## 2 Materials and Methods
### Antioxidant Activity and Total Phenolic Content  
Extracts were prepared by grinding 0.5 g of lyophilized tomato material and diluting them with 5 mL of 80% methanol, and the extract was incubated for 24 h at four degrees Celsius in a refrigerator, then centrifuged, and the resulting extract was used for further analysis. Each of the three biological replicates consisted of at least five conjugated fruits  
Figure 1: Tomato maturity stages: 80–100% typical size—BBCH 70–79 (**a**); 40–50% ripeness—BBCH 84–85 (**b**); and 100% ripeness—BBCH 89 (**c**).  
and was repeated in three analytical replicates. The antioxidant properties of tomato fruits were evaluated as follows:  
The ABTS (2,2\({}^{\prime}\)-azino-bis (3-ethylbenzothazoline-6-sulphonic acid) radical cation was obtained by incubating the 7 mM ABTS stock solution with 2.45 mM potassium persulfate (K\({}_{2}\)S\({}_{2}\)O\({}_{8}\); final concentration) and allowing the mixture to stand in the dark at room temperature for 12-16 h before use [41]. After that, 20 \(\upmu\)L of the prepared sample was mixed with 290 \(\upmu\)L of ABTS solution (ABTS stock solution was diluted 1:7) and the absorbance was measured after 11 min (plateau phase) at 734 nm (Spectrostar Nano, BMG Labtech microplate reader, Ortenberg, Germany). The ABTS scavenging activity of tomato fruit extracts was calculated as the difference between the initial absorbance and after reacting for 10 min.  
For the DPPH (2-diphenyl-1-picrylhydrazyl) assay, a stable 126.8 \(\upmu\)M DPPH (100% purity; Sigma-Aldrich, Burlington, MA, USA) solution was prepared in methanol [42]. Subsequently, 290 \(\upmu\)L of the DPPH solution was transferred to a test tube and mixed with 20 \(\upmu\)L of the tomato fruit extract. The absorbance was scanned at 515 nm (Spectrostar Nano, BMG Labtech microplate reader, Germany) while reacting for 16 min. The free radical scavenging capacity in tomato fruits was expressed as \(\upmu\)mol of DPPH radicals scavenged per 1 g of fresh weight (\(\upmu\)mol g\({}^{-1}\) FW).  
The Fe\({}^{2+}\)-reducing antioxidant power (FRAP) method is based on reducing ferric ions (Fe\({}^{3+}\)) to ferrous ions (Fe\({}^{2+}\)). The fresh working solution was prepared by mixing 300 mM, pH 3.6 acetate buffer, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl, and 20 mM FeCl\({}_{3}\)\(\times\) 6H\({}_{2}\)O at 10:1:1 (\(v/v\)/\(v\)) [43]. 20 \(\upmu\)L of the sample was mixed with 290 \(\upmu\)L of working solution and incubated in the dark for 30 min. Readings of the colored product (ferrous tripyridyl-triazine complex) were then taken at 593 nm with Spectrostar Nano BMG Labtech microplate reader (Germany). A calibration curve was determined using Fe\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) (iron (III) sulfate; 97% purity; Sigma-Aldrich, Burlington, MA, USA) as an external standard with a range of concentrations from 0.005 to 0.5 mM (R\({}^{2}\) = 0.99). The antioxidant power of tomato fruit was expressed as Fe\({}^{2+}\) antioxidant capacity (Fe\({}^{2+}\)\(\upmu\)mol g\({}^{-1}\) FW).  
Total phenolic compound content (TPC) was determined as gallic acid equivalent. A 20 \(\upmu\)L aliquot of the sample extract was mixed with 20 \(\upmu\)L of 10% (\(w/v\)) Folin-Ciocalteu reagent and 160 \(\upmu\)L of 1 M Na\({}_{2}\)CO\({}_{3}\) solution [44]. After incubation for 20 min in the dark, the absorbance was measured at 765 nm (Spectrostar Nano, BMG Labtech microplate reader, Germany). The total content of phenolic compounds (mg g\({}^{-1}\)) was calculated from the calibration curve of gallic acid (0.01-0.1 mg mL\({}^{-1}\), R\({}^{2}\) = 0.99).