Inhibitory Effect of Korean Fermented Soybean (Chungkookjang) Extract and Genistein Against Trp-P-1 Induced Genotoxicity in HepG2 Cells

Eun Jeong Song,Nam Yee Kim,Moon Young Heo 한국식품위생안전성학회 2017 한국식품위생안전성학회지 Vol.32 No.3

청국장추출물과 청국장의 주요한 플라보노이드의 하나인 genistein의 HepG2 세포에서 Trp-P-1 유도 세포독성과 DNA손상에 대한 보호효과를 평가하였다. 청국장추출물과 주요 플라보노이드성분 genistein은 Trp-P-1 유도 세포독성에 대하여 세포독성보호효과를 나타내었다. 청국장추출물 은 Trp-P-1 유도 DNA single strand breaks를 억제하였다. 한편, 청국장추출물은 HepG2 세포에서 Trp-P-1 유도에 의한 CYP1A1와 CYP1A2 발현의 억제를 나타내었다. 청국 장추출물과 genistein은 Trp-P-1에 의한 유도 세포독성과 DNA손상에 대하여 CYP1A1, CYP1A2 발현억제에 의하 여 보호효과가 나타나는 것으로 판단된다. 한국의 전통 콩 발효식품인 청국장은 게놈 불안정성(genomic instability)을 일으키는 heterocyclic amines (HCAs)과 같은 식품의 가열 조리로부터 올 수 있는 발암물질에 대한 유전독성을 예방할 수 있는 유망한 기능성물질로서 활용가능성이 있을 것으로 판단된다. This study evaluated the protective effect of Chungkookjang (CKJ) extract, a Korean traditional fermented soybean product made from Bacillus species in rice straw and boiled soybean, and one of its main flavonoids, genistein, against Trp-P-1 induced cytotoxicity and DNA damage in HepG2 cells. CKJ and genistein exhibited protective effect against Trp-P-1 induced cytotoxicity and Trp-P-1 induced DNA single strand breaks. CKJ and genistein inhibited Trp-P-1 induced CYP1A1 and CYP1A2 transcription in HepG2 cells. Our results indicated that CKJ and genistein have the protective effect against Trp-P-1 induced cytotoxicity and DNA damage. Via inhibiting expression of CYP1A1 and CYP1A2. CKJ can be used as a promising functional food material that prevents the genotoxicity induced by carcinogens produced by the heat treatment of foods such as heterocyclic amines (HCAs) that cause genomic instability.

말오줌나무 추출물의 미백활성 검증

유단희(Dan-Hee Yoo),김진태(Jin-Tae Kim),오민정(Min-Jeong Oh),염현지(Hyeon-Ji Yeom),이진영(Jin-Young Lee) 한국생명과학회 2019 생명과학회지 Vol.29 No.3

본 연구는 말오줌나무 70% 에탄올 추출물의 항산화 효과와 미백 효과를 검증하였다. 말오줌나무 추출물의 전자공여능 측정실험은 1,000 μg/ml에서 86.21%의 효과를 나타내었고, ABTS 소거능을 측정한 결과 1,000 μg/ml 농도에서 97.9%의 소거능을 보였다. Tyrosinase 저해활성 측정은 1,000 μg/ml 농도에서 37%의 효과를 보였다. 말오줌나무 추출물의 세포 생존율을 melanoma cell (B16F10)에서 확인하기 위해 MTT assay를 진행하였는데 세포 생존율을 측정한 결과, 100 μg/ml 농도에서 90% 이상의 생존율을 보였다. 말오줌나무 추출물의 MITF, TRP-1, TRP-2 및 tyrosinase의 단백질 발현 효과를 25, 50, 100 μg/ml 농도에서 측정하였으며 양성 대조군으로 β-actin을 사용하였다. 그 결과, MITF, TRP-1, TRP-2 및 tyrosinase의 각각 100 μg/ml 농도에서 34.5%, 45.6%, 58.4%, 79.6%의 단백질 발현 억제 효과를 보였다. MITF, TRP-1, TRP-2 및 tyrosinase의 mRNA발현을 RT-PCR로 25, 50, 100 μg/ml 농도에서 측정하였고, 양성 대조군으로 GAPDH를 사용하였다. 그 결과, 말오줌나무 추출물의 100 μg/ml 농도에서 각각 85.4%, 67.5%, 85.2%, 67.1%의 mRNA 발현이 감소함을 확인 할 수 있었다. 따라서, 말오줌나무 추출물이 미백 소재로서의 가능성을 확인할 수 있었다. This study evaluated the anti-oxidant and whitening effects of a 70% ethanol extract of the Sambucus sieboldiana var. pendula (Nakai) (SS). At 1,000 μg/ml concentration, the electron donating ability of this SS extract was found to be 86.21% and the ABTS+ radical scavenging ability was 97.9%. In terms of whitening activity, the tyrosinase inhibitory effect of the extract was 37%, also at 1,000 μg/ml concentration. To explore the extractefftoxicity to B16F10 melanoma cells, a 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide assay was performed. Results showed 90% or more cells remained viable at 100 μg/ml concentration. A Western blot of the SS extract was used to measure microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), tyrosinase relate protein-2 (TRP-2), and the tyrosinase protein expression inhibitory effect at 25, 50, 100 μg/ml concentrations; β-actin was used as a positive control. Consequently, the MITF, TRP-1, TRP-2, and the tyrosinase protein expression inhibitory effect were seen to decrease by 34.5%, 45.6%, 58.4%, and 79.6%, respectively, at 100 μg/ml concentration. These were also then measured by reverse transcription-polymerase chain reaction at 25, 50, 100 μg/ml concentrations with GAPDH as a positive control. As a result, the SS extract was seen to decrease MITF, TRP-1, TRP-2, and the tyrosinase protein expression inhibitory effect by 85.4%, 67.5%, 85.2%, 67.1%, respectively at the 100 μg/ml concentration. We therefore confirmed the possibility of Sambucus sieboldiana var. pendula (Nakai) extract as a whitening material.

2',4'-Dimethoxyflavone의 합성과 멜라닌 생성 저해 효과

이범천 ( Bum Chun Lee ),심관섭 ( Gwan Sub Sim ),김진화 ( Jin Hwa Kim ),김기호 ( Ki Ho Kim ),이화정 ( Hwa Jeong Lee ),표형배 ( Hyeong Bae Pyo ) 대한화장품학회 2006 대한화장품학회지 Vol.32 No.3

본 연구에서는 새로운 미백제를 개발하기 위하여 2',4'-dimethoxyflavone을 합성하였으며 멜라닌 형성 세포에서의 미백효과를 연구하였다 2',4'-Dimethoxyflavone은 프리 라디칼 소거활성(DPPH)이나 mushroom tyrosinase에 대한 직접적인 저해 효과는 없었다. 2',4'-Dimethoxyflavone을 48 h 처리한 B16 멜라노마 세포에서의 멜라닌 생합성 저해효과는 5μg/mL에서는 멜라닌생성이 27% 정도 감소되었다. 2',4'-Dimethoxyflavone은 농도에 비례해서 B16 멜라노마 세포내의 tyrosinase 활성을 저해하였으며, western blot을 이용하여 tyrosinase 단백질 발현도 감소시키는 것으로 확인되었다. 또한, RT-PCR을 이용하여 2',4'-dimethoxyflavone이 멜라닌 생성 과정에 관련하는 유전자 발현을 조사한 결과 tyrosinase와 tyrosinase 관련 단백질(TRP-1)의 mRNA발현을 억제 하였으며 TRP-2의 발현량에는 영향이 없었다. 이 결과로 2',4'-dimethoxyflavone은 멜라닌 형성세포에서 멜라닌 생성에 관여하는 타이로시나제와 TRP-1 유전자 발현 조절을 통하여 미백 효과를 나타내는 것으로 생각된다. We synthesized 2',4'-dimethoxyflavone and investigated the effects on melanogenesis. To determine the effects as a whitening agent, various in uitro tests were performed such as free radical scavenging activity, melanin assay, tyrosinase activity and expression of tyrosinase, TRP-1 and TRP-2 (western blot and RT-PCR) in Bl6 melanoma cells. 2',4'-Dimethoxyflavone showed neither free radical scavenging activities against 1,1- diphenyl -2-picrylhydrazvl (DPPH) radical and inhibition of mushroom tyrosinase activity, 2',4'-dimethoxyflavone significantly inhibited melanin production in B16 melanoma cells. 2',4'-Dimethoxyflavone treatment (48h) suppressed the biosynthesis of melanin up to 27% at 5μg/mL and reduced tyrosinase activity up to 20% at $5{\mu}g/mL$ in B16 melanoma cells. 2',4'-Dimethoxyflavone was also able to significantly inhibit tyrosinase and TRP-1 expression in protein and mRNA level. These results suggest that 2',4'-dimethoxyflavone inhibits melanin biosynthesis at the level of enzyme activity and protein mRNA expression B16 melanoma cells. Therefore, 2',4'-dimethoxyflavone may be useful as a new whitening agent in cosmetics.

Loss of EMP2 Inhibits Melanogenesis of MNT1 Melanoma Cells via Regulation of TRP-2

Enkhtaivan Enkhmend,Kim Hyun Ji,Kim Boram,Byun Hyung Jung,Yu Lu,Nguyen Tuan Minh,Nguyen Thi Ha,Do Phuong Anh,Kim Eun Ji,Kim Kyung Sung,Huy Hiệu Phùng,Rahman Mostafizur,Jang Ji Yun,Rho Seung Bae,이호,Kan 한국응용약물학회 2022 Biomolecules & Therapeutics(구 응용약물학회지) Vol.30 No.2

Melanogenesis is the production of melanin from tyrosine by a series of enzyme-catalyzed reactions, in which tyrosinase and DOPA oxidase play key roles. The melanin content in the skin determines skin pigmentation. Abnormalities in skin pigmentation lead to various skin pigmentation disorders. Recent research has shown that the expression of EMP2 is much lower in melanoma than in normal melanocytes, but its role in melanogenesis has not yet been elucidated. Therefore, we investigated the role of EMP2 in the melanogenesis of MNT1 human melanoma cells. We examined TRP-1, TRP-2, and TYR expression levels during melanogenesis in MNT1 melanoma cells by gene silencing of EMP2. Western blot and RT-PCR results confirmed that the expression levels of TYR and TRP-2 were decreased when EMP2 expression was knocked down by EMP2 siRNA in MNT1 cells, and these changes were reversed when EMP2 was overexpressed. We verified the EMP2 gene was knocked out of the cell line (EMP2 CRISPR/Cas9) by using a CRISPR/Cas9 system and found that the expression levels of TRP-2 and TYR were significantly lower in the EMP2 CRISPR/Cas9 cell lines. Loss of EMP2 also reduced migration and invasion of MNT1 melanoma cells. In addition, the melanosome transfer from the melanocytes to keratinocytes in the EMP2 KO cells cocultured with keratinocytes was reduced compared to the cells in the control coculture group. In conclusion, these results suggest that EMP2 is involved in melanogenesis via the regulation of TRP-2 expression.

Inhibitory Effect of Korean Fermented Soybean (Chungkookjang) Extract and Genistein Against Trp-P-1 Induced Genotoxicity in HepG2 Cells

Song, Eun Jeong,Kim, Nam Yee,Heo, Moon Young The Korean Society of Food Hygiene and Safety 2017 한국식품위생안전성학회지 Vol.32 No.3

청국장추출물과 청국장의 주요한 플라보노이드의 하나인 genistein의 HepG2 세포에서 Trp-P-1 유도 세포독성과 DNA손상에 대한 보호효과를 평가하였다. 청국장추출물과 주요 플라보노이드성분 genistein은 Trp-P-1 유도 세포독성에 대하여 세포독성보호효과를 나타내었다. 청국장추출물은 Trp-P-1 유도 DNA single strand breaks를 억제하였다. 한편, 청국장추출물은 HepG2 세포에서 Trp-P-1 유도에 의한 CYP1A1와 CYP1A2 발현의 억제를 나타내었다. 청국장추출물과 genistein은 Trp-P-1에 의한 유도 세포독성과 DNA손상에 대하여 CYP1A1, CYP1A2 발현억제에 의하여 보호효과가 나타나는 것으로 판단된다. 한국의 전통 콩발효식품인 청국장은 게놈 불안정성(genomic instability)을 일으키는 heterocyclic amines (HCAs)과 같은 식품의 가열조리로부터 올 수 있는 발암물질에 대한 유전독성을 예방할 수 있는 유망한 기능성물질로서 활용가능성이 있을 것으로 판단된다. This study evaluated the protective effect of Chungkookjang (CKJ) extract, a Korean traditional fermented soybean product made from Bacillus species in rice straw and boiled soybean, and one of its main flavonoids, genistein, against Trp-P-1 induced cytotoxicity and DNA damage in HepG2 cells. CKJ and genistein exhibited protective effect against Trp-P-1 induced cytotoxicity and Trp-P-1 induced DNA single strand breaks. CKJ and genistein inhibited Trp-P-1 induced CYP1A1 and CYP1A2 transcription in HepG2 cells. Our results indicated that CKJ and genistein have the protective effect against Trp-P-1 induced cytotoxicity and DNA damage. Via inhibiting expression of CYP1A1 and CYP1A2. CKJ can be used as a promising functional food material that prevents the genotoxicity induced by carcinogens produced by the heat treatment of foods such as heterocyclic amines (HCAs) that cause genomic instability.

Involvement of Transglutaminase-2 in a-MSH-Induced Melanogenesis in SK-MEL-2 Human Melanoma Cells

( Hyun Ji Kim ),( Hye Ja Lee ),( Mi Kyung Park ),( Kyung Jin Gang ),( Hyun Jung Byun ),( Jeong Ho Park ),( Mi Kyung Kim ),( Soo Youl Kim ),( Chang Hoon Lee ) 한국응용약물학회 2014 Biomolecules & Therapeutics(구 응용약물학회지) Vol.22 No.3

Skin hyperpigmentation is one of the most common skin disorders caused by abnormal melanogenesis. The mechanism and key factors at play are not fully understood. Previous reports have indicated that cystamine (CTM) inhibits melanin synthesis, though its molecular mechanism in melanogenesis remains unclear. In the present study, we investigated the effect of CTM on melanin production using ELISA reader and the expression of proteins involved in melanogenesis by Western blotting, and examined the involvement of transglutaminase-2 (Tgase-2) in SK-MEL-2 human melanoma cells by gene silencing. In the results, CTM dose-dependently suppressed melanin production and dendrite extension in a-MSH-induced melanogenesis of SK-MEL-2 human melanoma cells. CTM also suppressed a-MSH-induced chemotactic migration as well as the expressions of melanogenesis factors TRP-1, TRP-2 and MITF in a-MSH-treated SK-MEL-2 cells. Meanwhile, gene silencing of Tgase-2 suppressed dendrite extension and the expressions of TRP-1 and TRP-2 in a-MSH-treated SK-MEL-2 cells. Overall, these findings suggested that CTM suppresses a-MSH-induced melanogenesis via Tgase-2 inhibition and that therefore, Tgase-2 might be a new target in hyperpigmentation disorder therapy.

New selectable host–marker systems for multiple genetic manipulations based on TRP1, MET2 and ADE2 in the methylotrophic yeast Hansenula polymorpha

Cheon, Seon Ah,Choo, Jinho,Ubiyvovk, Vera M.,Park, Jeong-Nam,Kim, Moo Woong,Oh, Doo-Byoung,Kwon, Ohsuk,Sibirny, Andriy A.,Kim, Jeong-Yoon,Kang, Hyun Ah John Wiley Sons, Ltd. 2009 Yeast Vol.26 No.9

<P>Interest has been increasing in the thermotolerant methylotrophic yeast Hansenula polymorpha as a useful system for fundamental research and applied purposes. Only a few genetic marker genes and auxotrophic hosts are yet available for this yeast. Here we isolated and developed H. polymorpha TRP1, MET2 and ADE2 genes as selectable markers for multiple genetic manipulations. The H. polymorpha TRP1 (HpTRP1), MET2 (HpMET2) and ADE2 (HpADE2) genes were sequentially disrupted, using an HpURA3 pop-out cassette in H. polymorpha to generate a series of new multiple auxotrophic strains, including up to a quintuple auxotrophic strain. Unexpectedly, the HpTRP1 deletion mutants required additional tryptophan supplementation for their full growth, even on complex media such as YPD. Despite the clearly increased resistance to 5-fluoroanthranilic acid of the HpTRP1 deletion mutants, the HpTRP1 blaster cassette does not appear to be usable as a counter-selection marker in H. polymorpha. Expression vectors carrying HpADE2, HpTRP1 or HpMET2 with their own promoters and terminators as selectable markers were constructed and used to co-transform the quintuple auxotrophic strain for the targeted expression of a heterologous gene, Aspergillus saitoi MsdS, at the ER, the Golgi and the cell surface, respectively. The nucleotide sequences presented here were submitted to GenBank under Accession Nos AY795576 (HpTRP1), FJ226453 (HpMET2) and FJ493241 (HpADE2), respectively. Copyright © 2009 John Wiley & Sons, Ltd.</P>

태반추출물이 SK30 인체 악성 흑색종 세포주의 tyrosinase, TRP-1과 TRP-2 발현에 미치는 영향

김현진 ( Kim Hyeon Jin ),이진우 ( Lee Jin U ),김영일 ( Kim Yeong Il ),이무형 ( Lee Mu Hyeong ) 대한피부과학회 2003 대한피부과학회지 Vol.41 No.12

N/A Placental extract is a reservoir of a large number of bioactive molecules. It has been used in skin care cosmetics and soap, it is expected to have skin whitening effect. But, the effect of placental extract on melanogenesis is not clarified yet and there are several conflicting reports. To study the effect of the placental extract on proliferation and melanogenesis of pigment cells, we examined the proliferation and change of melanin amounts and expressions of tyrosinase, tyro-sinase-related protein (TRP)-1 and TRP-2 _mRNA in SK30 melanoma cells, which were irradiated or not. The results were as follows : 1. The placental extract inhibited the melanogenesis of SK30 melanoma cells. 2. The placental extract showed no significant effect on the proliferation of SK30 melanoma calls. 3. The placental extract showed antimelanogenic effect by inhibiting the synthesis of tyrosinase, TRP-1 and TRP-2 _mRNA. 4. The inhibitory effect of placental extract was more significant in UVB-irradiated SK30 melanoma cell lines. In conclusion, this study showed that the placental extract might be a good therapeutic regimens for UV-aggravated pigment disorders including melasma. Henceforth, further investigation is needed to identify and purify the active substance from the crude placental extract. (Korean J Dermatol 2003;41(12): 1612~1618)

Novel Mechanism of Massive Photoreceptor Degeneration Caused by Mutation in the trp Gene of Drosophila

Yoon, Jaeseung,Ben-Ami, Hagit Cohen,Hong, Young Seok,Park, Soyeon,Strong, Lydia L.R.,Bowman, John,Geng, Chaoxian,Baek, Kwanghee,Minke, Baruch,Pak, William L. 경희대학교 생명자원과학연구원 2000 硏究論文集 Vol.21 No.-

The Drosophila trp gene encodes a light-activated Ca^(2+) channel subunit, which is a prototypical member of a novel class of channel proteins. Previously identified trp mutants are all recessive, loss-of-function mutants characterized by a transient receptor potential and the total or near-total loss of functional TRP protein. Although retinal degeneration does occur in these mutants, it is relatively mild and slow in onset. We report herein a new mutant, Trp^(p365), that does not display the transient receptor potential phenotype and is characterized by a substantial level of the TAP protein and rapid, semi-dominant degeneration of photoreceptors. We show that, in spite of its unusual phenotypes, Trp^(p365) is a trp allele because a Trp^(p365) transgene induces the mutant phenotype in a wild-type back-ground, and a wild-type trp transgene in a Trp^(p365) background suppresses the mutant phenotype. Moreover, amino acid alterations that could cause the Trp^(p365) phenotype are found in the transmembrane segment region of the mutant channel protein. Whole-cell recordings clarified the mechanism underlying the retinal degeneration by showing that the TRP channels of Trp^(p365) are constitutively active. Although several genes, when mutated, have been shown to cause retinal degeneration in Drosophila, the underlying mechanism has not been identified for any of them. The present studies provide evidence for a specific mechanism for massive degeneration of photoreceptors in Drosophila. Insofar as some human homologs of TRP are highly expressed in the brain, a similar mechanism could be a major contributor to degenerative disorders of the brain.

회화나무(Sophora japonica L.) 꽃(괴화)과 열매(괴각)의 미백활성 평가

장태원,박재호 한국자원식물학회 2017 한국자원식물학회지 Vol.30 No.1

Sophora japonica has been used for treatment of liver and blood-related diseases in herbology. We evaluated whitening activities of Sophorae Flos and Fructus. Sophorae Flos and Fructus were extracted with methanol (MeOH) and divided into petroleum ether, ethyl acetate(EtOAC) and water fraction. For whitening effect by western blot in B16 F10 cells, we analyzed it by investigating the effect of tyrosinase, TRP-l (tyrosinase-related protein 1), TRP-2 (tyrosinase-related protein 2) and MITF (microphthalmia-associated transcription factor) protein expression. The protein expressions of tyrosinase, TRP-1, TRP-2 and MITF in B16 F10 cells treated with Sophorae Flos and Fructus extract were reduced in a dose-dependant manner. Therefore, these results suggest that Sophorae Flos and Fructus have useful plant resource to be developed as functional cosmetic. 회화나무는 한의학에서 간과 혈관계 질환의 치료에 사용되어온 약용 식물로, 그 꽃과 열매를 괴화와 괴각이라 한다. 괴화와 괴각은 메탄올 추출물로부터 에틸아세테이트 분획물을 얻어시료로 사용하였고, B16 F10 세포를 이용하여 Western blot을실행하였다. 미백효과를 확인하기 위해, tyrosinase, TRP-1, TRP-2, MITF 단백질을 확인하였다. 그 결과, 괴화와 괴각 추출물은 세포 생존율에는 영향이 미미하였으며, 모두 tyrosinase,TRP-1, TRP-2, MITF 단백질의 발현 억제효과를 나타냈다. 또한 농도 의존적으로 억제효과를 보였으며, 이를 통해 전통적으로 사용되어 왔던 괴화와 괴각은 기능성 화장품의 원료와 같은천연식물 유래 자원으로 상당한 가치를 나타냈다.