닌은 약성이 매우 온화하고 과량투여에 의한 독성이 없을 뿐만이 아니라 용혈작용도 거의 없다는 것이 밝혀졌다.This ginseng saponin was found to be different in chemical structure and pharmacological efficacy from the saponins of other plants contained in 750 kinds of plants. In particular, ginseng saponin was found to be very mild, not toxic by overdose, and have little hemolytic action.
또한 인삼 사포닌을 인지질과의 복합체인 리포좀(liposome)형태로 인체피부에 도포한 결과 노화된 피부에 활력을 주며, 탄력성증가, 수화성증가와 피부의 혈액순환 촉진 등의 효과가 있음이 보고되었다(Curri. SB, Gezz, Z, Longhi, MG, Castelpietra, R : Fitoterapia, 57, 217(1986)) (Gezzi, A, Longhi, MG,Mazzoleni, R, Curri, SB : Fitoterapia, 57, 15(1986)) (Bombardelli, E. Curri, SB, Gariboldi, PL : Proc. 5th Intl. Ginseng Sym. Seoul Korea, 11(1988))In addition, it was reported that ginseng saponin was applied to human skin in the form of liposome, a complex with phospholipids, to revitalize aged skin, increase elasticity, increase hydration, and promote blood circulation ( Curri.SB, Gezz, Z, Longhi, MG, Castelpietra, R: Fitoterapia, 57, 217 (1986)) (Gezzi, A, Longhi, MG, Mazzoleni, R, Curri, SB: Fitoterapia, 57, 15 (1986) (Bombardelli, E. Curri, SB, Gariboldi, PL: Proc. 5th Intl. Ginseng Sym. Seoul Korea, 11 (1988))
이후, 인삼 사포닌을 노화억제제품의 원료로 응용하기 위하여, 인삼 사포닌의 효능을 유지하고, 특히 피부 투과성을 증가시킨 생전환(bioconversion)된 인삼 어글리콘(aglycon)의 피부에서의 효능을 확인하였다.Then, in order to apply ginseng saponin as a raw material of the anti-aging product, the efficacy of ginseng saponin was maintained, and in particular, the efficacy in the skin of bioconversion ginseng aglycon (aglycon) that increased skin permeability was confirmed.
상기한 인삼추출물 및 인삼 사포닌을 이용한 사례로써, 화장료(미국특허 5,565,207, 5,567,419, 5,578,312, 5,663,160, 5,626,868, 5,753,242, 5,747,300, 5,853,705, 6,027,728, 6,063,366, 6,221,372, 6,228,378), 의약원료(미국특허 5,569,459, 5,571,516, 5,587,167, 5,674,488, 5,665,393, 5,629,316, 5,776,460, 5,739,165, 5,916,555, 6,071,521, 6,083,512, 6,255,313) 및 분리, 정제기술(미국특허 5,591,611, 5,591,612, 5,736,380, 5,789,392, 5,780,620, 5,922,580, 5,935,636, 6,132,726, 6,156,817, 6,207,164) 등에 관한 많은 연구결과가 보고되었다.As a case using the ginseng extract and ginseng saponin, cosmetics (US Patents 5,565,207, 5,567,419, 5,578,312, 5,663,160, 5,626,868, 5,753,242, 5,747,300, 5,853,705, 6,027,728, 6,063,366, 6,221,569, 6,221,569, 6,221, 569, 6,221, 569, 6,221, 569) 5,587,167, 5,674,488, 5,665,393, 5,629,316, 5,776,460, 5,739,165, 5,916,555, 6,071,521, 6,083,512, 6,255,313 Many studies have been reported.
그러나, 인삼 사포닌은 담마란 타입에 R1, R2 및 R3 위치의 알코올성 OH기에 당류가 에테르결합으로 연결된 구조를 가지고 있어 친수성이 크며, 분자량이 커짐에 따라, 이로 인해 피부 투과성 및 흡수성이 낮으며, 인삼 사포닌 자체에 친수성을 성질을 가지고 있어 피부의 각질층을 통과하지 못하여 인삼 사포닌이 피부 내부로의 유입이 어려운 문제점이 있었다.However, ginseng saponins have a structure in which sugars are linked by ether bonds to alcoholic OH groups at the R1, R2 and R3 positions in the dammaran type, and thus have high hydrophilicity, and as the molecular weight increases, the skin permeability and absorption are low. The saponin itself has a hydrophilic property, so it could not pass through the stratum corneum, so ginseng saponin was difficult to enter the skin.
한편, 최근에는 사포닌의 대사물에 관한 연구가 진행되면서, 인삼 사포닌의 효능이 사포닌 자체보다는 사포닌이 장내세균에 의해 분해된 장내세균 대사물이 활성본체임이 시사되어지고 있으며, 이에 인삼의 사포닌 성분 중 어글리콘에 당(글루코스)이 하나 붙은 구조로 이루어진 진세노사이드 Rh1, Rh2 및 F1과 화합물 K 등이 암세포증식 억제작용, 종양증식 억제작용, 항암제의 항암활성 증대작용 등의 약리작용이 있는 것으로 알려지고 있다.On the other hand, as research on metabolites of saponins has recently been conducted, it is suggested that the efficacy of ginseng saponins is an active body of enterobacterial metabolites in which saponins are degraded by intestinal bacteria rather than saponins themselves. Ginsenosides Rh1, Rh2 and F1 and compound K, which are composed of a sugar (glucose) attached to aglycone, are known to have pharmacological effects such as inhibiting cancer cell proliferation, inhibiting tumor growth, and increasing anticancer activity of anticancer drugs. ought.
인삼 사포닌으로부터 당의 일부를 제거하는 것에 의해 얻은 화합물 K에 관한 연구가 활발히 진행되고는 있으나, 아직까지 이를 피부에 도입시키는 기술 및 제형화 기술에 관하여는 연구된 바 없었으며, 본 발명자들은 화합물 K의 세포증식효과 및 콜라겐 생합성 효과를 이용한 피부노화방지용 화장료 및 의약료 조성물을 제공하고자 하였다. 또한 화합물 K의 생리활성효과를 응용하기 위하여 사용되는 제형화기술에는 제한이 없으나, 일반유화기술을 이용한 500nm에서 1000um이하의 일반유화입자 및 미세유화기술을 이용한 500nm이하 30nm이상의 미세유화입자, 화합물 K를 포집하여 캡슐화하는 방법 등이 가능할 것으로 예상되었다.Although studies on compound K obtained by removing a part of sugar from ginseng saponin have been actively conducted, there has been no research on the technique of introducing it into the skin and formulation technology. To provide a skin and anti-aging cosmetics and pharmaceutical compositions using a cell proliferation effect and collagen biosynthesis effect. In addition, there is no limitation in the formulation technology used to apply the physiological activity effect of the compound K, but the general emulsified particles of 500nm to less than 1000um using the general emulsification technology and microemulsion particles of more than 30nm, 500nm or less using the microemulsion technology, Compound K It is expected that the method of encapsulating and encapsulating it is possible.
이에 본 발명에서는 인삼 사포닌으로부터 생전환법에 의해 분리 정제된 화합물 K를 주요 유효성분으로 하여 피부친화성 유화제 및 나노유화기술을 사용하여 미세한 유화입자 및 리포좀 내부에 함유시킴으로써 우수한 피부세포 증식 및 콜라겐 생합성 촉진 효과를 갖는 피부노화 방지용 화장료 및 의약료 조성물을 제공하고자 한다.Therefore, in the present invention, by using the compound K purified from the ginseng saponin by the bioconversion method as a main active ingredient, the skin cell proliferation and collagen biosynthesis by containing the fine emulsified particles and liposomes inside the skin using an affinity emulsifier and a nanoemulsification technique. It is to provide a cosmetic and pharmaceutical composition for preventing skin aging having a promoting effect.
도 1은 비교예 1의 콜라겐 생합성 효능 측정 결과를 보여주는 피부세포 조직도이다.1 is a skin cell organization chart showing the results of measuring collagen biosynthesis efficacy of Comparative Example 1.
도 2는 실시예 1의 콜라겐 생합성 효능 측정 결과를 보여주는 피부세포 조직도이다.2 is a skin cell organization chart showing the results of measuring collagen biosynthesis efficacy of Example 1.
본 발명은 생전환(bioconversion)법에 의하여 제조된 인삼의 주요대사산물인 화합물 K를 나노유화기술을 사용하여 미세한 유화입자 및 리포좀 내에 함유시킴으로써 피부흡수를 극대화한 피부 외용제 조성물에 관한 것으로, 더욱 상세하게는 고압유화법, 용매추출법등의 나노유화기술을 이용하여 레시틴 등의 피부친화성이 우수한 유화제와 함께 미세한 유화입자 내에 화합물 K를 함유시키는 방법 및 이를 제형화함으로써 경피흡수율을 크게 향상시켜 우수한 피부세포 증식 및 콜라겐 생합성 촉진 효과를 갖는 피부 노화방지용 화장료 및 의약료 조성물에 관한 것이다.The present invention relates to a composition for external application for skin, in which compound K, which is a major metabolite of ginseng prepared by bioconversion method, is contained in fine emulsified particles and liposomes by using nanoemulsification technology to maximize skin absorption. Preferably, a method of containing compound K in fine emulsified particles together with emulsifiers such as lecithin and a skin emulsifier using nanoemulsification techniques such as a high pressure emulsification method and a solvent extraction method, and formulating the same, greatly improves transdermal absorption rate. The present invention relates to a skin anti-aging cosmetic and pharmaceutical composition having an effect of promoting cell proliferation and collagen biosynthesis.
상기의 화합물 K는 하기 화학식 1로 표현된다.The compound K is represented by the following formula (1).
일반적으로 효과적인 피부투과를 위해서는 친수성의 성질을 지니고 있는 물질보다는 소수성의 물질이 더 효과적이다. 이는 피부의 각질층 중에 분포되어 있는 세라미드를 포함한 세포간 지질 사이를 통과하기 위해서는 친수성의 물질보다는 소수성의 물질이 세포간 지질과의 상호작용에 더 효과적이며, 따라서 보다 자유롭게 피부 최외각 층을 통과할 수 있기 때문이다.In general, hydrophobic substances are more effective than hydrophilic substances for effective skin penetration. In order to pass between intercellular lipids, including ceramides, which are distributed in the stratum corneum of the skin, hydrophobic substances are more effective in interacting with intracellular lipids than hydrophilic substances, and thus more freely pass through the outermost layer of skin. Because there is.
상기의 화학식 1에서 보여주듯이 본 발명에서 사용하는 화합물 K는 일반적인인삼 사포닌에서 당의 일부를 제거함으로써 분자량을 줄이고, 소수성인 것을 특징으로 하고 있다.As shown in Chemical Formula 1, the compound K used in the present invention is characterized in that the molecular weight is reduced by removing a part of sugar from the common ginseng saponin and is hydrophobic.
이하 본 발명을 보다 상세하게 설명하면 다음과 같다.Hereinafter, the present invention will be described in more detail.
본 발명에서 사용된 화합물 K는 인삼 정제 사포닌을 산, 알칼리 또는 효소 등으로 가수분해하여 인삼 사포닌으로부터 당을 제거한 후 반응액을 실리카 칼럼에 통과하여 제조한다.Compound K used in the present invention is prepared by hydrolyzing ginseng purified saponin with acid, alkali or enzyme to remove sugar from ginseng saponin and then passing the reaction solution through a silica column.
여기에서 사용하는 효소는 사포닌 당결합을 분해하는 β-글루코스 분해효소(β-glucosidase), α,β-아라비노스 분해효소(α,β-arabinosidase), α,β-람노스 분해효소(α,β-rhamosidase) 등 엑소 당결합 분해효소 및 이들을 함유하고 있는 복합효소제 등이다.The enzymes used here include β-glucosidase, α, β-arabinosidase, and α, β-rhamnose enzymes (α, exo-glucose degrading enzymes such as β-rhamosidase), and complex enzymes containing them.
본 발명의 별도의 나노유화기술을 적용하지 않고, 일반적인 오일에 용해하여 제형화하는 것도 가능하며, 미세한 유화입자 및 리포좀의 제조시에는 내부에 화합물 K가 유화입자 및 리포좀 총 중량에 대하여 10-10∼70중량%의 양으로 함유되며, 바람직하게는 0.001∼30 중량%로 함유되는 것이 좋다. 또한 화합물 K를 함유한 미세 유화입자는 제법에 따라 차이가 있지만 화장료 및 의약료 조성물 총 중량에 대해서 10-10∼99.99중량%의 양으로 함유된다. 이와 같이 미세 유화입자 내부에 포집하는 기술을 적용하는 방법 외에 500nm에서 1000um의 입경을 가지는 일반적인 유화방법에 의한 유화입자 및 캡슐내부에 포집하거나, 화합물 K를 용해시킬 수 있는 오일에 용해하여 사용할 수 있다.Without applying a separate nanoemulsification technology of the present invention, it is also possible to formulate by dissolving in a general oil, in the preparation of fine emulsified particles and liposomes, the compound K inside the 10 -10 relative to the total weight of the emulsified particles and liposomes It is contained in an amount of -70% by weight, preferably 0.001-30% by weight. In addition, the fine emulsified particles containing the compound K are contained in an amount of 10 -10 to 99.99% by weight based on the total weight of the cosmetic and pharmaceutical composition, depending on the manufacturing method. In addition to the method of applying the technology to collect the fine emulsified particles in this way can be collected in the emulsified particles and capsules by a general emulsification method having a particle size of 500um to 1000um, or dissolved in an oil capable of dissolving compound K. .
본 발명의 미세한 유화입자는 그 입경이 500nm 내지 30nm 정도이고, 바람직하게는 300nm 내지 50nm 정도가 적당하다. 통상의 유화입자의 경우, 최소 500nm 이상의 크기를 갖지만, 본 발명의 미세 유화입자 및 리포좀은 피부와의 접촉면적이 상대적으로 증가함으로써 경피흡수 가능 면적도 증가하게 된다. 또한, 피부 각질층의 세포간 지질사이의 틈이 약 50nm 내외라는 점과 유화입자의 유화막이 유연성을 가진다는 점을 감안하여, 본 발명의 미세 유화입자가 세포간 지질 내로의 흡수, 확산이 용이하도록 하였다.The fine emulsified particles of the present invention have a particle diameter of about 500 nm to 30 nm, preferably about 300 nm to 50 nm. In the case of a conventional emulsified particles, the microemulsion particles and liposomes of the present invention have a size of at least 500 nm or more, and thus the percutaneous absorption available area also increases due to a relatively increased contact area with the skin. In addition, in consideration of the fact that the intercellular lipids in the stratum corneum layer is about 50 nm and the emulsification film of the emulsified particles is flexible, the fine emulsified particles of the present invention are easily absorbed and diffused into the intercellular lipids. It was.
즉, 나노유화기술에 의하여 제조된 평균입경 300nm 내지 50nm의 유화입자는 피부와의 접촉면적의 증가 및 세포간 지질로의 침투 및 확산이라는 두 가지 경로를 통해, 미세 유화입자 자체 또는 유화입자 내부의 화합물 K의 경피흡수율을 높여주게 된다.That is, emulsified particles having an average particle diameter of 300 nm to 50 nm prepared by nanoemulsification technology are obtained through the two paths of increasing the contact area with the skin and penetration and diffusion into intercellular lipids. It increases the percutaneous absorption of compound K.
한편, 본 발명에서 사용되는 유화제인 레시틴은 유화 입자 총 중량의 0.5∼5중량%, 바람직하게는 2∼3중량%가 함유되며, 레시틴의 구성성분은 포스파티딜콜린 (phosphatidylcholine), 라이조포스파티딜콜린(lysophosphatidylcholine), 포스파티딜에탄올아민(phosphatidylethanolamine) 등의 불포화 콜린계, 세린계, 에탄올아민계 화합물 및 이들의 수소첨가물 형태를 포함하고 있다.On the other hand, lecithin, which is an emulsifier used in the present invention, contains 0.5 to 5% by weight, preferably 2 to 3% by weight of the total weight of the emulsified particles, and the components of the lecithin include phosphatidylcholine, lysophosphatidylcholine, Unsaturated choline-based, serine-based, ethanolamine-based compounds such as phosphatidylethanolamine, and hydrogenated forms thereof.
또한, 본 발명에서 레시틴과 함께 사용되는 보조유화제로는 음이온계, 양이온계, 비이온계 또는 양성이온계 유화제가 가능하며, 사용되는 레시틴의 함량 및 구성성분에 따라 레시틴 함량 대비 0.5∼5배, 바람직하게는 1∼3배의 비율로 사용한다.In addition, the co-emulsifier used in conjunction with the lecithin in the present invention may be an anionic, cationic, nonionic or zwitterionic emulsifier, 0.5 to 5 times the lecithin content, depending on the content and composition of the lecithin used, Preferably it is used in the ratio of 1-3 times.
또 나노유화기술로서 고압유화방법(압력 500∼2,500bar) 및 용매추출방법을사용하여 화합물 K를 함유한 미세 유화 입자를 제조한다.In addition, fine emulsified particles containing compound K are prepared by using a high pressure emulsion method (pressure 500-2500 bar) and a solvent extraction method as nanoemulsification technology.
본 발명에 따른 화합물 K가 함유된 피부 노화방지용 화장료 및 의약료 조성물은 그 제형에 있어서 특별히 한정되는 바가 없다. 예를 들면, 유연화장수, 수렴화장수, 영양화장수, 영양크림, 마사지크림, 에센스, 아이크림, 아이에센스, 클렌징크림, 클렌징폼, 클렌징워터, 팩, 파우더, 보디로션, 보디크림, 보디오일 및 보디에센스, 메이컵 베이스, 파운데이션, 염모제, 샴푸, 린스, 보디 세정제, 치약, 구강청정액 등의 화장료 및 로션, 연고, 겔, 크림, 패취 또는 분무제 등의 의약료로 제형화될 수 있다.The skin anti-aging cosmetic and pharmaceutical composition containing the compound K according to the present invention is not particularly limited in its formulation. For example, supple cosmetics, astringent cosmetics, nourishing cosmetics, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil and body It can be formulated into cosmetics such as essences, makeup bases, foundations, hair dyes, shampoos, rinses, body cleansers, toothpastes, mouthwashes, and lotions, ointments, gels, creams, patches or sprays.
이하, 실시예 및 시험예를 들어 본 발명의 구성 및 효과를 보다 구체적으로 설명한다.Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to Examples and Test Examples.
[참고예 1] 인삼 정제 사포닌의 제조Reference Example 1 Preparation of Ginseng Purified Saponin
홍삼 2㎏에 물, 물을 포함한 에탄올 4ℓ를 넣고, 77℃에서 3회 환류 추출한 후, 15℃에서 6일간 침적시켰다. 그 후, 여과포 여과와 원심분리를 통해 잔사와 여액을 분리하고, 분리된 여액을 감압농축하여 얻은 엑기스를 물에 현탁한 후에, 에테르 1ℓ로 5회 추출하여 색소를 제거하고, 수층을 1-부탄올 500㎖로 3회 추출하였다. 이로부터 얻은 총 1-부탄올층을 5% KOH로 처리한 다음 증류수로 세척한 뒤, 감압농축하여 1-부탄올 엑기스를 얻고, 이를 소량의 메탄올에 녹인 다음, 대량의 에틸아세테이트에 추가하여, 생성된 침전물을 건조함으로써, 인삼 정제 사포닌 100g(수율: 5%)을 얻었으며, 동일조작을 10회 반복하여 총 1kg의 인삼 정제 사포닌을 준비하였다.4 g of ethanol including water and water was added to 2 kg of red ginseng, and the mixture was extracted under reflux three times at 77 ° C. and then deposited at 15 ° C. for 6 days. Thereafter, the residue and the filtrate were separated through filter cloth filtration and centrifugation. The extract obtained by concentrating the separated filtrate under reduced pressure was suspended in water, and then extracted five times with 1 L of ether to remove the pigment, and the aqueous layer was 1-butanol. Extracted three times with 500 ml. The total 1-butanol layer obtained therefrom was treated with 5% KOH, washed with distilled water, and then concentrated under reduced pressure to obtain 1-butanol extract, which was dissolved in a small amount of methanol and added to a large amount of ethyl acetate. By drying the precipitate, 100 g (yield: 5%) of ginseng purified saponin was obtained, and a total of 1 kg of ginseng purified saponin was prepared by repeating the same operation 10 times.
[참고예 2] 산 가수분해 방법에 의한 화합물 K의 제조Reference Example 2 Preparation of Compound K by Acid Hydrolysis Method
참고예 1에서 얻은 인삼 정제 사포닌 100g에 20배(v/w)의 7% 황산/50% 에탄올 용액(v/w)을 가하여, 100℃ 수욕조에서 6시간 동안 가열 환류시켜, 인삼 사포닌에 결합된 당결합을 가수분해시켰다. 반응액을 감압농축하여 용매를 제거하고, 잔사에 증류수(1,000㎖)를 가해 현탁시킨 후, 동량의 에테르로 3회 추출하였다. 총 에테르층을 증류수로 세척한 뒤, 무수황산마그네슘(MgSO4)으로 탈수, 여과, 농축하여 조생성물을 얻었다. 얻은 조생성물을 실리카겔 칼럼 크로마토그래피(클로로포름:메탄올=9:1 --> 4:1로 점차 극성을 증가시켜주면서 분리)로 분리하였으며, 각 분획에 대하여 박막크로마토그라피(클로로포름/메탄올/물 = 65/35/10, Rf = 0.73)를 사용하여 화합물 K의 분획을 분리, 회수하여 최종적으로 화합물 K 800mg (수율 0.8%)을 얻었다.20 g (v / w) of 7% sulfuric acid / 50% ethanol solution (v / w) was added to 100 g of purified ginseng saponin obtained in Reference Example 1, and the mixture was heated and refluxed in a 100 ° C. water bath for 6 hours to bind to ginseng saponin. Sugar bonds were hydrolyzed. The reaction solution was concentrated under reduced pressure, the solvent was removed, distilled water (1,000 ml) was added to the residue and suspended, and then extracted three times with the same amount of ether. The total ether layer was washed with distilled water and then dehydrated with anhydrous magnesium sulfate (MgSO 4 ), filtered and concentrated to obtain a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 9: 1-> 4: 1, gradually increasing polarity), and thin layer chromatography (chloroform / methanol / water = 65) for each fraction was performed. / 35/10, Rf = 0.73) was used to separate and recover the fraction of Compound K to obtain 800 mg (Yield 0.8%) of Compound K.
[참고예 3] 염기 가수분해 방법에 의한 화합물 K의 제조Reference Example 3 Preparation of Compound K by Base Hydrolysis Method
참고예 1에서 얻은 인삼 정제 사포닌 100g을 건조피리딘(5000㎖)에 녹이고, 여기에 소듐 메톡사이드(분말, 100g)를 가해 유욕상에서 8시간 동안 환류 반응시킴으로써, 사포닌의 당결합을 가수분해시켰다. 반응액을 감압농축하여 용매를 제거하고, 잔사에 증류수(1,000㎖)를 가해 현탁시킨 후, 동량의 에테르로 3회 추출하였다. 총 에테르층을 증류수로 세척한 뒤, 무수황산마그네슘(MgSO4)으로 탈수, 여과, 농축하여 조생성물을 얻었다. 얻은 조생성물을 실리카겔 칼럼 크로마토그래피(클로로포름:메탄올=9:1 --> 4:1로 점차 극성을 증가시켜주면서 분리)로 분리하였으며,각 분획에 대하여 박막크로마토그라피(클로로포름/메탄올/물 = 65/35/10, Rf = 0.73)를 사용하여 화합물 K의 분획을 분리, 회수하여 최종적으로 화합물 K 750mg (수율 0.75%)을 얻었다.100 g of ginseng purified saponin obtained in Reference Example 1 was dissolved in dry pyridine (5000 ml), and sodium methoxide (powder, 100 g) was added thereto to reflux for 8 hours on an oil bath to hydrolyze the sugar bond of saponin. The reaction solution was concentrated under reduced pressure, the solvent was removed, distilled water (1,000 ml) was added to the residue and suspended, and then extracted three times with the same amount of ether. The total ether layer was washed with distilled water and then dehydrated with anhydrous magnesium sulfate (MgSO 4 ), filtered and concentrated to obtain a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 9: 1-> 4: 1, gradually increasing polarity), and thin layer chromatography (chloroform / methanol / water = 65) for each fraction was performed. / 35/10, Rf = 0.73) was used to separate and recover the fraction of Compound K to give 750 mg (Yield 0.75%) of Compound K.
[참고예 4-1] 효소분해방법을 통한 화합물 K의 제조[Reference Example 4-1] Preparation of Compound K through Enzymatic Decomposition
참고예 1에서 얻은 인삼 정제 사포닌 100g을 1000㎖의 시트레이트 완충용액(pH 5.5)에 용해시키고, 여기에 페니실리움 속에서 분리한 나린지나제 효소 1g을 첨가하여 40℃ 수욕상에서 48시간 동안 교반시키면서 반응시켰다. 박층크로마토그래피에 의해 주기적으로 확인하여, 기질이 완전히 소실되면 열수중에서 10분간 가열하여 반응을 종료시킨 다음, 반응액은 동량의 에테르로 3회 추출, 농축하였다. 얻은 생성물을 실리카겔 칼럼 크로마토그래피(클로로포름:메탄올=9:1 --> 4:1로 점차 극성을 증가시켜주면서 분리)로 분리하였으며, 각 분획에 대하여 박막크로마토그라피(클로로포름/메탄올/물 = 65/35/10, Rf = 0.73)를 사용하여 화합물 K의 분획을 분리, 회수하여 최종적으로 화합물 K 4400mg (수율 4.4%)을 얻었다.100 g of ginseng purified saponin obtained in Reference Example 1 was dissolved in 1000 ml of citrate buffer (pH 5.5), and 1 g of naringinase enzyme isolated in penicillium was added and stirred for 48 hours in a 40 ° C water bath. The reaction was carried out. After periodic confirmation by thin layer chromatography, when the substrate was completely lost, the reaction was terminated by heating in hot water for 10 minutes, and then the reaction solution was extracted and concentrated three times with the same amount of ether. The obtained product was separated by silica gel column chromatography (chloroform: methanol = 9: 1-> 4: 1, gradually increasing polarity), and thin layer chromatography (chloroform / methanol / water = 65 / 35/10, Rf = 0.73) was used to separate and recover a fraction of Compound K to obtain 4400 mg (Yield 4.4%) of Compound K.
[참고예 4-2]Reference Example 4-2
인삼 정제 사포닌 100g을 15% 에탄올을 함유한 시트레이트 완충용액(pH 4.0) 1000㎖에 용해시키고, 여기에 페니실리움속에서 분리한 나린지나제 효소 0.5g을 첨가하여 40℃ 수욕상에서 48시간 동안 교반시키면서 반응시켰다. 박층크로마토그래피에 의해 주기적으로 확인하여, 기질이 완전히 소실되면 열수중에서 10분간 가열하여 반응을 종료시킨 다음, 반응액은 동량의 에틸아세테이트로 3회 추출, 농축하였다. 얻은 생성물을 실리카겔 칼럼 크로마토그래피(클로로포름:메탄올=9:1 -->4:1로 점차 극성을 증가시켜주면서 분리)로 분리하였으며, 각 분획에 대하여 박막크로마토그라피(클로로포름/메탄올/물 = 65/35/10, Rf = 0.73)를 사용하여 화합물 K의 분획을 분리, 회수하여 최종적으로 화합물 K 1500mg (수율1.5%)얻었다.100 g of ginseng purified saponin was dissolved in 1000 ml of citrate buffer solution (pH 4.0) containing 15% ethanol, and 0.5 g of naringinase enzyme isolated in penicillium was added thereto for 48 hours in a 40 ° C water bath. The reaction was carried out while stirring. After periodic confirmation by thin layer chromatography, when the substrate was completely lost, the reaction was terminated by heating in hot water for 10 minutes, and then the reaction solution was extracted and concentrated three times with the same amount of ethyl acetate. The obtained product was separated by silica gel column chromatography (chloroform: methanol = 9: 1-> 4: 1, gradually increasing polarity), and thin layer chromatography (chloroform / methanol / water = 65 / 35/10, Rf = 0.73) was used to separate and recover the fraction of Compound K to finally obtain 1500 mg of Compound K (yield 1.5%).
[참고예 4-3]Reference Example 4-3
인삼 정제 사포닌 100g을 15% 에탄올을 함유한 시트레이트 완충용액(pH 4.0) 1000㎖에 용해시키고, 여기에 아스퍼질러스 속에서 분리한 펙티나제 효소 2g을 첨가하여 30℃ 수욕상에서 48시간 동안 교반시키면서 반응시켰다. 박층크로마토그래피에 의해 주기적으로 확인하여, 기질이 완전히 소실되면 열수중에서 10분간 가열하여 반응을 종료시킨 다음, 반응액은 동량의 에틸아세테이트로 3회 추출, 농축하였다. 얻은 생성물을 실리카겔 칼럼 크로마토그래피((클로로포름:메탄올=9:1 --> 4:1로 점차 극성을 증가시켜주면서 분리)로 분리하였으며, 각 분획에 대하여 박막크로마토그라피(클로로포름/메탄올/물 = 65/35/10, Rf = 0.73)를 사용하여 화합물 K의 분획을 분리, 회수하여 최종적으로 화합물 K 800mg(수율 0.8%)을 얻었다.100 g of ginseng purified saponin was dissolved in 1000 ml of citrate buffer solution (pH 4.0) containing 15% ethanol, and 2 g of pectinase enzyme isolated from Aspergillus was added thereto and stirred for 48 hours in a 30 ° C water bath. The reaction was carried out. After periodic confirmation by thin layer chromatography, when the substrate was completely lost, the reaction was terminated by heating in hot water for 10 minutes, and then the reaction solution was extracted and concentrated three times with the same amount of ethyl acetate. The obtained product was separated by silica gel column chromatography ((chloroform: methanol = 9: 1-> 4: 1 with increasing polarity) and separated by thin layer chromatography (chloroform / methanol / water = 65). / 35/10, Rf = 0.73) was used to separate and recover the fraction of Compound K to obtain 800 mg (0.8% yield) of Compound K.
[참고예 4-4]Reference Example 4-4
인삼 정제 사포닌 100g을 1000㎖의 시트레이트 완충용액(pH 5.5)에 용해시키고, 여기에 아스퍼질러스 속에서 분리한 펙티나제 효소 2g을 첨가하여 30℃ 수욕상에서 48시간 동안 교반시키면서 반응시켰다. 박층크로마토그래피에 의해 주기적으로 확인하여, 기질이 완전히 소실되면 열수중에서 10분간 가열하여 반응을 종료시킨 다음, 반응액은 동량의 에테르로 3회 추출, 농축하였다. 얻은 생성물을 실리카겔 칼럼 크로마토그래피(클로로포름:메탄올=9:1 --> 4:1로 점차 극성을 증가시켜주면서 분리)로 분리하였으며, 각 분획에 대하여 박막크로마토그라피(클로로포름/메탄올/물 = 65/35/10, Rf = 0.73)를 사용하여 화합물 K의 분획을 분리, 회수하여 최종적으로 화합물 K 1800mg(수율 1.8%)을 얻었다.100 g of ginseng purified saponin was dissolved in 1000 ml of citrate buffer (pH 5.5), and 2 g of pectinase enzyme isolated in Aspergillus was added thereto and reacted with stirring for 30 hours in a 30 ° C water bath. After periodic confirmation by thin layer chromatography, when the substrate was completely lost, the reaction was terminated by heating in hot water for 10 minutes, and then the reaction solution was extracted and concentrated three times with the same amount of ether. The obtained product was separated by silica gel column chromatography (chloroform: methanol = 9: 1-> 4: 1, gradually increasing polarity), and thin layer chromatography (chloroform / methanol / water = 65 / 35/10, Rf = 0.73) was used to separate and recover a fraction of Compound K to obtain 1800 mg (Yield 1.8%) of Compound K.
상기 참고예로부터 얻은 화합물 K를 함유한 나노 유화 입자를 실시예 1 내지 실시예 3과 같이 제조하였다.Nano-emulsified particles containing Compound K obtained from the above Reference Example were prepared as in Examples 1 to 3.
[실시예 1]Example 1
레시틴, 수첨레시틴, 콜레스테롤, 콩기름 및 프로필렌글리콜에 용해시킨 화합물 K를 70∼75℃까지 가열하여 완전히 용해한 다음, 미리 가열된 수상파트(증류수, EDTA)와 혼합하여 일반 호모믹서로 선-유화(pre-emulsion)를 시키고(3분간, 3,000~6,000rpm) 고압유화기(Microfluidizer)를 사용하여 1,000Bar/3cycles로 시행하였다. 상기 성분에서, 수첨레시틴의 경우 에멀젼의 안정도는 우수하지만 피부흡수적인 측면에서 불포화 레시틴에 비해 피부친화도가 떨어지므로, 두 가지의 레시틴을 혼합하여 사용하였다.Compound K dissolved in lecithin, hydrogenated lecithin, cholesterol, soybean oil, and propylene glycol was dissolved completely by heating to 70-75 ° C, then mixed with pre-heated water part (distilled water, EDTA) and pre-emulsified by general homomixer (pre -emulsion) (3 min, 3,000 ~ 6,000rpm) and 1,000Bar / 3cycles using Microfluidizer. In the above components, hydrogenated lecithin has excellent emulsion stability, but in terms of skin absorption, the skin affinity is lower than that of unsaturated lecithin, and thus, two lecithins were mixed and used.
[실시예 2]Example 2
레시틴, PEG-5 레이프씨드(rapeseed) 스테롤, 카프릭/카프릴릭 트리글리세리드, BHT, a-토코페롤 및 펜틸렌글리콜과 에탄올에 용해시킨 화합물 K를 70∼75℃까지 가열하여 완전히 용해한 다음, 미리 가열된 수상파트(증류수, EDTA)와 혼합하여 일반 호모믹서로 선-유화를 시키고(3분간, 3,000∼6,000rpm), 고압유화기를 사용하여 1,000Bar/3cycles 로 시행하였다. 여기에서, 불포화 레시틴의 화학적 불안정성을 보완하기 위한 항산화제로써 BHT를 첨가하였으며, 유화 안정도를 증가시키기 위한 보조 유화제로써 PEG-5 레이프씨드(rapeseed) 스테롤을 첨가하였다.Compound K dissolved in lecithin, PEG-5 rapeseed sterols, capric / caprylic triglycerides, BHT, a-tocopherol and pentylene glycol and ethanol was heated to 70-75 ° C. to completely dissolve and then pre-heated The pre-emulsification was carried out with a general homomixer (3,000 min-6,000 rpm) by mixing with a water phase (distilled water, EDTA), and 1,000 bar / 3 cycles were performed using a high pressure emulsifier. Here, BHT was added as an antioxidant to compensate for the chemical instability of unsaturated lecithin, and PEG-5 rapeseed sterol was added as an auxiliary emulsifier to increase the emulsion stability.
[실시예 3]Example 3
수첨레시틴, 수첨 라이조포스파티딜콜린(HLPC) 및 프로필렌글리콜과 에탄올에 용해시킨 화합물 K를 70∼75℃까지 가열하여 완전히 용해한 다음, 미리 가열된 수상파트(증류수, EDTA, 글리세린, 베타인)와 혼합하여 일반 호모믹서로 유화시키고(3분간, 3,000∼6,000rpm) 실온으로 냉각하였다. 참고로, 상기 성분 중, 수첨 라이조포스파티딜콜린은 수첨레시틴을 구성하고 있는 성분 중 수첨 포스파티딜콜린(HPC)이 가수분해되면서 생기는 성분이며, HPC보다 유화력이 우수하다.Hydrogenated lecithin, hydrogenated lysophosphatidylcholine (HLPC) and compound K dissolved in propylene glycol and ethanol were dissolved completely by heating to 70-75 ° C, then mixed with pre-heated water-parts (distilled water, EDTA, glycerin, betaine) It was emulsified by a homomixer (3,000-6,000 rpm for 3 minutes) and cooled to room temperature. For reference, among the components, hydrogenated lysophosphatidylcholine is a component produced by hydrolysis of hydrogenated phosphatidylcholine (HPC) among the components constituting hydrogenated lecithin, and has better emulsifying power than HPC.
상기의 실시예 1 내지 실시예 3에서 제조한 화합물 K를 함유한 미세 유화입자 및 리포좀과 일반유화물을 제조하고, 각각의 경피흡수 차이를 비교하기 위하여 인삼 정제 사포닌을 함유한 비교예 1 내지 비교예 3을 제조하였으며, 그 내용을 정리하면 다음의 표 1과 같다. 하기의 표1에서 실시예 1과 실시예 2 및 비교예 1과 비교예 2는 미세유화입자를 제조하기 위하여 고압유화방식을 사용하였으며, 실시예 3 및 비교예 3은 일반 호모믹서를 사용하여 일반적인 유화입자가 형성되도록 하였다.Comparative Example 1 to Comparative Example containing ginseng purified saponin in order to prepare the fine emulsion particles and liposomes and the general emulsion containing the compound K prepared in Examples 1 to 3, and compare the transdermal absorption difference 3 was prepared, and the contents thereof are summarized in Table 1 below. In Table 1 below, Example 1, Example 2, and Comparative Example 1 and Comparative Example 2 used a high-pressure emulsification method to prepare microemulsified particles, and Example 3 and Comparative Example 3 were prepared using a general homo mixer. Emulsified particles were allowed to form.
이하, 화합물 K를 함유하는 미세 유화입자 및 리포좀 혹은 일반 유화입자를 제형화한 피부 외용제 조성물을 설명하면 다음과 같다.Hereinafter, the external preparation composition for formulating the fine emulsion particles and liposomes or general emulsion particles containing the compound K will be described.
[처방예 1] 크림제형[Prescription Example 1] Cream Preparation
[처방예 2] 영양화장수[Prescription 2] Nutritional Cosmetics
[처방예 3] 유연화장수[Prescription 3] Flexible Cosmetics
[처방예 4] 겔[Prescription Example 4] Gel
[처방예 5] 연고[Prescription Example 5] Ointment
[처방예 6] 분무제[Prescription Example 6] Spray
상기 처방을 펌프용기에 담아 사용한다.The prescription is used in a pump container.
[처방예 7] 패치[Example 7] Patch
[시험예 1] 경피흡수량 측정 시험Test Example 1 Percutaneous Absorption Measurement Test
피부 흡수는 기네아피그 피부를 대상으로 프란츠 투과셀을 이용하여 측정하였다. 시험 직전, 기네아피그의 복부 부분 피부를 채취하여, 평방 1㎠의 면적으로 절단한 후, 이를 투과경의 직경이 0.9㎝인 투과셀에 장치하고, 클램프로 고정하였다. 피부의 한쪽면(donor 용기)은 상기한 실시예 1∼3 및 비교예 1∼3를 0.05㎖ 취하여 0.5㎖가 되도록 증류수로 희석하고, 제형예 1∼15 및 비교제형예 1∼15의 경우에는 0.5㎖를 넣어주었다. 반대쪽면(receptor 용기)은 정제수와 에탄올이 4:1 중량비로 혼합된 용매와 접촉하도록 하였으며, 시험시 온도는 실제 피부 온도인 32℃를 유지하였다. 시험 시작 후, 일정 시간 간격으로 용매의 일부를 채취한 후, HPLC를 이용하여 피부에 흡수된 화합물 K의 양을 측정하여, 도포 농도당피부흡수량(㎍/㎠/중량%)으로 나타내었으며, 그 결과를 하기 표 2에 나타내었다. 인삼 정제사포닌에 대해서는 경피흡수된 사포닌의 함량을 정량하였으며, 화합물 K의 경우에는 경피흡수된 화합물 K의 함량을 정량 분석하여 총 경피흡수량을 계산하였다.Skin absorption was measured by using Franz permeation cells on guinea pig skin. Immediately before the test, abdominal skin of the guinea pigs was taken out, cut into square 1 cm 2 areas, and then mounted in a transmission cell having a diameter of 0.9 cm in the diameter of the penis and fixed with a clamp. One side of the skin (donor container) is diluted with distilled water to 0.05 ml of Examples 1 to 3 and Comparative Examples 1 to 3 described above, and in the case of Formulation Examples 1 to 15 and Comparative Formulation Examples 1 to 15, 0.5 ml was added. The other side (receptor vessel) was to contact the solvent mixed with purified water and ethanol in a 4: 1 weight ratio, the temperature was maintained at 32 ℃ the actual skin temperature during the test. After the start of the test, a portion of the solvent was collected at regular time intervals, and then the amount of Compound K absorbed into the skin was measured using HPLC, and expressed as the skin absorption amount (µg / cm 2 / wt%) per application concentration. The results are shown in Table 2 below. For ginseng purified saponin, the content of transdermal absorbed saponin was quantified, and in the case of compound K, total transdermal absorption was calculated by quantitative analysis of the content of transdermal absorbed compound K.
<HPLC 분석조건><HPLC Analysis Conditions>
- Column : C18(ODS)-Column: C18 (ODS)
- Solvent Flow : 1㎖/minSolvent Flow: 1ml / min
- Detection UV : 203㎚-Detection UV: 203nm
- Sample test concentration : 5㎎/㎖Sample test concentration: 5mg / ml
- Sample injection amount : 10㎍-Sample injection amount: 10㎍
- Eluent : Gradient conditionEluent: Gradient condition
- A : Acetonitrile/D.I. water=15/85A: Acetonitrile / D.I. water = 15/85
- B : Acetonitrile/D.I. water=80/20B: Acetonitrile / D.I. water = 80/20
<용매 구배 조건><Solvent Gradient Conditions>
상기의 시험결과로부터 나노유화기술을 적용하여 제조한 유화입자의 경우인 실시예 1∼2가, 일반유화 입자를 형성하는 실시예 3의 경우보다 우수한 경피흡수율을 보이고 있음을 확인할 수 있었으며, 사포닌을 나노유화기술을 사용하여 제조한 미세 유화 입자인 비교예 1 내지 비교예 2에 비해서 훨씬 높은 경피흡수율을 보임을 확인할 수 있었다. 특히, 화합물 K에 대해서 나노유화기술을 적용한 경우(실시예 1∼2)에는 극적인 경피흡수율의 증가가 있음을 확인할 수 있었다.From the above test results, it can be seen that Examples 1 and 2, which are the case of emulsified particles prepared by applying the nanoemulsification technique, showed better transdermal absorption than those of Example 3 forming general emulsified particles. Compared to Comparative Examples 1 to 2, which are microemulsified particles prepared using nanoemulsification technology, it was confirmed that the percutaneous absorption rate was much higher. In particular, when the nanoemulsification technology is applied to the compound K (Examples 1 to 2), it was confirmed that there is a dramatic increase in transdermal absorption.
또한, 각각의 대응하는 실시예 및 비교예를 통해서 볼 때, 화합물 K는 인삼사포닌보다 경피흡수율이 높음을 알 수 있으며, 이는 화합물 K의 화학적인 구조의 특징으로부터 유래한 것으로 볼 수 있다.In addition, through the corresponding examples and comparative examples, it can be seen that compound K has a higher percutaneous absorption rate than ginseng saponin, which may be derived from the characteristics of the chemical structure of compound K.
즉, 화합물 K는 인삼 정제 사포닌에 비해서 훨씬 높은 경피흡수율을 보이며, 특히, 피부친화성이 우수한 레시틴과 나노유화기술을 적용함으로써 월등한 경피흡수율을 나타냄을 확인할 수 있었다.In other words, Compound K showed a much higher transdermal absorption rate than ginseng purified saponin, and in particular, it was confirmed that excellent transdermal absorption rate was obtained by applying lecithin and nanoemulsification technology with excellent skin affinity.
또한, 이는 실시예 및 비교예를 제형화한 제형예 1∼15 및 비교제형예 1∼15를 통해서도 확인할 수 있으며, 나노유화기술 및 피부친화성이 우수한 레시틴의 사용으로 인한 화합물 K의 경피흡수율이 제형내에서도 그대로 재현, 적용되고 있음을 확인할 수 있었다.In addition, this can be confirmed through Formulation Examples 1 to 15 and Comparative Formulation Examples 1 to 15, which formulate examples and comparative examples, and the transdermal absorption rate of Compound K due to the use of lecithin excellent in nanoemulsification technology and skin affinity is increased. It was confirmed that the formulation is reproduced and applied as it is.
일반적으로 화장의 지속 시간이 4∼8시간임을 감안하면, 인삼 정제 사포닌을 함유한 실시예 및 제형예에 비해서 화합물 K를 함유한 실시예 및 제형예에서 경피흡수량이 월등히 우수함을 알 수 있다.Considering that the makeup duration is generally 4 to 8 hours, it can be seen that the percutaneous absorption in the Examples and Formulations containing Compound K is superior to the Examples and Formulations containing ginseng purified saponin.
[시험예 2] 섬유아세포(Fibroblast)의 증식효능 측정Test Example 2 Measurement of Proliferative Effect of Fibroblasts
3.5%의 우태아 혈청이 함유된 DMEM(Doubecco's Modified Eagle's Media)배지에서 배양한 인체 섬유아세포를 96공 평판배양기(96-well microtiter plate)에 5,000세포/well이 되도록 분주하고, 시료로서 실시예 1의 화합물 K, 비교예 1의 인삼 정제 사포닌을 각각 1%의 양으로 사용하고, 제형예 1 및 비교제형예 1을 각각 10%의 양으로 사용하여 배양배지로 1/10씩 순차적으로 희석하여 첨가한 후, 37℃ 온도에서 4일간 배양하였다. 배양 후 0.2% MTT(3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) 용액을 각 웰당 50㎕씩 첨가하고, 다시 37℃ 온도에서 4시간 동안 배양한 후 생성된 포르마잔(formazane)을 DMSO(Dimethyl sulfoxide)로 용해시켰다. 용해된 포르마잔의 흡광도를 평판배양측정기(microplate reader)를 이용하여 570nm에서 측정하였다. 이를 인삼 정제 사포닌과 바이오진을 처리하지 않은 대조군에 대하여 상기와 동일한 방법으로 실시하여 흡광도를 측정하였다. 인삼 정제 사포닌과 화합물 K를 함유한 시험군과 이를 함유하지 않은 대조군의 흡광도를 각각 비교한 후, 그 결과를 표 3에 나타내었다.Human fibroblasts cultured in DMEM (Doubecco's Modified Eagle's Media) medium containing 3.5% fetal calf serum were dispensed at a 96-well microtiter plate at 5,000 cells / well, and Example 1 was used as a sample. Of compound K, ginseng purified saponin of Comparative Example 1, respectively, in an amount of 1%, and using Formulation Example 1 and Comparative Formulation Example 1 in amounts of 10%, respectively, by diluting sequentially by 1/10 with a culture medium. After that, the cells were incubated at 37 ° C. for 4 days. After incubation, 50 μl of 0.2% MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) solution was added to each well, followed by incubation at 37 ° C. for 4 hours. Formazan was dissolved in dimethyl sulfoxide (DMSO). The absorbance of the dissolved formazan was measured at 570 nm using a microplate reader. This was carried out in the same manner as above for the control group not treated with ginseng purified saponin and biozine to measure the absorbance. After comparing the absorbance of the test group containing ginseng purified saponin and Compound K and the control group not containing them, the results are shown in Table 3.
표 3로부터, 인삼 정제 사포닌을 처리한 섬유아세포에 비하여, 화합물 K를 처리한 섬유아세포의 증식 효능이 훨씬 높음을 알 수 있었다. 또한, 각각의 실시예를 제형화한 제형예 1 및 비교 제형예 1의 경우에도 제형예 1이 비교 제형예 1보다 우수한 섬유아세포 증식효능이 있음을 확인할 수 있었다.Table 3 shows that the fibroblasts treated with Compound K had a much higher proliferative effect than the fibroblasts treated with ginseng purified saponin. In addition, in the case of Formulation Example 1 and Comparative Formulation Example 1 formulated in each example, it was confirmed that Formulation Example 1 has a superior fibroblast proliferation effect than Comparative Formulation Example 1.
[시험예 3] 각질형성세포(Keratinocyte)의 증식효능 측정Test Example 3 Measurement of proliferation effect of keratinocytes
각질형성세포를 사용하여 시험예 2에서와 동일한 방법으로 실시예 2와 비교예 2 및 제형예 2와 비교제형예 2에 대한 각질형성세포의 증식효능을 측정하여, 그 결과를 표 4 나타내었다.Using the keratinocytes to measure the proliferation effect of the keratinocytes in Example 2, Comparative Example 2 and Formulation Example 2 and Comparative Formulation Example 2 in the same manner as in Test Example 2, the results are shown in Table 4.
상기 표 4 서 알 수 있는 바와 같이, 인삼 정제 사포닌을 처리한 각질형성세포에 비하여, 화합물 K를 처리한 각질형성세포는 약 2배정도 향상된 증식 효능을 나타냄을 확인할 수 있었다. 이러한 결과는 나노유화기술과 피부친화성 레시틴을 사용하여 제조한 실시예 및 이를 함유한 제형에서 동일한 경향을 나타냄을 확인할 수 있었다.As can be seen in Table 4, compared to the keratinocytes treated with ginseng purified saponin, keratinocytes treated with Compound K showed about two times improved proliferative efficacy. These results were confirmed to show the same tendency in the formulation prepared using the nanoemulsification technology and skin-friendly lecithin and the formulation containing the same.
[시험예 4] 시험관내(in vitro) 콜라겐 생합성 효능 측정Test Example 4 In vitro Collagen Biosynthesis Efficacy Measurement
인체 섬유아세포를 24공 평판배양기에 배양한 후, 시험예 2와 동일한 방법을 사용하여 실시예 3, 비교예 3 대해서 배양배지로 1/100씩 순차적으로 희석하여 첨가하고, 제형예 3, 비교제형예 3 대해서 배양배지로 1/10씩 순차적으로 희석하여 첨가하였다. 배양 3일째 10%의 우태아 혈청이 함유된 DMEM배지를 각 웰당 0.5㎖씩 첨가한 후 L[2, 3, 4, 5-3H]-프롤린 10㎍ Ci를 첨가하였다. 24시간 경과 후, 각 웰에 들어있는 배지와 세포들을 긁어모아 5% 트리클로로아세틱엑시드(TCA; Trichloroacetic acid) 용액에 넣어 수세한 후, 2개의 시험관에 분주하고, 1개의 시험관에는 타입 I 콜라게나제(type I collagenase) 1unit/㎕를 넣고 37℃ 온도에서 90분간 배양하였으며, 다른 시험관은 4℃에서 보관하였다. 그 후, 모든 시험관에 50% TCA를 0.05㎖씩 첨가하고 4℃에서 20분간 방치한 다음, 각각 12,000rpm에서 10분간 원심분리하여, 각각의 상등액과 침전물을 액체 신틸레이션 계수기(LSC; Liquid Scintillation Counter)로 디피엠(DPM; decay per minute) 값을 얻어, 하기 수학식 1에 의거하여, 대조군과 시험군에 대해 콜라겐 생합성 값(RCB; Relative Collagen Biosynthesis)을 구하고, 그 결과를 하기 표 5 나타내었다.After culturing human fibroblasts in a 24-hole flat plate incubator, the mixture was diluted sequentially by 1/100 of the culture medium for Example 3 and Comparative Example 3, using the same method as Test Example 2, and then Formulation Example 3, Comparative Formulation. For Example 3, it was added by diluting sequentially in culture medium by 1/10. On the third day of culture, 0.5 ml of DMEM medium containing 10% fetal calf serum was added to each well, followed by 10 [mu] g of L [2, 3, 4, 5-3H] -proline. After 24 hours, the medium and cells in each well were scraped, washed in 5% Trichloroacetic acid (TCA) solution, and dispensed into two test tubes. 1 unit / μl of genase (type I collagenase) was added and incubated at 37 ° C. for 90 minutes, and other test tubes were stored at 4 ° C. Thereafter, 0.05 ml of 50% TCA was added to all test tubes and allowed to stand at 4 ° C. for 20 minutes, followed by centrifugation at 12,000 rpm for 10 minutes, respectively. To obtain a decay per minute (DPM) value, the collagen biosynthesis value (RCB; Relative Collagen Biosynthesis) for the control group and the test group was obtained based on Equation 1 below, and the results are shown in Table 5 below.
상기 표 5 결과로부터, 인삼 정제 사포닌을 처리한 섬유아세포에 비하여, 화합물 K를 처리한 섬유아세포는 약 2배에서3배정도의 콜라겐 생합성 촉진효능을 나타냄을 알 수 있었으며, 이러한 결과는 나노유화기술을 사용하지 않은 경우에도 화합물 K와 피부친화성 레시틴을 사용하여 제조한 실시예 및 이를 함유한 제형에서 동일한 경향을 나타냄을 확인할 수 있었다.From the results of Table 5, fibroblasts treated with Compound K showed collagen biosynthesis promoting activity about 2 to 3 times as compared to fibroblasts treated with ginseng purified saponin, and these results showed nanoemulsification technology. Even when not used, it was confirmed that the same trends were observed in the examples prepared using Compound K and skin-friendly lecithin and formulations containing the same.
[시험예 5] 생체내(in vivo)에서의 콜라겐 생합성 효능 측정[Test Example 5] Determination of collagen biosynthesis efficacy in vivo
탈모생쥐(Hairless mouse; 42주, female)에 화합물 K (부형제; EtOH:PG=7:3)를 도포, 3일간 첩포하고, 24시간 휴식기를 둔 후, 3일간 반복 첩포 하였다. 그 후 탈모생쥐의 피부를 생체검사(biopsy)하여 조직염색을 실시하였다. 조직염색은 타입 I pN 프로콜라겐(procollagen), MMP-1(MMP-1(Matrix Metalloproteinase-1)에 대한 면역염색 및 헤마톡실린 앤 에오신염색(Haematoxylin and Eosin Staining)을 통해 프로콜라겐, MMP-1의 발현량 정성(정량) 및 피부(표피)두께의 변화를 관찰하여, 그 결과를 도 1 및 도 2에 나타내었다.Compound K (excipient; EtOH: PG = 7: 3) was applied to hairless mice (42 weeks, female), patched for 3 days, allowed to rest for 24 hours, and then repeatedly patched for 3 days. After that, the skin of the bald mice was biopsyed and tissue stained. Tissue staining was performed using type I pN procollagen, MMP-1 (Immunostaining against Matrix Metalloproteinase-1) and Haematoxylin and Eosin Staining. The changes in the expression quantity qualitative (quantitative) and skin (epidermal) thickness were observed, and the results are shown in FIGS. 1 and 2.
도 1 및 도 2를 통해, 비교예 1보다는 실시예 1에서 콜라겐 생합성이 촉진되었음을 확인할 수 있었으며, 이는 피부친화성 레시틴 및 나노기술을 이용하여 화합물 K를 피부 내로 효과적으로 전달할 수 있음을 보여주는 결과이다.1 and 2, it was confirmed that the collagen biosynthesis was promoted in Example 1 rather than Comparative Example 1, which shows that the compound K can be effectively delivered into the skin using skin-friendly lecithin and nanotechnology.
상기 결과로 인삼 정제 사포닌은 피부에 효과적인 흡수가 되지 않으며, 결과적으로 실시예 및 제형예에서 콜라겐 생합성 효과가 미미한 것을 확인할 수 있었다.As a result, the ginseng tablet saponin is not effectively absorbed by the skin, and as a result, it was confirmed that the collagen biosynthesis effect is minimal in Examples and Formulation Examples.
이는 인삼 정제 사포닌이 구조적으로 피부 흡수가 어려우며, 이는 피부친화성 레시틴의 사용이나 나노유화기술을 이용한 제형화에 의해서도 쉽게 극복되지 않음을 보여주는 결과이다.This is a result showing that ginseng purified saponin is structurally difficult to absorb the skin, which is not easily overcome by the use of skin-friendly lecithin or formulation using nanoemulsification technology.
즉, 상기의 시험 결과로부터, 경피흡수가 용이한 구조를 가지는 화합물 K의 경피흡수에 있어 피부친화성 레시틴 및 나노유화기술을 사용함으로써, 극대화된 경피흡수가 가능하며, 결과적으로 효과적인 콜라겐 생합성이 이루어질 수 있음을 확인할 수 있었다.That is, from the above test results, by using the skin-friendly lecithin and nanoemulsification technology in the transdermal absorption of the compound K having an easy transdermal absorption structure, it is possible to maximize the transdermal absorption, resulting in effective collagen biosynthesis. Could confirm.
[시험예 6] 인체 피부를 대상으로 한 피부 주름개선 효과Test Example 6 Skin Wrinkle Improvement Effects on Human Skin
35∼45세의 안면주름이 있는 시험대상자 30명 대하여, 상기 제형예 1 및 비교제형예 1의 크림제형을 주고, 피부주름 개선효과를 비교평가하게 하였다. 피검자의 안면 좌부에는 제형예 1의 크림을, 우부에는 비교제형예 1의 크림을 3개월간 사용하게 하였으며, 크림 사용 이전에 안면 양쪽부의 피부 상태를 측정해 놓고, 3개월 후 동일부위를 재측정하는 방법으로 피부주름의 변화를 측정하였다. 피부측정은 온도 24℃, 상대습도 40%의 항온실습실에서 하였으며, 눈꼬리 부위의 주름을 레플리카(replica)로 떠서 비시오메타 시스템(Visiometer system; C+K사)으로 피부주름을 측정하였다. 피부주름의 변화량은 하기 수학식 2에 따라 계산하였다.Thirty-five subjects with facial wrinkles aged 35 to 45 years were given the cream formulation of Formulation Example 1 and Comparative Formulation Example 1, and the skin wrinkle improvement effect was evaluated. The cream of Formulation Example 1 was used on the left side of the subject and the cream of Comparative Formulation Example 1 was used on the right side for 3 months, and the skin condition was measured on both sides of the face before the use of the cream. The change in skin wrinkles was measured by the method. Skin measurement was performed in a constant temperature room with a temperature of 24 ° C. and a relative humidity of 40%. The wrinkles at the tail of the eyes were replicated, and skin wrinkles were measured using a Visiometer system (C + K). The amount of change in skin wrinkles was calculated according to Equation 2 below.
(상기 식에서, Tdi ; D90에서의 측정부위 값이며, Tdo ; D0에서의 측정부위 값이다.)(In the above formula, Tdi; measured value at D 90 , Tdo; measured value at D 0. )
상기 식에 따라, 계산한 결과를 하기 표 6에 나타내었다.According to the above formula, the calculated results are shown in Table 6 below.
상기의 결과에서도 알 수 있듯이, 본 발명에서는 인삼 사포닌의 구조적 특징으로 인한 낮은 경피흡수율의 단점을 극복하기 위하여 당이 제거된 화합물 K을 피부친화성 유화제 및 나노유화기술을 사용하여 미세한 유화입자 내부에 함유시킴으로써 경피흡수율을 극대화하였고, 주름감소 등 피부 노화 방지효과가 우수함을 알 수 있었다.As can be seen from the above results, in the present invention, in order to overcome the disadvantage of low transdermal absorption due to the structural characteristics of ginseng saponin, the sugar-removed compound K was used inside the fine emulsion particles using a skin-friendly emulsifier and a nanoemulsification technique. By maximizing the percutaneous absorption rate, it was found that the anti-aging effect such as wrinkle reduction is excellent.
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