The effect of desflurane on neuronal communication at a central synapse

Although general anesthetics are thought to modify critical neuronal functions, their impact on neuronal communication has been poorly examined. We have investigated the effect induced by desflurane, a clinically used general anesthetic, on information transfer at the synapse between mossy fibers and granule cells of cerebellum, where this analysis can be carried out extensively. Mutual information values were assessed by measuring the variability of postsynaptic output in relationship to the variability of a given set of presynaptic inputs. Desflurane synchronized granule cell firing and reduced mutual information in response to physiologically relevant mossy fibers patterns. The decrease in spike variability was due to an increased postsynaptic membrane excitability, which made granule cells more prone to elicit action potentials, and to a strengthened synaptic inhibition, which markedly hampered membrane depolarization. These concomitant actions on granule cells firing indicate that desflurane re-shapes the transfer of information between neurons by providing a less informative neurotransmission rather than completely silencing neuronal activity

Chromatographic techniques for metabolite fingerprinting of propolis polyphenols and volatile compounds

This study was aimed at developing advanced chromatographic methods for a complete phytochemical characterization of raw propolis polyphenols and volatile compounds, which are responsible for the biological activity of the extracts widely used in phytotherapy. The study of polyphenols was based on RP-HPLC coupled with DAD and MS detection, using the new fused-core column technology, which allowed a sensitive reduction in the total analysis time in comparison with conventional particulate stationary phases. The HPLC analyses were carried out on an Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 µm), with a mobile phase composed by 0.1% aqueous formic acid and ACN, under gradient elution. Different extraction methods were compared, including sonication and microwave extraction, in order to obtain a high recovery of polyphenols from raw propolis. The study of volatile compounds was based on GC coupled with MS detection. A suitable HS-SPME extraction procedure, whose experimental parameters were properly optimized, was developed using a 1 cm PDMS fiber. The GC analyses were carried out on an HP-5 MS cross-linked 5% diphenyl-95% dimethyl polysiloxane capillary column (30 m × 0.25 mm I.D., 1.00 µm film thickness), under programmed-temperature elution. The volatile compounds extracted from raw propolis were identified by comparing the MS fragmentation patterns with those of pure compounds and by mass spectrum database search using NIST. The chromatographic methods developed in this study were applied to samples of raw propolis collected in different Italian regions and allowed us to determine their metabolite fingerprinting, thus providing new and reliable tools for the complete phytochemical characterization of this material

Metabolite profiling of propolis polyphenols by microwave-assisted extraction combined with high-performance liquid chromatography using the fused-core technology

The chemical composition of propolis extracts is known to be very complex and influenced by several factors, such as the geographic origin. In propolis from temperate zones, the most representative biologically active compounds are polyphenols, including phenolic acids and flavonoids. Raw propolis can not be used as crude material, but it must be purified by solvent extraction to remove the useless material and preserve the active polyphenol fraction. In this study, a closed-vessel microwave-assisted extraction (MAE) technique was optimized for the first time for the extraction of phenolic acids and flavonoids from raw propolis. The optimal extraction conditions were determined by means of response surface experimental design methodology. In comparison with other extraction techniques, such as maceration, heat reflux extraction (HRE) and ultrasound-assisted extraction (UAE), the extraction with MAE was improved by shorter extraction time and lower volume of solvent needed. The HPLC analyses of propolis extracts were carried out for the first time on a fused-core Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 µm, Supelco), with a mobile phase composed by 0.1% formic acid in water and acetonitrile. Detection was performed by UV/DAD and ion trap mass analyzer. A total of thirty-eight polyphenol compounds were identified in propolis samples, on the basis of the UV, MS and MS2 data. In comparison with a conventional fully porous stationary phase, the fused-core one allowed a good separation of polyphenols and a reduction of both time and solvent usage. The method validation, performed in agreement with ICH guidelines, indicated that the correlation coefficients were > 0.999; the limit of detection (LOD) was in the range 0.5-0.8 ug/mL for phenolic acids and 1.2-3.0 ug/mL for flavonoids; the recovery range was 95.3-98.1% for phenolic acids and 94.1-101.3% for flavonoids; the intra- and inter-day %RSD values for retention times and peak areas were found to be ≤ 0.3 and 2.2%, respectively. The quali- and quantitative analysis of polyphenols in Italian samples of raw propolis was performed with the validated method. Total phenolic acids ranged from 5.0 to 120.8 mg/g and total flavonoids from 2.5 to 168.0 mg/g. The proposed MAE procedure and HPLC method can be considered reliable and useful tools for the comprehensive multi-component analysis of polyphenols in raw propolis extracts to be used in apitherapy

Development of advanced chromatographic methods for a complete phytochemical characterization of raw propolis polyphenols and volatile compounds

Propolis, or bee glue, is a dark-colored resinous substance collected by honeybees (Apis mellifera L.) from several tree species. Because of its biological and pharmacological properties, such as antibacterial, antiviral, antifungal, anticancer, anti-inflammatory, and immunomodulatory activities, propolis has attracted researchers’ interest in the last decades. Since propolis is used extensively, a rapid and efficient method for the extraction and analysis of propolis is certainly very useful. With the aim to determine polyphenols in propolis we have applied RP-HPLC coupled with DAD and MS detection, using the new fused-core column technology, which allowed a sensitive reduction in the total analysis time in comparison with conventional particulate stationary phases. The HPLC analyses were carried out on an Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 µm), with a mobile phase composed by 0.1% aqueous formic acid and acetonitrile, under gradient elution. Different extraction methods were compared, including sonication and microwave extraction, in order to obtain a high recovery of polyphenols from raw propolis. The other active component of propolis includes volatile compounds and its analysis was performed by GC coupled with MS detection. A suitable HS-SPME extraction procedure, whose experimental parameters were properly optimized, was developed using a 1 cm PDMS fiber. The GC analyses were carried out on an HP-5 MS cross-linked 5% diphenyl-95% dimethyl polysiloxane capillary column (30 m × 0.25 mm I.D., 1.00 m film thickness), under programmed-temperature elution. The volatile compounds extracted from raw propolis were identified by comparing the MS fragmentation patterns with those of pure compounds and by mass spectrum database search using NIST. The chromatographic methods developed in this study were applied to samples of raw propolis collected in different Italian regions and allowed us to determine their metabolite fingerprinting, thus providing new and reliable tools for the complete phytochemical characterization of this material

Metabolite fingerprinting of raw propolis from different Italian regions by innovative HPLC-ESI-MS and GC-MS

This study was aimed at a detailed chromatographic analysis of propolis samples by means of the development and application of innovative HPLC-ESI-MS and HS-SPME-GC-MS techniques to provide a complete fingerprinting of the biologically active components

Headspace solid-phase microextraction-gas chromatography-mass spectrometry characterization of propolis volatile compounds

In this study, a novel and efficient method based on headspace solid-phase microextraction (HS-SPME), followed by gas chromatography-mass spectrometry (GC-MS), was developed for the analysis of propolis volatile compounds. The HS-SPME procedure, whose experimental parameters were properly optimized, was carried out using a 100. μm polydimethylsiloxane (PDMS) fiber. The GC-MS analyses were performed on a HP-5 MS cross-linked 5% diphenyl-95% dimethyl polysiloxane capillary column (30 m × 0.25 mm I.D., 1.00 μm film thickness), under programmed-temperature elution.Ninety-nine constituents were identified using this technique in the samples of raw propolis collected from different Italian regions. The main compounds detected include benzoic acid (0.87-30.13%) and its esters, such as benzyl benzoate (0.16-13.05%), benzyl salicylate (0.34-1.90%) and benzyl cinnamate (0.34-3.20%). Vanillin was detected in most of the samples analyzed in this study (0.07-5.44%). Another relevant class of volatile constituents is represented by sesquiterpene hydrocarbons, such as δ-cadinene (1.29-13.31%), γ-cadinene (1.36-8.85%) and α-muurolene (0.78-6.59%), and oxygenated sesquiterpenes, such as β-eudesmol (2.33-12.83%), T-cadinol (2.73-9.95%) and α-cadinol (4.84-9.74%). Regarding monoterpene hydrocarbons, they were found to be present at low level in the samples analyzed in this study, with the exception of one sample from Southern Italy, where α-pinene was the most abundant constituent (13.19%). The results obtained by HS-SPME-GC-MS were also compared with those of hydrodistillation (HD) coupled with GC-MS.The HS-SPME-GC-MS method developed in this study allowed us to determine the chemical fingerprint of propolis volatile constituents, thus providing a new and reliable tool for the complete characterization of this biologically active apiary product

Innovative methods for the extraction and chromatographic analysis of honey bee products

In this study, new and efficient chromatographic techniques were developed for a complete chemical characterization of raw propolis polyphenols and volatile compounds, which are responsible for the biological activity of the extracts widely used in apitherapy. The analysis of polyphenols, including phenolic acids and flavonoids, was performed by RP-HPLC coupled with UV/DAD and MS detection, using the new fused-core technology. Microwave-assisted extraction (MAE) was carried out for these constituents and its parameters were optimized by means of response surface experimental design methodology. The characterization of propolis volatile compounds was based on GC coupled with MS detection. In this ambit, a suitable HS-SPME extraction procedure was developed for the first time using a PDMS fiber. Both these chromatographic techniques were applied to raw propolis samples collected in different Italian regions to determine their metabolite fingerprinting, thus providing new and reliable tools for the complete chemical characterization of this biologically active material

Metabolite profiling of polyphenols in the Tunisian plant Tamarix aphylla (L.) Karst

In this study, a detailed investigation on the composition of polyphenols of Tamarix aphylla (L.) Karst., consisting of phenolic acids and flavonoids, was carried out. In order to optimize the yield of secondary metabolites, three extraction techniques were compared, including dynamic maceration, ultrasound-assisted extraction and Soxhlet extraction. The latter technique provided the best results in terms of both recovery and selectivity, using ethyl acetate as extraction solvent for 2h. The analysis of T. aphylla polyphenols was performed by means of HPLC-UV/DAD, HPLC-ESI-MS and MS(2), using an ion trap mass analyzer. Phenolic acids and flavonoids were separated on an Ascentis C18 column (250mm×4.6mm I.D., 5μm), with a mobile phase composed of 0.1M formic acid in water and acetonitrile, under gradient elution. The proposed method was fully validated in agreement with ICH guidelines and then applied to the analysis of T. aphylla leaves and stems. A total of 14 phenolic compounds were characterized for the first time in this plant extracts by using UV, MS and MS(2) data. The amount of total phenolics was found to be 993.1±22.5μg/g in the leaves and 113.1±25.8μg/g in the stems, respectively. The most abundant constituents found in the leaves include ellagic acid (211.4±10.8μg/g), quercetin (125.7±4.7μg/g) and gallic acid (120.6±1.2μg/g), whereas those in the stems were ellagic acid (44.4±3.9μg/g), gallic acid (24.3±3.3μg/g) and kaempferol (16.3±1.6μg/g). The developed method can be considered a useful tool for the metabolite profiling of T. aphylla, which represents a potential source of bioactive compounds to be used in phytotherapy

SIMULTANEOUS METABOLITE PROFILING OF HYDROPHILIC AND LIPOPHILIC COMPOUNDS IN ECHINACEA PALLIDA BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH PHOTODIODE ARRAY AND ELECTROSPRAY IONIZATION-MASS SPECTROMETRY DETECTION

Echinacea spp. (family Asteraceae) herbal medicines and dietary supplements are traditionally used as immunostimulants in the prevention and treatment of inflammatory and viral diseases . Despite of many studies that have shown the chemical composition of alkamides in medicinally important species such as E. purpurea and E. angustifolia, in this study the attention was focused on secondary metabolites contained in E. pallida. In particular, caffeic acids derivatives, that have demonstrated to possess antioxidant, antiviral and anti-inflammatory activities, have been identified from the hydrophilic fractions of E. pallida extracts, while polyacetylenes and polyenes, that have shown interesting cytotoxic activities against a number of solid and leukemic cancer cell lines, have been isolated and characterized from lipophilic extracts of E. pallida roots.In this study, a detailed phytochemical characterization of E. pallida extracts and herbal medicines was carried out for the first time by developing advanced chromatographic techniques, based on RP-HPLC coupled with diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection, for the simultaneous analysis of the hydrophilic and lipophilic secondary metabolites. In the context of mass spectrometry detection, the performance of two mass analyzers, such as an ion trap and a triple quadrupole, were compared. The HPLC analyses were carried out on an Ascentis C18 column (250 mm × 4.6 mm I.D., 5 um), with a mobile phase composed by 0.1% aqueous formic acid and ACN with 0.1% of formic acid, under gradient elution. The flow rate was 1.0 mL/min and the column temperature was set at 30 °C. The UV spectra, in combination with MS and MS/MS data, allowed the identification of ten compounds, including caffeic acids derivatives, polyacetylenes and polyenes, in the analyzed samples. MS and MS/MS data were discussed in details and the typical fragmentation patterns of each class of secondary metabolites were identified.The RP-HPLC method was then fully validated in agreement with the ICH (Q2-R1) guidelines and then applied to real samples. The reference compounds used for quantification were isolated from the plant material and their structures were determined on the basis of the analysis of UV, IR, NMR and MS data. The quantitative analysis indicated that there was a great variability in the amount of the active compounds in the herbal medicines: the content of total caffeic acid derivatives ranged from 2.02 to 11.05 mg/g and the level of total polyacetylenes and polyenes from 9.62 to 39.12 mg/g. In the analyzed samples, the most abundant caffeic acid derivative was found to be echinacoside. Regarding polyacetylenes and polyenes, the most representative compounds were found to be pentadec-8-en-2-one, pentadeca-8,11-dien-2-one and tetradec-8-ene-11,13-diyn-2-one .The chemical standardization of E. pallida extracts is necessary to guarantee the quality, efficacy and safety of the corresponding herbal medicines. In this context, the proposed method, allowing the simultaneous determination of both the hydrophilic and lipophilic compounds contained in E. pallida, can be considered a useful and reliable tool for the metabolite profiling of plant material and the quality control of natural product

Caratterizzazione fitochimica della frazione volatile di propoli mediante tecnica HS-SPME-GC-MS

Dato il crescente interesse per i prodotti naturali, è importante riuscire a caratterizzarli per definirne nuove applicazioni per la salute, l'agricoltura e l'ambiente. Alcuni di essi, infatti, costituiscono alternative efficaci o complementari a composti sintetici dell'industria chimica, senza mostrarne gli effetti secondari. Fra questi prodotti di origine naturale è da ricordare la propoli, sostanza resinosa raccolta, trasformata e utilizzata dalle api (Apis mellifera L.) per sigillare i fori degli alveari, levigare le pareti interne e ripararsi dagli agenti esterni. Le api raccolgono queste resine da foglie, germogli e corteccia di alcune piante, quali pioppo (Populus sp.), betulla (Betula sp.), quercia (Quescus sp.), ippocastano (Aesculus hippocastanum), ontano (Alnus sp.) e conifere varie. Questa resina viene poi masticata, arricchita di enzimi salivari, parzialmente digerita e successivamente miscelata a cera d’api per essere usata nell’alveare. Per la propoli sono descritte numerose proprietà biologiche quali quella antibatterica, antimicotica, antivirale, antiossidante, anti-infiammatoria, antiproliferativa, immunostimolante. A dispetto di eventuali differenze di composizione dovute alle diverse fonti vegetali, la maggior parte dei campioni di propoli possiede una notevole somiglianza nella natura chimica complessiva. Essa è costituita da: 50% di resina (composta da polifenoli), 30% di cera, 10% di oli essenziali, 5% di polline, 5% di altri composti organici. I composti di interesse farmacologico si trovano nella frazione volatile e nella frazione polifenolica, le quali includono principalmente terpenoidi, steroidi, flavonoidi e acidi fenolici. Scopo del presente lavoro è la caratterizzazione quali e quantitativa dei componenti della frazione volatile della propoli: a tal fine sono state ottimizzate le condizioni di isolamento mediante tecnica SPME in spazio di testa (Head Space Solid Phase Micro Extraction) e quelle di analisi quali- e quantitativa con tecnica GC-MS (Gas Chromatography-Mass Spectrometry) e GC-MS (Gas Chromatography-Mass Spectrometry). In questo modo è stato possibile un confronto tra i campioni di propoli di origine diversa, sia di tipo qualitativo che semi-quantitativo