Wang Zhiqiang, Hwang Seung Hwan, Lim Soon Sung
College of Public Health, Hebei University, Baoding, 071002, China; Department of Food Science and Nutrition, Hallym University, 1 Hallymdaehak-gil, Chuncheon, 24252, Republic of Korea.
Department of Food Science and Nutrition, Hallym University, 1 Hallymdaehak-gil, Chuncheon, 24252, Republic of Korea.
J Chromatogr A. 2017 Dec 22;1529:63-71. doi: 10.1016/j.chroma.2017.11.008. Epub 2017 Nov 6.
In the present study, a novel hyphenation of ultrafiltration (UF), high-speed countercurrent chromatography (HSCCC), and high-performance liquid chromatography (HPLC) was developed for comprehensive profiling and characterization of the minor tyrosinase inhibitors from Gastrodia elata (GE). A small quantity of GE extract was first fractionated by HSCCC, using elution solvents with a wide range of polarities to enrich minor compounds; then, the fractions were profiled by UF-HPLC to generate a comprehensive 2D chromatogram of the distribution of bioactive components. To determine the binding affinities of these bioactive components, the binding degree (BD%) was calculated by peak area reduction, in which a higher BD% indicates a higher binding affinity to tyrosinase. Among the 212 metabolites, 49 were identified as tyrosinase ligands, 17 of which showed high binding affinity. According to the 2D chromatogram, these 17 candidates were isolated by semiprep-HPLC for characterization of their structure using off-line hyphenated ultraviolet (UV), electron ionized mass spectrometry (EIMS), proton nuclear magnetic resonance (H NMR). Their activities were further validated by functional assays. In conclusion, the approach developed here can comprehensively identify both major and minor bioactive constituents from natural products, and provide meaningful suggestions to direct further research. Compared to conventional approaches, this approach, developed by hyphenating several techniques, is a highly efficient means for comprehensive profiling of potent minor compounds extracted from natural products.
在本研究中，开发了一种将超滤（UF）、高速逆流色谱（HSCCC）和高效液相色谱（HPLC）联用的新技术，用于全面分析和表征天麻（GE）中的微量酪氨酸酶抑制剂。首先，采用具有广泛极性的洗脱溶剂，通过HSCCC对少量GE提取物进行分离，以富集微量化合物；然后，通过超滤-高效液相色谱（UF-HPLC）对各馏分进行分析，生成生物活性成分分布的综合二维色谱图。为了确定这些生物活性成分的结合亲和力，通过峰面积减少计算结合度（BD%），其中BD%越高表明对酪氨酸酶的结合亲和力越高。在212种代谢产物中，有49种被鉴定为酪氨酸酶配体，其中17种表现出高结合亲和力。根据二维色谱图，通过半制备高效液相色谱法分离出这17种候选物，使用离线联用紫外（UV）、电子电离质谱（EIMS）、质子核磁共振（H NMR）对其结构进行表征。通过功能测定进一步验证了它们的活性。总之，本文开发的方法可以全面鉴定天然产物中的主要和微量生物活性成分，并为进一步研究提供有意义的建议。与传统方法相比，这种通过联用多种技术开发的方法是一种高效的手段，可用于对从天然产物中提取的有效微量化合物进行全面分析。
