Differentiation of flavonol glucoside and galactoside isomers combining chemical isopropylidenation with liquid chromatography-mass spectrometry analysis.

Flavonol glycosides are important components of leaves from vascular plants. A lot of isomers of these compounds are produced by plants, making their analysis very difficult and causing many structural misinterpretations. Galactosides and glucosides as mono- or oligosaccharides yield many diastereoisomers, hindering the analysis by mass spectrometry. In order to enable the mass spectrometric distinctions of these isomers, in this work we combine an isopropylidene based chemical derivatization with liquid chromatography with multiple-stage mass spectrometry (LC-MS(n)) analysis. The isomers of flavonol triglycosides, after the reaction, yielded products with different molecular weight, therefore, they were no longer isomers, allowing their identification by MS(1) analysis. However, to the 4 isomers of flavonol diglycosides, only one yielded, after isopropylidenation, a product with different molecular weight. To the other 3 species, the incorporation of 2 isopropylidene groups retained them in the isomeric form. For such species, chromatographic separation and MS(n) detection targeting the lithium adducts of 3,4-O-isopropylidene-galactosyl or 4,6-O-isopropylidene-glucosyl residues (m/z 209.099) provided specific MS profile.

Chemical characterization of heteropolysaccharides from green and black teas (Camellia sinensis) and their anti-ulcer effect.

In order to obtain polysaccharides from green and black teas (Camellia sinensis), commercial leaves were submitted to infusion and then to alkaline extraction. The extracts were fractionated by freeze-thawing process, giving insoluble and soluble fractions. Complex arabinogalactan protein from the soluble fractions of both teas (GTPS and BTPS) were determined by methylation analysis and (1)H/(13)C-HSQC spectroscopy, showing a main chain of (1→3)-ß-Galp, substituted at O-6 by (1→6)-linked ß-Galp with side chains of α-Araf and terminal units of α-Araf, α-Fucp and α-Rhap. A highly branched heteroxylan from the insoluble fractions (GTPI and BTPI) showed in methylation analysis and (1)H/(13)C-HSQC spectroscopy the main chain of (1→4)-ß-Xylp, substituted in O-3 by α-Araf, ß-Galp and α-Glcp units. Evaluating their gastroprotective activity, the fractions containing the soluble heteropolysaccharides from green (GTPS) and black teas (BTPS) reduced the gastric lesions induced by ethanol. Furthermore, the fraction of insoluble heteropolysaccharides of green (GTPI) and black (BTPI) teas also protected the gastric mucosa. In addition, the maintenance of gastric mucus and reduced glutathione (GSH) levels was involved in the polysaccharides gastroprotection.

Gastroprotective bio-guiding fractionation of hydro-alcoholic extracts from green- and black-teas (Camellia sinensis).

Hydro-alcoholic extracts from leaves of Camellia sinensis (green- and black-tea leaves) were submitted to a fractionation, promoting the compound separation according to their polarity, and analyzed by ultra-high performance liquid chromatography-mass spectrometry. A wide range of compounds could be identified, such as catechins and their gallate (esters) or oxidation derivatives (theaflavins), glycosylated flavonoids and other phenolics, as well as lipids, saponins and alkaloids. Also have been developed, via bio-guided examination, the gastroprotective property of the compounds identified. The samples were assayed using the model of acute gastric lesions induced in rats by ethanol. Hydro-alcoholic extracts of green-tea and black-tea protected the gastric mucosa with ED50=3.6 and 10.2mg/kg, respectively, with participation of gastric mucus and reduced glutathione (GSH). The ethyl acetate fraction from green-tea and aqueous fraction from black-tea were, respectively, 6 and 10 times more effectiveness than the initial extracts. Moreover, the epigallocatechin gallate (EGCG, 0.204mg/kg), a main component of ethyl acetate fraction from green tea, reduced the gastric lesion by 56% and restored the mucus levels, however the rutin (0.0133mg/kg), a flavonoid found in the most active fraction of black-tea, was less significant at the natural concentrations. These results have confirmed that the different compounds present in green- and black-tea hydro-alcoholic extracts and partitioned fractions produce relevant gastroprotection mainly via maintenance of the protective factors, mucus and GSH.

Rhamnogalacturonan from Ilex paraguariensis: a potential adjuvant in sepsis treatment.

The present study evaluated the anti-inflammatory activity of a polysaccharide from maté, using a clinically relevant model of sepsis induced by cecal ligation and puncture (CLP). A polysaccharide from maté (SPI) was obtained from aqueous extraction followed by fractionation, being identified as a rhamnogalacturonan with a main chain of →4)-6-OMe-α-D-GalpA-(1→ groups, interrupted by α-L-Rhap units, substituted by a type I arabinogalactan. SPI was tested against induced-polymicrobial sepsis, at doses of 3, 7 and 10 mg/kg. Via oral administration, SPI prevented the late mortality of infected mice by a rate of 60% at 10 mg/kg, in comparison with untreated mice Dexamethasone, used as positive control, was slightly less effective, with an overall survival rate of 16.7% of mice at the end of the observation period. SPI also affected neutrophil influx, avoiding its accumulation in lungs, and significantly decreased tissue expression of iNOS and COX-2. In this context, maté is a potential nutraceutical, and its polysaccharide a promising adjuvant for sepsis treatment, being consumed as tea-like beverages with no related adverse effects.

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.

Comprehensive analysis of maté (Ilex paraguariensis) compounds: development of chemical strategies for matesaponin analysis by mass spectrometry.

Ilex paraguariensis (maté) is an important plant from southern Brazil, Paraguay, Uruguay and Argentina, where its leaves are widely used in hot or cold beverages. We now present a comprehensive ULPC-PDA-MS analysis of the compounds found in its leaves. The analysis was rapid, less than 13 min and many compounds were identified, among these, the chlorogenic acid series, and other quinic acid derivatives, xanthines, and several saponins, many of which have not been previously described, including many isomers. The isomerism could occur in the aglycone moiety, namely ursolic or oleanolic acid, or in the carbohydrate moiety. Additionally, many acetylated saponins were detected. The structure of known and novel saponins was determined using per-O-methylation with ESI-MS analysis, as well as with GC-MS of their partially O-methylated alditol acetate (PMAA) derivatives, along with ESI-MS analysis of the O-isopropylidene (IPP) derivatives provided fundamental information on interglycosidic linkages, avoiding the purification steps.