High-speed counter-current chromatography assisted preparative isolation of bioactive compounds from stem bark of Juglans mandshurica

High-speed counter-current chromatography was applied for the first time for the separation and purification of bioactive compounds contained in the stem bark of Juglans mandshurica Maxim. Silica gel column chromatography was first used to obtain three composition-enriched target fractions from a crude J. mandshurica methanol extract. Three independent high-speed counter-current chromatography processes were then used to further isolate 13 bioactive compounds, namely, six galloyl glucose derivatives, three flavonones, three naphthoquinones, and ethyl gallate. The isolates were identified by ultra high performance liquid chromatography with tandem mass spectrometry, and ultraviolet and NMR spectroscopy, and compared with literature data. Their purities were determined to be > 94.6% by ultrahigh performance liquid chromatography. Furthermore, based on the total phenolic content and results of a 2,2-diphenyl-1-picrylhydrazyl test, themethanol extract and two of the three initial fractions were observed to be rich in phenolic compounds and exhibit good free radical scavenging abilities, while nine of the isolated compounds exhibited remarkable antioxidant activity, superior to that of butyrate hydroxy-toluene and comparable to that of gallic acid. The results of this research confirm the effectiveness of high-speed counter-current chromatography for the separation of compounds contained in extremely complex samples, and provide a basis for the further utilization of J. mandshurica.