[논문]Piper methysticum 의 뿌리로부터 추출한 Flavokawain B와 C가 Melan-a 세포에서 멜라닌 합성에 미치는 영향

류종혁; 이정아; 고재영; 황재성

doi:10.15230/scsk.2022.48.1.11

Piper methysticum 의 뿌리로부터 추출한 Flavokawain B와 C가 Melan-a 세포에서 멜라닌 합성에 미치는 영향
Flavokawain B and C, Isolated from the Root of Piper methysticum, Inhibit Melanogenesis in Melan-a Cells 원문보기

大韓化粧品學會誌 = Journal of the society of cosmetic scientists of Korea, v.48 no.1, 2022년, pp.11 - 24

류종혁 (아모레퍼시픽, R&I 센터) , 이정아 (경희대학교 생명공학원 유전공학과) , 고재영 (아모레퍼시픽, R&I 센터) , 황재성 (아모레퍼시픽, R&I 센터)

초록
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Piper methysticum 뿌리의 에탄올 추출물은 멜라닌 세포 자극 호르몬 활성화 B16 흑색종 세포에서 멜라닌 생성을 억제하는 것으로 알려져 왔다. 이 추출물로부터 분리한 flavokawain B (FKB)와 flavokawain C (FKC)는 항멜라닌 생성 활성을 기반으로 멜라닌 생성을 억제하는 것으로 밝혀져 있다. 본 연구는 melan-a 세포에서의 멜라닌 합성에 대한 FKB 및 FKC의 억제 및 그 과정을 알아보고자 하였다. FKB와 FKC는 각각 10 μM, 5 μM 농도에서 melan-a 세포의 멜라닌 생성을 억제하였다. 그러나 FKB와 FKC 모두 세포 외 타이로시네이즈 활성은 억제하지 않았다. FKB는 타이로시네이즈 (tyrosinase, Tyr), 타이로시네이즈 연관 단백질 1 (tyrosinaserelated protein 1, TRP-1)과 2(tyrosinase-related protein 2, TRP-2), microphthalmia-associated transcription factor (MITF)의 단백질 발현량을 감소시켰으며 Tyr와 TRP-1의 mRNA 발현량을 감소시켰다. FKC는 TRP-2와 MITF의 단백질 발현량을 감소시켰으며 Tyr와 TRP-1의 mRNA 발현량을 감소시켰다. Tyr와 TRP-1의 감소된 발현은 주요 멜라닌 생성 단백질을 조절하는 MITF발현 감소로 인한 것임을 예상할 수 있었다. 다만 MITF의 mRNA 발현량은 FKB와 FKC 처리에 의해 변하지 않았기 때문에, MITF의 분해를 조절한다고 알려진 세포 외 신호 조절 키나아제(ERK)/AKT 인산화에 대한 FKB와 FKC의 영향을 확인하였다. FKB와 FKC는 AKT의 인산화를 증가시키지는 않았지만 ERK1/2의 인산화를 유의미하게 증가시켰다. 이러한 결과는 FKB와 FKC가 다양한 색소 침착 증상들에 대한 잠재적인 미백제로 도움이 될 수 있음을 말해준다.

Abstract ▼ AI-Helper

It has been reported that the ethanolic extract of the root of Piper methysticum (P. methysticum) inhibits melanogenesis in melanocyte stimulating hormone (MSH)-activated B16 melanoma cells. Flavokawain B (FKB) and Flavokawain C (FKC) isolated from this extract have been found to inhibit melanin production based on anti-melanogenesis activity. This study was designed to find out the inhibition and its process of FKB and FKC on melanin synthesis in melan-a melanocytes. FKB and FKC inhibited melanogenesis at 10 μM, 5 μM respectively in melan-a melanocytes. However, they did not inhibit extracellular tyrosinase activity from melan-a melanocytes. FKB reduced the protein level of tyrosinase (Tyr), tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein 2 (TRP-2), microphthalmia-associated transcription factor (MITF) and the mRNA level of Tyr and TRP-1. FKC reduced the protein level of TRP-2 and MITF and the mRNA level of TRP-1 and Tyr. The reduced expression of Tyr and TRP-1 might be resulted from the decreased MITF which regulates major melanogenic proteins. However, since the mRNA expression of MITF did not change by FKB and FKC treatment, the effects of FKB and FKC on extracellular signal regulating kinase (ERK)/AKT phosphorylation, known to regulate the degradation of MITF, were confirmed. FKB and FKC significantly increased the phosphorylation of ERK1/2, not in AKT. These results suggest that FKB and FKC may be helpful as a potential depigmenting agent for various hyper-pigmentary disorders.

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표/그림 (9)

그림 Figure 1. Structures of FKB and FKC.
표 Table 1. Primer Sequences Used for RT-PCR Analysis
그림 Figure 2. The effects of FKB and FKC on the viability of melan-a melanocytes. Cell viability was measured in melan-a melanocytes. Melan-a cells were seeded at 1.5 × 10⁴ cells/well in 96 well plates in RPMI-1640 media. After 24 h, melan-a melanocytes treated with diluted sample in RPMI-1640 media with 10% FBS, 1% P/S and 200 nM TPA for 72 h. Cell viability was measured using EZ-Cytox cell viability assay kit based on the cleavage of the tetrazolium salt to water soluble formazan by succinate-tetrazolium reductase. The results are shown as a percentage of untreated control and as the mean ± SD of three independent experiments. t- test ***p < 0.001 versus the untreated control.
그림 Figure 3. The effects of FKB and FKC on the melanin synthesis in melan-a melanocytes. Melan-a melanocytes were seeded at 1 × 10⁵ cells in 24 well plates. After incubation with DMSO, 2.5 / 5 / 10 μM FKB, 1.25 / 2.5 / 5 μM FKC and 50 μM PTU were treated 72 h and melanin content was subsequently measured. The results are shown as a percentage of untreated control and as the mean ± SD of three independent experiments. t- test *p < 0.05, **p < 0.01 , ***p < 0.001 versus the untreated control.
그림 Figure 4. The comparison of the melanin synthesis in melan-a melanocytes for 72 h. Melan-a melanocytes were seeded into 150 pi culture dishes (1 × 10⁷ cells/dish) for 24 h and treated with (A) DMSO, (B) 10 μM FKB, (C) 5 μM FKC and (D) 40 μM Melasolv^TM. And then observation at 0, 24, 48 and 72 h respectively using digital microscope. We also proceeded quantification of changed melanin contents by using an image analysis software program called'Image pro' ; (E-a) Control, (E-b) FKB 10 μM, (E-c) FKC 5 μM, (E-d) Melasolv™ 40 μM, (F) A change of quantified melanin contents by pixel per unit area.
그림 Figure 5. The effects of FKB and FKC on the extracellular tyrosinase activity in melan-a melanocytes. Melan-a cells were seeded into 150 pi culture dishes (1 × 10⁷ cells/dish) for 24 h. Cells were removed form culture dishes and lysed in tyrosinase extracts buffer and assayed for tyrosinase activity at various concentrations FKB (2.5, 5, 10 μM) and FKC (1.25, 2.5, 5 μM) as described in materials and methods. PTU was used as a positive control. The results are shown as a percentage of untreated control and as the mean ± SD of repeated three times. t- test **p < 0.01, ***p < 0.001 versus the untreated control.
그림 Figure 6. The effects of FKB and FKC on the expression of melanogenesis-related protein in melan-a melanocytes. (A) Melan-a melanocytes were seeded at 2 × 10⁵ cells i n 6 well plates a nd treated w ith DMSO, 2 .5 / 5 / 1 0 μM FKB, 1.25 / 2.5 / 5 μM FKC and 40 μM Melasolv^TM for 72 h and then subjected to Western blot analysis. β-actin was used as a loading control. (B) Semi-quantitative analysis of protein levels. The graph represents the image analysis for the Tyr, MITF, TRP-1 and TRP-2 at 72 h after treatment with FKB and FKC. The protein levels were expressed as the ratios of β-actin, respectively. The results are shown as a percentage of untreated control and as the mean ± SD of repeated three times. t- test ***p < 0 .001 , ^###p < 0.001, ^!!!p < 0.001 and ^///p < 0.001 versus the untreated control.
그림 Figure 7. The effects of FKB and FKC on the expression of melanogenesis-related gene expression in melan-a melanocytes. (A) Melan-a melanocytes were seeded at 2 × 10⁵ cells i n 6 well plates a nd treated w ith DMSO, 2 .5 / 5 / 1 0 μM FKB, 1.25 / 2.5 / 5 μM FKC and 40 μM Melasolv^TM for 72 h and then subjected to reverse transcription polymerase chain reaction (RT-PCR). PCR products were analyzed by 2% agarose gel electrophoresis. β-actin was used as a loading control. (B) Semi-quantitative analysis of mRNA levels. The graph represents the image analysis for the Tyr, MITF, TRP-1 and TRP-2 at 72 h after treatment with FKB and FKC. The mRNA levels were expressed as the ratios of β-actin, respectively. The results are shown as a percentage of untreated control and as the mean ± SD of repeated three times. t- test ^!p < 0.05, ***p < 0.001, ^###p < 0.001 , ^!!!p < 0.001 and ^///p < 0.001 versus the untreated control.
그림 Figure 8. The effects of FKB a nd FKC o n the Phosphorylation o f ERK1 /2 a nd AKT i n melan-a melanocytes. (A) Melan-a melanocytes were seeded into 150 pi culture dishes (1×10⁷ cells/dish) for 24 h and treated with 10 μM FKB for 0.5, 1.5, 3, 6, 12, 24, 48, 72 h. Western blot was done to determine the phosphorylation state of ERK(1/2) and AKT. (B-C) Semi-quantitative analysis of protein levels. The graph represents the image analysis for the protein levels of p-ERK/ERK (B) and p-Akt/Akt (C) after treatment with FKB at various times. The protein levels were expressed as the percentage ratios of 0 h, respectively. The results are shown as a percentage of untreated control and as the mean ± SD of repeated three times. t- test *p < 0.05 and ***p < 0 .001 versus the 0 h. (D) Melan-a melanocytes were seeded into 150pi culture dishes (1 × 10⁷ cells/dish) for 24 h and treated with 5 μM FKC for 0.5, 1.5, 3, 6, 12, 24, 48, 72 h. Western blot was done to determine the phosphorylation state of ERK(1/2) and Akt. (E-F) Semi-quantitative analysis of protein levels. The graph represents the image analysis for the protein levels of p-ERK/ERK (E) and p-Akt/Akt (F) after treatment with FKC at various times. The protein levels were expressed as the percentage ratios of 0 h, respectively. The results are presented as a percentage of untreated control and as the mean ± SD of repeated three times. t- test **p < 0.01 and ***p < 0 .001 versus the 0 h.

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