Lauro Mera de Souza

Application of acetate derivatives for gas chromatography–mass spectrometry: Novel approaches on carbohydrates, lipids and amino acids analysis

The structure of glycoconjugates has been determined by several chromatographic methods, however gas chromatography-mass spectrometry (GC-MS) has been widely used to identify and quantify the volatile trimethylsilyl and fluoroacyl derivatives. Adapting the reduction/acetylation strategies, we had performed the derivatization of all monosaccharide class, as well as amino acids and OH-fatty acids as from different glycoconjugates. Uronic acids gave characteristic ions at m/z 143, 156 and 173, and 19 amino acids derivatives, gave molecular ions [M]+ and daughter ions of [M-59]+ and [M-43]+ on electron impact (EI)-MS, which provide their rapid identification.

HPLC/ESI-MS and NMR analysis of flavonoids and tannins in bioactive extract from leaves of Maytenus ilicifolia

Maytenus ilicifolia is an important plant in Brazilian folk medicine, used in many gastric disorder treatments. Low molecular weight components present in the leaves have been characterized as afzelechin, epiafzelechin, catechin, epicatechin, gallocatechin and epigallocatechin, as detected by HPLC and ESI-MS. Condensed tannins have also been found, consisting of di-, tri-, tetra-, and penta-, hexa, and heptamers. ESI-MS analyses were performed in positive and negative ionization modes, and in contrast with other investigations, negative ionization improved sensitivity for obtaining molecular ions. Moreover, the tandem-MS profile with negative detection provided characteristic fragments, such as those found at m/z 543 [(epi)afzelechin-(epi)afzelechin], m/z 561 [(epi)afzelechin-(epi)catechin], and m/z 577 [(epi)catechin-(epi)catechin] or [(epi)afzelechin-(epi)gallocatechin]. The analysis of the fragments also indicated the presence of additional ether linkage between C2 and C7, present in A-Type proanthocyanidins, and were identified at m/z 559 [(epi)afzelechin-(epi)catechin], m/z 575 [(epi)catechin-(epi)catechin] or [(epi)afzelechin-(epi)gallocatechin] and at m/z 591, [(epi)catechin-(epi)gallocatechin]. CID-fragmentation was used for tannins sequencing, as well as 3D NMR HMQC-TOCSY and COSY, which provided fingerprint assignments for identification of cathechin at delta 4.55/82.1 (H-2/C-2), 3.96/68.1 (H-3/C-3) and 2.82-2.50/27.7 (H-4a-H-4b/C-4), and epicathechin delta 4.78/79.1 (H-2/C-2), 4.15/66.7 (H-3/C-3) and 2.82-2.73/28.5 (H-4a-H-4b/C-4). Since HMQC-TOCSY gives a high resolution heteronuclear connectivity, it is useful for identification of other cis-trans isomers present in complex flavonoid mixtures.

Analysis of Camellia sinensis green and black teas via ultra high performance liquid chromatography assisted by liquid-liquid partition and two-dimensional liquid chromatography (size exclusion × reversed phase).

Green and black teas (Camellia sinensis) contain compounds ranging from simple phenolics to complex glycosides, many of which have well-recognized health benefits. Here, we describe two methodologies aiming to achieve a comprehensive analysis of hydro-alcoholic extracts of C. sinensis. In the first step, the extracts were partitioned in water, n-butanol, ethyl acetate and chloroform to separate the compounds according to their polarity, yielding less complex samples to be analyzed by ultra high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). Additionally, a comprehensive two dimensional liquid chromatography (2D-LC) technique, employing size exclusion chromatography (SEC) × reversed phase (BEH-C18) was developed. The following compounds were identified on the basis of retention time, UV-spectra and MS fragmentation patterns: catechins, theaflavins and their gallate derivatives; kaempferol, quercetin and myricetin mono-, di-, tri- and tetraglycosides; esters of quinic acid and gallic or hydroxycinnamic acids; purine alkaloids, such as caffeine and theobromine and many lipids. Additionally, there were many novel compounds that were previously undescribed, such as saponin isomers and gallic acid esters of four glycosides of myricetin, quercetin and kaempferol.

Structure and degree of polymerisation of fructooligosaccharides present in roots and leaves of Stevia rebaudiana (Bert.) Bertoni

Fructooligosaccharides have been isolated from roots and leaves of Stevia rebaudiana, by hot aqueous extraction, followed by precipitation with ethanol. Their structure has been determined using methylation and NMR analysis, MALDI-TOF, and ESI-MS. Fructooligosaccharides contained almost exclusively (2→1)-linked β-fructofuranosyl, with terminal α-glucopyranosyl and β-fructofuranosyl units. MALDI-TOF and ESI-MS analyses showed the wide range of degree of polymerisation (DP) present in various extracts. From roots and leaves, three different fractions gave profiles of homologous series, with DPs ranging up to 17 with MALDI-TOF and 19 using ESI-MS. These inulin-type fructooligosaccharides were the major component of extracts from S. rebaudiana roots and significant components from the leaves.

Heart-cutting two-dimensional (size exclusion × reversed phase) liquid chromatography–mass spectrometry analysis of flavonol glycosides from leaves of Maytenus ilicifolia

Two-dimensional liquid chromatography-electrospray ionization mass spectrometry was employed to analyze flavonol glycosides present in leaves of Maytenus ilicifolia, frequently used in traditional Brazilian medicine. Since they contain many flavonol glycosides, including isomers, one-dimensional liquid chromatography did not give complete separation and identification, yielding overlapping of compounds with different molecular weights. Thus, employing size exclusion chromatography in the first and reversed phase in the second dimension, a great number of flavonol glycosides could be identified and its relative abundances determined. The majority of glycosides contained kaempferol or quercetin as aglycones, and glycosides with previously unreported structures were also present and characterized.

Glycolipids from macroalgae: potential biomolecules for marine biotechnology?

Brown, red and green algae from the Southeastern coast of Brazil were successively extracted with chloroform/methanol 2:1 and 1:2 (v/v). The crude lipid extract was partitioned according to Folch and the lower phase enriched in glycolipids was fractionated on a silica gel column chromatography eluted with chloroform, acetone and methanol. Three major orcinol-reactive bands present in the acetone and methanol fractions were detected by thin-layer chromatography with chromatographic mobilities corresponding to sulfoglycolipids and glycosyldiacylglycerols. These fractions exhibited potent antiviral activity against HSV-1-ACVs and HSV-1-ACVr and present low toxicity for cell cultures. Purification and identification of these bioactive glycolipids will be necessary in order to elucidate their primary structures and mechanism of action.

Ethanolic extract of roots from Arctium lappa L. accelerates the healing of acetic acid-induced gastric ulcer in rats: Involvement of the antioxidant system

We evaluate the curative efficacy of the ethanolic extract (EET) of roots from Arctium lappa (bardana) in healing of chronic gastric ulcers induced by 80% acetic acid in rats and additionally studies the possible mechanisms underlying this action. Oral administration of EET (1, 3, 10 and 30mg/kg) reduced the gastric lesion area in 29.2%, 41.4%, 59.3% and 38.5%, respectively, and at 10mg/kg promoted significant regeneration of the gastric mucosa, which was confirmed by proliferating cell nuclear antigen immunohistochemistry. EET (10mg/kg) treatment did not increase the gastric mucus content but restored the superoxide dismutase activity, prevented the reduction of glutathione levels, reduced lipid hydroperoxides levels, inhibited the myeloperoxidase activity and reduced the microvascular permeability. In addition, EET reduced the free radical generation and increased scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals in vitro. Furthermore, intraduodenal EET (10 and 30mg/kg) decreased volume and acidity of gastric secretion. Total phenolic compounds were high in EET (Folin-Ciocalteau assay) and the analysis by liquid chromatography-mass spectrometry revealed that the main compounds present in EET were a serie of hydroxycinnamoylquinic acid isomers. In conclusion, these data reveal that EET promotes regeneration of damaged gastric mucosa, probably through its antisecretory and antioxidative mechanisms.

Analysis of flavonol glycoside isomers from leaves of Maytenus ilicifolia by offline and online high performance liquid chromatography–electrospray mass spectrometry

Flavonol glycosides present in leaves of Maytenus ilicifolia, were examined after fractionation on silica-gel column. Flavonol mono-, di-, tri-, and tetraglycosides, containing kaempferol, quercetin or myricetin were identified by offline electrospray mass spectrometry. Increasing the cone energy induced to adducts variation, from H(+) to Na(+). Protonated ions were characteristically fragmented by sequentially removing the monosaccharide residues, whereas in the sodiated ions, the aglycone was firstly removed. Online high performance liquid chromatography-mass spectrometry, with simple gradients of water, acetonitrile and acetic acid indicated the presence of several isomers, which were further identified by gas chromatography-mass spectrometry as containing galactose or glucose.

Gastroprotective effect and structure of a rhamnogalacturonan from Acmella oleracea

The plant Acmella oleracea (L.) R.K.Jansen (Asteraceae), locally known as jambu, is widely used in Legal Amazon in local dishes and in folk medicine. A polysaccharide (SC) was isolated from this plant, following aqueous extraction, which contained uronic acid, galactose, arabinose, rhamnose, and glucose in a 15:2:1:1:0.5 molar ratio and had a M(w) 226,000 g/mol. Methylation analysis and NMR spectroscopy indicated that SC is a rhamnogalacturonan composed of a long chain of →4)-6-OMe-α-D-GalpA-(1→, interspersed with some α-L-Rhap residues, partly substituted by side-chains of type II arabinogalactans. SC significantly inhibited ethanol-induced gastric ulcers in rats with an ED₅₀ of 1.5 mg/kg, indicating that SC acts as gastroprotective agent.

Structural Characterization and Anti-HSV-1 and HSV-2 Activity of Glycolipids from the Marine Algae Osmundaria obtusiloba Isolated from Southeastern Brazilian Coast

Glycolipids were extracted from the red alga Osmundaria obtusiloba from Southeastern Brazilian coast. The acetone insoluble material was extracted with chloroform/methanol and the lipids, enriched in glycolipids, were fractionated on a silica gel column eluted with chloroform, acetone and then methanol. Three major orcinol-positive bands were found in the acetone and methanol fractions, being detected by thin layer chromatography. The structures of the corresponding glycolipids were elucidated by ESI-MS and 1H/13C NMR analysis, on the basis of their tandem-MS behavior and HSQC, TOCSY fingerprints. For the first time, the structure of sulfoquinovosyldiacylglycerol from the red alga Osmundaria obtusiloba was characterized. This molecule exhibited potent antiviral activity against HSV-1 and HSV-2 with EC50 values of 42 µg/mL to HSV-1 and 12 µg/mL to HSV-2, respectively. Two other glycolipids, mono- and digalactosyldiacylglycerol, were also found in the alga, being characterized by ESI-MS/MS. The structural elucidation of algae glycolipids is a first step for a better understanding of the relation between these structures and their biological activities.