Masi, Francesca (2022) Plant extracts of industrial interest: phytochemical characterization and optimization of extraction methods. [Tesi di dottorato]

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Abstract

Natural products have always played an important role in the pharmaceutical sciences both for their use as medicinal remedies and as a source of inspiration for drug discovery. In ancient times they were used in an unaltered state as concoctions or concentrated plant extracts for the treatment of ailments, but with the development of experimental procedures, the isolation and purification of the active ingredients from the phytocomplex have become a common practice. To date, the importance of natural products has been extensively recognized and natural sources have been significantly exploited in the development of new drugs, both as isolated natural products or their semi-synthetic derivatives. In addition, part of the drugs obtained by total synthesis have been inspired by the pharmacophore of active natural products. In recent years the role of herbal extracts has slightly changed and, in addition to their use as drugs or source of drugs, also a "support tool" for personal well-being has become important. In this framework, natural products used as health support and alternative strategies have been widely encouraged by a great number of research and studies, due to the benefits that products of natural origin can bring to human health, as well as to mitigate health problems. However, the complexity of natural matrices and their chemical composition should be considered. In fact, nature does not provide products with a standardized composition, and in-depth studies of plant extracts are required in several research areas. From the chemical point of view, the implementation of extraction and purification protocols is indispensable to obtain high-quality standardized products, as well as the implementation of analytical techniques to investigate the phytochemical profile. In this framework, the industrial PhD project was in collaboration with the industrial partner Indena Spa (Milan, Italy) and addressed both the characterization of commercial products obtained from different plant extracts and the development and optimization of the extraction method in the industrial laboratories. The phytochemical investigation involved the characterization of two commercial products obtained from two different plant species: i) Centella asiatica and ii) Rhodiola rosea. Thus, the second chapter of this thesis focuses on Centevita®, a nutraceutical extract obtained from the leaves of C. asiatica. It is standardized to 45% of triterpenes (glycosides), including mostly asiaticoside, madecassoside, asiatic acid, and madecassic acid. They represent the main bioactive compounds of the extract due to their antioxidant activity. My work first dealed with in the characterization of a previously unreported ursane saponin, that was called isomadecassoside, that emerged from preliminary analyses of a fraction enriched in madecassoside, obtained during the extract preparation of Centevita®. Then, the in-depth investigation of the full phytochemical profile of Centevita® through advanced analytical techniques resulted in the characterization of 24 secondary metabolites, including 10 polyphenols and 14 triterpenoids in the sapogenin or saponin form, and to the discovery of isoterminoloside, a new triglycoside saponin of the unprecedented 2α, 3β, 6β, 23-tetrahydroxyolean-13(18)-en-28-oic acid (isoterminolic acid). The third chapter reports on Rhodiola 5%, a nutraceutical extract obtained from the roots of Rhodiola rosea. It is standardized and purified to 5% of rosavins, the main bioactive compounds as tonic and adaptogen, and they include rosarin, rosavin, and rosin. The phytochemical investigation carried out resulted in: i) the characterization of 18 secondary metabolites including 13 polyphenols and 6 terpenoids, and ii) the discovery of rhodiosidin, a new compound, the first glycosylated derivative with both terpenoid and phenolic portions. In addition, the 5-lipoxygenase inhibition activity of the main components was characterized, revealing rosiridin, kenposide A and rosavins mainly responsible for the activity of the extract. The fourth chapter reports on the preliminary studies and the relevant scientific activities carried out at Indena SpA laboratories for the development and optimization of a procedure to obtain a standardized nutraceutical extract enriched in annurcoic acid, a triterpene acid from Malus domestica. Apple biomass was chosen among the waste of industrial apple juice processing. It was first subjected to a phytochemical investigation allowing the identification of seven terpene acids including: annurcoic acid, tormentic acid, maslinic acid, corosolic acid, oleanolic acid, ursolic acid, and pomolic acid. The laboratory R&D activities led to the development of a seven-step procedure obtaining a standardized extract characterized by a high content of triterpene acids, of which annurcoic acid was the main component (25% w/w). In parallel, a further procedure was set up to obtain annurcoic acid to be used as a reference standard. The research process led to the isolation of annurcoic acid (85% w/w), together with two minor compounds: tormentic acid (3.4% w/w) and isomer A (2.6% w/w). Results reported in this thesis have been in part already published in the following articles: - Chianese, G., Masi, F., Cicia, D., Ciceri, D., Arpini, S., Falzoni, M., Pagano, E., Taglialatela-Scafati, O. (2021). Isomadecassoside, a new ursane-type triterpene glycoside from Centella asiatica leaves, reduces nitrite levels in LPS-stimulated macrophages. Biomolecules, 11(4), 494. - Masi, F., Chianese, G., Peterlongo, F., Riva, A., Taglialatela-Scafati, O. (2022). Phytochemical profile of Centevita®, a Centella asiatica leaves extract, and isolation of a new oleanane-type saponin. Fitoterapia, 158, 105163.

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