Transfer of Caffeine and Its Major Metabolites to Chicken Eggs.

This study aimed to determine whether the eggs of laying hens fed caffeine contain this compound and its primary metabolites (theophylline, theobromine, and paraxanthine). Laying hens were distributed into four experimental groups fed rations containing 0 (control), 150, 300, or 450 µg/g of caffeine. For residual analysis, six eggs per group were collected after 4, 8, and 12 weeks. The concentrations of caffeine, theophylline, theobromine, and paraxanthine were determined in the white and yolk of each egg by a high-performance liquid chromatography with photodiode array detector (HPLC-PDA) method. All four compounds were detected in the white and yolk of eggs produced by hens fed caffeine, but their levels in the egg white were approximately twice those in the yolk. The major metabolite found in eggs was theophylline (57.5% of caffeine metabolites in the egg white and 58.5% in the yolk), followed by theobromine (39.9% in the egg white and 41.5% in the yolk), and paraxanthine (2.64% in the egg white and non-detected in the yolk). In summary, caffeine and its metabolites, theophylline, theobromine, and paraxanthine, are transferred to the chicken eggs.

Biological Properties and Antimicrobial Potential of Cocoa and Its Effects on Systemic and Oral Health.

Cocoa is considered a functional food because it is a natural source of macro- and micronutrients. Thus, cocoa is rich in vitamins, minerals, fiber, fatty acids, methylxanthines and flavonoids. In addition to favoring the metabolism of lipids and carbohydrates, the bioactive components of cocoa can have an antioxidant, anti-inflammatory and antimicrobial effect, providing numerous benefits for health. This literature review presents an overview of the effects of cocoa, fruit of the Theobroma cacao tree, on systemic and oral health. Several studies report that cocoa intake may contribute to the prevention of cardiovascular, neurodegenerative, immunological, inflammatory, metabolic and bone diseases, in addition to reducing the risk of vascular alterations and cognitive dysfunctions. On oral health, in vitro studies have shown that cocoa extract exerted an inhibitory effect on the growth, adherence and metabolism of cariogenic and periodontopathogenic bacteria, also inhibiting acid production, glycosyltransferase enzyme activity and the synthesis of insoluble polysaccharides. Additionally, administration of cocoa extract reduced biofilm accumulation and caries development in animals infected with cariogenic species. Clinical studies also reported that the use of mouthwashes containing cocoa extract reduced Streptococcus mutans counts in saliva and dental biofilm formation. In short, these studies highlight the nutritional value of cocoa, considering its clinical applicability, stability and economic accessibility.

Stability of Leaf Yerba Mate (Ilex paraguariensis) Metabolite Concentrations over the Time from the Prism of Secondary Sexual Dimorphism.

The yerba mate leaf metabolic composition depends mainly on genetics, sex, plant and leaf age, light intensity, harvest time, climate, and fertilization. In yerba mate, the secondary sexual dimorphism (SSD), the leaf metabolic SSD association with the frequency of leaf harvests, and the stability of the metabolites in the two genders over the years is not known. It was hypothesized that (1) the SSD in the metabolite segregation would differ among the winter and summer growth pauses, (2) females would show lower metabolite concentrations, and (3) the metabolic concentrations would show stability over the years on the same plants, not obligatorily associated with the SSD stability expression. Variations in theobromine, caffeine, chlorogenic and caffeic acids were correlated to the increasing time since the previous harvest, especially in females. However, the frequency of the metabolic SSD were associated with the studied growth pauses, rejecting the first hypothesis. No regular gender superiority was expressed in the yerba mate leaf secondary metabolites, rejecting our second hypothesis, even though more cases of superior female metabolite accumulation were identified. The stability of the leaf protein was preserved over the four years, with no SSD cases observed. The leaf methylxanthines were time stable, while the decrease in the phenolic content occurred with tree aging, which was not associated with the SSD expression, partially proving our third hypothesis. The novelty was related to the time stability of the leaf metabolic SSD observed over the winter and summer growth pauses, and over the four consecutive years without a regular expression of the male- or female-biased concentrations in the studied metabolites. To demystify the random metabolic gender responses in yerba mate, gender-orientated experiments with a high number of tree repetitions must be conducted, including clonal plants grown in various environments, such as monoculture and agroforestry, or on plantations in different climates and altitudes.

Antioxidant activity, total polyphenol content and methylxantine ratio in four materials of Theobroma cacao L. from Tolima, Colombia.

The International Cocoa Organization recognized Colombian cocoa as "fine aroma," but in recent years, clone CCN 51 has grown in popularity, widely due to its high yield. The Tolima department is the fourth producer of cacao in Colombia, but there is a lack of knowledge of the chemical properties of regional cocoa genotypes. The aim of this study was to evaluate the morphological, antioxidant activity, total polyphenol content and the methylxanthines ratio of four regional genotypes (UTLP02, UTVE01, UTGC01 and UTLM02) of Theobroma cacao L. from Tolima, Colombia. The universal clone of CCN51 was used as control. The highest values for the qualitative descriptors were obtained by the variants UTVE01 and CCN51 with FRAP and TPC ranging from 44.51 ± 0.90 to 106.77 ± 5.21 mg GAE/g and 27.13 ± 0.14 to 52.12 ± 4.71 mmol TE/g respectively. The genotypes with the highest values for FRAP and TPC were UTGC01 and CCN51. According to the methylxanthine ratio, UTVE01 was classified as Criollo, while UTLM02, UTGC01 and UTLP02, CCN51 are Trinitario and Forastero, respectively. Although CCN51 is considered a remarkable material in terms of productivity, the genotypes evaluated present good yields and interesting values of TPC and antioxidant activity, making them promising trees in local breeding programs.

How Climatic Seasons of the Amazon Biome Affect the Aromatic and Bioactive Profiles of Fermented and Dried Cocoa Beans?

In addition to the vast diversity of fauna and flora, the Brazilian Amazon has different climatic periods characterized by periods with greater and lesser rainfall. The main objective of this research was to verify the influence of climatic seasons in the Brazilian Amazon (northeast of Pará state) concerning the aromatic and bioactive profiles of fermented and dried cocoa seeds. About 200 kg of seeds was fermented using specific protocols of local producers. Physicochemical analyzes (total titratable acidity, pH, total phenolic compounds, quantification of monomeric phenolics and methylxanthines) and volatile compounds by GC-MS were carried out. We observed that: in the summer, the highest levels of aldehydes were identified, such as benzaldehyde (6.34%) and phenylacetaldehyde (36.73%), related to the fermented cocoa and honey aromas, respectively; and a total of 27.89% of this same class was identified during winter. There were significant differences (p ≤ 0.05, Tukey test) in the profile of bioactive compounds (catechin, epicatechin, caffeine, and theobromine), being higher in fermented almonds in winter. This study indicates that the climatic seasons in the Amazon affect the aromatic and bioactive profiles and could produce a new identity standard (summer and winter Amazon) for the cocoa almonds and their products.

Actividad antioxidante en genotipos de Theobroma spp. (Malvaceae) en México / Antioxidant activity in genotypes of Theobroma spp. (Malvaceae) in Mexico

Abstract Introduction: The organoleptic qualities of aromatic species and their derived products are directly related to some characteristics of flavor, color and nutritional value and depend largely on their genetic origin and content of secondary metabolites. Objective: The antioxidant activity of different genotypes of Theobroma spp. from Mexico was evaluated in order to distinguish promising qualities for genetic improvement, and to differentiate phylogenetic traits, considering biochemical variables. Methods: The amount of phenols, flavonoids and antioxidant activity was determined by ABTS and DDPH, in addition to the content of anthocyanins, theobromine and caffeine in four species of Theobroma L., and 50 genotypes derived from T. cacao. The results were analyzed using an analysis of variance, means test, principal component analysis and cladistic analysis. Results: There are highly significant differences between genotypes. The phenol content ranged from 7.5-85 mg g-1; flavonoids 6.57-69.6 mg g-1, antioxidant activity by ABTS of 17.3-86.1 and by DDPH of 40.0-53.3; anthocyanin content of 0.01-3, caffeine of 1.8-6.7-and theobromine of 2.9-9.8 mg g-1. Principal component and cladistic analysis helped explain the variation found and distinguish evolutionary characters and phylogenetic brotherhoods. The variation in content of phenols, flavonoids, antioxidant activity, anthocyanins, theobromine and caffeine was mainly due to the degree of domestication, while for the group of genotypes derived from T. cacao (forastero, trinitario and criollo) it was the origin of the seeds. Conclusions: The degree of domestication influences the content of phenols and antioxidant capacity. The results suggest that the evaluated variables can help to form criteria for genetic improvement in the complex derived from T. cacao oriented to the selection of higher phenol content and greater antioxidant activity.

Resumen Introducción: Las cualidades organolépticas de las especies aromáticas y sus productos derivados se relacionan directamente con algunas características del sabor, color y valor nutricional y dependen en gran medida de su origen genético y contenido de metabolitos secundarios. Objetivo: Se evaluó la actividad antioxidante de diferentes genotipos de Theobroma spp. de México, con el fin de distinguir cualidades promisorias para el mejoramiento genético, y diferenciar rasgos filogenéticos, considerando variables bioquímicas. Métodos: Se determinó la cantidad de fenoles, flavonoides y actividad antioxidante mediante ABTS y DDPH, además de contenido de antocianinas, teobromina y cafeína en cuatro especies de Theobroma L., y 50 genotipos derivados de T. cacao. Resultados: Los resultados fueron analizados mediante un análisis de varianza, prueba de medias, análisis de componentes principales y análisis cladístico. Existen diferencias altamente significativas entre genotipos. El contenido de fenoles varió de 7.5-85 mg g-1; flavonoides 6.57-69.6-mg g-1, actividad antioxidante por ABTS de 17.3-86.1 y por DDPH de 40.0-53.3; el contenido de antocianinas de 0.01-3, cafeína de 1.8-6.7 y teobromina de 2.9-9.8 mg g-1. El análisis de componentes principales y cladístico ayudó a explicar la variación encontrada y distinguir caracteres evolutivos y hermandades filogenéticas. La variación en contenido de fenoles, flavonoides, actividad antioxidante, antocianinas, teobromina y cafeína estuvo dada principalmente por el grado de domesticación, mientras que para el grupo de genotipos derivados de T. cacao (forastero, trinitario y criollo) fue el origen de las semillas. Conclusión: El grado de domesticación influye en el contenido de fenoles y actividad antioxidante. Los resultados sugieren que las variables evaluadas pueden ayudar a formar criterios para el mejoramiento genético en el complejo derivado de T. cacao orientado a la selección de mayor contenido de fenoles y mayor actividad antioxidante.

Simultaneous green extraction of fat and bioactive compounds of cocoa shell and protein fraction functionalities evaluation.

This work aimed to verify the feasibility of using alcoholic solvents under atmospheric pressure for the simultaneous extraction of fat and bioactive compounds from cocoa shell (CS), a byproduct of the cocoa industry, as well as to determine the influence of processing on the characteristics of defatted solids (DS). To this end, CS fat (CSF) extraction kinetics using ethanol or isopropanol as solvents were determined at 75 and 90 °C. Relative extraction yields of flavanols and alkaloids were determined, and protein functionalities such as the nitrogen solubility index were evaluated. CSF extraction yields from 36 to 70% were obtained with the highest figures related to absolute solvents. Conversely, hydrated alcohols were suitable to extract bioactive compounds, especially alkaloids, with extraction yields up to 73%. The best values of DS functionalities were obtained with the use of isopropanol, with a foaming capacity of (34 ± 2)% and stability of (57 ± 3)%; regarding emulsifying capability, the highest value was (126 ± 2) m2/g with a stability of (53 ± 4) min. CSF with a fatty acid composition similar to that of cocoa butter and DS with interesting protein functionalities were achieved, indicating that CS can be applied in food systems.

Assessment of remineralization potential of Theobromine and Sodium Fluoride gels on Artificial Caries like lesions / Avaliação do potencial de remineralização de géis de teobromina e fluoreto de sódio em lesões de cárie artificial

Objective: To compare the remineralization potential of theobromine and sodium fluoride gels on artificial caries like lesion. Materials and Methods: Forty longitudinal halves of human mandibular premolars were equally divided into 4 groups of 10 samples each: control group (C), samples were stored in distilled water during the experiment period. The remaining 30 specimens were subjected to demineralization protocol to create caries like lesions. samples were immersed for three days in a demineralization solution (pH 5.0) containing 0.2% carbopol and 0.1% lactic acid saturated with calcium phosphate tribasic. The samples were subdivided into 3 equal groups according to the treatment applied during the pH cycle. Demineralization group "D": no treatment applied. Group "F" treated with 2000 mg/liter sodium fluoride gel. Group "T" treated with 200 mg/liter theobromine gel. The specimens of the two studies groups were subjected to Demineralization- Remineralization PH Cycle Protocol for 5 days (Alternating four steps: 1: Treatment material, fluoride or theobromine ˜= 3 minutes. 2: Demineralizing solution 3 hours. 3: treatment material ˜= 3 minutes. 4: Remineralizing solution till the next cycle). The samples were investigated by scanning electron microscope (SEM) and energy dispersive x-ray analysis (EDXA). Results: The enamel of the demineralization group was porous with artificial caries like changes exposing the subsurface enamel rods with severe rod core defects. Theobromine gel and fluoride gel groups showed marked improvement in the surface characteristics in the enamel in both groups. Theobromine gel group showed more observable enamel surface improvement than the fluoride gel group. EDXA revealed that the calcium-phosphorus ratio displayed a descending order: (C > T > F > D). The differences between the two tested groups were not statistically significant. Conclusion: Theobromine gel had more effective remineralizing potential than fluoride gel as a result of its effect in improving the enamel surface characteristics of human teeth. (AU)

Objetivo: Comparar o potencial de remineralização dos géis de teobromina e fluoreto de sódio em lesões de cáries artificiais. Materiais e Métodos: Quarenta metades longitudinais de pré-molares inferiores humanos foram igualmente divididas em 4 grupos de 10 amostras cada: grupo controle (C), as amostras foram armazenadas em água destilada durante o período do experimento. As 30 amostras restantes foram submetidas ao protocolo de desmineralização paracriar lesões artificiais de cárie. As amostras foram imersas por três dias em uma solução de desmineralização (pH 5,0) contendo 0,2% de carbopol e 0,1% de ácido lático saturado com fosfato de cálcio tribásico. As amostras foram subdivididas em 3 grupos iguais, de acordo com o tratamento aplicado durante o ciclo do pH. Grupo de desmineralização "D": nenhum tratamento aplicado. Grupo "F" tratado com 2000 mg / litro de fluoreto de sódio em gel. Grupo & quot;T &q uot; tratado com 200 mg / litro de gel de teobromina. As amostras dos dois grupos de estudo foram submetidas ao protocolo de ciclo de desmineralização - remineralização por 5 dias (quatro etapas alternativas: 1: material de tratamento, flúor ou teobromina ˜= 3 minutos. 2: solução desmineralizante 3 horas. 3: material de tratamento ˜= 3 minutos 4: Solução de remineralização até o próximo ciclo). As amostras foram investigadas por microscopia eletrônica de varredura(MEV) e análise de raios-x dispersivos de energia (EDXA). Resultados: O esmalte do grupo de desmineralização era poroso, com cáries artificiais, como alterações que expunham as hastes de esmalte do subsolo com graves defeitos no núcleo da haste. Os grupos gel de teobromina e flúor apresentaram melhora acentuada nas características da superfície do esmalte nos dois grupos. O grupo gel de teobromina mostrou uma melhoria na superfície do esmalte mais observável do que o grupo gel de fluoreto. A EDXA revelou que a razão cálcio-fósforo exibia uma ordem decrescente: (C> T> F> D). As diferenças entre os dois grupos testados não foram estatisticamente significantes. Conclusão: O gel de teobromina teve um potencial remineralizante mais eficaz que o gel de flúor como resultado de seu efeito na melhoria das características da superfície do esmalte dos dentes humanos. (AU)

Extracción de compuestos solubles de la cascarilla de cacao con CO2 supercrítico. Caso de metilxantinas y grasa / Extraction of soluble compounds from cocoa shell with supercritical CO2 . A methilxantines and fat case

Resumen La extracción supercrítica es una técnica efectiva para separar diversos compuestos desde matrices naturales, por las propiedades que le confiere a los solventes como el CO2en estado supercrítico. El objetivo del presente trabajo fue la extracción supercrítica de teobromina, cafeína y grasa de la cascarilla de cacao con CO2supercrítico. Se propuso un diseño 22 con 5 puntos centrales, en condiciones estáticas de extracción, teniendo como factores de estudio, presión (2 000 psi a 6 000 psi) y temperatura (318 K a 333 K). Se analizaron los residuos en la cámara de extracción para evaluar los porcentajes de remoción y residual de grasa, cafeína y teobromina, respectivamente. Los resultados mostraron un rendimiento entre 1.72 % a 9.57 %, con una razón de remoción para grasa y cafeína de 61.31 % a 94.54 % y 38.52 % a 78.38 %, respectivamente. La teobromina se retuvo en el polvo de la cascarilla con una razón residual mayor a 90 %. Los efectos de las variables de temperatura y presión para la remoción de grasa fueron predichos a través de un polinomio de primer orden con efectos de interacción, no así para cafeína o teobromina. El método de extracción fue eficiente para remover grasa y cafeína, quedando un residuo en la cámara de extracción considerado como rico en compuestos funcionales.

Abstract Supercritical extraction is an effective technique for separating various compounds from natural matrices due to the properties from solvents, such as CO2, in supercritical state. In the present work, the supercritical extraction of theobromine, caffeine and fat from cocoa shell was carried out with supercritical CO2. A 22 design with 5 central points was proposed, with static operation conditions, having pressure (2 000 psi to 6 000 psi) and extraction temperature (318 K to 333 K) as study factors. The residues were analyzed in the extraction chamber to evaluate the removal and residual percentages of fat, caffeine and theobromine, respectively. The results showed a yield between 1.72 % to 9.57 %, with a removal rate for fat and caffeine of 61.31 % to 94.54 % and 38.52 % to 78.38 %, respectively. The theobromine was retained in the shell powder with a residual ratio greater than 90 %. The effects of temperature and pressure for fat were predicted through a first-order polynomial with interaction effects, but not for caffeine or theobromine. The extraction method was efficient to remove fat and caffeine, leaving a residue powder in the extraction chamber considered as high in functional compounds.

Effects of theobromine addition on chemical and mechanical properties of a conventional glass ionomer cement.

In vitro effect of 1% theobromine addition on the physical and chemical properties of conventional glass ionomer (GIC) cement was investigated. Conventional GIC (GIC-C) and 1% theobromine added to GIC (GIC-THEO) specimens were compared regarding the microhardness (n = 10), sorption (n = 5), solubility (n = 5), color change (n = 10), fluoride release in saliva (n = 10) and the amount of biofilm deposition (n = 20). Compared against conventional GIC, adding 1% theobromine increased microhardness (p < 0.05), while its sorption, solubility, color and fluoride release to saliva (p > 0.05) remained unchanged. On the other hand, Streptococcus mutans biofilm amount deposited on its surface decreased statistically when theobromine was added to GIC (p < 0.05). Based on the results, it could be concluded that 1% theobromine addition to GIC can be a good strategy as it keeps some of its properties and improves microhardness and biofilm deposits strengthening its role in the preventive approach of dentistry.

The Influence of a Xanthine-Catechin Chemical Matrix on in vitro Macrophage-Activation Triggered by Antipsychotic Ziprasidone.

Ziprasidone (ZIP) is an effective antipsychotic with low side effects than other second-generation antipsychotics. Despite this, there are reports of adverse events and previous studies associating the use of ZIP the inflammatory response. It is possible to infer that bioactive molecules present in some foods could attenuate peripheral inflammatory and oxidative stress potentially triggered ZIP. This is the case of guaraná xanthine-catechin chemical matrix (XC-Mix) that presents caffeine, theobromine, and catechin. The in vitro protocols using murine RAW 264.7 cell macrophages were ZIP-exposure in culture medium supplemented with chemical isolated and admixture of Caf, The, and Cat. Main results showed that supplementation with isolated and XC-mix had a lowering effect on 72 h macrophages proliferation. XC-mix with 1:1:1 proportion at 25 µg/mL of each caffeine, theobromine, and catechin, molecules present lowering effect on nitric oxide levels, oxidative stress markers (DNA oxidation quantified by 8-hydroxy-2' -deoxyguanosine), lipoperoxidation, and protein carbonylation. XC-mix also decreased protein levels and downregulated genes of proinflammatory cytokines (IL-1ß, IL-6, TNF-α). At contrary, XC-Mix increased levels and upregulated gene of anti-inflammatory IL-10 cytokine. The results suggest that XC-matrix could present some beneficial action on peripheral proinflammatory effects ZIP-triggered. Complementary in vivo studies could be useful to confirm these in vitro findings described here.

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.

Bioactive compounds in Mexican genotypes of cocoa cotyledon and husk.

A characterization of the phenolic profile of 25 cocoa genotypes established in a Mexican gene bank was carried out. From five different extraction methods commonly used for phenols, extraction with acidified methanol-water was chosen as the best to quantify the concentrations of theobromine and individual phenols in cocoa beans. High concentrations of individual and total phenols were found for genotypes native to Mexico (like RIM105, M031, and M033) or from Peru and Ecuador (INI10), but not the commercial mix (CAF), and were directly associated with their antioxidant activities. Despite the loss of some theobromine and phenols during fermentation, epicatechin remained in the fermented cotyledon in high concentrations. This study could help promote the commercialization of Mexican genotypes of cocoa and reports the possibility of upcycling fermented cocoa husks, which are rich in bioactive compounds and fiber, as novel functional extracts for use in food formulation or for nutraceutical purposes.

Relationship between aluminum stress and caffeine biosynthesis in suspension cells of Coffea arabica L.

Toxicity by aluminum is a growth-limiting factor in plants cultivated in acidic soils. This metal also promotes signal transduction pathways leading to the biosynthesis of defense compounds, including secondary metabolites. In this study, we observed that Coffea arabica L. cells that were kept in the dark did not produce detectable levels of caffeine. However, irradiation with light and supplementation of the culture medium with theobromine were the best conditions for cell maintenance to investigate the role of aluminum in caffeine biosynthesis. The addition of theobromine to the cells did not cause any changes to cell growth and was useful for the bioconversion of theobromine to caffeine. During a short-term AlCl3-treatment (500µM) of C. arabica cells kept under light irradiation, increases in the caffeine levels in samples that were recovered from both the cells and culture media were evident. This augmentation coincided with increases in the enzyme activity of caffeine synthase (CS) and the transcript level of the gene encoding this enzyme (CS). Together, these results suggest that actions by Al and theobromine on the same pathway lead to the induction of caffeine biosynthesis.

A chemical compound based on methylxanthine-polyphenols lowers nitric oxide levels and increases post-thaw human sperm viability.

We produced a new chemical compound based on methylxanthines and polyphenols (CCMP) present in the chemical matrix of guaraná (Paullinia cupana), a seed extract with antioxidant properties. After supplementation with the standard extract of resveratrol, a well documented antioxidant found in other plant sources, we investigated whether this resveratrol-enriched compound could improve sperm viability and modulate differentially reactive oxygen species (ROS) and nitric oxide (NO) levels in thawed sperm. Sperm samples obtained from healthy young donors were treated with different concentrations of guaraná extract (0.1, 1, 5 or 10 mg/ml) and cells were frozen at -80°C for 24 h. In addition, the potential protective effects of guaraná treatment on sperm treated with pro-oxidant compound (200 µM hydrogen peroxide, H2O2) were assessed. Samples were also exposed to three concentrations of CCMP before being frozen in liquid nitrogen (-196°C) or in an ultrafreezer (-80°C) for 24 h, and both pre-freezing and post-thaw measurements of viability and oxidative stress were performed. Guaraná supplementation at 10 mg/ml significantly increased post-thaw viability and decreased oxidative metabolism of the sperm. Moreover, selected concentrations of CCMP improved viability and oxidative metabolism in sperm samples pre-freezing. Furthermore, CCMP showed cryoprotective activity by increasing viability and decreasing oxidative stress in post-thaw samples. In summary, these findings suggested that CCMP supplementation acts as a cryoprotectant to modulate ROS and NO levels in thawed sperm. CCMP could be used to enhance sperm quality and reproductive success.

Theobromine increases NAD⁺/Sirt-1 activity and protects the kidney under diabetic conditions.

Reduction in sirtuin 1 (Sirt-1) is associated with extracellular matrix (ECM) accumulation in the diabetic kidney. Theobromine may reduce kidney ECM accumulation in diabetic rats. In the current study, we aimed to unravel, under diabetic conditions, the mechanism of kidney ECM accumulation induced by a reduction in Sirt-1 and the effect of theobromine in these events. In vitro, we used immortalized human mesangial cells (iHMCs) exposed to high glucose (HG; 30 mM), with or without small interfering RNA for NOX4 and Sirt-1. In vivo, spontaneously hypertensive rats (SHR) were rendered diabetic by means of streptozotocin and studied after 12 wk. The effects of treatment with theobromine were investigated under both conditions. HG leads to a decrease in Sirt-1 activity and NAD(+) levels in iHMCs. Sirt-1 activity could be reestablished by treatment with NAD(+), silencing NOX4, and poly (ADP-ribose) polymerase-1 (PARP-1) blockade, or with theobromine. HG also leads to a low AMP/ATP ratio, acetylation of SMAD3, and increased collagen IV, which is prevented by theobromine. Sirt-1 or AMPK blockade abolished these effects of theobromine. In diabetic SHR, theobromine prevented increases in albuminuria and kidney collagen IV, reduced AMPK, elevated NADPH oxidase activity and PARP-1, and reduced NAD(+) levels and Sirt-1 activity. These results suggest that in diabetes mellitus, Sirt-1 activity is reduced by PARP-1 activation and NAD(+) depletion due to low AMPK, which increases NOX4 expression, leading to ECM accumulation mediated by transforming growth factor (TGF)-ß1 signaling. It is suggested that Sirt-1 activation by theobromine may have therapeutic potential for diabetic nephropathy.

Spatial distribution of theobromine--a low MW drug--in tissues via matrix-free NALDI-MS imaging.

The ability of nano-assisted laser desorption-ionization mass spectrometry imaging (NALDI-IMS) to provide selective chemical monitoring with appropriate spatial distribution of a low molecular drug in a biological tissue was investigated. NALDI-IMS is a matrix-free laser desorption ionization (LDI) protocol based on imprinting of tissue constituents on a nanostructured surface. Using the accumulation of theobromine in rat kidney as a model, NALDI-IMS was found to provide well-resolved images of the special distribution of this low molecular weight (MW) drug in tissue.

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.