Benzenesulfonic acid

Contents of ·O₂⁻, H₂O₂ and MDA were determined according to the methods described by Hu et al. [17 ] with slight modifications. The generation rate of ·O₂⁻ was determined using hydroxylamine method. Banana peel samples (0.50 ± 0.05 g) were ground with 3 mL of 50 mM Tris-HCl buffer (pH7.8) and the homogenate was centrifuged at 12,000 g at 4°C for 30 min. The reaction mixture (0.5 mL) contained 50 mM Tris-HCl buffer (pH7.5), 0.5 mM XTT [sodium, 3-1- (phenylamino-carbonyl)-3, 4-tetrazolium-bis(4-methoxy-6- nitro), and benzenesulfonic acid hydrate], and 50 μL of sample extracts. Corrections were made for the background absorbance in the presence of 50 U of superoxide dismutase (SOD). The rate of ·O₂⁻ production was expressed as μg·g⁻¹ FW (fresh weight) · s⁻¹.
For determination of H₂O₂, banana peels (0.50 ± 0.05 g) were ground and extracted in 3 mL cold acetone. The homogenate was centrifuged at 10,000 g at 4°C for 30 min and 0.5 mL of the supernatant fraction was mixed with 1.5 mL of CHCl₃ and CCl₄ (1:3, V/V) mixture, then 2.5 mL of distilled water was added and the mixture centrifuged at 10,000 g for 1 min and the aqueous phase collected for H₂O₂ determination. The reaction system included 0.5 mL aqueous phase, 0.5 mL of buffer (phosphate-buffered saline, 200 mM, pH7.8), and 20 μL (0.5 unit) of catalase as control or inactive catalase protein (catalase inactivated by heating in boiling water for 5 min). After incubation at 37°C for 10 min, 0.5 mL of 200 mM titanium 4-(2-pyridylazo) resorcinol (Ti-PAR) was added to the reaction mixture for further incubation at 45°C for another 20 min. Absorbance at 508 nm was measured and H₂O₂ content was indicated as μmol·g⁻¹ FW (fresh weight).
For MDA analysis banana peel samples (0.50 ± 0.05 g) were ground in 3 mL of 0.1% trichloroacetic acid (TCA) and centrifuged at 10,000 g for 30 min, and 1.8 mL of the supernatant fraction was mixed with 1.8 mL of 20% TCA containing 0.5% thiobarbituric acid. The mixture was heated at 100°C for 30 min, cooled, and centrifuged at 15,000 g for 10 min. Absorbance was recorded at 532 nm and the value for nonspecific absorption at 600 nm was subtracted. An extinction coefficient of 155 mM⁻¹·cm⁻¹ was used to calculate MDA content and the content of MDA in banana peels was expressed as μmol·g⁻¹ FW (fresh weight).

Cell viability and potential cytotoxicity effects in RAW264.7 cells were assessed by measuring tetrazolium salt (XTT) conversion and lactate dehydrogenase (LDH) leakage, respectively, as previously reported [41 (link)]. For XTT conversion, XTT Cell Proliferation Kit II (Roche Applied Science, Almere, The Netherlands) was used. Briefly, after 48 h incubation of RAW264.7 cells with the compounds and LPS, supernatants were removed (and used for LDH determination) and fresh medium (100 μL) containing sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) (final concentration = 0.45 mM) and N-methyldibenzopyrazine methylsulfate (1.25 mM), was added to the cells. After incubating at 37 °C, the quantity of formazan formed in the medium was evaluated at 450 nm on a plate reader (Multiskan Ascent, ThermoLabsystem, Breda, The Netherlands). LDH leakage was measured using a Cytotoxicity Detection Kit (Roche Applied Science, Almere, The Netherlands). LDH was evaluated in culture supernatants (100 μL), previously taken and mixed with enzyme reagents (diaphorase/NAD mixture, 250 μL) and dye solutions (iodotetrazolium chloride and sodium lactate, 11.25 mL). After incubating for 30 min at 25 °C, the absorbance was measured at 492 nm.
Pancreatic INS-1 832/13 β-cell line was seeded in 96-well plates. After overnight attachment, cells were incubated for 24 h with test compounds at the final concentration of 2.5 µM, in duplicate. DMSO was used as negative control. Then, MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] solution in DPBS (3 mg mL⁻¹) was added and cells were incubated for another 6 h. Mitochondrial reductase enzymes in viable cells reduce the yellow tetrazolium MTT in its formazan, which has a purple color when dissolved in DMSO under basic condition [42 (link),43 (link)]. Media were finally replaced with 100 µL of DMSO and cell viability was evaluated by measuring absorbance of the colored wells at 545 nm, using Multi-Mode Microplate Reader (Synergy H1-BioTeck, Winooski, VT, USA).

Zr-BPT (34.6 mg, 0.01 mmol) or MOF-808 (17.1 mg, 0.01 mmol) was immersed in an aqueous solution (50.0 mL) containing both aryl (benzenesulfonic acid (BS) or phenylphosphonic acid (PP)) and alkyl acids (methanesulfonic acid (MS) or methylphosphonic acid (MP)) (5.0, 10.0, 20.0, 30.0, 50.0, 100 mM for each substance) for a day at 298 K. After removing the MOF, the concentration of the acid substances in the supernatant solution was determined using ¹H NMR, as described in the case of the screening process. Excess percentage for the aryl acid (BS or PP) was calculated using the following equationwhere q_{aryl acids} and q_{alkyl acids} correspond to the adsorption quantity of aryl acids (BS or PP) and alkyl acids (MS or MP), respectively.