Impact of Caffeine Intake Strategies on Heart Rate Variability During Post-Exercise Recovery: A Systematic Review and Meta-Analysis.

OBJECTIVES: The objective of this systematic review and meta-analysis is to evaluate the influence of caffeine (CAF) intake strategies, taking into account their form, timing, and dosage, on heart rate variability (HRV) indices in the post-exercise recovery period. METHODS: The meta-analysis adhered to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines and is registered in the PROSPERO database (CRD42023425885). A comprehensive literature search was carried out across MEDLINE, Web of Science, LILACS, and SCOPUS, concluding in May 2023. We concentrated on randomized clinical trials comparing CAF supplementation effects to placebo on HRV indices post-exercise in active adults aged 18 and above. The primary endpoint was the assessment of HRV indices, measured both prior to and following exercise. RESULTS: Of the 10 studies included, 7 were used for the meta-analysis, and all contributed to the systematic review. The research explored a variety of CAF strategies, spanning different forms (capsule, drink, gum), times (10, 45, 60 min) and doses (2.1 to 6.0 mg/kg). The outcomes revealed no substantial variations between the placebo and CAF conditions in terms of both the square root of the average of successive squared differences between adjacent RR intervals (RMSSD) (standardized mean difference (SMD) -0.03, 95% CI -0.265 to 0.197, p=0.77) and high frequency (HF) index (SMD -0.061, 95% CI -0.272 to 0.150, p=0.57). Furthermore, metaregression analysis, employing a fixed-effects model and accounting for the administered CAF doses, revealed no significant correlation between caffeine doses and HRV indices (p>0.05). CONCLUSION: In conclusion, there is moderate-certainty evidence suggesting that different CAF intake strategies, encompassing aspects such as form, time, and dose, do not have a significant impact on HRV indices recovery post-exercise (i.e., vagal modulation).

Natural products from Vitex polygama and their antimycobacterial and anti-inflammatory activity.

Tuberculosis (TB) remains a worldwide public health threat because of the emergence of resistant strains and subsequent inappropriate response to current therapy. We have been studying the restinga plants' antimycobacterial and anti-inflammatory potential. Dichloromethane fraction (DCM) from Vitex polygama Cham. showed high activity against Mycobacterium tuberculosis (Mtb) H37Rv. In this context, DCM fraction and isolated compounds were investigated against Mtb H37Rv and M299 (MDR strain) and for their immunomodulatory and cytotoxicity actions. Orientin showed the best antimycobacterial effect against Mtb M299 MDR strain (MIC50 15.4 ± 1.6 µg/mL), capacity of inhibiting NO production by macrophages (IC50 6.5 ± 1.2 µg/mL) and no significant cytotoxicity. The antimycobacterial effect of orientin was also observed on Mtb H37Rv intracellular growth in RAW 264.7 macrophages (MIC50 3.5 ± 1.1 and MIC90 9.1 ± 1.0 µg/mL). This is the first report describing the antimycobacterial effect of orientin, in both extra- and intracellular growth.[Formula: see text].

Caffeine increases performance and leads to a cardioprotective effect during intense exercise in cyclists.

The present study was designed to investigate the effects of different caffeine dietary strategies to compare the impact on athletic performance and cardiac autonomic response. The order of the supplementation was randomly assigned: placebo(4-day)-placebo(acute)/PP, placebo(4-day)-caffeine(acute)/PC and caffeine(4-day)-caffeine(acute)/CC. Fourteen male recreationally-trained cyclists ingested capsules containing either placebo or caffeine (6 mg kg-1) for 4 days. On day 5 (acute), capsules containing placebo or caffeine (6 mg kg-1) were ingested 60 min before completing a 16 km time-trial (simulated cycling). CC and PC showed improvements in time (CC vs PP, Δ - 39.3 s and PC vs PP, Δ - 43.4 s; P = 0.00; ƞ2 = 0.33) and in output power (CC vs PP, Δ 5.55 w and PC vs PP, Δ 6.17 w; P = 0.00; ƞ2 = 0.30). At the final of the time-trial, CC and PC exhibited greater parasympathetic modulation (vagal tone) when compared to the PP condition (P < 0.00; ƞ2 = 0.92). Our study provided evidence that acute caffeine intake (6 mg∙kg-1) increased performance (time-trial) and demonstrated a relevant cardioprotective effect, through increased vagal tone.

In vitro and in vivo anti-inflammatory activity and chemical composition of Renealmia petasites Gagnep.

The study aimed to investigate the chemical composition and the anti-inflammatory activity of the hydroethanolic rhizomes, stems, and leaf extracts of Renealmia petasites using in vitro and in vivo assays. The chemical composition of the extracts was characterized in a linear iron trap mass spectrometer. Total phenolic, flavonoid, and tannin content were determined by spectrophotometry analyses. In vitro anti-inflammatory activity was investigated in lipopolysaccharide-stimulated macrophages evaluating the influence on the production of superoxide anion (O2-), nitric oxide (NO), and the pro-inflammatory cytokines tumor necrosis factor (TNF-α) and interleukin-6 (IL-6). In vivo effects were determined using the air pouch model in which were inoculated carrageenan and thereafter treated with 50 mg/kg of the hydroethanolic extracts of R. petasites. After 4 and 24 h, the cellular influx, protein exudation, cytokines, and nitric oxide were evaluated. Eight compounds were tentatively identified in the R. petasites extracts, suggesting five diarylheptanoids, one flavonoid, and two fatty alcohols. The in vitro results showed that the extracts were capable of blocking free radicals and/or inhibiting their intracellular actions by inhibiting the production of important mediators of the inflammatory process, such as NO, O2-, TNF-α, and IL-6. In vivo, R. petasites significantly decrease the influx of leukocytes, mainly neutrophils, protein exudation, NO, TNF-α, and IL-6 concentration in the air pouch model. The results evidenced that R. petasites can be considered a promising alternative therapy for the treatment and management of osteoarthritis and other inflammatory diseases.

Caffeine Supplementation for 4 Days Does Not Induce Tolerance to the Ergogenic Effects Promoted by Acute Intake on Physiological, Metabolic, and Performance Parameters of Cyclists: A Randomized, Double-Blind, Crossover, Placebo-Controlled Study.

The present study investigated whether the caffeine supplementation for four days would induce tolerance to the ergogenic effects promoted by acute intake on physiological, metabolic, and performance parameters of cyclists. A double-blind placebo-controlled cross-over design was employed, involving four experimental trials; placebo (4-day)-placebo (acute)/PP, placebo (4-day)-caffeine (acute)/PC, caffeine (4-day)-caffeine (acute)/CC and caffeine (4-day)-placebo (acute)/CP. Fourteen male recreationally-trained cyclists ingested capsules containing either placebo or caffeine (6 mg∙kg-1) for 4 days. On day 5 (acute), capsules containing placebo or caffeine (6 mg∙kg-1) were ingested 60 min before completing a 16 km time-trial (TT). CC and PC showed improvements in time (3.54%, ES = 0.72; 2.53%, ES = 0.51) and in output power (2.85%, ES = 0.25; 2.53%, ES = 0.20) (p < 0.05) compared to CP and PP conditions, respectively. These effects were accompanied by increased heart rate (2.63%, ES = 0.47; 1.99%, ES = 0.34), minute volume (13.11%, ES = 0.61; 16.32%, ES = 0.75), expired O2 fraction (3.29%, ES = 0.96; 2.87, ES = 0.72), lactate blood concentration (immediately after, 29.51% ES = 0.78; 28.21% ES = 0.73 recovery (10 min), 36.01% ES = 0.84; 31.22% ES = 0.81), and reduction in expired CO2 fraction (7.64%, ES = 0.64; 7.75%, ES = 0.56). In conclusion, these results indicate that caffeine, when ingested by cyclists in a dose of 6 mg∙kg-1 for 4 days, does not induce tolerance to the ergogenic effects promoted by acute intake on physiological, metabolic, and performance parameters.

Chemical composition and anti-inflammatory activity of essential oil and ethanolic extract of Campomanesia phaea (O. Berg.) Landrum leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Campomanesia species are used in folk medicine for anti-inflammatory, -ulcerogenic, -diabetic, -obesity, and many other purposes. AIM OF THE STUDY: This study aimed to investigate the phytochemical profile and pharmacotherapeutic potential of the essential oil (EO) and ethanolic extract (EXT) of the leaves of Campomanesia phaea in relation to antioxidant and anti-inflammatory effects using chemical methods and in vitro bioassays in cell culture. MATERIALS AND METHODS: Gas and liquid chromatography techniques coupled to mass spectrometry were used to identify the main secondary metabolites. The antioxidant activity was determined by the chemical methods of radical sequestration of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and by ferric reducing antioxidant power (FRAP); in addition to the protective effect against cellular oxidative damage caused by hydrogen peroxide (H2O2) in macrophage culture. The anti-inflammatory and immunomodulatory activity was evaluated for the influence on the production of nitric oxide and superoxide anion (O2•-), and by the quantification of proinflammatory cytokines tumor necrosis factor (TNF alpha) and interleukin 6 (IL- 6) through Enzyme Linked Immuno Sorbent Assay (ELISA) technique and inhibition of nuclear factor kappa B (NF-κB) through chemiluminescence. RESULTS: A total of 41 compounds were identified in the essential oil (EO), being (E)-caryophyllene (14%) and caryophyllene oxide (6.9%) the major compounds. In the ethanolic extract (EXT), three flavonoids from the flavanones group were identified: alpinetin O-dideoxy-hexoside, 5,7-dimethoxyflavanone and alpinetin. The EO and EXT inhibited the production of O2•- (99.0% and 52.9%) at a concentration of 100 µg/mL, intracellular NO•- (50.0% and 51.9%) and proinflammatory cytokines IL-6 (41.0% and 82.9%) and TNF-α (74.7% and 87.9%) at a concentration of 50 µg/mL, respectively. In addition, inhibition of nuclear factor kappa B (EO 36.2% and EXT 40.9%) was observed at 20 µg/mL. CONCLUSIONS: Taken together, the results indicated that EO and EXT possess potent anti-inflammatory activities and it may hold therapeutic promise in the management of acute and chronic inflammatory conditions.

Delapril and Indapamide: Development and Validation of a Stability-Indicating Core-Shell LC Method and Its Application for Simultaneous Tablets Assay.

Background: The combination of delapril (DEL) and indapamide (IND) may be regarded as an optimal drug treatment for hypertensive patients. However, there is no published study concerning the suitable stability conditions and evaluation of drugs in the raw material and commercial product. Objective: The aim of the present study was to develop an innovative, high-throughput, and stability-indicating LC method for the simultaneous analysis of DEL and IND in combined dosage form. Methods: Analyses were performed using a core-shell C18 column (100 mm × 4.6 mm id, 2.6 µm) at 45°C using isocratic elution for the mobile phase composed of triethylamine solution (0.3%, pH 5.0)-acetonitrile-methanol (58 + 35 + 7, v/v/v). The separation was obtained within 3.5 min at a flow rate of 1.0 mL/min and UV detection set at 213 nm. Results: The specificity and stability-indicating capability of the method were proven through degradation studies, which also showed that there is no interference of the formulation excipients, showing that the peak is free from any co-eluting peak. Conclusions: The method showed adequate precision, with relative standard deviation values lower than 1.85%. Excellent values of accuracy were obtained, with a mean value of 98.64% for IND and 98.65% for DEL. Experimental design was used during validation to calculate and prove the method robustness. Highlights: The proposed LC method was successfully validated according to International Conference on Harmonisation requirements and applied for the simultaneous determination of DEL and IND in tablets, presenting suitability for stability studies and contributing to improve the QC of pharmaceuticals.

Biological activities and phytochemical profile of Passiflora mucronata from the Brazilian restinga

ABSTRACT In general, Passiflora species have been reported for their folk medicinal use as sedative and anti-inflammatory. However, P. caerulea has already been reported to treat pulmonary diseases. Severe pulmonary tuberculosis, generally caused by Mycobacterium tuberculosis strains resistant to multiple drugs, can lead to deleterious inflammation and high mortality, encouraging new approaches in drug discovery. Thus, the aim of this work was to evaluate the Passiflora mucronata Lam., Passifloraceae, potential for tuberculosis treatment. Specifically, related to antimycobacterial activity and anti-inflammatory related effects (based on inhibition of nitric oxide, tumor necrosis factor-alpha production and antioxidant potential), as well as the chemical profile of P. mucronata. High performance liquid chromatography coupled with diode-array ultraviolet and mass spectrometer analyses of crude hydroalcoholic extract and ethyl acetate fraction showed the presence of flavonoids. Ethyl acetate fraction showed to be as antioxidant as Ginkgo biloba standard extract with EC50 of 14.61 ± 1.25 µg/ml. One major flavonoid isolated from ethyl acetate fraction was characterized as isoorientin. The hexane fraction and its main isolated compound, the triterpene β-amyrin, exhibited significant growth inhibitory activity against Mycobacterium bovis BCG (MIC50 1.61 ± 1.43 and 3.93 ± 1.05 µg/ml, respectively). In addition, Passiflora mucronata samples, specially hexane and dichloromethane fractions, as well as pure β-amyrin, showed a dose-related inhibition of lipopolysaccharide (LPS)-induced nitric oxide production. In conclusion, Passiflora mucronata presented relevant biological potential and should be considered for further studies using in vivo pulmonary tuberculosis model.

Phytochemical Study of Tapirira guianensis Leaves Guided by Vasodilatory and Antioxidant Activities.

The aim of this research was to perform a phytochemical study of the methanol leaves extract of T. guianensis (MET) guided by vasodilatory and antioxidant activities. The chemical profile of MET and the ethyl acetate fraction (EA fraction) was determined by HPLC-UV-MS and EA fraction guided fractionation by reverse-phase chromatography. The vasorelaxant effects of MET, fractions, sub-fractions and constituents were assessed on rat aorta pre-contracted with phenylephrine. Antioxidant activity was evaluated by using a DPPH assay. The results show that MET-induced vasodilation was dependent on NO/cGMP; and that the PI3K/Akt pathway seems to be the main route involved in eNOS activation. The EA fraction showed greater vasodilatory and antioxidant potency and was submitted to further fractionation. This allowed the isolation and characterization of quercetin, quercetin 3-O-(6″-O-galloyl)-ß-d-galactopyranoside and 1,4,6-tri-O-galloyl-ß-d-glucose. Also, galloyl-HHDP-hexoside and myricetin deoxyhexoside were identified by HPLC-UV-MS. These compounds are being described for the first time for T. guianensis. 1,4,6-tri-O-galloyl-ß-d-glucose and quercetin 3-O-(6″-O-galloyl)-ß-d-galactopyranoside showed no vasodilatory activity. Quercetin and myricetin glycoside seems to contribute to the MET activity, since they have been reported as vasodilatory flavonoids. MET-induced vasodilation could contribute to the hypotensive effect of T. guianensis previously reported.

In Vitro and In Vivo Anti-inflammatory Effects of Struthanthus vulgaris.

Struthanthus vulgaris is probably the most common medicinal mistletoe plant in Brazil, and has been used in folk medicine as an anti-inflammatory agent and for cleaning skin wounds. Our proposal was to evaluate the anti-inflammatory activity of S. vulgaris ethanol leaf extract and provide further insights of how this biological action could be explained using in vitro and in vivo assays. In vitro anti-inflammatory activity was preliminarily investigated in lipopolysaccharide/interferon gamma-stimulated macrophages based on their ability to inhibit nitric oxide production and tumor necrosis factor-alpha. In vivo anti-inflammatory activity of S. vulgaris ethanol leaf extract was investigated in the mice carrageenan-induced inflammation air pouch model. The air pouches were inoculated with carrageenan and then treated with 50 and 100 mg/kg of S. vulgaris ethanol leaf extract or 1 mg/kg of dexamethasone. Effects on the immune cell infiltrates, pro- and anti-inflammatory mediators such as tumor necrosis factor-alpha, interleukin 1, interleukin 10, and nitric oxide, were evaluated. The chemical composition of S. vulgaris ethanol leaf extract was characterized by LC-MS/MS. In vitro S. vulgaris ethanol leaf extract significantly decreased the production of nitric oxide and tumor necrosis factor-alpha in macrophages and did not reveal any cytotoxicity. In vivo, S. vulgaris ethanol leaf extract significantly suppressed the influx of leukocytes, mainly neutrophils, protein exudation, nitric oxide, tumor necrosis factor-alpha, and interleukin 1 concentrations in the carrageenan-induced inflammation air pouch. In conclusion, S. vulgaris ethanol leaf extract exhibited prominent anti-inflammatory effects, thereby endorsing its usefulness as a medicinal therapy against inflammatory diseases, and suggesting that S. vulgaris ethanol leaf extract may be a source for the discovery of novel anti-inflammatory agents.

Metabolic profile and safety of piperlongumine.

Piperlongumine (PPL), a natural plant product, has been extensively studied in cancer treatment going up on clinical trials. Since the first report related to its use on cancer research (in 2011) around 80 papers have been published in less than 10 years, but a gap still remaining. There are no metabolism studies of PPL in human organism. For the lack of a better view, here, the CYP450 in vitro oxidation of PPL was described for the first time. In addition, the enzymatic kinetic data, the predicted in vivo parameters, the produced metabolites, the phenotyping study and possible piperlongumine-drug interactions in vivo is presented.

Caffeine Attenuates Decreases in Leg Power Without Increased Muscle Damage.

Ribeiro, BG, Morales, AP, Sampaio-Jorge, F, Barth, T, de Oliveira, MBC, Coelho, GMdO, and Leite, TC. Caffeine attenuates decreases in leg power without increased muscle damage. J Strength Cond Res 30(8): 2354-2360, 2016-Caffeine ingestion has been shown to be an effective ergogenic aid in several sports. Caffeine administration may increase exercise capacity, which could lead to a greater degree of muscle damage after exercise. This was a randomized, double-blind, placebo-controlled crossover study. Six male handball athletes ingested placebo (PLA) or caffeine (CAF) (6 mg·kg body mass) capsules on 2 different occasions. Sixty minutes after ingestion of the capsules, serum CAF levels were evaluated. Thereafter, all participants performed a protocol of vertical jumps (VJs). The protocol consisted of 4 sets of 30 seconds of continuous VJs with 60 seconds of recovery between sets. Blood lactate (LAC) and creatine kinase (CK) levels were determined before and after the protocol. We found significant differences in serum CAF levels between PLA (0.09 ± 0.18 µg·ml) vs. CAF (6.59 ± 4.44 µg·ml) (p < 0.001). Caffeine elicited a 5.23% (p ≤ 0.05) improvement in the leg power compared with PLA. The CAF trial displayed higher LAC (p ≤ 0.05) compared with PLA (6.26 ± 2.01 vs. 4.39 ± 2.42 mmol·L, respectively) after protocol of VJs, whereas no difference in CK was observed between trials (p > 0.05). These results indicate that immediate ingestion of CAF (6 mg·kg body weight) can reduce the level of muscle fatigue and preserve leg power during the test, possibly resulting in increase in LAC. There was no increase in muscle damage, which indicates that immediate administration of (6 mg·kg body weight) CAF is safe. Thus, nutritional interventions with CAF could help athletes withstand a greater physiological overload during high-intensity training sessions. The results of this study would be applicable to sports and activities that require repetitive leg power.

In vitro metabolism of the lignan (-)-grandisin, an anticancer drug candidate, by human liver microsomes.

(-)-grandisin is a tetrahydrofuran lignan that displays important biological properties, such as trypanocidal, anti-inflammatory, cytotoxic, and antitumor activities, suggesting its utility as a potential drug candidate. One important step in drug development is metabolic characterization and metabolite identification. To perform a biotransformation study of (-)-grandisin and to determine its kinetic properties in humans, a high performance liquid chromatography (HPLC) method was developed and validated. After HPLC method validation, the kinetic properties of (-)-grandisin were determined. (-)-grandisin metabolism obeyed Michaelis-Menten kinetics. The maximal reaction rate (Vmax ) was 3.96 ± 0.18 µmol/mg protein/h, and the Michaelis-Menten constant (Km ) was 8.23 ± 0.99 µM. In addition, the structures of the metabolites derived from (-)-grandisin were characterized via gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analysis. Four metabolites, 4-O-demethylgrandisin, 3-O-demethylgrandisin, 4,4'-di-O-demethylgrandisin, and a metabolite that may correspond to either 3,4-di-O-demethylgrandisin or 3,5-di-O-demethylgrandisin, were detected. CYP2C9 isoform was the main responsible for the formation of the metabolites. These metabolites have not been previously described, demonstrating the necessity of assessing (-)-grandisin metabolism using human-derived materials.

Evaluation of dispersive liquid-liquid microextraction in the stereoselective determination of cetirizine following the fungal biotransformation of hydroxyzine and analysis by capillary electrophoresis.

We developed a capillary electrophoresis (CE) and dispersive liquid-liquid microextraction (DLLME) method to stereoselectively analyze hydroxyzine (HZ) and cetirizine (CTZ) in liquid culture media. The CE analyses were performed on an uncoated fused-silica capillary; 50mmolL(-1) sodium borate buffer (pH 9.0) containing 0.8% (w/v) S-ß-CD was used as the background electrolyte. The applied voltage and temperature were +6 kV and 15 °C, respectively, and the UV detector was set to 214 nm. Chloroform (300 µL) and ethanol (400 µL) were used as the extraction and disperser solvents, respectively, for the DLLME. Following the formation of a cloudy solution, the samples were subjected to vortex agitation at 2000 rpm for 30s and to centrifugation at 3000 rpm for 5 min. The recoveries ranged from 87.4 to 91.7%. The method was linear over a concentration range of 250-12,500 ng mL(-1) for each HZ enantiomer (r>0.998) and 125-6250 ng mL(-1) for each CTZ enantiomer (r>0.998). The limits of quantification were 125 and 250 ng mL(-1) for CTZ and HZ, respectively. Among the six fungi studied, three species were able to convert HZ to CTZ enantioselectively, particularly the fungus Cunninghamella elegans ATCC 10028B, which converted 19% of (S)-HZ to (S)-CTZ with 65% enantiomeric excess.

Enantioselective analysis of ranolazine and desmethyl ranolazine in microsomal medium using dispersive liquid-liquid microextraction and LC-MS/MS.

BACKGROUND: An enantioselective bioanalytical method using dispersive liquid-liquid microextraction (DLLME) and LC-MS/MS was developed for the chiral analysis of ranolazine (RNZ) and one of its metabolites (desmethyl ranolazine [DRNZ]). RESULTS: The analytes were extracted from microsomal medium by DLLME, using chloroform as extractor solvent and acetone as dispersive solvent. The enantiomers of RNZ and DRNZ were analyzed simultaneously for the first time using a Chiralcel OD-H(®). Method validation showed recoveries in the order of 55 and 45%, and LLOQ of 25 and 10 ng ml(-1) for the enantiomers of RNZ and DRNZ, respectively. Linearity was established in the concentration range of 10 to 1000 and 25 to 2500 ng ml(-1) for each DRNZ and RNZ enantiomer, respectively. CONCLUSION: The unprecedented use of DLLME was demonstrated to be very useful for sample preparation of microsomal matrix. Furthermore, the in vitro metabolism of RNZ was enantioselective.

HPLC analysis of midodrine and desglymidodrine in culture medium: evaluation of static and shaken conditions on the biotransformation by fungi.

A high-performance liquid chromatography (HPLC) method is presented for the simultaneous determination of midodrine and desglymidodrine (DMAE) in Czapek-Dox culture medium, to be used in biotransformation studies by fungi. The HPLC analysis was conducted using a Lichrospher 100 RP18 column, acetonitrile-40 mmol/L formic acid solution (60:40, v/v) as mobile phase, and ultraviolet detection at 290 nm. The sample preparation was conducted by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 0.4-40.0 µg/mL for midodrine (r ≥ 0.9997) and DMAE (r ≥ 0.9998). Within-day and between-day precision and accuracy were evaluated by relative standard deviations (≤ 8.2%) and relative errors (-7.3 to 7.4%), respectively. The validated method was used to assess midodrine biotransformation by the fungi Papulaspora immersa Hotson SS13, Botrytis cinerea UCA 992 and Botrytis cinerea 2100 under static and shaken conditions. Under shaken conditions, the biotransformation of midodrine to DMAE was more efficient for all studied fungi, especially for the fungus Botrytis cinerea 2100, which converted 42.2% of midodrine to DMAE.

Chiral HPLC analysis of donepezil, 5-O-desmethyl donepezil and 6-O-desmethyl donepezil in culture medium: application to fungal biotransformation studies.

An high performance liquid chromatography (HPLC) method for the enantioselective determination of donepezil (DPZ), 5-O-desmethyl donepezil (5-ODD), and 6-O-desmethyl donepezil (6-ODD) in Czapek culture medium to be applied to biotransformation studies with fungi is described for the first time. The HPLC analysis was carried out using a Chiralpak AD-H column with hexane/ethanol/methanol (75:20:5, v/v/v) plus 0.3 % triethylamine as mobile phase and UV detection at 270 nm. Sample preparation was carried out by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 100-10,000 ng mL(-1) for each enantiomer of DPZ (r ≥ 0.9985) and of 100-5,000 ng mL(-1) for each enantiomer of 5-ODD (r ≥ 0.9977) and 6-ODD (r ≥ 0.9951). Within-day and between-day precision and accuracy evaluated by relative standard deviations and relative errors, respectively, were lower than 15 % for all analytes. The validated method was used to assess DPZ biotransformation by the fungi Beauveria bassiana American Type Culture Collection (ATCC) 7159 and Cunninghamella elegans ATCC 10028B. Using the fungus B. bassiana ATCC 7159, a predominant formation of (R)-5-ODD was observed while for the fungus C. elegans ATCC 10028B, DPZ was biotransformed to (R)-6-ODD with an enantiomeric excess of 100 %.

Assessment of the stereoselective fungal biotransformation of albendazole and its analysis by HPLC in polar organic mode.

A high-performance liquid chromatographic method using polar organic mode was developed to analyze albendazole (ABZ), albendazole sulfone (ABZSO(2)) and the chiral and active metabolite albendazole sulfoxide (ABZSOX, ricobendazole) that was further applied in stereoselective fungal biotransformation studies. The chromatographic separation was performed on a Chiralpak AS column using acetonitrile:ethanol (97:3, v/v) plus 0.2% triethylamine and 0.2% acetic acid as the mobile phase at a flow rate of 0.5 mL min(-1). The present study employed hollow fiber liquid-phase microextraction as sample preparation. The method showed to be linear over the concentration range of 25-5000 ng mL(-1) for each ABZSOX enantiomer, 200-10,000 ng mL(-1) for ABZ and 50-1000 ng mL(-1) for ABZSO(2) metabolite with correlation coefficient (r)>0.9934. The mean recoveries for ABZ, rac-ABZSOX and ABZSO(2) were, respectively, 9%, 33% and 20% with relative standard deviation below 10%. Within-day and between-day precision and accuracy assays for these analytes were studied at three concentration levels and were lower than 15%. This study opens the door regarding the possibility of using fungi in obtaining of the active metabolite ricobendazole. Nigrospora sphaerica (Sacc.) E. W. Mason (SS67), Pestalotiopsis foedans (VR8), Papulaspora immersa Hotson (SS13) and Mucor rouxii were able to stereoselectively metabolize ABZ into its chiral metabolite. Among them, the fungus Mucor rouxii was the most efficient in the production of (+)-ABZSOX.

Stereoselective liquid chromatographic determination of 1'-oxobufuralol and 1'-hydroxybufuralol in rat liver microsomal fraction using hollow-fiber liquid-phase microextraction for sample preparation.

A three-phase hollow-fiber liquid-phase microextraction (HF-LPME) method for the stereoselective determination of bufuralol metabolites 1'-oxobufuralol (1'-Oxo-BF) and 1'-hydroxybufuralol (1'-OH-BF) in microsomal preparations is described for the first time. The HPLC analysis was carried out using a Chiralcel OD-H column with hexane/2-propanol/methanol (97.5:2.0:0.5, v/v/v) plus 0.5% diethylamine as the mobile phase, and UV detection at 248 and 273 nm. The HF-LPME optimized conditions involved: n-octanol as the organic solvent, 0.2 mol/L acetic acid as the acceptor phase, donor phase pH adjusted to 13, sample agitation at 1500 rpm and extraction for 30 min. By using this extraction procedure, the recovery rates were in the range of 63-69%. The method was linear over the concentration range of 100-5000 ng/mL for each enantiomer of 1'-Oxo-BF (r>0.9978) and of 100-2500 ng/mL for each stereoisomer of 1'-OH-BF (r>0.9957). The quantification limits were 100 ng/mL for all analytes. The validated method was used to assess the in vitro biotransformation of bufuralol using rat liver microsomal fraction that demonstrated predominant formation of (S)-1'-Oxo-BF and (R,R)-1'-OH-BF.

Capillary electrophoresis and hollow fiber liquid-phase microextraction for the enantioselective determination of albendazole sulfoxide after biotransformation of albendazole by an endophytic fungus.

Hollow fiber liquid-phase microextraction and CE were applied for the determination of albendazole sulfoxide (ASOX) enantiomers in liquid culture medium after a fungal biotransformation study. The analytes were extracted from 1 mL of liquid culture medium spiked with the internal standard (rac-hydroxychloroquine) and buffered with 0.50 mol/L phosphate buffer, pH 10. The analytes were extracted into 1-octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the polypropylene hollow fiber. The electrophoretic separations were carried out in 0.05 mol/L tris(hydroxymethyl)aminomethane buffer, pH 9.3, containing 3.0% w/v sulfated-ß-CD (S-ß-CD) with a constant voltage of +15 kV and detection at 220 nm. The method was linear over the concentration range of 250-5000 ng/mL for each ASOX enantiomer. Within-day and between-day assay precision and accuracy for the analytes were studied at three concentration levels and the values of RSD% and relative error % were lower than 15%. The developed method was applied for the determination of ASOX after a biotransformation study employing the endophytic fungus Penicillium crustosum (VR4). This study showed that the endophytic fungus was able to metabolize the albendazole to ASOX enantioselectively. In addition, it was demonstrated that hollow fiber liquid-phase microextraction coupled to CE can be an excellent and environmentally friendly technique for the analysis of samples obtained in biotransformation studies.