Improved separation of furocoumarins of essential oils by supercritical fluid chromatography

Separation of furocoumarins has become of a great interest for cosmetic industry and human health, since the recent directive of the European Union. Furocoumarins are a class of compounds presenting varied substituents linked mainly in two positions to an identical skeleton made by a furan ring bonded to a coumarin nucleus (Psoralen). The substituents are mainly methoxy, or alkyl chains, which can contain double bonds, hydroxyl or epoxy groups. Due to the variety of compounds, and their subtle structure differences, their separation requires high-performance methods. Multi-gradient high-performance liquid chromatography (HPLC) and two-dimensional chromatography are usually applied. This paper describes a new approach, by using super/subcritical fluid chromatography (SFC), with a green mobile phase: CO₂–ethanol. The choice of the stationary phase from varied types of phases, and the effects of numerous analytical parameters (flow rate, modifier percentage, temperature and outlet pressure) are studied, described and discussed, on the basis of the separation of a complex sample: lemon residue. From these studies, isocratic conditions are determined to obtain a satisfactory separation in 10 min. A two-dimensional analysis was also investigated, by performing first a class fractionation of compounds on an ethylpyridine (EP) phase, then by separating each class on a pentafluorophenyl phase (Discovery HS F5) with the selected isocratic mobile phase. A gradient elution is also studied to improve separation of some minor compounds. Structure of the eluted compounds was determined by comparison with standards, HPLC-DAD, HPLC–MS analysis, and NMR analysis of collected fractions. All these approaches allow relating structure of compounds to retention behaviour, which is unusual due to the selected pentafluorophenyl stationary phase.     

类呋喃香豆素的合成与DNA的结合及细胞毒性研究

呋喃香豆素类化合物无紫外光辅助照射时抗肿瘤活性低,为提高其正常情况下的抗肿瘤活性,依据“最小嵌入”假说对其进行结构改造。把呋喃香豆素结构中的呋喃环拆分出来,使其与香豆素由原来稠环相并的结合方式转变为通过化学键相连,得到合成简化的类呋喃香豆素。利用DNA熔解曲线、吸收光谱、荧光发射光谱和粘度测试考察了这些类呋喃香豆素与DNA的相互作用。综合DNA溶解曲线、光谱法和粘度测试的结果,推测除目标产物5b是一DNA嵌入剂外,其他化合物嵌入DNA能力下降,5a以部分嵌入方式与DNA结合,5c和5d是通过极为少见、鲜有报道的“桥型结构”与DNA相结合。利用“罗丹明B蛋白染色法”考察了目标化合物的体外细胞毒性,测试结果显示,与对照品补骨脂素相比这些化合物对肿瘤细胞体外生长抑制作用明显增强,并且非经典嵌入结合的化合物活性增强更明显。该研究拓展了“最小嵌入”假说的应用范围,同时为呋喃香豆素类化合物结构改造提供了依据。     

Design,Synthesis and Biological Activity of Novel Furocoumarin Derivatives as Stimulators of Melanogenesis and Tyrosinase in B16 Cells

Sixteen novel and four known(4a, 4d, 4e, 4h) amine derivatives of furocoumarin were synthesized, then submitted to evaluation as stimulators of melanogenesis and tyrosinase in B16 murine cells. Among them, five compounds(4g, 4j-4m) showed potent activating effect on both melanogenesis and tyrosinase in vitro compared with positive control(8-MOP), the most widely used drugs for vitiligo in clinic. Noticeably, compounds 4h and 4j, which contain morpholine and piperazine, were recognized as the most effective stimulator of melanogenesis and tyrosianse in B16 cells separately. These derivatives may serve as lead compounds for further drug discovery for the treatment of vitiligo.     

A dual preconcentration method by combining micro matrix solid-phase dispersion extraction with field-enhanced sample injection and micelle to cyclodextrin stacking for sensitive analysis of neutral coumarins

A rapid, sensitive, environmental friendly dual preconcentration method by combining micro matrix solid-phase dispersion extraction with field-enhanced sample injection and micelle to cyclodextrin stacking has been developed for the determination of furocoumarins. Molecular sieve, KIT-6, was used as an adsorbent in micro matrix solid-phase dispersion process. The important parameters affecting off-line and online CE preconcentration efficiency were optimized. Under the optimal experimental conditions, all analytes showed good linearity (R² > 0.999). The LODs of notopterol, isoimperatorin, and imperatorin were 0.1 μg/mL, 1.2 mg/kg, and 1.0 mg/kg, respectively. Compared with the normal CE method, the enrichment times were up to 300. Moreover, Angelicae Dahuricae Radix was used as the mode of complex solid sample matrix to demonstrate the prospect of application of this methodology. The results showed the proposed strategy is promising for determining trace furocoumarins in complex matrix samples, which might be applied as a powerful and economic tool in monitoring illegal cosmetic adding.