## 2 Materials and Methods
### Methods
#### 2.3.9 Confocal Laser Scanning Microscopy (CLSM) Analysis
Estimation of Total Flavonoid Content (TFC), Total Polyphenol Content (TPC), and Antioxidant Capacity (DPPH)  
The method used to determine the TFC in RG is based on the acid reaction of AlCl\({}_{3}\) with the ketone group at position 4 of the heterocyclic ring C, or the hydroxylic groups at positions C\({}_{3}\) of the heterocyclic ring C or C\({}_{5}\) of the aromatic ring A of the rutin (the rutin structure is rendered in the additional material (Figure S1)), resulting in colored complexes that can be determined by colorimetry [61]. The mean TFC value of the RG plant was 22.28 mg RE/100 g DW, which is inconsistent with results obtained by other researchers: 26.87 (mg RE/g DW) for a methanolic extract and 27.72 (mg RE/g DW) for an ethanolic extract of RG, both from crops. Lower values were obtained for methanolic extract (4.18 mg RE/g DW) and ethanolic extract (7.10 mg RE/g DW) of the RG plant [10]. Similar results were obtained from a methanolic extract of the mixture from the stems, leaves, and fruits of RG (6.66 mg RE/g DW) [7]. In general, the number of flavonoids present in the plant is influenced by the maturity of the plant at harvest, growing conditions, soil type, and post-harvest treatments. From the analysis of the data obtained, it can be concluded that the RG extract had an appreciable content of TPC (37.98 mg GAE/g DW). This result is consistent with determinations made by other researchers, who studied the ethanolic extract obtained by RG ultrasonication, and the values found in that study are close to ours (39.90 mg GAE/g DW) [61]. The differences in the TPC of different types of plant extracts and fractions could be explained by the fact that the presence of polyphenols in RG may be affected by the plant species, its maturity at harvest, growing conditions, soil conditions, and post-harvest treatments [62]. The flavonoids and polyphenolic compounds present in the RG extract are compounds that, according to studies, have antioxidant capacity and can neutralize free radicals. The evaluation of the antioxidant capacity, in vitro, of the RG extract was carried out using the DPPH method, a spectrophotometric method that is very often used to test the capacity of some existing compounds in plant products to neutralize free radicals by donating protons [63]. Equation (2) was used to determine the IC\({}_{50}\) inhibition percentage of the extracts in relation to the DPPH reagent. The elimination capacity of 50% of the free radicals by performing the calculations corresponding to Equation (1) was IC\({}_{50}\) = 0.098 mg/mL, the extract concentration was 98.82 mg/mL, and the mean percentage inhibition of DPPH was 54.25%. The capture capacity of radicals is directly related to the donation capacity of the hydrogen atom of a compound and is not only correlated with the redox potentials [64]. These results are also due to the presence of a high content of polyphenolic compounds; there is a correlation between antioxidant activity and phenolic content [65; 66]. The observed antioxidant potential of the extract may be related to the presence of different functional groups, such as hydroxyl and carbonyl groups [67; 68]. Close results were obtained in other works, with 32.09% for an ethanolic extract obtained by ultrasonication at room temperature, the extract concentration of which was 0.05 mg/mL, and between 57.54% and 72.53% for an extraction performed with choline chloride and citric acid with stirring, at different time intervals and temperatures [69]. Other results, (73.50% and 72.52%), were obtained for extraction by the ultrasonication method, and the solvent used was acrylonitrile [70].