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1.
Anal Methods ; 16(15): 2278-2285, 2024 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-38525815

RESUMO

Sterols are unsaponifiable lipids resulting from plant metabolism that exhibit interesting bioactive properties. Microalgae are a major source of specific phytosterols, most of which are still not fully characterized. The similarity in sterol structures and the existence of positional isomers make the separation of phytosterols challenging. A method was developed based on an offline two-dimensional (2D) system, reversed-phase liquid chromatography (RPLC)-supercritical fluid chromatography (SFC)/quadrupole time-of-flight (Q-ToF) mass spectrometry, for the identification of sterols in microalgae. Subsequent positive-mode MS/MS was used to confirm the identified phytosterols. The 2D chromatogram exhibited a pattern related to the positions of the double bonds, which were confirmed by standard injection, enabling structural elucidation. The analysis of the unsaponifiable fraction of two algae, namely Scenedesmus obliquus, a freshwater microalgae, and Padina pavonica, a marine macroalgae, highlighted the ability of the method to distinguish a large number of sterol isomers.


Assuntos
Cromatografia com Fluido Supercrítico , Microalgas , Fitosteróis , Cromatografia de Fase Reversa/métodos , Fitosteróis/análise , Espectrometria de Massas em Tandem/métodos , Cromatografia com Fluido Supercrítico/métodos , Esteróis , Plantas
2.
J Sep Sci ; 44(16): 3070-3079, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34165880

RESUMO

Quil-A is a purified extract of saponins with strong immunoadjuvant activity. While specific molecules have been identified and tested in clinical trials, Quil-A is mostly used as a totum of the Quillaja Saponaria bark extract. Quality control of the extract stability is usually based on the monitoring of specific saponins, whereas the comparison of samples with an initial chromatogram seems more appropriate. A reference fingerprint based on comprehensive two-dimensional liquid chromatography offers a rapid detection of nonconform samples. To fulfill quality control constraints, off-line configuration using basic instrumentation was promoted. Hence, reversed-phase liquid chromatography × reversed-phase liquid chromatography and hydrophilic interaction chromatography × reversed-phase liquid chromatography methods with ultraviolet and single-quadrupole mass spectrometry detection were kinetically optimized. The reversed-phase liquid chromatography × reversed-phase liquid chromatography method used a pH switch between dimensions to maximize orthogonality. Despite diagonalization, it led to a high peak capacity of 831 in 2 h. On the other hand, the combination of hydrophilic interaction chromatography and reversed-phase liquid chromatography offered a larger orthogonality but a lower, yet satisfactory peak capacity of 673. The advantages of both methods were illustrated on degraded samples, where the reversed-phase liquid chromatography × reversed-phase liquid chromatography contour plot highlighted the loss of fatty acid chains, while the hydrophilic interaction chromatography × reversed-phase liquid chromatography method was found useful to evidence enzymatic loss of sugar moieties.


Assuntos
Técnicas de Química Analítica , Cromatografia Líquida/métodos , Quillaja/metabolismo , Saponinas/análise , Cromatografia de Fase Reversa/métodos , Cinética , Casca de Planta/metabolismo , Extratos Vegetais/análise , Controle de Qualidade , Saponinas de Quilaia/análise , Valores de Referência
3.
Biochimie ; 181: 34-41, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33242495

RESUMO

Dictyoglomus thermophilum ß-d-xylosidase DtXyl is attractive as a potential thermostable biocatalyst able to produce biologically active ginsenosides intermediates from ß-(1,2)-D-xylosylated compounds, including Notoginsenoside-R1. DtXyl was expressed as an active N-terminal His-tagged protein, and its crystal structure was solved in presence or absence of d-xylose product. Modelling of notoginsenoside R1 in DtXyl active site led to the identification of several hydrophobic residues interacting in close contact to the substrate hydrophobic core. Unlike other residues involved in substrate binding, these residues are not conserved among GH39 xylosidase family, and their physico-chemical properties can be correlated to the efficient binding and subsequent hydrolysis of Notoginsenoside R1.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Ginsenosídeos/química , Xilosidases/química , Bactérias/genética , Proteínas de Bactérias/genética , Cristalografia por Raios X , Hidrólise , Xilosidases/genética
4.
ACS Omega ; 4(1): 1916-1922, 2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-31459445

RESUMO

α-l-Rhamnosidases are catalysts of industrial tremendous interest, but their uses are still somewhat limited by their poor thermal stabilities and selectivities. The thermophilic DtRha from Dictyoglomus thermophilum was cloned, and the recombinant protein was easily purified to homogeneity to afford 4.5 mg/L culture of biocatalyst. Michaelis-Menten parameters demonstrated it to be fully specific for α-l-rhamnose. Most significantly, DtRha demonstrated to have a stronger preference for α(1 → 2) linkage rather than α(1 → 6) linkage when removing rhamnosyl moiety from natural flavonoids. This selectivity was fully explained by the difference of binding of the corresponding substrates in the active site of the protein.

5.
J Chromatogr A ; 1580: 126-133, 2018 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-30401539

RESUMO

Considering chemical complexity of plant crude extracts, purification of natural products is a rate limiting process to identify new compounds as well as to obtain standard references for quantitative or qualitative purposes. In the present work, a centrifugal partition chromatography (CPC) method was developed to isolate and produce high quality reference standards of valtrate and 7-homovaltrate from Centranthus ruber L. roots. These two compounds are controversial aglycon iridioids regulated by the legislation on plant-based dietary supplements. A new biphasic solvent system suitable for CPC separation of valepotriates was developed. It was composed of methanol/hexane/water (5/5/0.8, v/v/v). It yielded a partition coefficient near 1 and a theoretical selectivity of 1.3 between both targeted compounds. Optimization of CPC experimental parameters at the analytical scale (50 mL- and 100 mL-column capacity) enabled compounds' separation with a flow rate of 8 mL/min at 2500 rpm. Then a scale up from a 100 mL-column capacity to a 1000 mL-column capacity has been studied using the "free-space between peaks" concept. It allowed an injected quantity 16 times higher in comparison to the maximal loading capacity of the 100 mL-column. Both valtrate and 7-homovaltrate were recovered in one single step with a purity over 97%. Further MS and NMR characterization allowed to confirm unambiguously the compounds' structures. The highly efficient CPC separation developed in this work provides valepotriates in amounts suitable for further study and strong bases for future industrial development.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia Líquida , Iridoides/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Valerianaceae/química , Iridoides/química , Espectroscopia de Ressonância Magnética , Metanol/química , Extratos Vegetais/química , Solventes/química
6.
J Chromatogr A ; 1504: 55-63, 2017 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-28515006

RESUMO

The Edelweiss plant has been recognized as a very valuable source of anti-aging principles due to its composition of antioxidants compounds: leontopodic acid A and 3,5-dicaffeoylquinic acid. In this work, off-line multi-heart cutting CPC-LC separation was set up at industrial scale in order to isolate and produce new high quality reference material of these two antioxidants from Edelweiss. For this purpose, CPC and HPLC methods were developed and optimized at laboratory scale and a comprehensive CPCxHPLC analysis of the crude extract was established. Thereby, the CPC method led to a first separation of the target compounds according to their partition coefficient in the solvent system and the HPLC method was performed on the recovered fractions to lead to a second separation. A 2D CPCxHPLC plot was established in order to know the fractions to select at the industrial scale. Then, the CPC and HPLC methods were transferred at industrial scale and the multi-heart cutting CPC-LC was performed in off-line mode. Using CPC with methyl ter-butyl ether-water 1:1 (v/v) solvent system and LC with Denali C18 column, 2g of crude extract sample were injected and leontopodic acid A and 3,5-dicaffeoylquinic acid were recovered with purity over 97%. The compounds were identified by MS and NMR.


Assuntos
Antioxidantes/isolamento & purificação , Asteraceae/química , Centrifugação/métodos , Cromatografia Líquida de Alta Pressão/métodos , Extratos Vegetais/isolamento & purificação , Antioxidantes/análise , Centrifugação/instrumentação , Cromatografia Líquida de Alta Pressão/instrumentação , Espectroscopia de Ressonância Magnética , Extratos Vegetais/química
7.
Planta Med ; 82(11-12): 1110-6, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27286327

RESUMO

Over the last twenty years, tocotrienol analogues raised great interest because of their higher level and larger domain of biological activities when compared with tocopherols. Amongst the most promising therapeutic application, anti-inflammatory potency has been evaluated through the inhibition of various mediators of inflammation. Here, we worked on the isolation of two natural isoforms of garcinoic acid (i.e., δ and γ) from two different sources, respectively, Garcinia kola seeds and Garcinia amplexicaulis bark. We also developed semisynthetic strategies to access the other two non-natural α- and ß-garcinoic acid isoforms. In the next stage of our work, microsomal prostaglandin E2 synthase was defined as a target to evaluate the anti-inflammatory potential of the four garcinoic acid isomers. Both dimethylated isoforms, ß- and γ-garcinoic acid, exhibited the lowest IC50, 2.8 µM and 2.0 µM, respectively. These results showed that the affinity of tocotrienol analogues to microsomal prostaglandin E2 synthase-1 most probably contributes to the anti-inflammatory potential of this class of derivatives.


Assuntos
Benzopiranos/isolamento & purificação , Garcinia/química , Extratos Vegetais/isolamento & purificação , Prostaglandina-E Sintases/antagonistas & inibidores , Benzopiranos/síntese química , Benzopiranos/química , Linhagem Celular , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Humanos , Isomerismo , Casca de Planta/química , Extratos Vegetais/farmacologia
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