New insights on the diversity of phytocomplexes from Mediterranean shrubs species and their potential nutraceutical and industrial applications

Mediterranean plants are exposed to different environmental stresses, such as high temperatures, solar irradiance, and low water availability, that coexist together, especially during the summer period. To cope with such a combination of stresses, these plants have developed a suite of morpho-anatomical, physiological, and biochemical mechanisms. In particular, they increase the production of secondary metabolites with many defensive functions. Among them, polyphenolic compounds play an important ecological role, acting as UV screeners and antioxidants against reactive oxygen species (ROS) which are produced when the photosynthetic metabolism is impaired by abiotic constraints. The aim of this PhD project was to investigate four native Mediterranean wild species (Arbutus unedo L., Cistus x incanus L., Myrtus communis L. and Pistacia lentiscus L.) as sources of phytocomplexes that can be exploited for nutraceutical and other industrial applications. We have planned the set-up of green extractive methods to obtain polyphenolic rich extracts from their leaves and fruits, to perform a field-experiment for enhancing the polyphenolic production, and to test the potential utilization of these phytocomplexes as nutraceuticals. To achieve this, the initial part of the project was the setting up of efficient ultrasound-assisted (UAE) extractions using the Response Surface Methodology. In particular, a fractional factorial screening was performed to verify the effects of different levels of temperature, solvent volume, percentage of ethanol and time followed by an optimization step using a Box-Behnken design. Then, a field-experiment for enhancing the leaf polyphenolic and arbutin yield in A. unedo plants was performed through the application of water stress on a seasonal basis. Finally, the potential utilization of A. unedo and M. communis as nutraceuticals sources was also tested through the inclusion of their polyphenolic fruit extracts into whey milk, a dairy waste product. Regarding the optimization of the extractions, for P. lentiscus, higher content of total polyphenols can be achieved extracting the leaves using 50% of ethanol at 50 °C for 15 minutes with a solvent ratio of 0.13 L g-1. For A. unedo, a higher amount of arbutin was obtained using 75% of ethanol at ambient temperature for 15 minutes with a solvent ratio of 0.06 L g-1. Finally, for C. incanus the highest content of polyphenols was obtained using 60% EtOH at 40 °C for 60 minutes using 0.1 L g-1 solvent proportion. The results of the field experiment showed that the application of water stress during the cultivation of A. unedo plants triggered the biosynthesis of polyphenolic compounds in the leaves, increasing their content from summer until the beginning of autumn. Finally, polyphenolic fruit extracts of A. unedo and M. communis showed to be suitable for the inclusion in whey, maintaining their high polyphenolic content during storage thus proving their possible application as functional additives in the food industry. In conclusion, all the selected species provided extracts with high yields of polyphenols to be applied in the pharmaceutical and nutraceutical sectors, thus the cultivation of these plants may represent an alternative source of income for farmers in the Mediterranean drought-stressed areas