Cichorium intybus

To extract and quantify Glc, Fru and Suc, a circular shape (3.5 cm diameter) of leaf material with an approximate weight of 200 mg was taken from primed and infected leaves. In the case of infected leaves, a circular leaf disc (3.5 cm diameter) surrounding the lesion was sampled. This material was ground in liquid nitrogen and then heated at 90 °C in 0.5 mL water, and subsequently passed through Dowex^® anion and cation exchange resins. Rhamnose 20 µM was added as internal standard. The sample was then analyzed by HPAEC-IPAD Dionex 3000 (Thermofisher, Whaltam, MA, USA) according to Shiomi et al., [106 ].
BFOs were extracted from freeze-dried burdock (Arctium lappa) roots according to the protocol described by Hao et al. [107 ] with minor modifications. For enzymatic hydrolysis treatments, we used a -fructan 1-exohydrolase from chicory [108 (link)] immobilized on a Concanavalin-A Sephadex resin (GE Healthcare, Chicago, IL, USA). BFOs were dialyzed with 1 kDa cutoff Spectra/Por membrane (Repligen, Waltham, MA, USA).
OGs were extracted from polygalacturonic acid sodium salt (Sigma). 800 mg of powder were incubated with 10 mg of pectinase extracted from Aspergillus niger (0.71 units/mg, Polylab, Kundli, India) in 60 mL NaAc buffer pH 5 for 60 min at 40 °C. The solution was then boiled for 8 min at 90 °C and centrifuged (20 min, 40,000× g). The resulting supernatant was diluted in 40 mL acetone and centrifuged (15 min, 14,000× g). This step was repeated twice. Finally, the obtained pellet was washed two times in 80% acetone and dried with the use of a liofilizator (LSL Secfroid, Aclens, Switzerland).

Herbal products were purchased as one-component medicinal teas from specialized pharmaceutical units in Romania; the products comprise: artichoke leaves—Cynarae folium (CF), rosemary leaves—Rosmarini folium (RF), dandelion aerial parts—Taraxaci herba (TH), common chicory aerial parts—Cichorii herba (CH), and agrimony aerial parts—Agrimoniae herba (AH).
Based on previously reported studies [82 (link)], the solvents used for the extraction were 50% ethanol for CF, RF, CH, and AH, and 20% ethanol for TH. The choice of the solvent was motivated by the need to obtain the optimal extraction of phenolic compounds for all analyzed herbal products, and 20% ethanol was the best extraction solvent for dandelion aerial parts (the highest extraction yield for TF and TPA). The ethanol used as solvent in this section was purchased from Sigma-Aldrich, Darmstadt, Germany.
Aliquots of 50 g of each herbal product were subjected to two consecutive reflux extraction processes: the first one, using 1.5 L of solvent for 30 min, and the second one, using 750 mL of solvent, also for 30 min. The two extract solutions were mixed and concentrated in a rotary evaporator (Buchi, Vacuum Pump V-700) and then subjected to a lyophilization process (Christ Alpha 1-2/B Braun, BiotechInt). The dry extracts were conserved in a glass vacuum desiccator. The samples were marked as follows: CE (Cynarae extractum), RE (Rosmarini extractum), TE (Taraxaci extractum), CHE (Cichorii extractum), and AE (Agrimoniae extractum).
Other equipment and experimental conditions used in this study are presented in each stage of the experiments.

Eight-week-old C57Bl6/J male mice (n = 75; Janvier, France) were housed in pairs under specific conditions, i.e., specific and opportunistic pathogen-free conditions (SOPF) and in a controlled environment (12 h daylight cycle, temperature of 22 ± 2°C) with food and water ad libitum. After being acclimatized during one week with a control diet (CTRL) (D12450H, Research diet) and matched according to body weight and fat mass, mice were divided into 5 distinct dietary groups (n = 15/group): (1) control group of mice fed a control diet (D12450H, Research diets) containing 10% calories from fat (CTRL group); (2) mice fed a high-fat and high-sucrose diet (HFHS diet, D12451, Research diets) containing 45% calories from fat and 35% calories from carbohydrates (HFHS group); (3) mice fed a HFHS diet supplemented with rhubarb extract (0.3% w/w in the diet, Ortis, Belgium) (RHUB group), (4) mice fed a HFHS diet supplemented with inulin, a chicory root extract (20% food intake in water, Fibruline, Cosucra, Belgium) (ITF group); (5) mice fed a HFHS supplemented with rhubarb and inulin (0.3% rhubarb in the diet and 20% inulin in the drinking water) (RHUB+ITF group). Supplementation with either rhubarb, inulin or both started concomitantly with the introduction of HFHS diet. Water containing inulin was replaced every two days and the concentration of inulin was adapted depending on food intake for each cage. Body weight, food intake and water intake were recorded every week for the duration of the experiment (6 to 9 weeks). Body composition was assessed once a week by using 7,5-MHz time-domain nuclear magnetic resonance (LF50 minispec; Bruker; Rheinstetten, Germany). All mouse experiments were approved by and performed in accordance with the guideline of the local ethics committee (the ethics Committee of the Université catholique de Louvain for Animal Experiments specifically approved this study, agreement number 2017/UCL/MD/005).
At the end of the experiment and after 3 hours of fasting, all mice were anesthetized with isoflurane (Forene, Abbott, Queenborough, Ken, UK) and blood was sampled. After exsanguination, mice were euthanized by cervical dislocation. Organs and tissues were dissected, weighted, and directly immersed in liquid nitrogen before storage at −80°C for further analysis.

Freeze-dried tissues were grinded with mortar and pestle to fine powder. Semi polar metabolites were analysed by LC-MS [24 (link)]. To this end, 50 mg (±3 mg) of plant material was extracted using 950 µL 70% methanol supplemented with 0.13% formic acid followed by an incubation in the ultrasonic bath for 15 min. The plant debris was then separated from the extract by centrifugation at 14000 rpm in a tabletop centrifuge. LC-MS analysis was performed using the LC-PDA-LTQ-Orbitrap FTMS instrument (Thermo Scientific, Waltham, USA) equipped with an Acquity UPLC. Chromatographic separation and detection of metabolites was performed as described previously [5 (link)].
Targeted quantification of major chicory phenolic and terpene compounds was performed by comparison to a standard curve prepared from authentic standards of caftaric acid (Sigma-Aldrich Merck KGaA, Darmstadt, Germany), chicoric acid (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), isochlorogenic acid A (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), lactucin (Extrasynthese, Genay, France), and lactucopicrin (Extrasynthese, Genay, France). In case authentic standards were not available, compounds were relatively quantified and relative peak areas were presented. Metabolite analysis data were subjected to the analysis of variance (ANOVA) for statistical analysis using SPSS software (software version 25 for Windows, IBM). Tukey’s post hoc test (p > 0.05) was used to analyse differences between hairy root lines.