Ilex paraguariensis: the effect of genotypes and growth phase on biomass, secondary metabolism and antioxidant activity of in vitro cultivated calli / Ilex paraguariensis: el efecto de los genotipos y la fase de crecimiento sobre la biomasa, metabolismo secundario y la actividad antioxidante de los callos cultivados in vitro

Yerba mate ( Ilex paraguariensis ) produces several secondary metabolites of interest to the phar maceutical industry, such as chlorogenic acids and methylxanthines. These compounds have been produced in vitro by callus culture from different species. However, for I. paraguariensis , no studies upon the production of these compounds in vitro have been p erformed to date. In this work, we show that the concentration of secondary metabolites from I. paraguariensis callus is possible and highly dependent on the callus growth phase. We observed that the best phase for the production of secondary compounds in calli of yerba mate is the stationary growth phase on both genotypes tested. In this phase, higher levels of phenolic compounds, chlorogenic acid and 3,5 - dicaffeoylquinic acid and greater antioxidant activity were observed. Chlorogenic acid and 3,5 - dicaffe oylquinic acid presented positive correlation with antioxidant activity. For the first time, secondary compounds were reported in yerba mate calli cultivated in vitro .

La yerba mate ( Ilex paraguariensis ) produce varios metabolitos secundarios de interés para la industria farmacéutica, como los ácidos clorogénicos y las metilxantinas. Estos compuestos se han producido in vitro mediante cultivo de ca llos de diferentes especies. Sin embargo, para I. paraguariensis , hasta la fecha no se han realizado estudios sobre la producción de estos compuestos in vitro . En este trabajo, mostramos que la concentración de metabolitos secundarios desde callos de I. pa raguariensis es posible y altamente dependiente de la fase de crecimiento del callo. Observamos que la mejor fase para la producción de compuestos secundarios en callos de yerba mate es la fase de crecimiento estacionario en ambos genotipos probados. En es ta fase se observaron niveles más altos de compuestos fenólicos, ácido clorogénico y ácido 3,5 - dicafeoilquínico y una mayor actividad antioxidante. El ácido clorogénico y el ácido 3,5 - dicafeoilquínico presentaron correlación positiva con la actividad antio xidante. Por primera vez, se reportaron compuestos secundarios en callos de yerba mate cultivados in vitro .

Modified graphene-silica as a sorbent for in-tube solid-phase microextraction coupled to liquid chromatography-tandem mass spectrometry. Determination of xanthines in coffee beverages.

Given the increasing need for analyzing natural or contaminating compounds in complex food matrices in a simple and automated way, coupling miniaturized sample preparation techniques with chromatographic systems have become a growing field of research. In this regard, given the low extraction efficiency of conventional sorbent phases, the development of materials with enhanced extraction capabilities is of particular interest. Here we present several synthesized graphene-based materials supported on aminopropyl silica as sorbents for the extraction of xanthines. The synthesized materials were characterized by infrared spectroscopy and scanning electron microscopy. Aminopropyl silica coated with graphene oxide and functionalized with octadecylsilane/end-capped (SiGOC18ecap) showed the best performance for xanthines extraction. Hence, this material was employed as an in-tube solid phase microextraction (in-tube SPME) device coupled online with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and applied for the analysis of xanthines in roasted coffee samples. Extraction parameters and detection conditions were optimized. The method showed low limits of quantification (0.3-1.0 µg L-1), precision as relative standard deviation (RSD) values lower than 10%, recoveries between 73 and 109%, and pre-concentration factors from 5.6 to 7.2. Caffeine was determined in all ground roasted and instant coffee samples, in a wide range (0.9 to 36.8 mg g-1), and small amounts of theobromine and theophylline were also detected in some samples. This work demonstrated that functionalized graphene-based materials represent a promising new sorbent class for in-tube SPME, showing improved extraction capacity. The method was efficient, simple, and fast for the analysis of xanthines, demonstrating an excellent potential to be applied in other matrices.

Mass spectrometry imaging of thin-layer chromatography plates using laser desorption/low-temperature plasma ionisation.

Thin-layer chromatography (TLC) is a classic method for the separation and analysis of complex mixtures. Biological assays, chemical derivatisation and spectroscopy techniques are compatible with TLC and provide extra information about isolated compounds. However, coupling TLC to mass spectrometry is hampered by the difficulty to desorb the analytes from the silica surfaces. In this study, we used a multimodal ion source for laser desorption (LD) and low-temperature plasma (LTP) post-ionisation. Efficient desorption was reached by covering the TLC plates with activated carbon. Regions of interest can be analysed by spots, by lines or by area. We show the separation of methylxanthines from coffee, tea and cocoa preparations by TLC, with subsequent mass spectrometry imaging (MSI). Using a lateral resolution of 400 µm × 400 µm, allowed the acquisition of 21 895 spectra in 2.4 h (2.5 pixels per s). Further, we demonstrate the possibility of direct mass fragmentation studies and quantification. We mounted the system on an Open LabBot with a theoretical lateral resolution of 12.5 µm and performed the visualisation of ions of interest and the pixel-wise review of mass spectra with our free software RmsiGUI (). This non-proprietary and modular platform enables the cost-efficient adaption of the system and further development by the community.

Multivariate analysis of the composition of bioactive in tea of the species Camellia sinensis.

This study deals with the development of a separation and quantification method by high performance liquid chromatography with a diode array detector (HPLC- DAD), for the determination of phenolic acids and methylxanthines in tea (Camellia Sinensis) samples. Six phenolic acids (Gallic, trans-cinnamic, caffeic, ferulic and p-coumaric acids) and two methylxanthines (caffeine, theobromine) were studied, according to the principles of green chemistry, with the goal of comparing teas with different processing level. After development and validation of separation and extraction methods was applied to 11 extracts obtained by infusions of green and black tea samples in commercial areas of the city of Salvador-BA, showing feasible and efficient. Was employed multivariate data analysis and the Tukey test to correlate the chromatographic profile with the different samples. Principal component and analysis and hierarchical cluster analysis were used for the chromatographic analysis, allowing the visualization of two groups, formed by green and black tea samples.

Pressurized liquid extraction of flavanols and alkaloids from cocoa bean shell using ethanol as solvent.

Cocoa shell (CS) is a co-product of the cocoa industry used mainly as fuel for boilers but with secondary applications as fertilizer and in animal feed. Although it is known that this material is rich in flavanols and alkaloids, to date, a study has not been conducted that has quantitatively identified these compounds in CS. Thus, the aim of this work was to characterize CS in terms of its composition, regarding catechin, epicatechin, procyanidin B2, caffeine and theobromine, and to evaluate the extraction kinetics of the total flavanols using pressurized liquid extraction (PLE) with absolute ethanol. For the determination of the extraction kinetic data, the DMAC method was used, while each compound was quantified using a UPLC-MS/MS analysis. The major compounds found were theobromine and epicatechin (mean values of 9.89 and 3.5 mg/g CS, respectively). PLE proved to be quite effective; the flavanols extraction yield was enhanced by increasing the temperature and extraction time however, high extraction times and temperatures degraded the procyanidins B2. Peleg's model applied to extraction data description provided a reasonable agreement with the experimental results, which allows their application in modeling and optimization of solid-liquid extraction of the total flavanols from cocoa bean shell.

Characterization of secondary metabolites from green cocoa beans using focusing-modulated comprehensive two-dimensional liquid chromatography coupled to tandem mass spectrometry.

Proanthocyanidins as well as other secondary metabolites present in green cocoa beans were studied thanks to a new method involving the use of on-line comprehensive two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC × LC-MS/MS). In order to enhance the performance of previously developed methodologies, the use of different modulation strategies were explored. Focusing modulation clearly allowed the attainment of higher resolving power and peak capacity compared to non-focusing modulation set-ups. Moreover, the use of active modulation by the addition of a make-up flow efficiently helped to compensate for the solvent strength mismatch produced between dimensions. The optimized method was useful to successfully describe the secondary metabolite composition of green cocoa that was characterized by the presence of 30 main compounds, including 3 xanthines, 2 flavan-3-ols and 24 oligomeric procyanidins with a degree of polymerization up to 12. The obtained results showed that the proanthocyanidins found in the cocoa beans were exclusively B-type procyanidins. The existence of (epi)catechin subunits linked to sugar or galloyl moieties was not observed. The developed method produced a good separation of secondary metabolites allowing an improvement with respect to the available methodologies for the analysis of a complex food sample such as cocoa metabolites in terms of speed of analysis, resolution and peak capacity.

Ilex paraguariensis: Potential antioxidant on aluminium toxicity, in an experimental model of Alzheimer's disease.

Ilex paraguariensis, yerba mate is a native plant from the southern region of Brazil. Studies showed that yerba mate has an antioxidant potential, which could help to reduce the risk of developing neurodegenerative diseases, as Alzheimer's Disease (AD). It's known that I. paraguariensis grows in acid soils with aluminium (Al), which is bioavailable in these soils. Al has a neurotoxic potential related with the progression of neurological disorders. This study aim was to evaluate the potential of I. paraguariensis in the etiology of AD using strains of Caenorhabditis elegans and the concentration of Al and antioxidants in the yerba mate extract. The results of the I. paraguariensis infusions made at 65°C and at 75° C show that there was no significant difference between both temperatures when preparing the tea infusion in relation to the presence of Al, methylxanthines, phenolic compounds and flavonoids. Additionally, in the case of Al, there was no difference between the extracts prepared at both temperatures. The behavioral parameters of C. elegans were altered after a long-term exposure to both factors: I. paraguariensis extract and Al. Through the antioxidant levels results along with the Al content on the Acetylcholinesterase (AChE) activity it is possible to observe that the acute and chronic exposure to Al and I. paraguariensis leaves extract are very similar to wild-type worms. Moreover, we can observe that the results in both the transgenic strains long-term exposed to I. paraguariensis leaves extract and to the Al concentrations presented an increase in the AChE activity.

Daily Intake of Chlorogenic Acids from Consumption of Maté (Ilex paraguariensis A.St.-Hil.) Traditional Beverages.

The main objective of this study was to investigate the daily intake of chlorogenic acids (CGAs) and methylxanthines by consumers of maté traditional beverages (MTBs), terere and chimarrão (Ilex paraguariensis A.St.-Hill). In the studied population (450 citizens from Toledo, PR, Brazil), 63% consume the chimarrão and 37% terere, with weighted mean daily intakes estimated at 648-2160 and 244-746 mL, respectively. For every 100 mL of beverage consumed, the average amount of total phenol compounds extracted was 673.6 mg for chimarrão and 1184.9 mg for terere. Regarding CGAs composition, caffeoylquinic acids (CQAs) constitute about 38.4% for chimarrão and 55.3% for terere, and dicaffeoylquinic acids (diCQAs) represent 61.6 and 44.7% of the extracted compounds, respectively. The daily intake of phenolic compounds by MTB consumers was estimated for chimarrão (512.5-1708.5 mg/day) and terere (583.0-1779.7 mg/day). These results allow us to conclude that MTBs are important dietary sources of CGAs, mainly CQAs and di-CQAs.

Genetic and Phytochemical Analysis to Evaluate the Diversity and Relationships of Mate (Ilex paraguariensis A.St.-Hil.) Elite Genetic Resources in a Germplasm Collection.

The aim of this study was to evaluate the phytochemical and genetic diversity, relationships and identification of mate (Ilex paraguariensis A.St.-Hil.) elite genetic resources belonging to the Brazilian germplasm collection and mate breeding program. Mate has been studied due to the presence of phytochemical compounds, especially methylxanthines and phenolic compounds. The samples were collected from the leaves of 76 mate elite genetic resources (16 progenies × 5 localities). Total DNA was extracted from mate leaves and 20 random primers were used for DNA amplification. Methylxanthines (caffeine and theobromine) and phenolic compounds (chlorogenic, neochlorogenic, and criptochlorogenic acids) were quantified by HPLC. The genetic divergence estimated was higher within (92%) than among (8%) the different populations. Analysis of genetic distance between origins provided the formation of two groups by UPGMA cluster analysis, with higher polymorphism (94.9%). The average content of caffeine ranged from 0.01 to 1.38% and theobromine of 0.10 - 0.85% (w/w). The caffeoylquinic acids concentrations (1.43 - 5.38%) showed a gradient 3-CQA > 5-CQA > 4-CQA. The coefficient of genetic variation (CVg) was of low magnitude for all mono-caffeoylquinics acids. Significant correlations (positive and negative) were observed between the phytochemical compounds. Genetic diversity analysis performed by RAPD markers showed a greater intra-populational diversity; genetic resources with low caffeine and higher theobromine content were identified and can be used in breeding programs; the correlation between methylxanthines and phenolic compounds can be used as a good predictor in future studies.

Cocoa content influences chocolate molecular profile investigated by MALDI-TOF mass spectrometry.

BACKGROUND: Chocolate authentication is a key aspect of quality control and safety. Matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) mass spectrometry (MS) has been demonstrated to be useful for molecular profiling of cells, tissues, and even food. The present study evaluated if MALDI-TOF MS analysis on low molecular mass profile may classify chocolate samples according to the cocoa content. RESULTS: The molecular profiles of seven processed commercial chocolate samples were compared by using MALDI-TOF MS. Some ions detected exclusively in chocolate samples corresponded to the metabolites of cocoa or other constituents. This method showed the presence of three distinct clusters according to confectionery and sensorial features of the chocolates and was used to establish a mass spectra database. Also, novel chocolate samples were evaluated in order to check the validity of the method and to challenge the database created with the mass spectra of the primary samples. Thus, the method was shown to be reliable for clustering unknown samples into the main chocolate categories. CONCLUSION: Simple sample preparation of the MALDI-TOF MS approach described will allow the surveillance and monitoring of constituents during the molecular profiling of chocolates.

Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

Diversity of cacao trees in Waslala, Nicaragua: associations between genotype spectra, product quality and yield potential.

The sensory quality and the contents of quality-determining chemical compounds in unfermented and fermented cocoa from 100 cacao trees (individual genotypes) representing groups of nine genotype spectra (GG), grown at smallholder plantings in the municipality of Waslala, Nicaragua, were evaluated for two successive harvest periods. Cocoa samples were fermented using a technique mimicking recommended on-farm practices. The sensory cocoa quality was assessed by experienced tasters, and seven major chemical taste compounds were quantified by near infrared spectrometry (NIRS). The association of the nine, partially admixed, genotype spectra with the analytical and sensory quality parameters was tested. The individual parameters were analyzed as a function of the factors GG and harvest (including the date of fermentation), individual trees within a single GG were used as replications. In fermented cocoa, significant GG-specific differences were observed for methylxanthines, theobromine-to-caffeine (T/C) ratio, total fat, procyanidin B5 and epicatechin, as well as the sensory attributes global score, astringency, and dry fruit aroma, but differences related to harvest were also apparent. The potential cocoa yield was also highly determined by the individual GG, although there was significant tree-to-tree variation within every single GG. Non-fermented samples showed large harvest-to-harvest variation of their chemical composition, while differences between GG were insignificant. These results suggest that selection by the genetic background, represented here by groups of partially admixed genotype spectra, would be a useful strategy toward enhancing quality and yield of cocoa in Nicaragua. Selection by the GG within the local, genetically segregating populations of seed-propagated cacao, followed by clonal propagation of best-performing individuals of the selected GG could be a viable alternative to traditional propagation of cacao by seed from open pollination. Fast and gentle air-drying of the fermented beans and their permanent dry storage were an efficient and comparatively easy precondition for high cocoa quality.

Influence of the traditional Brazilian drink Ilex paraguariensis tea on glucose homeostasis.

In this study we examined the acute in vivo effect and short- and long-term in vitro effects of samples from native and commercial Ilex paraguariensis on glucose homeostasis. Also, the potential effect of I. paraguariensis on serum insulin secretion was investigated. The chemical identification and quantification of methyl xanthines and polyphenols in CH2Cl2, EtOAc and n-BuOH fractions of native I. paraguariensis as well as infusions of green and roasted I. paraguariensis from a commercial source was verified by high-performance liquid chromatography. The results for the serum glucose-lowering indicated that both fractions and both infusions were able to improve significantly the oral glucose tolerance curve. Additionally, both the EtOAc and n-BuOH fractions induced-insulin secretion, but EtOAc induced an early (at 15 min) and late (at 60 min) biphasic peak of insulin secretion similar to glipizide stimulatory effect. Both fractions increased liver glycogen content compared with fasted normal rats. Also, EtOAc and n-BuOH fractions inhibited in vitro disaccharidases activities after an acute treatment. The maximum inhibitory effect of the EtOAc and n-BuOH fractions on maltase activity (at 5 min) was around 35%. The evident reduction of protein glycation by glucose or fructose with EtOAc and n-BuOH fractions increased from 7 to 28 days of in vitro incubation. Inhibition of bovine serum albumin glycation by glucose and fructose, by around 50% and 90%, respectively, was observed. Additionally, the green and roasted mate infusions reduced the formation of AGEs in a characteristic long-term effect. In conclusion, this study shows that I. paraguariensis has an anti-hyperglycemic potential role able to improve the diabetic status and is probably a source of multiple hypoglycemic compounds.

Chemical and nutritional composition of tejate, a traditional maize and cacao beverage from the Central Valleys of Oaxaca, Mexico.

Foam-topped cacao and maize beverages have a long history in Mesoamerica. Tejate is such a beverage found primarily in the Zapotec region of the Central Valleys of Oaxaca, Mexico. Historically tejate has been ceremonially important but also as an essential staple, especially during periods of hard fieldwork. However, the nutritional contribution of traditional foods such as tejate has not been investigated. We analyzed tejate samples from three Central Valley communities, vendors in urban Oaxaca markets and one migrant vendor in California, USA for their proximate composition, amino acid content and scores, and mineral and methylxanthine content. Nutritional and chemical variation exists among tejate recipes, however, the beverage is a source of energy, fat, methylxanthines, K, Fe and other minerals although their availability due to presence of phytates remains to be determined. Tejate is a source of protein comparable to an equal serving size of tortillas, with protein quality similarly limited in both. Tejate provides the nutritional benefits of maize, and some additional ones, in a form appealing during hot periods of intense work, and year round because of its cultural significance. Its substitution by sodas and other high glycemic beverages may have negative nutritional, health and cultural consequences.

Phenolic acids and methylxanthines composition and antioxidant properties of mate (Ilex paraguariensis) residue.

UNLABELLED: Ilex paraguariensis is known to contain compounds with antioxidant properties, such as phenolic acids, and its stimulant properties are attributed to methylxanthines, such as caffeine. The aims of this study were to evaluate the phenolic, methylxanthinic, and tannin composition of a mate residue (mate powder), to compare the quali-quantitative phenolic composition and the antioxidant potential of extracts obtained from distinct solvent systems. Among the extracts prepared with different solvents, the 80% methanol extract showed the highest total polyphenol content (11.51 g/100 g) and antioxidant activity. HPLC analysis showed that 4,5 dicaffeoylquinic acid is the major component of the phenolic fraction of mate powder. The caffeine, theobromine, and tannin contents in mate powder were 1.01, 0.10, and 0.29 g/100 g, respectively. Consumption of mate powder would significantly contribute to antioxidant and stimulant intake, providing high amounts of phenolic acids, tannins, and methylxanthines with biological effects potentially beneficial for human health. PRACTICAL APPLICATION: This article contributes to the minimization of residues in yerba-mate processing.

Methylxanthines and phenolics content extracted during the consumption of mate (Ilex paraguariensis St. Hil) beverages.

"Chimarrao" and "terere" are popular beverages consumed in South America prepared using mate (Ilex paraguariensis St. Hil.). "Chimarrao" consists of a partial infusion where hot water is added, while "terere" is a total infusion, with addition of cold water. This study was designed to simulate preparation of these beverages for consumption, in order to estimate the total amount of xanthines and phenolic compounds in aqueous extracts that would be ingested by the consumer. Different commercial types of mate were employed for "chimarrao" preparation (native, smooth, traditional, and course-ground), and these were compared to "terere". In "chimarrao", beverages from coarse-ground mate showed the highest levels of xanthines. However, "terere" presented quantities 2.5 times higher than the beverage of the coarse-ground mate. Considering the total phenolics in "chimarrao", there was no difference between the types of herbs, but in "terere", the extraction of almost all of the phenolics was observed.

Consumption of yerba mate ( Ilex paraguariensis ) improves serum lipid parameters in healthy dyslipidemic subjects and provides an additional LDL-cholesterol reduction in individuals on statin therapy.

The objective of this study was to verify the effect of yerba mate (Ilex paraguariensis) consumption on lipid and lipoprotein levels in humans. One hundred and two individuals participated of this single-blind controlled trial. Normolipidemic (n = 15), dyslipidemic (n = 57), and hypercholesterolemic subjects on long-term statin therapy (n = 30) ingested 330 mL, 3 times/day, of green or roasted yerba mate infusions for 40 days. In normolipidemic subjects, yerba mate consumption reduced LDL-cholesterol by 8.7% (p < 0.05). Compared with the baseline period, yerba mate intake by dyslipidemic individuals for 20 and 40 days lowered LDL-cholesterol by 8.1 and 8.6% (p < 0.001) and non-HDL cholesterol by 5.4 and 6.5% (p < 0.01). After 20 days of yerba mate intake, apolipoprotein B was reduced by 6.0% (p < 0.05) and HDL-cholesterol was increased by 4.4% (p < 0.01). In all participants triglyceride levels remained unchanged. The consumption of yerba mate by hypercholesterolemic individuals on statin therapy promoted additional 10.0 and 13.1% reductions in LDL-C after 20 and 40 days, respectively (p < 0.001) and increased HDL-cholesterol by 6.2% after 40 days (p < 0.05). It was thus concluded that intake of yerba mate infusion improved the lipid parameters in normolipidemic and dyslipidemic subjects and provided an additional LDL-cholesterol reduction in hypercholesterolemic subjects on statin treatment, which may reduce the risk for cardiovascular diseases.

Quantitation of methylxanthinic alkaloids and phenolic compounds in mate (Ilex paraguariensis) and their effects on blood vessel formation in chick embryos.

Methylxanthinic alkaloids and phenolic compounds are related to the therapeutic properties of Ilex paraguariensis infusions. Considering the known vascular tropism of xanthines, an aqueous extract (mate) and caffeine were evaluated on blood vessel formation, in connection with the analysis of those secondary metabolites, which was performed in young and mature leaf samples collected in three cultivation systems located in the southern region in Brazil (Santa Catarina State). Samples of young and mature leaves from a monoculture cultivation system (MC) showed the highest content of phenolic compounds (149.68 microg/mL, young leaves; 135.50 microg/mL, mature leaves) and caffeine (young leaves, 148.07 microg/mL; mature leaves, 244.63 microg/mL) as compared to samples from agroforesty (AF) and shaded-native (NT) cultures. Theophylline was not detected in samples by reverse phase-high performance liquid chromatography, and mature leaves showed lower theobromine amounts (11.46 microg/mL). Treatments performed with mate aqueous extract and caffeine (1.03-4.12 microM/disk) in the yolk sac vascular membranes of 2-day-old chick embryos revealed pro-vasculo- and angiogenic properties as well as embryonic growth enhancement. These findings, uncoupled from any detectable embryotoxic effect, suggest a potential therapeutic and/or prophylactic use in cardiovascular disorders for caffeine and related constituents of mate plant extracts, an issue that waits further studies.

Determination of xanthines by high-performance liquid chromatography and thin-layer chromatography in horse urine after ingestion of Guaraná powder.

The seeds of Guaraná are rich in xanthines and are used for the preparation of guaraná powder which is very commonly given to horses as a 'tonic' in Brazil. In this paper, the xanthine content of guaraná powder was determined, in addition to its clearance time in horses. Thin-layer chromatography was used as a screening procedure and high-performance liquid chromatography was performed to quantify the drugs in both the powder and urine samples. The guaraná powder was found to contain 2.16, 1.10 and 36.78 mg g-1 of theobromine (TB), theophylline (TP) and caffeine (CF), respectively, and in urine it was possible to detect TB and TP up to 13 d and CF up to 9 d after the administration of guaraná powder.

Ritualistic use of the holly Ilex guayusa by Amazonian Jívaro Indians.

In Amazonian Peru and Ecuador leaf decoctions of the rainforest holly Ilex guayusa with high caffeine concentrations are used as a morning stimulant. After daily ingestion, ritualistic vomiting by male Achuar Indians, better known as Jívaros, reduces excessive caffeine intake, so that blood levels of caffeine and biotransformed dimethylxanthines do not cause undesirable CNS and other effects. Emesis is learned and apparently not due to emetic compounds.