Contribution à la connaissance du métabolisme secondaire des Thymelaeaceae et investigation phytochimique de l'une d'elles: "Gnidia involucrata" Steud. ex A. Rich

Dans le but de mieux connaître le métabolisme secondaire de la famille des Thymelaeaceae et de découvrir de nouveaux composés naturels à intérêt thérapeutique, 30 extraits provenant de 8 espèces africaines ont été soumis à un criblage chimique et biologique. Les cibles biologiques suivantes ont servi à l?évaluation de l?activité des extraits étudiés : la moisissure phytopathogène Cladosporium cucumerinum, la levure commensale Candida albicans, la bactérie opportuniste Bacillus subtilis, la larve du moustique vecteur de la fièvre jaune Aedes aegypti et l?hôte intermédiaire mollusque de la schistosomiase urinaire Biomphalaria glabrata. Les propriétés antiradicalaires et inhibitrices de l?acétylcholinestérase de ces extraits ont également été dépistées. Des analyses sur CCM avec révélation chimique, ainsi que des expériences LC/DAD-UV, ont permis demettre en évidence la présence de tanins, de flavonoïdes et de xanthones dans les extraits polaires. Sur la base des résultats de ces analyses préliminaires, l?investigation phytochimique des extraits méthanoliques des racines et des parties aériennes de Gnidia involucrata a été entreprise. Cette démarche a permis l?isolement de 8 composés naturels et leur caractérisation complète au moyen de méthodes spectroscopiques (UV, MS, CD, 1H- et 13C-NMR). Les activités de ces produits purs ont été évaluées et il est apparu qu?ils possédaient presque tous des propriétés antiradicalaires intéressantes, supérieures à celles du BHT, un antioxydant de synthèse (E 321) utilisé dans l?industrie alimentaire. Deux benzophénones simples, respectivement O- et C-glucosylées, ont été isolées des parties aériennes de G. involucrata au côté de la mangiférine, une C-glycosylxanthone ubiquitaire. Ces découvertes sont remarquables à plusieurs titres : (1) les benzophénones simples (nonprénylées) sont très rares dans la nature ; (2) c?est la première fois qu?une Oglycosylbenzophénone a été décrite ; (3) aucune xanthone n?avait été mise en évidence auparavant dans la famille et (4) les benzophénones semblent ne pas être que des produits intermédiaires dans la biosynthèse des xanthones. Trois 3,8??-biflavanones du type GB ont été isolées des racines et des parties aériennes de la même plante, dont deux stéréoisomères se trouvant en mélange. Une analyse LC/CD a permis d?attribuer les configurations absolues des quatre carbones asymétriques de chaque molécule. Cette classe de métabolites secondaires est réputée pour ses propriétés analgésiques et sa présence chez les Thymelaeaceae est prometteuse. Des techniques couplées de pointe ont été utilisées dans ce travail et ont montré leur apport inestimable dans le domaine de la recherche phytochimique. Une analyse LC/ MSn a ainsi permis de mettre en évidence on-line trois C-glycosylflavones ? l?isoorientine, l?isovitexine et la vitexine ? dans les extraits méthanoliques bruts de G. involucrata. De plus, les parties aériennes de cette même plante ont servi de matériel pour le développement d?une nouvelle méthode d?analyse d?extraits bruts : la LC/1H-NMR time-slice. Cette approche consiste à « découper » le temps d?analyse par des interruptions régulières du flux LC, durant lesquelles les données NMR nécessaires sont acquises. Le problème de la faible sensibilité relative de la LC/NMR a été partiellement résolu par ce biais et a permis d?envisager l?utilisation de la NMR au sein de systèmes de couplages multiples en série avec d?autres méthodes spectrales (UV, MS, IR, CD,?).<br/><br/>With the aim of acquiring a better knowledge of the secondary metabolism of the family Thymelaeaceae and of the discovering of new natural therapeutics, 30 extracts from 8 African plant species were submitted to chemical and biological screening. The following biological targets were used to estimate the activity of the extracts under study: the phytopathogenic fungus Cladosporium cucumerinum, the commensal yeast Candida albicans, the opportunistic bacteria Bacillus subtilis, larvae of the yellow fever-transmitting mosquito Aedes aegypti and the intermediate snail host of urinary schistosomiasis Biomphalaria glabrata. The antiradical and acetylcholinesterase-inhibiting properties of these extracts were also investigated. TLC analyses followed by chemical detection, together with LC/DAD-UV experiments, showed the presence of tannins, flavonoids and xanthones in the polar extracts. On the basis of these results, a phytochemical investigation of the methanol extracts of the roots and the aerial parts of Gnidia involucrata was undertaken. This procedure led to the isolation of 8 natural products, which were then characterised by spectroscopic means (UV, MS, CD, 1H- and 13C-NMR). The activities of the pure compounds were then further evaluated: almost all of them exhibited very interesting antiradical properties, superior to those of BHT, a synthetic antioxidant (E 321) used in the food industry. Two simple benzophenones, one O- and one C-glycosylated, were isolated from the aerial parts of G. involucrata, together with mangiferin, a ubiquitous C-glycosylxanthone. These findings are of multiple importance: (1) simple (non-prenylated) benzophenones are very rare in nature; (2) it is the first time that an O-glycosylbenzophenone has been described; (3) no xanthones have been previously reported in the family and (4) benzophenones do not seem to be exclusive intermediates in the biosynthesis of xanthones. Three 3,8??-biflavanones of the GB type were isolated from the roots and the aerial parts of the same plant, among them two stereoisomers in mixture. A LC/CD analysis allowed the assignment of the absolute configurations of all four stereocenters in both molecules. This class of secondary metabolite is well known for its analgesic properties and its presence in the Thymelaeaceae is very promising. Advanced hyphenated techniques were used in this work and showed their inestimable contribution to the field of phytochemical research. A LC/MSn analysis, for example, allowed the on-line characterisation of three C-glycosylflavones ? isoorientin, isovitexin and vitexin ? in the crude methanol extracts of G. involucrata. Furthermore, the aerial parts of this plant were used as material for the development of a new analytical method for crude plant extracts: time-slice LC/1H-NMR. This approach consisted in "slicing" the analytical procedure by interrupting the LC flow at given intervals, during which the necessary NMR data were acquired. The relative lack of sensitivity of LC/NMR was partially surmounted by this means, allowing one to envisage the use of NMR in a multiple hyphenated system, together with other spectroscopic methods (UV, MS, IR, CD,?)

Flavonoids affect host-microbiota crosstalk through TLR modulation

Interaction between host cells and microbes is known as crosstalk. Among other mechanisms, this takes place when certain molecules of the micro-organisms are recognized by the toll-like receptors (TLRs) in the body cells, mainly in the intestinal epithelial cells and in the immune cells. TLRs belong to the pattern-recognition receptors and represent the first line of defense against pathogens, playing a pivotal role in both innate and adaptive immunity. Dysregulation in the activity of such receptors can lead to the development of chronic and severe inflammation as well as immunological disorders. Among components present in the diet, flavonoids have been suggested as antioxidant dietary factors able to modulate TLR-mediated signaling pathways. This review focuses on the molecular targets involved in the modulatory action of flavonoids on TLR-mediated signaling pathways, providing an overview of the mechanisms involved in such action. Particular flavonoids have been able to modify the composition of the microbiota, to modulate TLR gene and protein expression, and to regulate the downstream signaling molecules involved in the TLR pathway. These synergistic mechanisms suggest the role of some flavonoids in the preventive effect on certain chronic diseases.

Flavonoids affect host-microbiota crosstalk through TLR modulation

Interaction between host cells and microbes is known as crosstalk. Among other mechanisms, this takes place when certain molecules of the micro-organisms are recognized by the toll-like receptors (TLRs) in the body cells, mainly in the intestinal epithelial cells and in the immune cells. TLRs belong to the pattern-recognition receptors and represent the first line of defense against pathogens, playing a pivotal role in both innate and adaptive immunity. Dysregulation in the activity of such receptors can lead to the development of chronic and severe inflammation as well as immunological disorders. Among components present in the diet, flavonoids have been suggested as antioxidant dietary factors able to modulate TLR-mediated signaling pathways. This review focuses on the molecular targets involved in the modulatory action of flavonoids on TLR-mediated signaling pathways, providing an overview of the mechanisms involved in such action. Particular flavonoids have been able to modify the composition of the microbiota, to modulate TLR gene and protein expression, and to regulate the downstream signaling molecules involved in the TLR pathway. These synergistic mechanisms suggest the role of some flavonoids in the preventive effect on certain chronic diseases.

Preparative separation of flavonoids from the medicinal plant Davilla elliptica St. Hill. by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) is a major tool for the fast separation of natural products from plants. It was used for the preparative isolation of the flavonoid monoglucosides present in the aerial parts of the Davilla elliptica St. Hill. (Dilleniaceae). This species is used in Brazilian folk medicine for the treatment of gastric disorders. The optimum solvent system used was composed of a mixture of ethyl acetate-n-propanol-water (140:8:80, v/v/v) and led to a successful separation of quercetin-3-O-alpha-L-rhamnopyranoside and myricetin-3-O-alpha-L-rhamnopyranoside in approximately 3.0 hours with purity higher than 95%. Identification was performed by ¹H NMR, 13C NMR and HPLC-UV-DAD analyses.

Flavonoids in Astragalus corniculatus

Nine flavonoids were identified in aerial parts of Astragalus corniculatus Bieb. (Fabaceae) by liquid chromatography coupled with ionspray mass spectrometry in the tandem mode (LC/MS/MS) with negative ion detection. Vitexin, orientin and eriodictyol-7-O-glucoside are obtained for the first time in genus Astragalus L, and isorhamnetin-3-O-glucoside in the species.

Flavonoids and a neolignan glucoside from Guarea macrophylla (Meliaceae)

This work describes the phytochemical study of the methanol extract obtained from leaves of Guarea macrophylla, leading to the isolation and identification of three flavonoid glycosides (quercetin 3-O-β-D-glucopyranoside, quercetin 3-O-b-D-galactopyranoside, kaempferol 7-O-β-D-glucopyranoside) and a neolignan glucoside, dehydrodiconiferyl alcohol-4-β-D-glucoside. All compounds were identified by a combination of spectroscopic methods (¹H, 1D, 2D NMR, 13C and UV), ESI-MS and comparison with the literature data. This is the first report of flavonoids in the genus Guarea and of a neolignan glucoside in the Meliaceae family.

Flavonoids and other bioactive phenolics isolated from Cenostigma macrophyllum (Leguminosae)

This work describes the phytochemical study of stem bark and leaves of Cenostigma macrophyllum Tul. (Leguminosae). Through usual chromatographic techniques were isolated bergenin as the primary compound of the stem bark of and from the leaves gallic acid, methyl gallate, ellagic acid, quercetin, quercetin-3-O-β-D-glucopyranoside, quercetin-3-O-(6"-O-galloyl)-β-D-glucopyranoside (tellimoside), quercetin-3-O-(6"-O-E-p-coumaroyl)-β-D-glucopyranoside (helichrysroside), agathisflavone and vitexin were obtained. The isolates were identified by spectroscopic data analysis, and bergenin showed dose-related antinociception when assessed in acetic acid-induced writhing in mice.

Chemical profiling of six samples of Brazilian propolis

Six samples of Brazilian propolis from Minas Gerais and Paraná states were analyzed to identify the constituents (GC/MS and HPLC/MS) and to determine their contents (HPLC and external standardization). All samples contained characteristic constituents of green propolis, but the samples from Minas Gerais had higher contents of prenylated phenylpropanoids and caffeoylquinic acids. Kaempferide and two other flavonoids were among the major constituents of the samples from Minas Gerais. Luteolin 5-O-methyl ether was detected only in samples from Paraná. Baccharis dracunculifolia was a source of resins for all samples analyzed, but the samples from Paraná had more complex plant origin.

LC/ESI-MS method applied to characterization of flavonoids glycosides in B. forficata subsp. pruinosa

Bauhinia forficata is used in folk medicine for its hypoglycemiant effect. In the south of Brazil, the subspecies pruinosa is found in a region with the characteristic flora, pampa biome. This species has been consumed by the local population as a tea for diabetes treatment. We studied the chemical composition of hydroethanolic extracts using LC/ESI-MS. The leaf extracts were prepared by percolation with 50% (v/v) ethanol. The chromatographic analyses were performed using a reverse-phase system, gradient elution with acetonitrile:phosphoric acid 0.05%, and ESI-MS in the positive ion mode. The chemical profile of the flavonoids was suggested to involve four quercetin and kaempferol glycosides.

Determination of flavonoids in stamen, gynoecium, and petals of Magnolia grandiflora L. and their associated antioxidant and hepatoprotection activities

Chromatographic analysis of flavonoids in ethyl acetate fractions of the stamen, gynoecium, and petal of Magnolia grandiflora L. by HPLC-PDA-MS/MS-ESI in the negative ionization mode was performed in this study. The results revealed the presence of eight flavonoids: apigenin 8-C-glucoside, luteolin 8-C-glucoside, quercetin 3-O-rutinoside, quercetin 3-O-galactoside, quercetin, 3-O-glucoside, kaempferol 3-O-rutinoside, isorhamnetin 3-O-glucoside, and isorhamnetin. Their quantification revealed that luteolin 8-C-glucoside is the major flavonoid and that the total phenolic content is concentrated primarily in the stamen. The antioxidant and hepatoprotective effects of ethanolic extract of the flower organs were evaluated against hepatotoxicity induced by CCl4, compared with the effects of silymarin.

HPLC-DAD based method for the quantification of flavonoids in the hydroethanolic extract of Tonina fluviatilis Aubl. (Eriocaulaceae) and their radical scavenging activity

This article describes the isolation and identification of flavonoids in the hydroethanolic extract of the aerial parts from Tonina fluviatilis and evaluation of their antiradical activity. A method based on HPLC-DAD was developed and validated for detecting and quantifying flavonoids in hydroethanolic extracts. The flavonoids identified and quantified in the extract were 6,7-dimethoxyquercetin-3-O-β-D-glucopyranoside (1), 6-hydroxy-7-methoxyquercetin-3-O-β-D-glucopyranoside (2), and 6-methoxyquercetin-3-O-β-D-glucopyranoside (3). The developed method presented good validation parameters, showing that the results obtained are consistent and can be used in ensuring the quantification of these constituents in the extracts. Compounds 2 and 3 showed strong antiradical activity when compared with the positive controls (quercetin and gallic acid).

Flavonoids extraction from Alpinia zerumbet (Pers.) Burtt et Smith leaves using different procedures

The current study aims to verify the best method for a rapid and efficient extraction of flavonoids from Alpinia zerumbet. Dried leaves were extracted using distillated water and ethanol 70% by extraction methods of shaking maceration, ultrasonic, microwave and stirring. By the application of TLC and reversed-phase HPLC techniques the rutin and kaempferol-3-O-glucuronide were detected. Ethanol 70% was more efficient for flavonoids extraction than water. No significant yielding variation was verified for ultrasonic, microwave and stirring methods using ethanol 70% (11 to 14%). Relative concentration of rutin and kaempferol-3-O-glucuronide, respectively, was higher by ultrasonic (1.5 and 5.62 mg g-1 dried leaves) and by microwave (1.0 and 6.64 mg g-1 dried leaves) methods using 70% ethanol. Rapid and simplified extraction proceeding optimize phytochemical work and acquisition of secondary metabolites.

Foliar flavonoids of nine species of Bauhinia

Foliar flavonoids of nine species of Bauhinia were isolated and identified. All the compounds correspond to glycosides derived from kaempferol, quercetin, isorhamnetin and myricetin. Derivatives of the latter aglyconhe seem to be rare in Bauhinia. Derivatives of isorhamnetin are commonly found in species of subgenus Bauhinia and were not detected in the two species of subgenus Phanera. Flavonoid patterns of species of the former subgenus are in general more complex than those of the latter.

Flavonoids and other phenolic compounds: characterization and interactions with lepidopteran and sawfly larvae

This thesis focuses on flavonoids, a subgroup of phenolic compounds produced by plants, and how they affect the herbivorous larvae of lepidopterans and sawflies. The first part of the literature review examines different techniques to analyze the chemical structures of flavonoids and their concentrations in biological samples. These techniques include, for example, ultraviolet-visible spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy. The second part of the literature review studies how phenolic compounds function in the metabolism of larvae. The harmful oxidation reactions of phenolic compounds in insect guts are also emphasized. In addition to the negative effects, many insect species have evolved the use of phenolic compounds for their own benefit. In the experimental part of the thesis, high concentrations of complex flavonoid oligoglycosides were found in the hemolymph (the circulatory fluid of insects) of birch and pine sawflies. The larvae produced these compounds from simple flavonoid precursors present in the birch leaves and pine needles. Flavonoid glycosides were also found in the cocoon walls of sawflies, which suggested that flavonoids were used in the construction of cocoons. The second part of the experimental work studied the modifications of phenolic compounds in conditions that mimicked the alkaline guts of lepidopteran larvae. It was found that the 24 plant species studied and their individual phenolic compounds had variable capacities to function as oxidative defenses in alkaline conditions. The excrements of lepidopteran and sawfly species were studied to see how different types of phenolics were processed by the larvae. These results suggested that phenolic compounds were oxidized, hydrolyzed, or modified in other ways during their passage through the digestive tract of the larvae.

Neuroprotection by flavonoids

The high morbidity, high socioeconomic costs and lack of specific treatments are key factors that define the relevance of brain pathology for human health and the importance of research on neuronal protective agents. Epidemiological studies have shown beneficial effects of flavonoids on arteriosclerosis-related pathology in general and neurodegeneration in particular. Flavonoids can protect the brain by their ability to modulate intracellular signals promoting cellular survival. Quercetin and structurally related flavonoids (myricetin, fisetin, luteolin) showed a marked cytoprotective capacity in in vitro experimental conditions in models of predominantly apoptotic death such as that induced by medium concentrations (200 µM) of H2O2 added to PC12 cells in culture. Nevertheless, quercetin did not protect substantia nigra neurons in vivo from an oxidative insult (6-hydroxydopamine), probably due to difficulties in crossing the blood-brain barrier. On the other hand, treatment of permanent focal ischemia with a lecithin/quercetin preparation decreased lesion volume, showing that preparations that help to cross the blood-brain barrier may be critical for the expression of the effects of flavonoids on the brain. The hypothesis is advanced that a group of quercetin-related flavonoids could become lead molecules for the development of neuroprotective compounds with multitarget anti-ischemic effects.