Ammonium thiocyanate

A negative pressure instrument (Electronic Diversities, Finksburg, MD, USA) constructed to produce standard suction blisters upon application of negative pressure, was used on healthy skin (ex vivo: abdominal skin; in vivo: lower forearm). Subcutaneous fat was partially removed from ex vivo skin using a scissor. Subsequently, skin (10 × 10 cm²) was placed (not fixed, not kept in medium) on a styrofoam lid that was covered with aluminium foil to provide (at least partial) backpressure. Suction chambers with 5 openings (Ø = 5 mm) on the orifice plate were attached to skin, topped with a styrofoam lid and pressed with 1 kg weight in order to avoid movement of the plate. A pressure of 200–250 millimeter (mm) mercury (Hg) (ex vivo) or 150–200 mm Hg (in vivo) caused the skin to be drawn through the openings creating typical suction blisters of different size within 6–8 h (ex vivo) and 1–2 h (in vivo). Suction blister fluid (~110 µl/5 blisters) was collected using a syringe with a needle. Cells within the fluid were counted and placed on adhesion slides for staining and analysis. Blister roof epidermis was cut with a scissor, fixed with ice-cold acetone (10 minutes) and used for staining. For comparison and control, epidermal sheets were prepared from unwounded skin biopsy punches (Ø = 6 mm; 3.8% ammonium thiocyanate (Carl Roth GmbH + Co. KG, Germany) in PBS (Gibco, Thermo Fisher, Waltham, MA, USA), 1 h, 37 °C). Removal of the blister roof created a wound area. Biopsies (Ø = 6 mm) from wounded and unwounded areas were cultivated for 12 days in either duplicates or triplicates in 12 well culture plates and Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco) and were cultured at the air-liquid interphase. Medium was changed every second day.

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 lipid peroxidation inhibition assay was conducted following the method described by Haenen and Bast [21 (link)]. Thiobarbituric acid (TBA)-reactive species were responsible for the lipid peroxidation (LPO) activity. To induce LPO, 0.005 mL of FeSO₄ (0.07 M) and approximately 1 mL of distilled water were added to the mixture, which was then incubated for 30 min. Afterward, the mixture was heated to 95 °C for 1 h. Subsequently, 1.5 mL of 0.8% (w/v) TBA, 1.5 mL of 20% acetic acid, 0.5 mL of 20% trichloroacetic acid (TCA), and 1.1% SDS were added. Another set of samples was treated similarly but without TBA. After cooling, each tube was filled with 5.0 mL of butanol and centrifuged for 10 min at 5000 rpm. The percentage of lipid peroxidation inhibition in the samples was calculated using the following formula: