Gut-derived metabolite 3-methylxanthine enhances cisplatin-induced apoptosis via dopamine receptor D1 in a mouse model of ovarian cancer.

Platinum-based chemotherapy failure represents a significant challenge in the management of ovarian cancer (OC) and contributes to disease recurrence and poor prognosis. Recent studies have shed light on the involvement of the gut microbiota in modulating anticancer treatments. However, the precise underlying mechanisms, by which gut microbiota regulates the response to platinum-based therapy, remain unclear. Here, we investigated the role of gut microbiota on the anticancer response of cisplatin and its underlying mechanisms. Our results demonstrate a substantial improvement in the anticancer efficacy of cisplatin following antibiotic-induced perturbation of the gut microbiota in OC-bearing mice. 16S rRNA sequencing showed a pronounced alteration in the composition of the gut microbiome in the cecum contents following exposure to cisplatin. Through metabolomic analysis, we identified distinct metabolic profiles in the antibiotic-treated group, with a notable enrichment of the gut-derived metabolite 3-methylxanthine in antibiotic-treated mice. Next, we employed a strategy combining transcriptome analysis and chemical-protein interaction network databases. We identified metabolites that shared structural similarity with 3-methylxanthine, which interacted with genes enriched in cancer-related pathways. It is identified that 3-methylxanthinesignificantly enhances the effectiveness of cisplatin by promoting apoptosis both in vivo and in vitro. Importantly, through integrative multiomics analyses, we elucidated the mechanistic basis of this enhanced apoptosis, revealing a dopamine receptor D1-dependent pathway mediated by 3-methylxanthine. This study elucidated the mechanism by which gut-derived metabolite 3-methylxanthine mediated cisplatin-induced apoptosis. Our findings highlight the potential translational significance of 3-methylxanthine as a promising adjuvant in conjunction with cisplatin, aiming to improve treatment outcomes for OC patients.IMPORTANCEThe precise correlation between the gut microbiota and the anticancer effect of cisplatin in OC remains inadequately understood. Our investigation has revealed that manipulation of the gut microbiota via the administration of antibiotics amplifies the efficacy of cisplatin through the facilitation of apoptosis in OC-bearing mice. Metabolomic analysis has demonstrated that the cecum content from antibiotic-treated mice exhibits an increase in the levels of 3-methylxanthine, which has been shown to potentially enhance the therapeutic effectiveness of cisplatin by an integrated multiomic analysis. This enhancement appears to be attributable to the promotion of cisplatin-induced apoptosis, with 3-methylxanthine potentially exerting its influence via the dopamine receptor D1-dependent pathway. These findings significantly contribute to our comprehension of the impact of the gut microbiota on the anticancer therapy in OC. Notably, the involvement of 3-methylxanthine suggests its prospective utility as a supplementary component for augmenting treatment outcomes in patients afflicted with ovarian cancer.

Tea (Camellia sinensis) cultivated in three agro-ecological regions of Bangladesh: Unveiling the variability of methylxanthine, bioactive phenolic compound, and antioxidant activity.

Tea (Camellia sinensis) is a widely consumed beverage known for its numerous health benefits, largely attributed to its rich content of quality determining secondary metabolites such as methylxanthine compounds and bioactive phenolic compounds. The goal of this study was to find out variations of the levels of methylxanthines, bioactive phenolic compounds, and antioxidant activity in methanolic and hot water extracts of 129 tea samples grown in three different ecological regions of Bangladesh named Panchagar, Sylhet, and Chattogram. Methylxanthine and bioactive phenolic compounds were determined by using HPLC-DAD, and the antioxidant profile was analysed by UV-vis spectrophotometric methods for methanol and hot water extracts of tea leaves. The IC50 values showed the trend as Panchagar > Sylhet > Chattogram and Sylhet > Chattogram > Panchagar for water and methanol extract, respectively. The results revealed significant (p < 0.05) variations in the levels of methylxanthines content: Panchagar > Chattogram > Sylhet. Caffeine was significantly higher (103.02 ± 5.55 mg/g of dry extract) in the methanolic extract of tea leaves of Panchagar district and lower (53.33 ± 4.30 mg/g of dry extract) in the hot water extract of Sylhet district. Panchagar and Chattogram possessed significantly (p < 0.05) higher catechin content for methanol (57.01 ± 5.50 mg/g dry extract) and hot water (55.23 ± 4.11 mg/g dry extract) extracts, respectively. The utilization of canonical discriminant functions yielded highly favorable outcomes in the classification of tea from three distinct cultivation origins in Bangladesh, relying on their inherent features. This study demonstrated the potential effects of geographical variations on the bioactive compounds and antioxidant properties of tea, emphasizing the importance of regional differences in tea cultivation for optimizing its health benefits as well as dispersing tea cultivation across the country.

Novel Insecticidal Butenolide-Containing Methylxanthine Derivatives: Synthesis, Crystal Structure, Biological Activity Evaluation, DFT Calculation and Molecular Docking.

The design of novel agrochemicals starting from bioactive natural products is one of the most effective ways in the discovery and development of new pesticidal agents. In this paper, a series of novel butenolide-containing methylxanthine derivatives (Ia-Ir) were designed based on natural methylxanthine caffeine and stemofoline, and the derivatized insecticide flupyradifurone of the latter. The structures of the synthesized compounds were confirmed via 1H-NMR, 13C NMR, HRMS and X-ray single crystal diffraction analyses. The biological activities of the compounds were evaluated against a variety of agricultural pests including oriental armyworm, bean aphid, diamondback moth, fall armyworm, cotton bollworm, and corn borer; the results indicated that some of them have favorable insecticidal potentials, particularly toward diamondback moth. Among others, Ic and Iq against diamondback moth possessed LC50 values of 6.187 mg ⋅ L-1 and 3.269 mg ⋅ L-1, respectively, - 2.5- and 4.8-fold of relative insecticidal activity respectively to that of flupyradifurone (LC50=15.743 mg ⋅ L-1). Additionally, both the DFT theoretical calculation and molecular docking with acetylcholine binding protein were conducted for the highly bioactive compound (Ic). Ic and Iq derived from the integration of caffeine (natural methylxanthine) and butenolide motifs can serve as novel leading insecticidal compounds for further optimization.

Raloxifene-driven benzothiophene derivatives: Discovery, structural refinement, and biological evaluation as potent PPARγ modulators based on drug repurposing.

By virtue of the drug repurposing strategy, the anti-osteoporosis drug raloxifene was identified as a novel PPARγ ligand through structure-based virtual high throughput screening (SB-VHTS) of FDA-approved drugs and TR-FRET competitive binding assay. Subsequent structural refinement of raloxifene led to the synthesis of a benzothiophene derivative, YGL-12. This compound exhibited potent PPARγ modulation with partial agonism, uniquely promoting adiponectin expression and inhibiting PPARγ Ser273 phosphorylation by CDK5 without inducing the expression of adipongenesis associated genes, including PPARγ, aP2, CD36, FASN and C/EBPα. This specific activity profile resulted in effective hypoglycemic properties, avoiding major TZD-related adverse effects like weight gain and hepatomegaly, which were demonstrated in db/db mice. Molecular docking studies showed that YGL-12 established additional hydrogen bonds with Ile281 and enhanced hydrogen-bond interaction with Ser289 as well as PPARγ Ser273 phosphorylation-related residues Ser342 and Glu343. These findings suggested YGL-12 as a promising T2DM therapeutic candidate, thereby providing a molecular framework for the development of novel PPARγ modulators with an enhanced therapeutic index.

Post-occlusive reactive hyperemia in habituated caffeine users: Effects of abstaining versus consuming typical doses.

BACKGROUND: Post-occlusive reactive hyperemia (PORH) typically requires caffeine abstinence. For habitual users, it is unknown if abstinence affects PORH. OBJECTIVE: Compare PORH after habitual users consume or abstain from caffeine. METHODS: On separate visits (within-subject), PORH was measured in 30 participants without abstinence from typical caffeine doses (CAFF) and with abstinence (ABS). Measurements included baseline and peak hyperemic velocity, tissue saturation index slopes during ischemia (Slope 1) and following cuff deflation (Slope 2), resting arterial occlusion pressure (AOP), heart rate (HR), systolic (SBP), and diastolic (DBP) blood pressure. All variables were compared using Bayesian paired t-tests. BF10 = likelihood of alternative vs null. Results are mean±SD. RESULTS: Comparing baseline velocity (cm/s) between CAFF (9.3±4.8) and ABS (7.5±4.9) yielded anecdotal evidence (BF10 = 1.0). Peak hyperemic velocity (cm/s) was similar (CAFF = 77.3±16.7; ABS = 77.6±19.0, BF10 = 0.20). For slopes (TSI% /s), CAFF Slope 1 = -0.11±0.04 and Slope 2 = 1.9±0.46 were similar (both BF10≤0.20) to ABS Slope 1 = -0.12±0.03 and Slope 2 = 1.8±0.42. SBP and DBP (mmHg) were both similar (CAFF SBP = 116.0±9.8, DBP = 69.6±5.8; ABS SBP = 115.5±10.7, DBP = 69.5±5.4; both BF10≤0.22). Comparing AOP (mmHg) (CAFF = 146.6±15.0; ABS = 143.0±16.4) yielded anecdotal evidence (BF10 = 0.46). HR (bpm) was similar (CAFF = 66.5±12.3; ABS = 66.9±13.0; BF10 = 0.20). CONCLUSIONS: In habitual users, consuming or abstaining from typical caffeine doses does not appear to affect post-occlusive reactive hyperemia.

Guarana (Paullinia cupana) but Not Low-Dose Caffeine Improves Cycling Time-Trial Performance Versus Placebo.

Guarana (GUA) seed extract, containing caffeine (CAF) and additional bioactive compounds, may positively affect mental performance, but evidence regarding exercise is limited. This investigation assessed acute GUA ingestion compared with CAF on endurance performance. Eleven endurance-trained noncyclists and cyclists (V˙O2peak = 49.7 ± 5.9, 60.4 ± 4.6 ml·kg·min-1) completed a double-blind, crossover experiment after ingesting (a) 100 mg CAF, (b) 500 mg GUA (containing 130 mg CAF), or (c) placebo (P) prior to 60-min fixed cycling workload (FIX) + 15-min time trial. Oxygen uptake, heart rate, respiratory exchange ratio, blood glucose, and lactate were not different (p ≥ .052) during FIX. A significant interaction (p = .042) for perceived exertion was observed at 50-min FIX with lower rating (p = .023) for GUA versus CAF but not compared with P. Work accumulated over 15-min time trial was greater (p = .038) for GUA versus P due to higher early (1-11 min) work outputs. Work performance favored (effect size = 0.18; 95% confidence interval [0.003, 0.355], p = .046) GUA (241.4 ± 39.9 kJ) versus P (232.1 ± 46.6 kJ), but CAF (232.3 ± 43.9) was not different from GUA (effect size = 0.19; 95% confidence interval [-0.022, 0.410], p = .079) or P. Postexercise strength loss was not attenuated with GUA (-5.6 ± 8.5%) or CAF (-8.3 ± 9.4%) versus P (-10.3 ± 5.1%). Acute GUA ingestion improved work performance relative to P, but effects were trivial to small and unrelated to altered substrate oxidation or muscular strength. Ergogenicity may involve central mechanisms reducing perceived effort with GUA (containing 130 mg caffeine). Due to issues related to identical matching of dosage, whether GUA confers additional benefits beyond its CAF content cannot be determined at present.

Production of 1-methylxanthine via the biodegradation of theophylline by an optimized Escherichia coli strain.

1-Methylxanthine is a high-value derivative of caffeine of limited natural availability with many potential pharmaceutical applications. Unfortunately, production of 1-methylxanthine through purely chemical methods of synthesis are unfavorable due to lengthy chemical processes and the requirement of hazardous chemicals, ultimately resulting in low yields. Here, we describe a novel biosynthetic process for the production of 1-methylxanthine from theophylline using engineered Escherichia coli whole-cell biocatalysts and reaction optimization. When scaled-up to 1590 mL, the simple biocatalytic reaction produced approximately 1188 mg 1-methylxanthine from 1444 mg theophylline, constituting gram-scale production of 1-methylxanthine in as little as 3 hours. Following HPLC purification and solvent evaporation, 1163 mg of dried 1-methylxanthine powder was collected, resulting in a 97.9 wt% product recovery at a purity of 97.8%. This is the first report of a biocatalytic process designed specifically for the production and purification of the high-value biochemical 1-methylxanthine from theophylline. This process is also the most robust methylxanthine N-demethylation process featuring engineered E. coli to date, capable of gram-scale production.

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.

Traversing the Cell Wall: The Chitinolytic Activity of Histoplasma capsulatum Extracellular Vesicles Facilitates Their Release.

Histoplasma capsulatum is the causative agent of histoplasmosis. Treating this fungal infection conventionally has significant limitations, prompting the search for alternative therapies. In this context, fungal extracellular vesicles (EVs) hold relevant potential as both therapeutic agents and targets for the treatment of fungal infections. To explore this further, we conducted a study using pharmacological inhibitors of chitinase (methylxanthines) to investigate their potential to reduce EV release and its subsequent impact on fungal virulence in an in vivo invertebrate model. Our findings revealed that a subinhibitory concentration of the methylxanthine, caffeine, effectively reduces EV release, leading to a modulation of H. capsulatum virulence. To the best of our knowledge, this is the first reported instance of a pharmacological inhibitor that reduces fungal EV release without any observed fungicidal effects.

Cardiorespiratory and Neuroprotective Effects of Caffeine in Neonate Animal Models.

Caffeine is widely used to improve neonatal health in animals with low vitality. Due to its pharmacokinetics and pharmacodynamics, caffeine stimulates the cardiorespiratory system by antagonism of adenosine receptors and alteration in Ca+2 ion channel activity. Moreover, the availability of intracellular Ca+2 also has positive inotropic effects by increasing heart contractibility and by having a possible positive effect on neonate vitality. Nonetheless, since neonatal enzymatic and tissular systems are immature at birth, there is a controversy about whether caffeine is an effective therapy for newborns. This review aims to analyze the basic concepts of caffeine in neonatal animal models (rat and mouse pups, goat kids, lambs, and piglets), and it will discuss the neuroprotective effect and its physiological actions in reducing apnea in newborns.

Caffeine Synthesis and Its Mechanism and Application by Microbial Degradation, A Review.

Caffeine is a metabolite derived from purine nucleotides, typically accounting for 2-5% of the dry weight of tea and 1-2% of the dry weight of coffee. In the tea and coffee plants, the main synthesis pathway of caffeine is a four-step sequence consisting of three methylation reactions and one nucleosidase reaction using xanthine as a precursor. In bacteria, caffeine degradation occurs mainly through the pathways of N-demethylation and C-8 oxidation. However, a study fully and systematically summarizing the metabolism and application of caffeine in microorganisms has not been established elsewhere. In the present study, we provide a review of the biosynthesis, microbial degradation, gene expression, and application of caffeine microbial degradation. The present review aims to further elaborate the mechanism of caffeine metabolism by microorganisms and explore the development prospects in this field.

Role of 7-methylxanthine in myopia prevention and control: a mini-review.

Myopia is becoming increasingly common. By 2050 around 10% of the world's population is expected to be highly myopic (<-5 diopters) and therefore particularly at risk of suffering from sight-threatening complications. Currently used myopia control treatments, such as multifocal soft contact lenses or spectacle lenses, orthokeratology, and atropine eyedrops, either do not completely arrest myopia progression or are associated with significant ocular and possibly systemic side effects. A new candidate for pharmaceutical control of myopia progression and excessive eye elongation, the non-selective adenosine antagonist 7-methylxanthine (7-MX), appears to be non-toxic and effective in reducing myopia progression and axial eye growth in experimental and clinical studies. The latest findings regarding 7-MX for myopia control and evaluate its potential as a supplement to existing treatment options were reviewed.

Could the serum glucose/potassium ratio offer an early reliable predictor of life-threatening events in acute methylxanthine intoxication?

Methylxanthines are widely used to manage pulmonary disorders, particularly in developing countries. Methylxanthines are unsafe due to their narrow therapeutic index and associated morbidity and mortality. The current study aimed to investigate the role of glucose/potassium ratio as a substantially useful early predictor of life-threatening events (LTEs) in the form of cardiovascular and neurological complications among methylxanthine users. A retrospective cohort study was conducted using medical records of patients diagnosed with acute methylxanthine intoxications and presented to an Egyptian Poison Control Center for 2 years. A total of 366 patients were enrolled. Of them, 59 patients (16.1%) were complicated with LTEs. The most frequent serious arrhythmia was T wave inversion (45.6% of patients with LTEs). Laboratory investigations that could significantly predict LTEs were the random blood glucose and potassium levels, glucose/potassium ratio, pH, liver transaminases, HCO3 level, hemoglobin, and platelet count (P < 0.05). The glucose/potassium ratio was the best predictor of LTEs (odds ratio = 2.92, and 95% confidence interval = 2.02-4.23). With an excellent area under the curve (0.906) and at a cutoff of 2.44, that ratio could correctly classify the patients based on their risk of LTEs with an overall accuracy of 73% (sensitivity of 88% and specificity of 70%). The current study endorsed an important, feasible, and easily obtainable ratio that could predictor stratify the patients according to severity and risk of LTEs, which guides the decision-making and prioritizes the treatment lines in methylxanthine intoxicated patients.

Resveratrol butyrate esters inhibit lipid biosynthesis in 3T3-L1 cells by AMP-activated protein kinase phosphorylation.

Resveratrol butyrate esters (RBEs), which are novel resveratrol-synthesized derivatives, exhibit increased biological activity. This study elucidated the effect of RBEs on fat metabolism and their anti-obesity characteristics. Their molecular mechanism was investigated in the 3T3-L1 murine preadipocyte cells and adipocytes. RBE doses of < 2 µM did not induce a significant change in the viability of 3T3-L1 adipocytes. After RBEs treatment, intracellular lipid droplet accumulation in 3T3-L1 adipocytes was stimulated by methylisobutylxanthine, dexamethasone, and insulin-containing medium. However, a significant dose-dependent reduction in intracellular lipid levels was observed. The mRNA levels of two adipogenic transcription factors (peroxisome proliferator-activated receptor [PPAR] and CCAAT/enhancer-binding proteins [C/EBP]) and lipogenic proteins (fatty acid-binding protein 4 [FABP4] and fatty acid synthase [FAS]) were significantly attenuated by RBE treatment in both MDI-stimulated and differentiated 3T3-L1 adipocytes. Moreover, the phosphorylation level of adenosine monophosphate-activated protein kinase (AMPK) also dramatically increased in the MDI + RBE-treated group compared to that in the MDI + vehicle-treated group. Collectively, our study provides strong evidence that RBEs inhibit adipogenesis by regulating adipogenic protein expression and increasing the p-AMPK/AMPK ratio. Future studies will be conducted on animal models to validate the application of RBEs as a functional food ingredient in improving human health. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05436-x.

Metabolism of guarana (Paullinia cupana Kunth var. sorbilis) plants and fruit production subjected to glyphosate doses.

Guarana (Paullinia cupana Kunth var. sorbilis) is a typically Amazonian plant of high economic value due to the compounds found in its seed. For guarana to reach the maximum productive potential, management practices such as weed control are necessary. The use of herbicides is a viable alternative, however, its drift may lead to adverse effects on the primary and secondary plant metabolisms and cause losses in crop production. This study evaluated the differential drift effects of glyphosate doses on the physiology of guarana plants and the production of compounds of economic interest in their seeds. Glyphosate doses (57.6, 115.2, 230.4, 460.8 g ae ha-1) were applied to adult guarana plants after the flowering period. The photosynthetic functions and metabolism effects were evaluated. Herbicide treatments led to oxidative stress due to increased lipid peroxidation and increased carbohydrate and amino acid in their leaflets. Despite this, glyphosate showed no effect on fruit production or the content of secondary metabolites of commercial interest in seeds.

Paraxanthine safety and comparison to caffeine.

Introduction: Caffeine, one of the most ubiquitous ingredients found in beverages and other ingested food products, has a long history of safe use. As a member of the methylxanthine class of stimulants, caffeine is not devoid of unwanted side effects at any serving level. Caffeine safety has been the subject of a safety workshop by FDA and the Institute of Medicine in the past decade. Thus, investigation into an alternate stimulant with similar pharmacology but improved safety is warranted. Paraxanthine (1,7-dimethylxanthine) is the predominant metabolite of caffeine in humans with similar stimulant properties. The few toxicity studies that are available for paraxanthine suggest that the molecule is relatively safe, although thorough characterization of its safety is required prior to widespread incorporation into foods/beverages. Methods: The aim of this study was to evaluate the toxicity of paraxanthine (Rarebird, Inc.) relative to caffeine through a battery of toxicological studies conducted in accordance with international guidelines. These studies evaluated the potential mutagenicity (bacterial reverse mutation, in vitro mammalian chromosomal aberration), genetic toxicity (in vitro mammalian cell gene mutation) and acute, sub-acute and sub-chronic oral toxicity of paraxanthine in Sprague Dawley rats. Results/Discussion: There was no evidence of genetic toxicity or mutagenicity in the in vitro studies. An acute oral LD50 of 829.20 mg/kg body weight (bw) was established. There was no mortality or treatment-related adverse effects in the 14-day repeat dose oral toxicity study, wherein rats received low, mid, or high doses of paraxanthine (50, 100, or 150 mg/kg bw, n = 5 rats/sex/group). The same findings were observed in the subchronic repeat-dose 90-day oral toxicity study at daily doses of paraxanthine of 100, 150, or 185 mg/kg bw which were compared to caffeine at 150 or 185 mg/kg bw (n = 10 animals/sex/group). However, mortality was reported in two animals in the high dose caffeine-treated animals. Therefore, the no observed adverse effect level (NOAEL) from the 90-day study was determined to be 150 mg/kg bw for caffeine and 185 mg/kg bw for paraxanthine for both male and female Sprague Dawley rats. These findings may suggest that paraxanthine could be a safer alternative to caffeine in humans.

Mixed culture biocatalytic production of the high-value biochemical 7-methylxanthine.

BACKGROUND: 7-Methylxanthine, a derivative of caffeine noted for its lack of toxicity and ability to treat and even prevent myopia progression, is a high-value biochemical with limited natural availability. Attempts to produce 7-methylxanthine through purely chemical methods of synthesis are faced with complicated chemical processes and/or the requirement of a variety of hazardous chemicals, resulting in low yields and racemic mixtures of products. In recent years, we have developed engineered microbial cells to produce several methylxanthines, including 3-methylxanthine, theobromine, and paraxanthine. The purpose of this study is to establish a more efficient biosynthetic process for the production of 7-methylxanthine from caffeine. RESULTS: Here, we describe the use of a mixed-culture system composed of Escherichia coli strains engineered as caffeine and theobromine "specialist" cells. Optimal reaction conditions for the maximal conversion of caffeine to 7-methylxanthine were determined to be equal concentrations of caffeine and theobromine specialist cells at an optical density (600 nm) of 50 reacted with 2.5 mM caffeine for 5 h. When scaled-up to 560 mL, the simple biocatalytic reaction produced 183.81 mg 7-methylxanthine from 238.38 mg caffeine under ambient conditions, an 85.6% molar conversion. Following HPLC purification and solvent evaporation, 153.3 mg of dried 7-methylxanthine powder was collected, resulting in an 83.4% product recovery. CONCLUSION: We present the first report of a biocatalytic process designed specifically for the production and purification of the high-value biochemical 7-methylxanthine from caffeine using a mixed culture of E. coli strains. This process constitutes the most efficient method for the production of 7-methylxanthine from caffeine to date.

Cyclic AMP Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.

The cyclic AMP assay is a functional assay that is commonly used to determine the pharmacological behavior (agonists, antagonists, and inverse agonists) of G-protein coupled receptor ligands. Here, we describe the cyclic AMP assay that is carried out with commercially available nonradioligand ready-to-use kits and CHO (Chinese Hamster Ovarian) cells stably transfected with the human cannabinoid CB2 receptor.

From Cocoa to Chocolate: Effect of Processing on Flavanols and Methylxanthines and Their Mechanisms of Action.

Despite the health benefits associated with the ingestion of the bioactive compounds in cocoa, the high concentrations of polyphenols and methylxanthines in the raw cocoa beans negatively influence the taste, confer the astringency and bitterness, and affect the stability and digestibility of the cocoa products. It is, therefore, necessary to process cocoa beans to develop the characteristic color, taste, and flavor, and reduce the astringency and bitterness, which are desirable in cocoa products. Processing, however, affects the composition and quantities of the bioactive compounds, resulting in the modification of the health-promoting properties of cocoa beans and chocolate. In this advanced review, we sought to better understand the effect of cocoa's transformational process into chocolate on polyphenols and methylxanthine and the mechanism of action of the original flavanols and methylxanthines. More data on the cocoa processing effect on cocoa bioactives are still needed for better understanding the effect of each processing step on the final polyphenolic and methylxanthine composition of chocolate and other cocoa products. Regarding the mechanisms of action, theobromine acts through the modulation of the fatty acid metabolism, mitochondrial function, and energy metabolism pathways, while flavanols mainly act though the protein kinases and antioxidant pathways. Both flavanols and theobromine seem to be involved in the nitric oxide and neurotrophin regulation.

Comparison of bioactive components and flavor volatiles of diverse cocoa genotypes of Theobroma grandiflorum, Theobroma bicolor, Theobroma subincanum and Theobroma cacao.

Theobroma grandiflorum, T. bicolor and T. subincanum represent underutilized genetic resources for cocoa quality. The bioactive components and flavor volatiles of different bean genotypes of these species were profiled and compared with those of T. cacao. T. bicolor and T. grandiflorum had different profiles of methylxanthines and polyphenols from T. bicolor and T. cacao. T. subincanum and T. grandiflorum were rich in theacrine and flavones. T. grandiflorum, T. bicolor and T. subincanum beans generally had less phenolics than T. cacao. Roasting decreased the concentrations of polyphenols and methylxanthines in the beans. Roasted T. grandiflorum and T. subincanum beans had higher concentrations of pyrazines and esters than T. cacao. T. grandiflorum and T. subincanum beans had more odor-active volatiles than T. cacao. Overall, the underutilized Theobroma species have potential to be exploited to improve the flavor and nutritional quality of cocoa products.