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In this study, a combined strategy with methanol gradient countercurrent chromatography and ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry was introduced for the fractionation and identification of active constituents from Curcuma longa L. The gradient countercurrent chromatography separation was performed using the heptane‐ethyl acetate‐methanol‐water (5:5:2:8, v/v) solvent system, in which the lower phase and methanol were used as the mobile phases. Constituents of turmeric with large partition coefficients were well resolved. Subsequent cytotoxicity analysis showed that the fractions 10, 11, 12, and 15 expressed significantly higher cytotoxicity against B16 mouse melanoma than the other fractions. Four compounds with potent activity, curcumin, demethoxycurcumin, and bisdemethoxycurcumin from fraction 11 and galanal A from fraction 15, were purified, and the half‐maximal inhibitory concentrations were 18.5 ± 1.3, 7.8 ± 0.4, 20.4 ± 1.3, and 14.1 ± 0.8 μM, respectively. Ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry was then applied for compound identification from fraction 10 with constituents in very low content. A total of 14 diarylheptanoids were identified, which are supposed to be cytotoxic constituents. It proved that the strategy based on the combination of bioassay‐guided methanol gradient countercurrent chromatography separation and ultra‐high‐performance liquid chromatography coupled with tandem mass spectrometry‐assisted peak identification could be an efficient method for natural product screening and new drug discovery. [ABSTRACT FROM AUTHOR]

Abstract: Gradient change of mobile phase composition is commonly used in liquid chromatography to shorten analysis duration. Gradient elution is possible in counter-current chromatography if it is demonstrated that the mobile phase composition changes are not associated with liquid stationary phase composition changes. Also, the solute distribution ratios in initial (1) and final (2) eluting systems must be known. If the solute distribution ratios during the changing mobile phase composition are modeled, the full gradient separation can be modeled using classical equations of liquid chromatography. Another approach allowed modeling empirically the gradient step. It considered the decreasing volume of mobile phase 1 and the increasing volume of mobile phase 2 used at each gradient time to derive a very simple equation. The two equations were compared computing the retention volumes of five test solutes. A remarkable agreement was observed considering the very different mathematical expressions of the two models. The step-gradient method was also modeled and its results compared to those of the linear gradient method. [Copyright &y& Elsevier]

Abstract: In this work, we have established a new stop-and-go two-dimensional chromatography coupling of counter-current chromatography and liquid chromatography (2D CCC×LC) for the preparative separation of two novel antioxidant flavonoids from the extract of alfalfa (Medicago sativa L.). The CCC column has been used as the first dimension to purify the target flavonoids using a solvent system of isopropanol and 20% sodium chloride aqueous solution (1:1, v/v) with the stop-and-go flow technique, and the LC column packed with macroporous resin has been employed as the second dimension for on-line absorption, desalination and desorption of the targeting effluents purified from the first CCC dimension. As a result, two novel flavonoids, 6,8-dihydroxy-flavone-7-O-β-d-glucuronide (15.3mg) and 6-methoxy-8-hydroxy-flavone-7-O-β-d-glucuronide (13.7mg), have been isolated from 126.8mg of crude sample pre-enriched by macroporous resin column. Their structures have been identified by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) and one- and two-dimensional nuclear magnetic resonance spectra (1D and 2D NMR). Further antioxidant assays showed that the first component possess a strong antioxidant activity. All the results demonstrated that the stop-and-go 2D CCC×LC method is very efficient for the separation of flavonoids of alfalfa and it can also be applied to isolate other comprehensive multi-component natural products. [Copyright &y& Elsevier]

Abstract: In this work, we have developed a novel hybrid two-dimensional counter-current chromatography and liquid chromatography (2D CCC×LC) system for the continuous purification of arctiin from crude extract of Arctium lappa. The first dimensional CCC column has been designed to fractionalize crude complex extract into pure arctiin effluent using a one-component organic/salt-containing system, and the second dimensional LC column has been packed with macroporous resin for on-line adsorption, desalination and desorption of arctiin which was effluent purified from the first CCC dimension. Thus, the crude arctiin mixture has been purified efficiently and conveniently by on-line CCC×LC in spite of the use of a salt-containing solvent system in CCC separation. As a result, high purity (more than 97%) of arctiin has been isolated by repeated injections both using the ethyl acetate–8% sodium chloride aqueous solution and butanol–1% sodium chloride aqueous solution. By contrast with the traditional CCC processes using multi-component organic/aqueous solvent systems, the present on-line CCC×LC process only used a one-component organic solvent and thus the solvent is easier to recover and regenerate. All of used solvents such as ethyl acetate, n-butanol and NaCl aqueous solution are low toxicity and environment-friendly. Moreover, the lower phase of salt-containing aqueous solution used as mobile phase, only contained minor organic solvent, which will save much organic solvent in continuous separation. In summary, our results indicated that the on-line hybrid 2D CCC×LC system using one-component organic/salt-containing aqueous solution is very promising and powerful tool for high-throughput purification of arctiin from fruits of A. lappa. [Copyright &y& Elsevier]