Copper exposure leads to changes in chlorophyll content and secondary metabolite profile in Lantana fucata leaves.

Cultivation of plants in environments polluted by metals at toxic levels can affect the biosynthesis of secondary metabolites. Here, we analysed the effect caused by excess copper on the concentration of chlorophylls a and b and the profile of secondary metabolites of Lantana fucata leaves. Five copper (Cu) treatments (mg Cukg-1 soil) were tested: T0, 0; T1, 210; T2, 420; T3, 630; and T4, 840. We found that the concentrations of chlorophylls in the plants decreased when compared to the control. However, this did not lead to a significant reduction in its growth, possibly due to the low translocation of the metal to shoots and the activation of plant defence systems to tolerate the environment in which they are exposed, increasing the emission of lateral roots and activating pathways for the production of secondary metabolites. Therefore, we found a decrease in the concentration of two key compounds in secondary metabolism, p -coumaric and cinnamic acids in treatments with higher copper concentrations. We also found an increase in phenolics. Decreases in p -coumaric and cinnamic acids may have occurred because these are precursors in the synthesis of phenolic compounds, which are increased in the high Cu treatments. Six secondary metabolites were characterised, described for the first time for this plant species. Thus, the presence of excess Cu in the soil may have triggered an increase in the amount of reactive oxygen species in the plants, which that led to the synthesis of antioxidant compounds, as a defence strategy.

The chemical composition of different leaf extracts of Lantana fucata Lindl. influences its cytotoxic potential: A study using the Allium cepa model.

ETHNOPHARMACOLOGICAL RELEVANCE: One of the most popular plants used to treat diseases in Brazil is Lantana fucata. Like most herbal medicines, its consumption is based on popular knowledge, which, despite being considered effective, may cause side effects. AIM OF THE STUDY: Since the scientific data on the pharmacological properties of L. fucata are still incipient, this research aimed to evaluate the cytotoxic and genotoxic potential of different types of extracts (infusion, aqueous and hydroalcoholic), characterizing them chemically. MATERIALS AND METHODS: The cytotoxicity assay was performed by the A. cepa model. The cytotoxicity parameters studied were number of dividing cells and percentage mitotic index (%MI). RESULTS: The result of the A. cepa assay showed that there was a decrease in the number of dividing cells and the percentage mitotic index as concentrations increased, for all extracts, indicating cytotoxicity. However, the hydroalcoholic extract was the most cytotoxic. Chromatography analysis allowed the characterization of secondary metabolites in the extracts, which were very similar. However, a greater abundance of flavonoids and triterpenoids was observed in the hydroalcoholic extract, suggesting that these compounds are responsible for its greater toxicity. CONCLUSIONS: Since the highest doses of extracts showed to have a cytotoxic effect, it is suggested that the ingestion of this species occurs in a moderate way.

Acid-gastric antisecretory effect of the ethanolic extract from Arctium lappa L. root: role of H+, K+-ATPase, Ca2+ influx and the cholinergic pathway.

BACKGROUND: Arctium lappa L., popularly known as burdock, is a medicinal plant used worldwide. The antiulcer and gastric-acid antisecretory effects of ethanolic extract from roots of Arctium lappa (EET) were already demonstrated. However, the mechanism by which the extract reduces the gastric acid secretion remains unclear. Therefore, this study was designed to evaluate the antisecretory mode of action of EET. MATERIALS AND METHODS: The effects of EET on H+, K+-ATPase activity were verified in vitro, whereas the effects of the extract on cholinergic-, histaminergic- or gastrinergic-acid gastric stimulation were assessed in vivo on stimulated pylorus ligated rats. Moreover, ex vivo contractility studies on gastric muscle strips from rats were also employed. RESULTS: The incubation with EET (1000 µg/ml) partially inhibited H+, K+-ATPase activity, and the intraduodenal administration of EET (10 mg/kg) decreased the volume and acidity of gastric secretion stimulated by bethanechol, histamine, and pentagastrin. EET (100-1000 µg/ml) did not alter the gastric relaxation induced by histamine but decreased acetylcholine-induced contraction in gastric fundus strips. Interestingly, EET also reduced the increase in the gastric muscle tone induced by 40 mM KCl depolarizing solution, as well as the maximum contractile responses evoked by CaCl2 in Ca2+-free depolarizing solution, without impairing the effect of acetylcholine on fundus strips maintained in Ca2+ -free nutritive solution. CONCLUSION: Our results reinforce the gastric antisecretory properties of preparations obtained from Arctium lappa, and indicate that the mechanisms involved in EET antisecretory effects include a moderate reduction of the H+, K+-ATPase activity associated with inhibitory effects on calcium influx and of cholinergic pathways in the stomach muscle.

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.

Identification of a dicaffeoylquinic acid isomer from Arctium lappa with a potent anti-ulcer activity.

Leaves of Arctium lappa contain several mono- and dicaffeoylquinic acids, as evaluated by liquid chromatography-mass spectrometry. In order to investigate the protection on gastric mucosa against ulcers, rats were treated with fractions from leaf extract prior to ethanol-induced ulcers. The original fraction obtained as ethanol soluble fraction from hot aqueous extract was able to protect de gastric mucosa, and this effect was retained in the ethyl acetate fraction, obtained from liquid/liquid fractionation. The main compound in this fraction was isolated and chemically characterized by nuclear magnetic resonance and mass spectrometry, assisted by isopropylidene derivatization which gave rise a mass increment of 40 units. Therefore, the underivatized compound that had m/z 515.119 [M-H](-) was shifted to m/z 555.151, being confirmed as 1,3-O-dicaffeoylquinic acid, which presented an ED50 of 57 µg kg(-1) on gastric protection, lesser than the therapeutic concentration of omeprazole (40 mg kg(-1)).

Total Phenolic Contents and Antioxidant Activity in Oxidized Leaves of Mate (Ilex paraguariensis St. Hil)

Ilex paraguariensis is a native species to South America. Its economic importance is in the use of its leaves and twigs in the processing of a product popularly known as "chimarrão". The aim of this study was to evaluate the correlations between some physico-chemical process parameters in the oxidation of I. paraguariensis leaves oxidation. The leaves of Maté with age 1.0, 6.5 and 12.0 months were subjected to an oxidation process for 3 h under different relative humidity conditions (80, 90 and 99%) at 26°C. The extracts were evaluated using the experimental design techniques to maximize the oxidation conditions for the total polyphenols, antioxidant capacity, water activity and color. The condition of 90% humidity and leaf age 6.5 months resulted in lower lightness and yellow color of the product. Green color was not significantly influenced by the studied parameters. The highest levels of total phenols and antioxidant potential were found when the leaf age was one month and humidity was 90%. Thus, it could be concluded that the air humidity of the chamber and leaf age influenced the oxidation process of the leaves of Maté as well as the antioxidant capacity of the extract obtained.

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.

Chemical and biological characterization of polysaccharides isolated from Ilex paraguariensis A. St.-Hil.

The potential gastroprotection of polysaccharides (SP) isolated from maté (Ilex paraguariensis) leaves of different growth stages, under different sunlight conditions and of processing methods were evaluated. The SP consist of type I arabinogalactan (AG1) containing a (1→4)-linked ß-Galp chain, with substituents of arabinosyl units at O-6. This arabinogalactan seems to be attached to rhamnosyl units from a RG1, via 1→4 linkage. Oral administration of SP1, SP9, SP10, SP11 and SP12 inhibited the gastric lesions induced by ethanol in rats. Altogether, the present data indicate the therapeutic role of maté polysaccharides against gastric lesion and propose its use or of its crude plant extract as a phytotherapic medicine.

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.

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 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.

Analysis of Flavonoids from Eugenia uniflora Leaves and Its Protective Effect against Murine Sepsis.

Eugenia uniflora, referred to as Pitanga cherry shrub, is largely distributed in tropical and subtropical America. This plant is cultivated in many countries and it is suitable for the production of juice, frozen pulp, and tea. Besides, it can be used as treatment for inflammatory diseases. We reported that a flavonoid-rich fraction (HE-Bu) obtained from leaves decreased the lethality induced by cecal ligation and puncture (CLP), a clinically relevant model of sepsis. The oral administration of HE-Bu reduced the late mortality rate by 30%, prevented neutrophil accumulation in lungs, decreased TNF-α and IL-1ß serum levels, and markedly decreased iNOS and COX-2 protein expression by ileum cells. Chemical investigation showed myricetin and quercetin rhamnosides as the major components of this fraction. The results showed that HE-Bu protected mice from sepsis and indicated that this edible plant produces compounds that could be considered as potential adjuvants for sepsis treatment.

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.

Assessment of oxidation of leaves of Ilex paraguariensis (St. Hil)

This work aimed to study the oxidation of leaves of Ilex paraguariensis St. Hil, in a process similar to the oxidation of Camellia sinensis, with the purpose of developing new products based on yerba maté. So far, no previous systematic study about the oxidation of yerba maté has been found elsewhere. A Plackett-Burman experimental design was used for the screening of the significant parameters that affected the oxidation. The oxidation was indirectly quantified by following the changes in the product color, water activity, and pH with the time of processing. The factors that most influenced the process were the age of the leaves and the relative humidity of the oxidation chamber. The differences between the treatments were more evident after 3, 6 12 and 24 h of oxidation. The results could contribute to the developing of new products based on I. paraguariensis St. Hil, increasing the profitability of cultivation of this native species.

Standardized extract of Dicksonia sellowiana Presl. Hook (Dicksoniaceae) decreases oxidative damage in cultured endothelial cells and in rats.

AIMS: Aging and a variety of pathologies, including cancer, diabetes, cardiovascular and inflammatory diseases have been associated with reactive oxygen species (ROS), such as superoxide anion (O2·â»), hydroxyl radical (·OH) and hydrogen peroxide (H2O2) generation. Plant polyphenols bear radical scavenging/antioxidant activity. A phytomedicinal preparation obtained from aerial parts of Dicksonia sellowiana (Dicksoniaceae), a native plant from Central and South America, has been widely used in Brazil against asthma and presents beneficial effects in several other diseases, including cardiovascular disturbance. In this work, we investigated whether Dicksonia sellowiana, which is also known to contain high levels of polyphenols, presents antioxidant activity. METHODS: The antioxidant activity of the hydroalcoholic extract obtained from Dicksonia sellowiana leaves (HEDS) was investigated by in vitro and in vivo tests. RESULTS: HEDS (0.1-100 µg/mL) exhibited a strong scavenging activity against all reactive species tested (DPPH, O2·â»,·OH and H2O2; IC50=6.83±2.05, 11.6±5.4, 2.03±0.4, and 4.8±0.4 µg/mL, respectively). HEDS strongly protected endothelial cells against H2O2-induced oxidative stress by mechanisms other than increasing catalase activity. In addition, HEDS protected cell membrane from oxidative damage. HEDS, (20 and 40 mg/kg) inhibited lipid peroxidation in vivo (29.8% and 24.5%, respectively). CONCLUSIONS: According to our results, we can speculate that the traditional uses of Dicksonia sellowiana for cardiovascular diseases, asthma and skin diseases could be, at least in part, related to the potent antioxidant and endothelial protective activities of the plant.