Abstract

1. Introduction

Agricultural waste, such as crop residues, peels, leaves, and roots, constitutes a significant portion of the organic waste produced, with an estimated 2800 to 3800 million tonnes/year globally [1]. Without proper disposal or reutilization, this waste can significantly contribute to soil and water pollution and can emit greenhouse gases during decomposition [2]. Therefore, it is essential to develop effective strategies for reusing and valorizing this waste, transforming it into valuable resources like compost, biogas, or ingredients for new food products or animal feedstock [3].

In addition to greater environmental sustainability, the transition to a circular food system brings direct economic benefits through the valorization of waste and indirect benefits through the expansion of the production chain [4].

With this work, the authors aim to address the agricultural waste problem by implementing a new way of thinking about crops, summarizing this type of approach as “Agroprospecting”. In this context, the term agroprospecting refers to the exploration of agrobiodiversity co-products and organic waste, which are from intensive, extensive, local, regional, traditional, or technological crops, in search of small- or macro-molecules that could be developed into materials of commercial value for agriculture, bioremediation, cosmetics, nanotechnology, or pharmaceutical and food industries. Agroprospecting (agro-exploration) is fundamental to closing the loop into a circular and sustainable food system; it analyzes the production chain and assesses the potential reuse, recycling, or transformation of organic residues, thus minimizing waste production.

Leveraging the principles of agroprospecting, the present study focuses on the artichoke cultivar Tema (Cynara scolymus L., Asteraceae) and addresses the considerable waste typical of artichoke cultivation; we aim to transform these byproducts into valuable co-products within a sustainable circular economy.

Tema cultivar is a commercial artichoke hybrid that represents a variety of Violetto artichoke and is characterized by a cylindrically shaped head lined with purple bracts ending in a short thorn [5]. The bracts are fleshy and tender with little bitter flavor. Its excellent cold resistance allows an extended harvesting period from November until March, depending on the year. Notably, this cultivar showed good resistance to the browning processes [6], a feature that makes it suitable for obtaining a fourth-range product.

However, due to the processing steps, it is characterized by a large amount of waste, namely the leaf portions of the stem and especially the outermost bracts. These aspects are common in artichoke cultivation; in fact, the edible portion of the artichoke is the immature flower head, which undergoes further processing to remove the outer bracts, leaves, and stems. Additionally, waste production in the artichoke’s production chain increases due to common agricultural practices involving the removal of secondary and tertiary flower heads. At the end of processing, approximately 80–85% of the total biomass is discarded [7]. Given the high waste generation, intensive processing, low yields, and significant field requirements (such as pesticides, fertilizers, and irrigation), artichoke cultivation is considered one of the least environmentally sustainable agricultural practices. This results in significant greenhouse gas emissions, and recent studies have calculated that the carbon footprint along the production chain is around 8000 kg CO₂eq per hectare [8].

Despite growing concerns about the environmental footprint of the artichoke supply chain due to high waste generation and resource requirements, the Mediterranean basin hosts the largest global production of artichokes, primarily in Egypt, Italy, and Spain [9]. Italy ranks as the world’s second-largest producer, with 378,110 tonnes in 2022, corresponding to a cultivated area of approximately 38,170 hectares [10]. Furthermore, Italy is recognized as the “primary cultivated gene pool” due to its significant reservoir of autochthonous germplasm. Numerous recognized cultivars exhibit variations in characteristics such as morphology (e.g., Spinoso, Romanesco, Catanese, and Violetto) or production period, which enables early production in autumn–winter and late production and allows harvesting until spring. Furthermore, ongoing research, which focuses on new hybrid varieties propagated from seed, has been introduced; these include Tema, Apollo, Madrigal, and Concerto [11,12].

In addition, artichoke cultivation offers valuable health benefits through its content of fibers and bioactive compounds like phenolic acids, terpenes, and flavonoids, which contribute to its status as a potent functional food [13,14,15,16]. Collectively, these compounds reduce the inflammatory processes, regulate sugar and fat metabolism and digestive processes, and decrease the risk of chronic diseases [17,18,19]. Among its primary bioactive molecules are chlorogenic acid, cynarin, and cynaropicrin, along with various isomers of caffeoylquinic acid and glycosidic derivatives of luteolin and apigenin [17,20,21].

In particular, chlorogenic acid is associated with antidiabetic and antilipidemic effects; it helps regulate blood glucose levels by modulating glucose absorption and metabolism, contributing to lipid-lowering and cholesterol-lowering actions that improve cardiovascular health by managing lipid profiles and reducing the risk of atherosclerosis [22,23]. Another fundamental bioactive metabolite is the sesquiterpene lactone cynaropicrin. Despite its low concentration, it is reported to possess robust anti-inflammatory effects by inhibiting pro-inflammatory cytokines and modulating immune responses, partly through suppression of the key pro-inflammatory NF-κB pathway [24]. Furthermore, cynaropicrin exhibits various metabolic regulatory activities, such as choleretic and anti-hyperlipidemic properties, thus supporting digestive health and fat metabolism [25]. It shows a broad spectrum of pharmacological actions, including anti-trypanosomal, anti-malarial, antifeedant, antispasmodic, anti-aging, anti-tumor and anti-Hepatitis C properties [26,27,28].

In this context, the comprehensive analysis and characterization of biowaste can enable the valorization of their bioactive molecules. Indeed, these matrices demonstrated diverse biological activities such as antimicrobial, antifungal, anticancer, and skin-protective functions [19,29,30]. This approach not only mitigates waste generation but also enhances the capacity of edible plant materials to exert positive effects on human, animal, and ecosystem health. By utilizing these bioactive properties, agricultural byproducts can be transformed into valuable resources, thereby advancing sustainability and delivering health benefits across various sectors.

Therefore, the aim of this study is to characterize and explore the biological activities of Tema cultivar biowaste to further increase its industrial value. The waste material was collected over the harvesting period and underwent a comprehensive chemical characterization to determine its concentration in its main bioactive molecules. Subsequently, the study investigates the waste’s efficacy on pivotal features of skin aging, namely wound healing (cell migration) and cellular senescence.

Through the chemical characterization and biological evaluation of artichoke waste, the authors attempt to unveil, for the first time, an innovative process for the sustainable use of agricultural byproducts of Tema cultivar, thus presenting new opportunities for a sustainable environmental and economic management of natural resources.

2. Results

2.2. Principal Component Analysis (PCA)

The metabolites were further analyzed using multivariate techniques, including Principal Component Analysis (PCA), showed in Figure 1. The first two principal components explain a significant portion of the variance (54.6% and 23.1%, respectively), indicating clear metabolic profiles associated with different collection periods (Figure 1A). Dimension 1 (PC1) effectively differentiated the batches according to their collection time. This indicates that the primary source of variation captured by Dimension 1 is closely related to the temporal changes in metabolite concentrations, reflecting seasonal influences or maturation stages.

Open in a separate window

Figure 1

Biplot and PCA analysis of the detected compounds in the globe artichoke agro-wastes, from batches A to H. In the biplot (A), each dot represents a batch of replicates. The PCA (B) indicates the variable contribution. Colors tending toward red indicate better-represented variables, while gradually lighter colors toward blue shades indicate less-represented variables.

Dimension 2 (PC2) contributed to further differentiation, explaining a smaller yet significant proportion of the total variability. In particular, PC2 focuses on metabolites that deviate from the main seasonal trend, such as apigenin and cynaropicrin, capturing less dominant differences, such as microenvironmental differences or unique responses to biotic or abiotic stressors.

The loading of variables (Figure 1B) further explains the most characterizing variables in the two principal dimensions. As previously highlighted, the most abundant compounds—chlorogenic acid, 3,4-di-O-caffeoylquinic acid, cynaroside, and scolymoside—exhibit high loadings and contribute to PC1. The prominence of these compounds in the loading plot confirms their substantial impact on the overall data structure and highlights their relevance in driving the main trends observed in the dataset.

PC2 is distinguished by variables such as apigenin, cynaropicrin, 1,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid, with apigenin and cynaropicrin providing a more substantial contribution. This differential contribution of PC2 explains the separation between batches B and A. In fact, B exhibits higher levels of cynaropicrin and 1,5-di-O-caffeoylquinic acid, whereas sample A shows lower values for these compounds. This distinction highlights how PC2 captures unique variations not accounted for by the main seasonal trend, illustrating specific deviations in metabolite profiles that could be driven by physiological or environmental conditions affecting these metabolites.

Together, these dimensions provide a comprehensive view of how the metabolite profiles vary with both time and additional influencing factors, thus offering deeper insights into the complex dynamics of metabolite synthesis and accumulation.

Based on quantification data and multivariate analysis, specific batches have been chosen to assess the biological potential of the average sample. Batch A, representing the beginning of the collection, along with batches E and F, representing the second half of the collection and the beginning of the increasing trend, have been selected.

2.3. Biological Activities

2.3.1. Effect of Cynara Agro-Wastes on Cell Viability

The effect of the hydroalcoholic extracts obtained from the three selected batches (A, E, and F) on fibroblasts (3T3 cell line) viability, a main target for skin anti-aging therapy, was assessed. Overall, as shown in Figure 2, no toxicity was observed at the tested concentrations (800–25 μg/mL), thus highlighting a very safe profile for all the extracts.

Open in a separate window

Figure 2

Safety profile of the hydroalcoholic extract from globe artichoke agro-wastes (batches A, E, and F) on fibroblasts assessed by the resazurin assay.

2.3.2. Effect of Cynara Byproducts on Cell Migration

Bearing in mind the overall safe profile observed for the tested extracts, we selected the intermediate concentrations of 100 and 200 μg/mL to further assess the bioactive potential of these byproducts on features related to skin aging. First, the effect of the extracts on cell migration, a feature highly compromised in aged skin, was tested. As shown in Figure 3, no significant effects on fibroblast migration were observed, although samples E and F at 200 μg/mL showed a slight tendency to increase this feature (Figure 3A,B).

Open in a separate window

Figure 3

Effect of hydroalcoholic extracts from globe artichoke agro-wastes (batches A, E, and F) on fibroblasts migration assessed by the wound healing assay. (A) Images acquired by phase-contrast microscopy immediately after wound induction (0 h) and after 18 h of incubation; (B) histograms showing the quantified wound area of the different extracts compared with the control (CT). Scale bar: 100 µm.

2.3.3. Effect of Cynara Byproducts on Cellular Senescence

To assess the effect of the extracts on cellular senescence, three complementary markers were considered, related to lysosomal activity, cell cycle arrest, and DNA damage. Regarding the first, senescence-associated (SA) β-galactosidase activity was determined in NIH/3T3 fibroblasts. The senescence inducer etoposide was used to induce an increase in this marker, as observed in Figure 4A,B. Interestingly, all the tested extracts, at 200 µg/mL, significantly inhibited this increase, pointing out a potential anti-senescent effect of the extracts, with extracts A and F being more effective (Figure 4B).

Open in a separate window

Figure 4

Effect of the hydroalcoholic extracts obtained from globe artichoke agro-wastes (batches A, E, and F) on NIH/3T3 fibroblasts senescence-associated β-galactosidase activity (A). Histograms showing the quantitative analysis using ImageJ software (B). Statistical significance * p < 0.05, ** p < 0.01 determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. Eto—etoposide (12.5 µM); Scale bar: 100 µm.

Similarly, the extracts significantly decreased the phosphorylation and nuclear accumulation of γH2AX induced by etoposide (Figure 5A,B), thus pointing out a protective effect on double-strand DNA damage.

Open in a separate window

Figure 5

Effect of the hydroalcoholic extracts from globe artichoke agro-wastes (batches A, E, and F) on NIH/3T3 fibroblasts nuclear accumulation of the phosphorylated form of H2AX (γ-H2AX). Representative confocal images (A) and corresponding histograms from quantitave analysis (B). Statistical significance ** p < 0.01, *** p < 0.001 by one-way ANOVA followed by Dunnett’s multiple comparisons test. Eto—etoposide (12.5 µM); Scale bar: 10 µm.

To corroborate these results, the effect of the extracts on cell cycle arrest was determined by evaluating their capacity to modulate the p53/p21 signaling pathway (Figure 6), which plays a key role in the initiation of senescence. Overall, the extracts show a tendency to decrease the protein levels of both p53 (Figure 6A,B) and p21, with extract F attaining statistical significance for the latter (Figure 6A,C).

Open in a separate window

Figure 6

Effect of the hydroalcoholic extracts from globe artichoke agro-wastes (batches A, E, and F) on protein levels of p53 (A,B) and p21 (A,C). Tubulin used as loading control and results normalized to etoposide. * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA, followed by Dunnett’s multiple comparison test. Eto—etoposide (12.5 µM).

3. Discussion

In the present work, the authors provide a characterization of the variation of the main phenolic acids, flavonoids, and cynaropicrin of Tema globe artichoke biowaste in relation to harvest time. Moreover, aiming at a potential valorization of these wastes, their protective effect against skin aging was investigated, pointing out novel applications in the cosmeceutical or pharmaceutical fields. Firstly, the outer bracts of the Tema cultivar were selected as biowaste. This selection was driven by two factors: the amount of available waste and the location of the collection. While some organic waste is typically left in the field (e.g., secondary or tertiary head flowers), the outer bracts of this cultivar are processed on the farm as part of the fourth-range processing, therefore simplifying the collection operations [7]. The information available in the literature reports different advantages of the Tema cultivar: Lombardo et al. [5] found the highest number of polyphenols in Tema out of the other seventeen artichoke cultivars tested. Accordingly, a high polyphenolic content was found by Cabezas-Serrano et al. [6], which, however, highlighted a low vitamin C content and a related low antioxidant activity. This cultivar is also sufficiently resistant to browning, making it suitable for processed products and rendering its waste particularly amenable to further utilization [6].

The chemical characterization here performed unveiled 11 main compounds belonging to the classes of phenolic acids and derivatives of caffeoylquinic acids, flavonoids, and the sesquiterpene lactone cynaropicrin. Based on the literature, these bioactive molecules are typically more abundant in the heart of the artichoke [31]. In contrast, higher values were found here when compared to those typically found in the outer bracts, [30], which differences may be attributed to the characteristics of this hybrid cultivar.

From a quantitative point of view, the most abundant compounds were chlorogenic acid (7.14 mg/g in Batch E) and 3,4-di-O-caffeoylquinic acid (8.89 mg/g in Batch G). Followed by 3,5-di-O-caffeoylquinic acid, scolymoside, and cynaroside. Chlorogenic acid, the most abundant compound found here and typically abundant in artichoke, is reported to have potent antioxidant and anti-inflammatory activities [22,29,32].

In addition, the analysis of the biowaste during the harvesting period underscores a significant variation in polyphenol content. This parallels prior findings on the content of chlorogenic acid in other crops and their byproducts, including potato [33], faba bean [34], and tomato [35]. In particular, it was interesting to note a visible trend for the majority of compounds, resulting in an increase in concentration in the late harvest lots. Specifically, this was evident in 7 out of 11 compounds, leading to an overall increase of 15.13 mg/g for chlorogenic acid derivatives and 1.30 mg/g for cynaroside derivatives. Multivariate analysis clearly highlighted this phenomenon. It clusters the batches based on harvest time in the first dimension and accounts for 54% of the variability. Many studies investigate artichoke’s polyphenol variation among different cultivars and different parts of the plant (e.g., leaves, head, inner and outer bracts), while little is known about phenol trends during artichoke seasonality [5,6,11,19]. Lombardo et al. (2010) demonstrated that Romanesco artichokes exhibit higher phenol content in the spring harvest compared to the winter harvest, with a slight increase in the outer bracts and a pronounced increase toward the inner parts of the flower head, reaching a maximum in the floral stem (values up to 16 times higher). Moreover, various forms of stress, such as drought, are known to elevate artichoke polyphenol levels [36]. Therefore, the increased polyphenol content can be attributed to progressive lignification during maturation, as well as to biotic and abiotic stresses, in order to enhance physical resistance: these polyphenols, including chlorogenic acids and its derivatives, are part of the phenylpropanoid pathway, which are utilized for lignin production in the outer bracts, whereas in the innermost portions, they remain more abundant as precursors [30].

Cynaropicrin, a sesquiterpene lactone characterized by a tricyclic structure featuring a butyrolactone ring, did not show this seasonal pattern: its concentration varied widely across different batches (the highest concentration was found in batch B, at 405 µg/g, while it was absent in late batches except for G, at 153.34 µg/g). This observation aligns with the literature, which reports higher levels of cynaropicrin in the leaves [37]. In contrast, it is typically present in low quantities in the inflorescence receptacle at the earliest stages of development, while it disappears at more advanced stages (0.05 mg/g when reaching the commercial stage) [25]. In contrast, the literature reports no cynaropicrin detection in the outer bracts. This may indicate a difference in cultivar characteristics; for example, Tema is a particularly tender cultivar suitable for raw consumption, whereas Romanesco, as tested by Eljounaidi et al. [25], is generally larger and fibrous, then suitable for cooked consumption.

In particular, in-vitro tests have demonstrated antiproliferative effects at very low concentrations (5, 7.5, and 10 micrograms per milliliter) against colorectal cancer cells [38]. Additionally, there is evidence of activity against lung, multiple myeloma, and melanoma cancer cell lines [39,40,41]. Based on the analyses performed, the outer bracts could still be a potential target for the industrial recovery of natural bioactive compounds to replace synthetic ones.

The bioactive studies focused on the anti-aging potential of the hydroalcoholic extracts of three selected batches (A, E, and F), particularly on features related to skin aging. Overall, significant anti-senescence effects were observed, thus highlighting a potential health-promoting effect for these agro-wastes. Indeed, particularly for extract F, one of the extracts representing the second half of the harvest period, significant inhibition was attained in all senescence complementary markers, namely in lysosomal activity (β-galactosidase activity), in cell cycle arrest (p53/p21 pathway) and in DNA damage (nuclear accumulation of γH2AX). Cynara scolymus is known for its health benefits due to its high polyphenolic content, as recently reviewed by Porro and colleagues [42] that pointed out anti-inflammatory, antioxidant, liver-protective, bile-expelling, antimicrobial, and lipid-lowering neuroprotective effects. Although the artichoke biowastes showed a tendency to promote wound healing here, this effect has been shown for other species of the genus. For example, an ointment made with C. humilis powder extract was able to increase neovascularization, collagen deposition, and re-epithelialization in an animal model of skin burn [43]. Moreover, in a different study, the wound healing effects of both ethanolic and aqueous extracts of this species promoted wound surface recovery, re-epithelization, and collagen deposition, with a concurrent decrease in wound scarring [44].

Regarding the anti-senescence effects of globe artichoke biowaste herein reported, the results are in line with a previous study that also highlighted the potential of an artichoke extract on skin anti-age effects, although resorting to a different approach. This study focused on the effect of the extract on endothelial cell integrity and functionality [45]. In addition, Mileo and colleagues showed that long-term exposure to polyphenols from artichoke induced cell cycle arrest, as indicated by an increase in p16 and p21 expressions [46]. Although these results seem contradictory, the study was performed on a human breast cancer cell line, in which inducing senescence is beneficial to avoid cell proliferation, thus pointing out a potential application in cancer prevention/treatment.

The main compounds present in artichoke biowaste, namely chlorogenic acid and their derivatives as well as cynaroside, also gather relevant bioactive potential, namely in what concerns delaying skin aging. For example, it was shown that 4,5-dicaffeoylquinic acid [47] and chlorogenic acid [48] promote wound healing. In addition, the presence of 3,5-dicaffeoylquinic acid ameliorated spatial learning and memory in a senescence-accelerated-prone-mice model [49], suggesting that this compound might have anti-senescent properties. Moreover, chlorogenic acid and several dicaffeoylquinic acids increased the lifespan of C. elegans, with 3,5-dicaffeoylquinic acid downregulating the insulin/insulin-like growth factor signaling (IIS) pathway [50]. Interestingly, this pathway seems to be responsible for the lifespan-enhancing properties of cynaroside (luteolin-7-O-glucoside) [51], a compound present in all agro-waste batches tested. This compound, in a model of UVA-induced photoaging on keratinocytes, decreased metalloproteinase-1 (MMP-1) production by modulation of the mitogen-activated protein kinases (MAPKs) and activator protein-1 (AP-1) signaling pathways [52], thus reinforcing its protective effect. Another relevant compound in artichoke is cynaropicrin. This compound is known to suppress skin photoaging by inhibiting nuclear factor-kappa B transcription activity [53], as well as the AhR-Nrf2-Nqo1 pathway [54]. However, this compound is not present in sample F and, therefore, does not contribute to the anti-skin aging effects herein reported.

4. Materials and Methods

5. Conclusions

The present study not only provides the chemical characterization of the Tema cultivar but also provides, through the evaluation of its agro-waste biological activity, a useful insight into its valorization. The application of the agroprospecting concept involved a crucial step of analyzing the crops’ production chain and their derived biological waste. In this case, the main advantage was the choice of an artichoke intended for sale as a fourth-range product, for which on-farm processing allows simplified waste collection.

Chemical characterization provided confirmation of phenolic acid and flavonoid content, while seasonal monitoring revealed their variability during the harvest season. For this reason, biological tests covered early and late harvest samples, which reveal similar action in wound healing, while greater efficacy of late batch F in antisenescence action highlights the potential anti-aging effects. These findings reveal the need for analysis of agro-waste during seasonality to make better utilization (e.g., as a supplement, human food or animal feed, or pharmaceutical or nutraceutical products).

In conclusion, this approach aims to foster a truly sustainable circular economy. An intriguing direction for future research is to determine how chemical composition varies in response to different factors such as cultivation environment, crop management practices, industrial processing, and marketing, all of which can affect the quality of biomolecules in these crop species. Furthermore, the same concept can easily be exported to different types of crops and their corresponding agro-wastes.

Acknowledgments

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules29163960/s1, Figure S1: UV spectra of Chlorogenic Acid and Cynaroside, used for the quantification and identification (from Diode Array analysis). Figure S2: (a) Globe artichoke of Tema cultivar. (b) Tema fourth range product. Figure S3: (A) Cynaroside calibration curve; (B) Chlorogenic acid calibration curve. Table S1. Gradient programme of HPLC-DAD analysis.

Funding Statement

Part of this work was supported by national funds from the Portuguese FCT within the projects UIDB/00102/2020, UIDP/00102/2020.

Author Contributions

Conceptualization, A.M., M.Z. and L.S.; methodology, A.M., D.M. and J.M.A.-S.; software, E.C. and D.F.; validation, A.M., E.C., D.F., M.Z. and L.S.; formal analysis, A.M., D.M., E.C., J.M.A.-S. and M.Z.; investigation, A.M., D.M., D.F. and J.M.A.-S.; resources, A.M.; data curation, D.F., E.C., A.M., D.M., J.M.A.-S. and M.Z.; writing—original draft preparation, A.M., E.C., J.M.A.-S. and M.Z.; writing—review and editing, A.M., E.C., M.Z., J.M.A.-S. and L.S.; visualization, E.C., D.M., J.M.A.-S. and M.Z.; supervision, A.M., M.Z. and L.S.; project administration, V.G., A.M., M.Z. and L.S.; funding acquisition, A.M., V.G., M.Z. and L.S. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author/s.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

References