마우스 흑색종세포에서 Purpurogallin의 멜라닌 생성 억제 효과

김한혁(Han-Hyuk Kim),김태훈(Tae Hoon Kim) 한국식품영양과학회 2015 한국식품영양과학회지 Vol.44 No.12

천연 미백소재 개발과 관련하여 많은 연구들이 멜라닌 합성저해 및 활성 메커니즘을 규명하는 데 초점이 맞춰졌으며, 이러한 이유로 tyrosinase 저해제 개발이 다양하게 이루어져 왔다. 본 연구는 배 유래의 polyphenol oxidase를 이용하여 천연에 존재하는 단순 폴리페놀인 pyrogallol의 산화축합반응을 유도하여 purpurogallin을 효율적으로 생합성하였으며, 본 화합물에 대해서 미백 활성을 평가하였다. 먼저 MTT assay를 통해 세포독성이 없는 농도구간을 설정하였으며, purpurogallin은 25 μM 농도의 melanoma 세포 내에서 tyrosinase 활성을 20% 이상 저해하는 것을 확인하였다. 또한 25 μM의 시험 농도에서 purpurogallin은 약 20%이상의 melanin 생합성 저해 활성을 나타내었다. 미백 관련 전사인자인 MITF, TRP-1, TRP-2, tyrosinase의 단백질 발현을 측정한 결과, 본 화합물은 B16F10 melanoma 세포에서 tyrosinase, TRP-1과 TRP-2의 단백질 생합성을 두 추출물 모두 억제하는 것을 확인하였다. Tyrosinase, TRP-1과 TRP-2의 발현을 조절하는 전사인자로는 MITF가 관여하는 것으로 알려져 있으며, 실제로 MITF는 melanin 생성과 관련된 여러 유전자의 발현을 조절하는 데 중요한 작용을 하고 있다. 따라서 purpurogallin은 melanin 생성과 관련된 중요한 세 가지 단백질의 생합성을 전사단계에서 조절 전사인자인 MITF의 단백질 발현을 억제하는 효과가 있음을 확인하였다. 이상의 결과로부터 멜라닌 생합성에 있어서 상위신호단계에 있는 전사인자 MITF의 활성을 억제함으로써 하위 신호전달 과정을 억제하는 것임을 시사하며, 향후 추가적인 검증작업을 통해 화장품 소재화가 가능할 것으로 판단된다. Melanin is one of the most important factors affecting skin color. Melanogenesis is the bioprocess of melanin production by melanocytes in the skin and hair follicles and is mediated by several enzymes, such as tyrosinase, tyrosinase related protein (TRP)-1, and TRP-2. Convenient enzymatic transformation of the simple phenol pyrogallol with polyphenol oxidase originating from pear to an oxidative product, purpurogallin, was efficient. The structure of the pyrogallol oxidation product was identified on the basis of spectroscopic methods. The biotransformation product purpurogallin showed significant inhibitory effects against both melanin synthesis and tyrosinase activity in a dose-dependent manner in B16 melanoma cells. In addition, purpurogallin significantly attenuated melanin production by inhibiting TRP-1, and TRP-2 expression through modulation of their corresponding transcription factors, and microphthalamia- associated transcription factor in B16 cells. Consequently, purpurogallin derived from convenient enzymatic transformation of pyrogallol might be a beneficial material for reducing skin hyperpigmentation.

섬쑥부쟁이(Aster glehni Fr. Schm.) 분획물의 항산화 및 항염증 활성

김한혁 ( Han Hyuk Kim ),박근혜 ( Gun Hye Park ),박강수 ( Kang Soo Park ),이진영 ( Jin Young Lee ),안봉전 ( Bong Jeun An ) 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 2010 한국미생물·생명공학회지 Vol.38 No.4

최근 연구에서 폴리페놀을 함유한 천연물은 질병의 위험과 노화의 원인인 ROS와 RNS를 감소시키는 것과 연관이 있다고 밝혔다. 본 연구에서는 섬쑥부쟁이 추출물과 분획물의 산화와 염증에 대한 저해 효과가 있는지에 대한 연구를 조사하였다. 섬쑥부쟁이 70% 메탄올 추출물은 헥산층, 에틸 아세테이트층, 부탄올층, 물층을 극성별로 분획하였다. 섬쑥부쟁이로부터 분획한 에틸아세테이트 추출물과 부탄올 추출물의 전자공여능은 50 μg/mL에서 각각 58.0%, 46.4%를 나타냈다. 에틸아세테이트 추출물의 superoxide anion radical 소거능은 50 μg/mL에서 64.65%를 나타냈고, 부탄올 추출물은 50 μg/mL의 농도에서 35.66%의 저해 효과를 나타내었다. 항염증 효과와 관련된 hyaluronidase와 lipoxygenase의 저해 활성에 대한 에틸아세테이트 분획물의 효과는 5 μg/mL의 농도에서 각각 24.37%, 29.5%의 저해 효과를 나타냈다. 이러한 에틸아세테이트 추출물의 항염증 효과는 nitric oxide 생성 억제에서도 유의했으며, Raw 264.7 cell 내 LPS에 의해 유도된 iNOS 단백질에서 50 μg/mL의 EtOAc 추출물을 처리시 81.5%의 발현을 억제하는 효과가 확인되었다. The Plants and their extracts containing polyphenol have been shown to be associated with decreased the cause of aging and variety of disease such as reaction oxygen species (ROS) and reactive nitrogen species (RNS) in several recent studies. We conducted to investigate whether the extracts and fractionation isolated from Aster glehni Fr. Schm. has an inhibitory effect association with oxidation or inflammation. The Aster glehni Fr. Schm. 70% aq. MeOH was fractioned according to polarity with n-hexane layer, EtOAc layer, n-BuOH layer and water layer. The electron donating ability of EtOAc, n-BuOH solvent fraction from Aster glehni Fr. Schm. was about 58.0%, 46.4% at 50 μg/mL, respectively. The superoxide anion radical inhibitory effect of EtOAc extracts was about 64.65% at 50 μg/mL, and n-BuOH extracts was 35.66% at 50 μg/mL. EtOAc layer to the inhibition activity of hyaluronidase and lipoxygenase were inhibited about 24.37%, 29.5% at 5 μg/mL. In the anti-inflammation effect of EtOAc layer inhibited the generation of nitric oxide. also, these results showed that EtOAc extract inhibited 81.5% at 50 μg/mL on the expressions of iNOS protein in Raw264.7 cell line.

섬쑥부쟁이(Aster glehni Fr. Schm.) 분획물의 미백 및 주름 개선 효과

Han-Hyuk Kim(김한혁),Gun-hye Park(박근혜),Kang-Soo Park(박강수),Jin-Young Lee(이진영),Tae-Hoon Kim(김태훈),Bong-Jeun An(안봉전) 한국생명과학회 2010 생명과학회지 Vol.20 No.7

천연의 식물과 추출물은 비용의 효율성과 풍부한 자원을 이점으로 식품, 약, 의약 분야와 마찬가지로 화장품 분야에서도 이용될 수 있다. 현재까지 섬쑥부쟁이는 항산화 활성을 제외하고는 그 효능에 대해 알려진 바가 없다. 따라서 본 연구에서는 울릉도의 대표적인 특산 식물인 섬쑥부쟁이 추출물을 이용하여 화장품 분야에서의 활용 방안에 대한 연구를 조사한바, 효소적 in vitro 검색법을 측정하여 이들 중 가장 우수한 EtOAc 추출물을 대상으로 이러한 저해능이 멜라닌 생성에 관련된 단백질 발현과도 연관성이 있는지를 확인하기 위해 B16F10 melanoma cell line 내 단백질 수준을 확인하였다. Tyrosinase, TRP-1의 항체를 이용한 western blot으로 관련 단백질의 발현량을 조사한 결과 섬쑥부쟁이 에틸아세테이트 분획물 100 ㎍/㎖을 처리한 군에서는 tyrosinase protein을 30.5%를 억제하였고, TRP-1은 41.5% 억제하였다. 한편, 섬쑥부쟁이 분획물의 생육 저해환 측정에서는 Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli에 대하여 헥산 분획물 2 ㎎/disc에서 각각 21.0 ㎜, 12.0 ㎜, 14.0 mm의 저해환을 나타내어 섬쑥부쟁이 헥산 분획물만이 유일하게 높은 항균활성을 나타났다. 이들 실험 결과를 미루어보아, 섬쑥부쟁이의 화장품 분야에서의 활용적 가치가 충분하다고 사료되나, 그 활성 성분에 대한 명확한 규명을 위한 추가적인 연구를 제시하는 바이다. Plants and their extracts can be utilized as inexpensive and rich resources of active constituents in the cosmetic field, as well as the food, pharmaceutical and medicinal fields. Until now, Aster glehni Fr. Schm. had no known active effect, except on anti-oxidation, that was found during investigations for application in the cosmetic field. In this study, we examined the inhibition of enzymatic reactions to protein levels in inclusive B16F10 melanoma cell lines. Significant inhibition of enzymatic reactions was observed in the EtOAc extract, which advanced to tyrosinase protein and TRP-1 in the B16F10 melanoma cell line. These results indicated that the effect of EtOAc extract inhibited expressions of tyrosinase protein and TRP-1 in the B16F10 melanoma cell line by 30.5% and 41.5% at 100㎍/㎖ respectively. On the other hand, antimicrobial activity was evaluated to the four fractions in normal flora of the skin. Hexane extract was only exhibited in the higher clear zone in all strains. In conclusion, any cosmeceutical effects of Aster glehni Fr. Schm. may have a potential meaning, as well as possible value for further studies regarding the effects of chemical constituents of Aster glehni Fr. Schm.

오미자 잎 분획물의 B16F10 멜라노마 세포 내 색소침착 억제 효과

김한혁(Han-Hyuk Kim),나형진(Hyung-Jin Na),정아름(A-Leum Jeong),서미자(Mi-Ja Seo) 한국인체미용예술학회 2020 한국인체미용예술학회지 Vol.21 No.1

The Melanin synthesis is controlled by a series of enzymatic reactions regulated at the level of tyrosinase in melanocytes and melanoma cells. In an ongoing search for new tyrosinase inhibitors from natural sources, the dried leaves of Schisandra chinensis were extracted with 70% acetone and separated into fractions 1-3 (Fr.1-Fr.3). All fractions were evaluated for their inhibitory activity potency against the tyrosinase, and Fr.2 showed potent tyrosinase activities with 34.7% inhibition concentration 1,000 μg/mL compared to the positive control, albutin (23.5%). Finally, melanin contents was measured to determine the whitening effect. Fr.2 was inhibited at 19.3% at 50μg/mL, respectively. We investigated the changes in protein level of tyrosinase, Melanocyte Inducing Transcription Factor (MITF) by using western blotting. Fr.2 was able to significantly inhibit tyrosinase expression in protein level. These results suggest that Fr.2 inhibited melanin production which may be dependent on tyrosinase activity and expression in B16F10 melanoma cells, and an effective whitening agent for the skin.

차전초 잎의 물 추출물이 고지방식이로 비만 유도된 C57BL/6 생쥐의 체지방 감소에 미치는 영향

김소영 ( So Young Kim ),정미진 ( Mi Jin Jeong ),김유진 ( Yoo Jin Kim ),이은탁 ( Un-tak Lee ),추성태 ( Sung-tae Choo ),김한혁 ( Han-hyuk Kim ),김미려 ( Mi Ryeo Kim ) 대한본초학회 2018 大韓本草學會誌 Vol.33 No.2

Objective : Plantago asiatica L. has been used for many years in Korea, China, and Japan for the treatment of many diseases such as anti-inflammatory, anti-asthma and diuretic. But the anti-obesity effect of Plantaginis asiaticae Folium has not been studied yet. Thus, this study aimed to demonstrate the anti-obesity efficacy of Plantaginis asiaticae Folium water extract (PAF) in high fat diet-induced obesity animal model. Method : Male, five-weeks old C57BL/6 mice were divided into 5 groups; ND (normal diet + 0.9% saline), HFD (highfat diet + 0.9% saline), PC (high-fat diet + Garcinia cambogia 500 ㎎/㎏), PAF 100, 300 (high-fat diet + PAF 100, 300 ㎎/㎏. Treatments were performed daily for 6 weeks per os. We evaluated the changes of body weight, white adipose tissues weight, hepatic and fecal lipids level, pancreatic lipase activity and lipid profiles in plasma. Result : Body weights, adipose tissue weights and FER were significantly lower in mice fed the high-fat diet with PAF treatment than in mice fed the high-fat diet alone. In plasma, TG, TC, LDL-C, ATS and ALT levels significantly decreased in PAF groups compared with HFD group. Also, hepatic triglyceride (TG) levels decreased and fecal TG increased in PAF groups compared to HFD group. In addition, the activity of pancreatic lipase was decreased in the PAF groups compared to that of HFD group. Conclusion: These results suggest PAF extract is effective in preventing and improvement obesity, so it might be a potential and safe material for anti-obesity agent of weight control or functional supplements.

황련(Coptis chinensis) 추출물의 항균, 항진균 효과와 BCOP 분석을 이용한 안전성 검증

김은희 ( Eun-hee Kim ),장영아 ( Young-ah Jang ),김솔비 ( Sol-bi Kim ),김한혁 ( Han-hyuk Kim ),이진태 ( Jin-tae Lee ) 한국응용생명화학회(구 한국농화학회) 2018 Journal of Applied Biological Chemistry (J. Appl. Vol.61 No.3

황련은 한의학에서 진정, 소염, 항균 및 해열에 쓰이고 있으며, 주성분인 berberine은 강력한 항균작용을 가지는 것으로 알려져 있다. 본 연구에서는 황련에 대한 피부상재균 및 주거환경의 실내 낙하균에 대한 항균효과와 주거 환경에서 검출빈도가 높은 곰팡이에 대한 항진균 효과 및 인체에 대한 안전성을 조사하였다. 황련 열수 추출물(CW)은 Propionibacterium acnes, Staphylococcus aureus, Staphylococcus epidermidis에 대해 항균 효과를 보였으며, 각기 다른 4곳에서 포집된 실내 낙하균에 대해서도 항균효과를 보였다. 100 mg/mL의 농도에서 42일까지 항균의 지속력과 열처리 후에도 항균력의 변화가 없는 열 안정성을 보여주었다. CW의 MIC와 MBC는 S. aureus는 0.03, 0.05 mg/mL, S.epidermidis는 0.50, 0.75 mg/mL, P. acne는 0.10, 0.15 mg/mL였다. 주변 환경에서 검출빈도가 높은 5종의 곰팡이 중 4종에 대해 항진균 활성을 보였으며, 균사 생장의 100%를 억제할 수 있는 최저 농도로 결정하여 MIC를 측정한 결과 Gliocladium virens는 65 mg/mL이었다. Aureobasidium pullulans에 대한 MIC는 90 mg/mL, Penicilium pinophilum 및 Chaetomium globosum에 대한 MIC는 100 mg/mL로 측정되었다. 피부자극테스트인 패치테스트와 안자극 테스트인 ocular mucous membrane irritation evaluation test에서도 CW는 자극을 보이지 않는 안전한 추출물로 간주되었다. 따라서, 이러한 결과는, 황련(Coptis chinensis)은 항균, 항진균 활성 및 인체 안전성을 가지는 화장품 및 주거 환경 산업의 소재 개발에 응용될 수 있음을 시사한다. Coptis chinensis is used in oriental medicine for soothing, anti-inflammation, antimicrobial and antipyretic properties, and its main ingredient berberine is known to have strong antibacterial activity. In this study, we investigated the antimicrobial effect of hot water extract of Coptis chinensis (CW) on skin related microorganism and the airborne microbe, the antifungal effects of fungi, which are frequently detected in residential environments. CW showed antibacterial effect against Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis, against the airborne microbe, which was collected in four different places. At the concentration of 100 mg/mL, the antimicrobial activity continued for 42 days, showed heat stability without change in the antimicrobial activity even after heat treatment. The MIC and MBC of CW against S. aureus was 0.03, 0.05 mg/mL, against S. epidermidis was 0.50, 0.75 mg/mL and against P. acne was 0.10, 0.15 mg/mL. As a result of measuring the MIC of four kinds of fungi with high detection frequency in the surrounding environment, Gliocladium virens was 65 mg/mL by determined as MIC which can inhibit one hundred percent of mycelial growth. The concentration 90 mg/mL was determined as MIC against Aureobasidium pullulans and 100 mg/mL against Penicilium pinophilum and Chaetomium globosum. CW was considered a safe extract that showed no irritation even in the ocular mucous membrane irritation evaluation test, a patch test. Therefore, these results suggest that Coptis chinensis has antimicrobial, antifungal and safety on human body and can be applied to the development of materials for cosmetic and residential environment industries.

추출조건에 따른 차전초 추출물이 3T3-L1 세포의 지방 축적에 미치는 영향

정미진 ( Mi-jin Jeong ),김소영 ( So-young Kim ),김유진 ( Yoo-jin Kim ),이은탁 ( Eun-tag Lee ),추성태 ( Sung-tae Choo ),김한혁 ( Han-hyuk Kim ),김미려 ( Mi Ryeo Kim ) 대한본초학회 2018 大韓本草學會誌 Vol.33 No.2

Objectives : Plantaginis asiaticae Folium (PA) has been widely used in Korean medicine for treatment of liver disease, stomach troubles and inflammation. We investigated the effect of PA on lipid accumulation in 3T3-L1 according to extraction conditions. Methods : The MTT assay was employed to evaluate the cytotoxicity of PA extracted by different solvents (water, 50% ethanol, and 95% ethanol) on 3T3-L1 preadipocytes. Oil red O staining was used to identify intracellular lipid accumulation in 3T3-L1. 3T3-L1 adipocytes were treated with PA at concentration ranging in 0.1, 0.2 and 0.4 ㎎/㎖. PA was extracted by different extraction conditions such as extraction solvents, extraction time, and extraction temperature. In addition, UPLC analysis was used for determination of candidates of active ingredients in PA. Results : 3T3-L1 preadipocytes were treated with PA extracted by different solvents (water, 50% ethanol, and 95% ethanol) and there was no cytotoxicity. Oil red O staining was employed to identify the effect of PA on lipid accumulation in 3T3-L1. In the present study, PA water extraction at 70℃ for 6 hours decreased greatly in lipid accumulation. The range of concentrations was 0.1, 0.2 and 0.4 ㎎/㎖. Concentration at 0.2 ㎎/㎖ was the most effective one among them. Candidates of active ingredients in PA were shown plantamajoside and acteoside through UPLC. Conclusions : These results suggest that the effect of PA water extraction at 70℃ on lipid accumulation in 3T3-L1 is superior to other extraction conditions. We suppose that plantamajoside and acteoside may be candidates of active ingredients in PA.

산벚나무(Prunus sargentii R.) 수피의 화장품활성에 관한 연구

박정미 ( Jung Mi Park ),이진영 ( Jin Young Lee ),박태순 ( Tae Soon Park ),현석준 ( Sok Jun Hyun ),김한혁 ( Han Hyuk Kim ),조영제 ( Young Je Cho ),권오준 ( O Jun Kwon ),손애량 ( Ae Ryang Son ),김동석 ( Dong Seok Kim ),안봉전 ( Bong 한국응용생명화학회 2008 Applied Biological Chemistry (Appl Biol Chem) Vol.51 No.1