Comparative Evaluation of Secondary Metabolite Chemodiversity of Citrus Genebank Collection in Greece: Can the Peel be More than Waste?

Citrus fruits are among the most economically important crops in the world. In the global market, the Citrus peel is often considered a byproduct but substitutes an important phenotypic characteristic of the fruit and a valuable source of essential oils, flavonoids, carotenoids, and phenolic acids with variable concentrations. The Mediterranean basin is a particularly dense area of autochthonous genotypes of Citrus that are known for being a source of healthy foods, which can be repertoires of valuable genes for molecular breeding with the focus on plant resistance and quality improvement. The scope of this study was to characterize and compare the main phenotypic parameters (i.e., peel thickness, fruit volume, and area) and levels of bioactive compounds in the peel of fruits from the local germplasm of Citrus in Greece, to assess their chemodiversity regarding their polyphenolic, volatile, and carotenoid profiles. A targeted liquid chromatographic approach revealed hesperidin, tangeretin, narirutin, eriocitrin, and quercetin glycosides as the major polyphenolic compounds identified in orange, lemon, and mandarin peels. The content of tangeretin and narirutin followed the tendency mandarin > orange > lemon. Eriocitrin was a predominant metabolite of lemon peel, following its identification in lower amounts in mandarin and at least in the orange peel. For these citrus-specific metabolites, high intra- but also interspecies chemodiversity was monitored. Significant diversity was found in the essential oil content, which varied between 1.2 and 3% in orange, 0.2 and 1.4% in mandarin, and 0.9 and 1.9% in lemon peel. Limonene was the predominant compound in all Citrus species peel essential oils, ranging between 88 and 93% among the orange, 64 and 93% in mandarin, and 55 and 63% in lemon cultivars. Carotenoid analysis revealed different compositions among the Citrus species and accessions studied, with ß-cryptoxanthin being the most predominant metabolite. This large-scale metabolic investigation will enhance the knowledge of Citrus peel secondary metabolite chemodiversity supported by the ample availability of Citrus genetic resources to further expand their exploitation in future breeding programs and potential applications in the global functional food and pharmaceutical industries.

In vitro evaluation of the effects of methanolic plant extracts on the embryonation rate of Ascaridia galli eggs.

The present study aims to find efficient alternatives to synthetic anthelmintics among ethno-veterinary herbs. Ascaridia galli eggs isolated from the worm uterus were exposed in vitro to methanolic extracts (ME) of nine plant species such as Achillea millefolium (AM), Artemisia absinthium (AA), Artemisia vulgaris (AV), Cicerbita alpina (CA), Cichorium intybus (CI), Inula helenium (IH), Origanum vulgare (OV), Tanacetum vulgare (TV), Tanacetum parthenium (TP). Flubendazole (FL), 0.5% formalin with dimethylsulfoxide and Petri dishes without the addition of reagents were used as positive, negative and untreated control respectively. The effects of the different ME at concentrations 0.500, 0.325, 0.200 mg/ml were assessed on the embryonic development (ED) of the eggs in duplicate. Logit analysis was used to calculate EC50 values. A generalized linear mixed model, having plant species and concentration as fixed effect and day as repeated measure, was used to determine differences in ED. Estimated EC50 was the lowest for FL at 0.11 mg/ml. CA and TV followed with 0.27 mg/ml and 0.32 mg/ml. ED for FL was significantly lower (25%) than that of CA (47%). The analysis showed 0.5 mg/ml of the ME of CA and TV significantly affected the ED at 35% and 42% inhibitions respectively. The ED for all ME showed similar pattern i.e., relatively higher efficacy in the first experimental week compared to the rest of the experimental period. The effect from all multicomponent extracts is time and dose dependent. The plants have promising results in inhibiting ED, contributing to the identification of alternative anthelmintic treatments.

Assessment of the chemical and genetic variability among accessions of Cicerbita alpina (L.) Wallr., an alpine plant with anthelmintic properties.

Cicerbita alpina (L.) Wallr, is a perennial alpine plant and a member of the Asteraceae family, typically found at altitudes above 1000 meters in the Italian Alps. Although previously utilized primarily as a local delicacy, recent studies have revealed strong antiparasitic activity through in vitro experiments. In Europe, numerous chemical drugs employed to combat nematodes - helminths that infest the digestive tract of livestock - are banned due to their environmental harm or show only reduced efficiency because of the development of resistance. Consequently, there is a growing demand for new alternative anthelmintic treatments in agricultural practices. Specialized metabolites found in the extracts of C. alpina could offer a sustainable and biological alternative to chemical drugs, specifically for nematode control. For this purpose, a unique germplasm collection originating from eight distinct natural populations in the Italian Alps was analyzed for its chemical diversity using state-of-the-art targeted LC-MS/MS spectrometry, including quantification based on multiple reaction monitoring. The predominant metabolites identified from the species were the caffeic acid derivatives chicoric acid, chlorogenic acid, and 3. 5-dicaffeoylquinic acid, the sesquiterpene lactone derivative 8-O-acetyl-15-ß-D-glucopyranosyl lactucin and the flavone glycosides, apigenin-7-O-glucoside and luteolin-7-O-glucoside, alongside their precursors apigenin and luteolin, respectively. Additionally, the genetic diversity of eighty individual plants within the germplasm collection was evaluated using ten DNA molecular markers (Simple Sequence Repeats), successfully transferred from two closely related species (Cichorium intybus and Tanacetum parthenium). This investigation unveiled a significant range of genetic diversity within the examined populations, resulting in the establishment of three distinct genetic groups. The findings were further correlated with the original ecological environment and local climate conditions spanning a biennial period, indicating substantial variations among the different accessions and the intricate interplay between genetic background and environmental factors. These results could serve as a basis for future domestication of the species through plant breeding programs ensuring product quality, but also facilitating the cultivation of C. alpina in more diverse geographic regions.