Cary 100 uv vis

Total soluble carbohydrates were determined on five leaves for each treatment by the anthrone method, as reported in Hedge and Hofreiter [104 ]. The absorbance was measured at 630 nm using a spectrophotometer (UV-VIS Cary 100, Agilent Technologies, Palo Alto, CA, USA). The amount of soluble carbohydrates in the extracts was expressed as mg Glucose equivalents g⁻¹ FW (mg Glu eq g⁻¹ FW) using a Glucose standard curve.
Protein extraction was carried out on five fresh leaf samples ground in liquid nitrogen, according to Wang et al. [105 (link)]. Total protein content was quantified by Bradford colorimetric assay [106 (link)], measuring the sample absorbance at 595 nm by a spectrophotometer (UV-VIS Cary 100, Agilent Technologies, Palo Alto, CA, USA). Using a BSA standard curve, the protein concentration was expressed as µg BSA (bovine serum albumin) equivalents g⁻¹ FW.

Total polyphenols were evaluated as described by Sun et al. (1998) [29 (link)]. An aliquot of methanolic extract was mixed with 1:1 (v/v) 10% Foli–-Ciocálteu and 1:5 (v/v) 700 mM Na₂CO₃ and the solution was stored in the dark for 2 h. The absorbance was detected by a spectrophotometer (UV-VIS Cary 100, Agilent Technologies, Palo Alto, CA, USA) at 765 nm. The total polyphenol content was calculated by means of a gallic acid standard curve and expressed as mg Gallic Acid Equivalents (GAE) g⁻¹ FW.
Flavonoids were quantified accordig to Moulehi et al. (2017) and Dewanto et al. (2002) [30 (link),31 (link)]. Briefly, an aliquot of supernatant was added to 75 μL of 5% NaNO₂ (sodium nitrite). After, 150 μL of 10% AlCl₃ (aluminum chloride) and 500 μL NaOH (1 M), distilled water was added to the mixture up to a final volume of 1.525 mL. The absorbance was quantified spectrophotometrically (spectrophotometer UV-VIS Cary 100, Agilent Technologies, Palo Alto, CA, USA) at 510 nm. Flavonoid concentration was estimated using a catechin standard curve as mg Catechin Equivalents per gram of fresh weight (mg CE g⁻¹ FW). Statistical analysis was performed on 8 technical replicates by t-test, using GraphPad Prism8 software Prism software, version 8.0 (GraphPad, San Diego, CA, USA).

Total polyphenol content was measured according to the reported procedure [31 (link)]. Briefly, 0.25 g of powered sample was extracted with aqueous 80% methanol, at 4 °C (for 30 min) and then centrifuged at 11,000 rpm for 5 min. Extracts were combined with 1:1 (v/v) 10% Folin–Ciocalteu phenol reagent and water. After 3 min, 700 mM Na₂CO₃ solution was added to the resulting mixture in 5:1 (v/v). Samples were incubated for 2 h in darkness. Then, the absorbance at 765 nm was measured with a spectrophotometer (UV-VIS Cary 100, Agilent Technologies, Palo Alto, CA, USA). Gallic acid was used as a standard. Calibration curve was constructed analysing standard solutions in the interval of concentration 5–500 ppm. The total polyphenols concentration was calculated and expressed as gallic acid equivalents (mg GAE g⁻¹ FW) from the calibration curve (R² = 0.996) using gallic acid.