Industrial hemp is a strain of the Cannabis Sativa species that is specifically grown for the industrial uses of its derivatives. Besides being a rich source of bioactive compounds, such as cannabinoids, polar lipids, proteins, vitamins and phenolic compounds, hemp is still less characterized than its drug-like counterpart. However, it has recently gained increasing attention for nutraceutical, pharmaceutical and cosmetic applications, since its usage is rapidly spreading in western economies. In literature, investigations of bioactive compounds occurring in hemp flowers are mainly focused on cannabinoids and their derivatives, whereas comprehensive knowledge on polar lipids and phenolic compounds is rather scarce. In order to maximize bioactive compound identification, several specific extraction procedures will be tested for both polar lipids and polyphenols. A ultra-high-pressure liquid chromatography system coupled via an electrospray source to a hybrid quadrupole¿Orbitrap mass spectrometer will be employed. Spectra will be acquired in data-dependent mode, and several parameters will be investigated to acquire the largest possible number of both mass spectrometry (MS) features and MS2 mass spectra for unique species. Finally, bioinformatic softwares will be employed for data analysis, i. e. Lipostar, a comprehensive platform-neutral cheminformatics tool for lipidomics, and Compound Discoverer, a mass spectrometry data analysis software for small molecules. Also, a home-made comprehensive database of flavonoids, anthocyanins, phenolic acids and aldehydes, ellagitannins and proantocyanidins derivatives will be compiled and implemented on Compound Discoverer for polyphenols detection and identification. The aim of the project is obtaining deeper knowledge of polar lipids and phenolic compounds content in industrial hemp flower.
The main goal of the project is applying innovative and improved technologies to the characterization of industrial hemp flowers, a plant matrix whose use is spreading more and more in the occidental economies for its versatility and bioactive compound abundance. In particular, with respect to the current state of the art.
1) Optimization of chromatographic conditions of polar lipids on stationary phases which have not been used for such purpose, before. A systematic evaluation of reversed phase and hydrophilic interaction chromatography stationary phases will be performed, to find the most suitable for separation of polar lipids and coupling with high resolution mass spectrometry.
2) The first comprehensive analytical platform for polar lipids in industrial hemp flowers. This section will take into account both lipid extract preparation and lipid enrichment. First, the extraction of polar lipids from hemp flowers will be performed following several procedures reported in literature and others employing greener solvents. Moreover, chlorophyll removal will be taken into account to avoid matrix effect during UHPLC-MS analysis. Two complementary lipid enrichment strategies, based on GCB solid phase extraction and offline two-dimensional chromatography, will be optimized in order to achieve a larger coverage of lipid species. Those enrichment strategies could be eventually used for lipid analysis in other plant matrices.
3) Lipids profile characterization of industrial hemp flowers, focusing in particular on the fosfo-, sulfo- and glycolipid species, and qualitative comparison among them will be carried out.
4) The first comprehensive analytical platform for polyphenols in industrial hemp flower extracts. Several procedures for polyphenol extractions will be devised by comparison of different protocols and adaptation to hemp flower matrix. In particular, non-psychoactive cannabinoid removal will be taken into consideration in order to reduce matrix effects, due to high abundance of those substances in hemp matrix.
5) An innovative analytical platform for polyphenol derivative analysis will be employed for identification and validation of polyphenols in industrial hemp flower extracts. In particular, a database of flavonoids, anthocyanins, phenolic acid and aldehydes derivatives and polymeric polyphenols will be compiled and then implemented on Compound Discoverer to ease and maximize polyphenols identification. This platform could eventually be employed for data analysis of these phytochemicals in other plant matrices.