Extraction technique and vegetal matrix

The ethanolic extract for subsequent analyses was obtained by the Randall method with modifications using a SER 148/3 VELP Scientifica device (VELP Scientifica Srl, Usmate Velate MB, Italy). Ethanol was selected as the solvent because of its high polar capacity and broad spectrum of solubilization of phytochemicals. For the extraction process, 1 kg of fresh or dry irradiated material was taken depending on the case and extraction temperature (Te) of 95 °C, together with an immersion time (I) of 45 min. Afterwards, the extraction thimble was removed from the solvent and left for a wash time (W) of 25 min. Finally, the extraction process ended with a solvent recovery phase of (R) 15 min, according to the methodology reported by ^{Pérez et al. (2013)}.

Since a drying process can influence the concentration of phytochemicals in a material that is extracted, this effect was studied with extractions in both the fresh matrix and the material subjected to the drying process, using 60±3°C as the drying temperature for 24 h or until constant weight of the material, according to ^{Sárosi et al. (2013)}. The drying process was carried out in a Memmert U30 forced convection oven (Memmert GmbH + Co. KG, Germany).

Quantity of total phenols

The Folin-Ciocalteu method was used according to the methodology reported by ^{Roby et al. (2013)}. A sample of the alcoholic extract was taken and placed in a test tube. Then, 10 mL of saturated sodium carbonate were added with 5 mL of the Folin-Ciocalteu reagent. The tubes were shaken for 15 s and placed in a dark place for 30 min. Next, the absorbance of each sample was determined at a wavelength of 760 nm. Gallic acid in solution was used as a standard at different concentrations. The content of total phenols was reported as percentage of gallic acid, using the equation proposed by ^{Roby et al. (2013)} (1)

where, %AGeM corresponds to the percentage of gallic acid in the sample, EAG is the gram equivalent of gallic acid and Ce is the weight of the extract in mg.

Quantity of total flavonoids

The total flavonoid content was measured using the methodology from ^{Ghasemi et al. (2014)}. In a 10 mL graduated balloon, 2.5 mg (± 0.1 mg) of dry extract were placed together with 2 mL of 80% ethanol, 200 μL of 10% aluminum nitrate solution, 200 μL of sodium acetate solution and potassium 1 M. Finally, the volume was completed with 80% ethanol. The volumetric balloons were left for 40 min, and the absorbance of the samples was determined at a wavelength of 415 nm. The concentration of flavonoids is usually reported in mg of quercetin, therefore, a calibration curve of solutions with this pattern was constructed using the following equation (2)

where, mgQRT/mL corresponds to mg of quercetin per ml of extract and A415 is the absorbance of the sample measured at 415 nm.

Quantity of total anthocyanins

The content of total anthocyanins in the samples was determined with the pH-differential spectroscopy method following the methodology reported by ^{Medina et al. (2016)}. 0.5 g of sample were taken, and 1.0 mL of water was added, which was agitated for 15 min. Next, the solution was filtered, and two 0.2 mL samples were taken. The first aliquot was diluted with a buffer solution with pH 1.0, and its absorbance was measured at 530 nm. The other aliquot was diluted in a buffer solution with pH 4.5, and its absorbance was taken at 700 nm. For the calculations, the molar extinction coefficient of the cyanidin-3-glucoside estimated at 26,900 was taken as reference, and the results were expressed in equivalent mg of glucoside per 100 g of sample, using the following expression (3)

where, Am corresponds to monomeric anthocyanins, ΔA is the change in absorbance at the two wavelengths and at the two pH levels, and FD is the respective dilution factor. The number 26900 is the molar extinction coefficient for cyanidin-3-glucoside at 530 nm, and 100 is the conversion factor of mg in 100 g of sample.

All the determinations of chemical parameters (quantity of phenols, flavonoids and anthocyanins) were carried out in triplicate, as well as the measurements in the control samples and the absorbances for all cases. Measurements were taken using a UV-VIS Genesys 5 spectrophotometer (Klab innovative solutions, Seoul, Korea).

Content of total phenols

In all cases, the use of UV-B radiation as a post-harvest treatment caused an increase in the total content of phenols with respect to the control samples (Tab. 1). The highest value of total phenols was observed in the plant material from cut plants (75 DAT) subjected to radiation dose 1 (0.05 Wh m^-2), with an adaptation time of 48 h and plant fresh extraction. It had an increase close to 37.84% as compared to the control plants. In contrast, the lowest change with respect to the control value (3.27%) was obtained in material from the plants in phenological state 92 DAT and subjected to radiation dose 2 (0.075 Wh m^-2), left in adaptation time for 24 h and extracted from the dried material. The best treatments were those with the highest ΔFT: percentage change of treatment as compared to the control.

At the statistical level, there were no significant differences; however, the drying process had a direct negative effect on the amount of phenols present in the alcohol extracts subsequently obtained. This may have been due to the thermolabile characteristic of the phenolic compounds.

The results were similar to those reported in the literature for various genres. The increase in the concentration of this class of compounds, especially thymol and carvacrol, have been reported by ^{Inostroza-Blancheteau et al.(2014)}.

Total flavonoids

In the case of total flavonoid content, the material from the plants with 92 DAT, subjected to radiation dose 2, left for 48 h at adaptation time and extracted from the fresh material, showed an increase of 80% with respect to the control plant and was the best among all combinations of treatments. In contrast, results with a lower increase were obtained in plants at the cut-off point (75 DAT), with a low radiation dose (0.05 Wh m^-2) and at both adaptation times (24 and 48 h), with increases of 10.96 and 14.08%, respectively (Tab. 2). The best treatments are those with the highest ΔFLT: percentage change of treatment as compared to the control.

The impact on the production of flavonoids in plant tissues subjected to ultraviolet radiation has been studied through the effect that this kind of radiation has on various metabolic pathways of plants. Ultraviolet radiation has been found to stimulate specific enzymes such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS) and chalcone isomerase (CHI). The stimulation of the previous enzymes has a direct effect on the production of secondary metabolites, especially flavonoids. ^{Jenkins (2014)} reported that the effect has been observed to be greater if the plant material is subjected to moderate doses of UV radiation, especially in the B range.

Content of total anthocyanins

The number of total anthocyanins quantified in the study was stimulated with the application of the treatments. The material from plants in phenological stage of 92 DAT, submitted to the highest radiation dose used in the study (0.075 Wh m^-2) and left in both adaptation times (48 and 24 h), showed a greater increase in the amount of anthocyanins. This plant material had increases of 77.39% and 61.33% with respect to the value obtained with the control plant material, as shown in table 3. The best treatments had the highest ΔA: percentage change of treatment as compared to the control.

These results can be explained by the metabolic pathways elucidated for plants(^{Tholl, 2015})and confirm that the synthetic routes of acetate-malonate and shikimic acid are stimulated with the use of UV-B radiation as a post-harvest treatment. This occurs because routes are responsible for the specific production of phenols. As shown in table 3, the concentration of anthocyanins was higher in the extracts obtained from the fresh irradiated material because of the low thermal stability exhibited by anthocyanins, which are degraded in the drying process.

In a study by ^{Cechin et al. (2012)}, passion fruit plants were subjected to high doses of UV-B radiation (about 8 W m^-2), and the amount of anthocyanins present was measured. Their results showed an increase with respect to their control values and coincided with the results obtained with Thymusvulgaris L. This demonstrates that the generation of this class of compound constitutes a natural defense method against a change in radiation perceived by the plant material.

In a recent study conducted by ^{Huyskens-Keil et al. (2020)}, post-harvest white asparagus with different sources of light and UV-C radiation showed that the application of UV-C radiation stimulates the enzymatic action of phenylalanine ammonia-lyase [PAL, EC 4.3.1.5] and guaiacol peroxidase [POD, EC 1.11.1.7]. This is reflected in a decrease in the concentration of anthocyanins in plant tissues. The authors stated that the use of different sources of light radiation affects the post-harvest metabolism of this product differently, even stimulating the production of anthocyanins.

The relationship between anthocyanin synthesis and UV-B radiation was recently studied by ^{Hu et al. (2020)} in research on the expression of the transcription factor MdWRKY72 in apples, which is one of the factors attributed to the regulation of anthocyanin biosynthesis. The present study sought to elucidate some of the mechanisms of gene transcription that was stimulated by UV-B radiation applications, which decisively influenced the production of anthocyanins. This provides tools for the total elucidation of the underlying synthesis of anthocyanins induced by UV-B mediated by MdWRKY72.