Alix Toribio

Preparative purification of antiamyloidogenic stilbenoids from Vitis vinifera (Chardonnay) stems by centrifugal partition chromatography

Five stilbenoids, E-resveratrol, E-piceatannol, (+) E-(epsilon)-viniferin, (+)-ampelopsin A and vitisin C were isolated from methyl tert-butyl ether (MtBE) stem extract of Vitis vinifera (Chardonnay cv). Their purification on a preparative scale was obtained by centrifugal partition chromatography (CPC) using quaternary Arizona solvent systems composed of n-heptane/ethyl acetate/methanol/water. We tested 23 Arizona solvent systems to partition the extract and found that systems K and M (Hept/EtOAc/MeOH/water, 1:2:1:2 and 5:6:5:6, respectively; v/v) were the best to separate the stilbenes mentioned above. This support-free liquid-liquid chromatographic procedure made it possible to isolate ampelopsin A from V. vinifera for the first time. The antiamyloidogenic activity of the isolated stilbenes was evaluated versus beta-amyloid fibrils. E-resveratrol and (+)-ampelopsin A were found to be the most active compounds with 63 and 46% inhibition at 10microM, respectively. These findings suggest that E-resveratrol and (+)-ampelopsin A may function as attractive new candidates for protecting against brain cell dysfunction in vivo in AD by inhibiting the aggregation of Abeta.

Bioactive molecules in Kalanchoe pinnata leaves: extraction, purification, and identification.

Kalanchoe pinnata (Lam.) Pers. (syn. Bryophyllum pinnatum; family Crassulaceae) is a popular plant used in traditional medicine in many temperate regions of the world and particularly in South America. In Guyana, the leaves are traditionally used as an anti-inflammatory and antiseptic to treat coughs, ulcers, and sores. The purpose of this study was to implement a method for targeting and identifying molecules with antimicrobial activity, which could replace chemical preservatives in cosmetic applications. The leaves were extracted by a method based on pressurized liquid extraction (PLE), using different solvents. A study of antimicrobial activity and cytotoxicity tests were performed to select the most interesting extract. To isolate one or more active molecules, the selected crude extract was fractionated by centrifugal partition chromatography (CPC) and then antimicrobial activity and cytotoxicity of each fraction were tested under the same procedure. The last step consisted of identifying the main compounds in the most active fraction by LC-MS/MS.

Centrifugal partition chromatography directly interfaced with mass spectrometry for the fast screening and fractionation of major xanthones in Garcina mangostana

Xanthones are well known for their interesting phytochemical properties, which make them attractive to the pharmaceutical and medicinal industry. We have therefore developed a method to analyse the major xanthones in Garcina mangostana. The xanthones were extracted by pressurized liquid extraction with ethanol and separated at the semi-preparative scale by centrifugal partition chromatography (CPC) with a biphasic solvent system consisting of heptane/ethyl acetate/methanol/water (2:1:2:1, v/v/v/v). A CPC-electrospray ionisation MS coupling was performed and used to simultaneously separate and identify the compounds. Thanks to a variable flow splitter and an additional stream of ethanol/1 mol L(-1) ammonium acetate (95:5, v/v), all the compounds were ionised, detected and monitored whatever the solvents used in mobile phase for the CPC separation. The dual mode or elution-extrusion which are less solvent-consuming and faster than the elution mode were used without loss of ionisation and detection.

Hyphenation of centrifugal partition chromatography with electrospray ionization mass spectrometry using an active flow‐splitter device for characterization of flavonol glycosides

Online coupling of centrifugal partition chromatography to electrospray ionization mass spectrometry (CPC/ESI-MS) was investigated for the separation and characterization of flavonol glycosides. Structural identification and purification monitoring of analytes on milligram scale were demonstrated to be possible by using an active flow-splitter device which transfers automatically and successively, at discrete frequencies, small aliquots of the chromatographic effluent to an independent auxiliary stream directed to an ESI quadrupole mass spectrometer. The CPC protocol used a biphasic solvent system composed of ethyl acetate/ethanol/water (4.5:1:4.5, v/v/v) in isocratic mode. During the separation process, continuous acquisition of mass spectral data of the isolated flavonols from the effluent was performed in the negative ion mode with an auxiliary stream composed of 50 mM ammonium acetate/ethanol (2:8, v/v) delivered by a secondary pump. To demonstrate the potential of this hyphenated technique, flavonol glycosides from an apple peel extract were identified, purified and quantitatively analyzed. Calibration curves and limits of detection are also detailed.

Purification of Rosmarinic Acid by Strong Ion‐Exchange Centrifugal Partition Chromatography

Abstract Ion‐Exchange centrifugal partition chromatography using benzalkonium chloride as a strong exchanger (SIXCPC) was successfully used to purify rosmarinic acid from a crude extract that was produced by callus culture. The purification process was carried out on a gram scale using the ternary biphasic system CHCl3: n‐BuOH∶water 4.5:1:4.5 v/v/v in the ascending mode (mobile aqueous phase and stationary organic phase). Two particular points are discussed: the influence of benzalkonium chloride on ternary solvent system stability and the advantage of injecting the analytes as sodium salts rather than molecular acids.

Pilot‐scale ion‐exchange centrifugal partition chromatography: Purification of sinalbin from white mustard seeds

The purification of p-hydroxybenzylglucosinolate (sinalbin) on a multigram scale from a crude aqueous extract of white mustard seeds (Sinapis alba var. concerta) was successfully achieved by scaling up a strong ion-exchange centrifugal partition chromatography (SIXCPC) laboratory procedure. Thus, the one-step sinalbin purification was performed with 2.35 g of crude extract in approximately 170 min (830 mg/h) up to 70.3 g in approximately 160 min (26.3 g/h) by switching from a 200 mL laboratory scale column to a 5.7 L pilot-scale column. The required biphasic solvent system contained ethyl acetate, n-butanol, and water in 3:2:5 v/v/v proportions, Aliquat 336 (trioctylmethyl ammonium chloride) was added to the organic stationary phase (80 mM) and acted as ion-exchanger. Potassium iodide in the aqueous mobile phase (80 mM) was used as sinalbin displacer. The 28.5 mass scale factor arose from the increase in mobile phase flow-rate (from 2 to 50 mL/min), from the higher mass of injected white mustard seed extract (from 12 to 350 g), and from the calculated productivity (from 830 mg to 26.3 g). These results demonstrate that industry scale production of glucosinolates is easily performed by SIXCPC, thus providing pure reference standards for pharmacology studies.

Purification of antibiotics from the biocontrol agent Streptomyces anulatus S37 by centrifugal partition chromatography

A novel actinomycete strain, Streptomyces anulatus S37, has been isolated from the rhizosphere of healthy Moroccan Vitis vinifera on the basis on its ability to promote grapevine growth and to induce natural defences against various phytopathogens. In the present work, the main bioactive metabolites produced by S. anulatus S37 were isolated. A crude n-BuOH extract of the S37 fermentation broth was firstly partitioned in a biphasic solvent system composed of n-heptane, methanol, and water (5:1.5:3.5, v/v). The most active organic fraction (1.1g) as revealed by TLC-bioautography was subsequently separated by a two-step centrifugal partition chromatography procedure. The first separation was performed in the ascending mode at 6mL/min with the biphasic solvent system n-heptane, ethyl acetate, methanol and water (2:1:2:1, v/v), to finally recover 40mg of a pure compound identified as streptochlorin by NMR spectroscopy. In a second separation, the solvent system n-heptane, acetonitrile, and water (5:5:4, v/v) was used in the ascending mode at 3mL/min to purify 135mg of nigericin and 53mg of piericidin A1. Assays performed with the three compounds have confirmed their inhibitory impact on the growth of Botryris cinerea in dual confrontation and also on V. vinifera L. plantlets.

Different Ways to On-Line Hyphenate Centrifugal Partition Chromatography and Mass Spectrometry: Application to Prenylated Xanthones from Garcinia mangostana.

Centrifugal partition chromatography is a liquid-liquid separation method well adapted for the fractionation or purification of natural compounds from plant extracts. However, following the preparative isolation, the fractions collected must be analysed by high-performance thin-layer chromatography or high-performance liquid chromatography to evaluate their composition and/or their purity. These additional steps are time-consuming and increase the risk of compound degradation. In order to get an instantaneous analysis of fraction content with structural information on the phytochemicals eluted, it is possible to hyphenate on-line centrifugal partition chromatography with mass spectrometry. Depending on the complexity of the extract, two different kinds of centrifugal partition chromatography-mass spectrometry coupling can be performed: centrifugal partition chromatography-mass spectrometry or centrifugal partition chromatography-high-performance liquid chromatography-mass spectrometry coupling. In the first case, one part of the centrifugal partition chromatography effluent is directly introduced in the mass spectrometry ionisation source to identify the eluted compounds, while in the second case, it is directed to a high-performance liquid chromatography-mass spectrometry system where compounds are first separated thanks to high-performance liquid chromatography and then identified using mass spectrometry.