Sodium oxalate

Extraction and quantification of water-soluble and total oxalate were performed according to a previously described method (2 (link)). Oxalate compounds were converted to oxalic acid using hydrochloric acid and analyzed on a Phenomenex HPLC system (Phenomenex, Lane Cove West, NSW, Australia) equipped with a Gilson UV/VIS 151 multiwavelength detector (Gilson, Middleton, WI, USA). A Phenomenex reversed phase column (Synergi 4 μm Hydro-RP 80A, 250 × 4.60 mm) (Phenomenex, Lane Cove West, NSW, Australia) was used with an isocratic mobile phase of 20 mM phosphate buffer (pH 2.4) and a flow rate of 0.5 ml/min. The injection volume was 20 μL and detection was carried out at 210 nm. Sodium oxalate was obtained from Sigma-Aldrich (St. Louis, MI, USA). Sodium oxalate at the concentrations of 1, 2, 3, 4, and 8% in 20 mM phosphate buffer (pH 2.4) was used to prepare an external calibration curve to quantify the levels of total and water-soluble oxalate in T. ferdinandiana tissue samples.

The scavengers used was sodium chromate (Cr(VI)), 2.5 mM, Sigma, 99.5%) for electron, superoxide dismutase (400 U mL⁻¹, Sigma, 99%)) for •O₂⁻, l-histidine for ¹O₂ (l-His, 2.5 mM, Sigma, 99%), catalase for H₂O₂ (300 U mL⁻¹, Sigma), isopropanol (2.5 mM, Sigma, 99.5%) for •OH and sodium oxalate (2.5 mM, Sigma, 99.5%) for hole. The scavengers were added into the bacteria suspension before illumination. The concentrations of bacteria in solution were measured at different time intervals by using standard spread plating techniques. Each sample was serially diluted and each dilution was plated in triplicate onto nutrient agar and incubated at 37 °C for 20 h.

Insoluble polyvinylpyrrolidone (PVP), acting as solid phase material, was purchased from SERVA (Heidelberg, Germany) labeled as Polyclar^® AT pract. Chemicals used for method establishment were purchased from Sigma-Aldrich (St. Louis, MO, USA), including gallic acid (97.5–102.5%), L-tyrosine (≥98%), D(+)-glucose (≥99.5%) and L-ascorbic acid (99%). For reference analysis, Folin–Ciocalteu phenol reagent (2 M with respect to acid), sodium carbonate (≥99.5%), potassium permanganate (≥99%), sodium oxalate (≥99.5%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Indigo carmine (≥80% p.a.), used as indicator for Löwenthal titration, was purchased from Carl Roth (Karlsruhe, Germany). HPLC analysis was carried out using methanol (HPLC grade, 99.9%) purchased from VWR Chemicals (Radnor, PA, USA) and diluted trifluoroacetic acid (99%) purchased from Sigma Aldrich (St. Louis, MO, USA).

COM crystallization assay was performed according to a protocol established previously18 (link)40 (link). Briefly, 0.5 ml of 10 mM calcium chloride (Sigma-Aldrich; St. Louis, MO) in basic buffer containing 10 mM Tris and 90 mM NaCl (VWR; Leuven, Belgium) was added into 24-well polystyrene plate (Corning Inc.; Corning, NY) in the absence or presence of purified caffeine (Sigma) with various concentrations of 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM. For the control, an equal volume of the basic buffer was added instead of caffeine. Thereafter, 0.5 ml of 1 mM sodium oxalate (Sigma-Aldrich) in basic buffer was added into each well of the mixture. After 60-min incubation at room temperature (RT) (set at 25 °C), the crystal images were taken using an inverted light microscope (Nikon eclipse Ti; Nikon; Tokyo, Japan). Crystal size was then analyzed using crystal area measured by NIS-element D V.4.11 software (Nikon) from at least 100 individual crystals in each well. Crystal number in each well was counted from 15 high-power fields (HPFs). Crystal mass was calculated using the formula: