miR828a-CsMYB114 Module Negatively Regulates the Biosynthesis of Theobromine in Camellia sinensis.

Theobromine is an important quality component in tea plants (Camellia sinensis), which is produced from 7-methylxanthine by theobromine synthase (CsTbS), the key rate-limiting enzyme in theobromine biosynthetic pathway. Our transcriptomics and widely targeted metabolomics analyses suggested that CsMYB114 acted as a potential hub gene involved in the regulation of theobromine biosynthesis. The inhibition of CsMYB114 expression using antisense oligonucleotides (ASO) led to a 70.21% reduction of theobromine level in leaves of the tea plant, which verified the involvement of CsMYB114 in theobromine biosynthesis. Furthermore, we found that CsMYB114 was located in the nucleus of the cells and showed the characteristic of a transcription factor. The dual luciferase analysis, a yeast one-hybrid assay, and an electrophoretic mobility shift assay (EMSA) showed that CsMYB114 activated the transcription of CsTbS, through binding to CsTbS promoter. In addition, a microRNA, miR828a, was identified that directly cleaved the mRNA of CsMYB114. Therefore, we conclude that CsMYB114, as a transcription factor of CsTbS, promotes the production of theobromine, which is inhibited by miR828a through cleaving the mRNA of CsMYB114.

Determination of temporal changes in hepatic drug-oxidizing capacity using plasma metabolite/caffeine ratios in non-pregnant and pregnant goats.

Caffeine (CF) is a metabolic probe drug used in the determination of the hepatic drug-oxidizing capacity. The aim of this study was to investigate temporal changes in the hepatic drug-oxidizing capacity using plasma metabolite/CF ratios in non-pregnant goats (n = 11) and pregnant goats (n = 23). CF (5 mg/kg, intravenous) was administered in six periods (Period 1-6) with 45 days between two periods. The plasma levels of CF and its metabolites, theophylline (TP), theobromine (TB) and paraxanthine (PX), were determined by HPLC-UV. To evaluate hepatic drug-oxidizing capacity in terms of enzymes that play a role in CF metabolism, the plasma metabolic ratios including TB/CF, PX/CF, TP/CF and TB + PX + TP/CF were determined at 10 h following CF administration. Plasma metabolite/CF ratios were similar between non-pregnant and pregnant goats. However, plasma metabolite/CF ratios in Period 3 (45 days in pregnant goats) were significantly higher than those other periods in both pregnant and non-pregnant goats. The effect of pregnancy may not be observed on drugs that are substrates of enzymes involved in CF metabolism in goats.

7-Methylxanthine Inhibits the Formation of Monosodium Urate Crystals by Increasing Its Solubility.

Gout is characterized by the formation of monosodium urate crystals in peripheral joints. We carried out laboratory studies to investigate the effect of adding nine different methylxanthines and two different methylated uric acid derivatives on the development of these crystals over the course of 96 h in a medium whose composition was similar to that of synovial fluid. Our results showed that 7-methylxanthine reduced or totally prevented crystal formation; 1-methylxanthine, 3-methylxanthine, 7-methyluric acid, and 1,3-dimethyluric acid had weaker effects, and the other molecules had no apparent effect. The presented results indicate that a 7-methylxanthine concentration of about 6 × 10-5 M (10 mg/L) prevented the formation of crystals for an initial urate concentration of 1.78 × 10-3 M (300 mg/L) in the presence of 0.4 M of Na+ for 96 h at 25 °C and a pH of 7.4. We attribute these results to alterations in thermodynamics, not kinetics. Our results suggest that prevention of crystallization in vivo could be achieved by direct oral administration of 7-methylxanthine or other methylxanthines that are metabolized to 7-methylxanthine. For example, the hepatic metabolism of theobromine leads to significant plasma levels of 7-methylxanthine (14% of the initial theobromine concentration) and 3-methylxanthine (6% of the initial theobromine concentration); however, 7-methyluric acid is present at very low concentrations in the plasma. It is important to consider that several of the specific molecules we examined (theobromine, caffeine, theophylline, dyphylline, etophylline, and pentoxifylline) did not directly affect crystallization.

Herbaspirillum sp. ZXN111 Colonization Characters to Different Tea Cultivars and the Effects on Tea Metabolites Profiling on Zijuan (Camellia sinensis var. assamica).

Herbaspirillum sp. ZXN111 and its mutants (Δacc, Δtyrb, and Δacc-tyrb), which show PGP activity on Zijuan, were tested for tea plants' colonization characteristics and the strain-dependent response of tea metabolites. The results showed that strain ZXN111 could widely colonize in different tea cultivars of Zijuan, Yunkang-10, Longjin 43, and Shuchazao, but with significant colonization preference to Zijuan, which might be ascribed to anthocyanins' chemotaxis. After 9 weeks of co-cultivation, l-theanine and theobromine in Zijuan leaves that were inoculated with wild-type ZXN111 were decreased, while theobromine, caffeine, and l-theanine that were inoculated with mutant Δacc were increased; especially l-theanine increased much significantly. Metabolomics analysis showed that tea metabolite profiling of inoculant groups was clearly separated from the control; therein, the flavanols were downregulated in ZXN111 and Δacc groups, but the l-theanine of the Δacc group was significantly upregulated compared to control and ZXN111 groups. These results indicated that strain ZXN111, especially of mutant Δacc, improved Zijuan tea flavor.

Flavan-3-ol-methylxanthine interactions: Modulation of flavan-3-ol bioavailability in volunteers with a functional colon and an ileostomy.

Flavan-3-ols, including the flavan-3-ol monomer (-)-epicatechin, are dietary bioactives known to mediate beneficial cardiovascular effects in humans. Recent studies showed that flavan-3-ols could interact with methylxanthines, evidenced by an increase in flavan-3-ol bioavailability with a concomitant increase in flavan-3-ol intake-mediated vascular effects. This study aimed at elucidating flavan-3-ol-methylxanthine interactions in humans in vivo by evaluating the specific contributions of theobromine and caffeine on flavan-3-ol bioavailability. In ileostomists, the effect of methylxanthines on the efflux of flavan-3-ol metabolites in the small intestine was assessed, a parameter important to an understanding of the pharmacokinetics of flavan-3-ols in humans. In a randomized, controlled, triple cross-over study in volunteers with a functional colon (n = 10), co-ingestion of flavan-3-ols and cocoa methylxanthines, mainly represented by theobromine, increased peak circulatory levels (Cmax) of flavan-3-ols metabolites (+21 ± 8%; p < 0.05). Conversely, caffeine did not mediate a statistically significant effect on flavan-3-ol bioavailability (Cmax = +10 ± 8%, p = n.s.). In a subsequent randomized, controlled, double cross-over study in ileostomists (n = 10), cocoa methylxanthines did not affect circulatory levels of flavan-3-ol metabolites, suggesting potential differences in flavan-3-ol bioavailability compared to volunteers with a functional colon. The main metabolite in ileal fluid was (-)-epicatechin-3'-sulfate, however, no differences in flavan-3-ol metabolites in ileal fluid were observed after flavan-3-ol intake with and without cocoa methylxanthines. Taken together, these results demonstrate a differential effect of caffeine and theobromine in modulating flavan-3-ol bioavailability when these bioactives are co-ingested. These findings should be considered when comparing the effects mediated by the intake of flavan-3-ol-containing foods and beverages and the amount and type of methylxanthines present in the ingested matrixes. Ultimately, these insights will be of value to further optimize current dietary recommendations for flavan-3-ol intake. CLINICAL TRIAL REGISTRATION NUMBER: This work was registered at clinicaltrials.gov as NCT03526107 (study part 1, volunteers with functional colon) and NCT03765606 (study part 2, volunteers with an ileostomy).

Target Metabolome and Transcriptome Analysis Reveal Molecular Mechanism Associated with Changes of Tea Quality at Different Development Stages.

This study aimed to explore the molecular mechanisms underlying the differential quality of tea made from leaves at different development stages. Fresh Camellia sinensis (L.) O. Kuntze "Sichuan Colonial" leaves of various development stages, from buds to old leaves, were subjected to transcriptome sequencing and metabolome analysis, and the DESeq package was used for differential expression analysis, followed by functional enrichment analyses and protein interaction analysis. Target metabolome analysis indicated that the contents of most compounds, including theobromine and epicatechin gallate, were lowest in old leaves, and transcriptome analysis revealed that DEGs were significantly involved in extracellular regions and phenylpropanoid biosynthesis, photosynthesis-related pathways, and the oleuropein steroid biosynthesis pathway. Protein-protein interaction analysis identified LOC114256852 as a hub gene. Caffeine, theobromine, L-theanine, and catechins were the main metabolites of the tea leaves, and the contents of all four main metabolites were the lowest in old leaves. Phenylpropanoid biosynthesis, photosynthesis, and brassinosteroid biosynthesis may be important targets for breeding efforts to improve tea quality.

Pentoxifylline attenuates nonalcoholic fatty liver by inhibiting hepatic macrophage polarization to the M1 phenotype.

BACKGROUND: Nonalcoholic fatty liver (NAFL), recognized as one of the most common causes of chronic liver diseases, is increasingly prevalent worldwide. Pentoxifylline, a derivative of theobromine extracted from Theobroma cacao and tea, has been studied for effects on blood viscosity, tissue oxygenation and inflammation. However, its effects on hepatic lipid accumulation and the potential mechanisms remain unclear. PURPOSE: This study aimed to investigate the therapeutic effects of pentoxifylline on high-fat diet-induced NAFL and to explore the corresponding molecular mechanisms. METHODS: NAFL mice were injected with or without 25, 50 or 100 mg/kg pentoxifylline for 2 weeks. Hepatic steatosis was observed by haematoxylin-eosin staining and Oil Red O staining, the levels of serum total cholesterol, triglyceride were detected by biochemical kits, and insulin resistance was evaluated by glucose and insulin tolerance tests. In addition, we measured the frequencies of macrophage and its polarization subsets in the liver using flow cytometry and immunofluorescence. The expressions of proteins associated with macrophage polarization signaling pathways were assessed by Western blotting and flow cytometry histograms. Molecular docking and cellular thermal shift assay were conducted to identify and verify the target protein of pentoxifylline in macrophage. RESULTS: Pentoxifylline significantly alleviated hepatic lipid accumulation, reduced blood lipid levels and improved insulin resistance. Strikingly, the excessive M1 macrophages in NAFL development was abolished by pentoxifylline. And pentoxifylline was further evidenced it failed to reduce hepatocyte lipid accumulation in the absence of macrophages in vitro. Mechanistically, pentoxifylline competed with LPS for binding to toll-like receptor 4, dramatically inhibiting the TLR4/MyD88/NF-κB signaling pathway. CONCLUSION: Pentoxifylline attenuated NAFL by inhibiting hepatic macrophage M1 polarization, indicating that pentoxifylline could be a therapeutic candidate for NAFL. This study first observed that M1 macrophages were increased in NAFL mice and then revealed the molecule targeted by pentoxifylline. In addition, we provided evidence that macrophage targeting may be an emerging strategy for NAFL treatment.

Discovery and Biochemical Characterization of N-methyltransferase Genes Involved in Purine Alkaloid Biosynthetic Pathway of Camellia gymnogyna Hung T.Chang (Theaceae) from Dayao Mountain.

In the present study, purine alkaloid analysis and transcriptome of Camellia gymnogyna Hung T. Chang (Theaceae) from Dayao Mountain were performed by high-performance liquid chromatography (HPLC) and RNA-Seq, respectively. The results showed that the major purine alkaloids accumulated in Camellia gymnogyna Hung T. Chang (Theaceae) were theobromine together with a small amount of theacrine and caffeine. Through polymerase chain reaction (PCR), three types of cDNA encoding N-methyltransferases were isolated from the leaves of Camellia gymnogyna Hung T. Chang (Theaceae) and designated GCS1, GCS2, and GCS3. We subsequently expressed GCS1, GCS2, and GCS3 in Escherichia coli and incubated lysates of the bacterial cells with a variety of xanthine substrates in the presence of S-adenosyl-L-methionine as the methyl donor. We found that the recombinant GCS1 proteins catalyzed 1,3,7-trimethyluric acid to produce theacrine, the recombinant GCS3 proteins catalyzed 7-methylxanthine to produce theobromine, while the recombinant GCS2 proteins did not catalyze any xanthine derivatives. Simultaneous analysis of the expressions of GCS1, GCS2, GCS3, and a caffeine synthase gene (TCS1) in Camellia gymnogyna Hung T. Chang (Theaceae) and other tea plants provided a reference for further research on the functions of these genes.

Effects of long-term treatment with dietary theobromine on rat skeletal muscles.

BACKGROUND: Plastic changes of skeletal muscles, such as hypertrophy and atrophy, are dependent on physiological activities and regulated by a variety of signaling pathways, including cyclic adenosine monophosphate (cAMP) pathway. The cAMP inducing agents, such as the ß2-adrenergic agonist clenbuterol, are known to induce muscle hypertrophy, and has been reported to induce slow-to-fast transitions in rat soleus muscle. Theobromine, one of the active components of cacao, functions as an inhibitor of phosphodiesterase and increases cAMP. This study hypothesized that theobromine, like clenbuterol, can induce muscle hypertrophy and influence contractile properties. METHODS AND RESULTS: Male Wistar rats were fed a normal diet or a diet containing 0.05% theobromine for 20 weeks. Using biochemical, anatomical, and physiological techniques, effects of dietary theobromine on skeletal muscles (soleus, extensor digitorum longus, plantaris, and gastrocnemius) were examined. There were no significant differences in body weight, serum levels of proteins and lipids, muscle weights, dry/wet ratio of muscle weights, mitochondrial oxidation enzyme activity of muscles, isometric contractile properties of muscles, and muscle fatigue between control and theobromine-fed rats. Quantitative analysis of mRNA, however, revealed upregulation of myosin heavy chain 2x and myogenic differentiation 1, as previously reported in clenbuterol-treated muscles. CONCLUSION: The long-term theobromine (0.05%) diet in rats had no effect in inducing muscle hypertrophy and in changing contractile properties, although it had some similar effects of clenbuterol on muscle gene expression.

Finger sweat analysis enables short interval metabolic biomonitoring in humans.

Metabolic biomonitoring in humans is typically based on the sampling of blood, plasma or urine. Although established in the clinical routine, these sampling procedures are often associated with a variety of compliance issues, which are impeding time-course studies. Here, we show that the metabolic profiling of the minute amounts of sweat sampled from fingertips addresses this challenge. Sweat sampling from fingertips is non-invasive, robust and can be accomplished repeatedly by untrained personnel. The sweat matrix represents a rich source for metabolic phenotyping. We confirm the feasibility of short interval sampling of sweat from the fingertips in time-course studies involving the consumption of coffee or the ingestion of a caffeine capsule after a fasting interval, in which we successfully monitor all known caffeine metabolites as well as endogenous metabolic responses. Fluctuations in the rate of sweat production are accounted for by mathematical modelling to reveal individual rates of caffeine uptake, metabolism and clearance. To conclude, metabotyping using sweat from fingertips combined with mathematical network modelling shows promise for broad applications in precision medicine by enabling the assessment of dynamic metabolic patterns, which may overcome the limitations of purely compositional biomarkers.

Simultaneous determination of caffeine and its metabolites in rat plasma by UHPLC-MS/MS.

Caffeine is a widely consumed psychostimulant with several mechanisms of action and various positive and negative effects on organisms. Caffeine undergoes extensive hepatic metabolism to form main metabolites such as theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid. However, interspecies diversities have been observed in caffeine metabolism. In the present study, we developed a sensitive and straightforward ultra-high-performance liquid chromatography-tandem mass spectrometry method to quantify caffeine and its primary metabolites, namely theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid in rat plasma. After extraction of analytes using micro solid-phase extraction plate, analytes were separated by elution gradient on the Acquity UPLC HSS T3 (50 × 2.1 mm, 1.8 µm) column over 4 min. The detection was done on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring modes using a positive electrospray ionization interface. The method was successfully validated according to the European Medicine Agency guideline over a concentration range of 5-1500 ng/ml for caffeine, 5-1200 ng/mL for theobromine, and 2.5-1200 ng/mL for theophylline, paraxanthine, and 1,3,7-trimethyluric acid. The developed method was applied to analyze samples from animal experiments focusing on the metabolism and effects of caffeine and caffeine-containing beverages.

Exploring the Association between Urine Caffeine Metabolites and Urine Flow Rate: A Cross-Sectional Study.

Examination of urine excretion of caffeine metabolites has been a simple but common way to determine the metabolism and effect of caffeine, but the relationship between urinary metabolites and urine flow rate is less discussed. To explore the association between urinary caffeine metabolite levels and urine flow rate, 1571 participants from the National Health and Nutrition Examination Survey (NHANES) 2011-2012 were enrolled in this study. We examined the association between urinary caffeine metabolites and urine flow rate with linear regression models. Separate models were constructed for males and females and for participants aged <60 and ≥60 years old. A positive association was found between concentrations of several urinary caffeine metabolites and urine flow rate. Three main metabolites, namely, paraxanthine, theobromine, and caffeine, showed significance across all subgroups. The number of caffeine metabolites that revealed flow-dependency was greater in males than in females and was also greater in the young than in the elderly. Nevertheless, the general weakness of NHANES data, a cross-sectional study, is that the collection is made at one single time point rather than a long-term study. In summary, urinary concentrations of several caffeine metabolites showed a positive relationship with the urine flow rate. The trend is more noticeable in males and in young subgroups.

3-Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2.

BACKGROUND: Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3-methylxanthine. RESULTS: Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3-Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3-Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3-methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe2+ promoted 3-methylxanthine production significantly (p < 0.05). CONCLUSIONS: This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3-methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.

Nutritional value and safety of animal feed supplemented with Talaromyces verruculosus-treated cocoa pod husks.

Theobromine exerts deleterious effects on animal physiology. Removal of theobromine from the millions of metric tons of cocoa pod husks (CPH) discarded annually could allow for the production of cheap, CPH-based animal feed. The aim of this study was to evaluate safety and nutritional value of bio-detheobrominated CPH in Sprague-Dawley rats. Theobromine was removed from CPH by treatment with an isolate of Talaromyces verruculosus (TvTD). Substituted feeds containing CPH were formulated by replacing 30% or 50% of the maize content of regular rat feed with TvTD-treated or inactivated TvTD-treated CPH. Feeding groups included control groups without or with theobromine administration. Effects of the feed formulations on water and feed intake, weight gain, blood biochemistry and organ-specific toxicity were assessed. Rats ingesting theobromine in inactivated TvTD-treated CPH-based diet or by oral gavage variably exhibited marked deleterious effects, mainly evident in body weight, thymus wet weight and tissue histology. In contrast, substitution with TvTD-treated CPH caused significant increase in body weight. Substitution at 30% did not cause mortality or organ-specific toxicity with reference to the testes, kidneys, spleen or liver, unlike substitution at 50%. The data demonstrate that detheobrominated CPH may safely replace up to 30% of maize in animal feed formulations.

On-line in-tube solid phase microextraction coupled to capillary liquid chromatography-diode array detection for the analysis of caffeine and its metabolites in small amounts of biological samples.

In-tube solid phase microextraction (IT-SPME) coupled on-line to capillary liquid chromatography with diode array detection provides a simple and fast analytical methodology for the simultaneous quantitation of caffeine and its three primary metabolites (theobromine, paraxanthine and theophylline) in micro samples of serum, saliva and urine matrices. The sample amount required for one analysis was only 2.5 µL of saliva, 6.25 µL of serum or 40 µL of urine, a requirement for its implementation in a hospital laboratory for preterm newborns, where sample availability is a major problem. In standard conditions, 25 µL of diluted saliva or serum (or 100 µL of urine) were processed by IT-SPME in 30 cm of commercially available capillary GC column coated with ZB-FFAP (100% nitroterephthalic modified polyethylene glycol). The retained compounds were desorbed from the IT-SPME capillary by the mobile phase (a gradient mixture of water and methanol) and the separation was carried out in a C18 column (150 mm × 0.5 mm i.d., 5 µm particle size). Analytes eluted before 14 min, at a flow rate of 15 µL min-1, and were detected by absorbance at 275 nm. The calibration graphs presented good linearity (R2 > 0.99), without the presence of matrix effect, and recoveries between 84 and 112% were obtained. Limits of detection (S/N = 3) were 0.1 µg·mL-1 in serum and 0.5 µg·mL-1 in saliva and urine samples, for all compounds, and the intra- and inter-day variation coefficients (n = 3) were between 3 and 17%. Analytical figures of merit were similar to those proposed by other methodologies, but using lower sample volume and a faster and simpler sample treatment and analysis. Paired samples of serum and saliva from preterm newborns treated with caffeine at the pediatric intensive care unit were analyzed by the method, with statistically equivalent results for caffeine concentrations.

Purification and characterization of theobromine synthase in a Theobromine-Enriched wild tea plant (Camellia gymnogyna Chang) from Dayao Mountain, China.

Camellia gymnogyna Chang (CgC), a wild tea plant, was discovered on Dayao Mountain, China. However, research regarding this tea plant is limited. Our study found that CgC contains theobromine, caffeine, and theacrine, among which theobromine content was the highest (14.37-39.72 mg/g). In addition, theobromine synthase (TS) was partially purified from CgC leaves, up to 35.87-fold, with consecutive chromatography, and its molecular weight was found to be approximately 62 kDa. The optimum reaction time, pH, and temperature for theobromine synthase from 7-methylxanthine was found to be 6 h, 4, and 45 °C, respectively. TS expression at both mRNA and protein stages was higher in the first than in the fourth leaf (P < 0.05). Subcellular localization of TS indicated that it was localized in the nucleus. These results indicate that CgC can be of scientific value and could lead to efficient utilization of this rare wild tea germplasm.

Caffeine Content and Related Gene Expression: Novel Insight into Caffeine Metabolism in Camellia Plants Containing Low, Normal, and High Caffeine Concentrations.

Caffeine is a crucial secondary metabolic product in tea plants. Although the presence of caffeine in tea plants has been identified, the molecular mechanisms regulating relevant caffeine metabolism remain unclear. For the elucidation of the caffeine biosynthesis and catabolism in Camellia plants, fresh, germinated leaves from four Camellia plants with low (2), normal (1), and high (1) caffeine concentrations, namely, low-caffeine tea 1 (LCT1, Camellia crassicolumna), low-caffeine tea 2 (LCT2, C. crassicolumna), Shuchazao (SCZ, C. sinensis), and Yunkang 43 (YK43, C. sinensis) were used in this research. Transcriptome and purine alkaloids analyses of these Camellia leaves were performed using RNA-Seq and liquid chromatography-mass spectrometry (LC-MS). Moreover, 15N-caffeine tracing was performed to determine the metabolic fate of caffeine in leaves of these plants. Caffeine content was correlated with related gene expression levels, and a quantitative real-time (qRT) PCR analysis of specific genes showed a consistent tendency with the obtained transcriptomic analysis. On the basis of the results of stable isotope-labeled tracer experiments, we discovered a degradation pathway of caffeine to theobromine. These findings could assist researchers in understanding the caffeine-related mechanisms in Camellia plants containing low, normal, and high caffeine content and be applied to caffeine regulation and breeding improvement in future research.

Role of Theobromine in Cocoa's Metabolic Properties in Healthy Rats.

Cocoa is rich in polyphenols and methylxanthines, and it has been reported that its consumption, among other properties, has beneficial effects on metabolism. This study aimed to investigate the role of theobromine in cocoa's metabolic properties in healthy rats. In addition to morphometric measurements, biochemical markers of lipids and glucose metabolism and gene expression of molecules related to immune cells in adipose and hepatic tissues were assessed after 7 or 18 days of diet. Additionally, a metabolomic analysis was carried out at day 7. This study revealed the presence of six discriminant metabolites in plasma due to the diets. Moreover, the results showed that theobromine is the main responsible factor for cocoa's effects on body weight gain as well as on lipid and glucose metabolism. The effects on body weight and lipids appeared as early as after 7 days of diet, whereas those affecting glucose metabolism required a longer intervention.

Study on the interaction and antioxidant activity of theophylline and theobromine with SOD by spectra and calculation.

Theophylline (TP) and theobromine (TB) are the methyl derivatives of xanthine. The antioxidation of TP and TB as well as the effect of the antioxidation and activity of copper­zinc superoxide dismutase (SOD) with TP and TB were investigated. The contents of MDA in cells showed that both TP (14.49 µmol/g) and TB (14.25 µmol/g) are active in oxidation resistance and closed to the antioxidant effect of SOD (13.77 µmol/g). With the formation of TP-SOD and TB-SOD, the antioxidant ability can be superimposed. The interactions between TP/TB and SOD were studied by ultraviolet spectrum, fluorescence spectrum and molecular docking. The results showed that the complex of TP/TB and SOD with 1:1 component was stabilized by hydrogen bonding and van der Waals forces. The analysis also indicated that the microenvironment and structure of SOD were changed. All of the results indicate that the complex formation of TP-SOD and TB-SOD can maintain their respective antioxidant effects without changes in the activity of SOD.

Chronic administration of theobromine inhibits mTOR signal in rats.

Theobromine is a caffeine derivative and the primary methylxanthine in Theobroma cacao. We have shown previously that theobromine inhibits the Akt-mammalian target of rapamycin (mTOR) signal in vitro. In this study, we investigated whether orally administered theobromine could inhibit mTOR activity in rats. mTOR is phosphorylated by Akt. Thus, the level of phosphorylated mTOR was used as an index of mTOR activity. Male Wistar rats were divided into two groups. The control group (CN) was fed a normal diet, while the theobromine group (TB) was fed a diet supplemented with 0.05% theobromine for 40 days. We measured body-weights and tissue weights, food and water intake, blood count, concentrations of theobromine in the plasma, liver and brain, and the levels of phosphorylated mTOR in the liver and brain. Orally administered theobromine did not affect the body-weights and tissue weights, food and water intake, and blood count as determined by comparison with levels in rats that were fed standard chow. Theobromine was detected in the plasma, liver and brain obtained from TB rats, but was not detected in tissues obtained from CN rats. The phosphorylated mTOR levels in the liver and brain were significantly lower in TB rats than in CN rats. The results suggest that oral theobromine inhibits mTOR signalling in vivo.