1,720,972 research outputs found
High-performance liquid chromatoghraphy/tandem mass spectrometry methods in the phytochemical analysis of hydro-alcoholic propolis extracts.
No full textPolyphenolic compounds are the major active constituents of propolis extracts. Due to its phytochemical composition, propolis extracts are widely used in phytotherapy, particularly as anti-bacterial, anti-inflammatory and antioxidant.The interest in propolis compounds and their biological activity is still increasing. In this ambit, the development and validation of suitable analytical methods aimed at the phytochemical analysis and quality control of propolis is very important.In this study, a detailed characterization of propolis hydro-alcoholic extracts was carried out by developing advanced chromatographic techniques, based on RP-HPLC coupled with diode array and mass spectrometry detection and analyzing commercially available hydro-alcoholic propolis extracts. 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, under gradient elution. The flow rate was 1.2 mL/min and the column temperature was set at 30°C. In view of UV spectra, MS and MS/MS data, forty polyphenolic compounds, including phenolic acids and flavonoids, were identified as typical components of the analyzed samples. The comparison of the performance of the selected mass analyzers indicated that the triple quadrupole system provided more structural information on target analytes than the ion trap.The HPLC method was then fully validated in agreement with the guidelines of the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use and then applied to real samples.Although the chromatographic profile of the analyzed samples was similar, the quantitative analysis indicated that there is a great variability in the amount of the active compounds: the content of total phenolic acids ranged from 0.17 to 16.72 mg/mL and the level of total flavonoids from 2.51 to 42.72 mg/mL. In the analyzed samples, the most abundant flavonoids were found to be chrysin, galangin, pinocembrin and pinobanksin (and its esters). Regarding phenolic acids, the most representative compounds were found to be caffeic acid derivatives.The chemical standardization of propolis extracts is necessary to guarantee its quality, efficacy and safety. In this context the proposed method, allowing the determination of the complete phenolic profile characterized by both phenolic acids and flavonoids, can be considered as a useful tool for metabolite profiling and quality control of propolis natural products
Simultaneous metabolite fingerprinting of hydrophilic and lipophilic compounds in Echinacea pallida by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection
No full textIn this study, a detailed phytochemical characterization of Echinacea pallida (Nutt.) Nutt. root extracts and dietary supplements was carried out for the first time by developing advanced chromatographic techniques, based on HPLC with diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection (with ion trap and triple quadrupole mass analyzers), for the simultaneous analysis of hydrophilic and lipophilic secondary metabolites. 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 H2O and ACN both containing 0.1% formic acid, under gradient elution.
The UV spectra, in combination with MS and MS/MS data, allowed the identification of fourteen compounds, including caffeic acids derivatives, polyacetylenes and polyenes, in the analyzed samples. MS and MS/MS data were discussed in detail and the typical fragmentation patterns of each class of secondary metabolites were identified. For the first time, a hydroperoxide intermediate was characterized as an oxidation product of one of E. pallida monocarbonilyc acetylenes, providing a confirmation of the mechanism that leads to the generation of hydroxilated derivatives.
The HPLC method was fully validated in agreement with ICH guidelines and then applied to real samples. The quantitative analysis indicated that there was a great variability in the amount of the active compounds in the dietary supplements containing E. pallida root extracts: the content of total caffeic acid derivatives ranged from 2.31 to 11.45 mg/g and the amount of total polyacetylenes and polyenes from 6.38 to 30.54 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 tetradec-(8Z)-ene-11,13-diyn-2-one, pentedeca-(8Z,11Z)-dien-2-one and pentadec-(8Z)-en-2-one.
The developed method can be considered suitable for metabolite fingerprinting and quality control of E. pallida plant material and natural products
Studio di stabilità dei costituenti biologicamente attivi di Echinacea pallida
No full textRecenti studi hanno dimostrato che i metaboliti secondari (poliacetileni e polieni), isolati dall’estratto grezzo delle radici di Echinacea pallida1,2, possiedono buona attività citotossica su alcune linee cellulari tumorali (MIA PaCa-2 e COLO320)3. Tali composti sono presenti nella radice in forma non ossidata; tuttavia, durante i processi di essiccamento e successiva lavorazione, si nota una variazione della loro concentrazione, che è stata attribuita ad un possibile processo di ossidazione1.Considerata l’importanza dell’attività biologica di tali composti, scopo della presente ricerca è la valutazione della loro stabilità mediante RP-HPLC-DAD2. L’obiettivo più specifico è costituito dalla determinazione della stabilità di suddetti componenti in condizioni simili a quelle cellulari. Lo studio è stato inizialmente condotto su un estratto grezzo lipofilo ottenuto dalle radici di E. pallida ed è stato successivamente focalizzato sui singoli composti isolati e purificati mediante tecniche cromatografiche preparative. I risultati hanno evidenziato che, nel corso delle 72 ore di monitoraggio nelle diverse condizioni sperimentali, l’estratto grezzo subisce una progressiva ossidazione, con incremento dei derivati idrossilati. Le successive prove di stabilità condotte sui tre metaboliti isolati dall’estratto grezzo di E. pallida (tetradec-(8Z)-ene-11,13-diyn-2-one (1), pentadec-(8Z)-ene-11,13-diyn-2-one (2), pentadeca-(8Z,13Z)-dien-11-yn-2-one(3)) hanno confermato che i suddetti composti subiscono un processo di ossidazione, tramite la formazione di un intermedio idroperossido. Dei composti 1-3 è stata quindi studiata la cinetica di ossidazione nelle diverse condizioni sperimentali
Stability study on a cytotoxic dienynone isolated from Echinacea pallida
No full textRecent studies have demonstrated that some of the secondary metabolites (polyacetylenes and polyenes) isolated from the raw lipophilic extract of Echinacea pallida roots have good cytotoxic activity on cancer cell lines3. In particular, pentadeca-(8Z,13Z)-dien-11-yn-2-one was found to be the most active constituent, with an IC50 value of 32.17 ± 3.98 uM on pancreatic MIA PaCa-2 cancer cells and 2.34 ± 0.33 uM on colonic COLO320 cancer cells, after 72 h of exposure. This compound is present in the roots in a not-oxidized form but, during the drying process and the subsequent storage, a change in its concentration was observed, which has been attributed to a possible oxidation process. As part of our ongoing research on bioactive metabolites from Echinacea, the aim of this research was the evaluation of the stability of this secondary metabolite by RP-HPLC-DAD2 under the experimental conditions applied during the biological assays. The study was initially carried out on the lipophilic crude extract obtained from E. pallida roots and then it was focused on the individual component isolated and purified from the plant material by preparative chromatographic techniques. The results showed a moderate change in the HPLC profiles of E. pallida crude extract in 72 h, with an increase of hydroxylated derivatives. The oxidation kinetic of the most active compound, pentadeca-(8Z,13Z)-dien-11-yn-2-one, was found to be quite slow, since the % area of the intermediate hydroperoxide was about 25% after 72 h of exposure. The final product of the oxidation process, i.e. the hydroxilated derivative (i.e. 8-hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one, available thanks to a synthetic approach), was not observed in the HPLC chromatogram after 72 h. Considering that during the first 24 h of exposure, the % area of the hydroperoxide intermediate is below 15%, the observed cytotoxic activity can be mainly attributed to the genuine not oxidized compound
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
No full textEchinacea 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
Study on the enantiomerization of synephrine by chiral high-performance liquid chromatography
No full textSynephrine, the active phenethylamine alkaloid of Citrus aurantium L., is a biologically active compound that has effects on human metabolism that could help to reduce fat mass in obese people, since it stimulates lipolysis, raises the metabolic rate and promotes the oxidation of fat through increased thermogenesis. In the light of this, C. aurantium extracts are commonly used in the formulation of phytotherapic products employed in the treatment of obesity.
It is well known that synephrine is a chiral compound and its enantiomers have shown different pharmacological activity on alpha- and beta-adrenoreceptors. In particular, R-(−)-synephrine is from 1 to 2 orders of magnitude more active than its S-(+)-counterpart. R-(−)-Synephrine has been isolated and identified in Citrus fruits. However, a certain amount of S-(+)-synephrine has been detected in C. aurantium dry extracts and dietary supplements (1). The presence of S-(+)-synephrine in C. aurantium natural products has been attributed to a possible enantiomerization of R-(-)-synephrine during the industrial production of the fruit extracts, using a high temperature and a long period of refluxing.
To evaluate the enantiomerization kinetic parameters of R-(-)-synephrine, an off-column HPLC method based on a chiral stationary phase (CSP) with cellobiohydrolase as the chiral selector (Chiral-CBH) was developed. Analyses were carried out on a Chiral-CBH column (100 × 4.0 mm i.d., 5 um), with a mobile phase consisting of 2-propanol (5%, w/w) in sodium phosphate buffer (pH 6.0; 10 mM) and disodium EDTA (50 uM). The flow rate was 0.8 mL/min. The column was thermostatted at 20°C. Detection was set at 225 nm.
The individual enantiomers of the studied compound were isolated by fractional crystallization of the diastereomeric salts and subsequent ion-exchange. The rate constants and the free energy barriers of enantiomerization were determined in different solvents and buffer solutions at pH 1-11. The results generated by the off-line method were used to determine the influence of solvents and pH values on the enantiomerization rate of (+) and (-)-synephrine and to gain further insight into the enantiomerization mechanism of chiral phenethylamine type alkaloids in relation to the pKa values
Metabolite fingerprinting of bioactive compounds in Echinacea pallida by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection
No full textThis study was aimed at developing a novel, reliable and fully validated method for the simultaneous analysis of biologically active caffeic acid derivatives and polyacetylenes/polyenes in Echinacea pallida (Nutt.) Nutt. plant material and natural products by HPLC-UV/DAD, HPLC-ESI-MS and MS/MS using ion trap (IT) and triple quadrupole (TQ) mass analyzers. Even if it is more difficult to optimize the separation of such different constituents in one run, a method for the simultaneous determination of both caffeic acid derivatives and polyacetylenes/polyenes in E. pallida is useful because it can reduce both the time and the sample size required for the analysis. In addition, a technique able to provide a complete fingerprint of the secondary metabolites is of great interest in the ambit of phytochemical analysis and quality control of E. pallida raw material and derivatives, widely used in phytotherapy. 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 H2O and ACN both containing 0.1% formic acid, under gradient elution. A relevant novel aspect of the proposed technique is that the combination of MS and MS/MS data with retention times and UV spectra made the peak identification very reliable. The fragmentation patterns of caffeic acid derivatives and polyacetylenes/polyenes obtained by IT and TQ are discussed in detail for the first time in the present work. The oxidation mechanism of monocarbonylic acetylenes is demonstrated for the first time in this study using MS and MS/MS data. The practical applicability of the technique was demonstrated by the quantitative analysis of E. pallida root extracts and commercially available dietary supplements to provide reliable chromatographic fingerprints of their bioactive secondary metabolites
HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts
No full textIn this study, the composition of polyphenols (phenolic acids and flavonoids) in propolis extracts was investigated by HPLC-DAD and HPLC-ESI-MS/MS by comparing the performance of ion trap and triple quadrupole mass analyzers. The 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% formic acid in water and acetonitrile. Overall, the UV spectra, the MS and MS/MS data allowed the identification of 40 compounds. In the case of flavonoids, the triple quadrupole mass analyzer provided more collision energy if compared with the ion trap, originatingproduct ions at best sensitivity.The HPLC method was validated in agreement with ICH guidelines: the correlation coefficients were >0.998; the limit of detection was in the range 1.6–4.6 ug/ml; the recovery range was 96–105%; the intra- and inter-day %RSD values for retention times and peak areas were found to be <0.3 and 1.9%, respectively.The developed technique was applied to the analysis of hydroalcoholic extracts of propolis available on the Italian market. Although the chromatographic profile of the analyzed samples was similar, the quantitative analysis indicated that there is a great variability in the amount of the active compounds: the content of total phenolic acids ranged from 0.17 to 16.67 mg/ml and the level of total flavonoids from 2.48 to 41.10 mg/ml. The proposed method can be considered suitable for the phytochemical analysis of propolis extracts used in phytotherapy
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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