Folin-Ciocalteau reagent

Protein extraction from the legumes was carried by a subsequent process. Primarily, the samples were cleaned followed by grinding and sieving. After, a known amount of the sieved sample was mixed with Milli-Q water (5 mL), and the pH of the sample solution was regulated to be pH 9 using sodium hydroxide (NaOH, 0.1 N) at 25 °C. Then, the solution was shaken for 50 min at room temperature followed by centrifugation (HERMLE, model Z32 HK, Wehingen, Germany) at 10,000 rpm for 10 min. The sample extraction and centrifugation methods were repeated twice for the residue to achieve better yields. In order to precipitate the protein, sample extracts were collected together and the pH value (4.5) was adjusted using hydrochloric acid HCl (1 N). Proteins was obtained by eradication of the sample supernatant by means of decantation. The achieved protein mass was cleaned two times with Milli-Q water and further centrifuged (10,000 rpm, 15 min). The obtained protein was then freeze-dried using a freeze-dryer model BenchTop Pro with Omnitronics (SP Scientific, New York, NY, USA), and used for further analysis [46 ,47 (link)].
Lowry’s reagent A: (2% Na₂CO₃ in 0.1 N NaOH) was prepared by the addition of NaOH (2 g) and Na₂CO₃ (10 g) in distilled water (5 mL) followed by dilution to 500 mL with distilled water. Lowry’s reagent B₁ was prepared with the addition of CuSO₄ (1 g) and distilled water (5 mL), diluted to 100 mL with distilled water. Lowry’s reagent B₂ was prepared with the addition of sodium potassium tartrate (2 mL) and distilled water (5 mL), diluted to 100 mL with distilled water. Lowry’s reagent C was freshly prepared with the addition of Lowry’s reagent B₁ (2 mL) and Lowry’s reagent B₂ (2 mL) while stirring solution was added to Lowry’s reagent A (200 mL). Then, 2 g of the extracted protein sample were added to reagent C followed by incubation for 45 min with continuous stirring in a dark room at room temperature. Then, reagent E (1 mL) was added to the sample, and incubated again for 45 min. Finally, the amounts of protein in the samples were determined using a spectrophotometer.
This method was performed by measuring the absorbance of all standards and samples using a spectrophotometer. The phenolic group of tyrosine and tryptophan residues (amino acid) in a protein produced a blue purple color complex, which had a maximum absorption in the region of the 660 nm wavelength with Folin–Ciocalteau reagent, which consists of sodium tungstate molybdate and phosphate. Thus, the intensity of the color depends on the amount of these aromatic amino acids present and will thus vary for different proteins. The reaction is dependent on pH and a working range of pH 9 to 10.5 is essential.

Total phenolic content (TPC) were estimated using the Folin–Ciocalteau’s (FC) method with modifications [15 (link)]. Briefly, an aliquot (5 mL) of the gallic acid solution was transferred to a glass tube; reactive 10⁻¹ diluted FC reagent (20 mL) is added after 5 min; sodium carbonate (Na₂CO₃, 5 mL, 7.5% w/v) was added and the mixture shaken. After 30 min of incubation at ambient temperature in the dark, 200 µL samples were placed in 96-well plates. Finally, the absorbances were measured in a spectrophotometer (Infinite Pro M200 series, Tecan^TM, Männedorf, Switzerland) at 765 nm and compared to a gallic acid calibration curve for TPC (prepared using 0 to 1 mg/mL concentration gallic acid solution). Results were expressed as mg gallic acid equivalent (GAE)/100 mL. All measurements were done in duplicate.
Gallic acid content (GAC) was determined using HPLC (Agilent 1100 system, Agilent Technologies, Santa Clara, CA, USA) equipped with a Zorbax SB-C18 column according to the methodology presented in [16 (link)]. This was carried out to measure the changes in gallic acid concentration due to photodegradation. Results were expressed as mg GAC/100 mL solution.
To determine the antioxidant activity (AA) of gallic acid solutions, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was used. A standard curve was constructed using Trolox^TM (20 µM) solution. For sample wells, gallic acid (20 µL) was added. In both standard and sample wells of a 96-well microtiter plate, 1 mM DPPH (20 µL) was added. The blank well consisted of HPLC grade methanol (200 µL). The plate was incubated for 10 min at room temperature in the dark. Then the plate absorbances were read at 519 nm by a microtiter plate reader (Tecan^TM Infinite M200 Pro). All reagents were dissolved in HPLC grade methanol. Antioxidant capacity reported in mM Trolox^TM equivalents (TE) per mL of solution.

The TPC was quantified by the Folin-Ciocalteau method (17 (link)) with some modifications. Briefly, 200 μl of properly diluted samples or standard were mixed with 100 μl of Folin-Ciocalteau reagent. After 5 min, 300 μl of Na₂CO₃ (7.5%, w/v) and 1.0 ml of water were added. After 30 min, the absorbance at 765 nm was measured using a microplate reader (Biotek, Vermont, USA). The TPC was calculated based on the calibration curve plotted using gallic acid (0–200 μg/ml) and the results were expressed as μg of gallic acid equivalents per gram of extract [(μg GAE)/g E].

Briefly, the total phenolic contents of each ethanolic extract or serial concentrations of GA (standard) were mixed with Folin–Ciocalteau reagent and sodium carbonate. Phenolic concentration (mg/100 g plant tissue) was estimated using the standard curve of GA^{30 (link)}.
The total flavonoid contents of each ethanolic extract or serial concentrations of RU (standard) were mixed with sodium nitrite and aluminum chloride solution. Flavonoid concentration (mg/100 g plant tissue) was estimated using the standard curve of RU^{31 (link)}.

Folin–Ciocalteau reagent, gallic acid (GA), rutin (RU), sulfanilamide, naphthylethylenediamine dihydrochloride, Diphenyl–α-picrylhydrazyl (DPPH), nitroblue tetrazolium (NBT), riboflavin, and vitamin C were supplied from Riedel-de Haën, Germany. DMEM medium and fetal bovine serum (FBS) were obtained from Lonza (USA). Other chemicals were obtained with a high grade from Sigma-Aldrich (St. Louis, MO, USA). Primers for P53, BCL2, Cyclin D, MMP9 and VEGF were purchased from Bioneer, Korea as shown in Table 1 in supplementary materials.