Medicinal and food plants as sources of biopesticides and biologically active compounds: a focus on Carlina acaulis and Acmella oleracea

Botanical products have been employed since the early human history as foods, medicinal and insecticidal agents and this is ascribed to their richness in biologically active compounds. Regarding the use as insecticidal agents, they are nowadays gaining increasing scientific interest since the misuse and overuse of conventional pesticides led to the onset of resistance and negative effects for human health and the environment. Botanical pesticides, also known as biopesticides, are considered as alternatives to traditional pesticides due to the lack of persistence into the environment, selectivity towards non-target pests, and low toxicity on humans. From this scenario derives the increasing motivation of researching new and effective botanical pesticides obtainable by extraction from the plant matrix or also by synthesis of more active derivatives. This was one main purpose of the work carried out by our research group and herein reported. The main studies conducted on the Asteraceae plants Carlina acaulis L. and Acmella oleracea (L.) R. K. Jansen are described in the first two Chapters of this Thesis. In detail, Chapter I reports all the scientific investigations carried out on C. acaulis, which is a traditional medicinal plant growing in the European mountainous regions. The most interesting product of this plant is its roots essential oil (EO) (0.97% w/w yield), characterized by the predominance of the polyacetylene carlina oxide (> 95%). In this regard, an optimization of the EO extraction procedure through Microwave-Assisted Extraction (MAE) was performed using a one-step statistical approach (Design of Experiments, DoE) and compared to the traditional hydrodistillation (HD). This study pointed out that MAE is more efficient than conventional HD in terms in terms of EO yield (0.65 and 0.49% for MAE and HD, respectively) and extraction time (210 min for MAE). C. acaulis EO, carlina oxide, and an EO-nanoemulsion (EO-NE) were also tested on several bacterial and fungal strains. Indeed, they were extremely active on the Gram-positive bacteria strains tested, with a complete inefficacy against Gram-negative bacteria for the EO and carlina oxide. Nevertheless, the main studies carried out on C. acaulis were focused on its promising pesticidal activity. Indeed, its derived products were assayed on vectors, agricultural, and stored-product pests. Regarding vectors, C. acaulis-derived products (EO-microemulsion (EO-ME), EO-NE, and EO) were assayed on Culex quinquefasciatus Say larvae, the main vector of lymphatic filariaris and Zika virus. This study proved the high efficacy of the EO-ME on the vector, also with sublethal doses. The EO was also tested on human keratinocytes and fibroblasts and Winstar rats showing mild toxicity (LD50 on rats of 1098 mg kg-1 ). The LD50 on rats increased above 5 g/kg when the EO was encapsulated into ME. Concerning the agricultural pests, different C. acaulis-derived products were assayed on Lobesia botrana (Denis & Schiffermüller, 1775), Bactrocera oleae (Rossi), Ceratitis capitata (Wiedemann), Meloidogyne incognita, Xylosandrus compactus (Eichhoff), and Tetranychus urticae Koch, which are pests affecting different crops worldwide and leading to severe economic losses. From these studies, a marked activity on all the agricultural pests assayed was detected. In addition, diverse C. acaulis-derived products were tested on stored-product pests as Acarus siro L., Alphitobius diaperinus (Panzer), Oryzaephilus surinamensis L., Prostephanus truncatus (Horn), Rhyzopertha dominica (F.), Sitophilus oryzae L., Tribolium confusum Jacquelin du Val, Tribolium castaneum (Herbst), Tenebrio molitor L., and Trogoderma granarium Everts. These pests usually affect stored products leading to economic damages but also to negative effects on the consumers’ health. The bioactivity detected was extremely promising against all pests taken into consideration. During these studies, also the non-target toxicity on Neoseiulus californicus (McGregor) and Daphnia magna Straus was assessed resulting moderate. The development of a synthetic approach to produce carlina oxide analogues and the assessment of their insecticidal activity and toxicity are also reported in Chapter I. Between the analogues synthetized, the m-chloro substituted resulted more active on Cx. quinquefasciatus and less toxic on human keratinocytes (HaCaT cell line) than its precursor carlina oxide. On the other hand, Chapter II reports all the research carried out on A. oleracea, which is a traditional medicinal plant native to Brazil but spread worldwide as a crop. The A. oleracea-derived products’ (EO, EO-NE, and extracts) biological activities have been mainly linked to the presence of N-alkylamides, with a focus on spilanthol that resulted the most abundant. Firstly, MAE and HD were used for the EO extraction, and a comparison of the obtained products was performed. This study pointed out that MAE is more efficient than HD for the obtaining of the EO in terms of yield (0.47 and 0.22% w/w, respectively) and spilanthol content (11.7 and 2.3%, respectively). The EO, EO-NE, and a n-hexane-extract were efficient against Cx. quinquefasciatus larvae, with spilanthol and the n-hexane extract as the most active products. In addition, the safety for mammal cells of the EO and spilanthol was also proved together with the protection from lipopolysaccharide (LPS)- induced inflammation. Based on these findings, an optimization of the formulative parameters of the n-hexane extract and spilanthol into MEs and NEs was performed and from this study the optimal formulative parameters were found only for NEs. Moreover, seen the promising activity displayed by the n-hexane extract, an optimization study for the extraction of spilanthol from the raw material was assessed. In this regard, Soxhlet extraction with methanol resulted the best extractive procedure, even if the n-hexane extract displayed the highest concentration of spilanthol. These methanol and n-hexane extracts were assayed on several stored-product pests as O. surinamensis, T. granarium, T. castaneum, T. confusum, T. molitor, A. diaperinus, A. siro, and Cryptolestes ferrugineus (Stephens). This study pointed out the higher effectiveness of the n- hexane extract than the methanol extract and this result confirmed that the concentration of spilanthol is fundamental for the biological activity. Lastly, Chapter III reports the results of the research activity carried out abroad at the Laboratory of Organic Chemistry of Prof. Nuno Maulide (University of Vienna) for a period of four months. In detail, the goal was to develop a new synthetic approach to produce α-aminated amides through electrophilic amide activation with triflic anhydride. Initially, the reaction was investigated for protected α-aminated amides and, from the preliminary screening of protecting groups (PGs), benzoyl (Bz) resulted the best in terms of yield. The reaction displayed good functional group tolerance, except for bulky amides. Moreover, a protocol was developed and applied for the synthesis of ‘free’ α-aminated amides, and the reaction was screened on different substrates with acceptable yields.