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 (¹H 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，开发了一种新型的超滤联用（UF），高速逆流色谱（HSCCC）和高效液相色谱（HPLC），用于对天麻中的次要酪氨酸酶抑制剂进行综合分析和表征。（GE）。少量的GE提取物首先通过HSCCC进行分馏，使用极性范围广的洗脱溶剂富集少量化合物。然后，通过UF-HPLC对馏分进行分析，以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，通过峰面积减少来计算结合度（BD％），其中更高的BD％表示对酪氨酸酶的更高的结合亲和力。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2D色谱图，使用离线联用紫外（UV），电子电离质谱（EIMS），质子核磁共振（¹ H NMR）。通过功能测定进一步证实了它们的活性。总之，本文开发的方法可以从天然产物中全面识别主要和次要生物活性成分，并提供有意义的建议以指导进一步的研究。与常规方法相比，通过连接多种技术开发的这种方法是一种高效的方法，可对从天然产物中提取的有效次要化合物进行综合分析。