KR102543123B1 - Composition for skin moisturizing comprising Aloe flower extract thereof as an active ingredient - Google Patents
Composition for skin moisturizing comprising Aloe flower extract thereof as an active ingredient Download PDFInfo
- Publication number
- KR102543123B1 KR102543123B1 KR1020210067992A KR20210067992A KR102543123B1 KR 102543123 B1 KR102543123 B1 KR 102543123B1 KR 1020210067992 A KR1020210067992 A KR 1020210067992A KR 20210067992 A KR20210067992 A KR 20210067992A KR 102543123 B1 KR102543123 B1 KR 102543123B1
- Authority
- KR
- South Korea
- Prior art keywords
- skin
- moisturizing
- composition
- aloe vera
- flower extract
- Prior art date
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9794—Liliopsida [monocotyledons]
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
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Abstract
본 발명은 알로에베라 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 조성물에 관한 것으로, 상기 조성물은 피부 보습 인자의 발현을 조절하여 피부 보습을 향상시키고, UVB로 인한 피부 손상으로 저하된 피부 보습을 향상시키는 효과를 나타내는바, 피부 보습을 위한 화장료 조성물, 의약외품, 피부외용제 또는 식품 조성물로 사용될 수 있다.The present invention relates to a skin moisturizing composition containing an aloe vera flower extract or a compound isolated therefrom as an active ingredient, wherein the composition improves skin moisturizing by controlling the expression of skin moisturizing factors and prevents skin damage caused by UVB Since it exhibits an effect of improving skin moisturization, it can be used as a cosmetic composition, a quasi-drug, an external skin preparation, or a food composition for skin moisturizing.
Description
본 발명은 알로에 꽃 추출물을 유효성분으로 함유하는 피부 보습용 조성물에 관한 것으로, 보다 상세하게는 알로에베라(Aloe vera) 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 조성물에 관한 것이다.The present invention relates to a skin moisturizing composition containing an aloe flower extract as an active ingredient, and more particularly, to a skin moisturizing composition containing an Aloe vera flower extract or a compound isolated therefrom as an active ingredient. will be.
피부는 우리 몸에서 가장 큰 기관으로 표피, 진피, 피하조직이라고 불리는 세 개의 층을 가지고 있다. 표피의 각질층은 물리적, 화학적, 환경적 자극에 대한 일차적인 방어벽 역할을 한다. 특히 표피층의 세포 내 지질과 천연 보습 인자는 표피 수분 손실을 방지하여 피부 보습을 유지하는데 도움이 된다. 수분 부족 및 열악한 환경 조건으로 인한 각질층의 손상은 건조한 피부를 만들고, 이는 피부염, 가려움증, 건선 및 노화와 같은 여러 피부 질환이 발생하기 쉬운 상태로 만든다.Skin is the largest organ in our body and has three layers called epidermis, dermis, and subcutaneous tissue. The stratum corneum of the epidermis serves as a primary barrier against physical, chemical, and environmental stimuli. In particular, intracellular lipids and natural moisturizing factors in the epidermal layer help to maintain skin moisture by preventing epidermal water loss. Damage to the stratum corneum due to lack of moisture and poor environmental conditions results in dry skin, which makes it prone to various skin diseases such as dermatitis, itching, psoriasis and aging.
피부 보습과 관련된 분자 신호 경로를 기반으로, 여러 유형의 표피 단백질이 피부장벽 기능을 조절하는데 관여하는 것으로 보고되었다. 히알루로난 합성효소(Hyaluronan synthase, HASes)와 분해효소(hyaluronidase, HYALs)에 의해서 조절되는 히알루로난(HA, Hyaluronan)은 피부 보습 조절제이다. 프로필라그린(profilaggrin), 아쿠아포린(aquaporins, AQP), 인볼루크린(involucrin)은 피부의 강력한 물리적 장벽 및 투과장벽 기능에 기여한다. 이들 모두는 피부 보습과 피부 무결성을 유지하는데 중요한 역할을 한다.Based on molecular signaling pathways related to skin hydration, several types of epidermal proteins have been reported to be involved in regulating skin barrier function. Hyaluronan (HA, Hyaluronan), which is regulated by hyaluronan synthase (HASes) and degrading enzymes (hyaluronidase (HYALs)), is a skin moisturizing regulator. Profilaggrin, aquaporins (AQP), and involucrin contribute to the strong physical and permeation barrier function of the skin. All of these play an important role in maintaining skin hydration and skin integrity.
천연 원료 보습제는 전통적으로 여러 피부 질환을 치료하는데 사용해왔다. 알로에베라, 병풀, 해초, 동백은 보습 효과로 잘 알려진 식물이다. 아스포델루스과(Asphodelaceae)에 속하는 알로에베라(Aloe vera, Aloe barbadensis Mill.과 동의어)는 다년생 관목으로 장미와 같은 패턴으로 줄기를 둘러싼 잎이 있고, 중앙에는 꽃이 자란다. 알로에베라는 보습, 항염, 항균, 상처 치유 기능으로 보고되어 다양한 화장품에 사용되어 왔다.Natural sourced moisturizers have traditionally been used to treat a number of skin conditions. Aloe vera, centella asiatica, seaweed, and camellia are plants known for their moisturizing properties. Aloe vera (synonymous with Aloe barbadensis Mill.), belonging to the Asphodelaceae family, is a perennial shrub with leaves surrounding a stem in a rose-like pattern, with a flower growing in the center. Aloe vera has been reported for its moisturizing, anti-inflammatory, antibacterial, and wound healing properties and has been used in various cosmetics.
인체 적용시험을 포함해 대부분의 연구는 알로에베라 잎 추출물, 겔, 특정 성분 등에 대해서 수행되어 왔다. 반면에 알로에베라 꽃에 대한 문헌은 거의 없는 상황이며, 연구 내용은 항산화, 항균 특성을 가진 화학적 성분을 식별하는데 주로 초점을 맞추고 있다.Most studies, including human application tests, have been conducted on aloe vera leaf extract, gel, and specific ingredients. On the other hand, there is little literature on aloe vera flowers, and research is mainly focused on identifying chemical components with antioxidant and antibacterial properties.
이에, 본 발명자들은 천연 원료 보습제를 개발하기 위해 노력한 결과, 알로에베라 꽃 추출물 및 이로부터 유래한 화합물의 피부 보습 기능을 확인하였다. 이에, 알로에베라 꽃 추출물 또는 이로부터 유래한 화합물을 피부 보습용 조성물의 유효성분으로 유용하게 이용할 수 있다는 점을 밝힘으로써, 본 출원에 이르렀다.Accordingly, the present inventors, as a result of efforts to develop a natural raw material moisturizer, confirmed the skin moisturizing function of an aloe vera flower extract and a compound derived therefrom. Accordingly, the present application has been reached by revealing that an aloe vera flower extract or a compound derived therefrom can be usefully used as an active ingredient in a skin moisturizing composition.
본 발명의 목적은 알로에베라 꽃 추출물 또는 이로부터 유래한 화합물의 피부 보습 효과에 대한 분자신호전달경로 연구를 통해 피부 보습을 위한 조성물을 제공하는 것이다.An object of the present invention is to provide a composition for moisturizing the skin by studying the molecular signal transduction pathway for the skin moisturizing effect of an aloe vera flower extract or a compound derived therefrom.
본 발명의 목적을 달성하기 위하여, 본 발명은 알로에베라(Aloe vera) 꽃 추출물 및 이로부터 유래한 화합물이 피부 보습 인자들의 발현을 조절하고, UVB로 인한 손상으로 저하된 피부 보습을 향상시키는 효과가 있다는 것을 발견하였다.In order to achieve the object of the present invention, the present invention has the effect of aloe vera ( Aloe vera ) flower extract and a compound derived thereregulating the expression of skin moisturizing factors and improving skin moisturizing reduced by damage caused by UVB found that there is
이에, 본 발명은 알로에베라(Aloe vera) 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 화장료 조성물, 의약외품, 피부 외용제 및 식품 조성물을 제공한다.Accordingly, the present invention provides a cosmetic composition for moisturizing the skin, a quasi-drug, an external skin preparation, and a food composition containing an Aloe vera flower extract or a compound isolated therefrom as an active ingredient.
본 발명의 알로에베라 꽃 추출물 및 이로부터 분리한 화합물은 PKC와 MAPK 신호전달경로를 활성화 시켜 피부장벽 기능에 기여하는 인볼루크린(involucrin)의 발현을 증가시키고, 핵심적 피부 보습 인자인 AQP3, 필라그린, HA의 발현을 증가시키며, UVB로 인한 피부 손상으로 저하된 피부 보습을 향상시키는 효과를 나타내므로, 상기 알로에베라 꽃 추출물 또는 이로부터 분리한 화합물은 피부 보습를 목적으로 하는 화장품, 의약외품, 피부 외용제, 식품 조성물 등의 유효성분으로 유용하게 이용될 수 있다.The aloe vera flower extract of the present invention and the compounds isolated therefrom activate the PKC and MAPK signaling pathways to increase the expression of involucrin, which contributes to the skin barrier function, and the key skin moisturizing factors AQP3 and filaggrin , Since it increases the expression of HA and exhibits an effect of improving skin moisturizing reduced by skin damage caused by UVB, the aloe vera flower extract or a compound isolated therefrom is used in cosmetics, quasi-drugs, skin external agents for the purpose of moisturizing skin, It can be usefully used as an active ingredient in food compositions and the like.
도 1은 본 발명의 일 실시예에 따른 알로에베라 꽃 추출물의 유효성분을 확인한 도로서, 도 1A는 건조한 알로에베라 꽃을 나타내고, 도 1B는 본 발명의 일 실시예에 따른 알로에베라 꽃 수(AFWE), 100% 에탄올(AE), 50% 에탄올(EE) 추출물 및 이의 기능성분의 TLC 패턴을 나타내며, 도 1C는 AFWE의 HPLC 크로마토그램을 나타내고, 도 1D는 표준 유효성분의 HPLC 크로마토그램을 나타낸다.
도 2는 본 발명의 일 실시예에 따른 알로에베라 꽃 수 추출물(AFWE)의 HaCaT 세포에서 피부 수화(hydration) 관련 단백질 발현 조절 효과를 확인한 도로서, 도 2A는 MTT 분석을 통한 AFWE의 세포 독성 확인 결과를 나타내고, 도 2B는 ELISA 분석을 통한 히알루로난 함량 확인 결과를 나타내며, 도 2C는 Western blot 분석을 통한 HAS1, HYAL1 및 AQP3 발현 확인 결과를 나타내고, 도 2D 내지 도 2F는 농도계 분석을 통한 HAS1(도 2D), HYAL1(도 2E), AQP3(도 2F) 발현 확인 결과를 나타낸다.
도 3은 본 발명의 일 실시예에 따른 알로에베라 꽃 수 추출물(AFWE)의 HaCaT 세포에서 피부장벽 기능 관련 단백질 발현 향상 효과를 확인한 도로서, 도 3A는 Western blot 분석을 통한 인볼루크린(involucrin) 및 필라그린(filaggrin) 발현 확인 결과를 나타내고, 도 3B 내지 도 3C는 농도계 분석을 통한 인볼루크린(도 3B) 및 필라그린(도 3C) 발현 확인 결과를 나타내며, 도 3D는 qRT-PCR 분석을 통해 인볼루크린의 상대적 mRNA 발현 확인 결과를 나타내고, 도 3E는 Western blot 분석을 통한 인볼루크린 조절에 관여하는 신호전달경로 단백질 PCK 및 MAPK(pP38/p38, ERK1/2) 발현 확인 결과를 나타내며, 도 3F 내지 도 3H는 농도계 분석을 통한 PCK 및 MAPK(pP38/p38, ERK1/2) 확인 결과를 나타낸다.
도 4는 본 발명의 일 실시예에 따른 HaCaT 세포에 UVB 조사 후 알로에베라 꽃 수 추출물(AFWE) 처리시 피부 보습 인자들, 구체적으로 피부 수화 관련 단백질, 피부장벽 기능 관련 단백질 발현 조절 효과를 확인한 도로서, 도 4A는 MTT 분석을 통한 UVB 조건에서 AFWE의 세포 독성 확인 결과를 나타내고, 도 4B는 Western blot 분석을 통한 UVB 조건에서 AFWE 처리시 인볼루크린, HAS1, HYAL1, 프로필라그린, 필라그린 및 AQP3 발현 확인 결과를 나타내며, 도 4C 내지 도 4G는 농도계 분석을 통한 UVB 조건에서 알로에베라 꽃 수 추출물(AFWE) 처리시 인볼루크린(도 4C), HAS1(도 4D), HYAL1(도 4E), 필라그린(도 4F) 및 AQP3(도 4G) 발현 확인 결과를 나타낸다.
도 5는 본 발명의 일 실시예에 따른 HaCaT 세포에 UVB 조사 후 알로에베라 꽃 수 추출물(AFWE) 처리시 형태학적 변화를 확인한 도이다.
도 6은 본 발명의 일 실시예에 따른 알로에베라 꽃 수(AFWE), 100% 에탄올(AE), 50% 에탄올(EE) 추출물을 처리한 HaCaT 세포에서 피부 보습 인자들, 구체적으로 피부 수화 관련 단백질, 피부장벽 기능 관련 단백질 발현 조절 효과를 확인한 도로서, 도 6A는 MTT 분석을 통한 AFWE, AE, EE의 세포 독성 확인 결과를 나타내고, 도 6B는 ELISA 분석을 통한 히알루로난 함량 확인 결과를 나타내며, 도 6C는 Western blot 분석을 통한 인볼루크린, HAS1, HYAL1, 프로필라그린, 필라그린, AQP3 발현 확인 결과를 나타내고, 도 6D 내지 도 6H는 농도계 분석을 통한 인볼루크린, HAS1, HYAL1, 필라그린, AQP3 발현확인 결과를 나타낸다.
도 7은 본 발명의 일 실시예에 따른 알로에베라 꽃 추출물에 함유된 활성성분들, 구체적으로 이소오리엔틴(isoorientin, IO), 비텍신(vitexin, V), 이소비텍신(isovitexin, IV)의 HaCaT 세포에서 피부 보습 인자들 발현 효과를 확인한 도로서, 도 7A는 MTT 분석을 통한 IO, V, IV의 세포 독성 확인 결과를 나타내고, 도 7B는 ELISA 분석을 통한 히알루로난 함량 확인 결과를 나타내며, 도 7C는 Western blot 분석을 통한 인볼루크린, HAS1, HYAL1, 프로플라그린, 필라그린, AQP3 발현 확인 결과를 나타내고, 도 7D 내지 도 7H는 농도계 분석을 통한 인볼루크린(도 7D), HAS1(도 7E), HYAL1(도 7F), 필라그린(도 7G), AQP3(도 7H) 발현 확인 결과를 나타낸다.
도 8은 본 발명의 일 실시예에 따른 IO와 피부 보습 인자들의 분자결합 분석을 나타낸 도로서, 도 8A는 IO와 인볼루크린의 결합구조를 나타내고, 도 8B는 IO와 PKC의 결합구조를 나타내며, 도 8C는 IO와 P38의 결합구조를 나타낸다. 도 8D는 IO와 ERK1의 결합구조를 나타내고, 도 8E는 IO와 필라그린의 결합구조를 나타내며, 도 8F는 IO와 AQP3의 결합구조를 나타내며, 도 8G는 도 8A 내지 도 8F에 나타낸 결합체 형성의 결합 점수를 나타낸다.
도 9는 본 발명의 일 실시예에 따른 IO와 인볼루크린의 분자결합 분석을 확인한 도로서, IO와 인볼루크린의 결합포켓(도 9, 왼쪽) 및 IO와 인볼루크린 사이에 형성된 결합의 종류(도 9, 오른쪽)를 나타낸다.
도 10은 본 발명의 일 실시예에 따른 IO와 PKC 및 IO와 P38의 분자결합 분석을 확인한 도로서, 도 10A는 IO와 PKC의 결합포켓(도 10A, 왼쪽) 및 IO와 PKC 사이에 형성된 결합의 종류(도 10A, 오른쪽)를 나타내고, 도 10B는 IO와 P38의 결합포켓(도 10B, 왼쪽) 및 IO와 P38 사이에 형성된 결합의 종류(도 10B, 오른쪽)를 나타낸다.
도 11은 본 발명의 일 실시예에 따른 IO와 ERK 및 IO와 필라그린의 분자결합 분석을 확인한 도로서, 도 11A는 IO와 ERK의 결합포켓(도 11A, 왼쪽) 및 IO와 ERK 사이에 형성된 결합의 종류(도 11A, 오른쪽)를 나타내고, 도 11B는 IO와 필라그린의 결합포켓(도 11B, 왼쪽) 및 IO와 필라그린 사이에 형성된 결합의 종류(도 11B, 오른쪽)를 나타낸다.
도 12는 본 발명의 일 실시예에 따른 IO와 AQP3 및 IO와 HYAL1의 분자결합 분석을 확인한 도로서, 도 12A는 IO와 AQP3의 결합포켓(도 12A, 왼쪽) 및 IO와 AQP3 사이에 형성된 결합의 종류(도 12A, 오른쪽)를 나타내고, 도 12B는 IO와 HYAL1의 결합포켓(도 12B, 왼쪽) 및 IO와 HYAL1 사이에 형성된 결합의 종류(도 12B, 오른쪽)를 나타낸다.
도 13은 본 발명의 일 실시예에 따른 알로에베라 꽃 추출물 및 이에 함유된 활성성분이 신호전달경로를 통해 피부보습을 향상시키는 기전을 도식적으로 나타낸 도이다.1 is a diagram confirming the active ingredient of an aloe vera flower extract according to an embodiment of the present invention, FIG. 1A shows dried aloe vera flowers, and FIG. 1B shows the number of aloe vera flowers (AFWE) according to an embodiment of the present invention. ), 100% ethanol (AE), 50% ethanol (EE) extracts and TLC patterns of functional components thereof, FIG. 1C shows the HPLC chromatogram of AFWE, and FIG. 1D shows the HPLC chromatogram of the standard active ingredient.
2 is a diagram confirming the effect of Aloe vera flower extract (AFWE) on skin hydration-related protein expression regulation in HaCaT cells according to an embodiment of the present invention, and FIG. 2A is confirmation of AFWE's cytotoxicity through MTT assay The results are shown, Figure 2B shows the results of confirming the hyaluronan content through ELISA analysis, Figure 2C shows the results of confirming the expression of HAS1, HYAL1 and AQP3 through Western blot analysis, Figures 2D to 2F show HAS1 through densitometric analysis (FIG. 2D), HYAL1 (FIG. 2E), and AQP3 (FIG. 2F) expression confirmation results are shown.
Figure 3 is a diagram confirming the effect of enhancing the expression of proteins related to skin barrier function in HaCaT cells of Aloe vera flower extract (AFWE) according to an embodiment of the present invention, and Figure 3A is involucrin through Western blot analysis And filaggrin expression confirmation results are shown, and FIGS. 3B to 3C show involucrin (FIG. 3B) and filaggrin (FIG. 3C) expression confirmation results through densitometry analysis, and FIG. 3D shows qRT-PCR analysis. Figure 3E shows the results of confirming the expression of PCK and MAPK (pP38/p38, ERK1/2) signaling pathway proteins involved in involucrin regulation through Western blot analysis. 3F to 3H show PCK and MAPK (pP38/p38, ERK1/2) confirmation results through densitometry analysis.
4 is a diagram confirming the effect of modulating the expression of skin moisturizing factors, specifically proteins related to skin hydration and proteins related to skin barrier function, when HaCaT cells according to an embodiment of the present invention are treated with aloe vera flower extract (AFWE) after UVB irradiation 4A shows the results of confirming the cytotoxicity of AFWE under UVB conditions through MTT analysis, and FIG. 4B shows involucrin, HAS1, HYAL1, profilagrin, filaggrin and 4C to 4G show the results of confirming the expression of AQP3, involucrin (FIG. 4C), HAS1 (FIG. 4D), HYAL1 (FIG. 4E), HAS1 (FIG. 4D), HYAL1 (FIG. 4E), Filaggrin (FIG. 4F) and AQP3 (FIG. 4G) expression confirmation results are shown.
5 is a diagram confirming morphological changes when HaCaT cells according to an embodiment of the present invention are treated with Aloe vera flower extract (AFWE) after UVB irradiation.
Figure 6 shows skin moisturizing factors, specifically skin hydration-related proteins, in HaCaT cells treated with aloe vera flower water (AFWE), 100% ethanol (AE), and 50% ethanol (EE) extracts according to an embodiment of the present invention. , Fig. 6A shows the results of confirming the cytotoxicity of AFWE, AE, and EE through MTT analysis, and Fig. 6B shows the results of confirming the hyaluronan content through ELISA analysis, 6C shows the results of confirming the expression of involucrin, HAS1, HYAL1, profilagrin, filaggrin, and AQP3 through Western blot analysis, and FIGS. 6D to 6H show involucrin, HAS1, HYAL1, and filaggrin through densitometry analysis. , AQP3 expression confirmation results are shown.
Figure 7 shows the active ingredients contained in the aloe vera flower extract according to an embodiment of the present invention, specifically isoorientin (IO), vitexin (vitexin (V), isovitexin (isovitexin (IV)) As a diagram confirming the expression effect of skin moisturizing factors in HaCaT cells, FIG. 7A shows the results of confirming the cytotoxicity of IO, V, and IV through MTT analysis, and FIG. 7B shows the results of confirming the hyaluronan content through ELISA analysis, 7C shows the results of confirming the expression of involucrin, HAS1, HYAL1, proflagrin, filaggrin, and AQP3 through Western blot analysis, and FIGS. 7D to 7H show involucrin (FIG. 7D), HAS1 ( 7E), HYAL1 (FIG. 7F), filaggrin (FIG. 7G), and AQP3 (FIG. 7H) expression confirmation results are shown.
Figure 8 is a diagram showing the molecular bond analysis of IO and skin moisturizing factors according to an embodiment of the present invention, Figure 8A shows the bonding structure of IO and involucrin, Figure 8B shows the bonding structure of IO and PKC , Figure 8C shows the bonding structure of IO and P38. FIG. 8D shows the binding structure of IO and ERK1, FIG. 8E shows the binding structure of IO and filaggrin, FIG. 8F shows the binding structure of IO and AQP3, and FIG. 8G shows the conjugate formation shown in FIGS. 8A to 8F. represents the combined score.
9 is a diagram confirming the molecular bond analysis between IO and involucrin according to an embodiment of the present invention, showing the binding pocket between IO and involucrin (Fig. 9, left) and the bond formed between IO and involucrin. Indicates the type (Fig. 9, right).
Figure 10 is a diagram confirming the molecular bond analysis of IO and PKC and IO and P38 according to an embodiment of the present invention, Figure 10A is a bond formed between the binding pocket of IO and PKC (Fig. 10A, left) and IO and PKC Shows the type of (Fig. 10A, right), Fig. 10B shows the binding pocket of IO and P38 (Fig. 10B, left) and the type of bond formed between IO and P38 (Fig. 10B, right).
Figure 11 is a diagram confirming the molecular binding analysis of IO and ERK and IO and filaggrin according to an embodiment of the present invention, Figure 11A is formed between the binding pocket of IO and ERK (Fig. 11A, left) and IO and ERK The type of binding (Fig. 11A, right) is shown, and Fig. 11B shows the binding pocket between IO and filaggrin (Fig. 11B, left) and the type of binding formed between IO and filaggrin (Fig. 11B, right).
Figure 12 is a view confirming the molecular bond analysis of IO and AQP3 and IO and HYAL1 according to an embodiment of the present invention, Figure 12A is a bond formed between the binding pocket of IO and AQP3 (Fig. 12A, left) and IO and AQP3 shows the type of (Fig. 12A, right), and Fig. 12B shows the binding pocket of IO and HYAL1 (Fig. 12B, left) and the type of bond formed between IO and HYAL1 (Fig. 12B, right).
13 is a diagram schematically illustrating a mechanism in which an aloe vera flower extract according to an embodiment of the present invention and an active ingredient contained therein improve skin moisturizing through a signal transduction pathway.
이하, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 실시형태를 들어 상세히 설명한다. 본 발명의 실시형태는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 더욱 완전하게 설명하기 위해서 제공되는 것이다. 따라서, 본 발명의 실시형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시형태로 한정되는 것은 아니다.Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. Accordingly, the embodiments of the present invention can be modified in many different forms, and the scope of the present invention is not limited to the embodiments described below.
본 발명의 명세서 전체에서, 어떤 부분이 어떤 구성 요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성 요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Throughout the specification of the present invention, when a part "includes" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.
본 발명은 알로에베라(Aloe vera) 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 조성물을 제공한다.The present invention provides a skin moisturizing composition containing an Aloe vera flower extract or a compound isolated therefrom as an active ingredient.
본 발명에서 사용되는 용어, "추출물(extract)"은 상기 알로에 꽃의 추출 처리에 의하여 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출액을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다. 상기 추출물은 알로에 꽃의 천연, 잡종, 또는 변종 식물로부터 추출될 수 있고, 식물 조직 배양물로부터도 추출이 가능하다.As used herein, the term "extract" refers to an extract obtained by the extraction treatment of the aloe flower, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a refined or purified product of the extract, or It includes extracts of all formulations that can be formed using the extract itself and the extract, such as mixtures thereof. The extract may be extracted from natural, hybrid, or mutant plants of aloe flowers, and may also be extracted from plant tissue culture.
본 발명에서, 상기 알로에베라 꽃은 재배한 것 또는 시판되는 것을 제한없이 사용할 수 있다.In the present invention, cultivated or commercially available aloe vera flowers may be used without limitation.
본 발명에서, 상기 추출물은 물, 유기용매 또는 이들의 혼합용매를 사용할 수 있고, 상기 유기용매는 C1 내지 C5의 저급 알코올, 에틸아세테이트 및 아세톤 등의 극성용매, 헥산 및 디클로로메탄 등의 비극성 용매 또는 이들의 혼합용매를 사용할 수 있으며, 바람직하게는 물, C1 내지 C4 저급알코올 또는 이들의 혼합용매로 추출된 추출물일 수 있다. 상기 알코올은 메탄올, 에탄올, 프로판올, 부탄올 및 이소프로판올일 수 있고, 바람직하게는 에탄올일 수 있다. 보다 바람직하게는 물을 사용하여 추출된 추출물일 수 있다. 추출방법으로는 초음파추출, 진탕추출, Soxhelt 추출 또는 환류 추출방법을 이용할 수 있으나, 이에 한정되는 것은 아니다. 상기 추출용매를 세척하고 잘 건조된 알로에베라 꽃 분량의 1 내지 20배 첨가하여 추출하는 것이 바람직하고, 5 내지 20배 첨가하여 추출하는 것이 더욱 바람직하나, 이에 한정되는 것은 아니다. 또한, 추출시간은 1 내지 72시간이 바람직하며, 1 내지 48시간이 더욱 바람직하나, 이에 한정되는 것은 아니다. 아울러 추출 횟수는 1 내지 5회인 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the extract may use water, an organic solvent, or a mixed solvent thereof, and the organic solvent is a C 1 to C 5 lower alcohol, a polar solvent such as ethyl acetate and acetone, and a non-polar solvent such as hexane and dichloromethane. A solvent or a mixed solvent thereof may be used, and preferably may be an extract extracted with water, C 1 to C 4 lower alcohol, or a mixed solvent thereof. The alcohol may be methanol, ethanol, propanol, butanol and isopropanol, preferably ethanol. More preferably, it may be an extract extracted using water. As the extraction method, ultrasonic extraction, shaking extraction, Soxhelt extraction or reflux extraction method may be used, but is not limited thereto. It is preferable to extract by adding 1 to 20 times the amount of washed and well-dried aloe vera flowers with the extraction solvent, and more preferably to extract by adding 5 to 20 times, but is not limited thereto. In addition, the extraction time is preferably 1 to 72 hours, more preferably 1 to 48 hours, but is not limited thereto. In addition, the number of times of extraction is preferably 1 to 5 times, but is not limited thereto.
본 발명에서, 상기 화합물은 C-글리코사이드 플라보노이드(glycoside flavonoids) 배당체일 수 있고, 상기 C-글리코사이드 플라보노이드 배당체는 이소오리엔틴(issoorientin), 비텍신(vitexin) 또는 이소비텍신(isovitexin)일 수 있고, 구체적으로 이소오리엔틴일 수 있다.In the present invention, the compound may be a C-glycoside flavonoid glycoside, and the C-glycoside flavonoid glycoside may be issoorientin, vitexin, or isovitexin. And, specifically, it may be isoorientin.
본 발명에서, 상기 조성물은 피부 보습 인자를 조절함으로써, 피부 보습 효과를 나타내고, UVB로 인한 피부 손상으로 저하된 피부 보습을 향상할 수 있다.In the present invention, the composition may exhibit a skin moisturizing effect by controlling a skin moisturizing factor, and improve skin moisturizing reduced due to skin damage caused by UVB.
구체적으로 상기 조성물은 피부 보습 인자로 피부 수화(hydration) 관련 단백질 또는 피부장벽 기능 관련 단백질을 조절함으로써, 피부 보습 효과를 나타내고, UVB로 인한 피부 손상으로 저하된 피부 보습을 향상할 수 있다.Specifically, the composition can exhibit a skin moisturizing effect by controlling skin hydration related proteins or skin barrier function related proteins as skin moisturizing factors, and can improve skin moisturizing reduced due to skin damage caused by UVB.
보다 구체적으로 피부 수화 관련 단백질은 HAS1, HYAL1, 히알루로난 또는 AQP3이고, 상기 조성물은 HAS1, 히알루로난 또는 AQP3 발현을 증가시키고, HYAL1 발현을 감소시킴으로써, 피부 보습 효과를 나타내고, UVB로 인한 피부 손상으로 저하된 피부 보습을 향상할 수 있다.More specifically, the skin hydration-related proteins are HAS1, HYAL1, hyaluronan, or AQP3, and the composition increases HAS1, hyaluronan, or AQP3 expression and decreases HYAL1 expression, thereby exhibiting a skin moisturizing effect and reducing UVB-induced skin hydration. It can improve skin moisturization that has been reduced due to damage.
또한, 상기 피부장벽 기능 관련 단백질은 인볼루크린(involuclin) 또는 필라그린(filaggrin)이고, 상기 조성물은 인볼루크린 또는 필라그린 발현을 증가시킴으로써, 피부 보습 효과를 나타내고, UVB로 인한 피부 손상으로 저하된 피부 보습을 향상할 수 있다.In addition, the protein related to the skin barrier function is involucrin or filaggrin, and the composition increases the expression of involucrin or filaggrin, thereby exhibiting a skin moisturizing effect and reducing skin damage caused by UVB. can improve skin hydration.
또한, 본 발명은 알로에베라 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 화장료 조성물을 제공한다.In addition, the present invention provides a cosmetic composition for moisturizing skin containing an Aloe vera flower extract or a compound isolated therefrom as an active ingredient.
본 발명에서, 상기 추출물, 추출방법, 화합물에 대한 내용은 상기 알로에베라 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 조성물에서 설명한 내용과 동일하므로, 구체적인 설명은 상기 내용을 원용하고, 이하에서는 화장료 조성물의 특유한 구성에 대해서만 설명하도록 한다.In the present invention, the contents of the extract, the extraction method, and the compound are the same as those described in the skin moisturizing composition containing the aloe vera flower extract or a compound isolated therefrom as an active ingredient, so the detailed description refers to the above contents. And, hereinafter, only the unique configuration of the cosmetic composition will be described.
본 발명에서 사용되는 용어, "화장료 조성물"은 신체의 구조 또는 기능에 영향을 주지 않으면서 인간 피부에 국소적으로 도포되어 피부의 외관 및 건강을 개선시키는 조성물이다.As used herein, the term "cosmetic composition" is a composition that is topically applied to human skin to improve the appearance and health of the skin without affecting the structure or function of the body.
본 발명에서, 상기 화장료 조성물은 피부 점착 타입의 화장료 제형, 예를 들어, 기초제품 화장료(화장수, 크림, 에센스, 클렌징 폼, 클렌징 워터, 팩, 비누), 바디제품 화장료(바디 로션, 바디 오일, 바디 젤, 비누), 색조제품 화장료(파운데이션, 립스틱, 마스카라, 메이크업 베이스), 두발제품 화장료(샴푸, 린스, 헤어 컨디셔너, 헤어 젤) 등을 가질 수 있다. 또한, 경피 투여형 제형, 예를 들어, 연고제, 액제, 드레싱제, 패취제 또는 스프레이제 등으로 제조될 수 있으나, 이로 한정되는 것은 아니다.In the present invention, the cosmetic composition is a skin-adhesive cosmetic formulation, for example, basic product cosmetics (toilet, cream, essence, cleansing foam, cleansing water, pack, soap), body product cosmetics (body lotion, body oil, Body gel, soap), color product cosmetics (foundation, lipstick, mascara, makeup base), hair product cosmetics (shampoo, conditioner, hair conditioner, hair gel), etc. In addition, transdermal dosage forms such as ointments, liquids, dressings, patches, or sprays may be prepared, but are not limited thereto.
상기 화장료 조성물은 화장품 제제에 있어서 수용가능한 담체를 포함할 수 있고, 상기 담체는 화장품 제제에 포함될 수 있는 이미 공지되어 사용되고 있는 화합물 또는 조성물이거나 앞으로 개발될 화합물 또는 조성물로서 피부와의 접촉시 인체가 적응 가능한 이상의 독성, 불안정성 또는 자극성이 없는 것을 말한다. 상기 담체는 화장료 조성물의 전체 중량에 대하여 약 1 중량% 내지 약 99.99 중량%, 바람직하게는 화장료 조성물의 중량의 약 90 중량% 내지 약 99.99 중량%로 포함될 수 있다. 그러나 상기 비율은 본 발명의 피부 외용제 조성물이 제조되는 전술한 바의 제형에 따라 또 그것의 구체적인 적용 부위(얼굴, 목 등)나 그것의 바람직한 적용량 등에 따라 달라지는 것이기 때문에, 상기 비율은 어떠한 측면으로든 본 발명의 범위를 제한하는 것으로 이해되어서는 안 된다.The cosmetic composition may include a carrier acceptable in cosmetic formulations, and the carrier is a compound or composition that is already known and used, or a compound or composition to be developed in the future that can be included in a cosmetic formulation, and the human body adapts when in contact with the skin. It means that there is no more toxicity, instability or irritation than possible. The carrier may be included in about 1% to about 99.99% by weight based on the total weight of the cosmetic composition, preferably about 90% to about 99.99% by weight of the weight of the cosmetic composition. However, since the above ratio varies according to the above-described formulation in which the composition for external application for skin of the present invention is prepared, and also according to its specific application site (face, neck, etc.) or its preferred application amount, the above ratio is It should not be construed as limiting the scope of the invention.
한편, 상기 담체로서는 알코올, 오일, 계면활성제, 지방산, 실리콘 오일, 습윤제, 보습제, 점성 변형제, 유제, 안정제, 자외선 차단제, 발색제, 향료 등이 예시될 수 있다. 상기 알코올, 오일, 계면활성제, 지방산, 실리콘 오일, 습윤제, 보습제, 점성 변형제, 유제, 안정제, 자외선 차단제, 발색제, 향료로 사용될 수 있는 화합물/조성물 등은 이미 당업계에 공지되어 있기 때문에 당업자라면 적절한 해당 물질/조성물을 선택하여 사용할 수 있다.Meanwhile, as the carrier, alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscosity modifier, emulsion, stabilizer, sunscreen, coloring agent, fragrance, and the like may be exemplified. Compounds/compositions that can be used as alcohols, oils, surfactants, fatty acids, silicone oils, humectants, humectants, viscosity modifiers, emulsions, stabilizers, sunscreens, coloring agents, fragrances, etc. are already known in the art, so those skilled in the art An appropriate corresponding material/composition can be selected and used.
또한, 본 발명은 알로에베라 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 피부외용제를 제공한다.In addition, the present invention provides an external skin preparation for skin moisturizing containing an aloe vera flower extract or a compound isolated therefrom as an active ingredient.
본 발명에서, 상기 추출물, 추출방법, 화합물에 대한 내용은 상기 알로에베라 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 조성물에서 설명한 내용과 동일하므로, 구체적인 설명은 상기 내용을 원용하고, 이하에서는 피부 외용제의 특유한 구성에 대해서만 설명하도록 한다.In the present invention, the contents of the extract, the extraction method, and the compound are the same as those described in the skin moisturizing composition containing the aloe vera flower extract or a compound isolated therefrom as an active ingredient, so the detailed description refers to the above contents. In the following, only the unique composition of external skin preparations will be described.
본 발명에서 사용되는 용어, "외용제"는 외용으로 제공되는 제제이다.As used herein, the term "external use agent" is a preparation provided for external use.
본 발명에서, 피부외용제는 외용산제, 외용정제, 외용액제, 연고제, 크림제, 겔제, 경고제, 드레싱제, 패취제, 스프레이제 및 좌제로 구성된 군으로부터 선택되는 제형으로 제형화할 수 있다.In the present invention, the external skin preparation may be formulated into a dosage form selected from the group consisting of powders for external use, tablets for external use, liquids for external use, ointments, creams, gels, warning agents, dressings, patches, sprays, and suppositories.
또한, 상기 피부외용제는 상용되는 무기 또는 유기의 담체, 부형제 및 희석제를 가하여 고체, 반고체 또는 액상의 형태로 제제화된 비경구 투여제일 수 있다. 상기 비경구 투여를 위한 제재로는 점적제, 연고, 로션, 겔, 크림, 패취, 스프레이, 현탁제 및 유제로 이루어진 군에서 선택되는 경피 투여형 제형일 수 있으나, 이에 제한되지 않는다. 상기 외용제에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 올리고당, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.In addition, the external skin preparation may be a parenteral preparation formulated in a solid, semi-solid or liquid form by adding a commercially available inorganic or organic carrier, excipient, and diluent. The preparation for parenteral administration may be a transdermal dosage form selected from the group consisting of drops, ointments, lotions, gels, creams, patches, sprays, suspensions and emulsions, but is not limited thereto. Carriers, excipients and diluents that may be included in the external preparation include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명은 알로에베라 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 의약외품을 제공한다.The present invention provides a quasi-drug for skin moisturizing containing an aloe vera flower extract or a compound isolated therefrom as an active ingredient.
본 발명에서, 상기 추출물, 추출방법, 화합물에 대한 내용은 상기 알로에베라 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 조성물에서 설명한 내용과 동일하므로, 구체적인 설명은 상기 내용을 원용하고, 이하에서는 의약외품의 특유한 구성에 대해서만 설명하도록 한다.In the present invention, the contents of the extract, the extraction method, and the compound are the same as those described in the skin moisturizing composition containing the aloe vera flower extract or a compound isolated therefrom as an active ingredient, so the detailed description refers to the above contents. In the following, only the unique configuration of quasi-drugs will be described.
본 발명에서 사용되는 용어, "의약외품"은 사람이나 동물의 질병을 진단, 치료, 개선, 경감, 처치 또는 예방할 목적으로 사용되는 물품들 중 의약품보다 작용이 경미한 물품들을 의미하는 것으로, 예를 들어 약사법에 따르면 의약외품이란 의약품의 용도로 사용되는 물품을 제외한 것으로 사람이나 동물의 질병 치료나 예방에 쓰이는 제품, 인체에 대한 작용이 경미하거나 직접 작용하지 않는 제품일 수 있다.As used herein, the term "quasi-drugs" refers to items with a milder effect than pharmaceuticals among items used for the purpose of diagnosing, treating, improving, mitigating, treating or preventing diseases of humans or animals, for example, according to the Pharmaceutical Affairs Act. According to the Act, quasi-drugs are products excluding items used for pharmaceutical purposes, and may be products used to treat or prevent diseases in humans or animals, or products with minor or no direct action on the human body.
상기 의약외품 조성물은 바디 클렌저, 소독 청결제, 세정제, 주방용 세정제, 청소용 세정제, 치약, 가글제, 물티슈, 세제, 비누, 핸드 워시, 헤어세정제, 헤어 유연제, 가습기 충진제, 마스크, 연고제 및 필터 충진제로 구성된 군에서 선택되는 제형으로 제조할 수 있다.The quasi-drug composition is a body cleanser, disinfectant cleanser, detergent, kitchen detergent, cleaning detergent, toothpaste, gargle, wet tissue, detergent, soap, hand wash, hair wash, hair softener, humidifier filler, mask, ointment and filter filler in the group consisting of It can be prepared in the formulation of your choice.
또한, 본 발명은 알로에베라 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for moisturizing the skin containing an aloe vera flower extract or a compound isolated therefrom as an active ingredient.
본 발명에서, 상기 추출물, 추출방법, 화합물에 대한 내용은 상기 알로에베라 꽃 추출물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 피부 보습용 조성물에서 설명한 내용과 동일하므로, 구체적인 설명은 상기 내용을 원용하고, 이하에서는 식품 조성물의 특유한 구성에 대해서만 설명하도록 한다.In the present invention, the contents of the extract, the extraction method, and the compound are the same as those described in the skin moisturizing composition containing the aloe vera flower extract or a compound isolated therefrom as an active ingredient, so the detailed description refers to the above contents. And, hereinafter, only the specific configuration of the food composition will be described.
본 발명에서 사용되는 용어, "식품 조성물"은 각각의 조성물을 먹거나 마시는 데에 독성증상을 유발함이 없이 먹고/먹거나 마실 수 있는 임의 종류의 조성물을 지칭한다.As used herein, the term "food composition" refers to any kind of composition that can be eaten and/or drunk without causing toxicity symptoms when eating or drinking each composition.
본 발명에서 사용되는 용어, "건강기능식품(functional food)"은 특정보건용 식품(food for special health use, FOSHU)와 동일한 용어로, 영양 공급 외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료 효과가 높은 식품이다.The term used in the present invention, "functional food" is the same term as food for special health use (FOSHU), medicine processed to efficiently display bioregulatory functions in addition to nutritional supply, It is a food with high medical effect.
본 발명에서, 상기 식품 조성물은 음료, 육류, 소시지, 빵, 비스킷, 떡, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타면류, 껌류, 아이스크림류를 포함한 낙농제품, 스프, 음료수, 알코올 음료 및 비타민 복합제, 유제품 및 유가공 제품, 통상적인 의미에서의 가공 식품 및 건강기능식품일 수 있으나, 이에 한정되는 것은 아니다. 상기 음료조성물은 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러가지 향미제(천연 향미제, 예를 들어 타우마틴, 스테비아추출물, 레바우디오시드 A, 글리시르히진 등; 및 합성 향미제, 예를 들어 사카린, 아스파르탐 등) 또는 천연 탄수화물(모노사카라이드, 예를들어, 포도당, 과당등; 디사카라이드, 예를 들어 말토스, 슈크로스등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜) 등을 추가 성분으로서 함유할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 g 당 일반적으로 약 1 g 내지 20 g, 바람직하게는 약 5 g 내지 10 g이다.In the present invention, the food composition is a beverage, meat, sausage, bread, biscuits, rice cake, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, soup, beverages, alcoholic beverages and vitamin complexes, dairy products and milk-processed products, processed foods in a conventional sense, and health functional foods, but are not limited thereto. The beverage composition is not particularly limited in other ingredients, and as in conventional beverages, various flavors (natural flavors such as thaumatin, stevia extract, rebaudioside A, glycyrrhizin, etc.; and synthetic flavors, such as For example, saccharin, aspartame, etc.) or natural carbohydrates (monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, Conventional sugars such as cyclodextrin and sugar alcohols such as xylitol, sorbitol, and erythritol) may be contained as additional components. The proportion of the natural carbohydrate is generally about 1 g to 20 g, preferably about 5 g to 10 g per 100 g of the composition of the present invention.
또한, 상기 외에 본 발명의 식품은 영양제, 비타민, 광물(전해질), 합성 풍미제, 천연 풍미제, 착색제, 중진제(치즈, 초콜릿 등), 펙트산, 팩트산 염, 알긴산, 알긴산염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산화제 및 과육 등을 함유할 수 있다, 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있으며, 이러한 첨가제의 비율 또한 당업자에 의해 적절히 선택될 수 있다. 상기 첨가제의 비율은 본 발명의 조성물 100 g 당 일반적으로 약 0.1 g 내지 20 g, 바람직하게는 약 1 g 내지 20 g일 수 있다.In addition, in addition to the above, the food of the present invention contains nutrients, vitamins, minerals (electrolytes), synthetic flavors, natural flavors, colorants, enhancers (cheese, chocolate, etc.), pectic acid, pectic acid salts, alginic acid, alginates, organic acids. , protective colloidal thickener, pH adjusting agent, stabilizer, preservative, glycerin, alcohol, carbonating agent and fruit flesh, etc. These components may be used independently or in combination, and the ratio of these additives may also be determined appropriately by those skilled in the art. can be chosen The proportion of the additives may be generally about 0.1 g to 20 g, preferably about 1 g to 20 g per 100 g of the composition of the present invention.
또한, 상기 건강기능식품은 유효성분을 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 발명의 건강기능식품은 원료에 대하여 바람직하게 15 중량 % 이하, 바람직하게는 10 중량 % 이하의 양으로 첨가될 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있다.In addition, the health functional food may be added to food as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient can be suitably determined depending on the purpose of its use (for prevention or improvement). In general, when preparing food or beverage, the health functional food of the present invention may be added in an amount of preferably 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range.
이하, 본 발명을 실시예를 통해 보다 상세히 설명한다. 다만 하기 실시예는 본 발명의 이해를 돕기 위한 것이지 본 발명의 권리범위를 이로 한정하는 것을 의도하지 않는다.Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to aid understanding of the present invention and are not intended to limit the scope of the present invention thereto.
화학물 및 시약chemicals and reagents
건조된 알로에베라(Aloe vera) 꽃(도 1A)은 Univera Co., Ltd.(서울, 한국)로부터 제공 받았다. DMEM(Dulbecco's modified Eagle's medium) 배지, 소태아혈청(fetal bovine serum, FBS), 페니실린과 스트렙토마이신은 Gibco-BRL(Grand Island, NY, USA)로부터 구입하였다. 히알루로난 탐지를 위한 ELISA 키트는 R&D System Inc.(Minneapolis, MN, USA)로부터 구입하였다. PRO-PREPTM 단백질 추출 용액, ECL(Enhanced chemiluminescence) 탐지 키트는 인트론(성남, 한국)으로부터 구입하였다. GAPDH, 인볼루크린(involucrin), HAS1, HYAL1, AQP3, 필라그린(filaggrin), PKC, P38, ERK 1/2의 항체, HRP(horseradish peroxidase)와 접합된 이차 항체는 Santa Cruz(Dallas, TX, USA), Cell Science(Canton, MA, USA), Cell Signaling Technology(Beverly, MA, USA)로부터 구입하였다. 모든 그 밖의 화학물질들은 Sigma-Aldrich( Steinheim, Germany)로부터 구입하였다.Dried Aloe vera flowers (FIG. 1A) were provided by Univera Co., Ltd. (Seoul, Korea). DMEM (Dulbecco's modified Eagle's medium) medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA). An ELISA kit for hyaluronan detection was purchased from R&D System Inc. (Minneapolis, Minn., USA). PRO-PREP ™ protein extraction solution and ECL (Enhanced chemiluminescence) detection kit were purchased from Intron (Seongnam, Korea). Antibodies of GAPDH, involucrin, HAS1, HYAL1, AQP3, filaggrin, PKC, P38,
세포 배양cell culture
인간 각질형성세포(keratinocyte)인 HaCaT 세포는 한국 세포주 은행(서울, 한국)에서 구입하였다. 세포를 10% 열-비활성 소태아혈청(heat-inactivated FBS)와 1 % 페니실린-스트렙토마이신을 포함하는 high-glucose DMEM 배지로 37℃, 5% CO2 조건에서 배양하였다.HaCaT cells, human keratinocytes, were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in high-glucose DMEM medium containing 10% heat-inactivated fetal bovine serum (heat-inactivated FBS) and 1% penicillin-streptomycin at 37°C and 5% CO 2 conditions.
통계적 분석statistical analysis
실험군들을 비교하기 위해서 GraphPad Prism5(La Jolla, CA, USA)를 사용하여 1차원 분산 분석(ANOVA)을 하였고, p-value <0.05는 통계적으로 의미가 있는 것으로 하였다. 세 개의 독립적인 실험에 대해 평균±표준오차를 표시하였다.To compare the experimental groups, a one-dimensional analysis of variance (ANOVA) was performed using GraphPad Prism5 (La Jolla, CA, USA), and a p -value <0.05 was considered statistically significant. Mean±standard error is expressed for three independent experiments.
<실시예 1> 알로에베라 꽃 추출물 제조<Example 1> Preparation of aloe vera flower extract
추출용매를 달리하여 3종의 알로에베라 꽃 추출물을 제조하였다.Three kinds of aloe vera flower extracts were prepared by using different extraction solvents.
구체적으로, 건조하여 분말화한 알로에베라 꽃 1 g 각각을 물(AFWE), 100% 에탄올(AE), 50% 에탄올(EE) 10 mL에 넣고, 40℃에서 60분 동안 초음파로 추출한 후 원심분리 및 여과하였다. 이후 각 추출물은 회전감압농축기를 사용하여 감압농축한 후 동결건조하였다. 제조한 추출물은 사용 시까지 -4℃에서 보관하였다.Specifically, 1 g of each dried and powdered aloe vera flower was put into 10 mL of water (AFWE), 100% ethanol (AE), and 50% ethanol (EE), extracted by ultrasonic waves at 40 ° C for 60 minutes, and then centrifuged. and filtered. Thereafter, each extract was concentrated under reduced pressure using a rotary vacuum concentrator and then lyophilized. The prepared extract was stored at -4°C until use.
<실시예 2> 알로에베라 꽃 추출물에 포함된 활성성분 분석<Example 2> Analysis of active ingredients contained in aloe vera flower extract
알로에베라 꽃 추출물에 포함된 성분을 분석하기 위하여, 상기 실시예 1에서 제조한 3종의 알로에베라 꽃 추출물을 이용하여 TLC와 HPLC를 수행하였다. TLC는 종전의 방법에 따라 수행하였다(The Scientific World Journal Volume 2014, Article ID 724267, 6 page). HPLC 분석은 분리 모듈(e2695), 포토다이오드 어레이 검출기를 갖춘 Water system(Water Corp., Milford, MA, USA)을 사용하여 종전의 방법에 따라 수행하였다(Phytochemical Analysis Volume 16, Issue 5, pages 295-301). 내용물의 분석을 위해서 상기 실시예 1의 각 추출물들을 10 mg/ml씩 분리하였다. 활성성분으로 C-글리코사이드 플라보노이드(glycoside flavonoids) 배당체인 오리엔틴(orientin, O), 이소오리엔틴(isoorientin, IO), 비텍신(vitexin, V) 및 이소비텍신(isovitexin, IV)을 표준 성분으로 사용하였고, 1 mg/mL의 농도로 사용하였다.In order to analyze the components contained in the aloe vera flower extract, TLC and HPLC were performed using the three types of aloe vera flower extracts prepared in Example 1. TLC was performed according to a conventional method (The Scientific World Journal Volume 2014, Article ID 724267, page 6). HPLC analysis was performed according to a conventional method using a separation module (e2695) and a water system (Water Corp., Milford, MA, USA) equipped with a photodiode array detector (Phytochemical Analysis Volume 16,
그 결과, 도 1B 내지 도 1D에 나타낸 바와 같이, 물(AFWE), 100% 에탄올(AE), 50% 에탄올 수용액(EE)으로 추출한 알로에베라 꽃 추출물 모두에 이소오리엔틴(isoorientin, IO), 비텍신(vitexin, V), 이소비텍신(isovitexin, IV)가 함유되어 있고, 특히 AFWE에서 다른 추출물보다 이들 유효성분의 함량이 더 높다는 것을 확인하였다. 뿐만 아니라 이들 유효성분들 중에서 IO의 농도가 더 높았다. AFWE에서 IO의 함량은 22.24 %였고, IO>V>IV 순으로 함유하는 것을 확인하였다.As a result, as shown in FIGS. 1B to 1D, isoorientin (IO), B. It was confirmed that vitexin (V) and isovitexin (IV) were contained, and in particular, the content of these active ingredients was higher in AFWE than in other extracts. In addition, the concentration of IO was higher among these active ingredients. The content of IO in AFWE was 22.24%, and it was confirmed that it was contained in the order of IO>V>IV.
<실시예 3> 샘플 제조 및 세포 처리<Example 3> Sample preparation and cell treatment
상기 실시예 1의 각 추출물들을 PBS에 용해하고, 여과하여 10 mg/mL의 농도의 스톡을 만들었다. 그 후, 세포에 투여하기 위하여 상기 스톡을 이용하여 25 ㎍/mL의 추출물 샘플을 제조하고, 무혈청 배지에 희석시킨 5 ㎛의 IO, V, IV 샘플을 제조하였다. 상기 실시예 1의 추출물들 중에서 AFWE의 농도 의존적 효과를 평가하기 위하여 AFWE 스톡을 이용해 1, 2.5, 5 ㎍/mL 농도의 AFWE 샘플을 추가로 제조하였다. 또한, 양성 대조군으로 사용하기 위하여 피부 보습을 향상시킨다고 알려진 병풀(Centella asiatica) 에탄올 추출물Postepy Dermatologii Alergologii 2013: XXX, 1: 46-49) 5 ㎍/mL 샘플을 제조하였다. 또한, 추출물을 처리하지 않은 세포군을 음성 대조군으로 하였다.Each of the extracts of Example 1 was dissolved in PBS, and filtered to make a stock at a concentration of 10 mg/mL. Thereafter, a 25 μg/mL extract sample was prepared using the stock for administration to cells, and 5 μm IO, V, and IV samples diluted in serum-free medium were prepared. In order to evaluate the concentration-dependent effect of AFWE among the extracts of Example 1, AFWE samples at concentrations of 1, 2.5, and 5 μg/mL were additionally prepared using AFWE stock. In addition, to use as a positive control, a centella asiatica ethanol extract known to improve skin moisturizing (Postepy Dermatologii Alergologii 2013: XXX, 1: 46-49) 5 μg/mL sample was prepared. In addition, the cell group not treated with the extract was used as a negative control.
<실시예 4> AFWE의 세포 독성 분석<Example 4> Cytotoxicity analysis of AFWE
AFWE의 세포 독성을 확인하기 위해 MTT assay를 수행하였다.MTT assay was performed to confirm the cytotoxicity of AFWE.
구체적으로, HaCaT 세포(4×104 cells/well)를 10% FBS를 포함하는 배양 배지가 든 96 웰 플레이트에 분주하고, 24시간 배양 후, 세포에 상기 실시예 3의 AFWE 샘플들을 각각 투여하였다. 24시간 배양한 후 배양액을 제거하고, 0.5 mg/mL의 MTT 용액을 첨가한 후 37℃, 5% CO2 조건에서 1시간 동안 배양하였다. 그 후, 각 웰에서 MTT 용액을 제거하고 100 ㎕ DMSO를 첨가한 후 microplate reader(Morecular Device, CA, USA)를 이용하여 570 nm에서 흡광도를 측정하였다.Specifically, HaCaT cells (4×10 4 cells/well) were seeded in a 96-well plate containing a culture medium containing 10% FBS, and after culturing for 24 hours, the AFWE samples of Example 3 were administered to the cells, respectively. . After culturing for 24 hours, the culture medium was removed, and 0.5 mg/mL of MTT solution was added, followed by incubation at 37°C and 5% CO 2 for 1 hour. Then, after removing the MTT solution from each well and adding 100 μl DMSO, the absorbance was measured at 570 nm using a microplate reader (Molecular Device, CA, USA).
그 결과, 도 2A에 나타낸 바와 같이, 정상 상태에서 AFWE 처리군은 음성 대조군(NCtr) 또는 양성 대조군(PCtr)과 비교해서 세포 독성이 나타나지 않음을 확인하였다.As a result, as shown in FIG. 2A, it was confirmed that the AFWE-treated group did not exhibit cytotoxicity compared to the negative control group (NCtr) or the positive control group (PCtr) under normal conditions.
<실시예 5> AFWE의 피부 보습 인자 조절 효과<Example 5> Skin moisturizing factor control effect of AFWE
AFWE가 피부 보습에 미치는 영향을 알아보기 위하여, HaCaT 세포에 AFWE 처리 후 피부 보습 인자로 피부 수화(hydration) 관련 단백질인 히알루로난(HA), HSA1, HYAL1 및 AQP3, 피부장벽 기능 관련 단백질인 필라그린(filaggrin)의 분비를 확인하였다.In order to investigate the effect of AFWE on skin moisturizing, HaCaT cells were treated with AFWE, and then hyaluronan (HA), HSA1, HYAL1, and AQP3, proteins related to skin hydration, and skin barrier function-related proteins, pillars, were used as skin moisturizing factors. Secretion of filaggrin was confirmed.
<실시예 5-1> AFWE의 히알루로난 분비 조절 효과<Example 5-1> Hyaluronan secretion regulating effect of AFWE
AFWE의 히알루로난(HA) 분비 조절 효과를 알아보기 위해 ELISA(Enzyme Linked-Immuo sorbent assay)를 수행하였다.ELISA (Enzyme Linked-Immuo sorbent assay) was performed to examine the effect of AFWE on the regulation of hyaluronan (HA) secretion.
구체적으로, 상기 실시예 4에 기재된 방법과 동일한 방법으로 HaCaT 세포에 AFWE를 처리 및 배양한 후, 상층액을 모아 제조사의 설명에 따라 ELISA를 수행하였다.Specifically, after treating and culturing HaCaT cells with AFWE in the same manner as described in Example 4, the supernatant was collected and ELISA was performed according to the manufacturer's instructions.
그 결과, 도 2B에 나타낸 바과 같이, AFWE의 농도(1, 2.5, 5 ㎍/ml)에 따라 히알루로난 분비가 농도 의존적으로 증가하였다.As a result, as shown in FIG. 2B, hyaluronan secretion increased in a concentration-dependent manner according to the concentration of AFWE (1, 2.5, or 5 μg/ml).
<실시예 5-2> AFWE의 HAS1, HYAL1 조절 효과<Example 5-2> HAS1, HYAL1 regulatory effect of AFWE
히알루로난 분비를 조절하는 HAS1 및 HYAL1에 대한 AFWE의 조절 효과를 알아보기 위해 Western blot 및 농도계 분석(densitometric analysis)을 수행하였다.To investigate the regulatory effect of AFWE on HAS1 and HYAL1 that regulate hyaluronan secretion, Western blot and densitometric analysis were performed.
구체적으로, 상기 실시예 4에 기재된 방법과 동일한 방법으로 HaCaT 세포에 AFWE를 처리 및 배양한 후 HaCaT 세포를 회수하였다. 회수한 HaCaT 세포를 차가운 PBS 용액으로 세척한 후 단백질분해효소(protease)와 인산가수분해효소 억제제(phosphatase inhibitor)가 포함된 PRO-PREPTM에 용해하여 단백질을 추출하였다. 추출한 단백질을 종전에 확립된 방식(Phytomedicine 28, 19-26)으로 Western blot 및 농도계 분석을 수행하였다(Enhanced chemiluminescence from ECL, detection kit from Intron., Sungnam, Korea).Specifically, after treating and culturing HaCaT cells with AFWE in the same manner as described in Example 4, HaCaT cells were recovered. The recovered HaCaT cells were washed with a cold PBS solution, and proteins were extracted by dissolving in PRO-PREP TM containing protease and phosphatase inhibitors. Western blot and densitometry analysis of the extracted proteins was performed according to a previously established method (Phytomedicine 28, 19-26) (Enhanced chemiluminescence from ECL, detection kit from Intron., Sungnam, Korea).
그 결과, 도 2C 내지 도 2E에서 나타낸 바와 같이, 음성 대조군(NCtr)과 양성 대조군(PCtr)과 비교해서 HAS1 발현은 AFWE의 농도에 따라 농도 의존적으로 증가한 반면, HYAL1은 양성 대조군에 비해 감소하였다.As a result, as shown in FIGS. 2C to 2E, compared to the negative control group (NCtr) and the positive control group (PCtr), HAS1 expression increased in a concentration dependent manner according to the concentration of AFWE, whereas HYAL1 decreased compared to the positive control group.
<실시예 5-3> AFWE의 AQP3 및 필라그린(filaggrin) 조절 효과<Example 5-3> Modulating effect of AFWE on AQP3 and filaggrin
AQP3 및 피부장벽 기능 조절에 관여하는 필라그린(filaggrin)에 대한 AFWE의 조절 효과를 알아보기 위해 Western blot 및 농도계 분석(densitometric analysis)을 수행하였다.Western blot and densitometric analysis were performed to investigate the regulatory effect of AFWE on AQP3 and filaggrin involved in the regulation of skin barrier function.
구체적으로, 상기 실시예 5-2에서 추출한 단백질을 종전에 확립된 방식(Phytomedicine 28, 19-26)으로 Western blot 및 농도계 분석을 수행 하였다(Enhanced chemiluminescence from ECL, detection kit from Intron., Sungnam, Korea).Specifically, the protein extracted in Example 5-2 was subjected to Western blot and densitometric analysis (Enhanced chemiluminescence from ECL, detection kit from Intron., Sungnam, Korea) in a previously established manner (Phytomedicine 28, 19-26). ).
그 결과, 도 2C 및 도 2F에 나타낸 바와 같이, AFWE의 농도에 따라 AQP3의 발현이 농도 의존적으로 증가하였다.As a result, as shown in Figures 2C and 2F, the expression of AQP3 increased in a concentration-dependent manner according to the concentration of AFWE.
또한, 도 3A 및 도 3C에 나타낸 바와 같이, AFWE의 농도에 따라 필라그린의 발현도 양성 대조군에 비해 농도 의존적으로 증가하였다.In addition, as shown in Figures 3A and 3C, the expression of filaggrin was also increased in a concentration-dependent manner compared to the positive control according to the concentration of AFWE.
<실시예 6> AFWE의 PKC와 MAPK 신호전달경로를 통한 인볼루크린(involucrin) 발현 조절<Example 6> Regulation of involucrin expression through the PKC and MAPK signaling pathways of AFWE
AFWE의 피부장벽 기능 관련 단백질인 인볼루크린(involucrin) 조절 기전을 알아보기 위해 아래의 분석을 실시하였다.The following analysis was conducted to investigate the regulation mechanism of involucrin, a protein related to the skin barrier function of AFWE.
<실시예 6-1> AFWE의 인볼루크린 발현 조절<Example 6-1> Regulation of involucrin expression of AFWE
AFWE에 의한 인볼루크린 조절 효과를 알아보기 위해 Western blot 및 농도계 분석(densitometric analysis)을 수행하였다.Western blot and densitometric analysis were performed to examine the effect of involucrine regulation by AFWE.
구체적으로, 상기 실시예 5-2에서 추출한 단백질을 종전에 확립된 방식(Phytomedicine 28, 19-26)으로 Western blot 및 농도계 분석을 수행 하였다(Enhanced chemiluminescence from ECL, detection kit from Intron., Sungnam, Korea).Specifically, the protein extracted in Example 5-2 was subjected to Western blot and densitometric analysis (Enhanced chemiluminescence from ECL, detection kit from Intron., Sungnam, Korea) in a previously established manner (Phytomedicine 28, 19-26). ).
그 결과, 도 3A 및 도 3B에 나타낸 바와 같이, AFWE에 의해 인볼루크린의 발현이 농도 의존적으로 증가한다는 것을 확인하였다.As a result, as shown in FIGS. 3A and 3B , it was confirmed that the expression of involucrin was increased in a concentration-dependent manner by AFWE.
<실시예 6-2> AFWE의 mRNA 수준에서의 인볼루크린 조절<Example 6-2> Involucrin regulation at the mRNA level of AFWE
AFWE에 의한 mRNA 수준에서 인볼루크린 조절 효과를 알아보기 위해 qRT-PCR(Quantitative real time-reverse transcription-polymerase chain reaction)을 수행하였다.Quantitative real time-reverse transcription-polymerase chain reaction (qRT-PCR) was performed to examine the effect of involucrin regulation on the mRNA level by AFWE.
구체적으로, 상기 실시예 5-2에서 회수한 HaCaT 세포에서 RNA 추출 키트(KeyGEN BioTECH, Nanjing, China)를 이용하여 mRNA를 추출하였다. 상기 추출한 mRNA로부터 cDNA를 합성하고, PrimeScript RT regent 키트(Takara, Beijing, China)를 사용하여 제조사의 설명에 따라 qRT-PCR을 수행하였다. qRT-PCR에 사용된 프라이머 서열은 다음과 같다: IVL, forward: 5'-GTGGGGGAGAGAGGGAATTA-3'; reverse, 5'-CTCACCTGAGGTTGGGATTG-3'; GAPDH, foward: 5'-TCCACTGGCGTCTTCACC-3', reverse: 5'-GGCAGAGATGATGACCCTTTT-3'.Specifically, mRNA was extracted from the HaCaT cells recovered in Example 5-2 using an RNA extraction kit (KeyGEN BioTECH, Nanjing, China). cDNA was synthesized from the extracted mRNA, and qRT-PCR was performed using the PrimeScript RT agent kit (Takara, Beijing, China) according to the manufacturer's instructions. The primer sequences used for qRT-PCR were as follows: IVL, forward: 5'-GTGGGGGAGAGAGGGAATTA-3'; reverse, 5′-CTCACCTGAGGTTGGGATTG-3′; GAPDH, forward: 5′-TCCACTGGCGTCTTCACC-3′, reverse: 5′-GGCAGAGATGATGACCCTTTT-3′.
그 결과, 도 3D에 나타낸 바와 같이, AFWE 처리군과 양성 대조군의 인볼루크린 mRNA 발현 수준이 유사한 것을 확인하였다.As a result, as shown in FIG. 3D, it was confirmed that the involucrin mRNA expression levels of the AFWE-treated group and the positive control group were similar.
<실시예 6-3> PKC와 MAPK 신호전달경로를 통한 인볼루크린 발현 조절<Example 6-3> Regulation of involucrin expression through PKC and MAPK signaling pathway
인볼루크린의 발현 조절에서 PKC의 활성화와 P38, MAPK의 ERK1/2 신호전달경로가 중요한 역할을 한다. 이에, AFWE가 PKC와 MAPK 신호전달경로에 미치는 영향을 알아보기 위해 Western blot 및 농도계 분석(densitometric analysis)을 수행하였다.In regulating the expression of involucrin, the activation of PKC, P38, and the ERK1/2 signaling pathway of MAPK play an important role. Therefore, Western blot and densitometric analysis were performed to investigate the effect of AFWE on the PKC and MAPK signaling pathways.
구체적으로, 상기 실시예 5-2에서 추출한 단백질을 종전에 확립된 방식(Phytomedicine 28, 19-26)으로 Western blot 및 농도계 분석을 수행하였다(Enhanced chemiluminescence from ECL, detection kit from Intron., Sungnam, Korea).Specifically, Western blot and densitometry analysis was performed on the protein extracted in Example 5-2 in a previously established manner (Phytomedicine 28, 19-26) (Enhanced chemiluminescence from ECL, detection kit from Intron., Sungnam, Korea ).
그 결과, 도 3E 및 도 3F에 나타낸 바와 같이, 양성 대조군과 비교하여 AFWE 처리군에서 PKC 발현이 증가하였다.As a result, as shown in Figures 3E and 3F, PKC expression increased in the AFWE-treated group compared to the positive control group.
또한, 도 3E, 도 3G 및 도 3H에 나타낸 바와 같이, AFWE 처리군에서 음성 대조군에 비해 P38 인산화 및 ERK1/2 탈인산화가 증가하는 것을 확인하였다.In addition, as shown in Figures 3E, 3G and 3H, it was confirmed that P38 phosphorylation and ERK1/2 dephosphorylation increased in the AFWE-treated group compared to the negative control group.
<실시예 7> UVB 조건에서 AFWE의 피부 보습 향상 효과<Example 7> Skin moisturizing improvement effect of AFWE under UVB conditions
UVB 노출은 피부 손상을 유발하고, 이로 인해 피부 보습이 저하된다. 이에, UVB 조건에서 AFWE가 피부 보습 인자 발현을 유도하여 UVB로 인한 피부 손상으로 저하된 피부 보습을 향상시킬 수 있는지 확인하기 위해 아래의 분석을 실시하였다.UVB exposure causes skin damage, which reduces skin moisturization. Accordingly, the following analysis was conducted to confirm whether AFWE could induce skin moisturizing factor expression under UVB conditions to improve skin moisturizing caused by UVB-induced skin damage.
<실시예 7-1> UVB 조사<Example 7-1> UVB irradiation
HaCaT 세포(4×104 cells/well)를 10% FBS를 포함하는 배양 배지가 든 96 웰 플레이트에 분주하고, 24시간 배양 후, UVB 조사기계(Bio-Link BLX-312; Vilber Lourmat GmbH, Marne-la-Vallee, France)를 사용해 종전의 방식(Pharmaceutical Biology, 55:1, 1032-1040)으로 UVB(200 mj/cm2)를 조사하였다. 그 후, 즉시 상기 실시예 1의 AFWE 추출물을 처리하고, 24시간 배양하였다. UVB 조사하지 않은 세포는 음성 대조군으로 사용하였다.HaCaT cells (4 × 10 4 cells/well) were dispensed into a 96-well plate containing a culture medium containing 10% FBS, and after culturing for 24 hours, a UVB irradiation machine (Bio-Link BLX-312; Vilber Lourmat GmbH, Marne -la-Vallee, France) was irradiated with UVB (200 mj/cm 2 ) in a conventional manner (Pharmaceutical Biology, 55:1, 1032-1040). After that, the AFWE extract of Example 1 was immediately treated and cultured for 24 hours. Cells not irradiated with UVB were used as a negative control.
<실시예 7-2> UVB 조건에서 AFWE의 세포독성 분석<Example 7-2> Cytotoxicity analysis of AFWE under UVB conditions
UVB 조건에서 AFWE의 세포 독성을 확인하기 위해 MTT assay를 수행하였다.MTT assay was performed to confirm the cytotoxicity of AFWE under UVB conditions.
구체적으로, 상기 실시예 7-2에서 AFWE 추출물 처리 및 배양 완료 후, 상기 실시예 4에 개시된 방법과 동일한 방법으로 MTT assay를 수행하였다.Specifically, after completion of the treatment and culture of the AFWE extract in Example 7-2, the MTT assay was performed in the same manner as described in Example 4.
그 결과, 도 4A에 나타낸 바와 같이, UVB 조건에서 AFWE의 세포 독성이 나타나지 않음을 확인하였다.As a result, as shown in FIG. 4A, it was confirmed that AFWE did not exhibit cytotoxicity under UVB conditions.
또한, 도 5에 나타낸 바와 같이, UVB 조건에서 AFWE 처리군은 세포 형태학적 변화를 보이지 않았다.In addition, as shown in Figure 5, the AFWE-treated group under UVB conditions showed no change in cell morphology.
<실시예 7-3> UVB 조건에서 AFWE의 피부 보습 인자 조절 효과 확인<Example 7-3> Confirmation of skin moisturizing factor control effect of AFWE under UVB conditions
UVB 조건에서 AFWE에 의한 피부 보습 인자 조절 효과를 알아보기 위해 Western blot 및 농도계 분석(densitometric analysis)을 수행하였다.Western blot and densitometric analysis were performed to investigate the effect of AFWE on skin moisturizing factor regulation under UVB conditions.
구체적으로, 상기 실시예 7-2에서 AFWE 추출물 처리 및 배양 완료 후, 상기 실시예 5에 기재된 방법과 동일한 방법으로 Western blot 및 농도계 분석을 수행하였다.Specifically, after completion of AFWE extract treatment and culture in Example 7-2, Western blot and densitometric analysis were performed in the same manner as described in Example 5.
그 결과, 도 4B 내지 도 4G에 나타난 바와 같이, AFWE 처리군에서는 정상 조건에서 관찰된 것과 유사하게 인볼루크린 발현이 농도 의존적으로 증가하였다. 뿐만 아니라 HAS1 발현은 농도 의존적으로 증가하고, HYAL1 발현은 농도 의존적으로 감소하였다. 또한, 필라그린 및 AQP3 발현이 증가하였다. As a result, as shown in FIGS. 4B to 4G , involucrin expression was increased in a concentration-dependent manner similar to that observed in the normal condition in the AFWE-treated group. In addition, HAS1 expression increased in a concentration-dependent manner, and HYAL1 expression decreased in a concentration-dependent manner. In addition, filaggrin and AQP3 expression were increased.
<실시예 8> 추출용매에 따른 알로에베라 꽃 추출물의 피부 보습 효과 비교<Example 8> Comparison of skin moisturizing effect of aloe vera flower extract according to the extraction solvent
추출용매에 따른 알로에베라 꽃 추출물의 피부 보습 효과를 비교하기 위하여, MTT assay, Western blot 및 농도계 분석(densitometric analysis)을 수행하였다.In order to compare the skin moisturizing effect of the aloe vera flower extract according to the extraction solvent, MTT assay, Western blot and densitometric analysis were performed.
구체적으로, HaCaT 세포(4×104 cells/well)를 10% FBS를 포함하는 배양 배지가 든 96 웰 플레이트에 분주하고, 24시간 배양 후, 세포에 상기 실시예 3의 샘플들을 각각 투여하였다. 그 후, 실시예 4에 기재된 방법과 동일한 방법으로 MTT assay를 수행하고, 실시예 5에 기재된 방법과 동일한 방법으로 Western blot 및 농도계 분석을 수행하였다.Specifically, HaCaT cells (4×10 4 cells/well) were seeded in a 96-well plate containing a culture medium containing 10% FBS, and after culturing for 24 hours, the samples of Example 3 were administered to the cells, respectively. Thereafter, MTT assay was performed in the same manner as in Example 4, and Western blot and densitometric analysis were performed in the same manner as in Example 5.
그 결과, 도 6A에 나타낸 바와 같이, 알로에베라 꽃 100% 에탄올 추출물(AE)를 제외하고, 50% 에탄올 추출물(EE) 및 수 추출물(AFWE)에서 세포 독성의 없음을 확인하였다.As a result, as shown in FIG. 6A, it was confirmed that there was no cytotoxicity in the 50% ethanol extract (EE) and the water extract (AFWE), except for the 100% ethanol extract (AE) of Aloe vera flowers.
또한, 도 6B 내지 도 6H에 나타낸 바와 같이, 3종의 추출물 모두 피부 보습 인자 조절에 있어서 유사한 양상을 보였고, 이 중 AFWE가 피부 보습 인자 조절 효과가 가장 우수한 것으로 나타났다.In addition, as shown in Figures 6B to 6H, all three extracts showed similar aspects in skin moisturizing factor control, and among them, AFWE was found to have the best skin moisturizing factor control effect.
<실시예 9> 알로에베라 꽃 추출물에 함유된 유효성분들의 피부 보습 효과 비교<Example 9> Comparison of skin moisturizing effect of active ingredients contained in aloe vera flower extract
알로에베라 꽃 추출물에서 함유된 유효성분들의 기능과 피부 보습 효과를 비교하기 위해 아래의 분석을 실시하였다.The following analysis was conducted to compare the function of the active ingredients contained in the aloe vera flower extract and the skin moisturizing effect.
<실시예 9-1> 알로에베라 꽃 추출물에 함유된 유효성분들의 세포 독성 분석<Example 9-1> Cytotoxicity analysis of active ingredients contained in aloe vera flower extract
실시예 2에서 확인한 유효성분 IO, V, IV의 세포 독성을 확인하기 위해 MTT assay를 수행하였다.MTT assay was performed to confirm the cytotoxicity of the active ingredients IO, V, and IV confirmed in Example 2.
구체적으로, HaCaT 세포(4×104 cells/well)를 10% FBS를 포함하는 배양 배지가 든 96 웰 플레이트에 분주하고, 24시간 배양 후, 세포에 상기 실시예 3의 IO, V, IV 샘플들을 각각 투여하였다. 그 다음, 24시간 배양한 후 실시예 4에 기재된 방법과 동일한 방법으로 MTT assay를 수행하였다.Specifically, HaCaT cells (4×10 4 cells/well) were seeded in a 96-well plate containing a culture medium containing 10% FBS, and after culturing for 24 hours, the IO, V, and IV samples of Example 3 were added to the cells. were administered respectively. Then, after culturing for 24 hours, MTT assay was performed in the same manner as described in Example 4.
그 결과, 도 7A에 나타낸 바와 같이, IO, V, IV 처리군은 음성 대조군과 비교해 세포 생존력이 유사함을 확인하였다.As a result, as shown in FIG. 7A, it was confirmed that the cell viability of the IO, V, and IV treatment groups was similar to that of the negative control group.
<실시예 9-2> 알로에베라 꽃 추출물에 함유된 유효성분들의 피부 보습 인자 조절 효과 비교<Example 9-2> Comparison of skin moisturizing factor control effect of active ingredients contained in aloe vera flower extract
실시예 2에서 확인한 유효성분 IO, V, IV에 의한 피부 보습 인자 조절 효과를 비교하기 위하여 Western blot 및 농도계 분석(densitometric analysis)을 수행하였다.Western blot and densitometric analysis were performed to compare the effect of regulating skin moisturizing factors by the active ingredients IO, V, and IV confirmed in Example 2.
구체적으로, 상기 실시예 9-1에서 각각의 샘플 처리 및 배양 완료한 세포를 회수하였다. 그 다음, 회수한 세포를 이용하여 상기 실시예 5에 기재된 방법과 동일한 방법으로 Western blot 및 농도계 분석을 수행하였다.Specifically, cells that had been treated and cultured for each sample in Example 9-1 were recovered. Then, using the recovered cells, Western blot and densitometric analysis were performed in the same manner as described in Example 5 above.
그 결과, 도 7B 내지 도 7H에 나타낸 바와 같이, 음성 대조군에 비해 IO 처리군 및 IV 처리군에서 인볼루크린의 발현이 증가하는 것을 확인하였다. 또한, IO 처리군, V 처리군 및 IV 처리군에서 HYAL1의 발현이 감소하여 히알루로난 분비가 증가한 것을 확인하였다. 또한, IO 처리군, V 처리군 및 IV 처리군에서 필라그린의 발현이 유의적으로 증가하는 것을 확인하였다. 특히, IO 처리군에서 상기 피부 보습 인자들의 조절 효과가 가장 우수한 것을 확인하였다.As a result, as shown in FIGS. 7B to 7H , it was confirmed that the expression of involucrin increased in the IO-treated group and the IV-treated group compared to the negative control group. In addition, it was confirmed that hyaluronan secretion increased as the expression of HYAL1 decreased in the IO-treated group, the V-treated group, and the IV-treated group. In addition, it was confirmed that the expression of filaggrin significantly increased in the IO-treated group, the V-treated group, and the IV-treated group. In particular, it was confirmed that the control effect of the skin moisturizing factors was the most excellent in the IO treatment group.
<실시예 10> 알로에베라 꽃 추출물에 함유된 유효성분 IO의 피부 보습 인자 결합친화도 분석<Example 10> Analysis of skin moisturizing factor binding affinity of active ingredient IO contained in aloe vera flower extract
상기 실시예 9-2에서 피부 보습 인자 조절 효과가 가장 우수한 IO와 피부 보습 인자 간 결합 친화도를 알아보기 위해, IO와 피부 보습 인자에 대해 Molecular docking study를 실행하였다.In order to examine the binding affinity between IO and skin moisturizing factor, which have the most excellent skin moisturizing factor control effect in Example 9-2, a molecular docking study was performed on IO and skin moisturizing factor.
구체적으로, IO와 피부 보습 인자 간 분자결합은 종전의 방법(Phytomedicine 31, 1-9)에 따라 AutoDock Vina를 사용하여 확인하였다.Specifically, the molecular binding between IO and skin moisturizing factors was confirmed using AutoDock Vina according to the previous method (Phytomedicine 31, 1-9).
그 결과, 도 8 내지 도 12에 나타낸 바와 같이, IO와 피부 보습 인자 간 강한 결합 친화도를 나타내는 것을 확인하였다.As a result, as shown in Figs. 8 to 12, it was confirmed that the IO and the skin moisturizing factor exhibit strong binding affinity.
상기의 결과를 통해 도 13의 모식도와 같이 본 발명의 알로에베라 꽃 추출물 및 이로부터 유래된 화합물이 피부 보습 인자, 구체적으로 피부장벽 기능 관련 단백질 및 피부 수화(hydration) 관련 단백질을 조절하여 피부 보습 효과를 나타냄을 알 수 있다. 또한, UVB에 의한 피부 손상으로 저하된 피부 보습 향상에서 우수한 효과를 나타냄을 알 수 있다.Through the above results, as shown in the schematic diagram of FIG. 13, the aloe vera flower extract of the present invention and the compound derived therefrom modulates skin moisturizing factors, specifically proteins related to skin barrier function and proteins related to skin hydration, and has a skin moisturizing effect. It can be seen that it represents In addition, it can be seen that it exhibits an excellent effect in improving skin moisturization reduced by skin damage caused by UVB.
Claims (13)
상기 화합물은 이소오리엔틴(Isoorientin), 비텍신(Vitexin) 및 이소비텍신(Isovitexin)으로 이루어진 군으로부터 선택되는 1종 이상인, 피부 보습용 화장료 조성물.
A cosmetic composition for moisturizing skin containing a compound isolated from an Aloe vera flower extract as an active ingredient,
The compound is at least one selected from the group consisting of isoorientin, vitexin, and isovitexin, a cosmetic composition for moisturizing the skin.
상기 알로에베라 꽃 추출물은 물, C1 내지 C4의 저급 알코올 또는 이들의 혼합용매로 추출한 것인, 피부 보습용 화장료 조성물.
The method of claim 1,
The aloe vera flower extract is extracted with water, C 1 to C 4 lower alcohol or a mixed solvent thereof, a cosmetic composition for moisturizing the skin.
상기 알로에베라 꽃 추출물은 물로 추출한 것인, 피부 보습용 화장료 조성물.
The method of claim 1,
The aloe vera flower extract is extracted with water, a cosmetic composition for moisturizing the skin.
상기 조성물은 피부 보습 인자를 조절하는 것인, 피부 보습용 화장료 조성물.
The method of claim 1,
The composition is to adjust the skin moisturizing factor, a cosmetic composition for moisturizing the skin.
상기 피부 보습 인자는 피부 수화(hydration) 관련 단백질 또는 피부장벽 기능 관련 단백질인, 피부 보습용 화장료 조성물.
The method of claim 6,
The skin moisturizing factor is a skin hydration related protein or a skin barrier function related protein, a cosmetic composition for moisturizing the skin.
상기 피부 수화 관련 단백질은 HAS1, HYAL1, 히알루로난 또는 AQP3이고, 상기 조성물은 HAS1, 히알루로난 또는 AQP3 발현을 증가시키고, HYAL1 발현을 감소시키는 것인, 피부 보습용 화장료 조성물.
The method of claim 7,
The skin hydration-related protein is HAS1, HYAL1, hyaluronan or AQP3, and the composition increases HAS1, hyaluronan or AQP3 expression, and reduces HYAL1 expression, a cosmetic composition for moisturizing the skin.
상기 피부장벽 기능 관련 단백질은 인볼루크린(involucrin) 또는 필라그린(filaggrin)이고, 상기 조성물은 인볼루크린 또는 필라그린 발현을 증가시키는 것인, 피부 보습용 화장료 조성물.
The method of claim 7,
The skin barrier function-related protein is involucrin or filaggrin, and the composition increases the expression of involucrin or filaggrin, a cosmetic composition for moisturizing the skin.
상기 조성물은 UVB로 인한 피부 손상으로 저하된 피부 보습을 향상하는 것인, 피부 보습용 화장료 조성물.
The method of claim 1,
The composition is a cosmetic composition for skin moisturizing, which improves skin moisturizing reduced by skin damage caused by UVB.
상기 화합물은 이소오리엔틴, 비텍신 및 이소비텍신으로 이루어진 군으로부터 선택되는 1종 이상인, 피부 보습용 의약외품.
A quasi-drug for skin moisturizing containing a compound isolated from an aloe vera flower extract as an active ingredient,
The compound is at least one selected from the group consisting of isoorientin, vitexin and isobitexin, a quasi-drug for skin moisturizing.
상기 화합물은 이소오리엔틴, 비텍신 및 이소비텍신으로 이루어진 군으로부터 선택되는 1종 이상인, 피부 보습용 피부 외용제.
As an external skin preparation for skin moisturizing, containing a compound isolated from an aloe vera flower extract as an active ingredient,
The compound is at least one selected from the group consisting of isoorientin, bitexin and isobitexin, external skin preparation for skin moisturizing.
상기 화합물은 이소오리엔틴, 비텍신 및 이소비텍신으로 이루어진 군으로부터 선택되는 1종 이상인, 피부 보습용 식품 조성물.
A food composition for moisturizing the skin, containing a compound isolated from an aloe vera flower extract as an active ingredient,
The compound is at least one selected from the group consisting of isoorientin, bitexin and isobitexin, a food composition for moisturizing the skin.
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| PCT/KR2022/006170 WO2022250313A1 (en) | 2021-05-27 | 2022-04-29 | Composition for moisturizing skin, promoting skin regeneration, and treating wounds, comprising aloe vera flower extract, or aloe vera flower extract and aloe vera polysaccharides, as active ingredient(s) |
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| KR100720973B1 (en) * | 2005-03-18 | 2007-05-22 | 주식회사 유니젠 | Composition comprising isoorientin for suppressing histamine |
| CN108635301A (en) * | 2018-07-25 | 2018-10-12 | 云南万绿生物股份有限公司 | A kind of aloe flower extract and preparation method thereof |
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| KR102181893B1 (en) * | 2018-12-31 | 2020-11-23 | 대봉엘에스 주식회사 | Cosmetic and pharmaceutical compositin comprising aloe extract, upland rice extract and ceramide |
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| CN108635301A (en) * | 2018-07-25 | 2018-10-12 | 云南万绿生物股份有限公司 | A kind of aloe flower extract and preparation method thereof |
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