Investigation of alternative two-phase solvent systems for purification of natural products by centrifugal partition chromatography.

INTRODUCTION: Centrifugal partition chromatography (CPC) is a liquid-liquid chromatography characterised by its solvent flexibility. The compounds undergoing separation are subjected to a continuous partition process between two immiscible phases in a column space free of solid support. In the context of green chemistry, it is important to substitute halogenated and petroleum-based solvents commonly used in purification processes. OBJECTIVES: The main goal of the current study was to replace classical solvents used in CPC (e.g., hexane and methanol) by green and renewable alternatives. METHODS: Solvents were first selected based on literature. Their commercial availability, price, recyclability, toxicity and ability to form two phases were particularly sought after. KEY FINDINGS: The new two-phase solvent systems were evaluated for the purification of two compounds of interest: piperine and cannabidiol. Using these alternative two-phase solvent systems allows us to isolate natural products with a high purity level (> 95%). CONCLUSION: Substituting petroleum-based solvents with bio-sourced, renewable alternatives reduces the environmental impact of CPC. Herein, new biphasic solvent systems were built using hexamethyldisiloxane, ethyl isobutyrate and 2-methyl tetrahydrofuran in combination with ethanol and water. Furthermore, this research provides a scientific basis for developing new and sustainable solvent systems in CPC.

Superior anticancer activity is demonstrated by total extract of Curcuma longa L. as opposed to individual curcuminoids separated by centrifugal partition chromatography.

Three curcuminoids: bisdemethoxycurcumin, demethoxycurcumin, and curcumin from turmeric were successfully separated by a high capacity solvent system composed of heptane: chloroform: methanol: water mixture (5: 6: 3: 2 v/v/v/v) tailored for centrifugal partition chromatographs at K-values of 0.504, 1.057, 1.644, respectively. These three ferulic acid derivatives obtained at a purity rate exceeding 95% were analysed by an HPLC-MS spectrometer. Turmeric extract inhibited the proliferation/viability of A549 human lung cancer, HT29 colon cancer, and T98G glioblastoma cell lines in (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay (MTT). Single curcuminoids significantly decreased the viability/proliferation of lung cancer cells in a dose-dependent manner. However, total extract displayed the superior anticancer activity in the investigated cell lines. Crude extract in combination with cisplatin augmented the decrease in the viability of cancer cells compared with single compound treatment in A549 lung cancer cells. Total extract of Curcuma longa could be regarded as being more effective against lung cancer cells in vitro than its separated compounds.

Off-line coupling of new generation centrifugal partition chromatography device with preparative high pressure liquid chromatography-mass spectrometry triggering fraction collection applied to the recovery of secoiridoid glycosides from Centaurium erythraea Rafn. (Gentianaceae).

A purification sequence including a Gilson CPC 250 PRO device coupled to PrepHPLC hyphenated with a MS triggering fraction collector was applied to isolate secoiridoid glycosides from a complex methanolic extract of Centaurium erythraea. This species is widely used for ethnomedicinal purposes around the Mediterranean Sea. The solvent system ethyle acetate/ethanol/water 7.5/3/5 was determined using shake-flask method targeting swertiamarin, the major secoiridoid of the extract. Optimization of CPC experimental parameters enabled the injection of 4g of extract with a flow rate of 40mL/min at 3000rpm to provide a secoiridoid glycosides enriched fraction. 130mg of this latter was submitted to a second step of purification by preparative HPLC (gradient water/formic acid (19:1) (A) and methanol (B) as follows: 0min, 85% A; 8min, 60% A; 12min, 55% A; 35min, 55% A; 40min, 10% A; 50min, 10% A; 52min, 85% A; 55min, 85% A) to give swertiamarin (36mg, yield 27.7%, purity 98.2%). Other secoiridoid glycosides (sweroside, gentiopicroside, secologanol, secoxyloganin) were also isolated in minor amounts. As these monoterpene derivatives are responsible for several biological activities, their quick recovery with high yield and purity may serve as a model for further scale-up and industrial development.

Purification of vandaterosides from Vanda teres (Orchidaceae) by stepwise gradient centrifugal partition chromatography.

Vandaterosides are polar glucosyloxybenzyl eucomate derivatives found in Vanda teres (Orchidaceae), which display biological activities that slow the skin ageing process. In order to obtain larger quantities to allow us to go further in the bioassays, the hydroalcoholic extract of aerial parts (leaves and stems) of V. teres were fractionated by centrifugal partition chromatography, combining isocratic, gradient, and dual elution modes. The first fractionation was performed on the extract maintained in the stationary phase as water saturated in butanol, while increasing the polarity of the mobile phase by changing the proportions of ethyl acetate/1-butanol/water, in order to obtain two enriched fractions. Vandateroside I was then purified by isocratic mode with ethyl acetate/ethanol/water (46:14:40), while vandateroside II was obtained by combining isocratic elution with ethyl acetate/isopropanol/water (30:20:50) followed by a multiple dual mode with ethyl acetate/ethanol/water (46:14:40). In this manner, hundreds of milligrams of vandateroside I and II were recovered from 10 g of V. teres extract.

Comparison of twin-cell centrifugal partition chromatographic columns with different cell volume.

Two twin-cell centrifugal partition chromatographic columns (SCPC 250 and SCPE-250-BIO, Armen Instrument, France) with the same column volume but different cell size and number were compared in terms of stationary phase retention and column efficiency. The columns were tested with two types of solvent systems: a commonly used organic solvent based biphasic system from the ARIZONA solvent system family and a polymer/salt based aqueous two phase system (ATPS). The efficiency of the columns was evaluated by pulse injection experiments of two benzenediols (pyrocatechol and hydroquinone) in the case of the ARIZONA system and a protein mixture (myoglobin and lysozyme) in the case of the ATPS. As result of high stationary phase retention, the column with the lower number of larger twin-cells (SCPE-250-BIO) is suitable for protein separations using ATPS. On the other hand, due to higher column efficiency, the column with the greater number of smaller cells (SCPC 250) is superior for batch elution separations performed with standard liquid-liquid chromatography organic solvent based biphasic systems.

Orthogonal Analysis Underscores the Relevance of Primary and Secondary Metabolites in Licorice.

Licorice botanicals are produced from the roots of Glycyrrhiza species (Fabaceae), encompassing metabolites of both plant and rhizobial origin. The composition in both primary and secondary metabolites (1°/2°Ms) reflects the physiologic state of the plant at harvest. Interestingly, the relative abundance of 1°Ms vs 2°Ms in licorice extracts remains undetermined. A centrifugal partition chromatography (CPC) method was developed to purify liquiritin derivatives that represent major bioactive 2°Ms and to concentrate the polar 1°Ms from the crude extract of Glycyrrhiza uralensis. One objective was to determine the purity of the generated reference materials by orthogonal UHPLC-UV/LC-MS and qHNMR analyses. The other objectives were to evaluate the presence of 1°Ms in purified 2°Ms and define their mass balance in a crude botanical extract. Whereas most impurities could be assigned to well-known 1°Ms, p-hydroxybenzylmalonic acid, a new natural tyrosine analogue, was also identified. Additionally, in the most polar fraction, sucrose and proline represented 93% (w/w) of all qHNMR-quantified 1°Ms. Compared to the 2°Ms, accounting for 11.9% by UHPLC-UV, 1°Ms quantified by qHNMR defined an additional 74.8% of G. uralensis extract. The combined orthogonal methods enable the mass balance characterization of licorice extracts and highlight the relevance of 1°Ms, and accompanying metabolites, for botanical quality control.

[From the disorganization of care to the support of nursing sciences]. / De la désorganisation des soins a l'apport des sciences infirmières.

From the disorganisation of care to the support of nursing sciences. The early communal wards providing shelter for the poor led to the development of mass care. The emergence of concepts regarding the provision of care, combined with the increasing demands of patients in terms of quality and safety inspired a more individualised approach to nursing care. The evolution of determining healthcare factors will lead to new organisations within which nursing sciences will have a role to play.

Multi-grams scale purification of xanthanolides from Xanthium macrocarpum. Centrifugal partition chromatography versus silica gel chromatography.

Plants of the Asteraceae family are known as a source of sesquiterpene lactones with interesting biological activities. The purification of several xanthanolides (xanthathin, 4-epi-xanthanol and 4-epi-isoxanthanol) was realized in one step, directly from the crude chloroformic extract of the leaves of X. macrocarpum by silica gel chromatography and for the first time by liquid/liquid chromatography (counter-current chromatography/centrifugal partition chromatography), using a FCPC 5L (Fast centrifugal partition chromatograph, Kromaton Technologies, Angers, France). Recovery, purity of xanthathin and solvent consumption were improved with the liquid/liquid chromatography compared to solid/liquid chromatography.