KR102646678B1 - An antioxidant, anti-inflammatory, or moisturizing cosmetic composition containing Liquidambaris Fructus extract as an active ingredient - Google Patents

An antioxidant, anti-inflammatory, or moisturizing cosmetic composition containing Liquidambaris Fructus extract as an active ingredient Download PDF

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KR102646678B1
KR102646678B1 KR1020230171792A KR20230171792A KR102646678B1 KR 102646678 B1 KR102646678 B1 KR 102646678B1 KR 1020230171792 A KR1020230171792 A KR 1020230171792A KR 20230171792 A KR20230171792 A KR 20230171792A KR 102646678 B1 KR102646678 B1 KR 102646678B1
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강경아
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/007—Preparations for dry skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95

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Abstract

천연 추출물을 함유한 화장료 조성물로서 동물실험 및 세포실험을 통해 항산화, 항염증 및 보습 효과가 검증된 화장료 조성물이 개시된다. 본 발명은 노로통 추출물을 유효성분으로 포함하는 항산화, 항염증 또는 보습용 화장료 조성물을 제공한다.Disclosed is a cosmetic composition containing natural extracts whose antioxidant, anti-inflammatory and moisturizing effects have been verified through animal and cell experiments. The present invention provides an antioxidant, anti-inflammatory, or moisturizing cosmetic composition containing a Norotong extract as an active ingredient.

Description

노로통 추출물을 유효성분으로 포함하는 항산화, 항염증 또는 보습용 화장료 조성물{An antioxidant, anti-inflammatory, or moisturizing cosmetic composition containing Liquidambaris Fructus extract as an active ingredient}An antioxidant, anti-inflammatory, or moisturizing cosmetic composition containing Liquidambaris Fructus extract as an active ingredient}

본 발명은 천연 추출물을 포함하는 화장료 조성물에 관한 것으로, 보다 상세하게는 노로통 추출물을 유효성분으로 포함하는 항산화, 항염증 또는 보습용 화장료 조성물에 관한 것이다.The present invention relates to a cosmetic composition containing natural extracts, and more specifically, to a cosmetic composition for antioxidant, anti-inflammatory or moisturizing purposes containing norotong extract as an active ingredient.

염증은 외부 유기체, 즉 병원균, 바이러스, 독성 물질 등으로부터 우리 몸을 보호하기 위한 생리적 반응이다(비특허문헌 1 참조). 최근 연구에서는 염증이 비만, 당뇨병, 심혈관 질환, 자가면역 질환, 염증성 장 질환, 관절염 및 암과 같은 다양한 만성 질병의 진행에서 중요한 역할을 하는 것으로 보고되었다(비특허문헌 2 참조). 염증은 주로 다양한 세포 기전과 염증 과정에 따라 급성과 만성으로 나뉘며, 급성 염증은 몇 분부터 수일까지 지속되는 혈관 외부 영역으로의 혈장 단백질 또는 액체의 유출과 백혈구의 이동이 일어나는 것에 특징이 있다(비특허문헌 3 및 4 참조). 또한 감염 유형(세균, 바이러스, 기생충 등)에 따라 대상 조직, 센서 및 매개체에서 염증 반응이 다르게 나타난다(비특허문헌 5 참조). 그 중 미생물이 생성하는 PAMPs(pathogen-associated molecular patterns)은 TLRs (Toll-like receptors)와 같은 선천적 면역 시스템의 수용체를 통해 인식된다 (비특허문헌 6 참조). 또한 손상된 세포나 죽은 세포에서 방출되는 일부 DAMPs (damage-associated molecular patterns)은 TLR을 포함한 PRR(pattern recognition receptors)에 결합한다(비특허문헌 7 및 8 참조). PAMPs의 가장 잘 알려진 예는 LPS(lipopolysaccharide)이다. LPS는 그램-음성균 세포벽의 외부에서 발견되며 일반적으로 내독소로 알려져 있다(비특허문헌 9 참조). LPS를 포함한 PAMPs에 의한 PRR의 참여는 염증 경로를 활성화한다(비특허문헌 10 참조). 염증 반응의 개시와 연관되는 대식세포(macrophage)는 선천적 면역 및 후천적 면역 모두에서 중요한 역할을 한다(비특허문헌 11 참조). 대식세포는 미생물 위협에 대한 반응으로, 병원체 제거 및 조직 수복를 위한 다양한 분자를 생성한다 (비특허문헌 12 참조). 순환하는 단핵구(monocyte)에서 유래한 대식세포가 손상된 조직으로 모여서, 노출된 자극의 패턴에 따라 다른 표현형을 나타내는 것으로 알려져 있다. 이를 토대로, 대식세포는 염증성 대식세포(M1 표현형) 또는 항염증/상처 치유 대식세포(M2 표현형)로 크게 분류된다. 이는 대식세포의 폭넓은 다양성과 가소성을 볼 수 있다(비특허문헌 13 내지 15 참조). 특히 NF-кB(nuclear factor-kappa B) 또는 MAPK(mitogen-activated protein kinase) 경로와 관련된 M1 표현형은 염증성 사이토카인(cytokine)인 TNFα(tumor necrosis factor alpha), IL-1β(interleukin-1β), 키모카인(chemokine) 및 산화 질소(nitric oxide, NO)의 생성을 자극한다(비특허문헌 16 참조). 지금까지 수행된 연구는 NF-кB 또는 MAPK 경로와 같은 두 주요 염증 경로의 억제로 질병 진행이 완화되었음을 명확히 보여주었다(비특허문헌 17 내지 18 참조).Inflammation is a physiological response to protect our body from external organisms, such as pathogens, viruses, and toxic substances (see Non-Patent Document 1). Recent studies have reported that inflammation plays an important role in the progression of various chronic diseases such as obesity, diabetes, cardiovascular disease, autoimmune disease, inflammatory bowel disease, arthritis, and cancer (see Non-Patent Document 2). Inflammation is mainly divided into acute and chronic depending on various cellular mechanisms and inflammatory processes. Acute inflammation is characterized by the outflow of plasma proteins or fluid and the migration of white blood cells to the extravascular area, lasting from several minutes to several days. (see Patent Documents 3 and 4). Additionally, inflammatory responses appear differently in target tissues, sensors, and mediators depending on the type of infection (bacteria, virus, parasite, etc.) (see Non-Patent Document 5). Among them, PAMPs (pathogen-associated molecular patterns) produced by microorganisms are recognized through receptors of the innate immune system, such as TLRs (Toll-like receptors) (see Non-Patent Document 6). Additionally, some DAMPs (damage-associated molecular patterns) released from damaged or dead cells bind to PRRs (pattern recognition receptors), including TLRs (see Non-Patent Documents 7 and 8). The best known example of PAMPs is lipopolysaccharide (LPS). LPS is found outside the cell wall of Gram-negative bacteria and is generally known as an endotoxin (see Non-Patent Document 9). Participation of PRRs by PAMPs, including LPS, activates inflammatory pathways (see Non-Patent Document 10). Macrophages, which are associated with the initiation of inflammatory reactions, play an important role in both innate and adaptive immunity (see Non-Patent Document 11). In response to microbial threats, macrophages produce various molecules for pathogen removal and tissue repair (see Non-Patent Document 12). It is known that macrophages derived from circulating monocytes gather in damaged tissues and exhibit different phenotypes depending on the pattern of stimulation to which they are exposed. Based on this, macrophages are broadly classified into inflammatory macrophages (M1 phenotype) or anti-inflammatory/wound healing macrophages (M2 phenotype). This shows the wide diversity and plasticity of macrophages (see Non-Patent Documents 13 to 15). In particular, the M1 phenotype related to the nuclear factor-kappa B (NF-кB) or mitogen-activated protein kinase (MAPK) pathway is associated with inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β), Stimulates the production of chemokines and nitric oxide (NO) (see Non-Patent Document 16). Studies conducted so far have clearly shown that disease progression is alleviated by inhibition of two major inflammatory pathways, such as the NF-кB or MAPK pathway (see Non-Patent Documents 17 to 18).

최근 연구에 따르면 활성산소종(reactive oxygen species, ROS)의 과도한 생성이 산화 스트레스를 일으켜, 염증 반응 진행에 중요한 역할을 하는 것으로 알려져 있다(비특허문헌 19 내지 20 참조). ROS는 적어도 하나의 산소 원자와 하나 이상의 홀전자를 포함하는 산소 자유 라디칼이다. 손상된 영역에는 백혈구(Leukocyte) 및 비만 세포(mast cell)가 존재하며 "호흡 폭발(respiratory burst)"의 결과로 산소 흡수가 증가하고 따라서 염증 반응 중에 ROS의 생성 및 방출이 증가한다(비특허문헌 21 내지 22 참조). 특히 산화 에너지 대사 과정에서 부산물로 생성된 ROS는 신경퇴행성 질환, 허혈/재관류 손상, 폐쇄성 수면 무호흡, 동맥경화, 당뇨병, 암, 류마티스 관절염 등의 질병의 발병과 관련이 있는 것으로 알려져 있다(비특허문헌 23 참조).According to recent studies, excessive production of reactive oxygen species (ROS) causes oxidative stress and is known to play an important role in the progression of inflammatory reactions (see Non-Patent Documents 19 to 20). ROS are oxygen free radicals containing at least one oxygen atom and one or more unpaired electrons. Leukocytes and mast cells are present in the damaged area, and oxygen uptake increases as a result of a “respiratory burst,” thus increasing the production and release of ROS during the inflammatory response (Non-patent Document 21 to 22). In particular, ROS generated as a by-product during oxidative energy metabolism is known to be associated with the development of diseases such as neurodegenerative diseases, ischemia/reperfusion injury, obstructive sleep apnea, arteriosclerosis, diabetes, cancer, and rheumatoid arthritis (non-patent literature) 23).

피부는 인체에서 가장 외부에 위치하여 병원성 세균, 오염물질 등의 침입을 차단하는 선천성 면역계의 물리적 장벽 역할을 하며, 피부에서 이러한 물리적 장벽 역할을 원활하게 수행하기 위해서는 피부의 수분 함량이 적절히 유지되는 것이 중요하다.The skin is the outermost part of the human body and serves as a physical barrier to the innate immune system, blocking the invasion of pathogenic bacteria and pollutants. In order for the skin to function as a physical barrier smoothly, the skin's moisture content must be properly maintained. It is important.

노로통(Liquidambaris Fructus, LF)은 중국 약초에서 Lu Lu Tong로도 불리는 것으로, Liquidambar Formosana Hance의 건조한 열매를 말한다. LF는 유방 기능뿐만 아니라 간 및 족양명위경(stomach meridian)과도 관련이 깊다고 알려져 있다(비특허문헌 24 참조). 따라서 LF는 모유분비부전증(hypogalactia), 유선 증식, 유선염 및 유선관 확장증과 같은 일부 유방 질환을 치료하는 데 사용되었다(비특허문헌 25 참조). 이전 연구에서는 LF에서 추출된 베틸로닉산(betulonic acid) 및 트리테르페노이드(triterpenoid)와 같은 효과적인 성분이 인간 유방암 세포의 증식을 억제하고 세포 주기 중단을 통한 세포사멸 캐스케이드(apoptosis cascade)를 활성화한다고 보고되었다(비특허문헌 26 참조). 또한 LF는 관절통을 완화하는 데 사용되는 것으로 보고되었다(비특허문헌 27 참조). Norotong (Liquidambaris Fructus, LF), also called Lu Lu Tong in Chinese herbal medicine, refers to the dried fruit of Liquidambar Formosana Hance. LF is known to be closely related not only to breast function but also to the liver and stomach meridian (see Non-Patent Document 24). Therefore, LF has been used to treat some breast diseases such as hypogalactia, mammary gland hyperplasia, mastitis, and mammary duct ectasia (see Non-Patent Document 25). Previous studies have shown that effective components such as betulonic acid and triterpenoids extracted from LF inhibit the proliferation of human breast cancer cells and activate the apoptosis cascade through cell cycle arrest. It was reported (see non-patent document 26). Additionally, LF has been reported to be used to relieve joint pain (see Non-Patent Document 27).

그러나 노로통의 염증 및 피부 보습에 미치는 영향에 대한 구체적인 효과 및 그 기전에 관한 연구는 알려진 바가 없다.However, there is no known research on the specific effects and mechanisms of inflammation and skin moisturizing in norotonia.

[비특허문헌][Non-patent literature]

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본 발명은 천연 추출물을 함유한 화장료 조성물로서 동물실험을 통해 항산화, 항염증 및 보습 효과가 검증된 화장료 조성물을 제공하고자 한다.The present invention seeks to provide a cosmetic composition containing natural extracts whose antioxidant, anti-inflammatory and moisturizing effects have been verified through animal testing.

상기 과제를 해결하기 위하여 본 발명은, 노로통 추출물을 유효성분으로 포함하는 항산화, 항염증 또는 보습용 화장료 조성물을 제공한다.In order to solve the above problems, the present invention provides an antioxidant, anti-inflammatory, or moisturizing cosmetic composition containing a Norotong extract as an active ingredient.

또한 상기 노로통 추출물은 건조된 노로통을 50 내지 80 %(v/v) 에탄올로 10 내지 30℃에서 18 내지 36 시간 동안 1 내지 5회 반복하여 추출하여 수득된 것을 특징으로 하는 항산화, 항염증 또는 보습용 화장료 조성물을 제공한다. In addition, the Norotong extract is an antioxidant and anti-inflammatory extract obtained by repeatedly extracting dried Norotong with 50 to 80% (v/v) ethanol at 10 to 30°C for 18 to 36 hours 1 to 5 times. Alternatively, a moisturizing cosmetic composition is provided.

또한 상기 노로통 추출물은 SOD 활성 및 GSH 함량을 향상시킬 수 있는 것을 특징으로 하는 항산화용 화장료 조성물을 제공한다.In addition, the Norotong extract provides a cosmetic composition for antioxidants, characterized in that it can improve SOD activity and GSH content.

또한 상기 노로통 추출물은 NOS2, TNF-α 및 IL-6의 발현을 억제시킬 수 있는 것을 특징으로 하는 항염증용 화장료 조성물을 제공한다.In addition, the Norotong extract provides an anti-inflammatory cosmetic composition characterized in that it can inhibit the expression of NOS2, TNF-α, and IL-6.

또한 상기 노로통 추출물은 80 내지 100℃의 열수 추출물이고, 100 내지 800 ㎍/mL의 농도로 포함되고, 히알루론산(Hyaluronic acid)의 생성량을 향상시킬 수 있는 것을 특징으로 하는 보습용 화장료 조성물을 제공한다.In addition, the Norotong extract is a hot water extract at 80 to 100°C, is contained at a concentration of 100 to 800 ㎍/mL, and provides a moisturizing cosmetic composition characterized in that it can improve the amount of hyaluronic acid produced. do.

본 발명에 따른 화장료 조성물은 노로통 추출물을 유효성분으로 포함하여 LPS로 유발된 염증 동물모델에서 SOD 활성 및 GSH 함량 향상, PI3K/Akt/NF-κB 경로에 의한 염증 개선 효과 및 HaCaT 세포 실험을 통해 히알루론산 생산량 증가에 대한 유의적인 결과를 얻어 항산화, 항염증 및 보습 효과가 있음을 확인하였다.The cosmetic composition according to the present invention contains Norotong extract as an active ingredient, improves SOD activity and GSH content in an animal model of LPS-induced inflammation, has an effect of improving inflammation by the PI3K/Akt/NF-κB pathway, and has the effect of improving inflammation through HaCaT cell experiments. Significant results were obtained regarding the increase in hyaluronic acid production, confirming that it has antioxidant, anti-inflammatory and moisturizing effects.

도 1은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 RAW 264.7 세포의증식 정도를 측정한 결과를 나타낸 그래프이다.
도 2는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 RAW 264.7 세포의p-Akt 단백질 발현량을 측정한 결과를 나타낸 사진 및 그래프이다.
도 3은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 RAW 264.7 세포의p-p38, p-JNK 및 p-ErK의 발현량을 측정한 결과를 나타낸 사진 및 그래프이다.
도 4는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 증식 정도를 측정한 결과를 나타낸 그래프이다.
도 5는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 TLR4 mRNA 발현량을 측정한 결과를 나타낸 그래프이다.
도 6은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 MD2 및 CD14 mRNA 발현량을 측정한 결과를 나타낸 그래프이다.
도 7은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 Nos2, TNFα 및 IL-6의 mRNA 발현량을 측정한 결과를 나타낸 그래프이다.
도 8은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 IL-10 및 Arg1의 mRNA 발현량을 측정한 결과를 나타낸 그래프이다.
도 9a는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 TLR4 발현 세포를 IHC로 염색한 결과를 나타낸 사진이다.
도 9b는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 TLR4 발현 세포 수를 측정한 결과를 나타낸 그래프이다.
도 10a는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 NF-κB가 활성화된 세포를 IHC로 염색한 결과를 나타낸 사진이다.
도 10b는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 NF-κB가 활성화된 세포 수를 측정한 결과를 나타낸 그래프이다.
도 11a는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 iNOS 발현 세포를 IHC로 염색한 결과를 나타낸 사진이다.
도 11b는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 iNOS 발현 세포 수를 측정한 결과를 나타낸 그래프이다.
도 12a는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 IL-1β 발현 세포를 IHC로 염색한 결과를 나타낸 사진이다.
도 12b는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 마우스 복막 대식세포의 IL-1β 발현 세포 수를 측정한 결과를 나타낸 그래프이다.
도 13은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 증식 정도를 측정한 결과를 나타낸 그래프이다.
도 14는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 DCFDA 수준을 측정한 결과를 나타낸 그래프이다.
도 15는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 DHE 수준을 측정한 결과를 나타낸 그래프이다.
도 16은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 SOD 활성을 측정한 결과를 나타낸 그래프이다.
도 17은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 GSH 함량을 측정한 결과를 나타낸 그래프이다.
도 18은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 세포 사멸 정도를 측정한 결과를 나타낸 사진 및 그래프이다.
도 19는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 ROS 강도를 측정한 결과를 나타낸 사진 및 그래프이다.
도 20은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 TUNEL 양성 세포 수를 측정한 결과를 나타낸 사진 및 그래프이다.
도 21은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 γH2Ax 수준을 측정한 결과를 나타낸 사진 및 그래프이다.
도 22는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 53BP1 수준을 측정한 결과를 나타낸 사진 및 그래프이다.
도 23은 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 MDA 강도를 측정한 결과를 나타낸 사진 및 그래프이다.
도 24는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 p47phox 수준을 측정한 결과를 나타낸 사진 및 그래프이다.
도 25는 본 발명의 실험예에서 ELF 및 WLF 처리에 따른 Huh7 세포의 4-HNE 수준을 측정한 결과를 나타낸 사진 및 그래프이다.
도 26은 본 발명의 실험예에서 WLF 처리에 따른 HaCaT 세포의 증식 정도를 측정한 결과를 나타낸 그래프이다.
도 27은 본 발명의 실험예에서 WLF 처리에 따른 HaCaT 세포의 히알루론산 생성량을 측정한 결과를 나타낸 그래프이다.
도 28은 본 발명의 실험예에서 WLF 처리에 따른 HaCaT 세포의 DCFDA 생성량을 측정한 결과를 나타낸 그래프이다.
도 29는 본 발명의 실험예에서 WLF 처리에 따른 HaCaT 세포의 TNF-α, IL-1β, IL-6 및 IL-8 생성량을 측정한 결과를 나타낸 그래프이다.
도 30은 본 발명의 실험예에서 WLF 처리에 따른 HaCaT 세포의 COX-2, HAS2 및 filaggrin 단백질 발현량을 측정한 결과를 나타낸 사진 및 그래프이다.Figure 1 is a graph showing the results of measuring the degree of proliferation of RAW 264.7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 2 is a photograph and graph showing the results of measuring the p-Akt protein expression level in RAW 264.7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 3 is a photograph and graph showing the results of measuring the expression levels of p-p38, p-JNK, and p-ErK in RAW 264.7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 4 is a graph showing the results of measuring the degree of proliferation of mouse peritoneal macrophages according to ELF and WLF treatment in an experimental example of the present invention.
Figure 5 is a graph showing the results of measuring the TLR4 mRNA expression level of mouse peritoneal macrophages according to ELF and WLF treatment in an experimental example of the present invention.
Figure 6 is a graph showing the results of measuring the MD2 and CD14 mRNA expression levels of mouse peritoneal macrophages according to ELF and WLF treatment in an experimental example of the present invention.
Figure 7 is a graph showing the results of measuring the mRNA expression levels of Nos2, TNFα, and IL-6 in mouse peritoneal macrophages according to ELF and WLF treatment in an experimental example of the present invention.
Figure 8 is a graph showing the results of measuring the mRNA expression levels of IL-10 and Arg1 in mouse peritoneal macrophages according to ELF and WLF treatment in an experimental example of the present invention.
Figure 9a is a photograph showing the results of IHC staining of TLR4-expressing cells of mouse peritoneal macrophages following ELF and WLF treatment in an experimental example of the present invention.
Figure 9b is a graph showing the results of measuring the number of TLR4-expressing cells in mouse peritoneal macrophages according to ELF and WLF treatment in an experimental example of the present invention.
Figure 10a is a photograph showing the results of IHC staining of NF-κB-activated cells of mouse peritoneal macrophages following ELF and WLF treatment in an experimental example of the present invention.
Figure 10b is a graph showing the results of measuring the number of NF-κB-activated cells in mouse peritoneal macrophages according to ELF and WLF treatment in an experimental example of the present invention.
Figure 11a is a photograph showing the results of IHC staining of iNOS-expressing cells of mouse peritoneal macrophages following ELF and WLF treatment in an experimental example of the present invention.
Figure 11b is a graph showing the results of measuring the number of iNOS-expressing cells in mouse peritoneal macrophages according to ELF and WLF treatment in an experimental example of the present invention.
Figure 12a is a photograph showing the results of IHC staining of IL-1β expressing cells of mouse peritoneal macrophages following ELF and WLF treatment in an experimental example of the present invention.
Figure 12b is a graph showing the results of measuring the number of IL-1β expressing cells in mouse peritoneal macrophages according to ELF and WLF treatment in an experimental example of the present invention.
Figure 13 is a graph showing the results of measuring the degree of proliferation of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 14 is a graph showing the results of measuring the DCFDA level of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 15 is a graph showing the results of measuring the DHE level of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 16 is a graph showing the results of measuring SOD activity of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 17 is a graph showing the results of measuring the GSH content of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 18 is a photograph and graph showing the results of measuring the degree of cell death of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 19 is a photograph and graph showing the results of measuring the ROS intensity of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 20 is a photograph and graph showing the results of measuring the number of TUNEL-positive cells in Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 21 is a photograph and graph showing the results of measuring the γH2Ax level of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 22 is a photograph and graph showing the results of measuring the 53BP1 level of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 23 is a photograph and graph showing the results of measuring the MDA intensity of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 24 is a photograph and graph showing the results of measuring the p47 phox level of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 25 is a photograph and graph showing the results of measuring the 4-HNE level of Huh7 cells according to ELF and WLF treatment in an experimental example of the present invention.
Figure 26 is a graph showing the results of measuring the degree of proliferation of HaCaT cells according to WLF treatment in an experimental example of the present invention.
Figure 27 is a graph showing the results of measuring the amount of hyaluronic acid produced by HaCaT cells according to WLF treatment in an experimental example of the present invention.
Figure 28 is a graph showing the results of measuring the amount of DCFDA produced by HaCaT cells according to WLF treatment in an experimental example of the present invention.
Figure 29 is a graph showing the results of measuring the amount of TNF-α, IL-1β, IL-6, and IL-8 produced by HaCaT cells according to WLF treatment in an experimental example of the present invention.
Figure 30 is a photograph and graph showing the results of measuring the protein expression levels of COX-2, HAS2, and filaggrin in HaCaT cells according to WLF treatment in an experimental example of the present invention.

이하 바람직한 실시예를 통하여 본 발명을 상세히 설명하기로 한다. 이에 앞서, 본 명세서 및 청구범위에 사용된 용어나 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 아니 되며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여, 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다. 따라서, 본 명세서에 기재된 실시예의 구성은 본 발명의 가장 바람직한 일실시예에 불과할 뿐이고 본 발명의 기술적 사상을 모두 대변하는 것은 아니므로, 본 출원시점에 있어서 이들을 대체할 수 있는 다양한 균등물과 변형예들이 있을 수 있음을 이해하여야 한다. 또한, 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한, 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있음을 의미한다.Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, the terms or words used in this specification and claims should not be construed as limited to their usual or dictionary meanings, and the inventor should appropriately define the concept of terms in order to explain his or her invention in the best way. Based on the principle of definability, it must be interpreted with meaning and concept consistent with the technical idea of the present invention. Therefore, the configuration of the embodiments described in this specification is only one of the most preferred embodiments of the present invention and does not represent the entire technical idea of the present invention, so various equivalents and modifications can be substituted for them at the time of filing the present application. You must understand that there may be. Additionally, throughout the specification, when a part is said to “include” a certain element, this means that, unless specifically stated to the contrary, it does not exclude other elements but may further include other elements.

본 발명은 노로통(Liquidambaris Fructus, LF) 추출물을 유효성분으로 포함하는 항산화, 항염증 또는 보습용 화장료 조성물을 개시한다.The present invention discloses an antioxidant, anti-inflammatory, or moisturizing cosmetic composition containing a Norotong (Liquidambaris Fructus, LF) extract as an active ingredient.

본 발명에서 상기 추출물의 형태나 성상에는 제한이 없으며, 용액, 농축물일 수도 있고, 추출물 제조에 사용된 용매를 제거한 고형분 또는 분말일 수도 있다.In the present invention, there is no limitation on the form or nature of the extract, and it may be a solution, a concentrate, or a solid or powder obtained by removing the solvent used in preparing the extract.

상기 추출물은 당 업계에 공지된 통상의 추출 방법, 예컨대, 용매 추출법을 사용하여 제조될 수 있다. 용매 추출법을 이용한 추출물 제조 시 사용되는 추출 용매는 물, 탄소 수가 1 내지 4인 저급 알코올(메탄올, 에탄올, 프로판올 및 부탄올) 또는 이들의 혼합물인 함수 저급 알코올, 프로필렌글리콜, 1,3-부틸렌글리콜, 글리세린, 아세톤, 디에틸에테르, 에틸아세테이트, 부틸아세테이트, 디클로로메탄, 클로로포름, 헥산 및 이들의 혼합물로 구성된 군으로부터 선택될 수 있고, 이중 물, 알코올 또는 이들의 혼합물에서 선택되는 것이 바람직하다. 또한, 추출 용매로 물을 사용할 경우 열수 추출이 바람직하고, 알코올을 사용하는 경우 알코올은 탄소 수가 1 내지 4인 저급알코올인 것이 바람직하고, 저급 알코올은 에탄올인 것이 더욱 바람직하다.The extract can be prepared using conventional extraction methods known in the art, such as solvent extraction. The extraction solvent used when preparing the extract using the solvent extraction method is water, lower alcohols with 1 to 4 carbon atoms (methanol, ethanol, propanol, and butanol) or mixtures thereof such as hydrous lower alcohols, propylene glycol, and 1,3-butylene glycol. , glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane, and mixtures thereof, preferably selected from water, alcohol, or mixtures thereof. In addition, when water is used as the extraction solvent, hot water extraction is preferable, and when alcohol is used, the alcohol is preferably a lower alcohol with 1 to 4 carbon atoms, and the lower alcohol is more preferably ethanol.

또한, 추출 용매로 함수 알코올을 사용하는 경우 알코올 함량은 10 내지90 %(v/v)일 수 있고, 바람직하게는 40 내지 85 %(v/v)일 수 있고, 더욱 바람직하게는 50 내지 80 %(v/v)일 수 있다. In addition, when using hydrous alcohol as an extraction solvent, the alcohol content may be 10 to 90% (v/v), preferably 40 to 85% (v/v), and more preferably 50 to 80% (v/v). It may be %(v/v).

한편, 본 발명에서 상기 추출물은 상기한 추출 용매뿐만 아니라, 다른 추출 용매를 이용하여도 실질적으로 동일한 효과를 나타내는 추출물이 얻어질 수 있다는 것은 당업자에게 자명한 것이다. 또한, 본 발명에서 상기 추출물은 상술한 추출 용매에 의한 추출물뿐만 아니라, 통상적인 다른 추출 방법을 통해 얻어진 추출물 내지 정제 및 발효 과정을 거친 추출물도 포함한다. 예컨대, 이산화탄소에 의한 감압, 고온에 의한 초임계 추출법에 의한 추출, 초음파를 이용한 추출법에 의한 추출, 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 이용한 분리, 다양한 크로마토그래피 (크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의해 분리하거나 자연상태나 각종 미생물을 이용한 발효산물에 의한 추출물 등, 추가적으로 실시된 다양한 정제 및 추출방법을 통해 얻어진 활성 분획도 본 발명의 추출물에 포함된다.Meanwhile, it is obvious to those skilled in the art that in the present invention, the extract showing substantially the same effect can be obtained by using not only the above-described extraction solvent but also other extraction solvents. In addition, in the present invention, the extract includes not only extracts using the above-described extraction solvents, but also extracts obtained through other conventional extraction methods or extracts that have undergone purification and fermentation processes. For example, decompression with carbon dioxide, extraction by supercritical extraction at high temperature, extraction by ultrasonic extraction, separation using an ultrafiltration membrane with a certain molecular weight cut-off value, and various chromatographies (size, charge, hydrophobicity or affinity). Active fractions obtained through various additional purification and extraction methods, such as separation by (manufactured for separation according to gender) or extracts from fermentation products in natural state or using various microorganisms, are also included in the extract of the present invention.

본 발명에서 추출물 제조에 사용되는 노로통은 특정 상태로 한정되지 않으며, 노로통 건조물이 사용될 수 있고, 파우더 형태로 용매 추출에 사용되는 것이 바람직하다.In the present invention, the noro barrel used to produce the extract is not limited to a specific state, and the dried noro barrel can be used, and it is preferably used for solvent extraction in powder form.

또한 상기 노로통을 에탄올로 추출하는 경우, 예컨대, 70 %(v/v) 에탄올을 노로통 건조물의 5 내지 20 배 중량으로 첨가하고, 10 내지 30℃에서 18 내지 36시간 동안 추출하되, 1 내지 5회 반복하여 추출할 수 있고, 바람직하게는 상기 에탄올을 8 내지 15 배 중량으로 첨가하고, 15 내지 25℃에서 20 내지 30 시간 동안 추출하되, 2 내지 4회 반복하여 추출할 수 있다.In addition, when extracting the noro barrel with ethanol, for example, 70% (v/v) ethanol is added in an amount 5 to 20 times the weight of the noro barrel dried material, and extracted at 10 to 30° C. for 18 to 36 hours. Extraction can be repeated 5 times, preferably by adding 8 to 15 times the weight of ethanol and extracting at 15 to 25°C for 20 to 30 hours. Extraction can be repeated 2 to 4 times.

또한 상기 노로통을 열수로 추출하는 경우, 예컨대, 증류수를 노로통 건조물의 5 내지 20 배 중량으로 첨가하고, 70 내지 120℃로 1 내지 10 시간 동안 추출하되, 1 내지 5회 반복하여 추출할 수 있고, 바람직하게는 상기 증류수를 8 내지 15 배 중량으로 첨가하고, 80 내지 100℃로 2 내지 5 시간 동안 추출하되, 2 내지 4회 반복하여 추출할 수 있다.In addition, when extracting the noro barrel with hot water, for example, distilled water is added in an amount 5 to 20 times the weight of the dried noro barrel, and extraction is performed at 70 to 120 ° C. for 1 to 10 hours, and the extraction can be repeated 1 to 5 times. Preferably, 8 to 15 times the weight of the distilled water is added, and extraction is performed at 80 to 100° C. for 2 to 5 hours, and the extraction may be repeated 2 to 4 times.

상기 노로통 추출물은 항산화, 항염증 및 보습 효과를 구현하며, 구체적으로, LPS로 유발한 염증 동물모델의 복막에서 채취한 대식세포를 대상으로 한 실험에서, SOD 활성 및 GSH 함량 향상에 의한 항산화 활성 및 NOS2, TNF-α 및 IL-6 발현 억제에 의한 항염증 활성이 확인되었고, HaCaT 세포를 대상으로 한 실험에서, 히알루론산(Hyaluronic acid)의 생성량 향상에 의한 피부 보습 활성이 확인되었다.The Norotong extract has antioxidant, anti-inflammatory and moisturizing effects, and specifically, in experiments targeting macrophages collected from the peritoneum of an LPS-induced inflammation animal model, antioxidant activity by improving SOD activity and GSH content. And anti-inflammatory activity was confirmed by suppressing the expression of NOS2, TNF-α and IL-6, and in an experiment with HaCaT cells, skin moisturizing activity was confirmed by improving the production of hyaluronic acid.

또한 본 발명에서 상기 노로통의 에탄올 추출물은 상기 항산화 및 항염증 활성이 상기 노로통의 열수 추출물 대비 향상되는 것으로 후술하는 실험예를 통해 확인되었다. In addition, in the present invention, it was confirmed through an experimental example described later that the antioxidant and anti-inflammatory activities of the ethanol extract of Noro Tong were improved compared to the hot water extract of Noro Tong.

또한 본 발명에서 상기 노로통의 열수 추출물은 100 내지 800 ㎍/mL로 상기 화장료 조성물에 포함될 수 있고, 피부 보습 및 항염증 효과 극대화 측면에서, 바람직하게는 200 내지 600 ㎍/mL으로 포함될 수 있고, 더욱 바람직하게는 300 내지 500 ㎍/mL으로 포함될 수 있다.Additionally, in the present invention, the hot water extract of Noro Tong may be included in the cosmetic composition at 100 to 800 μg/mL, and in terms of maximizing skin moisturizing and anti-inflammatory effects, it may preferably be included at 200 to 600 μg/mL, More preferably, it may be contained at 300 to 500 μg/mL.

본 발명에 따른 화장료 조성물은 젤 타입, 스킨 타입, 크림 타입, 연고 타입 등으로 적용될 수 있지만, 이들만으로 한정되는 것은 아니다. 상기의 조성물은 그것의 타입에 따라 적절한 통상의 연화제, 유화제, 증점제 또는 당업계에 공지되어 있는 기타 물질들을 첨가하여, 공지의 방법에 의해 적절하게 제조될 수 있다.The cosmetic composition according to the present invention can be applied in gel type, skin type, cream type, ointment type, etc., but is not limited to these. The above composition can be appropriately prepared by known methods by adding appropriate conventional softeners, emulsifiers, thickeners or other substances known in the art depending on its type.

상기 젤 타입 조성물은 트리메틸올프로판, 폴리에틸렌 글리콜 또는 글리세린 등의 연화제, 프로필렌 글리콜, 에탄올, 이소세틱알콜 등의 용매, 정제주 등을 첨가하여 제조할 수 있다.The gel-type composition can be prepared by adding a softener such as trimethylolpropane, polyethylene glycol or glycerin, a solvent such as propylene glycol, ethanol, or isocetic alcohol, and refined alcohol.

상기 스킨 타입 조성물은 스테아릴 알콜, 미리스틸 알콜, 베헤닐 알콜, 아라키딜 알콜, 이소스테아릴 알콜, 이소세틸 알콜 등의 지방 알콜, 부틸렌 글라이콜, 글리세린, 알란토인, 메틸 파라벤, 이디티에이-2-소디움, 잔탄검, 디메티콘, 폴리 에틸렌 글라이콜-60 하이드로제네이트 카스톨 오일, 폴리 소르베이트 60 및 정제수 등을 첨가하여 제조할 수 있다.The skin type composition contains fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, and isocetyl alcohol, butylene glycol, glycerin, allantoin, methyl paraben, EDTA- It can be prepared by adding 2-sodium, xanthan gum, dimethicone, polyethylene glycol-60 hydrogenate castor oil, polysorbate 60, and purified water.

상기 크림 타입 조성물은 스테아릴 알콜, 미리스틸 알콜, 베헤닐 알콜, 아라키딜 알콜, 이소스테아릴 알콜, 이소세틸 알콜 등의 지방 알콜, 레시틴, 포스파티딜콜린, 포스파티딜에탄올아민, 소프파티질세린, 소프파티딜이노시톨 등의 리피드, 이들의 유도체, 글리세릴 스테아레이트, 소르비탄 팔미테이트, 소리비탄 스테아레이트 등의 유화제, 아보카도 오일, 살구 오일, 바바수 오일, 유리지치 오일, 동백 오일 등의 천연 지방 또는 오일, 프로필렌글리콜 등의 용매 및 정제수 등을 첨가하여 제조할 수 있다.The cream-type composition contains fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, and isocetyl alcohol, lecithin, phosphatidylcholine, phosphatidylethanolamine, sophatidylserine, and sophphatidyl. Lipids such as inositol, their derivatives, emulsifiers such as glyceryl stearate, sorbitan palmitate, and sorbitan stearate, natural fats or oils such as avocado oil, apricot oil, babassu oil, borage oil, and camellia oil, It can be manufactured by adding solvents such as propylene glycol and purified water.

상기 연고 타입 조성물은 연화제, 유화제 및 마이크로크리스탈린납, 파라핀, 세레신, 밀납, 경납, 바세린 등의 왁스를 첨가하여 제조할 수 있다.The ointment-type composition can be prepared by adding softeners, emulsifiers, and waxes such as microcrystalline wax, paraffin, ceresin, beeswax, hard wax, and vaseline.

본 발명에서 상기 화장료 조성물은 유효성분 이외에 추가로 동일 또는 유사한 기능을 나타내는 성분을 1종 이상 추가로 함유할 수 있다. 이의 비제한적인 예시로는 비타민 C, 레티노산, TGF, 동물 태반 유래의 단백질, 베튤린산 및 클로렐라 추출물로 구성되는 군으로부터 선택되는 어느 하나 이상인 것일 수 있으나, 이에 제한되는 것은 아니다. 또한, 본 발명의 화장료 조성물은 형광물질, 살진균제, 굴수성 유발물질, 보습제, 방향제, 방향제 담체, 단백질, 용해화제, 당 유도체, 일광차단제, 비타민, 식물 추출물 등을 포함하는 부형제를 추가로 함유할 수 있다.In the present invention, the cosmetic composition may further contain one or more ingredients that exhibit the same or similar functions in addition to the active ingredient. Non-limiting examples thereof may include, but are not limited to, any one or more selected from the group consisting of vitamin C, retinoic acid, TGF, protein derived from animal placenta, betulinic acid, and chlorella extract. In addition, the cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrotropes-inducing substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, etc. You can.

이하, 실시예 및 실험예를 들어 본 발명을 상세히 설명하기로 한다.Hereinafter, the present invention will be described in detail through examples and experimental examples.

실시예 1Example 1

추출물 제조에 사용된 노로통(Liquidambaris Fructus, LF) 건조물은 대성약국(Daegu, Korea)에서 구입하였다. 노로통 건조물의 9 배 중량에 해당하는 증류수를 첨가한 후 99℃에서 3 시간 동안 추출하되, 3회 반복하여 추출하였다. 얻어진 추출물은 filter paper(No. 20 qualitative filter paper, Hyundai micro Co., Ltd., Korea)로 여과되었고, 농축된 후 냉동 건조되어, 최종적으로 분말(노로통 열수 추출물, 이하 WLF, 수율 5.7%)을 얻었으며, 4℃에서 냉장 보관하였다가 실험 직전에 증류수에 녹여 샘플로 사용하였다.The dried liquid of Norotong (Liquidambaris Fructus, LF) used to prepare the extract was purchased from Daesung Pharmacy (Daegu, Korea). After adding distilled water equivalent to 9 times the weight of the dried Noro barrel, extraction was performed at 99°C for 3 hours, and the extraction was repeated three times. The obtained extract was filtered through filter paper (No. 20 qualitative filter paper, Hyundai micro Co., Ltd., Korea), concentrated, then freeze-dried, and finally powdered (Norotong hot water extract, hereinafter referred to as WLF, yield 5.7%). was obtained, stored in refrigeration at 4°C, dissolved in distilled water immediately before the experiment, and used as a sample.

실시예 2Example 2

실시예 1에서 증류수 대신 70 %(v/v) 에탄올을 사용하고, 상온에서 24 시간 동안 추출하되, 3회 반복하여 추출한 것을 제외하고는 실시예 1과 동일하게 제조하여 분말 상태의 노로통 에탄올 추출물(이하 ELF, 수율 6.5%)을 얻었으며, 4℃에서 냉장 보관하였다가 실험 직전에 증류수에 녹여 샘플로 사용하였다.Norotong ethanol extract in powder form was prepared in the same manner as in Example 1, except that 70% (v/v) ethanol was used instead of distilled water and extraction was performed at room temperature for 24 hours, and the extraction was repeated three times. (hereinafter referred to as ELF, yield 6.5%) was obtained, stored in refrigeration at 4°C, dissolved in distilled water immediately before the experiment, and used as a sample.

실험예 1: 항산화 및 항염증 효능 평가Experimental Example 1: Evaluation of antioxidant and anti-inflammatory efficacy

[실험 방법][Experimental Method]

(1) 시약(1) Reagent

본 실험에 사용된 LPS(CAT. L2630), dimethyl sulfoxide(DMSO), phosphate-buffered saline(PBS), thioglycolate, Dulbecco's modified Eagle's medium(DMEM), Fetal Bovine Serum(FBS), penicillin-streptomycin, 2',7'-Dichlorofluorescein diacetate(DCFDA), Dihydroethidium(DHE), paraformaldehyde, diaminobenzidine(DAB)는 MilliporeSigma(Burlington, MA, USA)에서 구입하였다. protease inhibitor mixture solution 및 ethylenediaminetetraacetic acid(EDTA)는 Wako Pure Chemical Industries, Ltd.(Osaka, Japan)에서 구입하였다. Radio-Immunoprecipitation Assay(RIPA) lysis and extraction buffer, Halt?? protease and phosphatase inhibitor cocktail 및 PierceTM bicinchoninic acid(BCA) protein assay kit는 Thermo Fisher Scientific(Waltham, MA, USA)에서 구입하였다. PVDF membrane는 MilliporeSigma에서 얻었다. ECL Western Blotting Detection Reagents는 Cyanagen Srl(Bologna, Italy)에서 구입하였다. Goat anti-rabbit(GTX213110-01), rabbit anti-goat(GTX228416-01) 및 goat anti-mouse(GTX213111-01) immunoglobulin G(IgG) horseradish peroxidase(HRP)-conjugated secondary antibodies는 GeneTex, Inc.(Irvine, LA, USA)에서 구입하였다. WST-1 measurement kit(WST-1 Cell Proliferation Assay Kit)는 Cayman Chemical(Item No. 10008883, Ann Arbor, MI, USA)에서 구입하였다.LPS (CAT. L2630), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), thioglycolate, Dulbecco's modified Eagle's medium (DMEM), Fetal Bovine Serum (FBS), penicillin-streptomycin, 2', used in this experiment. 7'-Dichlorofluorescein diacetate (DCFDA), dihydroethidium (DHE), paraformaldehyde, and diaminobenzidine (DAB) were purchased from MilliporeSigma (Burlington, MA, USA). Protease inhibitor mixture solution and ethylenediaminetetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Radio-Immunoprecipitation Assay (RIPA) lysis and extraction buffer, Halt?? Protease and phosphatase inhibitor cocktail and PierceTM bicinchoninic acid (BCA) protein assay kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). PVDF membrane was obtained from MilliporeSigma. ECL Western Blotting Detection Reagents were purchased from Cyanagen Srl (Bologna, Italy). Goat anti-rabbit (GTX213110-01), rabbit anti-goat (GTX228416-01), and goat anti-mouse (GTX213111-01) immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated secondary antibodies were manufactured by GeneTex, Inc. (Irvine). , LA, USA). WST-1 measurement kit (WST-1 Cell Proliferation Assay Kit) was purchased from Cayman Chemical (Item No. 10008883, Ann Arbor, MI, USA).

(2) 세포 배양 및 처리(2) Cell culture and processing

1) 사용된 세포주(cell line) 및 배양 조건1) Cell line and culture conditions used

RAW 264.7 세포는 ATCC(American-type culture collection, Rockville, MD, USA)에서 구입하였다. 세포 배양은 37℃ 및 5% CO2의 세포 배양기에서 10% FBS가 포함된 DMEM에서 수행되었고, 1% 페니실린이 배지에 첨가되었다. RAW 264.7 cells were purchased from ATCC (American-type culture collection, Rockville, MD, USA). Cell culture was performed in DMEM containing 10% FBS in a cell incubator at 37°C and 5% CO 2 , and 1% penicillin was added to the medium.

또한 Huh7 세포는 MilliporeSigma에서 구입되어 본 실험에 사용되었다.Additionally, Huh7 cells were purchased from MilliporeSigma and used in this experiment.

2) 마우스 복막 대식세포의 분리2) Isolation of mouse peritoneal macrophages

복막 대식세포의 분리를 위해 수컷 ICR 마우스(36 내지 38 g)을 DBL Co., Ltd.(Eumseong, Korea)에서 구입하였다. 마우스는 12 시간의 주/야 주기에서 23±2℃의 실온과 50±10%의 습도에서 유지되었다. For isolation of peritoneal macrophages, male ICR mice (36 to 38 g) were purchased from DBL Co., Ltd. (Eumseong, Korea). Mice were maintained at room temperature of 23 ± 2°C and humidity of 50 ± 10% with a 12-hour day/night cycle.

본 실험은 동물실험의 윤리적, 과학적 타당성 검토 및 효율적인 관리를 위하여 대구한의대학교 동물실험윤리 위원회(Institutional Animal Care and Use Committee, IACUC)의 승인(승인번호: DHU2023-017)을 받아 진행되었다.This experiment was conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of Daegu Haany University (approval number: DHU2023-017) to review the ethical and scientific validity and efficient management of animal experiments.

Thioglycolate는 복강 내 주사로 3 mL 주입되었고, 마취 후 복강 내 주사를 통해 Avertin(250 mg/kg)이 투여되었다. 마취된 마우스의 경동맥 근처 피부에 접근하여, 경동맥을 절단하고 혈액을 배출하였다. 10 mM PBS(pH 7.3)가 복강 내 주사로 주입되었다. 약 10분 후 얼음으로 덮고 마사지를 한 후, 대식세포가 분리되었고, 부유 물질이 원심분리를 통해 분리되었고, 최종적으로 복막 대식세포가 획득되어 본 실험에 사용되었다.Thioglycolate was injected in an amount of 3 mL by intraperitoneal injection, and Avertin (250 mg/kg) was administered by intraperitoneal injection after anesthesia. The skin near the carotid artery of an anesthetized mouse was approached, the carotid artery was cut, and blood was drained. 10 mM PBS (pH 7.3) was injected intraperitoneally. About 10 minutes later, after covering with ice and massaging, the macrophages were separated, the floating material was separated through centrifugation, and finally, peritoneal macrophages were obtained and used in this experiment.

(3) 세포 증식 분석(Cell proliferation assay)(3) Cell proliferation assay

독성 및 세포 증식/분열을 확인하기 위한 세포 증식 분석(Cell proliferation assay)은 RAW 264.7 세포주와 Huh7 세포주를 사용하여 진행되었다. 먼저, RAW 264.7 세포는 농도가 2Х103 cells/well로 96 well-microplate에 분주되었고, 각 시료를 두 농도로 처리 후 24시간 동안 정치되었다. 세포 배양기에서 WST-1 Cell Proliferation Assay Kit로 1 시간 동안 배양한 후 Microplate reader(VersaMax, Molecular Device, Germany)를 사용하여 450 nm 및 600 nm의 두 파장에서 측정되었다. Huh7 세포도 RAW 264.7 세포와 동일한 방식으로 처리되었으며, 샘플의 전처리 후 6시간 뒤에 500 μM H2O2(CAS: 7722-84-1, Thermo Fisher Scientific)가 적용되어 약물의 산화 손상에 대한 보호 효과를 확인하였다. 24 시간 후에 10% 농도의 WST-1 시약이 첨가되고, 1시간 동안 배양한 후 450 nm 및 600 nm의 두 파장에서 측정되었다.Cell proliferation assay to check toxicity and cell proliferation/division was performed using RAW 264.7 cell line and Huh7 cell line. First, RAW 264.7 cells were distributed in 96 well-microplates at a concentration of 2Х10 3 cells/well, and each sample was treated with two concentrations and left for 24 hours. After culturing for 1 hour with the WST-1 Cell Proliferation Assay Kit in a cell incubator, it was measured at two wavelengths of 450 nm and 600 nm using a Microplate reader (VersaMax, Molecular Device, Germany). Huh7 cells were also treated in the same manner as RAW 264.7 cells, and 500 μM H 2 O 2 (CAS: 7722-84-1, Thermo Fisher Scientific) was applied 6 hours after pretreatment of the samples to ensure the protective effect of the drug against oxidative damage. was confirmed. After 24 hours, a 10% concentration of WST-1 reagent was added, incubated for 1 hour, and then measured at two wavelengths of 450 nm and 600 nm.

(4) 세포 사멸 신호 측정(The measurement of cell death signaling)(4) The measurement of cell death signaling

LIVE/DEAD cell assay 관련 실험은 Thermo Fisher Scientific에서 상업적으로 구매한 제품(L3224)을 사용하여 측정되었다. 면역 형광 염색 이미지는 형광 현미경(ZEISS, Jena, Germany)을 사용하여 측정되었다. 또한, 세포 사멸 신호 분석은 terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling(TUNEL) assay kit(ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit, S7100, MilliporeSigma)를 사용하여 제조사의 지침에 따라 분석되었다.LIVE/DEAD cell assay-related experiments were measured using a commercially purchased product (L3224) from Thermo Fisher Scientific. Immunofluorescence staining images were measured using a fluorescence microscope (ZEISS, Jena, Germany). Additionally, cell death signaling was analyzed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay kit (ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit, S7100, MilliporeSigma) according to the manufacturer's instructions.

(5) 활성산소종 측정(5) Measurement of reactive oxygen species

활성산소종(ROS)은 세포에서 생성된 ROS의 양을 측정하기 위해 DCFDA 및 DHE가 사용되었다. 각 세포 내 단백질 수준은 형광 파장을 사용하여 측정되었으며 특히, 형광 염색 이미지는 DCFDA probe kit(Fluorometric Intracellular Ros Kit, CAT. MAK143, MilliporeSigma) 사용 후 형광 현미경으로 측정되었다.For reactive oxygen species (ROS), DCFDA and DHE were used to measure the amount of ROS generated in cells. Protein levels within each cell were measured using fluorescence wavelengths, and in particular, fluorescence staining images were measured using a fluorescence microscope after using the DCFDA probe kit (Fluorometric Intracellular Ros Kit, CAT. MAK143, MilliporeSigma).

(6) SOD 활성 및 GHS 함량 측정(6) Measurement of SOD activity and GHS content

LF의 항산화 효과를 확인하기 위해 Huh7 세포를 사용하여 생화학적 실험이 수행되었다. 65 mm 직경의 세포 배양용 접시에 5Х106 개의 Huh7 세포를 분주한 후, 세포 배양기(incubator)에서 배양되었으며, 다음 날에 세포를 세척 후 각 샘플은 6 시간 동안 전처리되었고, 이후 500 μM hydrogen peroxide로 12 시간 처리되었다. 세포를 PBS로 세척한 후 RIPA 버퍼를 사용하여 단백질을 분리했다. 분리된 세포의 단백질에서 superoxide dismutase(SOD)의 활성 및 glutathione (GSH) 함량은 상업적으로 구매한 SOD assay kit(MAK379, MilliporeSigma) 및 GSH assay kit (CAT. CS0260, MilliporeSigma)를 사용하여 측정되었다.To confirm the antioxidant effect of LF, biochemical experiments were performed using Huh7 cells. Six 5Х10 Huh7 cells were dispensed onto a 65 mm diameter cell culture dish and cultured in a cell incubator. The next day, the cells were washed and each sample was pretreated for 6 hours, then treated with 500 μM hydrogen peroxide. It was processed for 12 hours. After washing the cells with PBS, proteins were separated using RIPA buffer. The activity of superoxide dismutase (SOD) and glutathione (GSH) content in the proteins of isolated cells were measured using commercially purchased SOD assay kit (MAK379, MilliporeSigma) and GSH assay kit (CAT. CS0260, MilliporeSigma).

(7) 면역조직학적 분석(Analysis of immunohistochemistry)(7) Analysis of immunohistochemistry

IHC(immunohistochemistry)는 표준 면역염색 방법에 따라 수행하였다. 5Х104 cells/well(24 well plates)의 복막에서 분리한 대식세포를 유리 커버(12 mm 원형 타입) 위에 분주한 후 세포를 하루 동안 배양하고, 다음 날 배지를 10 mM PBS로 교환한 후, 세포를 샘플로 처리하였고, 6시간 후 0.2 μg/mL의 LPS를 처리하였다. 30분 후, 세포를 수집하고 면역염색을 실시하였다.Immunohistochemistry (IHC) was performed according to standard immunostaining methods. Macrophages isolated from the peritoneum of 5Х10 4 cells/well (24 well plates) were spread on a glass cover (12 mm round type), the cells were cultured for one day, and the next day, the medium was exchanged with 10 mM PBS, and the cells were cultured. was treated as a sample, and after 6 hours, 0.2 μg/mL of LPS was treated. After 30 minutes, cells were collected and immunostained.

먼저, 회수된 세포를 PBS로 세척하고, 4% 파라포름알데하이드(paraformaldehyde)로 고정한 후 PBS로 2회 세척한 후 PBST를 통해 투과성을 테스트하였다. 테스트 후, 1차 항체를 2.5% Normal Horse Serum(CAT. S-2012-5, NHS, Vector Laboratories Inc. Newark, CA, USA)(표 1 참조)으로 블로킹한 후 동일한 용액을 이용하여 처리하였고, 1차 항체를 4℃에서 약 18시간 동안 배양한 후 1차 항체를 0.05% PBST로 세척한 후 2차 항체를 처리하여 같은 방법으로 세척하고 diaminobenzidine(DAB)을 사용하여 염색을 종결하였다. Hematoxylin을 사용하여 핵을 대조염색(counterstain)으로 염색하였고 광학현미경 조건에서 400 배율로 이미지를 획득하였다. 또한, 양성 신호의 정성 분석을 위해 Image J software ver. 5.12 (NIH, Bethesda, MD, USA)를 사용하여 4가지 서로 다른 실험을 통해 평균값을 기록하여 통계처리를 수행하였다.First, the recovered cells were washed with PBS, fixed with 4% paraformaldehyde, washed twice with PBS, and then permeability was tested through PBST. After the test, the primary antibody was blocked with 2.5% Normal Horse Serum (CAT. S-2012-5, NHS, Vector Laboratories Inc. Newark, CA, USA) (see Table 1) and then treated with the same solution. After incubating the primary antibody at 4°C for about 18 hours, the primary antibody was washed with 0.05% PBST, treated with secondary antibody, washed in the same manner, and staining was completed using diaminobenzidine (DAB). Nuclei were counterstained using hematoxylin, and images were acquired at 400x magnification under light microscope conditions. Additionally, for qualitative analysis of positive signals, Image J software ver. Statistical processing was performed by recording average values from four different experiments using 5.12 (NIH, Bethesda, MD, USA).

본 IHC 실험에서 사용된 1차 항체에 관한 정보는 하기 표 1에 나타내었다.Information on the primary antibodies used in this IHC experiment is shown in Table 1 below.

Cell typeCell type ContentsContents HostHost CompanyCompany Catcat RatioRatio VehicleVehicle Mouse peritoneal macrophagesMouse peritoneal macrophages TLR4TLR4 RabbitRabbit CSTCST #14358#14358 1:2001:200 2.5% NHS2.5% NHS NF-κBNF-κB RabbitRabbit CSTCST #8242#8242 1:2001:200 2.5% NHS2.5% NHS InosInos RabbitRabbit InvitrogenInvitrogen #PA1-036#PA1-036 1:2001:200 2.5% NHS2.5% NHS IL-1βIL-1β MouseMouse CSTCST #12242#12242 1:1001:100 2.5% NHS2.5% NHS γH2AxγH2Ax RabbitRabbit CSTCST #60566#60566 1:1001:100 2.5% NHS2.5% NHS Huh7 cellsHuh7 cells 53BP153BP1 RabbitRabbit InvitrogenInvitrogen PA1-16565PA1-16565 1:1001:100 2.5% NHS2.5% NHS 47phox 47 phox RabbitRabbit CSTCST #4301#4301 1:1001:100 2.5% NHS2.5% NHS MDAMDA MouseMouse InvitrogenInvitrogen MA5-27559MA5-27559 1:2001:200 2.5% NHS2.5% NHS 4-HNE4-HNE MouseMouse RNDRND MAB3249MAB3249 1:2001:200 2.5% NHS2.5% NHS

(8) mRNA 분리 및 실시간 중합효소연쇄반응(real-time PCR)(8) mRNA isolation and real-time polymerase chain reaction (real-time PCR)

복막에서 분리한 대식세포를 직경 65 mm의 세포 배양 접시에 2Х106개 cells/dish로 분주하였고, 다음 날 각각의 시료를 6시간 동안 전처리 후 처리하였다. 그런 다음 LPS를 0.2 ㎍/mL(15분)로 처리하고 30분 후에 PBS로 세척하였다. 그 후 TRIZOL을 이용하여 mRNA를 분리하였다. mRNA 분리 후, 시판되는 제품(SuperScript?? One-Cycle cDNA Kit(CAT. A10752030, Thermo Fisher Scientific))을 이용하여 cDNA 합성을 수행하고, 실시간 중합효소연쇄반응(real-time PCR)을 수행하였다.Macrophages isolated from the peritoneum were distributed at 2Х10 6 cells/dish in cell culture dishes with a diameter of 65 mm, and each sample was pretreated for 6 hours the next day. Then, it was treated with LPS at 0.2 μg/mL (15 minutes) and washed with PBS after 30 minutes. Afterwards, mRNA was isolated using TRIZOL. After mRNA isolation, cDNA synthesis was performed using a commercially available product (SuperScript?? One-Cycle cDNA Kit (CAT. A10752030, Thermo Fisher Scientific)), and real-time polymerase chain reaction (real-time PCR) was performed.

본 real-time PCR실험에서 사용된 프라이머 서열은 하기 표 2에 나타내었다.Primer sequences used in this real-time PCR experiment are shown in Table 2 below.

GeneGene Primer Sequence(5'→ 3')Primer Sequence(5'→ 3') 서열 식별번호sequence identifier Arg1Arg1 ForwardForward 5′- CCC AAT GAG TAG GCT GGA GA -3′5′- CCC AAT GAG TAG GCT GGA GA -3′ 1One ReverseReverse 5′- TCT GGA CCC ATT CCT TCT TG -3′5′- TCT GGA CCC ATT CCT TCT TG -3′ 22 TLR4TLR4 ForwardForward 5′- AGC TTC TCC AAT TTT TCA GAA CTT C-3′5′- AGC TTC TCC AAT TTT TCA GAA CTT C-3′ 33 ReverseReverse 5′- TGA GAG GTG GTG TAA GCC ATG C-3′5′- TGA GAG GTG GTG TAA GCC ATG C-3′ 44 Nos2Nos2 ForwardForward 5′-GAG ACA GGG AAG TCT GAA GCA C-3′5′-GAG ACA GGG AAG TCT GAA GCA C-3′ 55 ReverseReverse 5′-CCA GCA GTA GTT GCT CCT CTT C-3′5′-CCA GCA GTA GTT GCT CCT CTT C-3′ 66 CD14CD14 ForwardForward 5′-TTG AAC CTC CGC AAC GTG TCG T-3′5′-TTG AAC CTC CGC AAC GTG TCG T-3′ 77 ReverseReverse 5′-CGC AGG AAA AGT TGA GCG AGT G-3′5′-CGC AGG AAA AGT TGA GCG AGT G-3′ 88 MD2MD2 ForwardForward 5′-CTG AAC CCT GCA TAA GAC TGA GG-3′5′-CTG AAC CCT GCA TAA GAC TGA GG-3′ 99 ReverseReverse 5′-CTT CCT TAC GCT TCG GCA ACT C-3′5′-CTT CCT TAC GCT TCG GCA ACT C-3′ 1010 IL-6IL-6 ForwardForward 5′-CAA AGC CAG AGT CCT TCA GAG-3′5′-CAA AGC CAG AGT CCT TCA GAG-3′ 1111 ReverseReverse 5′-GAG CAT TGG AAA TTG GGG TA-3′5′-GAG CAT TGG AAA TTG GGG TA-3′ 1212 IL-10IL-10 ForwardForward 5′- CAG AGC CAC ATG CTC CTA GA-3'5′- CAG AGC CAC ATG CTC CTA GA-3’ 1313 ReverseReverse 5′- GCT TGG CAA CCC AAG TAA CC-3'5′- GCT TGG CAA CCC AAG TAA CC-3’ 1414 TNFαTNFα ForwardForward 5'- TGT GAA ATG CCA CCT TTT GA-3'5'- TGT GAA ATG CCA CCT TTT GA-3' 1515 ReverseReverse 5'- GGT CAA AGG TTT GGA AGC AG-3'5'- GGT CAA AGG TTT GGA AGC AG-3' 1616 β-actinβ-actin ForwardForward 5′- GGC ACCACACCTTCTACAATGA-3′5′- GGC ACCACACCTTCTACAATGA-3′ 1717 ReverseReverse 5′- ATCTTTTCACGGTTGGCCTTAG-3′5′-ATCTTTTCACGGTTGGCCTTAG-3′ 1818

(9) 웨스턴블롯(Western blotting)(9) Western blotting

RAW 264.7 세포를 RIPA 버퍼를 사용하여 용해시키고 40분 동안 4℃에서 12,000 rpm으로 원심분리기(Centrifuge 5414R, Eppendorf, Hamburg, Germany)로 원심분리하였다. 1분간 원심분리하여 단백질 상층액을 추출한 후 Pierce?? BCA(bicinchoninic acid)를 이용한 정량화에 의한 샘플링을 진행하였다. 각 샘플을 4 내지 20% Poly-acrylamide gradient GEL에 50㎍씩 분주한 후 전기영동하였고, 이동된 단백질은 polyvinylidene difluoride(PVDF) 멤브레인으로 전송(transfer)되었고, 전송된 단백질은 5% skim milk (PBST + Skim milk powder)로 전송되고, bovine serum albumin (BSA)(0.05% PBST + BSA powder)을 사용하여 블로킹(blocking)하였다.RAW 264.7 cells were lysed using RIPA buffer and centrifuged at 12,000 rpm at 4°C for 40 minutes (Centrifuge 5414R, Eppendorf, Hamburg, Germany). After centrifugation for 1 minute to extract the protein supernatant, Pierce?? Sampling was performed through quantification using BCA (bicinchoninic acid). 50㎍ of each sample was dispensed into 4 to 20% Poly-acrylamide gradient GEL and then electrophoresed. The transferred protein was transferred to a polyvinylidene difluoride (PVDF) membrane, and the transferred protein was mixed with 5% skim milk (PBST). + Skim milk powder) and blocked using bovine serum albumin (BSA) (0.05% PBST + BSA powder).

본 웨스턴블롯(Western blotting) 실험에 프로브(probe)로 사용된 1차 항체에 관한 정보는 하기 표 3에 나타내었다.Information on the primary antibody used as a probe in this Western blotting experiment is shown in Table 3 below.

상기 1차 항체로 탐침을 수행한 다음 2차 항체를 각 막에 처리한 후 ECL 시약을 사용하여 시각화하였다. 밴드는 Image J software를 사용하여 감지되고 정량되었다.After probing was performed with the primary antibody, each membrane was treated with a secondary antibody and visualized using ECL reagent. Bands were detected and quantified using Image J software.

ContentsContents HostHost CompanyCompany Catcat RatioRatio PurposePurpose VehicleVehicle AktAkt RabbitRabbit CSTCST #4060#4060 1:10001:1000 WBWB 5% FBS5%FBS p-Aktp-Akt RabbitRabbit CSTCST #4691#4691 1:10001:1000 WBWB 5% FBS5%FBS p38p38 RabbitRabbit CSTCST #8690#8690 1:10001:1000 WBWB 5% FBS5%FBS p-p38p-p38 RabbitRabbit CSTCST #4511#4511 1:10001:1000 WBWB 5% FBS5%FBS JNKJNK RabbitRabbit CSTCST #9252#9252 1:10001:1000 WBWB 5% FBS5%FBS p-JNKp-JNK RabbitRabbit CSTCST # 9255#9255 1:10001:1000 WBWB 5% FBS5%FBS ErkErk RabbitRabbit CSTCST # 4695#4695 1:10001:1000 WBWB 5% FBS5%FBS p-Erkp-Erk RabbitRabbit CSTCST #4370#4370 1:10001:1000 WBWB 5% FBS5%FBS α-actininα-actinin RabbitRabbit SCBTSCBT sc-17829sc-17829 1:20001:2000 WBWB 5% FBS5%FBS

(10) 통계 분석(10) Statistical analysis

모든 수치는 평균±표준오차로 표시하였으며, SPSS(Version 26.0, IBM, Armonk, NY, USA)를 사용하여 one-way analysis of variance(ANOVA) test를 실시한 후, 통계적 유의성은 Tukey's post-hoc test를 통해 평가되었고, p<0.05의 값은 유의미한 것으로 평가되었다.All values were expressed as mean ± standard error, and after performing a one-way analysis of variance (ANOVA) test using SPSS (Version 26.0, IBM, Armonk, NY, USA), statistical significance was determined using Tukey's post-hoc test. It was evaluated through, and a value of p<0.05 was evaluated as significant.

[실험 결과] [Experiment result]

(1) RAW 264.7 세포에서의 LF의 항염증 효과(1) Anti-inflammatory effect of LF in RAW 264.7 cells

1) RAW 264.7 세포의 증식에 미치는 영향1) Effect on proliferation of RAW 264.7 cells

RAW 264.7에서 WLF 및 ELF의 처리에 따른 세포 증식에 미치는 영향을 확인하기 위해 도 1에 표시된 농도의 WLF 및 ELF로 처리한 후 24시간 동안 세포 증식을 수행하였고, 세포 증식 정도를 WST-1 분석으로 측정한 결과를 도 1에 나타내었다.To confirm the effect on cell proliferation according to the treatment of WLF and ELF in RAW 264.7, cell proliferation was performed for 24 hours after treatment with WLF and ELF at the concentrations shown in Figure 1, and the degree of cell proliferation was measured by WST-1 analysis. The measured results are shown in Figure 1.

도 1을 참조하면, RAW 264.7 세포에서는 WLF200 군을 제외한 다른 군에서 세포 증식 정도가 70% 이상으로 높은 것을 확인할 수 있다.Referring to Figure 1, it can be seen that in RAW 264.7 cells, the degree of cell proliferation was high at more than 70% in other groups except the WLF200 group.

따라서 이후 실험에서는 세포 증식 정도가 저해되지 않는 100μg/mL의 농도를 이용하여 실험을 진행하였다.Therefore, in subsequent experiments, the experiment was conducted using a concentration of 100 μg/mL, which does not inhibit cell proliferation.

2) RAW 264.7 세포의 p-Akt 단백질 발현에 미치는 영향2) Effect on p-Akt protein expression in RAW 264.7 cells

대식세포는 TLR4를 포함하는 TLR family의 구성원을 통해 활성화될 수 있다. 활성화된 TLR4는 phosphoinositide 3 kinases(PI3K/Akt)를 포함한 특정 세포 내 경로의 활성화를 유도한다. 인산화된 Akt는 대식세포 분극화(macrophage polarization)에 참여한다(비특허문헌 33 참조).Macrophages can be activated through members of the TLR family, including TLR4. Activated TLR4 induces the activation of specific intracellular pathways, including phosphoinositide 3 kinases (PI3K/Akt). Phosphorylated Akt participates in macrophage polarization (see Non-Patent Document 33).

RAW 264.7 세포에서 p-Akt 단백질 발현에 대한 LF의 영향을 조사하기 위해 Western blotting을 수행하였고, 그 결과를 도 2에 나타내었다.Western blotting was performed to investigate the effect of LF on p-Akt protein expression in RAW 264.7 cells, and the results are shown in Figure 2.

도 2를 참조하면, 대조군(LPS group)의 p-Akt는 정상군(Normal group)과 대비하여 유의하게 증가한 반면(2.07배, P<0.01), WLF 처리군은 대조군 대비 46.38% 감소하였고, ELF 처리군은 대조군 대비 65.40% 감소하였으며, 특히, ELF 처리군은 WLF 처리군 대비 35.48% 감소하였다.Referring to Figure 2, p-Akt in the control group (LPS group) significantly increased compared to the normal group (2.07 times, P<0.01), while the WLF treatment group decreased by 46.38% compared to the control group, and ELF The treatment group decreased by 65.40% compared to the control group, and in particular, the ELF-treated group decreased by 35.48% compared to the WLF-treated group.

3) RAW 264.7 세포의 MAPK 단백질 발현에 미치는 영향3) Effect on MAPK protein expression in RAW 264.7 cells

p38, c-Jun N-terminal kinase (JNK) 및 extracellular signal-regulated kinase(Erk) 키나제(kinase)를 포함한 MAPK 구성원은 세포 외부 자극에 반응하여 활성화된다. MAPK 신호 전달 경로는 전염증 매개체(pro-inflammatory mediator)의 발현을 유도하는 전형적인 염증 관련 신호이다(비특허문헌 34 참조). 또한 상기 신호 전달 경로는 세포 주기, 증식 및 세포 사멸과 같은 광범위한 세포 반응을 조절하는 데 중요한 역할을 한다.MAPK members, including p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (Erk) kinase, are activated in response to extracellular stimuli. The MAPK signaling pathway is a typical inflammation-related signal that induces the expression of pro-inflammatory mediators (see Non-Patent Document 34). Additionally, the signaling pathway plays an important role in regulating a wide range of cellular responses such as cell cycle, proliferation, and apoptosis.

MAPK 경로에 대한 LF의 영향을 알아보기 위해 p-p38, p-JNK 및 p-ErK의 발현을 확인하였고, 그 결과를 도 3에 나타내었다. To determine the effect of LF on the MAPK pathway, the expression of p-p38, p-JNK, and p-ErK was confirmed, and the results are shown in Figure 3.

도 3을 참조하면, 상기 p-p38, p-JNK 및 p-ErK 단백질의 발현은 정상군과 대비하여 대조군에서 유의하게 증가하였다(p-p38, 2.57배; p-JNK, 2.65배; 및 p-ErK, 2.82배). 여기서, WLF 처리군 및 ELF 처리군 모두에서 p-ErK 수준은 대조군과 대비하여 현저하게 감소하였다(WLF, 66.87%, p<0.001; ELF, 75.11%, p<0.001). 그러나 p-p38및 p-JNK의 발현은 ELF 처리군에서만 유의하게 감소하였다(p-p38, p<0.01; p-JNK, p<0.001). 특히 ELF100 처리군(ELF 100μg/mL 처리군)의 p-p38과 p-JNK 수치는 대조군 대비 각각 최대 53.07% 및 83.85% 감소되어, 유의미한 감소를 보였다. 반면 WLF 처리군은 대조군과 대비하여 p-p38 및 p-JNK의 발현에 유의미한 차이를 보이지 않았다.Referring to Figure 3, the expression of p-p38, p-JNK, and p-ErK proteins was significantly increased in the control group compared to the normal group (p-p38, 2.57 times; p-JNK, 2.65 times; and p -ErK, 2.82 times). Here, p-ErK levels in both the WLF-treated group and the ELF-treated group were significantly decreased compared to the control group (WLF, 66.87%, p<0.001; ELF, 75.11%, p<0.001). However, the expression of p-p38 and p-JNK was significantly decreased only in the ELF-treated group (p-p38, p<0.01; p-JNK, p<0.001). In particular, p-p38 and p-JNK levels in the ELF100 treated group (ELF 100 μg/mL treated group) decreased by up to 53.07% and 83.85%, respectively, compared to the control group, showing a significant decrease. On the other hand, the WLF treatment group showed no significant difference in the expression of p-p38 and p-JNK compared to the control group.

(2) 마우스 복막 대식세포에서의 LF의 항염증 효과(2) Anti-inflammatory effect of LF on mouse peritoneal macrophages

1) 마우스 복막 대식세포의 증식에 미치는 영향1) Effect on proliferation of mouse peritoneal macrophages

마우스 복막 대식세포에서 WLF 및 ELF 처리에 의한 세포 증식에 미치는 영향을 확인하기 위해, 도 4에 표시된 농도의 WLF 및 ELF로 처리한 후 24시간 동안 세포 증식을 수행하였고, 세포 증식 정도를 WST-1 분석으로 측정한 결과를 도 4에 나타내었다.To determine the effect on cell proliferation by WLF and ELF treatment in mouse peritoneal macrophages, cell proliferation was performed for 24 hours after treatment with WLF and ELF at the concentrations shown in Figure 4, and the degree of cell proliferation was measured using WST-1. The results measured by the analysis are shown in Figure 4.

도 4를 참조하면, 모든 군에서 유의미한 차이를 보이지 않았다.Referring to Figure 4, there was no significant difference in all groups.

2) 마우스 복막 대식세포의 TLR4 mRNA 발현에 미치는 영향2) Effect on TLR4 mRNA expression in mouse peritoneal macrophages

대다수의 염증 매개체의 생산은 주로 TLR의 활성화에 의해 매개된다. The production of the majority of inflammatory mediators is primarily mediated by activation of TLRs.

TLR의 발현을 real-time PCR을 이용하여 측정한 결과를 도 5에 나타내었다.The results of measuring TLR expression using real-time PCR are shown in Figure 5.

도 5를 참조하면, 정상군과 대비하여 대조군에서 TLR4 발현이 높게 유도되었다(p<0.001). 이는 TLR4 발현이 세균 외독소(bacterial exotoxin)에 의해 유발되는 염증에 관여하고 있음을 나타낸다. Referring to Figure 5, TLR4 expression was highly induced in the control group compared to the normal group (p<0.001). This indicates that TLR4 expression is involved in inflammation caused by bacterial exotoxin.

대조군에서 증가된 TLR4 수준은 ELF 처리군에서만 유의하게 조절되었다(p<0.001). 특히, ELF100 처리군의 TLR4 수치는 대조군 대비 65.2% 감소하는 것으로 확인되었다(p<0.001).The increased TLR4 level in the control group was significantly regulated only in the ELF-treated group (p<0.001). In particular, TLR4 levels in the ELF100-treated group were found to decrease by 65.2% compared to the control group (p<0.001).

3) 마우스 복막 대식세포의 MD2 및 CD14 mRNA 발현에 미치는 영향3) Effect on MD2 and CD14 mRNA expression in mouse peritoneal macrophages

복합 리간드인 TLR4-myeloid differentiation factor 2(MD2) heterodimer는 cluster of differentiation 14(CD14)를 통해 내독성 LPS를 인식하여 염증 반응을 유도한다. The complex ligand TLR4-myeloid differentiation factor 2 (MD2) heterodimer recognizes endotoxic LPS through cluster of differentiation 14 (CD14) and induces an inflammatory response.

MD2 및 CD14의 발현을 real-time PCR을 이용하여 측정한 결과를 도 6에 나타내었다.The results of measuring the expression of MD2 and CD14 using real-time PCR are shown in Figure 6.

도 6을 참조하면, 대조군에서 MD2 및 CD14의 발현이 정상군(무처리군) 대비 각각 1.5배 및 1.4배 유의하게 증가하였다(MD2, p<0.05, CD14, p<0.01). 한편, 대조군에서의 이러한 증가는 ELF 처리에 의해 유의미한 감소를 나타내었다(MD2, p<0.01; CD14, p<0.05). 구체적으로, ELF 처리군의 MD2 및 CD14 수준은 대조군 대비 각각 33.0% 및 19.2% 감소되어, 현저하게 발현이 억제된 것을 확인할 수 있다.Referring to Figure 6, the expression of MD2 and CD14 in the control group was significantly increased by 1.5-fold and 1.4-fold, respectively, compared to the normal group (untreated group) (MD2, p<0.05, CD14, p<0.01). Meanwhile, this increase in the control group was significantly reduced by ELF treatment (MD2, p<0.01; CD14, p<0.05). Specifically, MD2 and CD14 levels in the ELF-treated group were reduced by 33.0% and 19.2%, respectively, compared to the control group, confirming that expression was significantly suppressed.

4) 마우스 복막 대식세포의 M1 유형의 분극화 생체지표에 미치는 영향4) Effect on M1 type polarization biomarkers of mouse peritoneal macrophages

대식세포는 단핵 식세포(mononuclear phagocyte) 그룹에 속하며 면역 반응에서 중요한 역할을 한다. 활성화 후, 대식세포는 광범위하게 전염증성(pro-inflammatory) 또는 항염증성(anti-inflammatory) 생체지표(biomarker)를 방출할 수 있으며, 이는 동료 면역 세포를 더욱 활성화시킨다. 즉, 대식세포는 서로 다른 기능을 수행하는 두 그룹으로 분극화되는데, 전형적으로 활성화된 M1형 대식세포(pro-inflammation 반응)와 대안적으로 활성화된 M2형 대식세포(anti-inflammation 반응)로 분극화된다(비특허문헌 35 참조).Macrophages belong to the mononuclear phagocyte group and play an important role in immune responses. After activation, macrophages can release a wide range of pro-inflammatory or anti-inflammatory biomarkers, which further activate fellow immune cells. That is, macrophages are polarized into two groups that perform different functions: typically activated M1 macrophages (pro-inflammation response) and alternatively activated M2 macrophages (anti-inflammation response). (Refer to Non-Patent Document 35).

대식세포 분극화 조절에 대한 LF의 효과를 이해하기 위해 M1형 대식세포에 대한 Nitric oxide synthase 2(Nos2), TNFα 및 IL-6를 포함한 분극화 생체지표의 수준을 qPCR(real time PCR)로 측정하였고, 그 결과를 도 7에 나타내었다.To understand the effect of LF on the regulation of macrophage polarization, the levels of polarization biomarkers, including nitric oxide synthase 2 (Nos2), TNFα, and IL-6, in M1-type macrophages were measured by real-time PCR (qPCR). The results are shown in Figure 7.

도 7을 참조하면, 대조군에서 Nos2, TNFα 및 IL-6의 수준이 정상군 대비 상향 조절된 것을 확인할 수 있다. 구체적으로, 대조군에서 Nos2, TNFα 및 IL-6의 mRNA 발현은 정상군(무처리군) 대비 각각 1.6배, 1.5배 및 1.6배 유의하게 증가하였다(Nos2, p<0.05; TNFα, p<0.05; IL-6, p<0.01). Referring to Figure 7, it can be seen that the levels of Nos2, TNFα, and IL-6 in the control group were upregulated compared to the normal group. Specifically, the mRNA expression of Nos2, TNFα, and IL-6 in the control group was significantly increased by 1.6-fold, 1.5-fold, and 1.6-fold, respectively, compared to the normal group (untreated group) (Nos2, p<0.05; TNFα, p<0.05; IL-6, p<0.01).

한편, 이러한 증가는 ELF 처리에 의해 현저하게 감소된 것을 확인할 수 있다(Nos2, p<0.01; TNFα, p<0.01; IL-6, p<0.02). 구체적으로, ELF 처리군의 Nos2, TNFα 및 IL-6 수준은 대조군 대비 각각 38.7%, 37.9% 및 52.2% 감소되어, M1형 대식세포에 대한 분극화 생체지표가 효과적으로 억제되었다. 특히, TNFα와 IL-6는 ELF 처리에 의해 정상군 수준 이하로 하향 조절되었다. 반면, WLF 처리는 대조군과 대비하여 Nos2, TNFα 및 IL-6의 발현에 유의미한 차이를 나타내지 않았다.Meanwhile, it can be seen that this increase was significantly reduced by ELF treatment (Nos2, p<0.01; TNFα, p<0.01; IL-6, p<0.02). Specifically, the levels of Nos2, TNFα, and IL-6 in the ELF-treated group were reduced by 38.7%, 37.9%, and 52.2%, respectively, compared to the control group, effectively suppressing polarization biomarkers for M1-type macrophages. In particular, TNFα and IL-6 were downregulated below normal levels by ELF treatment. On the other hand, WLF treatment did not show significant differences in the expression of Nos2, TNFα, and IL-6 compared to the control group.

5) 마우스 복막 대식세포의 M2 유형의 분극화 생체지표에 미치는 영향5) Effect on M2 type polarization biomarkers of mouse peritoneal macrophages

M2형 대식세포가 사이토카인 IL-4 및 IL-13에 대한 반응으로 분극화되면 IL-10과 같은 항염증 사이토카인을 분비하고 Arginase 1(Arg1)의 발현이 상향 조절된다. Arg1은 L-아르기닌을 요소 및 L-오르니틴으로 대사한다. 여기서 L-오르니틴은 세포 증식 및 콜라겐 합성과 같은 필수적인 역할을 수행할 수 있는 프롤린을 부분적으로 합성한다(비특허문헌 37 참조). When M2-type macrophages are polarized in response to the cytokines IL-4 and IL-13, they secrete anti-inflammatory cytokines such as IL-10 and the expression of Arginase 1 (Arg1) is upregulated. Arg1 metabolizes L-arginine to urea and L-ornithine. Here, L-ornithine partially synthesizes proline, which can perform essential roles such as cell proliferation and collagen synthesis (see Non-Patent Document 37).

대식세포 분극화 조절에 대한 LF의 효과를 확인하기 위해 M2형 대식세포에 대한 IL-10 및 Arg1을 포함한 분극화 생체지표의 수준을 qPCR을 이용하여 측정하였고, 그 결과를 도 8에 나타내었다.To confirm the effect of LF on the regulation of macrophage polarization, the levels of polarization biomarkers, including IL-10 and Arg1, for M2-type macrophages were measured using qPCR, and the results are shown in Figure 8.

도 8을 참조하면, IL-10 및 Arg1의 mRNA 발현은 대조군에서 정상군 대비 각각 37.83% 및 60.61% 유의하게 감소하였다(IL-10, p=0.05; Arg1, p<0.05). 이러한 감소는 ELF 처리에 의해 현저하게 증가하는 것으로 확인되었다(IL-10 및 Arg1, p<0.01). 구체적으로 ELF 처리군의 IL-10 및 Arg1 수준은 대조군 대비 각각 2.1배 및 1.7배 효과적으로 상향 조절되었다.Referring to Figure 8, the mRNA expression of IL-10 and Arg1 was significantly decreased by 37.83% and 60.61% in the control group compared to the normal group, respectively (IL-10, p=0.05; Arg1, p<0.05). This decrease was confirmed to be significantly increased by ELF treatment (IL-10 and Arg1, p<0.01). Specifically, IL-10 and Arg1 levels in the ELF-treated group were effectively upregulated by 2.1-fold and 1.7-fold, respectively, compared to the control group.

6) 마우스 복막 대식세포의 TLR4 발현 세포 수에 미치는 영향6) Effect on the number of TLR4-expressing cells in mouse peritoneal macrophages

TLR4가 일련의 특성화된 하위 신호 전달을 통해 염증 반응을 개시하므로, 면역조직화학(immunohistochemistry, IHC)을 사용하여 TLR4의 발현을 검출하였고, 면역조직화학적 염색 결과 사진은 도 9a에 나타내었으며, TLR4 발현 세포 수를 계수한 결과는 도 9b에 나타내었다.Since TLR4 initiates the inflammatory response through a series of characterized downstream signaling, the expression of TLR4 was detected using immunohistochemistry (IHC), and the immunohistochemical staining results are shown in Figure 9a, TLR4 expression The results of counting the number of cells are shown in Figure 9b.

도 9a 및 도 9b를 참조하면, 대조군의 TLR4 발현 세포수는 정상군 대비 17.5배 유의하게 증가하였다(##p<0.01). 상기 증가된 수준은 ELF 처리에 의해 현저하게 억제되었으나(74.29%, *p<0.05), WLF 처리는 대조군 대비 감소하는 경향만 보였다.Referring to Figures 9A and 9B, the number of TLR4-expressing cells in the control group was significantly increased by 17.5 times compared to the normal group ( ## p<0.01). The increased level was significantly suppressed by ELF treatment (74.29%, * p < 0.05), but WLF treatment only showed a tendency to decrease compared to the control group.

7) 마우스 복막 대식세포에서 NF-κB 핵 세포 수에 미치는 영향7) Effect on NF-κB nuclear cell number in mouse peritoneal macrophages

LPS는 숙주 TLR4 수용체에 결합하여 NF-κB 신호 전달을 활성화한다.LPS binds to the host TLR4 receptor and activates NF-κB signaling.

면역조직화학(immunohistochemistry, IHC)을 사용하여 NF-κB 핵 세포를 검출하였고, 면역조직화학적 염색 결과 사진은 도 10a에 나타내었으며, NF-κB 핵 세포 수를 계수한 결과는 도 10b에 나타내었다.NF-κB nuclear cells were detected using immunohistochemistry (IHC). A photograph of the immunohistochemical staining results is shown in Figure 10a, and the results of counting the number of NF-κB nuclear cells are shown in Figure 10b.

도 10a 및 도 10b를 참조하면, 대조군의 NF-κB 핵 세포 수는 정상군 대비 6.77배 유의하게 증가하였다(##p<0.01). 이러한 증가된 수준은 ELF 처리에 의해 현저하게 억제되었다(55.62%, **p<0.01). 그러나 대조군과 WLF100 처리군 간에는 유의미한 차이가 없는 것으로 확인되었다.Referring to Figures 10A and 10B, the number of NF-κB nuclear cells in the control group was significantly increased by 6.77 times compared to the normal group ( ## p<0.01). These increased levels were significantly suppressed by ELF treatment (55.62%, ** p < 0.01). However, it was confirmed that there was no significant difference between the control group and the WLF100 treatment group.

8) 마우스 복막 대식세포의 iNOS 발현 세포 수에 미치는 영향8) Effect on the number of iNOS-expressing cells in mouse peritoneal macrophages

염증 진행이나 억제는 염증 세포에서 합성되는 유도성 효소와 같은 inducible nitric oxide synthase(iNOS)에 의해 제어된다.The progression or suppression of inflammation is controlled by inducible nitric oxide synthase (iNOS), an inducible enzyme synthesized in inflammatory cells.

면역조직화학(immunohistochemistry, IHC)을 사용하여 iNOS의 발현을 검출하였고, 면역조직화학적 염색 결과 사진은 도 11a에 나타내었으며, iNOS 발현 세포 수를 계수한 결과는 도 11b에 나타내었다.The expression of iNOS was detected using immunohistochemistry (IHC). A photo of the immunohistochemical staining results is shown in Figure 11a, and the results of counting the number of iNOS-expressing cells are shown in Figure 11b.

도 11a 및 도 11b를 참조하면, 대조군의 iNOS 발현 세포 수는 정상군 대비 2.85배 유의하게 증가하였다(##p<0.01). 이러한 증가 수준은 ELF 처리에 의해 효과적으로 억제되었으나(83.78%, **p<0.01), WLF 처리군은 대조군 대비 감소하는 경향만 보였다.Referring to Figures 11A and 11B, the number of iNOS-expressing cells in the control group was significantly increased by 2.85 times compared to the normal group ( ## p<0.01). This increase level was effectively suppressed by ELF treatment (83.78%, ** p < 0.01), but the WLF treatment group only showed a tendency to decrease compared to the control group.

9) 마우스 복막 대식세포의 IL-1β 발현 세포 수에 미치는 영향9) Effect on the number of IL-1β expressing cells in mouse peritoneal macrophages

염증 진행 또는 억제는 염증 세포에서 방출되는 염증성 사이토카인에 의해 조절된다.The progression or suppression of inflammation is regulated by inflammatory cytokines released from inflammatory cells.

면역조직화학(immunohistochemistry, IHC)을 사용하여 IL-1β의 발현을 검출하였고, 면역조직화학적 염색 결과 사진은 도 12a에 나타내었으며, IL-1β 발현 세포 수를 계수한 결과는 도 12b에 나타내었다.The expression of IL-1β was detected using immunohistochemistry (IHC). A photo of the immunohistochemical staining results is shown in Figure 12a, and the results of counting the number of cells expressing IL-1β are shown in Figure 12b.

도 12a 및 도 12b를 참조하면, 대조군의 IL-1β 발현 세포 수는 정상군 대비 4.41배 유의하게 증가하였다(###p<0.001). 이러한 증가된 수준은 ELF 처리에 의해 현저하게 억제되었으며(78.46%, ***p<0.001), 이에 따라 ELF100 처리군의 IL-1β 발현 세포 수는 정상군과 유사한 수준으로 확인되었다. 그러나 대조군과 WLF100 처리군 간에는 유의미한 차이가 없는 것으로 확인되었다.Referring to Figures 12A and 12B, the number of IL-1β expressing cells in the control group was significantly increased by 4.41 times compared to the normal group ( ### p<0.001). This increased level was significantly suppressed by ELF treatment (78.46%, *** p<0.001), and accordingly, the number of IL-1β expressing cells in the ELF100 treated group was confirmed to be similar to that of the normal group. However, it was confirmed that there was no significant difference between the control group and the WLF100 treatment group.

(3) Huh7 세포에서의 LF의 항산화 효과(3) Antioxidant effect of LF in Huh7 cells

1) Huh7 세포의 증식에 미치는 영향1) Effect on proliferation of Huh7 cells

Huh7 세포주는 간세포암종(hepatocellular carcinoma)을 앓고 있는 57세 남성과 영구 세포주로부터 유래되었다. The Huh7 cell line was derived from a 57-year-old man with hepatocellular carcinoma and a persistent cell line.

WLF 및 ELF 처리에 따른 Huh7 세포의 세포 증식에 미치는 영향을 확인하기 위하여 도 13에 표시된 농도의 WLF 및 ELF로 처리한 후 24시간 동안 세포 증식을 수행하고, WST-1 분석을 사용하여 세포 증식 정도를 측정하였고, 그 결과를 도 13에 나타내었다.To confirm the effect of WLF and ELF treatment on cell proliferation of Huh7 cells, cell proliferation was performed for 24 hours after treatment with WLF and ELF at the concentrations shown in Figure 13, and the degree of cell proliferation was measured using WST-1 analysis. was measured, and the results are shown in Figure 13.

도 13을 참조하면, WLF 200 처리군을 제외한 다른 군에서 세포 증식 정도가 70% 이상으로 높은 것을 확인할 수 있다.Referring to Figure 13, it can be seen that the degree of cell proliferation was high at more than 70% in other groups except the WLF 200 treatment group.

따라서 이후 실험에서는 세포 증식 정도가 저해되지 않는 100μg/mL의 농도를 이용하여 실험을 진행하였다.Therefore, in subsequent experiments, the experiment was conducted using a concentration of 100 μg/mL, which does not inhibit cell proliferation.

2) Huh7 세포의 DCFDA 수준에 미치는 영향2) Effect on DCFDA levels in Huh7 cells

형광성 염료인 DCFDA는 세포 내 hydroxyl(ㆍOH), peroxyl(ROOㆍ) 및 기타 ROS 활성을 측정하는 데 사용된다(비특허문헌 38 참조).DCFDA, a fluorescent dye, is used to measure intracellular hydroxyl (ㆍOH), peroxyl (ROOㆍ) and other ROS activities (see Non-Patent Document 38).

H2O2로 유도된 Huh7 세포에서 WLF 및 ELF 처리에 따른 DCFDA 수준의 변화를 측정하였고, 그 결과를 도 14에 나타내었다.Changes in DCFDA levels according to WLF and ELF treatment were measured in Huh7 cells induced with H 2 O 2 , and the results are shown in Figure 14.

도 14를 참조하면, 대조군(H2O2만 유도한 그룹)의 DCFDA 수준은 정상군(무처리군) 대비 2.5배 유의하게 증가하였다. 이러한 증가된 DCFDA 수치는 ELF 처리에 의해 약 21.33% 현저하게 감소하였다. 그러나 대조군과 WLF 처리군 사이에는 유의미한 차이가 없는 것으로 확인되었다.Referring to Figure 14, the DCFDA level of the control group (group induced only with H 2 O 2 ) was significantly increased by 2.5 times compared to the normal group (untreated group). These increased DCFDA levels were significantly reduced by approximately 21.33% by ELF treatment. However, it was confirmed that there was no significant difference between the control group and the WLF treatment group.

3) Huh7 세포의 DHE 수준에 미치는 영향3) Effect on DHE levels in Huh7 cells

DHE는 널리 사용되는 에티듐(ethidium) 기반의 산화환원 민감성 형광 프로브이고, superoxide(O2ㆍ-)에 의해 산화되는 것으로 알려져 있다. 라디칼 반응의 개시제인 superoxide는 특히 강력한 산화제로서 높은 반응성을 가지고 있다(비특허문헌 39 참조).DHE is a widely used ethidium-based redox-sensitive fluorescent probe, and is known to be oxidized by superoxide (O2 ㆍ- ). Superoxide, an initiator of radical reactions, is a particularly strong oxidizing agent and has high reactivity (see Non-Patent Document 39).

H2O2로 유도된 Huh7 세포에서 WLF 및 ELF 처리에 따른 DHE 수준의 변화를 측정하였고, 그 결과를 도 15에 나타내었다.Changes in DHE levels according to WLF and ELF treatment were measured in Huh7 cells induced with H 2 O 2 , and the results are shown in Figure 15.

도 15를 참조하면, 대조군의 DHE 수준은 정상군 대비 2.27배 유의하게 증가하였다. 이러한 증가된 DHE 수준은 ELF 처리에 의해 약 23.52% 현저하게 감소하였다. 그러나 대조군과 WLF 처리군 사이에는 유의미한 차이가 없는 것으로 확인되었다.Referring to Figure 15, the DHE level of the control group was significantly increased by 2.27 times compared to the normal group. These increased DHE levels were significantly reduced by about 23.52% by ELF treatment. However, it was confirmed that there was no significant difference between the control group and the WLF treatment group.

4) Huh7 세포의 SOD 활성에 미치는 영향4) Effect on SOD activity in Huh7 cells

스트레스 조건에서 ROS의 과잉 생산은 세포 손상을 유발한다. superoxide의 제거는 superoxide에서 H2O2로의 dismutation을 촉매하는 상위 효소인 SOD를 통해 이루어질 수 있다. SOD는 산화 스트레스에 민감한 모든 호기성 유기체에 존재하는 것으로 알려져 있다(비특허문헌 40 참조).Overproduction of ROS under stress conditions causes cell damage. Removal of superoxide can be achieved through SOD, an upstream enzyme that catalyzes the dismutation of superoxide into H 2 O 2 . SOD is known to exist in all aerobic organisms sensitive to oxidative stress (see Non-Patent Document 40).

H2O2로 유도된 Huh7 세포에서 WLF 및 ELF 처리에 따른 SOD 활성의 변화를 측정하였고, 그 결과를 도 16에 나타내었다.Changes in SOD activity according to WLF and ELF treatment were measured in Huh7 cells induced with H 2 O 2 , and the results are shown in FIG. 16.

도 16을 참조하면, 대조군의 SOD 활성은 정상군 대비 63.20% 유의하게 감소하였고, 감소된 SOD 활성은 ELF 처리에 의해 대조군 대비 약 1.83배 유의하게 증가하였다. 그러나 대조군과 WLF 처리군 사이에는 유의미한 차이가 없는 것으로 확인되었다.Referring to Figure 16, the SOD activity of the control group was significantly reduced by 63.20% compared to the normal group, and the reduced SOD activity was significantly increased by about 1.83 times compared to the control group by ELF treatment. However, it was confirmed that there was no significant difference between the control group and the WLF treatment group.

5) Huh7 세포의 GSH 함량에 미치는 영향5) Effect on GSH content in Huh7 cells

GSH는 호기성 세포에서 가장 풍부한 항산화제이며 체액에는 마이크로몰(μM) 농도로, 조직에는 밀리몰(mM) 농도로 존재한다. GSH는 활성산소 제거제 역할을 하고 지질 과산화를 억제하며 H2O2 해독에 참여하여 산화 스트레스로부터 조직을 보호한다(비특허문헌 41 참조).GSH is the most abundant antioxidant in aerobic cells and exists at micromolar (μM) concentrations in body fluids and millimolar (mM) concentrations in tissues. GSH acts as a free radical scavenger, inhibits lipid peroxidation, and participates in H 2 O 2 detoxification to protect tissues from oxidative stress (see Non-Patent Document 41).

H2O2로 유도된 Huh7 세포에서 WLF 및 ELF 처리에 따른 GSH 함량의 변화를 측정하였고, 그 결과를 도 17에 나타내었다.Changes in GSH content according to WLF and ELF treatment were measured in Huh7 cells induced with H 2 O 2 , and the results are shown in FIG. 17.

도 17을 참조하면, 대조군의 GSH 함량은 정상군 대비 66.33% 유의하게 감소하였다. 감소된 GSH 함량은 ELF 처리에 의해 약 1.65배 유의하게 증가하였다. 그러나 WLF 처리군의 GSH 함량은 대조군 대비 감소한 것으로 확인되었다.Referring to Figure 17, the GSH content of the control group was significantly decreased by 66.33% compared to the normal group. The reduced GSH content was significantly increased by about 1.65 times by ELF treatment. However, the GSH content of the WLF treatment group was confirmed to be decreased compared to the control group.

6) Huh7 세포의 사멸에 미치는 영향6) Effect on Huh7 cell death

LIVE/DEAD® Viability/Cytotoxicity Kit는 원형질막 무결성 및 에스테라제 활성을 기반으로 세포의 생존율을 결정하는 쉽고 빠른 2색 분석(two-color assay)이다(비특허문헌 42 참조).LIVE/DEAD ® Viability/Cytotoxicity Kit is an easy and fast two-color assay that determines cell viability based on plasma membrane integrity and esterase activity (see Non-Patent Document 42).

Huh7 세포에서 WLF 및 ELF 처리에 따른 세포 사멸에 미치는 영향을 확인하기 위하여 상기 LIVE/DEAD® Viability/Cytotoxicity Kit를 사용하여 생존한 세포(초록색) 및 사멸한 세포(주황색)를 측정하였고, 그 결과를 도 18에 나타내었다. To confirm the effect on cell death caused by WLF and ELF treatment in Huh7 cells, surviving cells (green) and dead cells (orange) were measured using the LIVE/DEAD ® Viability/Cytotoxicity Kit, and the results were It is shown in Figure 18.

도 18을 참조하면, 대조군에서 사멸한 세포(%)는 정상군 대비 57.39배 유의하게 증가하였다.Referring to Figure 18, the dead cells (%) in the control group significantly increased 57.39 times compared to the normal group.

한편, 대조군에서 증가된 사멸 세포의 수치는 ELF 처리에 의해 69.35% 현저하게 감소하였다. 그러나 대조군과 WLF100 처리군 간에는 유의미한 차이가 없는 것으로 확인되었다.Meanwhile, the number of dead cells increased in the control group was significantly reduced by 69.35% by ELF treatment. However, it was confirmed that there was no significant difference between the control group and the WLF100 treatment group.

7) Huh7 세포의 ROS 강도에 미치는 영향7) Effect on ROS intensity in Huh7 cells

H2O2로 유도된 Huh7 세포에서 WLF 및 ELF 처리에 따른 ROS 강도(intensity)의 변화를 측정하기 위하여 면역형광염색법으로 염색 후 형광현미경으로 관찰하였고, 그 결과를 도 19에 나타내었다. To measure changes in ROS intensity according to WLF and ELF treatment in Huh7 cells induced with H 2 O 2 , the cells were stained with immunofluorescence staining and observed with a fluorescence microscope, and the results are shown in FIG. 19.

도 19를 참조하면, ROS에 영향을 받지 않은 생존 세포는 핵이 파란색으로 나타나고, ROS의 강도가 강하면 세포가 파쇄되어 초록색의 신호가 나타났다. 대조군의 ROS 강도(생존 세포의 백분율(%))는 정상군 대비 324.8배 유의하게 증가하였다.Referring to Figure 19, surviving cells that were not affected by ROS had blue nuclei, and when the intensity of ROS was strong, the cells were destroyed and a green signal appeared. The ROS intensity (percentage (%) of viable cells) of the control group significantly increased 324.8 times compared to the normal group.

한편, 증가된 ROS 강도는 ELF 처리에 의해 70.01% 현저하게 감소하였다. 그러나 대조군과 WLF 처리군 간에는 유의미한 차이가 없는 것으로 확인되었다.Meanwhile, the increased ROS intensity was significantly reduced by 70.01% by ELF treatment. However, it was confirmed that there was no significant difference between the control group and the WLF treatment group.

(4) Huh7 세포에서의 LF의 세포 사멸 억제(Anti-apoptotic) 효과(4) Anti-apoptotic effect of LF in Huh7 cells

1) Huh7 세포의 TUNEL 양성 세포 수에 미치는 영향1) Effect on the number of TUNEL-positive cells in Huh7 cells

TUNEL에 의한 DNA 단편화의 검출은 세포 사멸의 말기 단계에서 세포 사멸 세포를 식별하기 위해 사용되는 기술로, TUNEL 분석은 유세포 분석기(flow cytometer)를 사용하고 TUNEL에 대해 양성인 세포를 계산한다(비특허문헌 43 참조).Detection of DNA fragmentation by TUNEL is a technique used to identify apoptotic cells in the late stages of cell death. The TUNEL assay uses a flow cytometer and counts cells positive for TUNEL (non-patent literature) 43).

H2O2로 유도된 Huh7 세포에서 WLF 및 ELF 처리에 따른 TUNEL 양성 세포 수의 변화를 유세포 분석기로 측정하였고, 그 결과를 도 20에 나타내었다.Changes in the number of TUNEL-positive cells in Huh7 cells induced with H 2 O 2 according to WLF and ELF treatment were measured using flow cytometry, and the results are shown in FIG. 20.

도 20을 참조하면, 대조군의 TUNEL 양성 세포 수는 정상군 대비 47.33배 유의하게 증가하였다.Referring to Figure 20, the number of TUNEL positive cells in the control group was significantly increased by 47.33 times compared to the normal group.

한편, 이러한 TUNEL 양성 세포 수의 증가는 ELF 처리에 의해 65.49% 현저하게 감소하였다. 그러나 WLF 처리군은 대조군 대비 3.52% 감소하였으나 유의미한 차이는 없는 것으로 확인되었다.Meanwhile, this increase in the number of TUNEL-positive cells was significantly reduced by 65.49% by ELF treatment. However, the WLF treatment group decreased by 3.52% compared to the control group, but it was confirmed that there was no significant difference.

(5) Huh7 세포에서의 LF의 DNA 손상 억제 효과(5) DNA damage inhibition effect of LF in Huh7 cells

1) Huh7 세포의 γH2Ax 수준에 미치는 영향1) Effect on γH2Ax levels in Huh7 cells

DNA 손상 반응은 전리 방사선(ionizing radiation)이나 DNA 손상 치료제와 같은 다양한 스트레스 신호에 반응하여 시작될 수 있다. gamma H2A histone family member x(γ-H2Ax)는 histone H2Ax의 인산화된 버전(histone H2A 변이체) 이며 DNA 손상으로 인한 double-stranded breaks(DSBs)에 대한 마커(marker)이다. 여기서 γ-H2Ax는 현미경을 이용한 면역염색을 통해 검출될 수 있다(비특허문헌 44 참조).The DNA damage response can be initiated in response to a variety of stress signals, such as ionizing radiation or DNA damage therapeutics. gamma H2A histone family member Here, γ-H2Ax can be detected through immunostaining using a microscope (see Non-Patent Document 44).

H2O2로 유도된 Huh7 세포에서 WLF 및 ELF 처리에 따른 γH2Ax 수준의 변화를 측정하였고, 그 결과를 도 21에 나타내었다.Changes in γH2Ax levels following WLF and ELF treatment were measured in Huh7 cells induced with H 2 O 2 , and the results are shown in Figure 21.

도 21을 참조하면, 대조군의 γH2Ax 수준은 정상군 대비 34.5배 유의하게 증가하였다.Referring to Figure 21, the level of γH2Ax in the control group was significantly increased by 34.5 times compared to the normal group.

한편, 증가된 γH2Ax 수치는 ELF 처리에 의해 71.01% 현저하게 감소하였다. 그러나 WLF 처리군은 대조군 대비 0.72% 감소하였으나 유의미한 차이는 없는 것으로 확인되었다.Meanwhile, the increased γH2Ax level was significantly reduced by 71.01% by ELF treatment. However, the WLF treatment group decreased by 0.72% compared to the control group, but it was confirmed that there was no significant difference.

2) Huh7 세포의 53BP1 수준에 미치는 영향2) Effect on 53BP1 levels in Huh7 cells

p53-binding protein 1(53BP1)은 DNA DBS 신호 전달 및 복구와 관련된 중요한 구성 요소이며 게놈 무결성(genomic integrity)을 보존하고 세포 항상성을 유지하는 데 중요한 역할을 한다. 따라서 53BP1은 DNA DSB에 대한 세포 반응의 핵심 조절자 역할을 한다(비특허문헌 45 참조).p53-binding protein 1 (53BP1) is an important component involved in DNA DBS signaling and repair and plays an important role in preserving genomic integrity and maintaining cellular homeostasis. Therefore, 53BP1 serves as a key regulator of cellular response to DNA DSB (see Non-Patent Document 45).

H2O2로 유도된 Huh7 세포에서 WLF 및 ELF 처리에 따른 53BP1 수준의 변화를 측정하였고, 그 결과를 도 22에 나타내었다.Changes in 53BP1 levels following WLF and ELF treatment were measured in Huh7 cells induced with H 2 O 2 , and the results are shown in Figure 22.

도 22를 참조하면, 대조군의 53BP1 수준은 정상군 대비 37.75배 유의하게 증가하였다.Referring to Figure 22, the 53BP1 level of the control group was significantly increased 37.75 times compared to the normal group.

한편, 증가된 53BP1 수치는 ELF 처리에 의해 58.28% 현저하게 감소하였다. 그러나 WLF 처리군은 대조군 대비 9.27% 감소하였으나 유의미한 차이는 없는 것으로 확인되었다.Meanwhile, the increased 53BP1 level was significantly reduced by 58.28% by ELF treatment. However, the WLF treatment group decreased by 9.27% compared to the control group, but it was confirmed that there was no significant difference.

(6) Huh7 세포에서의 LF의 산화 스트레스 억제 효과(6) Oxidative stress inhibitory effect of LF in Huh7 cells

1) Huh7 세포의 MDA 강도에 미치는 영향1) Effect on MDA intensity in Huh7 cells

Malondialdehyde(MDA)는 불포화지방산의 과산화에 따른 반응성이 높고 세포독성이 높은 부산물이며 산화 스트레스의 생체지표로 알려져 있다(비특허문헌 46 참조).Malondialdehyde (MDA) is a highly reactive and highly cytotoxic by-product of the peroxidation of unsaturated fatty acids and is known as a biomarker of oxidative stress (see Non-Patent Document 46).

H2O2로 유도된 Huh7 세포에서 WLF 및 ELF 처리에 따른 MDA 강도의 변화를 측정하였고, 그 결과를 도 23에 나타내었다.Changes in MDA intensity according to WLF and ELF treatment were measured in Huh7 cells induced with H 2 O 2 , and the results are shown in Figure 23.

도 23을 참조하면, 대조군의 MDA 강도는 정상군 대비 56.1배 유의하게 증가하였다.Referring to Figure 23, the MDA intensity of the control group was significantly increased by 56.1 times compared to the normal group.

한편, 증가된 MDA 강도는 ELF 처리에 의해 76.48% 현저하게 감소하였다. 그러나 WLF 처리군과 대조군 간에는 유의미한 차이가 없는 것으로 확인되었다.Meanwhile, the increased MDA intensity was significantly reduced by 76.48% by ELF treatment. However, it was confirmed that there was no significant difference between the WLF treatment group and the control group.

2) Huh7 세포의 p47phox 수준에 미치는 영향2) Effect on p47 phox levels in Huh7 cells

NADPH 산화효소(NOX)라고도 알려진 Nicotinamide adenine dinucleotide phosphate oxidase (NADPH) 산화효소 계열은 ROS 생성의 주요 원인인 다양한 하위 요소를 포함한다. NOX는 2개의 transmembrane components(p22phox 및 gp91phox)와 4개의 cytosolic components(p47phox, p67phox, p40phox 및 Rac-1/2)로 구성된다. 이들은 세포 활성화 시 막 부위에서 조립되며 이러한 과정은 p47phox의 인산화에 의존한다(비특허문헌 47 참조).Nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase family, also known as NADPH oxidase (NOX), contains various subcomponents that are major contributors to ROS generation. NOX consists of two transmembrane components (p22 phox and gp91 phox ) and four cytosolic components (p47 phox , p67 phox , p40 phox , and Rac-1/2). They are assembled at the membrane site upon cell activation, and this process depends on the phosphorylation of p47 phox (see Non-Patent Document 47).

H2O2로 유도된 Huh7 세포에서 WLF 및 ELF 처리에 따른 p47phox 수준의 변화를 측정하였고, 그 결과를 도 24에 나타내었다.Changes in p47phox levels according to WLF and ELF treatment were measured in Huh7 cells induced with H 2 O 2 , and the results are shown in Figure 24.

도 24를 참조하면, 대조군의 p47phox 수준은 정상군 대비 8.76배 유의하게 증가하였다.Referring to Figure 24, the p47 phox level of the control group was significantly increased by 8.76 times compared to the normal group.

한편, 증가된 p47phox 수치는 ELF 처리에 의해 72.28% 현저하게 감소하였다. 그러나 WLF 처리군과 대조군 간에는 유의미한 차이가 없는 것으로 확인되었다.Meanwhile, the increased p47 phox level was significantly reduced by 72.28% by ELF treatment. However, it was confirmed that there was no significant difference between the WLF treatment group and the control group.

3) Huh7 세포의 4-HNE 수준에 미치는 영향3) Effect on 4-HNE levels in Huh7 cells

산화 스트레스는 세포막에서 불포화지방산의 지질 과산화를 가속화하여 MDA 및 4-hydroxy-2-nonenal(4-HNE)과 같은 알데하이드의 생성을 초래한다(비특허문헌 48 참조).Oxidative stress accelerates lipid peroxidation of unsaturated fatty acids in cell membranes, resulting in the production of aldehydes such as MDA and 4-hydroxy-2-nonenal (4-HNE) (see Non-Patent Document 48).

H2O2로 유도된 Huh7 세포에서 WLF 및 ELF 처리에 따른 4-HNE 수준의 변화를 측정하였고, 그 결과를 도 25에 나타내었다.Changes in 4-HNE levels according to WLF and ELF treatment were measured in Huh7 cells induced with H 2 O 2 , and the results are shown in Figure 25.

도 25를 참조하면, 대조군의 4-HNE 수준은 정상군 대비 14.86배 유의하게 증가하였다.Referring to Figure 25, the 4-HNE level of the control group was significantly increased by 14.86 times compared to the normal group.

한편, 증가된 4-HNE 수준은 ELF 처리에 의해 57.67% 현저하게 감소하였다. 그러나 WLF100 처리군과 대조군 간에는 유의미한 차이가 없는 것으로 확인되었다.Meanwhile, the increased 4-HNE level was significantly reduced by 57.67% by ELF treatment. However, it was confirmed that there was no significant difference between the WLF100 treatment group and the control group.

실험예 2: 보습 및 항염증 효능 평가Experimental Example 2: Evaluation of moisturizing and anti-inflammatory efficacy

본 실험예에서는 실시예 1에 따른 노로통 열수 추출물(WLF)를 대상으로 보습 효능을 다음과 같이 평가하였다.In this experimental example, the moisturizing efficacy of Norotong thermal water extract (WLF) according to Example 1 was evaluated as follows.

[실험 방법][Experimental Method]

(1) 세포 독성(1) Cytotoxicity

세포를 96-well plate에 4Х103 cells/well이 되도록 180 ㎕ 분주하고, 실시예 1에 따른 노로통 열수 추출물(WLF)을 농도별로 제조한 후, 20 ㎕ 첨가하여 5% CO2 incubator에서 24 시간 배양하였다. 여기에 WLF를 농도별로 처리하고, WST-8 용액을 사용하여 10 ㎕ 첨가하여 1 시간 배양한 후 상층액을 100 ㎕를 취합하여, ELISA reader(Power Wave XS, BioTekInstruments, Inc., Winooski, VT, USA)로 450 nm에서 흡광도를 측정하였다.180 ㎕ of cells were distributed in a 96-well plate to make 4Х10 3 cells/well, and after preparing Norotong hot water extract (WLF) according to Example 1 at each concentration, 20 ㎕ was added and incubated in a 5% CO 2 incubator for 24 hours. Cultured. Here, WLF was treated according to concentration, 10 ㎕ of WST-8 solution was added, incubated for 1 hour, and 100 ㎕ of the supernatant was collected and analyzed using an ELISA reader (Power Wave USA), the absorbance was measured at 450 nm.

WLF 시료 용액의 처리군과 무처리군의 흡광도 감소율을 토대로 세포 증식 정도를 환산하였다.The degree of cell proliferation was calculated based on the rate of decrease in absorbance of the treated and untreated groups of the WLF sample solution.

(2) 히알루론산(Hyaluronic acid)의 생성량(2) Amount of hyaluronic acid produced

HaCaT 세포 배양액 내의 히알루론산(Hyaluronic acid)의 생성량은 ELISA kit (Abcam)를 이용하여 측정하였다. HaCaT 세포는 DMEM 배지를 이용하여 2Х105 cells/mL로 조절한 후 24 well plate에 접종하고, 5% CO2 incubator에서 24 시간 배양하였다. 세포에 TNF-α/IFN-γ 와 농도별로 제조한 WLF를 처리한 후 24 시간 배양하였고, 배양이 끝난 후 상등액을 취하여 Hyaluronic acid의 생성량을 측정하였다.The amount of hyaluronic acid produced in HaCaT cell culture was measured using an ELISA kit (Abcam). HaCaT cells were adjusted to 2Х10 5 cells/mL using DMEM medium, then inoculated into a 24 well plate and cultured in a 5% CO 2 incubator for 24 hours. The cells were treated with TNF-α/IFN-γ and WLF prepared at different concentrations and cultured for 24 hours. After the culture was completed, the supernatant was taken and the amount of hyaluronic acid produced was measured.

(3) Reaction Oxygen Species(ROS) 생성량(3) Reaction Oxygen Species (ROS) production amount

HaCaT 세포를 배양한 후 96 well plate에 5Х103 cells/well로 접종하고 24 시간 안정화하였다. 동일 배지로 교체한 후 20 mJ/cm2의 UVB로 자극한 뒤, WLF를 처리한 후 24 시간 배양하였고, DCF_DA 및 AmplexRed Hydrogen oxide quantitative kit를 사용하여 ROS 생성량을 측정하였다.After culturing HaCaT cells, they were inoculated into a 96 well plate at 5Х10 3 cells/well and stabilized for 24 hours. After replacing with the same medium, stimulation with 20 mJ/cm 2 of UVB, WLF was treated and cultured for 24 hours, and ROS production was measured using DCF_DA and AmplexRed Hydrogen oxide quantitative kit.

(4) TNF-α, IL-1β, IL-6 및 IL-8 생성량(4) Amount of TNF-α, IL-1β, IL-6, and IL-8 production

세포를 24 well plate에 2Х105 cells/mL 로 24 시간 배양 후 TNF-α/IFN-γ와 농도별로 제조한 WLF를 처리한 후 18 시간 뒤 세포 배양액을 취하여 염증성 cytokine인 TNF-α, IL-1β, IL-6 및 IL-8의 생성량을 측정하였다. TNF-α, IL-1β, IL-6 및 IL-8의 함량은 ELISA kit (R&D system, Minneapolis, MN, USA)를 사용하여 측정하였으며, 함량은 표준물질의 반응으로부터 얻어진 표준곡선을 이용하여 환산하였다.The cells were cultured in a 24 well plate at 2Х10 5 cells/mL for 24 hours, treated with TNF-α/IFN-γ and WLF prepared at different concentrations, and 18 hours later, the cell culture medium was taken and inflammatory cytokines TNF-α and IL-1β were collected. , the production amounts of IL-6 and IL-8 were measured. The contents of TNF-α, IL-1β, IL-6, and IL-8 were measured using an ELISA kit (R&D system, Minneapolis, MN, USA), and the contents were converted using a standard curve obtained from the reaction of standard substances. did.

(5) COX-2, HAS2 및 filaggrin 단백질 발현(5) COX-2, HAS2 and filaggrin protein expression

세포는 60 mm tissue culture dish에 2Х106 cells의 농도로 접종 및 배양하여 세포 밀도가 80%에 도달할 때, 무혈청 배지로 교환하고 24 시간 배양한 후 WLF를 농도별로 처리하였다. WLF 처리 후 TNF-α/IFN-γ를 정상군을 제외한 모든 dish에 넣어 자극시키고 24 시간 배양하였다. 그 후 상층액을 제거하고 PBS 로 2 회 세척한 후에 scrapper로 세포를 수확하였다. 수확된 각 세포에 lysis buffer를 well 당 100 ㎕를 첨가하고 1 시간동안 냉장하여 세포를 용해시킨 후 원심분리(12,000 rpm, 4℃ 및 10 min)하여 세포막 성분들을 제거하였다. 원심 분리하여 얻은 단백질은 BCA로 정량하였으며, 20 ㎕의 단백질을 10%의 sodium dodecyl sulfate polyacrylamide gel electrophoresis를 이용하여 전기영동한 후, 항체의 비특이적 결합을 억제시키기 위해 PVDF membrane에 옮긴 다음 transfer하였다.Cells were inoculated and cultured in a 60 mm tissue culture dish at a concentration of 2Х10 6 cells. When the cell density reached 80%, the medium was replaced with serum-free medium and cultured for 24 hours, and then WLF was treated at each concentration. After WLF treatment, TNF-α/IFN-γ was stimulated by adding TNF-α/IFN-γ to all dishes except the normal group and cultured for 24 hours. Afterwards, the supernatant was removed, washed twice with PBS, and the cells were harvested using a scraper. 100 ㎕ of lysis buffer per well was added to each harvested cell, refrigerated for 1 hour to lyse the cells, and then centrifuged (12,000 rpm, 4°C and 10 min) to remove cell membrane components. Protein obtained by centrifugation was quantified using BCA, and 20 μl of protein was electrophoresed using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a PVDF membrane to inhibit non-specific binding of antibodies.

Transfer 가 끝나면 5% skim milk에 1 내지 2 시간동안 방치하여 background를 제거시켰다. 1ХTBST 로 3회 washing 후 1 차 antibody(1:1000)를 24 시간 붙인 후 1ХTBST 로 3회 washing 후 2 차 antibody(1:1000)를 2 시간 반응한 후 ECL kit(Amersham Pharmacia, England)를 이용하여 film에 옮겨 측정하였고, Image Quant LAS-4000 (GE life sciences, Taiwan)으로 Band density를 확인하였다.After transfer was completed, the background was removed by leaving it in 5% skim milk for 1 to 2 hours. After washing 3 times with 1ХTBST, attach primary antibody (1:1000) for 24 hours, wash 3 times with 1ХTBST, react with secondary antibody (1:1000) for 2 hours, then use ECL kit (Amersham Pharmacia, England) It was measured by transferring it to film, and the band density was confirmed with Image Quant LAS-4000 (GE life sciences, Taiwan).

[실험 결과][Experiment result]

(1) 세포 독성(1) Cytotoxicity

WLF 처리에 따른 세포 독성을 평가하기 위하여 도 26에 표시된 농도로 WLF를 처리하고 24시간 동안 세포 증식을 수행하고, WST-8을 사용하여 세포 증식 정도를 측정하였고, 그 결과를 도 26에 나타내었다.To evaluate the cytotoxicity of WLF treatment, WLF was treated at the concentration shown in Figure 26 and cell proliferation was performed for 24 hours. The degree of cell proliferation was measured using WST-8, and the results are shown in Figure 26. .

도 26을 참조하면, WLF를 처리하지 않은 무처리군과 대비하여 WLF100 처리군, WLF200 처리군 및 WLF400 처리군에서는 유의미한 차이를 보이지 않았고, WLF800 처리군만 무처리군 대비 감소된 것으로 확인되었다.Referring to Figure 26, compared to the untreated group without WLF treatment, there was no significant difference in the WLF100-treated group, WLF200-treated group, and WLF400-treated group, and only the WLF800-treated group was confirmed to be reduced compared to the untreated group.

즉, WLF의 처리 농도가 800 ㎍/mL 이하에서는 세포 독성이 나타나지 않는 것으로 확인되었다.In other words, it was confirmed that no cytotoxicity occurred when the treatment concentration of WLF was 800 ㎍/mL or less.

(2) 히알루론산(Hyaluronic acid)의 생성량(2) Amount of hyaluronic acid produced

WLF 처리에 따른 히알루론산(Hyaluronic acid)의 생성량을 측정하였고, 그 결과를 도 27에 나타내었다.The amount of hyaluronic acid produced according to WLF treatment was measured, and the results are shown in Figure 27.

도 27을 참조하면, 대조군의 히알루론산 생성량이 정상군과 대비하여 유의하게 감소한 것을 확인할 수 있다.Referring to Figure 27, it can be seen that the amount of hyaluronic acid produced in the control group was significantly reduced compared to the normal group.

한편, WLF100 처리군을 제외한 모든 군에서 대조군 대비 유의하게 히알루론산 생성량이 증가하였고, 특히 WLF400 처리군에서 히알루론산 생성량이 현저하게 증가하였다.Meanwhile, the amount of hyaluronic acid production increased significantly compared to the control group in all groups except the WLF100 treatment group, and in particular, the amount of hyaluronic acid production increased significantly in the WLF400 treatment group.

(3) ROS 생성량(3) ROS production amount

WLF 처리에 따른 ROS의 생성량 변화를 확인하기 위하여 ROS 생성량과 비례 관계에 있는 DCFDA 생성량을 측정하였고, 그 결과를 도 28에 나타내었다.In order to confirm the change in the amount of ROS production according to WLF treatment, the amount of DCFDA production, which is proportional to the amount of ROS production, was measured, and the results are shown in Figure 28.

도 28을 참조하면, 대조군에서의 DCF_DA의 생성량이 정상군 대비 유의하게 증가한 것으로 확인되었다.Referring to Figure 28, it was confirmed that the production amount of DCF_DA in the control group was significantly increased compared to the normal group.

한편, WLF 처리 시 농도 의존적으로 DCFDA의 생성이 억제되는 것으로 확인되었고, 특히 WLF400 처리군에서 처리 농도 대비 DCFDA 생성 억제 효능이 가장 우수한 것으로 확인되었다.Meanwhile, it was confirmed that the production of DCFDA was suppressed in a concentration-dependent manner during WLF treatment, and in particular, the WLF400 treatment group was confirmed to have the highest efficacy in suppressing DCFDA production compared to the treatment concentration.

따라서, WLF 처리군에서 ROS 생성을 효과적으로 억제할 수 있는 것으로 확인되었다.Therefore, it was confirmed that ROS generation could be effectively suppressed in the WLF treatment group.

(4) TNF-α, IL-1β, IL-6 및 IL-8 생성량(4) Amount of TNF-α, IL-1β, IL-6, and IL-8 production

WLF 처리에 따른 염증성 사이토카인 생성량의 변화를 확인하기 위하여 TNF-α, IL-1β, IL-6 및 IL-8 생성량을 측정하였고, 그 결과를 도 29에 나타내었다.To determine changes in inflammatory cytokine production following WLF treatment, TNF-α, IL-1β, IL-6, and IL-8 production were measured, and the results are shown in Figure 29.

도 29를 참조하면, 대조군의 TNF-α, IL-1β, IL-6 및 IL-8의 생성량이 정상군 대비 유의하게 증가하였다. Referring to Figure 29, the production amounts of TNF-α, IL-1β, IL-6, and IL-8 in the control group were significantly increased compared to the normal group.

한편, WLF 처리 시 농도 의존적으로 TNF-α, IL-1β, IL-6 및 IL-8 생성량이 감소하는 것으로 확인되었다.Meanwhile, it was confirmed that the production of TNF-α, IL-1β, IL-6, and IL-8 decreased in a concentration-dependent manner upon WLF treatment.

(5) COX-2, HAS2 및 filaggrin 단백질 발현(5) COX-2, HAS2 and filaggrin protein expression

WLF 처리에 따른 COX-2 및 filaggrin 단백질 발현량의 변화를 측정하였고, 그 결과를 도 30에 나타내었다.Changes in COX-2 and filaggrin protein expression levels according to WLF treatment were measured, and the results are shown in Figure 30.

도 30을 참조하면, 대조군의 COX-2 발현이 정상군 대비 유의하게 증가하였으나, WLF200 처리군 및 WLF400 처리군에서 대조군 대비 유의하게 COX-2 발현이 감소하였다.Referring to Figure 30, COX-2 expression in the control group was significantly increased compared to the normal group, but COX-2 expression was significantly decreased in the WLF200-treated group and WLF400-treated group compared to the control group.

또한 대조군의 HAS2 발현이 정상군 대비 유의하게 감소하였으나, WLF200 처리군 및 WLF400 처리군에서 대조군 대비 유의하게 HAS2 발현이 증가하였다.In addition, HAS2 expression in the control group was significantly decreased compared to the normal group, but HAS2 expression was significantly increased in the WLF200-treated group and WLF400-treated group compared to the control group.

또한 대조군의 filaggrin 발현이 정상군 대비 유의하게 감소하였으나, WLF400 처리군에서 대조군 대비 유의하게 filaggrin 발현이 증가한 것으로 확인되었다.In addition, filaggrin expression in the control group was significantly decreased compared to the normal group, but filaggrin expression was found to be significantly increased in the WLF400-treated group compared to the control group.

염증은 다양한 병원체의 침입에 대한 신체의 방어로 인해 발생하는 생리적 반응이다. 축적된 증거들은 염증이 신경퇴행성질환, 심혈관질환, 관절염, 당뇨병, 암 등 많은 만성질환의 발생 및 발병 과정과 밀접한 관련이 있음을 시사하고 있다(비특허문헌 54 참조). 또한, 최근 많은 연구에서는 항염증 및 항산화 효과를 나타내는 천연 소재가 피부 개선 효과가 있다는 보고가 있다(비특허문헌 55 및 56 참조). 따라서 본 발명에서는 물, 70 %(v/v) 에탄올 등 다양한 추출 용매에 따른 LF의 항염증 효과와 항산화 효과의 분자적 메커니즘을 규명하고자 하였다.Inflammation is a physiological response that occurs as a result of the body's defense against invasion by various pathogens. Accumulated evidence suggests that inflammation is closely related to the occurrence and pathogenesis of many chronic diseases, such as neurodegenerative diseases, cardiovascular diseases, arthritis, diabetes, and cancer (see Non-Patent Document 54). In addition, many recent studies have reported that natural materials with anti-inflammatory and antioxidant effects have skin-improving effects (see Non-Patent Documents 55 and 56). Therefore, the present invention sought to identify the molecular mechanisms of the anti-inflammatory and antioxidant effects of LF according to various extraction solvents such as water and 70% (v/v) ethanol.

대식세포는 중요한 면역 조절 세포이며 염증 반응에도 중요한 역할을 한다. 대식세포가 그람 음성균의 세포 외피로 구성된 LPS에 노출되었을 때(비특허문헌 58 참조) 염증과 관련된 신호 전달이 시작되는 특징이 있다(비특허문헌 59 참조). NO 생성 증가, 전염증성 사이토카인 분비, 항염증성 사이토카인 억제, 살균/종양 활성 억제 등 다양한 염증 반응이 연속적으로 밀접하게 발생한다(비특허문헌 60 참조). 따라서 이러한 신호 전달 경로의 조절은 세포 외 외래 유기체에 대한 선천적 면역 반응의 초기 단계를 제어하는 데 중요한 요소이다.Macrophages are important immune regulatory cells and also play an important role in inflammatory responses. When macrophages are exposed to LPS composed of the cell envelope of Gram-negative bacteria (see Non-Patent Document 58), signaling related to inflammation begins (see Non-Patent Document 59). Various inflammatory reactions occur in close succession, such as increased NO production, secretion of pro-inflammatory cytokines, inhibition of anti-inflammatory cytokines, and inhibition of bactericidal/tumor activity (see Non-Patent Document 60). Therefore, regulation of these signaling pathways is an important factor in controlling the early stages of the innate immune response against extracellular foreign organisms.

LPS(내독소로 알려짐)는 박테리아와 숙주 사이의 숙주 면역 반응을 방해하여 위험한 역할을 수행한다(비특허문헌 61 참조). 많은 연구에서 LPS가 PI3K-Akt 신호 전달 경로를 활성화한다고 보고되었다. 이는 PI3K/Akt 경로의 억제가 산화 스트레스 수준을 감소시키고 LPS 유발 감염에서 염증을 하향 조절할 수 있음을 의미한다(비특허문헌 62 및 63 참조). 또한, ERK1/2, JNK, p38 및 ERK5를 포함하는 4개의 별개의 MAPK 계통을 갖는 MAPK 신호 전달 경로는 세포 증식, 분화 및 이동과 연관되어 있다. 게다가 대식세포 활성화와 분극화를 통해 염증성 질환의 주요 조절자 역할을 동시에 수행한다(비특허문헌 64 참조). 따라서, LF의 항염증 가능성을 평가하기 위한 in vitro 연구는 LPS로 자극된 RAW 264.7 세포를 사용하여 수행되었다. 세포 증식을 확인하기 위해 WLF 및 ELF를 RAW 264.7 세포에서 100 및 200 μg/mL로 24시간 동안 처리하였다. 세포 증식은 정상군(0h 군)과 대비하여 WLF 200 μg/mL 처리군을 제외한 다른 군에서 70% 이상 높은 것으로 확인되었다(도 1 참조). 따라서 이후 실험에서는 100μg/mL 농도를 이용하여 실험을 진행하였다. LPS (known as endotoxin) plays a dangerous role by interfering with the host immune response between bacteria and the host (see Non-Patent Document 61). Many studies have reported that LPS activates the PI3K-Akt signaling pathway. This means that inhibition of the PI3K/Akt pathway can reduce the level of oxidative stress and downregulate inflammation in LPS-induced infection (see non-patent documents 62 and 63). Additionally, the MAPK signaling pathway, with four distinct MAPK lineages including ERK1/2, JNK, p38, and ERK5, is associated with cell proliferation, differentiation, and migration. In addition, it simultaneously acts as a major regulator of inflammatory diseases through macrophage activation and polarization (see Non-Patent Document 64). Therefore, an in vitro study to evaluate the anti-inflammatory potential of LF was performed using RAW 264.7 cells stimulated with LPS. To confirm cell proliferation, WLF and ELF were treated at 100 and 200 μg/mL in RAW 264.7 cells for 24 hours. Cell proliferation was confirmed to be more than 70% higher in the other groups except the WLF 200 μg/mL treatment group compared to the normal group (0h group) (see Figure 1). Therefore, in subsequent experiments, a concentration of 100 μg/mL was used.

여기에서 상기 결과는 ELF가 LPS에 의해 유도된 Akt의 인산화를 억제한 다음(도 2 참조) p38, Erk 및 JNK와 같은 세 가지 MAPK의 활성화를 억제한다는 것을 보여주었다. 그러나 WLF 군의 p-p38 및 p-JNK 발현은 LPS 군과 비교하여 p-Erk 발현을 제외하고는 유의한 억제를 나타내지 않았다(도 3 참조).Here, the results showed that ELF inhibited the phosphorylation of Akt induced by LPS (see Figure 2) and then inhibited the activation of three MAPKs, such as p38, Erk, and JNK. However, p-p38 and p-JNK expression in the WLF group did not show significant inhibition compared to the LPS group, except for p-Erk expression (see Figure 3).

대식세포의 LPS 반응과 관련된 수많은 신호 전달 경로가 있으나, 그 중에서 가장 빠르게 유도된 신호전달 반응은 막 수용체 CD14와 상호작용할 가능성이 높다. LPS에 대한 다기능 수용체는 55-kD의 글리코실-포스파티딜이노시톨 결합 막 단백질(glycosyl-phosphatidylinositol(GPI)-linked membrane protein)인 CD14이다(비특허문헌 65 참조). CD14는 처음으로 확인된 PRR이었지만 나중에 CD14는 병원체 관련 분자 패턴(pathogen-associated molecular patterns, PAMP) 검출을 위한 TLR4 공동 수용체로 알려졌다. LPS 반응을 시작하는 CD14는 세포 실험과 동물 모델에서 입증되었다. CD14를 과발현하는 형질전환 마우스는 LPS에 과민한 반면(비특허문헌 66 참조), CD14가 결핍된 쥐는 저용량 LPS 자극에 어떤 반응도 보이지 않았다(비특허문헌 67 참조). LPS와 CD14의 고친화력 결합은 혈액 내 LPS 결합 단백질(LBP)의 존재에 의해 상당히 증가된다(비특허문헌 68 참조).There are numerous signaling pathways involved in the LPS response of macrophages, but the most rapidly induced signaling response is likely to interact with the membrane receptor CD14. The multifunctional receptor for LPS is CD14, a 55-kD glycosyl-phosphatidylinositol (GPI)-linked membrane protein (see Non-Patent Document 65). CD14 was the first PRR identified, but later CD14 became known as a TLR4 co-receptor for detection of pathogen-associated molecular patterns (PAMPs). CD14 initiating the LPS response has been demonstrated in cell experiments and animal models. Transgenic mice overexpressing CD14 were hypersensitive to LPS (see Non-Patent Document 66), while mice deficient in CD14 did not show any response to low-dose LPS stimulation (see Non-Patent Document 67). The high-affinity binding of LPS and CD14 is significantly increased by the presence of LPS binding protein (LBP) in the blood (see Non-Patent Document 68).

LBP 및 CD14는 LPS가 TLR4-MD2 복합체로 전달되는 것을 촉매한다(비특허문헌 69 및 70 참조). 본 발명에서는 TLR4-MD2 복합체 및 CD14 mRNA 발현에 대한 LF의 염증 개시 억제 가능성을 실험을 통해 확인하였다. 복강 내 대식세포의 real-time qPCR 분석에서 TLR4, MD2 및 CD14의 mRNA 발현은 LPS 처리 15분 후에 눈에 띄게 증가하였다. 그러나 ELF(100 μg/ml)로 6시간 동안 전처리한 세포는 LPS만 처리한 세포와 대비하여 TLR4, MD2 및 CD14의 mRNA 발현이 현저하게 감소한 것으로 확인되었다(도 4 및 도 5 참조).LBP and CD14 catalyze the transfer of LPS to the TLR4-MD2 complex (see Non-Patent Documents 69 and 70). In the present invention, the possibility of LF to inhibit inflammation initiation on TLR4-MD2 complex and CD14 mRNA expression was confirmed through experiments. In real-time qPCR analysis of intraperitoneal macrophages, the mRNA expression of TLR4, MD2, and CD14 was noticeably increased after 15 minutes of LPS treatment. However, cells pretreated with ELF (100 μg/ml) for 6 hours were found to have significantly reduced mRNA expression of TLR4, MD2, and CD14 compared to cells treated only with LPS (see Figures 4 and 5).

대식세포는 대식세포 분극화라고 불리는 외부 자극과 신호에 대한 반응으로 엄격하게 구분되는 두 가지 표현형을 가지고 있다(비특허문헌 71 참조). 대식세포 극성은 염증 단계에 따라 촉진되거나 억제될 수 있다(비특허문헌 72 참조). 대식세포는 전형적으로 두 가지 표현형과 역할로 나뉜다: (i) TNFα, IL-1β, IL-6 및 iNOS를 포함한 사이토카인을 방출하는 "전형적으로 활성화된" 대식세포, 전염증성 또는 "M1 표현형" 대식세포 및 (ii) 혈관신생, ECM 복구, 항염증 사이토카인 방출을 촉진하는 "대안적으로 활성화된" 대식세포, 항염증제 또는 "M2 표현형" 대식세포(비특허문헌 73 참조). 대식세포가 호중구를 식균할 때 그들의 표현형은 M1에서 M2로 변하는데, 이는 호중구에서 분비된 매개체를 통해 조절되는 과정이다(비특허문헌 74 참조). 본 발명에서는 다른 연구(비특허문헌 75 및 76 참조)와 유사하게, LPS 자극이 Nos2, TNFα 및 IL-6를 포함한 분극화 생체지표를 크게 증가시킨 것을 확인하였다. 한편, 본 발명에서는 ELF 전처리가 M1 표현형 대식세포에 대한 상기 분극화 생체지표를 현저하게 억제한다는 것을 확인하였다 (도 6 참조). ELF 처리에 의한 억제 효과는 정상군 이하로 개선되었다. LPS 자극에 의해 감소된 IL-10 및 Arg1 수준은 ELF 처리군에서 효과적으로 증가하였다(도 7 참조). 이러한 결과는 ELF 처리가 M2 표현형 대식세포에 대한 IL-10 및 Arg1을 포함한 분극화 생체지표에 유의미한 영향을 미칠 수 있는 것으로 생각된다.Macrophages have two phenotypes that are strictly distinguished in response to external stimuli and signals, called macrophage polarization (see Non-Patent Document 71). Macrophage polarity can be promoted or suppressed depending on the stage of inflammation (see Non-Patent Document 72). Macrophages are typically divided into two phenotypes and roles: (i) “typically activated” macrophages, pro-inflammatory or “M1 phenotype”, which release cytokines including TNFα, IL-1β, IL-6, and iNOS; macrophages and (ii) “alternatively activated” macrophages, anti-inflammatory or “M2 phenotype” macrophages that promote angiogenesis, ECM repair, and anti-inflammatory cytokine release (see Non-Patent Document 73). When macrophages phagocytose neutrophils, their phenotype changes from M1 to M2, a process regulated through mediators secreted by neutrophils (see Non-Patent Document 74). In the present invention, similar to other studies (see non-patent documents 75 and 76), it was confirmed that LPS stimulation significantly increased polarization biomarkers including Nos2, TNFα, and IL-6. Meanwhile, in the present invention, it was confirmed that ELF pretreatment significantly suppressed the polarization biomarker for M1 phenotype macrophages (see Figure 6). The inhibitory effect by ELF treatment was improved below the normal group. IL-10 and Arg1 levels decreased by LPS stimulation were effectively increased in the ELF-treated group (see Figure 7). These results suggest that ELF treatment may have a significant effect on polarization biomarkers, including IL-10 and Arg1, on M2 phenotype macrophages.

또한 LPS로 자극된 RAW 264.7 세포에서 MAPK의 인산화는 TLR4 과발현을 통해 NF-κB의 전사 활성화를 자극할 수도 있다(비특허문헌 77 참조). 또한, NF-κB의 억제는 전염증성 사이토카인 및 매개체와 같은 하위 유전자를 억제하는 것으로 알려져 있다(비특허문헌 78 및 79 참조). 본 발명에서는 이러한 변화를 IHC 염색을 통해 비교 분석하였다(도 8 내지 11 참조). IHC 결과에 따르면 LPS 자극에 의한 TLR4 세포 수가 증가하면 NF-κB 세포 수가 증가하는 것으로 확인되었다. 이는 TLR4 활성화가 NF-κB의 핵 전위를 촉진하고 이후 활성화로 이어진다는 것을 의미한다(비특허문헌 80 참조). 반면, TLR4와 NF-κB 모두의 증가된 세포 수는 ELF 처리 후에 크게 억제되었다. NF-κB 활성화 차단으로 인해 iNOS 및 IL-1β가 감소하였다.Additionally, phosphorylation of MAPK in RAW 264.7 cells stimulated with LPS may stimulate transcriptional activation of NF-κB through TLR4 overexpression (see Non-Patent Document 77). In addition, inhibition of NF-κB is known to inhibit downstream genes such as pro-inflammatory cytokines and mediators (see Non-Patent Documents 78 and 79). In the present invention, these changes were compared and analyzed through IHC staining (see Figures 8 to 11). According to IHC results, it was confirmed that an increase in the number of TLR4 cells by LPS stimulation increased the number of NF-κB cells. This means that TLR4 activation promotes nuclear translocation of NF-κB and leads to its subsequent activation (see Non-Patent Document 80). On the other hand, the increased cell numbers of both TLR4 and NF-κB were significantly suppressed after ELF treatment. Blockade of NF-κB activation resulted in a decrease in iNOS and IL-1β.

ROS(O2 ·-, H2O2, ·OH 등)의 과도한 생성은 ROS 스트레스를 증가시켜 최종적으로 세포 사멸을 유발한다. 본 발명에서는 H2O2로 자극된 Huh7 세포를 사용하여 LF의 항산화 효과를 평가하기 위해 추가 실험을 수행하였다. 세포 증식을 확인하기 위해 Huh 7 세포에 WLF 및 ELF를 100 및 200 μg/mL로 24시간 동안 처리하였다(도 12 참조). ELF 처리는 WLF 처리 대비 세포 증식에 있어서 우수한 것으로 확인되었다. 산화 스트레스로 인한 세포 사멸은 여러 세포 신호 전달에 의해 제어된다(비특허문헌 81 및 82 참조). 본 발명에서는 ROS와 관련된 요인을 조사하였다. H2O2 처리 후 증가된 DCFDA 및 DHE 수준은 ELF 처리군에서만 농도 의존적으로 현저하게 감소하였다(도 13 및 도 14 참조). 반면 H2O2 처리 후 감소된 SOD 활성과 GSH 함량은 ELF 처리군에서만 농도 의존적으로 유의하게 증가하였다(도 15 및 도 16 참조). 즉, ELF 처리군은 WLF 처리군 대비 우수한 항산화 효과가 있는 것으로 확인되었다.Excessive production of ROS (O 2 ·- , H 2 O 2 , ·OH, etc.) increases ROS stress and ultimately causes cell death. In the present invention, additional experiments were performed to evaluate the antioxidant effect of LF using Huh7 cells stimulated with H 2 O 2 . To confirm cell proliferation, Huh 7 cells were treated with 100 and 200 μg/mL of WLF and ELF for 24 hours (see Figure 12). ELF treatment was confirmed to be superior to WLF treatment in terms of cell proliferation. Cell death due to oxidative stress is controlled by several cell signaling (see Non-Patent Documents 81 and 82). In the present invention, factors related to ROS were investigated. The increased levels of DCFDA and DHE after H 2 O 2 treatment were significantly reduced in a concentration-dependent manner only in the ELF-treated group (see Figures 13 and 14). On the other hand, the reduced SOD activity and GSH content after H 2 O 2 treatment significantly increased in a concentration-dependent manner only in the ELF-treated group (see Figures 15 and 16). In other words, the ELF-treated group was confirmed to have superior antioxidant effect compared to the WLF-treated group.

또한, IF 염색을 통한 Dead cell과 ROS 강도와 관련하여 ELF 처리군에서 WLF 처리군 대비 향상된 효과를 나타내는 것으로 확인되었다(도 17 및 도 18 참조). terminal deoxynucleotidyl TUNEL assay을 사용하여 Apoptotic Huh7 세포를 얻었다(비특허문헌 83 참조). 내인성 또는 외인성 DNA 손상이 발생하면 Huh7 세포에서 유전독성 스트레스(γH2AX)가 증가하는 것으로 알려져 있다(비특허문헌 84 참조). γH2AX는 히스톤 H2AX의 인산화를 의미한다(비특허문헌 85 참조). 53BP1은 DNA 말단의 핵분해 제거를 억제하여 DNA DSB의 복구를 조절하는 염색질 결합 단백질이다(비특허문헌 86 참조). 여기서 DNA DSB는 방사선 및 특정 화학물질을 포함한 외인성 물질에 의해 또는 생리학적 과정 중에 생성될 수 있는 세포독성 DNA 형태이다(비특허문헌 87 참조). 이러한 DSB의 복구는 세포 항상성을 유지하고 게놈 불안정성을 예방하는 데 중요하다. 53BP1은 DNA DSB 복구 메커니즘에서 중요한 역할을 한다(비특허문헌 85 참조). 병리생리학적 반응에서 발생하는 산화 스트레스의 최종 산물인 지질 과산화는 MDA를 측정하여 평가한다(비특허문헌 88 참조). 또한, 숙주 방어에서 NADPH 산화효소의 세포질 단백질 중 하나인 p47phox(phox: phagocyte oxidase)의 중요성은 만성 육아종증(CGD), 죽상경화증, 류마티스 관절염 및 간 섬유증과 같은 다양한 질병과 연관된 것으로 알려져 있다. 따라서 p47phox 인산화의 조절은 염증성 질환에서 ROS 과잉 생산을 하향 조절하는 데 필요하다(비특허문헌 89 참조). n-6 지방산 산화의 주요 α, β-불포화 알데하이드 생성물이라고 불리는 4-HNE는 대사 질환, 암 및 신경퇴행성 질환과 같은 여러 병리학에 관련이 있다 (비특허문헌 90 참조). 본 발명에서는 Huh7 세포에서 세포 사멸(TUNEL+), DNA 손상(γH2AX 및 53BP1) 및 산화 스트레스(MDA 및 p47phox)와 관련된 요인에 대한 효과를 확인하였다. H2O2 처리 후 TUNEL+, γH2AX, 53BP1, MDA 및 p47phox는 급격하게 증가하였으나, ELF 처리는 WLF 처리와 달리 유의미한 억제 효과를 나타내었다(도 19 내지 24 참조).In addition, it was confirmed that the ELF-treated group showed an improved effect compared to the WLF-treated group in relation to dead cells and ROS intensity through IF staining (see Figures 17 and 18). Apoptotic Huh7 cells were obtained using terminal deoxynucleotidyl TUNEL assay (see non-patent document 83). It is known that genotoxic stress (γH2AX) increases in Huh7 cells when endogenous or exogenous DNA damage occurs (see Non-Patent Document 84). γH2AX refers to phosphorylation of histone H2AX (see Non-Patent Document 85). 53BP1 is a chromatin-binding protein that regulates the repair of DNA DSBs by inhibiting nucleolytic removal of DNA ends (see Non-Patent Document 86). Here, DNA DSB is a cytotoxic form of DNA that can be generated by exogenous substances, including radiation and certain chemicals, or during physiological processes (see Non-Patent Document 87). Repair of these DSBs is important for maintaining cellular homeostasis and preventing genomic instability. 53BP1 plays an important role in the DNA DSB repair mechanism (see Non-Patent Document 85). Lipid peroxidation, which is the end product of oxidative stress occurring in pathophysiological reactions, is evaluated by measuring MDA (see Non-Patent Document 88). Additionally, the importance of p47 phox (phox: phagocyte oxidase), one of the cytosolic proteins of NADPH oxidase, in host defense is known to be associated with various diseases such as chronic granulomatous disease (CGD), atherosclerosis, rheumatoid arthritis, and liver fibrosis. Therefore, regulation of p47 phox phosphorylation is necessary to downregulate ROS overproduction in inflammatory diseases (see Non-Patent Document 89). 4-HNE, referred to as the major α, β-unsaturated aldehyde product of n-6 fatty acid oxidation, is involved in several pathologies such as metabolic diseases, cancer, and neurodegenerative diseases (see Non-Patent Document 90). In the present invention, the effects on factors related to cell death (TUNEL + ), DNA damage (γH2AX and 53BP1), and oxidative stress (MDA and p47 phox ) were confirmed in Huh7 cells. After H 2 O 2 treatment, TUNEL + , γH2AX, 53BP1, MDA and p47 phox increased rapidly, but ELF treatment showed a significant inhibitory effect, unlike WLF treatment (see Figures 19 to 24).

또한 본 발명에서는 노로통 열수 추출물(WLF)의 처리에 따른 보습 효과를 평가하기 위하여 HaCaT 세포를 대상으로 히알루론산 생성량, 히알루론산 생성 효소(HAS2)의 발현량 및 filaggrin 발현량을 측정하였고, WLF 처리에 따른 피부에서의 HaCaT 세포를 대상으로 항염증 효과를 평가하기 위하여 ROS 생성량, 염증성 사이토카인 생성량 및 피부 염증에 따른 주요 인자(COX-2)의 발현량을 측정하였으며, 그 결과 WLF 처리에 의해 히알루론산 생성량, HAS2의 발현량 및 filaggrin 발현량이 향상되었고, ROS 생성량, 염증성 사이토카인 생성량 및 피부 염증에 따른 주요 인자(COX-2)의 발현량이 유의하게 감소하는 것으로 확인되었다.In addition, in the present invention, in order to evaluate the moisturizing effect of treatment with Norotong thermal water extract (WLF), the amount of hyaluronic acid production, expression level of hyaluronic acid synthase (HAS2), and filaggrin expression were measured in HaCaT cells, and WLF treatment In order to evaluate the anti-inflammatory effect of HaCaT cells in the skin, the amount of ROS production, the amount of inflammatory cytokines production, and the expression level of the key factor (COX-2) associated with skin inflammation were measured. As a result, hyaluronic acid was induced by WLF treatment. It was confirmed that the production of ronic acid, the expression level of HAS2, and the expression level of filaggrin were improved, and the amount of ROS production, inflammatory cytokine production, and expression of a key factor associated with skin inflammation (COX-2) were significantly decreased.

이상에서 설명한 본 발명의 바람직한 실시예들은 기술적 과제를 해결하기 위해 개시된 것으로, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면 본 발명의 사상 및 범위 안에서 다양한 수정, 변경, 부가 등이 가능할 것이며, 이러한 수정 변경 등은 이하의 특허청구범위에 속하는 것으로 보아야 할 것이다.The preferred embodiments of the present invention described above were disclosed to solve technical problems, and those skilled in the art will be able to make various modifications, changes, additions, etc. within the spirit and scope of the present invention. , such modifications and changes should be regarded as falling within the scope of the patent claims below.

Claims (5)

노로통 추출물을 유효성분으로 포함하는 보습용 화장료 조성물로서,
상기 노로통 추출물은 열수 추출물이고, 300 내지 500 ㎍/mL의 농도로 포함되고, 히알루론산(Hyaluronic acid)의 생성량을 향상시킬 수 있는 것을 특징으로 하는 보습용 화장료 조성물.A moisturizing cosmetic composition containing Norotong extract as an active ingredient,
The Norotong extract is a hot water extract, is contained at a concentration of 300 to 500 μg/mL, and is a moisturizing cosmetic composition that is capable of improving the amount of hyaluronic acid produced. 삭제delete 삭제delete 삭제delete 삭제delete
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014169256A (en) * 2013-03-05 2014-09-18 Kose Corp Anti-oxidant containing liquidambar formosana extract
KR20180090123A (en) * 2017-02-02 2018-08-10 주식회사 엘지생활건강 Composition for skin improvement containing liquidambaric lactone

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014169256A (en) * 2013-03-05 2014-09-18 Kose Corp Anti-oxidant containing liquidambar formosana extract
KR20180090123A (en) * 2017-02-02 2018-08-10 주식회사 엘지생활건강 Composition for skin improvement containing liquidambaric lactone

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