Inhibitory Effects of 2’-Hydroxy-Dimethoxychalcones on Inflammation and Melanogenesis in Mouse Cell Lines

Abstract

Chalcone은 식용 식물에 풍부하며 항암, 항균, 항산화 등 다양한 생물학적 할성을 나타내는 플로보노이드 전구체이다. 칼콘 유사체의 합성 및 생물학적 평가에 관한 성공 사례가 꾸준히 보고되면서 이러한 화합물과 의약 응용 분야에서의 활용 가능성에 대한 관심이 높아지고 있다. 그러나 2′-hydroxy-dimethoxychalcone이 염증과 멜라닌 생성에 미치는 영향은 아직 명확하게 규명되지 않았다. 따라서 이 연구의 목적은 2′-hydroxy-3,4′-dimethoxychalcone (3,4′-DMC), 2′-hydroxy- 4,4′-dimethoxychalcone (4,4′-DMC), 2′-hydroxy-3′,4′-dimethoxychalcone (3′,4′-DMC), 2′-hydroxy-4′,6′-dimethoxychalcone (4′,6′-DMC), 2′-hydroxy- 2,4-dimethoxychalcone (2,4-DMC), 2′-hydroxy-2,5′-dimethoxy- chalcone (2,5′-DMC), 2′-hydroxy-2,6′-dimethoxychalcone (2,6′-DMC) 및 2′-hydroxy- 3,6′-dimethoxychalcone (3,6′-DMC)의 항염증 및 멜라닌 생성 억제 효과를 평가하는 것이다. 2′-hydroxy-dimethoxychalcone 유도체 중 4′,6′-DMC, 2,6′-DMC, 3,6′-DMC는 강력한 항염증과 멜라닌 생성 억제 효과를 나타내었다. 여러 생물학적 분석에서 확인된 바와 같이 4′,6′-DMC, 2,6′-DMC, 3,6′-DMC는 세포 독성을 나타내지 않았으며 4′,6′-DMC, 2,6′-DMC 및 3,6′-DMC로 처리하면 LPS에 의해 유도된 NO, 염증성 cytokine 및 iNOS, COX-2 단백질 발현 수준이 감소하는 것으로 나타났다. 종합적으로 4′,6′-DMC, 2,6′-DMC, 3,6′-DMC는 NF-kB, p38, ERK 단백질 인산화 및 NF-kB/p65의 핵 전위를 감소시켜 LPS에 의한 손상을 약화시키는 것으로 나타났다. 또한 tyrosinase, TRP-1 및 TRP-2 효소의 발현을 감소시켰을 뿐만 아니라 MITF, GSK-3β, β-catenin, AKT, PKA, CREB, p38 및 JNK의 하향조절과, ERK 및 p-β-catenin의 상향 조절을 통해 B16F10 흑색종 세포에서 멜라닌 함량 및 세포 내 tyrosinase 활성을 감소시켰다. 마지막으로 인체피부 일차자극 시험을 통해 4′,6′-DMC, 2,6′-DMC, 3,6′-DMC의 국소 적용 가능성을 확인한 결과, 4′,6′-DMC, 2,6′-DMC, 3,6′-DMC는 저자극으로 판단되어 부작용을 유발하지 않는 것으로 확인되었다. 이러한 결과는 4′,6′-DMC, 2,6′-DMC, 3,6′-DMC가 피부 질환 치료법에 새로운 시사점을 제공할 수 있음을 보여준다. | Chalcones are flavonoid precursors that are abundant in edible plants and exhibit a wide range of biological activities, including anticancer, antimicrobial, and antioxidant. As success stories concerning the synthesis and biological evaluation of chalcones analogs are steadily being reported, interest in these compounds and their potential use in medicinal applications is increasing. However, the effect of 2′-hydroxy-dimethoxychalcone on anti-inflammation and anti-melanogenic has not been fully investigated. The aims of this study were to evaluate the anti-inflammatory and anti-melanogenic effect of 2′-hydroxy-3,4′-dimethoxychalcone (3,4′-DMC), 2′-hydroxy-4,4′-dimethoxychalcone (4,4′-DMC), 2′-hydroxy-3′,4′-dimethoxychalcone (3′,4′-DMC), 2′-hydroxy-4′,6′-dimethoxychalcone (4′,6′-DMC), 2′-hydroxy-2,4-dimethoxychalcone (2,4-DMC), 2′-hydroxy-2,5′-dimethoxychalcone (2,5′-DMC), 2′-hydroxy-2,6′-dimethoxychalcone (2,6′-DMC), and 2′-hydroxy-3,6′-dimethoxychalcone (3,6′-DMC). Among the 2′-hydroxy-dimethoxychalcone derivatives, 4′,6′-DMC, 2,6′-DMC, and 3,6′-DMC had the potent anti-inflammatory and anti-melanogenic effect. As confirmed by multiple biological assays, 4′,6′-DMC, 2,6′-DMC, and 3,6′-DMC exhibited no cytotoxicity and the results demonstrated that treatment with 4′,6′-DMC, 2,6′-DMC, and 3,6′-DMC profoundly attenuated the lipopolysaccharide (LPS) stimulated levels of nitric oxide and pro-inflammatory cytokines, and protein expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Collectively, 4′,6′-DMC, 2,6′-DMC, and 3,6′-DMC treatment notably weakened LPS-induced damage by reducing the phosphorylation of NF-kB, p38, JNK, ERK proteins, and NF-kB/p65 nuclear translocation. In addition, significantly reduced the expression of tyrosinase, TRP-1, and TRP-2 enzymes as well as intracellular tyrosinase activity and melanin content in B16F10 melanoma cells via MITF, GSK-3β, β-catenin, AKT, PKA, CREB, p38 and JNK-dependent downregulation, and upregulating ERK and p-β-catenin cascades. Finally, we tested the topical applicability of 4′,6′-DMC, 2,6′-DMC, and 3,6′-DMC through primary human skin irritation experiments and found that 4′,6′-DMC, 2,6′-DMC and 3,6′-DMC did not induce any adverse effects. These results suggest that 4′,6′-DMC, 2,6′-DMC, and 3,6′-DMC may provide new insights into the development of therapies for skin diseases.