TNF-α inhibition, antioxidant effects and chemical analysis of extracts and fraction from Brazilian guaraná seed powder.

Paullinia cupana Kunth., commonly named Guaraná, is a plant from Brazil used as stimulant. The aim of this study was to evaluate the potential of extracts and tannins-rich and methylxanthines-free fraction from guaraná in the anti-inflammatory and antioxidant effect in vitro. Extract 1 obtained good yields of tannins and methylxanthines and was used to identify a type-A procyanidin trimer by LC-ESI-MS. Fraction 4 was rich in tannins and absent of methylxanthines. The extracts and fraction exhibited strong capacity for scavenging DPPH radical with IC50 between 5.88 and 42.75-µg/mL and inhibited TNF-α release by LPS-activated THP-1 cells when compared with control cells and did not present toxicity to THP-1 cells. The fraction 4, rich in tannins, was highly active, with IC50 5.88 µg/mL by DPPH method and inhibited TNF-α release in 83.50% at 90 µg/mL. These results reinforced potential anti-inflammatory of guaraná and data for new therapeutic approaches.

Microwave-assisted modified synthesis of C8-analogues of naturally occurring methylxanthines: Synthesis, biological evaluation and their practical applications.

An efficient, microwave-assisted, oxidant-interceded, transition-metal-free, cross-dehydrogenative Csp2-Csp3 coupling of C8-Caffeine 2/Theobromine 3/theophylline 4 with substituted aliphatic alcohols 11a-lvia CH bond activation for the preparation of series of substituted C8-(hydroxymethyl) Caffeine 12a-l/theobromine 13a-c/theophylline 14a-b has been developed using microwave irradiation upto 98% yield. The reaction proceeds smoothly in the presence of tert-butyl hydroperoxide (TBHP) under solvolysis condition at 120 °C for 20 min to corresponding substituted C8-(hydroxymethyl)-methylxanthine derivatives in good to excellent yields. The good substrate scope, control experiments, gram-scale synthesis, and practical synthetic transformations further highlights the practicality of this methodology. These C8-(hydroxymethyl) Caffeine 12a-l, 13a-c and 14a-b have been found to show promising in vitro antioxidant as well as antiplatelet activities.

Insoluble-Bound Polyphenols Released from Guarana Powder: Inhibition of Alpha-Glucosidase and Proanthocyanidin Profile.

The Brazilian Food Supplement Law recently recognized that guarana (Paullinia cupana) contains bioactive substances, hence supporting its role as a functional food ingredient. The health benefits of guarana are associated, at least in part, to its phenolic compounds. However, to the best of our knowledge, there is no literature addressing the presence of phenolic compounds in the fraction containing insoluble-bound compounds and its contribution in terms of alpha-glucosidase inhibition. The concentration of phenolic extracts released from the insoluble-bound fraction required to inhibit 50% of alpha-glucosidase (IC50) activity was 5.8-fold lower than that present in the soluble counterpart. Both fractions exhibited a mixed inhibition mode. Fourteen proanthocyanidins (dimers to tetramers) present in the insoluble-bound fraction were tentatively identified by MALDi-TOF-MS. Future studies aiming at increasing the concentration of the soluble counterpart are deemed necessary. The results presented here enhance the phenolic database of guarana and have a practical impact on the procurement of nutraceuticals and functional ingredients related to the prevention and/or management of type 2 diabetes. The Brazilian normative on food supplements has been recently revised. This study lends support to the future inclusion of guarana powder in the list of sources of proanthocyanidins for the industry of food supplements.

Production and characterization of solid lipid microparticles loaded with guaraná (Paullinia cupana) seed extract.

Guaraná is a native fruit of the Amazon rainforest, which presents high levels of phenolic compounds. However, these bioactive compounds may be unstable in food processing and gastrointestinal conditions. Thus, this work aimed to characterize guaraná seed extract (GSE) followed by microencapsulation using a spray-chilling method and with vegetable fat as carrier, as well as to evaluate the particles. Phenolic-rich GSE was produced using 50% (w/w) hydroalcoholic solution and dehydrated by spray drying and lyophilization. Powdered GSE was characterized in relation to its inhibitory activity on digestive enzymes. Solid lipid microparticles (SLM) were evaluated for the retention of bioactive compounds and the release profile of phenolic compounds in simulated gastrointestinal conditions. Powdered GSE showed anti-obesity potential due to the high inhibitory activity of lipase. Regarding the retention of phenolic compounds, at least 75% were detected after 90 days at 25 °C in SLM. Moreover, SLM loaded with 7.5% GSE released approximately 99% of phenolic compounds in simulated gastrointestinal conditions. These results show the efficiency of spray chilling for protection and release of phenolic compounds from GSE, allowing future application in food.

Cocoa bean husk: industrial source of antioxidant phenolic extract.

BACKGROUND: Cocoa bean husk (CBH) is the principal by-product of the cocoa industry and a significant agro-industrial residue. In this study, using different hydrothermal treatments of CBH, it is shown that CBH is an important source of bioactive compounds, including theobromine, epicatechin and catechin. RESULTS: Treatment over 150 °C significantly increased the yield of total and individual phenols and theobromine as well as the antioxidant capacity of the liquid fraction. A total of 52 different genotypes of CBH harvested in two seasons of production were analyzed. Overall, higher amounts of total phenols, theobromine and epigallocatechin were detected in samples from the 2015 season, while samples from 2014 had higher quantities of catechin and similar quantities of epicatechin. CONCLUSION: CBH treatment at 170 °C for 30 min produces an antioxidant-rich extract high in phenols (55 mg g-1 ), sugars (220 mg g-1 ) and theobromine (56 mg g-1 ) that is suitable for applications in the food, cosmetic and nutraceutical industries. © 2018 Society of Chemical Industry.

Guarana (Paullinia cupana Mart.) alters gut microbiota and modulates redox status, partially via caffeine in Wistar rats.

Microbiota alterations are observed in pathological conditions, and their regulation is a subject of great interest. Gut microbes are affected by diet, and plant polyphenols may have positive effect on gut microbiota; however, plant-derived extracts may have toxic effects. Guarana (Paullinia cupana Mart.) is a nontraditional medicinal plant applied worldwide. Guarana yields an alkaloid and polyphenol-rich seed with antimicrobial, antioxidant, and anti-inflammatory properties, where caffeine is the major compound. We evaluated the effects of guarana seed powder (GSP) and purified caffeine on gut microbial composition and redox and inflammatory parameters in Wistar rats after 21 days of treatment. Fecal microbiota was analyzed utilizing 16S rDNA sequencing. Antioxidant enzymes activities from liver, kidney, and colon, as well as oxidative damage markers, were evaluated. Total nonenzymatic antioxidant potential was also evaluated. Microbiota was altered by both treatments, GSP and caffeine, without loss of diversity. In the liver, the kidney, and the colon, we observed a decrease in the antioxidant enzymes activities in the GSP group with no increase in the expression of oxidative damage markers, although some enzymes were also regulated by caffeine. Taken together, these results suggested that GSP ameliorates redox parameters but negatively affected gut microbiota, partially via caffeine.

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

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

An aptamer nanopore-enabled microsensor for detection of theophylline.

This paper reports an aptamer-based nanopore thin film sensor for detecting theophylline in the buffer solution and complex fluids including plant extracts and serum samples. Compared to antibody-based detection, aptamer-based detection offers many advantages such as low cost and high stability at elevated temperatures. Experiments found that this type of sensor can readily detect theophylline at a concentration as low as 0.05µM, which is much lower than the detection limit of current lab-based equipment such as liquid chromatography (LC). Experiments also found that the aptamer-based sensor has good specificity, selectivity, and reasonable reusability with a significantly improved dynamic detection range. By using the same nanopore thin film sensors as the reference sensors to further mitigate the non-specific binding effect, the theophylline in plant extracts and serum has been detected. Only a small amount (~1µL) of plant extracts or serum samples is required to measure theophylline. Its low cost and ease-of-operation make this type of sensor suitable for point-of-care application to monitor the theophylline level of patients in real time.

Electronic nose and chiral-capillary electrophoresis in evaluation of the quality changes in commercial green tea leaves during a long-term storage.

Electronic nose and capillary electrophoresis were applied in quality control of green tea samples subjected to long-term storage. Twelve representative green teas were considered, available as an "aged" (tea leaves stored during a long-term period of two years) and/or "not aged" (fresh products) samples. Their infusions were analyzed by an electronic nose, equipped with an array of six metal oxide semiconductor (MOS) sensors to obtain olfactive fingerprints of the volatile compounds in the infusions headspace. Upon training and chemometric analysis of acquired data (linear discriminant analysis), the electronic nose was found to be able in correctly classifying unknown samples as "aged" or "not aged". Concomitantly, the infusion samples were analyzed by Cyclodextrin-modified Micellar Electrokinetic Chromatography (CD-MEKC) for determination of catechins. The analysis of seven most represented catechins and the methylxanthines theobromine and caffeine revealed a general loss of the polyphenols in each of the considered aged samples (up to 45%, w/w). In addition, the applied enantioselective method based on (2-hydroxypropyl)-ß-cyclodextrin (HP-ßCD) as chiral selector, was exploited for the estimation of (+)-Gallocatechin in the presence of (-)-Gallocatechin; the latter, as the non-native enantiomer, can be associated to the epimerisation of (-)-Epigallocatechin and was assumed as a marker occurring in case of uncorrected storage conditions of tea leaves. Interestingly, it was observed that epimerization did not significantly occur during aging. The application of CD-MEKC and electronic nose allowed for a fast characterization of green teas taking into account that the aroma is a decisive parameter for the acceptance of the product, whereas the catechins content is associated to the biological value.

Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

UHPLC determination of catechins for the quality control of green tea.

An ultra-high performance liquid chromatography (UHPLC) with UV detection method was developed for the fast quantitation of the most represented and biologically important green tea catechins and caffeine. UHPLC system was equipped with C18 analytical column (50mm×2.1mm, 1.8µm), utilizing a mobile phase composed of pH 2.5 triethanolamine phosphate buffer (0.1M) and acetonitrile in a gradient elution mode; under these conditions six major catechins and caffeine were separated in a 3min run. The method was fully validated in terms of precision, detection and quantification limits, linearity, accuracy, and it was applied to the identification and quantification of catechins and caffeine present in green tea infusions. In particular, commercially available green tea leaves samples of different geographical origin (Sencha, Ceylon Green and Lung Ching) were used for infusion preparations (water at 85°C for 15min). The selectivity of the developed UHPLC method was confirmed by comparison with UHPLC-MS/MS analysis. The recovery of the main six catechins and caffeine on the three analyzed commercial tea samples ranged from 94 to 108% (n=3). Limits of detection (LOD) were comprised in the range 0.1-0.4µgmL(-1). An orthogonal micellar electrokinetic (MEKC) method was applied for comparative purposes on selectivity and quantitative data. The combined use of the results obtained by the two techniques allowed for a fast confirmation on quantitative characterization of commercial samples.

Guarana powder polysaccharides: characterisation and evaluation of the antioxidant activity of a pectic fraction.

Guarana is a fruit from the Amazon whose seeds are used to produce guarana powder. Guarana powder is consumed by the population mainly for its stimulant activity. It has been shown that guarana seeds contain low-molar-mass compounds; however, no data have been reported concerning the polysaccharides. In this work, the polysaccharides present in guarana powder were investigated. A pectic fraction and a xylan were isolated and characterised. Antioxidant activity tests were performed with a methanolic extract and the pectic fraction at concentrations of 0.1-10 mg/ml. The methanolic extract exhibited a strong capacity for scavenging DPPH radicals (90.9% at 10 mg/ml). At the same concentration, the polysaccharide showed a DPPH(·)-scavenging activity of 68.4%. At a higher concentration, the methanolic extract and the polysaccharide exhibited similar hydroxyl radical-scavenging effects (~70%). The results suggest that the polysaccharides present in guarana can contribute to the possible biological effects of guarana powder.

Cocrystals of quercetin with improved solubility and oral bioavailability.

Flavonoids have been studied extensively due to the observation that diets rich in these compounds are associated with lower incidences of many diseases. One of the most studied flavonoids, quercetin, is also the most abundant of these compounds in the plant kingdom. Numerous therapeutic bioactivities have been identified in vitro. However, its in vivo efficacy in pure form is limited by poor bioavailability, primarily due to its low solubility and consequent low absorption in the gut. Cocrystallization has gained attention recently as a means for improving the physicochemical characteristics of a compound. Here, we synthesized and evaluated four new cocrystals of quercetin (QUE): quercetin:caffeine (QUECAF), quercetin:caffeine:methanol (QUECAF·MeOH), quercetin:isonicotinamide (QUEINM), and quercetin:theobromine dihydrate (QUETBR · 2H(2)O). Each of these cocrystals exhibited pharmacokinetic properties that are vastly superior to those of quercetin alone. Cocrystallization was able to overcome the water insolubility of quercetin, with all four cocrystals exhibiting some degree of solubility. The QUECAF and QUECAF·MeOH cocrystals increased the solubility of QUE by 14- and 8-fold when compared to QUE dihydrate. We hypothesized that this improved solubility would translate into enhanced systemic absorption of QUE. This hypothesis was supported in our pharmacokinetic study. The cocrystals outperformed QUE dihydrate with increases in bioavailability up to nearly 10-fold.

Determination of caffeine in coffee products by dynamic complexation with 3,4-dimethoxycinnamate and separation by CZE.

A method based on the formation of pi-complexes with chlorogenate-like species was proposed for the determination of caffeine in regular (nondecaffeinated) and decaffeinated coffee. Both caffeate and 3,4-dimethoxycinnamate were able to transform caffeine--a neutral species in aqueous solutions--into an anionic species. The usage of 3,4-dimethoxycinnamate in the running electrolyte is advantageous, because of its greater chemical stability and the improved resolution of the peaks of caffeine, theobromine, and theophylline. Negative peaks were registered with a capacitively coupled contactless conductivity detector when solutions of these alkylxanthines were analyzed with a BGE composed of 20 mmol/L 3,4-dimethoxycinnamic acid and pH adjusted to 8.5 with Tris. This behavior was expected, because the complex is larger and thus should move slower than the free anion. Caffeine was determined in ground and instant coffee with precision and accuracy that meet Brazilian norms about such products. The LOD was estimated as 33 mg/L, which corresponds to 0.8 and 0.3 mg of caffeine per gram of dry instant coffee and ground coffee, respectively. For the case of decaffeinated coffee, ten times preconcentration with dichloromethane was carried out to allow the quantitation of caffeine, which should not exceed the concentration of 1 mg/g in dry matter.

Effect of drug solubility on release behavior of calcium polysaccharide gel-coated pellets.

The aim of this study was to investigate the effect of drug solubility on the release behavior from calcium polysaccharide gel (CaPG)-coated pellets. Three different drugs with similar chemical structure, but different water solubility, namely caffeine (CAF), theophylline (TPL) and theobromine (TBR), were used. Drug-loaded spherical pellets were manufactured by an extrusion-spheronization method. The CaPG was applied on the pellets loaded with different drugs by interfacial complexation coating. The encapsulation efficiency of coated pellets was found to vary from 57.6 to 84.3%, depending on the solubility of the active drug and polysaccharide type. Drug release from different uncoated pellets was relatively unaffected by pH and release media but depended mainly on drug solubility. Release behavior was significantly modified in the pellets coated with CaPG, for all of the drugs tested. Drug release from coated pellets of the different drugs showed different release kinetics. The difference in the drug release is probably due to the difference in the drug dissolution within the core, before its partition and diffusion through the CaPG coat. The CAF dissolved faster and achieved a higher concentration in solution, which drove diffusion. The release of TBR from the coated pellets was much slower than that of the CAF or TPL because of its low solubility. However, the release of all drugs was about four- to sixfold slower for coated than uncoated pellets, suggesting that the coating influenced the retardation of drug release from the coated pellets. Therefore, the CaPG coating may provide a sustained release delivery system for all drugs tested.

Fluid bed drying of guarana (Paullinia cupana HBK) extract: effect of process factors on caffeine content.

The aim of this study was to study the convective drying of the hydroalcoholic extracts obtained from powdered guarana seeds in a spouted bed dryer. The influence of process variables, such as the convective airflow rate, extract feed rate, and air inlet temperature, on the quality of the dry extract was determined using the caffeine and moisture content for the process evaluation. The caffeine content in the alcoholic and dried extracts was determined by capillary gas chromatography. The experiments were performed following a 3(3) factorial design and the data analyzed by response surface. The analysis of dry extract showed that the air and extract feed rates did not significantly affect (25% level) the caffeine content, but that drying temperature is a major factor to consider when the extract is submitted to fluid bed drying. Caffeine losses were significant (1% level) for drying temperatures above 120 degrees C, while moisture content was lower than 3% for temperatures above 120 degrees C. The data showed that there is an optimum temperature for the drying of guarana extracts in spouted beds, and under the conditions used in this study it was 120 degrees C.