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.

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.

TRPA1, TRPV1, and Caffeine: Pain and Analgesia.

Caffeine (1,3,7-trimethylxanthine) is a naturally occurring methylxanthine that acts as a potent central nervous system stimulant found in more than 60 different plants and fruits. Although caffeinated beverages are widely and casually consumed, the application of caffeine beyond dietary levels as pharmacologic therapy has been recognized since the beginning of its recorded use. The analgesic and vasoactive properties of caffeine are well known, but the extent of their molecular basis remains an area of active research. There is existing evidence in the literature as to caffeine's effect on TRP channels, the role of caffeine in pain management and analgesia, as well as the role of TRP in pain and analgesia; however, there has yet to be a review focused on the interaction between caffeine and TRP channels. Although the influence of caffeine on TRP has been demonstrated in the lab and in animal models, there is a scarcity of data collected on a large scale as to the clinical utility of caffeine as a regulator of TRP. This review aims to prompt further molecular research to elucidate the specific ligand-host interaction between caffeine and TRP by validating caffeine as a regulator of transient receptor potential (TRP) channels-focusing on the transient receptor potential vanilloid 1 (TRPV1) receptor and transient receptor potential ankyrin 1 (TRPA1) receptor subtypes-and its application in areas of pain.

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.

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.

Biocatalytic production of 7-methylxanthine by a caffeine-degrading Escherichia coli strain.

7-Methylxanthine, a derivative of caffeine (1,3,7-trimethylxanthine), is a high-value compound that has multiple medical applications, particularly with respect to eye health. Here, we demonstrate the biocatalytic production of 7-methylxanthine from caffeine using Escherichia coli strain MBM019, which was constructed for production of paraxanthine (1,7-dimethylxanthine). The mutant N-demethylase NdmA4, which was previously shown to catalyze N3 -demethylation of caffeine to produce paraxanthine, also retains N1 -demethylation activity toward paraxanthine. This study demonstrates that whole cell biocatalysts containing NdmA4 are more active toward paraxanthine than caffeine. We used four serial resting cell assays, with spent cells exchanged for fresh cells between each round, to produce 2,120 µM 7-methylxanthine and 552 µM paraxanthine from 4,331 µM caffeine. The purified 7-methylxanthine and paraxanthine were then isolated via preparatory-scale HPLC, resulting in 177.3 mg 7-methylxanthine and 48.1 mg paraxanthine at high purity. This is the first reported strain genetically optimized for the biosynthetic production of 7-methylxanthine from caffeine.

Phosphodiesterases S-sulfhydration contributes to human skeletal muscle function.

The increase in intracellular calcium is influenced by cyclic nucleotides (cAMP and cGMP) content, which rating is governed by phosphodiesterases (PDEs) activity.Despite it has been demonstrated a beneficial effect of PDEs inhibitors in different pathological conditions involving SKM, not much is known on the role exerted by cAMP-cGMP/PDEs axis in human SKM contractility. Here, we show that Ssulfhydration of PDEs modulates human SKM contractility in physiological and pathological conditions. Having previously demonstrated that, in the rare human syndrome Malignant Hyperthermia (MH), there is an overproduction of hydrogen sulfide (H2S) within SKM contributing to hyper-contractility, here we have used MH negative diagnosed biopsies (MHN) as healthy SKM, and MH susceptible diagnosed biopsies (MHS) as a pathological model of SKM hypercontractility. The study has been performed on MHS and MHN human biopsies after diagnosis has been made and on primary SKM cells derived from both MHN and MHS biopsies. Our data demonstrate that in normal conditions PDEs are S-sulfhydrated in both quadriceps' biopsies and primary SKM cells. This post translational modification (PTM) negatively regulates PDEs activity with consequent increase of both cAMP and cGMP levels. In hypercontractile biopsies, due to an excessive H2S content, there is an enhanced Ssulfhydration of PDEs that further increases cyclic nucleotides levels contributing to SKM hyper-contractility. Thus, the identification of a new endogenous PTM modulating PDEs activity represents an advancement in SKM physiopathology understanding.

7-Methylxanthine Influences the Behavior of ADORA2A-DRD2 Heterodimers in Human Retinal Pigment Epithelial Cells.

INTRODUCTION: The goal of this study was to investigate the presence of ADORA2A-DRD2 heterodimers in human retinal pigment epithelial (RPE) cells; determine if 7-methylxanthine (7-MX), a nonselective adenosine receptor antagonist which was used to control myopia progression, can influence the behavior of RPE cells through the ADORA2A-DRD2 receptor pathway; and assess the changes in the expression of signaling molecules during cellular signal transduction. METHODS: Human RPE cells were cultured in vitro in the presence or absence of 7-MX. Cell proliferation was evaluated with the CCK-8 assay. Apoptosis and necrosis rates were determined by annexin V-FITC/propidium iodide staining and flow cytometry. Immunofluorescence and coimmunoprecipitation were used to examine the protein expression and colocalization of ADORA2A and DRD2 in RPE cells. ADORA2A and DRD2 were knocked down with small interfering RNAs (siRNAs). Changes in the protein expression of ERK1/2 and phospho-ERK1/2 (pERK 1/2), which are signaling molecules downstream of dopamine receptors, were evaluated by Western blot analysis. RESULTS: Immunofluorescence and coimmunoprecipitation showed that ADORA2A and DRD2 were colocalized in RPE cells. The expression of ADORA2A in RPE cells was inhibited by treatment with 50 µmol/L 7-MX for 48 h, and the expression of DRD2, ERK1/2, and pERK1/2 was increased after treatment with 50 µmol/L 7-MX for 48 h. After siRNA-mediated knockdown of DRD2 in RPE cells and further treatment with 50 µmol/L 7-MX for 48 h, the expression of DRD2 was nearly restored to the level observed in the native control. At the experimental concentrations, 7-MX and siRNAs did not affect the proliferation or apoptosis of human RPE cells. CONCLUSIONS: ADORA2A and DRD2 heterodimers were present in RPE cells. 7-MX may affect the behaviors of RPE cells through the ADORA2A-DRD2 receptor pathway. 7-MX is an inhibitor of ADORA2A receptors that can prevent inhibition of the DRD2 receptor pathway and increase DRD2 receptor pathway activity. This phenomenon may explain the mechanism of action through which 7-MX can control myopia progression.

Sub-chronic and chronic toxicity evaluation of 7-methylxanthine: a new molecule for the treatment of myopia.

Myopia (nearsightedness) is a vision disorder with a blurring of far objects, affect millions worldwide. 7-methylxanthine (7-MX) is a molecule that is presently under clinical investigation for the treatment of myopia. In the present study, we have investigated sub-chronic and chronic toxicity of 7-MX in comparison to other clinically used methylxanthines i.e., caffeine and theobromine as per OECD guidelines 408 and 452. 7-MX was administered orally for 90 days at three different doses of 250, 500, and 1000 mg/kg for sub-chronic toxicity evaluation, and at a limit dose of 1000 mg/kg in 180 days chronic toxicity evaluation in rats. In sub-chronic treatment, 7-MX showed no mortality and signs for toxicity in any group, whereas 10% and 40% mortality with signs for toxicity were observed in caffeine and theobromine treated groups, respectively. A similar, safety profile was observed with 7-MX in 180 days of chronic toxicity study. Further, to confirm any morphological changes in organs; ultrasound and X-rays analysis were performed and no changes in the size of organs, cyst formation, fluid retention, or crystal formation was observed. Thus, the repeated dose study of 7-MX for 180 days may augment the possibility of using 7-MX clinically for the safe and effective treatment of myopia.

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.

Neuroprotective effects of theobromine in transient global cerebral ischemia-reperfusion rat model.

Transient global cerebral ischemia (tGCI) is a cerebrovascular disorder characterized by a brief decline in blood flow, neurological deficits, and is often predictive of stroke. Theobromine (TBR) is consumed worldwide in chocolates, tea, and cocoa products. TBR is a natural stimulant and vasoactive alkaloid that may protect against ischemic injury. In this study, neuroprotective potential of theobromine (TBR) was evaluated in 2-vessel occlusion transient global cerebral ischemia-reperfusion (tGCI/R) rat model. Rats were treated with TBR (50, 100 mg/kg, p.o.) for 7 successive days, and subjected to bilateral common carotid artery occlusion (20 min) or sham surgery after last dose of TBR. Severe neurological deficits accompanied by brain infarction, blood-brain barrier abnormalities, and oedema were noted in rats subjected to tGCI/R, and these effects were prevented by TBR. TBR protected against lipid peroxidation and enhanced glutathione level in brain against tGCI/R. TBR pre-treatment for 7 days prevented tGCI/R induced cell death (lactate dehydrogenase, caspase-3), vascular injury (MMP-9), and inflammation (TNF-α, NFκB) in rat whole brain. TBR protected against glutamate excitotoxicity and GABAergic decline in the brain of rats against tGCI/R injury. Findings of this study showed that TBR can alleviate chances of stroke by preventing acute episodes of cerebral ischemia.

Combined methazolamide and theophylline improves oxygen saturation but not exercise performance or altitude illness in acute hypobaric hypoxia.

NEW FINDINGS: What is the central question of this study? Does the combination of methazolamide and theophylline reduce symptoms of acute mountain sickness (AMS) and improve aerobic performance in acute hypobaric hypoxia? What is the main finding and its importance? The oral combination of methazolamide (100 BID) and theophylline (300 BID) improved arterial oxygen saturation but did not reduce symptoms of AMS and impaired aerobic performance. We do not recommend this combination of drugs for prophylaxis against the acute negative effects of hypobaric hypoxia. ABSTRACT: A limited number of small studies have suggested that methazolamide and theophylline can independently reduce symptoms of acute mountain sickness (AMS) and, if taken together, can improve aerobic exercise performance in normobaric hypoxia. We performed a randomized, double-blind, placebo-controlled, cross-over study to determine if the combination of oral methazolamide and theophylline could provide prophylaxis against AMS and improve aerobic performance in hypobaric hypoxia (∼4875 m). Volunteers with histories of AMS were screened at low altitude (1650 m) and started combined methazolamide (100 mg BID) and theophylline (300 mg BID) treatment, or placebo, 72 h prior to decompression. Baseline AMS (Lake Louise Questionnaire), blood (haemoglobin, haematocrit), cognitive function, ventilatory and pulse oximetry ( SpO2 ) measures were assessed at low altitude and repeated between 4 and 10 h of exposure to hypobaric hypoxia (PB  = 425 mmHg). Aerobic exercise performance was assessed during a 12.5 km cycling time trial (TT) after 4 h of hypobaric hypoxia. Subjects repeated all experimental procedures after a 3-week washout period. Differences between drug and placebo trials were evaluated using repeated measures ANOVA (α = 0.05). The drugs improved resting SpO2 by ∼4% (P < 0.01), but did not affect the incidence or severity of AMS or cognitive function scores relative to placebo. Subjects' performance on the 12.5 km TT was ∼3% worse when taking the drugs (P < 0.01). The combination of methazolamide and theophylline in the prescribed dosages is not recommended for use at high altitude as it appears to have no measurable effect on AMS and can impair aerobic performance.

Identification of the anti-fungal drug fenticonazole nitrate as a novel PPARγ-modulating ligand with good therapeutic index: Structure-based screening and biological validation.

In this study, SB-VHTS of the old drug library was conducted to seek for novel PPARγ ligand. In the end, an antifungal drug, FN, was identified in vitro and in vivo as a new and potent PPARγ-modulating ligand to demonstrate significantly anti-diabetic and anti-NAFLD efficacies with minimized side effects induced by PPARγ full agonists TZDs drugs. Further mechanistic investigations revealed that FN showed such desired pharmacological properties mainly through selectively activating the expressions of Adiponectin and GLUT4, effectively promoting the Akt Ser473 phosphorylation, inhibiting the expressions of proinflammatory genes including TNF-α, IL-1ß and IL-6 and blocking the PPARγ Ser273 phosphorylation mediated by CDK5 without leading to adipogenesis and increasing the expressions of key adipogenic genes CD36, AP2, LPL, C/EBPα, FASN and PPARγ. Subsequently, a molecular docking study revealed an interesting binding mode between FN and PPARγ LBD including the hydrogen-bonding network among oxygen atom, sulfur atom and nitrogen atom in FN respectively with the PPARγ residues Cys285, Tyr327 and Ser342, which gave proof of concept for the above anti-diabetic action mechanism. Taken together, our findings not only suggest that FN can serve as the new, safe and highly efficacious anti-diabetic and anti-NAFLD agents for clinical use, they can also provide a molecular basis for the future development of PPARγ modulators with a high therapeutic index and the possibility to explore new uses of old drugs for immediate drug discovery.

Tea and coffee consumption and pathophysiology related to kidney stone formation: a systematic review.

OBJECTIVE: To explore the mechanisms behind the potential protective effect of coffee and tea consumption, regarding urinary stone formation, previously demonstrated in large epidemiological studies. METHODS: A systematic review was performed using the Medline, Cochrane library (CENTRAL) and Scopus databases, in concordance with the PRISMA statement. English, French and Spanish language studies, regarding the consumption of caffeinated and decaffeinated coffee and tea, and the relationship to urinary stone formation were reviewed. Meta-analyses, systematic reviews, case reports and letters, unpublished studies, posters and comments abstracts were excluded. RESULTS: As per the inclusion criteria, 13 studies were included in the final review. The major findings show that caffeine increases urinary excretion of calcium, sodium and magnesium, in addition to a diuretic action with consumption > 300-360 mg (approximately four cups of coffee). Together with other components of coffee, this beverage might have potential protective effects against the formation of urinary stones. Tea exerts many protective effects against stone formation, through the accompanying water intake, the action of caffeine and the effects of components with antioxidant properties. CONCLUSION: Caffeine has a hypercalciuric effect, balanced partially by a diuretic effect which appears after consumption of large quantities of caffeine. The current available literature supports in general, a potentially protective role for tea against stone formation, mainly for green tea. Additional standardization in this field of research, through specification of tea and coffee types studied, and their respective compositions, is needed for further clarification of the relation between coffee, tea and urinary stones.

Pre-clinical and cellular toxicity evaluation of 7-methylxanthine: an investigational drug for the treatment of myopia.

The present study entails the toxicity evaluation of 7-methyl xanthine (7-MX), first of its kind molecule found effective in phase II clinical trials for the treatment of myopia, in comparison to other clinically used xanthines i.e., caffeine and theobromine. For acute toxicity evaluation, 7-MX was administered orally in two rodent species (rat and mice) at the doses of 300 mg/kg and 2000 mg/kg and for repeated dose 28-d oral toxicity, at 250, 500, and 1000 mg/kg in rats. Further, cellular toxicity was evaluated in normal breast epithelial (fR2), rat brain C6 glioma (C6 glioma) and human colorectal (Caco-2) cell lines. Also, the cell uptake assay to determine the intestinal permeability of drug was performed in Caco-2 cells. In acute toxicity, 7-MX treatment showed no mortality and toxicity, whereas 66.6% (mice) and 33.3% (rat) mortality was observed in both caffeine and theobromine treatment groups. In repeated dose 28-d oral toxicity, 7-MX treatment was found to have no-observed-adverse-effect level up to the dose of 1000 mg/kg in the present study conducted as per OECD guidelines 407. Also, very high IC50 value of 305.5 and 721 µg/mL was observed for 7-MX in fR2 and C6 glioma cells, respectively. In Caco-2 cells, linear bioavailability and high % cell viability was observed. Thus, 7-MX may be classified as Globally Harmonized System (GHS) category 5 drug with LD50 >2000-5000 mg/kg. Also, the repeated dose 28-d oral toxicity study demonstrated 7-MX to be nontoxic in nature, with cell line toxicity results further endorsing its nontoxic nature.

The possible effect of pentoxifylline on development and severity of retinopathy of prematurity.

BACKGROUND AND AIM: Retinopathy of prematurity (ROP) is the major ocular problem of preterm infants that occurs with abnormal proliferation of immature retinal vessels. Although pentoxifylline (PTX) was reported to inhibit vasculogenesis and neovascularization in experimental studies, there is no clinical data about the effects of PTX treatment on the development and severity of ROP. This clinical study aimed to investigate the possible effects of PTX on the development of ROP. MATERIALS AND METHODS: A single-centre retrospective study was conducted including preterm infants who were hospitalised in the neonatal intensive care unit between 2015-2017 years. Infants were divided into two groups in terms of PTX administration for adjuvant therapy, as PTX and non-PTX groups. RESULTS: A total of 211 infants were included in the study [gestational age 29 (27-31) weeks, birth weight 1140 (960-1340) g]. From these, 97 infants (46%) were given PTX treatment. The two groups were similar in terms of demographic data and baseline clinical characteristics. Any stage of ROP was detected in 47.4% of infants in the PTX group, which was significantly higher than those in the non-PTX group (27.2%) (p = 0.002). The incidence of advanced-stage ROP in the PTX group (10.3%) was also higher than in the non-PTX group (2.6%) (p = 0.021). Repeated usage of PTX was not found to be related to the development of ROP (p = 0.059). The time of PTX administration was similar between the ROP and no-ROP groups (median; one vs one week, p = 0.825). Surfactant therapy, duration of hospital stay, and PTX treatment were found as significant risk factors for ROP in the logistic regression analysis. CONCLUSIONS: In contrast to the experimental studies and also promising results of PTX treatment in some neonatal morbidities, it may be associated with increased incidence and stage of ROP.

Selected morphological and mechanical features of the femur of pregnant rats after exposure to caffeine applied at various temperatures Caffeine in the nutrition of pregnant rats.

It was assumed that the temperature at which caffeine is consumed may cause changes in the mechanical and morphological properties of bones and affect the bone metabolism of pregnant female. The aim of the study was to assess the effect of caffeine used at different temperatures on selected morphological and mechanical properties of the femur as well as biochemical indicators of bone formation in female rats in pregnancy. It was use test solution at the temperature 10, 25 and 45oC from 8 to 21 day during pregnancy, once a day. It was found that the temperature of administrated caffeine may have an effect on changing the morphometric properties and on the bone metabolism of pregnant female rats. The application of caffeine solution, was administrated at 10oC caused the most constrained growth of the femur and weakened resistance to load, and caused increased susceptibility to cracks. Analysis of bone metabolism indicators showed that caffeine administered in the form of a solution at 10oC and 25oC caused the most negativity effects for bone formation and bone turnover indicators. The administration of caffeine at 10oC causes the largest negative changes in bone morphological and strength indicators and hasn't a beneficial effect on the bone metabolism of pregnant female rats.

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.

Isolation, characterization and application of theophylline-degrading Aspergillus fungi.

BACKGROUND: Caffeine, theobromine and theophylline are main purine alkaloid in tea. Theophylline is the downstream metabolite and it remains at a very low level in Camellia sinensis. In our previous study, Aspergillus sydowii could convert caffeine into theophylline in solid-state fermentation of pu-erh tea through N-demethylation. In this study, tea-derived fungi caused theophylline degradation in the solid-state fermentation. The purpose of this study is identify and isolate theophylline-degrading fungi and investigate their application in production of methylxanthines with theophylline as feedstock through microbial conversion. RESULTS: Seven tea-derived fungi were collected and identified by ITS, ß-tubulin and calmodulin gene sequences, Aspergillus ustus, Aspergillus tamarii, Aspergillus niger and A. sydowii associated with solid-state fermentation of pu-erh tea have shown ability to degrade theophylline in liquid culture. Particularly, A. ustus and A. tamarii could degrade theophylline highly significantly (p < 0.01). 1,3-dimethyluric acid, 3-methylxanthine, 3-methyluric acid, xanthine and uric acid were detected consecutively by HPLC in A. ustus and A. tamarii, respectively. The data from absolute quantification analysis suggested that 3-methylxanthine and xanthine were the main degraded metabolites in A. ustus and A. tamarii, respectively. 129.48 ± 5.81 mg/L of 3-methylxanthine and 159.11 ± 10.8 mg/L of xanthine were produced by A. ustus and A. tamarii in 300 mg/L of theophylline liquid medium, respectively. CONCLUSIONS: For the first time, we confirmed that isolated A. ustus, A. tamarii degrade theophylline through N-demethylation and oxidation. We were able to biologically produce 3-methylxanthine and xanthine efficiently from theophylline through a new microbial synthesis platform with A. ustus and A. tamarii as appropriate starter strains.

Novel caffeine degradation gene cluster is mega-plasmid encoded in Paraburkholderia caffeinilytica CF1.

The widespread use of caffeine in food and drug industries has caused great environmental pollution. Herein, an efficient caffeine-degrading strain Paraburkholderia caffeinilytica CF1 isolated from a tea garden in China can utilize caffeine as its sole carbon and nitrogen source. Combination of chromatographic and spectrophotometric techniques confirmed that strain CF1 adopts N-demethylation pathway for caffeine degradation. Whole genome sequencing of strain CF1 reveals that it has two chromosomes with sizes 3.62 Mb and 4.53 Mb, and a 174-kb mega-plasmid. The plasmid P1 specifically harbors the genes essential for caffeine metabolism. By analyzing the sequence alignment and quantitative real-time PCR data, the redundant gene cluster of caffeine degradation was elucidated. Genes related to catalyzing the N1-demethylation of caffeine to theobromine, the first step of caffeine degradation were heterologously expressed, and methylxanthine N1-demethylase was purified and characterized. Above all, this study systematically unravels the molecular mechanism of caffeine degradation by Paraburkholderia. KEY POINTS: • Caffeine degradation cluster in Paraburkholderia caffeinilytica CF1 was located in mega-plasmid P1. • The whole genome and the caffeine degrading pathway of P. caffeinilytica CF1 were sequenced and elucidated, respectively. • This study succeeded in heterologous expression of methylxanthine N1-demethylase (CdnA) and Rieske oxygenase reductase (CdnD) and illuminated the roles of CdnA and CdnD in caffeine degradation of P. caffeinilytica CF1.