Ferricyanide

Postnatal day 30 (P30) TWI mice and their WT littermates (5 for each experimental group processed in 5 different experimental sessions, every TWI with its WT littermate) and one P15 TWI mouse versus its WT littermate were perfused with a fixative solution (4% paraformaldehyde and 0.1%–1%–2.5% glutaraldehyde in phosphate buffer, pH 7.4). Sciatic nerves, spinal cords and gastrocnemius muscles were dissected and post-fixed for 4 hours at room temperature in the same fixative solution.
Spinal cords were dissected in the lumbar region, isolating four 1-mm-thick sections in the lumbar enlargement region and the gastrocnemius muscles were cut in small portions, approximately 1 mm³ in volume. Sciatic nerves were processed without further sectioning.
The selected tissues were further treated for epoxy resin embedding as previously described⁴³ . Briefly, the samples were deeper fixed in 2–2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4). After rinsing, specimens were post-fixed with osmium tetroxide (1%)-potassium ferricyanide (1%) in cacodylate buffer, rinsed again, en bloc stained with 3% uranyl acetate in ethanol, dehydrated and embedded in epoxy resin, that was baked for 48 h at 60 °C. Thin sections were obtained with an ultramicrotome (UC7, Leica Microsystems, Vienna, Austria) and collected on G300Cu grids (EMS). Finally, sections were examined with a Zeiss LIBRA 120 plus transmission electron microscope equipped with an in-column omega filter.

Determination of total phenolic content (TPC): Amount of TP were assessed using the Folin-Ciocalteu reagent [25 ]. Briefly, the crude extract (50 mg) was mixed with Folin-Ciocalteu reagent (0.5 mL) and deionized water (7.5 mL). The mixture was kept at room temperature for 10 min, and then 20% sodium carbonate (w/v, 1.5 mL) was added. The mixture was heated in a water bath at 40 ^oC for 20 min and then cooled in an ice bath; absorbance was read at 755 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). Amounts of TP were calculated using gallic acid calibration curve within range of 10-100 mgL^-1(R² = 0.9986). The results were expressed as gallic acid equivalents (GAE) g/100g of dry plant matter. All samples were analyzed thrice and the results averaged. The results are reported on dry weight basis (DW).
Determination of total flavonoid contents (TFC): The TFC were measured following a previously reported spectrophotometric method [26 (link)]. Briefly, extracts of each plant material (1 mL containing 0.1 mg/mL) were diluted with water (4 mL) in a 10 mL volumetric flask. Initially, 5% NaNO₂ solution (0.3 mL) was added to each volumetric flask; at 5 min, 10% AlCl₃ (0.3 mL) was added; and at 6 min, 1.0 M NaOH (2 mL) was added. Water (2.4 mL) was then added to the reaction flask and mixed well. Absorbance of the reaction mixture was read at 510 nm. TFC were determined as catechin equivalents (g/100g of dry weight). Three readings were taken for each sample and the results averaged.
Determination of reducing power: The reducing power of the extracts was determined according to the procedure described earlier [27 ], with a slight modification. Concentrated extract (2.5-10.0 mg) was mixed with sodium phosphate buffer (5.0 mL, 0.2 M, pH 6.6) and potassium ferricyanide (5.0 mL, 1.0%); the mixture was incubated at 50 ^oC for 20 min. Then 10% trichloroacetic acid (5 mL) was added and the mixture centrifuged at 980 g for 10 min at 5 °C in a refrigerated centrifuge (CHM-17; Kokusan Denki, Tokyo, Japan). The upper layer of the solution (5.0 mL) was decanted and diluted with 5.0 mL of distilled water and ferric chloride (1.0 mL, 0.1%), and absorbance read at 700 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). All samples were analyzed thrice and the results averaged.
DPPH^. scavenging assay: 1, 1^’–diphenyl–2-picrylhydrazyl (DPPH) free radical scavenging activity of the extracts was assessed using the procedure reported earlier [28 (link)]. Briefly, to extract (1.0 mL) containing 25 μg/mL of dry matter in methanol, freshly prepared solution of DPPH (0.025 g/L, 5.0 mL) was added. Absorbance at 0, 0.5, 1, 2, 5 and 10 min was measured at 515 nm using a spectrophotometer. The scavenging amounts of DPPH radical (DPPH^.) was calculated from a calibration curve. Absorbance read at the 5th min was used for comparison of radical scavenging activity of the extracts.
Determination of antioxidant activity in linoleic acid system: The antioxidant activity of the tested plant extracts was also determined by measuring the oxidation of linoleic acid [28 (link)]. Five mg of each plant extract were added separately to a solution of linoleic acid (0.13 mL), 99.8% ethanol (10 mL) and 0.2 M sodium phosphate buffer (pH 7, 10 mL). The mixture was made up to 25 mL with distilled water and incubated at 40 ^oC up to 360 h. Extent of oxidation was measured by peroxide value applying thiocyanate method as described by Yen et al. [27 ]. Briefly, ethanol (75% v/v, 10 mL ), aqueous solution of ammonium thiocyanate (30% w/v, 0.2 mL), sample solution (0.2 mL) and ferrous chloride (FeCl₂) solution (20 mM in 3.5% HCl; v/v, 0.2 mL) were added sequentially. After 3 min of stirring, the absorption was measured at 500 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). A control contained all reagents with exception of extracts. Synthetic antioxidants butylated hydroxytoluene (BHT) was used as a positive control. Percent inhibition of linoleic acid oxidation was calculated with the following equation: 100 – [(increase in absorbance of sample at 360 h / increase in absorbance of control at 360 h) × 100], to express antioxidant activity.

The NADH–ferricyanide reductase activity was measured with minor modification, as recently published [31 (link)]. The medium of the cells was exchanged against 1 mL buffer (50 mM Tris-HCl pH 8.0, 500 μM β-NADH) providing 500 μM potassium ferricyanide (III) (K₃Fe(CN)₆) (Riedel-de-Haen—Honeywell Specialty Chemicals Seelze GmbH, Seelze, Germany) per 35 mm dish and the cells were incubated for 60 min at 37 °C and 5% CO₂. Ferricyanide was reduced to ferrocyanide. Subsequently, an aliquot of the buffer was drawn to measure the absorbance at 420 nm with a photometer (UV1202, Shimadzu, Kyoto, Japan). The NADH–ferricyanide reductase activity was expressed as reduced ferricyanide in nmol/min/4 × 10⁵ cells. Per the experiment, each condition was measured in duplicates.

The ferricyanide-reducing antioxidant activity of the methanol and aqueous extracts was determined using [29 (link)], method with minor modifications. Potential antioxidant interacts with a colourless Fe³⁺ complex to reduce it into intense blue Fe²⁺, which is the basis of the ferric-reducing antioxidant power assay (FRAP) [30 (link)].
Exactly 1 ml of the test solutions or L-Ascorbic acid was combined with 2.5 ml of 0.03 M potassium ferricyanide and 2.5 ml of phosphate buffer (0.2 M, pH 6.6). The concentrations of the extract solution or L-Ascorbic acid ranged from 0.32 μg/ml to 1000 μg/ml. The mixtures were then incubated in a water bath at 50 ℃ for 20 min. After cooling 2.5 ml of 0.6M Trichloroacetic Acid (TCA) was added to each of the test solutions. To an aliquot of 2 ml of each solution, 0.5 ml of FeCl₃ solution (0.01%) and 2 ml of distilled water were added. The absorbance of the test solutions was measured at 700 nm by a double-beam Uv Vis spectrophotometer [29 (link)].