Agnès Chartier

Optimization of the isolation and quantitation of kahweol and cafestol in green coffee oil

Kahweol and cafestol are two diterpenes that exist mainly as esters of fatty acids in green coffee oil. To recover them under their free form they have to be either saponified or trans-esterified. These two compounds are well known to be sensitive to heat, and reagents, therefore experimental conditions used in the transesterification reaction are critical. In this paper, a Doehlert experimental design plan is used to optimize the transesterification conditions using some key variables such as the temperature of the reaction, the reagent base concentration and the duration of the reaction. Therefore, the optimal parameters determined from the Doehlert design are equal to 70 °C, temperature of the reaction; 1.25 mol L(-1) concentration of the reagent base; and 60 min reaction time. The contour plots show that the extracted quantity of kahweol and cafestol can depend greatly from the experimental conditions. After transesterification, the free form of the diterpernes is extracted from the lipid fraction using liquid-liquid extraction and analyzed using GC-FID without prior derivatization. The amount of kahweol and cafestol obtained from green coffee oil obtained by cold mechanical press of Catuai coffee bean is equal to 33.2±2.2 and 24.3±2.4 g kg(-1)oil, respectively. In an attempt to streamline the process, the transesterification reaction is performed in an in-flow chemistry reactor using the optimal conditions obtained with the Doehlert experimental design. The amount of kahweol and cafestol obtained from the same green coffee oil is equal to 43.5 and 30.072 g kg(-1)oil, respectively. Results are slightly higher compared to the ones obtained with the batch procedure. This can be explained by a better mixing of the coffee oil with the reagents and a faster transesterification reaction.

Optimization of the derivatization protocol of pentacyclic triterpenes prior to their gas chromatography-mass spectrometry analysis in plant extracts.

This paper focuses on the application of a two-level full factorial design to optimize the key derivatization step before the GC-FID and GC-MS analysis of pentacyclic triterpenes. The derivatization reaction was screened for influential factors and statistically significant parameters with a p value less than 0.05. A multi-response optimization based on a desirability function was then applied, while simultaneously considering overall detection enhancement of compounds. Results showed that derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) in pyridine (22:13:65v/v/v) for 2h at 30°C was the most efficient method of derivatizing all the hydroxyl and carboxylic acid groups contained in the triterpene structures. The validity of the method was demonstrated using GC-MS analyzes of a mixture containing eleven standards (β-amyrin, α-amyrin, lupeol, erythrodiol, uvaol, betulin, oleanolic acid, betulinic acid, ursolic acid, maslinic acid and corosolic acid). These compounds are representative of different classes of terpene compounds bearing different functional groups such as alcohols, diols, and carboxylic acids. The derivatization procedure was then tested on four plant extracts: apple pomace, salvia sclarea (dried leaves and flowers), sea buckthorn (Hyppophae rhammnoides L.) berries, and B. serrata resin. The identification of triterpenes was based on the comparison of their retention time and mass spectra to those of standards. The presence of compounds already identified in the literature was confirmed and new ones such as maslinic and corosolic acids were identified in apples, sea buckthorn and salvia sclarea.

Microalgae amino acid extraction and analysis at nanomolar level using electroporation and capillary electrophoresis with laser-induced fluorescence detection

Amino acids play a key role in food analysis, clinical diagnostics, and biochemical research. Capillary electrophoresis with laser-induced fluorescence detection was used for the analysis of several amino acids. Amino acid labeling with fluorescein isothiocyanate was conducted using microwave-assisted derivatization at 80°C (680 W) during only 150 s. Good electrophoretic resolution was obtained using a background electrolyte composed of sodium tetraborate buffer (100 mM; pH 9.4) and β-cyclodextrin (10 mM), and the limits of quantification were 3-30 nM. The developed capillary electrophoresis with laser-induced fluorescence method was used to analyze amino acids in Dunaliella salina green algae grown under different conditions. A simple extraction technique based on electroporation of the cell membrane was introduced. A home-made apparatus allowed the application of direct and alternating voltages across the electrochemical compartment containing a suspension of microalgae in distilled water at 2.5 g/L. A direct voltage of 12 V applied for 4 min gave the optimum extraction yield. Results were comparable to those obtained with accelerated-solvent extraction. The efficiency of electroporation in destroying microalgae membranes was shown by examining the algae surface morphology using scanning electron microscopy. Stress conditions were found to induce the production of amino acids in Dunaliella salina cells.

Highly convergent synthesis and antiviral activity of (E) -but-2-enyl nucleoside phosphonoamidates

Abstract Several hitherto unknown (E)-but-2-enyl nucleoside phosphonoamidate analogs (ANPs) were prepared directed with nitrogen reagents by cross-metathesis in water-under ultrasound irradiation. Two diastereoisomers were formally identified by X-ray diffraction. These compounds were evaluated against a large spectrum of DNA and RNA viruses. Among them, the phosphonoamidate thymine analogue 19 emerged as the best prodrug against varicella-zoster virus (VZV) with EC50 values of 0.33 and 0.39 μM for wild-type and thymidine kinase deficient strains, respectively, and a selectivity index ≥200 μM. This breakthrough approach paves the way for new purine and pyrimidine (E)-but-2-enyl phosphonoamidate analogs.

Development of a gas chromatography-mass spectrometry method to monitor in a single run, mono- to triterpenoid compounds distribution in resinous plant materials.

A new procedure based on gas chromatography coupled to mass spectrometry (GC-MS) was developed for the simultaneous determination of mono- to triterpenoid compounds in resinous materials. Given the difference of volatility and polarity of the studied compounds some critical steps in this methodology had to be identified and investigated. The recovery of volatile compounds after sample extraction was studied. A recovery range from 30% to 100% from the more volatile monoterpene to the least one was observed. Then the mandatory derivatization step for the analysis of pentacyclic triterpenes bearing hydroxyl and carboxyl groups was optimized. Results showed that derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) in pyridine (22:13:65 v/v/v) for 2h at 30 °C was the most efficient method of derivatizing all the hydroxyl and carboxylic acid groups contained in the triterpene structures. After choosing the best injection parameters for these compounds, the selectivity of the GC column towards the separation of these terpenoids was investigated using statistical tools (principal component analysis and desirability functions). A separation with a good resolution was achieved on an HP-5ms column using a programmed temperature vaporizing injector (PTV). The method was pre-validated in terms of detection limits (LOD from 100 μg L(-1) to 200 μg L(-1) depending on the compound), linearity and repeatability using seven compounds representative of mono- and triterpenoid classes. An exhaustive characterization of various types of resins (di-, triterpenic and oleo-gum resins) was achieved.