HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts
Introduction
Propolis, also called bee glue, is a dark-coloured resinous material collected by honeybees (Apis mellifera L.) from leaf buds and cracks in the bark of several tree species [1]. Once collected, this material is enriched with salivary and enzymatic secretions [1]. Several pharmacological activities have been attributed to propolis extracts, mainly antibacterial, antiviral, antifungal, anti-inflammatory, antioxidant, antiproliferative, immunostimulating, anti-ulcerous etc. [2]. Current applications of propolis include herbal products for cold syndrome and dermatological preparations [3]. In addition, propolis is used to prevent and treat oral inflammations [3]. Most of these preparations contain ethanolic extracts of propolis [3].
The detailed chemical composition of propolis is known to be very complex [1], [4]. In propolis from temperate zones, the most important class of biologically active compounds is characterized by polyphenols, including flavonoids, phenolic acids and their esters [1]. In contrast, propolis from tropical areas has shown to contain prenylated phenylpropanoids and non-typical propolis flavonoids (Brazil) or polyisoprenylated benzophenones (Cuba) [5]. The content of polyphenols in “poplar type” propolis extracts may vary as a function of the origin of samples and these differences can affect the biological activity of preparations and therefore their pharmacological effects [1]. In this context, the development of analytical methods for the phytochemical analysis propolis is of great interest.
Several methods have been described in the literature for the analysis of polyphenols in propolis, based on non-separation techniques, such as UV–vis spectrophotometry [1] and NMR [6] or on separation techniques, including GC, HPTLC, HPLC and HPCE [1]. Of these methods, the spectrophotometric ones are considered to be useful especially for the routine control of propolis samples [1], [7]. HPLC in combination with spectroscopic and spectrometric detection has significantly improved the analysis of phenolic compounds in natural products derived from bees, providing definitive information for the identification and quantification of these biologically active constituents [3], [5], [6], [8], [9], [10], [11]. However, most of these methods have not been validated in agreement with ICH guidelines [12] for comprehensive multi-component analysis of phenolic acids and flavonoids in propolis samples.
In this context, this paper aims to provide a reliable and fully validated method for the phytochemical analysis of propolis hydroalcoholic extracts by HPLC-DAD and HPLC-ESI-MS/MS with ion trap (IT) and triple quadrupole (TQ) mass analyzers. By using HPLC-ESI-MS/MS, it was possible to obtain the quasi-molecular ions and the MS/MS spectra, which, in combination with retention times and UV data, made the peak identification of target analytes very reliable. The fragmentation patterns of flavonoids and caffeic acids obtained by IT and TQ are discussed in the present work. The practical applicability of the technique was demonstrated by the analysis of propolis extracts representative of the Italian market to provide a reliable phytochemical profiling of their health-promoting secondary metabolites.
Section snippets
Chemicals and solvents
Caffeic acid, p-coumaric acid, ferulic acid, quercetin, pinocembrin, cinnamic acid, apigenin, kaempferol and galangin were from Sigma–Aldrich–Fluka (Milan, Italy). Isorhamnetin and luteolin were from Roth (Karlsruhe, Germany). Chrysin was from Extrasynthese (Genay, France).
Quercetin-3-methyl-ether, pinobanksin, galangin-5-methyl-ether, quercetin-7-methyl-ether, caffeic acid phenylethyl ester (CAPE) and pinobanksin-3-O-acetate were kindly donated by Prof. Dr. Eckhard Wollenweber, Institut für
Identification of propolis secondary metabolites
The HPLC-DAD analysis of a typical commercial sample of propolis (PE-9) available on the Italian market at 290 nm indicated a very complex composition, as shown in Fig. 1. The corresponding peak identification is described in Table 2A, Table 2B, Table 2C. Considering the complexity of the sample, the overall chromatographic separation can be considered satisfactory. The only limitation is the separation of caffeic acid phenylethyl ester (CAPE) (peak 28) and pinobanksin-3-O-acetate (peak 29):
Conclusion
The proposed HPLC method, based on the use of UV, MS and MS/MS data, allowed the identification and quantification of 40 compounds, including phenolic acids and flavonoids, in hydroalcoholic extracts of propolis on sale on the Italian market. Under the applied conditions, the TQ mass analyzer provided a higher fragmentation degree of the target analytes in comparison with the IT and, therefore, more structural information.
The method validation indicated that this technique represents a reliable
Acknowledgments
The authors acknowledge the “Fondazione Cassa di Risparmio di Modena” for funding the HPLC-ESI-IT and HPLC-ESI-TQ systems at the Centro Interdipartimentale Grandi Strumenti (CIGS) of the University of Modena and Reggio Emilia.
The authors are particularly grateful to Prof. Dr. Eckhard Wollenweber (Institut für Botanik, Darmstadt, Germany) for kindly providing several compounds used to confirm the identity of propolis constituents.
The authors are also grateful to Dr. Alessandro Biancardi
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