Cinnamic

About 100 g of air-dried powder aerial parts of the two samples of H. curassavicum (Coastal and Inland) were extracted in 70% hydro-methanol at room temperature (27 ± 2°C), filtered, and dried under vacuum to give dark black gum (1.6 and 1.85 g, respectively).
The standard phenolic, gallic, cinnamic, chlorogenic, ferulic, coffeic, syringic, ellagic, coumaric acids, vanillin, caffeine, propyl gallate, and flavonoids, quercetin, rutin, catechin, pyrocatechol, naringenin, 4‘.7-dihydroxy isoflavone, were purchased from Sigma-Aldrich (Germany). Trifloroacetic acid and acetonitrile (HPLC gradient grades) were purchased from Sigma-Aldrich (Germany). The used di-distilled water in HPLC was obtained by Hamilton water distillation apparatus (Hamilton Laboratory Glass Ltd., Kent, England).
HPLC analysis was performed using an Agilent 1260 series. The separation was carried out using a C18 column (4.6 × 250 mm i.d., 5 μm). The mobile phase contained water (A) and 0.02% trifloroacetic acid in acetonitrile (B) with a flow rate of 1 mL min⁻¹. The mobile phase was automated successively in a linear gradient as follows: 0 min (80% A), 0–5 min (80% A), 5–8 min (40% A), 8–12 min (50% A), 12–14 min (80% A), and 14–16 min (80% A). The multi-wavelength detector was monitored at 280 nm. The injection volume was 10 μL for each of the sample solutions. The column temperature was maintained at 35 °C.

Twelve reference fungal species from a fungal pool assayed on different OMWWs in previous researches [6 (link),7 (link)] (Table 1) were studied to assess their growth on media containing either vanillic (code 94770, purum ≥ 97.0%, HPLC, Sigma-Aldrich) or cinnamic (code 133760, 97%, Sigma-Aldrich) acids.

Metabolites were extracted from whole fracture hematoma/callus tissue and blood serum as described before^{60 (link)}, with some modifications as described in the following. Frozen fracture tissue was homogenized as described under 4.3 animal sacrifice and weight. Per 50 mg frozen tissue 1 ml of methanol-chloroform-water (MCW; 5:2:1 v/v/v, Merck, Germany) was added, supplemented with 2 µg/ml cinnamic (Sigma-Aldrich, MI, USA) acid serving as internal standard. MCW-sample solution was further homogenized by ultra-sonication for twice for 30 s. Samples were shaken for 30 min at 10 °C, 200 rpm, water was added and shaken again for 5 min before centrifuged for 10 min at 10,000 x g, 4 °C to separate the polar metabolites from the lipid metabolites/phase. The aqueous, polar phase was collected and vacuum dried. For derivatization, 20 µl methoxyamine hydrochloride solution (40 mg/ml in pyrimidine) (Sigma-Aldrich, MI, USA) was added to the dried fracture tissue extracts and incubated for 90 min at 30 °C. Next, 80 µl of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA, VWR, PA, USA), including a retention index mixture (nine alcanes) was added to the mixture and incubated for 60 min at 37 °C, while shaking constantly. Mixture was centrifuged for 5 min at maximum speed and supernatant was split and transferred into glass vials for GC-MS measurement.

Deionized water was produced using Millipore Simplicty UV station (Merck Millipore, Burlington, MA, USA). Acetonitrile, formic acid, pyridine, ethanol, methanol, hydroxylamine hydrogen chloride, acetic anhydride, dichlorethane, hydrochloric acid, heptane, ethyl acetate, formalin, tetrachlormethane was purchased from VWR (Radnor, PA, USA). Chlorogenic acid, rutin, pyrogallol were purchased from Carl Roth (Karlsruhe, Germany). Hydroxyphenylacetate, caffeic, syringic, p-coumaric, ferulic, sinapic, cinnamic, quinic acids and rutin, quercetin-3-D-glucoside (isoquercitrin), naringin, neohesperidin, quercetin, naringenin, kaempferol, sorbitol, luteolin and aluminum chloride were purchased from Sigma-Aldrich (Sant Louis, MI, USA).

Phenylalanine, cinnamic, ferulic, caffeic, p-coumaric, chlorogenic and gallic acids, rutin, and quercetin standard were purchased from Sigma (Italy), naringenin from Aldrich (Italy), naringenin-7-O-glucoside from Infodine (USA). Methanol, formic acid, and water HPLC grade were obtained from Merck (Darmstadt, Germany). Deionized water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). Chromatographic solvents were degassed for 20 min using a Branson 5200 (Branson Ultrasonic, Corp., USA) ultrasonic bath.

Methyl chloroformate and a linear alkane series from heptane to triacontane were obtained from Sigma Aldrich (Sigma-Aldrich Pty Ltd., Sydney, Australia). Optima LC-MS grade methanol and analytical grade pyridine, chloroform, sodium acetate and sodium hydroxide were from Thermo Fisher (Thermo Fisher Scientific Inc., Auckland, New Zealand). Ultrapure water was obtained from a Purite Select Fusion system (Total Lab Systems, Auckland, New Zealand). Analytical grade standards of 2-hydroxybutyric, 3-hydroxybenzoic, adipic, benzoic, cis-aconitic, citric, fumaric, itaconic, lactic, malic, malonic, 4-toluic, succinic, syringic, cinnamic and vanillic acids, as well as d₄-alanine, were obtained from Sigma Aldrich (Sigma-Aldrich Pty Ltd., Sydney, Australia).

The analysis was carried out just on the VE aqueous extracts at 10 mg/mL. The samples were filtered through a 45 μm filter to remove impurities, and they were directly injected into the equipment. An Agilent Technologies liquid chromatograph 1200 (Santa Clara, CA, USA) was used for the study.
Several reference substances were used as standards, more particularly the flavonoids rutin, quercetin, kaempferol-3-O-glucoside, apigenin, catechin, epicatechin and phenolic acids (ferulic, caffeic, cinnamic and gallic acids, 98% purity, Sigma-Aldrich, St Louis, MO, USA).
The separations were carried out on a Kinetex EVO C18 (5 µm, 250 mm × 4.6 mm) column. The elution was carried out at a flow rate of 1 mL/min, the injection volume was 20 μL, and a pressure of 25 μPa was used. The oven temperature was 25 °C. The detection was achieved with a diode array detector (DAD) in the range 200–400 nm. The mobile phase consisted of water with 0.1% formic acid (solvent A) and acetonitrile-methanol (7:3) with 0.1% formic acid (solvent B), using a gradient program of 5% B in 0–5 min, 5–100% B in 5–50 min, 100% B in 50–60 min and 5% B in 60–65 min for re-equilibration.