Giulia Orlandini

HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts.

In 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 C(18) column (250mm×4.6mm I.D., 5μm), 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, originating product 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 1.6-4.6μg/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 16.67mg/ml and the level of total flavonoids from 2.48 to 41.10mg/ml. The proposed method can be considered suitable for the phytochemical analysis of propolis extracts used in phytotherapy.

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.

In 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 C(18) column (250 mm × 4.6 mm I.D., 5 μm), with a mobile phase composed by H(2)O 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 acid 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 monocarbonylic acetylenes, providing a confirmation of the mechanism that leads to the generation of hydroxylated 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.

Chromatographic methods for metabolite profiling of virus- and phytoplasma-infected plants of Echinacea purpurea.

This study was focused on the effects of virus and phytoplasma infections on the production of Echinacea purpurea (L.) Moench secondary metabolites, such as caffeic acid derivatives, alkamides, and essential oil. The identification of caffeic acid derivatives and alkamides was carried out by means of high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-electrospray ionization-mass spectrometry (ESI-MS), and MS(2). Quantitative analysis of these compounds was carried out using HPLC-DAD. The results indicated that the presence of the two pathogens significantly decreases (P < 0.05) the content of cichoric acid, the main caffeic acid derivative. Regarding the main alkamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, a significant decrease (P < 0.05) in the content of this secondary metabolite was observed in virus-infected plants in comparison with healthy plants, while in the phytoplasma-infected sample the variation of this secondary metabolite was not appreciable. The % relative area of the E/Z isomers of this alkamide was also found to change in infected samples. The gas chromatography (GC) and GC-MS analysis of E. purpurea essential oil enabled the identification of 30 compounds. The main significant differences (P < 0.05) in the semiquantitative composition were observed for three components: limonene, cis-verbenol, and verbenone. The results indicate that the presence of virus and phytoplasma has an appreciable influence on the content of E. purpurea secondary metabolites, which is an important issue in defining the commercial quality, market value, and therapeutic efficacy of this herbal drug.

Gas chromatography combined with mass spectrometry, flame ionization detection and elemental analyzer/isotope ratio mass spectrometry for characterizing and detecting the authenticity of commercial essential oils of Rosa damascena Mill.

RATIONALE The essential oil of Rosa damascena Mill. is known for its fine perfumery application, use in cosmetic preparations and for several pharmacological activities. Due to its high value, it can be easily adulterated with flavors or cheaper oils. This study is aimed at a detailed phytochemical characterization of commercial samples of R. damascena essential oil and at their authenticity assessment. METHODS Nineteen commercial samples of R. damascena essential oil of different geographic origin and an additional authentic one, directly extracted by hydro-distillation from fresh flowers, were considered. GC/MS and GC/FID techniques were applied for the phytochemical analysis of the samples. EA/IRMS (Elemental Analyzer/Isotope Ratio Mass Spectrometry) and GC/C (Combustion)/IRMS were used to determine the δ(13)C composition of bulk samples and of some specific components. RESULTS Citronellol (28.7-55.3%), geraniol (13.5-27.3%) and nonadecane (2.6-18.9%) were the main constituents of Bulgarian and Turkish essential oils, while those from Iran were characterized by a high level of aliphatic hydrocarbons (nonadecane: 3.7-23.2%). The δ(13)C values of bulk samples were between -28.1 and -26.9‰, typical for C3 plants. The δ(13)C values of specific components were in the usual range for natural aromatic substances from C3 plants, except for geranyl acetate, which displayed higher values (up to -18‰). These unusual δ(13)C values were explained by the addition of a natural cheaper oil from a C4 plant (Cymbopogon martinii, palmarosa), which was found to occur in most of the essential oils. CONCLUSION GC/C/IRMS, in combination with GC/MS and GC/FID, can be considered as an effective and reliable tool for the authenticity control of R. damascena essential oil.

Cytotoxic activity and G1 cell cycle arrest of a Dienynone from Echinacea pallida.

In the present study, a further investigation of the cytotoxic activity of an acetylenic constituent of Echinacea pallida roots, namely, pentadeca-(8 Z,13 Z)-dien-11-yn-2-one, was performed, revealing a concentration-dependent cytotoxicity on several human cancer cell lines, including leukemia (Jurkat and HL-60), breast carcinoma (MCF-7), and melanoma (MeWo) cells. As part of its mechanism of action, the ability of this constituent to arrest the cell cycle in the G1 phase was demonstrated on HL-60 cells. Furthermore, a stability test of the target compound over 72 h was carried out, indicating that the cytotoxic activity can be attributed mainly to the genuine, not oxidized, molecule.