Gallic acid

A methanolic extract of plant tissue (0.5 g) was used to quantify the phenolic content by Folin-Ciocalteu reagent at 755 nm, according to Kim et al.^{57 (link)}. The results were expressed as equivalents of gallic acid (Scharlau, Barcelona, Spain) per g of plant dry weight (mg of GAE/g dry weight).
Total flavonoids were assayed according to the aluminum chloride colorimetric method^{57 (link)}, and the absorbance was recorded at 510 nm. The content of total flavonoids is expressed as rutin equivalents (mg rutin/g dry tissue).

Ascorbic acid, Folin–Ciocalteu’s reagent, FeCl₃, FeSO₄, HCl, NaNO₂, NaOH, fructose, glucose, and sucrose were provided from Merck (Darmstadt, Germany). 2,4,6-Tris(2-pyridyl)-1,3,5-triazine (TPTZ) and 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), were purchased from Sigma (Darmstadt, Germany). AlCl₃ (Fluka, Seelze, Germany). In addition, 2,2-diphenyl-1-picrylhydrazyl (DPPH) (CalBiochem, Darmstadt, Germany), Gallic acid (Scharlau, Barcelona, Spain), Na₂CO₃ (AppliChem, Darmstadt, Germany), potassium persulfate (Bernd Kraft, Duisburg, Germany), and Trolox (Cayman, Ann Arbor, MI, USA) were used. Methanol and ethanol were purchased from Chemsolute (Hamburg, Germany) and were HPLC grade.

Ascorbic acid, Folin–Ciocalteu’s reagent, FeCl₃, FeSO₄, NaOH, HCl, and NaNO₂, were purchased from Merck (Darmstadt, Germany). 2,4,6-Tris(2-pyridyl)-1,3,5-triazine (TPTZ) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), were provided from Sigma (Darmstadt, Germany). Gallic acid (Scharlau, Barcelona, Spain), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (CalBiochem, Darmstadt, Germany), AlCl₃ (Fluka, Seelze, Germany), Na₂CO₃ (AppliChem, Darmstadt, Germany), potassium persulfate (Bernd Kraft, Duisburg, Germany), Trolox (Cayman, Ann Arbor, MI, USA), were used. Caffeic acid, myricetin, p-coumaric acid, and quercetin (HPLC grade) were purchased from Sigma (Darmstadt, Germany). Ferrulic acid, Gallic acid, kaempherol, and rutin (HPLC grade) were supplied from Carl Roth GmbH (Karlsruhe, Germany). Methanol and ethanol from Chemsolute (Hamburg, Germany), acetic acid (AppliChem, Darmstadt, Germany), and acetonitrile (J.T.Baker, Hamburg, Germany) purchased were HPLC grade.

To assess the total phenolic content, the extract solution was diluted 100 times. Then, 40 μL of the test solution was taken and transferred into a microcentrifuge tube, and 400 μL of distilled water was added to it. Next, 20 μL of Folin–Ciocâlteu reagent (PanReac AppliChem, Hesse, Germany) was added to the test tube, and the tube was protected from light before adding 500 μL of 20% Na₂CO₃ (99%, Katayama Chemical Co., Ltd.) solution. After vortexing, the solutions were incubated in the dark for 20 min. The absorbance of the solution was measured at 750 nm. A standard curve was developed using various concentrations of gallic acid (99%, Scharlab, Barcelona, Spain) (0, 6.25, 12.5, 25, 50, and 100 μg/g) [31 (link)].
For analysis of flavonoid content, 0.1 mL of the diluted sample of the extraction solution was poured into a microcentrifuge tube, and then 0.3 mL of 99% ethanol (99%, Merck & Co., Inc., Darmstadt, Germany), 0.02 mL of 10% AlCl₃ (97%, Katayama Chemical Co., Ltd.), 0.02 mL of 1 M CH₃COOK (99%, PanReac AppliChem, Chicago, IL, USA), and 0.56 mL of deionized water were sequentially added. Thereafter, the solution was left to react at room temperature for 40 min. Absorbance was then detected at 415 nm. A standard curve was developed with quercetin, and the results were expressed as mg QE/g dw. The analyses were carried out in triplicate [6 (link)].