Valeria Cinquepalmia, Ilario Lositoa,b, Andrea Castellanetaa, Beniamino Leonic, Massimiliano Rennac, Pietro Santamariac, Cosima Damiana Calvanoa,b, Tommaso R.I. Cataldia,b

aDipartimento di Chimica, Università degli Studi di Bari Aldo Moro, 70126 Bari, Italy;
bCentro Interdipartimentale SMART, Università degli Studi di Bari Aldo Moro, 70126 Bari, Italy
cDip. di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi di Bari Aldo Moro, 70126 Bari, Italy

Plant sterols (PSs) are secondary metabolites occurring in plants in various forms, including free sterols (FSs), esters of fatty acids, steryl glycosides and acyl steryl glycosides. PSs are mainly located within the raft-domains and play an essential role in cells by preserving integrity and regulating the fluidity of the lipid bilayer [1]. PSs are bioactive substances due to their ability to lower blood cholesterol levels and to exert anti-obesity, anti-diabetic and anti-inflammatory activities [2]. In the last decade, with the increasing attention to healthy eating and lifestyle, the interest in fresh, ready to eat innovative vegetables, such as microgreens, has been on the rise. The characterization of FSs in these products using reversed-phase liquid chromatography coupled to high-resolution Fourier-transform mass spectrometry with atmospheric pressure chemical ionization (RPLC-APCI-FTMS) has thus been recently undertaken in our laboratories. 

The focus is on innovative products of the Apulian Brassicaceae species, such as broccoli raab and kale, already known as sources of bioactive compounds [2], including PSs, recently identified in these vegetables using GC-MS [3]. In the present work, the developed RPLC-APCI-FTMS method, fine-tuned on a mixture of appropriate standards, allowed to detect FSs in extracts of microgreens, micro leaves, baby leaves and, for comparison, in the canonical adult forms of both broccoli raab and kale. Major FSs such as β-sitosterol and campesterol, as well as stigmasterol, brassicasterol and their isomers, were identified in the real samples. In particular, using a combined characterization based on HCD and CID tandem MS, also minor isomeric species for which no standards were available were recognized. The RPLC-APCI-FTMS method was also validated to quantify FSs and thus evaluate concentration variations related to different growth stages. The obtained results show that β-sitosterol and campesterol are the most abundant FSs in the examined vegetables; specific minor FSs are principally present in microgreens, thus corroborating the nutraceutical potential of these innovative vegetable products.

Acknowledgements: Italia Domani – Piano Nazionale di Ripresa e Resilienza 

Keywords: Sterols, RPLC, APCI, FTMS

References

1. Valitova J.N., Sulkarnayeva, A. G., et al., Biochemistry, 81, (2016), pag. 819-834. DOI: 10.1134/S0006297916080046. 

2. Poudel P., Petropoulos S.A., et al., Natural Secondary Metabolities: From Nature, Through Science, to Industry, (2023), pag. 285-319. DOI: 10.1007/978-3-031-18587-8_8. 

3. Castellaneta A. Losito I., et al., Journal of Steroid Biochemistry and Molecular Biology, 232 (2023), pag. 1-10. DOI: 10.1016/j.jsbmb.2023.106361.