Lactucin Synthase Inactivation Boosts the Accumulation of Anti-inflammatory 8-Deoxylactucin and Its Derivatives in Chicory (Cichorium intybus L.).

For several sesquiterpene lactones (STLs) found in Asteraceae plants, very interesting biomedical activities have been demonstrated. Chicory roots accumulate the guaianolide STLs 8-deoxylactucin, lactucin, and lactucopicrin predominantly in oxalated forms in the latex. In this work, a supercritical fluid extract fraction of chicory STLs containing 8-deoxylactucin and 11ß,13-dihydro-8-deoxylactucin was shown to have anti-inflammatory activity in an inflamed intestinal mucosa model. To increase the accumulation of these two compounds in chicory taproots, the lactucin synthase that takes 8-deoxylactucin as the substrate for the regiospecific hydroxylation to generate lactucin needs to be inactivated. Three candidate cytochrome P450 enzymes of the CYP71 clan were identified in chicory. Their targeted inactivation using the CRISPR/Cas9 approach identified CYP71DD33 to have lactucin synthase activity. The analysis of the terpene profile of the taproots of plants with edits in CYP71DD33 revealed a nearly complete elimination of the endogenous chicory STLs lactucin and lactucopicrin and their corresponding oxalates. Indeed, in the same lines, the interruption of biosynthesis resulted in a strong increase of 8-deoxylactucin and its derivatives. The enzyme activity of CYP71DD33 to convert 8-deoxylactucin to lactucin was additionally demonstrated in vitro using yeast microsome assays. The identified chicory lactucin synthase gene is predominantly expressed in the chicory latex, indicating that the late steps in the STL biosynthesis take place in the latex. This study contributes to further elucidation of the STL pathway in chicory and shows that root chicory can be positioned as a crop from which different health products can be extracted.

Targeting proteases involved in the viral replication of SARS-CoV-2 by sesquiterpene lactones from chicory (Cichorium intybus L.).

SARS-CoV-2 is a highly transmissible and pathogenic coronavirus causing a respiratory disease that emerged in 2019, leading to a public health emergency situation which continues to date. The treatment options are still very limited and vaccines available are less effective against new variants. SARS-CoV-2 enzymes, namely main protease (Mpro) and papain-like protease (PLpro), play a pivotal role in the viral life cycle, making them a putative drug target. Here, we described for the first time the potential inhibitory activity of chicory extract against both proteases. Besides, we have identified that the four most abundant sesquiterpene lactones in chicory inhibited these proteases, showing an effective binding in the active sites of Mpro and PLpro. This paper provides new insight for further drug development or food-based strategies for the prevention of SARS-CoV-2 by targeting viral proteases.

Evaluation of Microwave-Assisted Extraction as a Potential Green Technology for the Isolation of Bioactive Compounds from Saffron (Crocus sativus L.) Floral By-Products.

The saffron flower stigmas are used for the saffron spice production while the remaining saffron floral by-products, that are a valuable source of natural bioactive compounds, remain underutilized. The aim of this study was to evaluate the microwave-assisted extraction (MAE) through response surface methodology to obtain high value-added compounds from saffron tepals as ingredients with potential application in the food, pharmaceutical and/or cosmetic industries. A central composite design was applied to optimize process variables: temperature, time and ethanol solvent concentration. Extracts were characterized in terms of total phenolic and total flavonoid content, and antioxidant capacity (ORAC and HOSC assays), being the maximum values obtained: 126.20 ± 2.99 mg GAE/g dry matter; 8.05 ± 0.11 mg CE/g dry matter; 6219 ± 246 µmol TEAC/dry matter; 3131 ± 205 µmol TEAC/dry matter, respectively. Results indicated that the optimal extraction conditions were the combination of low temperature (25 °C)-high extraction time (5 min) using ethanol as solvent (100%). MAE revealed to be an efficient technique to isolate bioactive compounds from saffron floral by-products with a low energy footprint.

Chicory Extracts and Sesquiterpene Lactones Show Potent Activity against Bacterial and Fungal Pathogens.

Chicory (Cichorium intybus L.) is an important industrial crop cultivated mainly to extract the dietary fiber inulin. However, chicory also contains bioactive compounds such as sesquiterpene lactones and certain polyphenols, which are currently discarded as waste. Plants are an important source of active pharmaceutical ingredients, including novel antimicrobials that are urgently needed due to the global spread of drug-resistant bacteria and fungi. Here, we tested different extracts of chicory for a range of bioactivities, including antimicrobial, antifungal and cytotoxicity assays. Antibacterial and antifungal activities were generally more potent in ethyl acetate extracts compared to water extracts, whereas supercritical fluid extracts showed the broadest range of bioactivities in our assays. Remarkably, the chicory supercritical fluid extract and a purified fraction thereof inhibited both methicillin-resistant Staphylococcus aureus (MRSA) and ampicillin-resistant Pseudomonas aeruginosa IBRS P001. Chicory extracts also showed higher antibiofilm activity against the yeast Candida albicans than standard sesquiterpene lactone compounds. The cytotoxicity of the extracts was generally low. Our results may thus lead to the development of novel antibacterial and antifungal preparations that are both effective and safe for human use.

Lipid-based nanostructures as a strategy to enhance curcumin bioaccessibility: Behavior under digestion and cytotoxicity assessment.

The aim of this study was to evaluate the behavior of different lipid-based nanostructures during in vitro digestion, in particular on curcumin's bioaccessibility, and to access their potential toxicity. Solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and nanoemulsions (NE) were submitted to harmonized static in vitro digestion and their cytotoxicity and cellular transport were evaluated using Caco-2 cell line. NE presented the highest curcumin's bioaccessibility followed by NLC and SLN, 71.1%, 63.7% and 53.3%, respectively. Free fatty acids percentage increased in the following order: NLC ≤ NE < SLN. Non-digested nanostructures and excipients presented no cytotoxicity; however, digested NE and NLC presented cytotoxicity due to MCT oil, which presented cytotoxicity after digestion. The apparent permeability coefficient of NLC was higher than SLN and NE. These results showed that lipid-based nanostructures' physical state and composition have a high influence on particles' behavior during digestion, and on their cytotoxicity/intestinal permeability, and highlights the importance of conducting cytotoxicity assessments after in vitro digestion. This work contributes to a better understanding of the behavior of lipid-based nanostructures under digestion/adsorption, and this knowledge will be useful in design of nanostructures that afford both safety and an increased bioactive compounds' bioavailability.

Supercritical CO2 Extraction as a Tool to Isolate Anti-Inflammatory Sesquiterpene Lactones from Cichorium intybus L. Roots.

Cichorium intybus L. or chicory plants are a natural source of health-promoting compounds in the form of supplements such as inulin, as well as other bioactive compounds such as sesquiterpene lactones (SLs). After inulin extraction, chicory roots are considered waste, with most SLs not being harnessed. We developed and optimized a new strategy for SL extraction that can contribute to the conversion of chicory root waste into valuable products to be used in human health-promoting applications. In our work, rich fractions of SLs were recovered from chicory roots using supercritical CO2. A response surface methodology was used to optimize the process parameters (pressure, temperature, flow rate, and co-solvent percentage) for the extraction performance. The best operating conditions were achieved at 350 bar, 40 °C, and 10% EtOH as a co-solvent in a 15 g/min flow rate for 120 min. The extraction with supercritical CO2 revealed to be more selective for the SLs than the conventional solid-liquid extraction with ethyl acetate. In our work, 1.68% mass and a 0.09% sesquiterpenes yield extraction were obtained, including the recovery of two sesquiterpene lactones (8-deoxylactucin and 11ß,13-dihydro-8-deoxylactucin), which, to the best of our knowledge, are not commercially available. A mixture of the abovementioned compounds were tested at different concentrations for their toxic profile and anti-inflammatory potential towards a human calcineurin/NFAT orthologue pathway in a yeast model, the calcineurin/Crz1 pathway. The SFE extract obtained, rich in SLs, yielded results of inhibition of 61.74 ± 6.87% with 50 µg/mL, and the purified fraction containing 8-deoxylactucin and 11ß,13-dihydro-8-deoxylactucin inhibited the activation of the reporter gene up to 53.38 ± 3.9% at 10 µg/mL. The potential activity of the purified fraction was also validated by the ability to inhibit Crz1 nuclear translocation and accumulation. These results reveal a possible exploitable green technology to recover potential anti-inflammatory compounds from chicory roots waste after inulin extraction.