Isolation of Achyrobichalcone from Achyrocline satureioides by High- Speed Countercurrent Chromatography

Abstract

Achyrobichalcone is a biflavonoid recently found in Achyrocline satureioides. This substance has unprecedented chemical structure and occurrence, but resembles other bioactive bichalcones, which have important pharmacological properties, such as anticancer activity. The major challenge for evaluation of the physicochemical and biological properties of this new molecule is the isolation step, which affects the purity and yield of the isolated product. Thus, the objective of this work was to develop a semi-preparative method for achyrobichalcone isolation from Achyrocline satureioides by high-speed countercurrent chromatography. The high-speed countercurrent chromatography separation was achieved in two steps. In the first step, an enriched fraction of achyrobichalcone from the freeze-dried extract was obtained, using the solvent system hexane-ethyl acetate-methanol-water 0.8:1:0.8:1, v/v. The purification of achyrobichalcone from the enriched fractions was achieved by further high-speed countercurrent chromatography fractionation with hexane-ethyl acetate-methanol-water 0.9:0.9:0.8:1, v/v. The final isolated product was obtained using preparative thin layer chromatography and crystallization procedure. A yellow semi-crystalline solid with purity close to 90% was obtained as the final product. The mass recovery of achyrobichalcone isolation was near 67%. The structural identification from spectroscopic and chromatographic techniques confirmed the achyrobichalcone structure. This is the first report of achyrobichalcone isolation on a semi-preparative scale by high-speed countercurrent chromatography. This method afforded achyrobichalcone in good yield and purity for further biopharmaceutical studies.

Semi‐preparative isolation and purification of phenolic compounds from Achyrocline satureioides (Lam) D.C. by high‐performance counter‐current chromatography

“…In order to ensure the successful separation and purification of target compounds by the HPCCC method, an appropriate two‐phase solvent system is required with a suitable K D . The solvent systems used were tested at different volume ratios in order to obtain enriched fractions and isolated products from the crude extract of A. satureioides inflorescences, without the need for additional purification techniques such as chromatography in column, thin layer chromatography or even recrystallisation, as previously described …”

“…Figure (B) presents the chromatographic profile of F 2 at 362 nm, where three main peaks can be observed at retention times of 5.7, 6.4, and 7.4 min, corresponding to QCT, LUT, and 3 O MQ, respectively. The corresponding diode array spectra of ACB, 3 O MQ, QCT, and LUT are shown in Figure (C), with λ max (maximum absorption) at 198/367, 253/358, 257/268/371, and 254/267/371, respectively, which are in agreement with the values reported in the literature …”

“…Then 6 mL were injected into the loop. Three independent experiments were carried out with the solvent system Hex/EtOAc/MeOH/H 2 O at different ratios (1.0:1.0:1.0:1.0, 0.8:1.0:0.8:1.0, and 0.9:0.9:0.8:1, v / v / v / v ) . The upper phase (mobile phase) was then pumped into the column at a flow rate of 6 mL/min, while the apparatus was rotated at 1600 rpm and column temperature was kept at 30°C.…”

“…The quantification of ACB, 3OMQ, QCT, and LUT was performed 25 The upper phase (mobile phase) was then pumped into the column at a flow rate of 6 mL/min, while the apparatus was rotated at 1600 rpm and column temperature was kept at 30°C. After hydrodynamic equilibrium was established, the sample was injected through the sampler port.…”

“…23,24 However, since the occurrence of ACB has only recently been described 12 and due to the difficulty to isolate the compound with high purity and yield, its properties have not been clarified. Carini et al 25 isolated ACB with 90% purity using a multi-step ethanol extraction method of A. satureioides constituents.…”