Spekol 1300

DPPH (2,2‐Diphenyl‐1‐Picrylhydrazyl) and the standard substance ascorbic acid were used to evaluate the antioxidant properties of the extracts. First, 2 mg of the extracts was dissolved in 1 ml of methanol and then different concentrations (20, 40, 60, 80, 100 μg/ml) were prepared. 550 μl of DPPH solution was added to 50 μl of each of the prepared solutions and placed in the dark for 30 min. The absorption of all solutions and control samples was then measured using a spectrophotometer (Analytik Jena, Spekol 1,300, Germany) at 517 nm (Hazrati et al., 2019). Radical scavenging activity was evaluated based on the following equation: $\text{Percentage of radical scavenging activity}=\left(\text{Abs control}‐\text{Abs sample}\right)/\text{Abs control}\times 100$

The PP (Pisum sativum) is an ancient, economical, small sphere-shaped native seed or pod of fruit belonging to the Leguminosae family, mostly common in the northern part of Africa and the western part of Asia as well as other parts of the world. Like every other leguminous plant, PP encompasses symbiotic bacteria known as rhizobia inside the root lumps of their root arrangements. Rhizobia have the distinct capability of fixing nitrogen from molecular nitrogen into ammonia. It comprises a high proportion of consumable proteins, vitamin A and vitamin C, and it is also rich in some essential minerals like calcium and phosphorus (Singh et al. 2020 ). However, the PP used for this study was acquired from an indigenous market and was utilized for the production of the activated TETA-PP. Sulphuric acid (H₂SO₄, MW = 98.07 g, 99%) and potassium dichromate (K₂Cr₂O₇, MW = 294.19 g, 99 %) and TETA were obtained from Sigma-Aldrich. The standard stock solution of Cr⁶⁺ ions was prepared from K₂Cr₂O₇. 1,5-Diphenylcarbazide was obtained from BDHZ Chemicals LTD, Poole, UK, as a composite reagent for Cr⁶⁺ ion analysis. The “Analytik Jena SPEKOL 1300 UV/visible digital spectrophotometer” instrument with matching glass cells of a 1.00-cm optical path, a shaker (“JSOS-500”) and a pH meter (“JENCO 6173”) were used for the experiments and analysis.

Gallic acid was used as the standard for measuring the content of total phenolic content compounds. A mixture of 100 μl of the extract (2 mg/ml) was mixed with 500 μl of Folin–Ciocalteu (10% by volume/volume). After 5 min, 500 ml of 7% sodium carbonate was added to the mixture and the absorption of the samples (after 2 hr in the dark) was measured at 765 nm by spectrophotometer (Analytik Jena, Spekol 1,300, Germany) (Haghighi et al., 2012).

Samples of dried plant material (5 mg DW) were ground to fine powder and hydrolyzed in sulfuric acid according to [87 (link)]. The content of total carbohydrates in the hydrolysates was determined spectrophotometrically using anthrone reagent [88 (link)]. Briefly, 3 mL of anthrone reagent was added to 0.3 mL of the sample (diluted as necessary). The mixture was boiled for 7 min and then immediately cooled in the ice bath. The extinction was measured at 620 nm using SPEKOL 1300 spectrophotometer (Analytik Jena AG, Jena, Germany), and the amount of total carbohydrate was calculated based on a calibration curve with glucose as standard.

Lichtenthaler's (1987) method was applied to measure chlorophyll and carotenoids content. To extract these pigments, 0.2 g of leaf samples was immersed in test tubes containing 80% acetone. The samples were filtered through filter paper and finally their absorption was read at 646.8, 663.2 and 470 nm by spectrophotometer (Analytik Jena, Spekol 1,300, Germany).

For chlorophyll analysis, fragments of algal thalli (10 mg FW) were ground using a mortar and pestle in 100% acetone with small quantities of Na₂SO₄ and NaHCO₃. Several rounds of extraction were completed with additional acetone until the extract was colorless. The concentration of acetone was adjusted to 90% (in distilled water), and the content of chlorophyll a was calculated [91 (link)] from data obtained with a SPEKOL 1300 spectrophotometer (Analytik Jena, Jena, Germany).
For determination of phycoerythrin content, fragments of algal thalli (50 mg FW) were homogenized in potassium phosphate buffer (0.1 M, pH 6.8) using a TissueRuptor II homogenizer (QIAGEN, Germany) and left soaking at 4 °C overnight. Then the extracts were centrifuged (5000× g, 10 min), and the content of phycoerythrin was determined spectrophotometrically (SPEKOL 1300, Analytik Jena) [92 (link)].

Quercetin was used as the standard for measuring the content of total flavonoid compounds. A mixture of 100 μl of the extract (2 mg/ml) was mixed with 50 μl of aluminum chloride (2% by volume/volume) and 100 μl of ammonium acetate (1 mol). After 10 min, the absorption of the samples was measured at 426 nm by spectrophotometer (Analytik Jena, Spekol 1,300, Germany) (Haghighi et al., 2012).