Freezone 6

The frozen dialysates samples were evaporated to dryness in a vacuum concentrator (Jouan RC10.10, Thermo Fisher Scientific) with a freeze dry system (FreeZone 6, LABCONCO). All evaporated microdialysates were treated identically. The content of OT in each dialysate was quantified with a highly sensitive (0.1 pg OT/100 μl sample) and selective radioimmunoassay (RIA, minimal affinity for arginine-vasopressin, RIAgnosis, Munich, Germany), as described previously (Neumann et al., 1993 (link); Ross et al., 2009 (link); Bosch et al., 2016 (link)). Cross-reactivity of the polyclonal antiserum with arginine–vasopressin and other related peptides was <0.7%. Intra- and inter-assay coefficients of variation were <8 and <11%, respectively. Due to the high total number of microdialysis samples (560), microdialysates were analyzed during four RIAs and each assay contained balanced number of samples from different treatment groups.

For the extraction, previously described protocols were modified [12 (link),25 (link)]. Two grams of dried leaves were grounded in the presence of liquid nitrogen and subsequently extracted with 20 mL petroleum ether (three times) and 20 mL 70% (aq.) methanol (three times) for 20 min each time in an ultrasonic bath (120 W, 40 kHz) at a temperature under 40 °C. Two biological replicates per population were extracted. The petroleum ether (PE) extracts obtained were condensed, dried over anhydrous sodium sulfate, and stored at −20 °C. The hydroalcoholic extracts obtained were freeze-dried (Freezone 6, Labconco, MO, USA) and stored at −20 °C.

The culture was filtered through a cellulose filter and centrifuged at 11 000 rpm for 20 min. The supernatant was concentrated on a reverse osmosis column. The EPS was precipitated by addition of 96% ethanol:water (1:1 v/v) and left for 24 h at 4 °C. The 1:1 v/v ethanol:water ratio was selected in preliminary tests in which the efficiency of precipitation was checked using ethanol in a ratio of 1:1 to 1:4. Then, the precipitate was centrifuged at 11 000 rpm for 15 min, vacuum-dried in a lyophilizer (Freezone 6, Labconco, Kansas City, MO, USA) for 3 days, and stored in a desiccator until further investigation.

Cryogels were produced as previously described (Rodrigues et al., 2013 (link); Salgado et al., 2016 (link), 2019 (link)). Briefly, bovine collagen Type I (Sigma-Aldrich, Germany) was homogenized (Ultra Turrax T25, IKA) at 10000 rpm, in 5 mM HCl (36.5–38% grade, Sigma-Aldrich, Germany) at a concentration of 2% (w/v). Collagen-nanoHA biocomposites were prepared by mixing the collagen solution with 1% nanoHA (particle size 5.0 ± 1.0, nanoXIM.HAp202, FLUIDINOVA, S.A, Portugal), final composition Collagen-nanoHA 50:50 w/w%). O-phospho-L-serine (OPS, ≥98% grade, Sigma-Aldrich, Germany) was added to the nanoHA suspension (0.5% w/w%) with the final mass proportion of 1:1:0.5 for the Coll-nanoHA/OPS scaffold. For the preparation of cryogels, materials were crosslinked with 10 mM of N-hydroxysuccinimide (NHS, 98% grade, Sigma-Aldrich, Germany) and 20 mM of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, ≥98% grade, Sigma-Aldrich, Germany), at the final mass proportion of 1:0.012:0.031 (collagen/NHS/EDC), and were kept in a freezer at −18°C for 24 h to complete the crosslinking. Afterward, materials were thawed at room temperature and the scaffolds were washed with distilled water and finally dried in a freeze-dryer (Labconco, FreeZone 6) at −80°C for 24 h.

Mangoes (cv. Ataulfo) were purchased from the local market in Tepic, Nayarit State, México. The fruits were washed with tap water, disinfected (Biopur, Mexico) and sliced (peel with pulp) 3 mm thick (Tor-Rey, R-300, México). Fruit slices were dried in an oven with air-forced convection at 70 °C for 22 h; (Scorpion Scientific, A-52055, Mexico). The dried slices were ground in a food processor (NutriBullet, NBR-0804B, Los Angeles, CA, USA). To prepare the mango-based bars, the ground fruit was molded with a binding agent (Nutriose FB, Tecnovam, Mexico), and the bars were dried at 60 °C for 3 h (Scorpion Scientific, A-52055, Mexico). The bars were prepared without any sweetening additive or preservative. The bar samples were freeze-dried (FreeZone 6, Labconco, Kansas City, MO, USA), and subsequently ground (NutriBullet), sieved (0.5 microns), and stored in sealed bags at −20 °C until analysis.