Secondary metabolism in evergreen species with high tolerance to osmotic stress: what is the role for these species as biofactories?

Vegetation in Mediterranean areas is exposed to different sources of environmental stresses, especially during the summer when high temperatures, high solar irradiance and low water availability in the soil prevail for long periods. To cope with such combination of stresses, which are commonly defined as drought, Mediterranean plants have evolved a suite of morpho-anatomical, biochemical, and physiological mechanisms. In particular, these plants are able to synthesize a broad spectrum of secondary metabolites. Among these, polyphenols play a key-role in the defence against reactive oxygen species (ROS), which are inevitably produced when aerobic or photosynthetic metabolism is impaired by environmental stresses. The antioxidant activity of polyphenols is also maintained in human cells and in the last years their use in the medicinal and nutraceutical fields has considerably grown. In this context, the knowledge of Mediterranean plants of pharmaceutical interest becomes crucial and represents a prerequisite for their rational valorization and protection. The present PhD research was developed under this scenario. In particular, the general objectives of this thesis are: 1) to explore the physiological and biochemical responses of three selected Mediterranean species (Phillyrea latifolia L., Pistacia lentiscus L. and Cistus incanus L.) in their natural habitat; 2) to assess the relative significance of these species as “biofactories”, i.e. potential sources of health-promoting polyphenols. In order to achieve these goals, a comparative field study was carried out in 2014 and 2015 on the coastal dunes of Castiglione della Pescaia (Gr), where all the above species co-occurred. We measured both physiological (gas exchange, water relations and PSII photochemistry) and biochemical parameters (carotenoids, polyphenols and abscisic acid in its free and conjugated form) on a daily and seasonal basis. Furthermore, the effects of climatic factors (temperature, global irradiance and precipitation) on the physiological and biochemical traits were also assessed. Then, to investigate the examined species as “biofactories”, we identified the most abundant polyphenols present in their leaves throughout the HPLC–DAD–MS/MS (Liquid Chromatography-tandem Mass Spectrometry) technique. Moreover, we optimized an extraction protocol to obtain different polyphenolic-rich extracts (flavonoid-rich fractions and tannin-rich fractions) from each plant. Finally, in order to compare their antioxidant and cytotoxic effects, the total extracts and the obtained fractions were tested throughout in vitro and ex vivo assays. These analyses were conducted in collaboration with the Department of Molecular Medicine and Development at the University of Siena. Specifically, the antioxidant activity was examined using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging test, while the study of cytotoxicity was carried out on mouse fibroblast (NIH 3T3) cell line. Overall, the results obtained from this PhD project were the following: 1. definition of the main physiological and biochemical response mechanisms of P. latifolia, P. lentiscus and C. incanus to multiple stress typical of the Mediterranean environment on a daily, seasonal and inter-annual basis; 2. phytochemical characterization of the polyphenolic-enriched extracts obtained from C. incanus and P. lentiscus leaves; 3. characterization of the in vitro antioxidant activity and the ex vivo cytotoxicity of the extracts, in order to evaluate their pharmacological potential.