Ethyl acetate

Analytical grade cordycepin (purity ≥98.0%), adenosine (purity ≥99.0%), l-ascorbic acid (purity ≥99.0%), epigallocatechin gallate (EGCG; purity ≥95.0%), oleanolic acid (purity ≥97.0%), sodium chloride (NaCl), sodium phosphate (Na₃PO₄), sodium dihydrogen phosphate (NaH₂PO₄), disodium phosphate (Na₂HPO₄), sodium carbonate (Na₂CO₃), tricine, tris base, elastase from porcine pancreas lyophilised powder (E–E.C.3.4.21.36), N-succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN), metalloproteinase-1 (MMP-1) from Clostridium histolyticum (Weinberg and Seguin 1916) (ChC–EC.3.4.23.3), N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA; purity ≥99.0%), hyaluronidase from bovine testes (E.C.3.2.1.3.5), hyaluronic acid, bovine serum albumin (BSA), and sodium lauryl sulphate (SLS) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Analytical grade ethanol, hexane, ethyl acetate, acetic acid, and dimethyl sulfoxide (DMSO) were purchased from Labscan (Dublin, Ireland). HPLC-grade methanol was purchased from Labscan (Dublin, Ireland).

All chemicals and solvents were analytical grade. Standard catechin was purchased from Sigma^® (Sigma‐Aldrich, USA). Stock solutions of Standard catechin were freshly prepared for daily use. Stationary phase was TLC plates precoated with silica gel 60 F₂₅₄ (20x10 cm) and thickness of 0.2 mm purchased from Merck (USA). Ethanol, hexane, dichloromethane, ethyl acetate, and mEthanol were purchased from Labscan (Dublin, Ireland). 2,20‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS), 2,4,6 tripyridyl‐s‐triazine (TPTZ), 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (Trolox), and linoleic acid were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Tris base was purchased from Fisher Chem Alert (Fair Lawn, NJ, USA). Potassium persulfate, ferric chloride, ferrous sulfate, and sodium acetate were purchased from Fisher Chemicals (Loughborough, UK). Hydrochloric acid, acetic acid, trichloroacetic acid, thiobarbituric acid, and ascorbic acid were purchased from Merck (Darmstadt, Germany).

Aliquots collected during the incubation of isolated strains, were extracted and analyzed by HPLC-DAD-ESI-Q (MS) as previously described (García-Villalba et al., 2016 (link)). Briefly, fermented medium (5 ml) was extracted with ethyl acetate (5 ml) (Labscan, Dublin, Ireland) acidified with 1.5% formic acid (Panreac), vortexed for 2 min and centrifuged at 3500 g for 10 min. The organic phase was separated and evaporated and the dry samples were then re-dissolved in methanol (250 μl) (Romil, Barcelona, Spain). An HPLC system (1200 Series, Agilent Technologies, Madrid, Spain) equipped with a photodiode-array detector (DAD) and a single quadrupole mass spectrometer detector in series (6120 Quadrupole, Agilent Technologies, Madrid, Spain) was used as previously described (García-Villalba et al., 2016 (link)). Calibration curves were obtained for EA, urolithin C as well as urolithin M6 and isourolithin A (Villapharma SL, Murcia, Spain) with good linearity (R² > 0.998). Isourolithin A and urolithin C were quantified at 305 nm, while urolithin M5, urolithin M6, and EA were quantified at 360 nm, all with their own standards.