Showing papers on "Theobromine published in 2018"

Theobromine, a Methylxanthine in Cocoa Bean, Stimulates Thermogenesis by Inducing White Fat Browning and Activating Brown Adipocytes

Abstract: Natural medicinal compounds to treat obesity have recently attracted a great deal of attention because of the serious side effects of synthetic anti-obesity drugs. Recent advances have been made to identify natural products showing thermogenic activity, which is responsible for energy expenditure in brown or brown-like (beige) adipocytes. Here, we explored the thermogenic effects of theobromine, one of the most abundant methylxanthines in cocoa, on 3T3-L1 white adipocytes and HIB1B brown adipocytes. Theobromine markedly increased the expression levels of brown-fat signature proteins (PGC-1α, PRDM16, and UCP1) and beige-specific genes (Cd137, Cidea, Cited1, Tbx1, and Tmen26) in 3T3-L1 white adipocytes and remarkably elevated the expression levels of brown fatspecific genes (Cidea, Lhx8, Ppargc1, Prdm16, Ucp1, and Zic1) in HIB1B brown adipocytes. Theobromine also reduced the expression of the key adipogenic transcription factors, C/EBPα and PPARγ, in white adipocytes, while enhancing their expression in HIB1B cells. In addition, theobromine regulated lipolytic events and fat oxidation by upregulating the expression of pACC, ATGL, pHSL, ACOX, and CPT1. Additional mechanistic study revealed that theobromine activates β3-AR and AMPK. In summary, our results provide evidence for the first time indicating that theobromine has a potential beneficial effect on browning of white adipocytes and improves lipid catabolic metabolism in both cultured white and brown adipocytes via β-adrenergic signaling and AMPK activation. Consumption of theobromine may be a feasible way to activate thermogenesis and improve systematic lipid metabolism to protect against obesity and other metabolic disorders.

Poly(para-phenyleneethynylene)-Sensor Arrays Discriminate 22 Different Teas.

Abstract: Two nine-element sensor arrays, consisting of either three cationic poly(para-phenyleneethynylene)s (PPE) or the same PPEs complexed by cucurbituril[8] (CB[8]) at pH 3, 7, and 13 in water, discriminate 22 different teas and some of their small molecule components, including caffeine, theobromine and theophylline. Both arrays distinguish all of the black, green and oolong teas. The discrimination occurs by differential fluorescence modulation of the components of the sensor array and the treatment of the collected data by linear discriminant analysis. The signal is generated by either simple quenching (PPE only array) or the disruption of the PPE/CB[8] complex and quenching of the complex's or the PPEs' fluorescence through the polyphenolic colorants of the teas. Added amino acids, theobromine, theophylline, and caffeine give a fluorescence turn on of the PPE-CB[8] array, due to the disruption of the self-assembled complex, while for the PPE-alone tongue only caffeine, theobromine, and theophylline elicited useful fluorescence response. Both tongues discriminate different teas without any problem.

Hongyacha, a Naturally Caffeine-Free Tea Plant from Fujian, China

Abstract: Hongyacha (HYC) is a type of new wild tea plant discovered in Fujian Province, China. This tea is helpful to the healing or prevention of disease in its original growing area. However, research on this tea is limited. Our results showed that HYC displayed obvious differences in its morphological characteristics compared with Cocoa tea ( Camellia ptilophylla Chang), a famous caffeine-free tea plant in China. Theobromine and trans-catechins, but not caffeine and cis-catechins, were the dominant purine alkaloids and catechins detected in HYC. HYC might contain abundant gallocatechin-(4 → 8)-gallocatechin gallate, 1,3,4,6-tetra- O-galloyl-β-d-glucopyranose, and (-)-gallocatechin-3,5-di- O-gallate, which were not detected in regular tea. We also found that the TCS1 of HYC was distinct, and the responding recombinant protein exhibited only theobromine synthase activity. The obtained results showed that HYC is a new kind of caffeine-free tea plant and may be used for scientific protection and efficient utilization in the future.

HPLC Method for Quantification of Caffeine and Its Three Major Metabolites in Human Plasma Using Fetal Bovine Serum Matrix to Evaluate Prenatal Drug Exposure.

Abstract: Caffeine is recognized as the first-line therapeutic agent for apnea of prematurity. The dosage regimen is 10 mg/kg loading dose and 2.5 mg/kg maintenance dose. However, the plasma concentration achieved, not always, is therapeutically useful. It makes necessary to increase the doses to reach plasma concentration up to 30 or 35 μg/mL or even higher to attain therapeutic effect. To study why neonates have these differences, and whether these effects are linked to prenatal caffeine exposure, we had to develop an analytical method for an accurate measurement of caffeine and metabolites concentration. The analysis was carried out using fetal bovine serum (FBS) as biological matrix in a high-performance liquid chromatography with an ultraviolet detector method. This method allows acceptable chromatographic resolution between analytes in 15 minutes. It was validated and proved to be linear in the 0.1–40 µg/mL range for caffeine, paraxanthine, theobromine, and theophylline in the same chromatographic analysis. Accuracy for quality control samples for intra- and interday assays was ranged from 96.5 to 105.2% and 97.1 to 106.2%. Precision had CV no more than 10% in all concentration levels for all analytes. No differences were observed between quantification in human and FBS. This method was applied to quantify plasma drug concentration in mothers and their newborns in a Mexican northeast population. In our study, we confirmed self-reported caffeine maternal intake in 85.2% ( ); meanwhile, in their newborn’s plasma, it was detected only in 78% ( ). Caffeine plasma concentrations in mother and newborn had a linear relationship, and no differences were observed between groups (mothers versus children). These results suggest that our analytical method and substitution of biological matrix was linear, precise, and accurate for caffeine quantification and could be used for measuring prenatal exposure and let us to study, in the future, concentration differences observed during apnea clinical treatment.

Quantitative Trait Loci Mapping for Theobromine and Caffeine Contents in Tea Plant (Camellia sinensis)

Abstract: Understanding the genetic basis of theobromine and caffeine accumulation in the tea plant is important due to their contribution to tea flavor. Quantitative trait loci (QTL) analyses were carried out to identify genetic variants associated with theobromine and caffeine contents and ratio using a pseudo-testcross population derived from an intervarietal cross between two varieties of Camellia sinensis. A total of 10 QTL controlling caffeine content (CAF), theobromine content (TBR), sum of caffeine and theobromine (SCT), and caffeine-to-theobromine ratio (CTR) were identified over four measurement years. The major QTL controlling CAF, qCAF1, was mapped onto LG01 and validated across years, explaining an average of 20.1% of the phenotypic variance. The other QTL were detected in 1 or 2 years, and of them there were four, two, and three for TBR, SCT, and CTR, respectively. The present results provide valuable information for further fine mapping and cloning functional genes and for genetic improvement in tea plant.

Effect of Consumption of Cocoa-Derived Products on Uric Acid Crystallization in Urine of Healthy Volunteers.

Abstract: The purpose of this study was to determine the effects of consumption of different cocoa-derived products on uric acid crystallization in urine of 20 healthy volunteers. Participants were requested to select the specific diet that they wished to follow during the 12 h prior to collection of urine. The only restriction was that the diet could not include any product with cocoa, coffee, or caffeine. On the first day, each volunteer followed their selected diet, and an overnight 12 h urine sample was collected as the baseline urine. After seven days on an unrestricted diet, each volunteer repeated the same diet with 20 g of milk chocolate, chocolate powder, or dark chocolate during breakfast and another 20 g during dinner. Overnight 12 h urine samples were then collected. Urine volume, pH, oxalate, creatinine, uric acid, theobromine, and a uric acid crystallization test were determined for each sample. The results for all 20 patients show that uric acid crystallization was significantly lower following the consumption of chocolate powder or dark chocolate relative to baseline or following the consumption of milk chocolate. The results indicated that increased concentrations of urinary theobromine reduced the risk of uric acid crystallization.

Theobromine Is Responsible for the Effects of Cocoa on the Antibody Immune Status of Rats.

Abstract: Background A 10% cocoa-enriched diet influences immune system functionality including the prevention of the antibody response and the induction of lower immunoglobulin (Ig) concentrations. However, neither cocoa polyphenols nor cocoa fiber can totally explain these immunoregulatory properties. Objectives This study aimed to establish the influence of cocoa theobromine in systemic and intestinal Ig concentrations and to determine the effect of cocoa or theobromine feeding on lymphoid tissue lymphocyte composition. Methods Three-week-old female Lewis rats were fed either a standard diet (AIN-93M; RF group), a 10% cocoa diet (CC group), or a 0.25% theobromine diet (the same amount provided by the cocoa diet; TB group) in 2 separate experiments that lasted 19 (experiment 1) or 8 (experiment 2) d. Serum IgG, IgM, IgA, and intestinal secretory IgA (sIgA) concentrations were determined. In addition, at the end of experiment 2, thymus, mesenteric lymph node (MLN), and spleen lymphocyte populations were analyzed. Results Both CC and TB groups in experiments 1 and 2 showed similar serum IgG, IgM, and IgA and intestinal sIgA concentrations, which were lower than those in the RF group (46-98% lower in experiment 1 and 23-91% lower in experiment 2; P < 0.05). In addition, in experiment 2, the cocoa and theobromine diets similarly changed the thymocyte composition by increasing CD4-CD8- (+133%) and CD4+CD8- (+53%) proportions (P < 0.01), changed the MLN composition by decreasing the percentage of T-helper (Th) lymphocytes (-3%) (P = 0.015), and changed the spleen composition by increasing the proportion of Th lymphocytes (+9%) (P < 0.001) after 1 wk of diet treatment. Conclusions The theobromine in cocoa plays an immunoregulatory role that is responsible for cocoa's influence on both systemic and intestinal antibody concentrations and also for modifying lymphoid tissue lymphocyte composition in young healthy Lewis rats. The majority of these changes are observed after a single week of being fed a diet containing 0.25% theobromine.

Characterization of Bitter Compounds via Modulation of Proton Secretion in Human Gastric Parietal Cells in Culture.

Abstract: Humans perceive bitterness via around 25 different bitter receptors. Therefore, the identification of antagonists remains a complex challenge. We previously demonstrated several bitter-tasting compounds such as caffeine to induce acid secretion in the stomach and in a human gastric tumor cell line (HGT-1). Here, the results of a fluorescent-based in vitro assay using HGT-1 cells and a human sensory panel testing nine selected potential bitter modulators, with or without the bitter compounds caffeine or theobromine, were compared. Of the bitter-modulating compounds tested, eriodictyol, matairesinol, enterolacton, lariciresinol, and homoeriodictyol reduced the effect of caffeine on proton secretion by −163 ± 14.0, −152 ± 12.4, −74 ± 16.4, −58 ± 7.2, and −44.6 ± 16.5%, respectively, and reduced the bitter intensity of caffeine in the human sensory panel. In contrast, naringenin and 5,7-dihydroxy-4(4-hydroxyphenyl)chroman-2-one neither reduced the caffeine-induced proton secretion in HGT-1 cells nor showed an...

New fluorescence spectroscopic method for the simultaneous determination of alkaloids in aqueous extract of green coffee beans

Abstract: There is no fluorescence spectroscopic method for the determination of trigonelline and theobromine in green coffee beans. Therefore, the objective of this study was to develop a new fluorescence spectroscopic method to determine the alkaloids simultaneously in the aqueous extract of green coffee beans. The calibration curves were linear in the range 2–6, 1–6, 1–5 mg/L for caffeine, theobromine and trigonelline, respectively, with R2 ≥ 0.9987. The limit of detection and limit of quantification were 2, 6 and 7 µg/L and 40, 20 and 20 µg/L for caffeine, theobromine and trigonelline, respectively. Caffeine and trigonelline exhibited well separated fluorescence excitation spectra and therefore the two alkaloids were selectively quantified in the aqueous extract of green coffee. While theobromine showed overlapping fluorescence excitation spectra with caffeine and hence theobromine could not be determined in the aqueous extract of green coffee beans. The amount of caffeine and trigonelline in the three samples of green coffee beans were found to be 0.95–1.10 and 1.00–1.10% (w/w), respectively. The relative standard deviations (RSD ≤ 4%) of the method for the three compounds of interest were of very good. The accuracy of the developed analytical method was evaluated by spiking standard caffeine and trigonelline to green coffee beans and the average recoveries were 99 ± 2% for both the alkaloids. A fast, sensitive and reliable fluorescence method for the simultaneous determination of caffeine and trigonelline in the aqueous extract of green coffee beans was developed and validated. The developed method reflected an effective performance to the direct determination of the two alkaloids in the aqueous extract of green coffee beans.

Theobromine Does Not Affect Fasting and Postprandial HDL Cholesterol Efflux Capacity, While It Decreases Fasting miR-92a Levels in Humans.

Abstract: SCOPE Chocolate consumption lowers cardiovascular disease risk, which might be attributed to the methylxanthine theobromine. These effects may be mediated through effects on HDL-mediated cholesterol efflux, which may be affected by microRNA (miRNA) levels in the HDL particles. Therefore, the aim of this study is to investigate effects of theobromine consumption on fasting and postprandial cholesterol efflux and miRNAs levels. METHODS AND RESULTS Thirty overweight and 14 obese healthy men and women participated in this randomized, double-blind crossover study. Participants consumed 500 mg d-1 of theobromine or placebo for 4 weeks. ABCA1-mediated cholesterol efflux was measured using J774 macrophages. MiRNAs levels (miR-92a, miR-223, miR-135a*) were quantified in apolipoprotein B-depleted serum. Theobromine consumption did not affect fasting and postprandial cholesterol efflux. Fasting miR-223 and miR-135a levels were unchanged, while miR-92a levels were decreased (-0.21; p < 0.05). The high-fat meal increased postprandial cholesterol efflux capacity (+4.3 percentage points; p ≤ 0.001), miR-92a (+1.21; p < 0.001), and miR-223 (+1.79; p < 0.001) levels, while a trend was found for miR-135a (+1.08; p = 0.06). CONCLUSION Theobromine did not improve fasting and postprandial ABCA1-mediated cholesterol efflux capacity, but decreased fasting miR-92a levels. High-fat meal intake increased postprandial cholesterol efflux and the three selected miRNAs levels.

Associations of Urinary Caffeine and Caffeine Metabolites With Arterial Stiffness in a Large Population-Based Study

Abstract: Objective To assess the influence of caffeine on arterial stiffness by exploring the association of urinary excretion of caffeine and its related metabolites with pulse pressure (PP) and pulse wave velocity (PWV). Participants and Methods Families were randomly selected from the general population of 3 Swiss cities from November 25, 2009, through April 4, 2013. Pulse pressure was defined as the difference between the systolic and diastolic blood pressures obtained by 24-hour ambulatory monitoring. Carotid-femoral PWV was determined by applanation tonometry. Urinary caffeine, paraxanthine, theophylline, and theobromine excretions were measured in 24-hour urine collections. Multivariate linear and logistic mixed models were used to explore the associations of quartiles of urinary caffeine and metabolite excretions with PP, high PP, and PWV. Results We included 863 participants with a mean ± SD age of 47.1±17.6 years, 24-hour PP of 41.9±9.2 mm Hg, and PWV of 8.0±2.3 m/s. Mean (SE) brachial PP decreased from 43.5 (0.5) to 40.5 (0.6) mm Hg from the lowest to the highest quartiles of 24-hour urinary caffeine excretion ( P P P =.03). Similar associations were found for paraxanthine and theophylline, whereas no associations were found with theobromine. Conclusion Urinary caffeine, paraxanthine, and theophylline excretions were associated with decreased parameters of arterial stiffness, suggesting a protective effect of caffeine intake beyond its blood pressure–lowering effect.

Bio-detheobromination of cocoa pod husks: reduction of ochratoxin A content without change in nutrient profile

Abstract: Utilization of cocoa pod husks (CPH) in animal feed is hindered by the presence of theobromine, which is variably toxic to animals. Treatment of this agro-waste to remove theobromine, while preserving its nutrient content, would allow beneficial use of the millions of metric tonnes discarded annually. The aim of this study was to assess the suitability of selected theobromine-degrading filamentous fungi for use as bio-tools in degradation of theobromine in CPH. The candidate fungi assessed in this study were an Aspergillus niger (AnTD) and three Talaromyces spp. (TmTD-1, TmTD-2, TvTD) isolates. All the fungi eliminated CPH theobromine, 0.15% w/w starting concentration, within 7 days of start of treatment, and were capable of degrading caffeine and theophylline. The fungi decreased CPH ochratoxin A content by 31–74%. Pectin was not detectable in fungus-treated CPH whereas parameters assessed for proximate composition were not affected. The data provide ample evidence that the four isolates can be applied to CPH for the purpose of eliminating theobromine and decreasing ochratoxin A content without affecting nutrient profile. Comparatively, Talaromyces verruculosus TvTD was considered as most suitable for use as a bio-tool in detheobromination of CPH for animal feed.

Determination of Caffeine and Its Metabolites in Wastewater Treatment Plants Using Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry.

Abstract: For caffeine and its seven major metabolites (i.e., theobromine, theophylline, paraxanthine, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, and xanthine), an optimized analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for their detection in wastewater samples was developed in this study. Extraction of these compounds (recoveries ranged from 60.3 to 83.2%) was made possible by combining universal polymeric reversed-phase cartridge and polymeric strong cation exchange cartridge. This method was applied to the determination of caffeine and its metabolites in the influent and effluent of an anaerobic-anoxic-oxic (A2O) process. In the A2O influent, caffeine and its metabolites (except xanthine) ranged from 1.39 to 5.45 μg/L, and their concentrations in the A2O effluent ranged from 10.2 to 171.3 ng/L. The mass load of caffeine was 14.9 g/day/1000 inhabitants, considering the population served by the target wastewater treatment plant (WWTP). The concentration of caffeine derivatives in wastewater influent is a tool for estimating the population size in the area served by WWTPs.

Different Catabolism Pathways Triggered by Various Methylxanthines in Caffeine-Tolerant Bacterium Pseudomonas putida CT25 Isolated from Tea Garden Soil.

Abstract: The degradation efficiency and catabolism pathways of the different methylxanthines (MXs) in isolated caffeine-tolerant strain Pseudomonas putida CT25 were comprehensively studied. The results showed that the degradation efficiency of various MXs varied with the number and position of the methyl groups on the molecule (i.e., xanthine > 7-methylxanthine ≈ theobromine > caffeine > theophylline > 1-methylxanthine). Multiple MX catabolism pathways coexisted in strain CT25, and a different pathway would be triggered by various MXs. Demethylation dominated in the degradation of N-7-methylated MXs (such as 7-methylxanthine, theobromine, and caffeine), where C-8 oxidation was the major pathway in the catabolism of 1-methylxanthine, whereas demethylation and C-8 oxidation are likely both involved in the degradation of theophylline. Enzymes responsible for MX degradation were located inside the cell. Both cell culture and cell-free enzyme assays revealed that N-1 demethylation might be a rate-limiting step for the catabolism of the MXs. Surprisingly, accumulation of uric acid was observed in a cell-free reaction system, which might be attributed to the lack of activity of uricase, a cytochrome c-coupled membrane integral enzyme.

Xanthine urolithiasis: Inhibitors of xanthine crystallization.

Abstract: Objective To identify in vitro inhibitors of xanthine crystallization that have potential for inhibiting the formation of xanthine crystals in urine and preventing the development of the renal calculi in patients with xanthinuria. Methods The formation of xanthine crystals in synthetic urine and the effects of 10 potential crystallization inhibitors were assessed using a kinetic turbidimetric system with a photometer. The maximum concentration tested for each compound was: 20 mg/L for 3-methylxanthine (3-MX); 40 mg/L for 7-methylxanthine (7-MX), 1-methylxanthine (1-MX), theobromine (TB), theophylline, paraxanthine, and caffeine; 45 mg/L for 1-methyluric acid; 80 mg/L for 1,3-dimethyluric acid; and 200 mg/L for hypoxanthine. Scanning electron microscopy was used to examine the morphology of the crystals formed when inhibitory effects were observed. Results Only 7-MX, 3-MX, and 1-MX significantly inhibited xanthine crystallization at the tested concentrations. Mixtures of inhibitors had an additive effect rather than a synergistic effect on crystallization. Conclusion Two of the inhibitors identified here—7-MX and 3-MX—are major metabolites of TB. In particular, after TB consumption, 20% is excreted in the urine as TB, 21.5% as 3-MX, and 36% as 7-MX. Thus, consumption of theobromine could protect patients with xanthinuria from the development of renal xanthine calculi. Clinical trials are necessary to demonstrate these effects in vivo.

The acute effects on duodenal gene expression in healthy men following consumption of a low-fat meal enriched with theobromine or fat.

Abstract: Increasing apoA-I synthesis may improve HDL functionality and lower CVD risk. As theobromine and fat increase fasting apoA-I concentrations, and the intestine is involved in apoA-I production, the acute effects of both were studied on duodenal gene transcription to better understand underlying mechanisms. In this crossover study, 8 healthy men received once a low fat (LF) meal, a LF meal plus theobromine (850 mg), or a high fat (HF) meal. Five hours after meal intake duodenal biopsies were taken for microarray analysis. Theobromine and HF consumption did not change duodenal apoA-I expression. Theobromine did not change gene expression related to lipid and cholesterol metabolism, whereas those related to glycogen/glucose breakdown were downregulated. HF consumption increased gene expression related to lipid and cholesterol uptake and transport, and to glucose storage, while it decreased those related to glucose uptake. Furthermore, genes related to inflammation were upregulated, but inflammation markers in plasma were not changed. In healthy men, acute theobromine and fat consumption did not change duodenal apoA-I mRNA, but inhibited expression of genes related to glucose metabolism. Furthermore, HF intake activated in the duodenum expression of genes related to lipid and cholesterol metabolism and to inflammation.

Chocolate and Its Component's Effect on Cardiovascular Disease

Abstract: This review article examines and discusses the advantageous effects of chocolate and its various components on cardiovascular disease. Based on both observational and experimental studies, cocoa consumption is correlated with reduced risk of cardiovascular disease. Complementing this, cocoa consumption is also highly associated with reduced blood pressure, usually accounting for decreases in blood pressure by 3–5 mm Hg. A large contributing factor influencing these beneficial cardioprotective effects is chocolate's many components. For instance, chocolate contains many elements such as lipids, minerals, fiber, theobromine, and most notably flavonoids. Flavonoids are in many different plant-derived foods such as tea, grapes, wine, berries, and cocoa; chocolate is particularly rich in a subclass of flavonoids called flavanols. Usually, the percentage of cacao in chocolate influences the concentration of flavanols present; dark chocolate tends to contain more flavanols than milk chocolate. These flavanols are a highly significant component of cacao as they provide numerous cardiovascular health benefits. They have antihypertensive, antiinflammatory, and antioxidant qualities. The mechanisms of how cocoa consumption actually reduces the risk of cardiovascular disease are not completely known yet; however, many ongoing research studies show that the bioavailability of nitric oxide is significant.

ANovel Approach to Reduce Toxicities and to Improve Bioavailabilities of DNA/RNAof Human Cancer Cells–Containing Cocaine (Coke), Lysergide (Lysergic AcidDiethyl Amide or LSD), Δ9–Tetrahydrocannabinol (THC) [(–)–trans–Δ⁹–Tetrahydrocannabinol],Theobromine (

Abstract: The aim of the present study was to reduce toxicities and to improve bioavailabilities of DNA/RNA of human cancer cells–containing Cocaine (Coke), Lysergide (Lysergic Acid Diethyl Amide or LSD), Δ9–Tetrahydrocannabinol (THC) [(–)–trans–Δ⁹–Tetrahydrocannabinol], Theobromine (Xantheose), Caffeine, Aspartame (APM) (NutraSweet) and Zidovudine (ZDV) [Azidothymidine (AZT)] as anti–cancer Nano drugs by coassembly of dual anti–cancer Nano drugs to inhibit DNA/RNA of human cancer cells drug resistance.

Determination of theobromine and caffeine in some Malaysian beverages by liquid chromatography-time-offlight mass spectrometry

Abstract: Purpose: To determine the concentration of theobromine (TB) and caffeine (CF) in tea and other beverages using liquid chromatography-time of flight-mass spectrometry (LC-TOF-MS).Methods: The extract of caffeine and theobromine from tea and other beverages was filtered by 0.45 μm nylon micro-syringe and then injected into a LC-ToF-MS system. Theobromine and caffeine were separated using Thermo Scientific C18-column (length 250 mm, width 2.1 mm and diameter 5 μm). Acetonitrile-methanol (ACN – MeOH, 3:1 v/v) was used as mobile phase B, while mobile phase A was 0.1 % FA in DIW. The volume injected was 30 μL at a rate of 0.3 mL/min.Results: Good linearity was obtained in the range of 0.3 – 400 and 0.2 – 200 mg/L for theobromine and caffeine, respectively (regression coefficient (R2) > 0.970). The limits of detection were 0.15 and 0.05 μg/mL for theobromine and caffeine, respectively. The highest concentrations of caffeine and theobromine determined in tea samples were 159.1 and 255.8 mg/L, respectively.Conclusion: Theobromine and caffeine have been successfully analysed in tea, coffee and soft drinks. LC-TOF-MS is an accurate and promising instrument for the determination of the studied compounds in beverages.Keywords: Theobromine, Caffeine, Tea, Coffee, LC-TOF/MS

UHPLC-HRMS Analysis of Theobromine in Theobroma cacao and its Products

Abstract: Theobroma cacao seed is the major ingredient in all types of chocolate products and contains Theobromine. Theobromine is a bitter alkaloid beneficial in the treatment of hypertension, arteriosclerosis and angina pectoris. Of all chocolate brand samples, Sample J containing 70% Cacao had the highest amount of theobromine. Sample A containing 11% Cacao had the least amount of theobromine. The 100% Cacao Chocolate Bar had the highest concentration of theobromine in comparison to roasted cocoa having the lowest concentration of theobromine. Quantitative analysis of Theobromine was completed on the Thermo Scientific UHPLC and LTQ Orbit rap Discovery equipped with an ESI ion source. A three-minute gradient method with a flow rate of 300 μL/min was developed on the UHPLC-HRMS using HPLC-grade water and acetonitrile. Ethyl ether was used to remove cacao fats and water was used to isolate theobromine. To obtain the precision of the theobromine extraction process, the recovery analysis was 86%.

Application of Aspergillus sydowii NRRL250 to Degrade Caffeine in Pu-erh Tea

Abstract: Pu-erh tea is produced by a solid-state fermentation. The natural microbiota presented in pu-erh tea influence caffeine level. In previous study, one effective fungi was selected from pu-erh tea and identified as Aspergillus sydowii NRRL250, which could lead caffeine degradation. In this paper, A. sydowii NRRL250 was inoculated into a liquid medium with different initial caffeine concentrations (600, 1200 and 1800 mg/L of caffeine, respectively) to explore caffeine degradation products. The solid-state fermentation of sun-dried tea leaves and submerged fermentation of tea infusion were carried out to investigate the application of A. sydowii NRRL250 through an inoculation. Samples were collected periodically, and the contents of caffeine, theophylline, 3-methylxanthine and theobromine were determined by HPLC. In the substrate tests, caffeine degraded drastically, theophylline and 3-methylxanthine were detected and increased obviously with the degradation of caffeine, and theobromine was not detected. In the solid-state and submerged fermentation, caffeine decreased radically (p 0.05). Compared with the submerged fermentation without caffeine addition, the extra addition of caffeine could enhance the productions of theophylline and 3-methylxanthine significantly (p<0.05). Therefore, theophylline and 3-methylxanthine were the main degradation products from caffeine, caffeine concentration had a significant (p<0.05) effect on the productions of theophylline and 3-methylxanthine. And A. sydowii NRRL250 had great application potential to produce decaffeinated and high-theophylline tea through an inoculation.

Synthesis, physical-chemical and biological properties of 8-R-thioderivatives of 1-benzyltheobromine

Abstract: Key way for creating new medicinal drugs is structural modification of known and existent natural compounds with high biological activity. In this aspect researchers’ attention is drawn by xanthine derivatives which appear to be antagonists of adenosine receptors, phosphodiesterase inhibitors and histone deacetylase inducers. This resulted in their widespread application in medicine to cure asthma, bronchitis and chronic obstructive pulmonary disease. In addition, xanthine derivatives are used as diuretics, analgesics, heart pacemakers, anti-inflammatory, psychotropic and renal protective agents. The aim of this work lies in developing unique methods to synthesize undocumented in other scientific papers 8-thioderivatives of 1-benzylthiobromine and also studying of their physical, chemical and biological properties. Acute toxicity of synthesized compounds has been studied with the application of Kerber method. The study of diuretic activity of obtained compounds was carried out applying Berkhin method. Analgesic activity of synthesized xanthines was studied at ‘acetic acid writhing’ model. Anti-inflammatory activity was studied at ‘acute aseptic edema’ model. Antioxidant activity was studied in vitro using the method of non-enzymatic initiation of free-radical oxidation. Heating of 1-benzyl-8-bromotheobromine with double excess of sodium sulphidenonahydrate in dimethylformamide environment results in formation of 8-thiotheobromine. Reactions of thioxanthines with halogenketones and chloroacetamide proceed smoothly with their short-time heating in aqueous alcohol environment. By applying such computer programs as ALOGPS, DRAGON, GUSAR and ACD/Percepta Platform was established viability of further invitro and invivo research. Accessible laboratory methods have been elaborated to synthesize 8-thiosubstituted 1-n-methylbenzyltheobromine, their structure having been proved by elemental analysis, PMR-spectroscopy and mass-spectrometry data. Molecular and pharmacological descriptors to forecast properties of the obtained substances have been calculated, in addition to acute toxicity index. Also the study of acute toxicity, diuretic, analgesic, anti-inflammatory and anti-oxidant activity of synthesized compounds has been carried out. After additional research 1-benzyl-8-(2-oxo-2-phenylethylthio)theobromine can be used in medical practice as an antioxidant agent.