Glycosidic inhibitors of melanogenesis from leaves of Momordica charantia

Eight glycosidic compounds, 1-8, including two new compounds, (4ξ)-α-terpineol 8-O-[α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside] (5) and myrtenol 10-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside] (7), were isolated from the BuOH-soluble fraction of a MeOH extract of Momordica charantia leaves. The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Upon evaluation of compounds 1-8 on the melanogenesis in B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), these compounds were found to exhibit inhibitory activities with 7.1-27.0% and 23.6-46.4% reduction of melanin content at 30 μM and 100 μM, respectively, with no or almost no toxicity to the cells (80.0-103.5% of cell viability at 100 μM). Western blot analysis showed that compound 7 reduced the protein levels of MITF, tyrosinase, TRP-1, and TRP-2 mostly in a concentration-dependent manner, suggesting that this compound inhibits melanogenesis on the α-MSH-stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of tyrosinase, TRP-1, and TRP-2.     

一种新型补骨脂素衍生物BSP-1诱导B16细胞中黑色素合成的作用机制

8-甲氧基补骨脂素（8-MOP）和5-甲氧基补骨脂素（5-MOP）等补骨脂素类药物在临床上常用于治疗白癜风,但同时具有诸多副作用。因此,发掘作用更强、毒性更小的补骨脂素类化合物用以治疗白癜风成为研究热点。在我们的前期研究中,本课题组设计合成了一系列结构新颖的补骨脂素席夫碱衍生物,并评价了它们的抗白癜风活性。本论文选取了其中一个补骨脂素席夫碱衍生物（BSP-1）,研究了它对小鼠B16细胞中黑色素合成的作用及其信号通路。利用CCK 法、L-Dopa 氧化法、NaOH溶解法及Western印迹法分别分析BSP-1对细胞增殖、黑色素含量、酪氨酸酶(TYR)活性及相关蛋白表达的影响。结果显示,BSP-1能够促进B16细胞内黑色素生成和TYR活性,上调 TYR、TRP-1、TRP-2和MITF的蛋白表达,并呈浓度依赖性。机制研究发现,BSP-1通过提高Akt和GSK-3β的磷酸化水平,上调细胞核中β 联蛋白的含量,最终使得小眼相关转录因子（MITF）的蛋白表达增加。综上所述,本研究提示BSP-1可通过调节Wnt/β-联蛋白信号通路来促进B16细胞内的黑色素合成。     

Molecular Characterisation of Small Molecule Agonists Effect on the Human Glucagon Like Peptide-1 Receptor Internalisation

The glucagon-like peptide receptor (GLP-1R), which is a G-protein coupled receptor (GPCR), signals through both Gαs and Gαq coupled pathways and ERK phosphorylation to stimulate insulin secretion. The aim of this study was to determine molecular details of the effect of small molecule agonists, compounds 2 and B, on GLP-1R mediated cAMP production, intracellular Ca²⁺ accumulation, ERK phosphorylation and its internalisation. In human GLP-1R (hGLP-1R) expressing cells, compounds 2 and B induced cAMP production but caused no intracellular Ca²⁺ accumulation, ERK phosphorylation or hGLP-1R internalisation. GLP-1 antagonists Ex(9–39) and JANT-4 and the orthosteric binding site mutation (V36A) in hGLP-1R failed to inhibit compounds 2 and B induced cAMP production, confirming that their binding site distinct from the GLP-1 binding site on GLP-1R. However, K334A mutation of hGLP-1R, which affects Gα_s coupling, inhibited GLP-1 as well as compounds 2 and B induced cAMP production, indicating that GLP-1, compounds 2 and B binding induce similar conformational changes in the GLP-1R for Gα_s coupling. Additionally, compound 2 or B binding to the hGLP-1R had significantly reduced GLP-1 induced intracellular Ca²⁺ accumulation, ERK phosphorylation and hGLP-1R internalisation. This study illustrates pharmacology of differential activation of GLP-1R by GLP-1 and compounds 2 and B.