Aurélie Urbain

Natural Product Inhibitors of Acetylcholinesterase

Various natural products, such as physostigmine, have long been recognized as inhibitors of the enzyme acetylcholinesterase. Since the recent approval of galanthamine for the treatment of Alzheimers disease by the blockage of acetylcholine degradation, attempts to find other inhibitors of the enzyme have multiplied, leading to promising candidates such as huperzine A. In this review, a listing is presented of natural product inhibitors, both alkaloid and non-alkaloid in origin. These have been isolated from plant, animal and microbial sources. Details of current testing methods on cholinesterases are given, including solution assays and screening techniques by TLC and HPLC.

Ultra‐performance liquid chromatography/time‐of‐flight mass spectrometry as a chemotaxonomic tool for the analysis of Gentianaceae species

INTRODUCTION The segregation between the genera Gentiana and Gentianella among the Gentianaceae family is poorly defined. In order to clarify the classification of these genera, some researchers have tried to incorporate data about the chemical constitution, but this has not yet been achieved in a comprehensive way. OBJECTIVE To develop a fast and reproducible analytical method for the observation of characteristic fingerprints of secondary metabolites of each genus. METHODOLOGY Seven species were investigated, three Gentianella and four Gentiana selected for their close taxonomic links within each genus. Ten xanthones previously isolated from one of these species were used as chemotaxonomic markers. A UPLC/ESI-TOF-MS method was developed to analyse the methanolic extracts. RESULTS The UPLC/TOF-MS provided clear metabolic fingerprints and elemental composition of the compounds. The profiles of the three Gentianella species were strikingly similar. On the contrary, metabolic profiles of Gentiana species were very different from the Gentianella chromatograms and also from each other. Several compounds were unique to each genus and therefore could be used as biomarkers. CONCLUSION UPLC/TOF-MS can be applied as a chemotaxonomic tool for the rapid screening of Gentianaceae species and for the distinction between closely related taxa from the Gentianaceae family.

Quercetin and kaempferol 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside]-7-O-α-L-rhamnopyranosides from Anthyllis hermanniae: structure determination and conformational studies.

The study reports the isolation and structural identification of two new flavonol triglycosides from the methanolic extract of Anthyllis hermanniae, exhibiting the same glycosylation pattern: quercetin 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside]-7-O-α-L-rhamnopyranoside (1) and kaempferol 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside]-7-O-α-L-rhamnopyranoside (2). A conformational study related to the central arabinoside moiety was carried out including the analysis of the contribution of NOE effects and acetylation to the elucidation of the 2-O-linked arabinoside configuration of the anomeric carbon. We also report the total synthesis of a model compound, quercetin 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside (3), which verifies the structures of the isolated compounds.

One-step isolation of γ-oryzanol from rice bran oil by non-aqueous hydrostatic countercurrent chromatography.

The value-added γ-oryzanol was purified in one step from crude rice bran oil (RBO) using a preparative hydrostatic countercurrent chromatography (hydrostatic CCC) method, operating in the dual mode. The fractionation was performed using a non-aqueous biphasic solvent system consisting of heptane-acetonitrile-butanol (1.8:1.4:0.7, v/v/v), leading rapidly to the target compounds. Transfer of the analytical CCC method to large-scale isolation was also carried out yielding a high quantity-high purity fraction of γ-oryzanol. In addition, a fraction of hydroxylated triterpene alcohol ferulates (polar γ-oryzanol) was clearly separated and obtained. Furthermore, a fast HPLC-APCI(±)-HRMS method was developed and applied for the identification of γ-oryzanol as well as the polar γ-oryzanol in RBO and the resulting fractions. The purity of γ-oryzanol fraction was estimated as 97% based on HPLC-APCI-HRMS analysis.

Preparative isolation of closely‐related xanthones from Gentianella amarella ssp. acuta by High‐speed countercurrent chromatography

INTRODUCTION A methanolic extract from Gentianella amarella ssp. acuta was shown to contain several xanthones exhibiting acetylcholinesterase inhibitory activity. These xanthones were difficult to separate by conventional LC techniques, which prevented the isolation of pure compounds in sufficient amounts to perform in-depth biological testing. OBJECTIVE To develop a suitable preparative method for the separation of closely related xanthones. METHODOLOGY The methanolic extract was first partitioned with solvents of increasing polarity, in order to separate glycosides from xanthone aglycones. High-speed countercurrent chromatography (HSCCC) methods were then optimised for the fractionation of both polar and non-polar extracts. RESULTS The use of HSCCC enabled the separation of xanthones which co-eluted by HPLC. Ten closely related xanthones--three of which were isomeric--were successfully isolated by developing suitable solvent systems. All compounds were obtained in sufficient amounts to allow further biological assays (e.g. up to 250 mg), including even minor compounds that were not detectable by analytical HPLC. CONCLUSION The orthogonality of HSCCC with HPLC and the absence of solid-phase supports enabled the detection, separation and preparative isolation of closely related compounds which were difficult to resolve by other techniques.

Identification of Phenanthrene Derivatives in Aerides rosea (Orchidaceae) Using the Combined Systems HPLC–ESI–HRMS/MS and HPLC–DAD–MS–SPE–UV–NMR

INTRODUCTION In our continued efforts to contribute to the general knowledge on the chemical diversity of orchids, we have decided to focus our investigations on the Aeridinae subtribe. Following our previous phytochemical study of Vanda coerulea, which has led to the identification of phenanthrene derivatives, a closely related species, Aerides rosea Lodd. ex Lindl. & Paxton, was chosen for investigation. OBJECTIVE To identify new secondary metabolites, and to avoid isolation of those already known, by means of the combined systems HPLC-DAD(diode-array detector) with high-resolution tandem mass spectrometry (HRMS/MS) and HPLC-DAD-MS-SPE(solid-phase extraction)-UV-NMR. METHODS A dereplication strategy was developed using a HPLC-DAD-HRMS/MS targeted method and applied to fractions from A. rosea stem extract. Characterisation of unknown minor compounds was then performed using the combined HPLC-DAD-MS-SPE-UV-NMR system. RESULTS The dereplication method allowed the characterisation of four compounds (gigantol, imbricatin, methoxycoelonin and coelonin), previously isolated from Vanda coerulea stem extract. The analyses of two fractions permitted the identification of five additional minor constituents including one phenanthropyran, two phenanthrene and two dihydrophenanthrene derivatives. The full set of NMR data of each compound was obtained from microgram quantities. CONCLUSION Nine secondary metabolites were characterised in A. rosea stems, utilising HPLC systems combined with high-resolution analytical systems. Two of them are newly described phenanthrene derivatives: aerosanthrene (5-methoxyphenanthrene-2,3,7-triol) and aerosin (3-methoxy-9,10-dihydro-2,5,7-phenanthrenetriol).

Pregnane Glycosides from Cynanchum marnierianum Stimulate GLP-1 Secretion in STC-1 Cells.

In the framework of the search for natural glucagon-like peptide-1 secretagogues, the bioassay-guided fractionation of the ethanolic extract from Cynanchum marnierianum led to the isolation of two new pregnane glycosides named marnieranosides A (1) and B (2). The structures were determined based on spectroscopic data and were established as 12β,20 S-O-dibenzoyl-pregn-6-en-5α,8β,14β,17β-tetraol-3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranoside (1) and 12β,20R-O-dibenzoyl-pregn-6-en-5α,8β,14β-triol-3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-canaropyranosyl-(1 → 4)-β-D-cymaropyranoside (2). They present structural analogies to pregnanes previously described in species known for their appetite suppressant and antihyperglycemic effects, such as P57 from Hoodia gordonii. Lupeol (3), a known dipeptidyl peptidase-4 inhibitor, and the insulinomimetic kaempferol-3-O-neohesperidoside (4) were also identified in C. marnierianum. In an in vitro assay on secretin tumor cell line-1 cells, compounds 1, 2, and P57 were found to stimulate the secretion of GLP-1 by 130 % (all tested at 100 µM). These results suggest that C. marnierianum could be of great interest in the treatment of type 2 diabetes, and that pregnane derivatives should be partly responsible via the stimulation of glucagon-like peptide-1 secretion.

Hydrostatic countercurrent chromatography and ultra high pressure LC: Two fast complementary separation methods for the preparative isolation and the analysis of the fragrant massoia lactones.

Using a one-step preparative hydrostatic countercurrent chromatography method, the fragrant massoia lactones were purified from the crude massoia bark oil, in less than 3 h. The fractionation was performed with the biphasic solvent system c-hexane-methanol-water (10:9:1, v/v/v), leading to target compounds with purity over 96%, as determined by GC-MS and ultra high pressure LC-MS analyses. Together with C-10, C-12 and C-14 massoia lactones, two other aromatic compounds used in perfumes, benzyl benzoate and benzyl salicylate, were also obtained as pure compounds. In parallel, an easy and efficient ultra high pressure LC method was developed for the ultra-fast analysis of massoia lactones, as an alternative to long GC-MS methods.

Pregnane glycosides from Cynanchum menarandrense.

Five new pregnane-type steroidal glycosides, named menarandrosides A-E (1-2, 5-7) were isolated from the aerial parts of Cynanchum menarandrense, together with three known compounds, carumbelloside I (3), carumbelloside II (4), and pregnenolone-3-O-gentiobioside (8). Their structures were determined on the basis of spectroscopic analyses including NMR and mass spectrometry, reporting C-21 steroids glycosylated only by one or two glucose moieties. Compounds were then investigated for their potential to stimulate glucagon-like peptide-1 (GLP-1) secretion in intestinal cells; although none of the pure compounds had any influence, the fraction enriched in pregnanes exhibited a significant activity, suggesting a possible synergistic effect. Furthermore, none of the purified compounds affected cell viability.

Profiling, Isolation, Chemical Characterisation and Distribution of Gentianaceae Constituents

The family Gentianaceae includes about 1700 species over 90 genera. These plants are known to biosynthesise, in particular, rare polyphenolic pigments known as xanthones. They also produce flavonoids, as well as monoterpene glycosides (secoiridoids) which are their bitter principles. Numerous phytochemical studies have involved these constituents due to their pharmacological and chemotaxonomic relevance. The strategies used for the isolation of the different constituents of the Gentianaceae as well as their chemical characterisation are discussed, and different analytical methods (LC-MS, LC-UV-PDA and LC-NMR) used for the profiling of these constituents in different species of the Gentianaceae are presented. Examples are also given how these approaches are used to establish chemotaxonomic relationships between species with discussion on chemotaxonomic aspects mainly focused on two subtribes, the Gentianinae and Swertiinae.