In a cytopathic effect inhibition assay, a standardized Rhodiola rosea root and rhizome extract, also known as roseroot extract (SHR-5), exerted distinct anti-influenza A virus activity against HK/68 (H3N2) (IC50 of 2.8 µg/mL) without being cytotoxic. For fast and efficient isolation and identification of the extract's bioactive constituents, a high-performance countercurrent chromatographic separation method was developed. It resulted in a three-stage gradient elution program using a mobile phase solvent system composed of ethyl acetate/n-butanol/water (1 : 4 : 5 → 2 : 3 : 5 → 3 : 2 : 5) in the reversed-phase mode. The elaborated high-performance countercurrent chromatographic method allowed for fractionation of the complex roseroot extract in a single chromatographic step in a way that only one additional orthogonal isolation/purification step per fraction yielded 12 isolated constituents. They cover a broad polarity range and belong to different structural classes, namely, the phenylethanoid tyrosol and its glucoside salidroside, the cinnamyl alcohol glycosides rosavin, rosarin, and rosin as well as gallic acid, the cyanogenic glucoside lotaustralin, the monoterpene glucosides rosiridin and kenposide A, and the flavonoids tricin, tricin-5-O-β-D-glucopyranoside, and rhodiosin. The most promising anti-influenza activities were determined for rhodiosin, tricin, and tricin-5-O-β-D-glucopyranoside with IC50 values of 7.9, 13, and 15 µM, respectively. The herein established high-performance countercurrent chromatographic protocol enables fast and scalable access to major as well as minor roseroot constituents. This is of particular relevance for extract standardization, quality control, and further in-depth pharmacological investigations of the metabolites of this popular traditional herbal remedy.

中文翻译：

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高效逆流色谱法获取红景天流感病毒抑制成分

在细胞病变效应抑制试验中，标准化的玫瑰红景天根和根茎提取物，也称为玫瑰根提取物 (SHR-5)，对 HK/68 (H3N2) 发挥独特的抗甲型流感病毒活性（IC50 为 2.8 µg/mL）没有细胞毒性。为了快速有效地分离和鉴定提取物的生物活性成分，开发了一种高性能逆流色谱分离方法。这导致使用由乙酸乙酯/正丁醇/水（1 : 4 : 5 → 2 : 3 : 5 → 3 : 2 : 5）组成的流动相溶剂系统在反相中进行三阶段梯度洗脱程序模式。精心设计的高性能逆流色谱方法允许在单个色谱步骤中对复杂的玫瑰根提取物进行分馏，每个馏分只需一个额外的正交分离/纯化步骤即可产生 12 种分离成分。它们涵盖广泛的极性范围并属于不同的结构类别，即苯乙醇酪醇及其葡萄糖苷红景天苷、肉桂醇糖苷 rosavin、rosarin 和松香以及没食子酸、含氰葡萄糖苷 lotaustralin、单萜葡萄糖苷 玫瑰红苷和A、黄酮类化合物tricin、tricin-5-O-β-D-吡喃葡萄糖苷和红景天苷。最有希望的抗流感活性被确定为红景天、tricin 和tricin-5-O-β-D-吡喃葡萄糖苷，IC50 值分别为 7.9、13 和 15 µM。本文建立的高性能逆流色谱协议能够快速和可扩展地访问主要和次要玫瑰根成分。这对于提取物标准化、质量控制以及对这种流行的传统草药代谢物的进一步深入药理学研究尤其重要。