Location of the experiment

The basil cultivars were established in March 2020 in a greenhouse with sliding plastic covers and anti-aphid mesh with semi-automatic control, located at the Polytechnic University of Gómez Palacio, Durango, 25° 38’ 19.83” north latitude, 103° 31’ 52.12” west longitude. The temperature and relative humidity values of the greenhouse were kept between 25 and 30 °C and 70-80%, respectively.

Establishment of cultivars

Three basil cultivars were planted, their seeds were from donations from organic basil plantations in Baja California Sur (Lemon, Cinnamon and Red Rubin). Sowing was carried out in polystyrene germination trays of 250 cavities and peat moss was used as a substrate. The trays were watered twice a day with water until the day of transplantation. When the seedlings had three to four true leaves and a height of 10 to 15 cm, they were transplanted into black plastic pots of one gallon capacity, which contained a mixture of sand:perlite (80:20).

Preparation of samples and obtaining extracts

At 35 days after transplantation, leaf samples of the three basil varieties were taken until samples of all the plants in the experiment were obtained. All samples were washed with distilled water and then the excess water was removed on kraft paper. The drying process was carried out at room temperature (25 ±2 °C) for 15 days. After the dehydration time, the samples went through a grinding process until obtaining a powder. This process was carried out in a blender (Hamilton Beach).

After that, the pulverized samples were stored at 5 °C for the subsequent obtaining of extracts (^{Abkhoo and Jhani, 2016}). Extracts were obtained by the solid-liquid extraction technique by adding 1 g of each of the pulverized samples placed in test tubes. Ten milliliters of each of the solvents used (hexane, methanol, petroleum ether, Jalmek^® ethanol) were added. An automated stirrer (Stuart^®) was used for 24 h to keep the mixture under stirring at room temperature (25 ±2 °C).

After the time established in stirring, the samples were left to stand in a rack for the phase change and precipitation of each sample. When the separation was observed, the extract was obtained, leaving the precipitated sample in the tubes. The extracts were concentrated to obtain the compounds of interest with a rotary evaporator (Buchí, model-210) and a water bath (RIOSA, Mexico) at 35-40 °C. The extracts obtained were stored in deep freezing at -20 °C until their analysis (^{Ramírez et al., 2019}).

Quantification of total phenolics (TPCs)

The determination of total phenolic compounds was performed by spectrophotometry (Genesys 10 UV equipment USA), based on the oxide-reduction colorimetric reaction according to ^{Zamora (2016}) with some modifications. To 30 μl of extract, 2 ml of distilled water, 250 μl of Folin-Ciocalteu reagent (analytical grade, Sigma-Aldrich, St. Louis MO, USA) were added, then 1 ml of Na₂CO₃ at 10% w/v was added, and it was measured to 5 ml with distilled water. The samples were read at a wavelength of 765 nm after staying 1 h at room temperature and in the absence of light. For the calculation of total phenolics, a calibration curve was performed with analytical grade gallic acid (Sigma-Aldrich St. Louis MO, USA). The phenolic content was expressed in mg GAE g^-1 of DW (mg of gallic acid equivalents per gram of dry weight).

Quantification of flavonoids (TFVs)

The flavonoids were quantified by a spectrophotometric analysis (Genesys 10 UV equipment USA) based on the formation of a complex between the Al (III) ions and the carbonyl and hydroxyl groups of the flavonoid, as described by ^{Zamora (2016}) with some modifications. Fifty microliters of extract were mixed with 100 μl of AlCl₃ at 5% w/v in ethanol, 100 μl of 1M sodium acetate, and they were measured to 5 ml with analytical grade methanol (JT Baker). The flavonoid compounds were measured at a wavelength of 425 nm after 30 minutes of staying in the absence of light. Prior to the reading, a calibration curve was made with analytical grade quercetin (Sigma-Aldrich St. Louis MO, USA). The flavonoid content is expressed in mg QE g^-1 of DW (mg of quercetin equivalents per gram of dry weight).

Determination of antioxidant capacity (AA)

Antioxidant capacity was measured according to the methodology proposed by ^{Chaves et al. (2016}) with modifications. The ABTS radical (2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) was obtained by the reaction of ABTS (7 mM) with potassium persulfate (K₂S₂O₈) 2.45 mM, then measured with water at a volume of 10 ml and incubated at room temperature (±25 °C) and in the dark for 18 h. The ABTS radical was diluted in ethanol until obtaining an absorbance value between 0.7 (±0.1) at a wavelength of 754 nm.

The extracts were dissolved in ethanol at a concentration of 200 mg L^-1. After that, 50 μl of sample and 1 950 μl of ABTS were placed in a test tube. They were stirred for 1 min and kept in the dark for 10 min. After the reaction, the absorbance was read at 754 nm (Genesys 10 UV spectrophotometer USA). The results are expressed as a percentage of inhibition in micromoles Trolox per each gram of sample in dry weight (μmol Trolox g^-1 DW).

Statistical analysis

The statistical analysis was carried out by means of an analysis of variance using the Statistical Release Software 7.0 and the comparison of means was performed using the Tukey test with a significance value of p≤ 0.05. A 3 x 4 factorial arrangement was used in a completely randomized design, where the factors were: genotype and extraction solvent. Three repetitions were performed per treatment.

Total flavonoids

The results of the TFV content are shown in Figure 1. The flavonoid content in this experiment ranged from 2.4 to 10.8 mg QE g^-1 DW, obtaining that the solvent with the highest efficiency for the extraction of these compounds was methanol for the three basil cultivars used, followed by the petroleum ether solvent and with the ethanol and hexane solvents registering the lowest extraction percentages (38 and 22%, respectively) with respect to the highest value of TFVs. This study coincides with the results obtained by ^{Do et al. (2014}), who reported that the extraction of flavonoids from plants was strongly influenced by the different solvents used.

Figure 1 Concentration of total flavonoids in extracts of three basil varieties obtained by four different solvents. The literals indicate the significant difference between the different extraction solvents for each variety of basil studied. 

The content of TFVs in aqueous extracts of the flower of M. acuminata (159 45 mg QE 100 g^-1) and P. tetragonolobus (172 44 mg QE 100 g^-1) was consistent with that reported by ^{Ng and See (2019}) (196 3 mg QE 100 g^-1) and ^{Gan et al. (2017}) (185 mg QE 100 g^-1). Compared to water extracts, methanol significantly increased the TFV yield of the flower of M. acuminata (24%) and P. tetragonolobus (73%), while n-hexane provided the lowest TFV content in both plants. The above results are similar to those obtained in this experiment, where the solvent with the highest extraction capacity was methanol and that with the lowest efficiency was hexane.

Flavonoids are the most abundant polyphenols with several reactive phenolic hydroxyl groups in benzene rings (^{Altemimi et al., 2017}), but reference is also made to the fact that the differences obtained in the different extractions by each solvent could be attributed to the variation in the biochemical composition of the plant, where the phenolics, in particular flavonoids, are the main extractable phytochemicals (^{Joseph et al., 2014}). Regarding the solvent with the highest extraction capacity (methanol), it is known that in tannin-protein complexes, most flavonoids exist in glycosides and methanol favors the extraction of these polar complexes of sugar-flavonoids (^{Ng et al., 2020}).

Total phenolic compounds

Phenolic compounds are ubiquitous in most medicinal plants and constitute an essential part of the human diet due to their antioxidant properties and many other beneficial health properties (^{Balasundram et al., 2006}). TPCs range between the values of 0.2 to 17.9 mg GAE g^-1 DW, showing the wide range of extraction between the different solvents used for the three varieties of basil analyzed. The following figure (Figure 2) shows the extraction efficiency of each solvent used for each variety of basil analyzed.

The methanol solvent was the most efficient extraction agent for TPCs; extraction values with this solvent ranged from 15.8, 16.7 and 17.9 mg GAE g^-1 DW for Lemon, Cinnamon and Red Rubin varieties, respectively, followed by hexane, ethanol and petroleum ether. According to the chemical structure of hexane, it is known that it is a solvent with lower polarity than petroleum ether (^{Nawaz et al., 2020}), which explains why it worked more efficiently in the extraction of TPCs than in the extraction of TFVs, since, according to the classification of phenolic compounds, flavonoids are a subgroup of phenols that do not always have affinity with low polar solvents (^{Nawaz et al., 2020}).

Figure 2 Concentration of total phenolic compounds in extracts of three basil varieties obtained by four different solvents. The literals indicate the significant difference between the different extraction solvents for each variety of basil studied. 

Authors such as ^{Orlando et al. (2019}) indicated that the extraction yields of polyphenols varied considerably between the different plant varieties. ^{Salih et al. (2020}) did not obtain a good extraction of polyphenols from macerated or cooked samples when using water as an extractant, compared to the use of methanol and ethanol. However, the use of other solvents leads to optimal extraction of bioactive compounds (^{Lezoul et al. 2020}).

In this sense, the polarity of methanol is greater than that of ethanol; for this reason, very polar phenolic acids, such as cinnamic or benzoic acids, could be extracted more easily, increasing the content of total polyphenols in the extracts obtained (^{Stalikas, 2007}). This explains the results of the present study, since, when methanol was used, the extraction of TPCs was greater compared to the rest of the solvents used in the different varieties of basil.

Antioxidant activity

In the present study, free radical scavenging properties were shown, and the values obtained showed indicators of antioxidant capacities based on the elimination of the free radical used in the ABTS technique. It was observed that the values for the free radical scavenging activity are higher when the solvent used was methanol for the Lemon and Cinnamon varieties, it was not the same case for the Red Rubin variety since, in these extracts, the greatest efficiency of antioxidant activity occurred when the solvent used was ethanol (Figure 3).

Table 1 mentions that the variety of basil with greater antioxidant activity, according to the averages yielded in the statistical analysis, is Cinnamon (249.5 μmol Trolox μg^-1 DW); however, when analyzing the factors in the interaction, it was observed that the AA is higher in the Red Rubin basil variety when the solvent used is ethanol with a value of 409.4 μmol Trolox μg^-1 DW. It should be noted that the Red Rubin basil variety has different characteristics in relation to the other two varieties, such as its pigmentation, and the quantity of secondary metabolites it has (^{Ramírez et al., 2019}).

Figure 3 Micromolar concentration of antioxidant activity by ABTS technique in extracts obtained by different solvents for three basil varieties. The literals indicate the significant difference between the different extraction solvents for each variety of basil studied. 

In the study conducted by ^{Dowlath et al. (2020}), it was reported that they found no significant differences between the extraction solvents petroleum ether and chloroform when they analyzed the antioxidant compounds (TPCs and AA) of Cardiospermum halicacabum; however, these solvents had a significant difference with respect to the ethanol solvent in the measurement of antioxidant capacity by the DPPH method. The above findings coincide with those obtained in this experiment since the solvent with the highest efficiency for free radical scavenging was ethanol and methanol (^{Dowlath et al., 2020}).

Because the solvent used for extraction shows variations in the radical scavenging capacity of the extracts (^{Dhanani et al., 2017}), it demonstrates the influence of the solvent on the antioxidant activity of the crude extracts (^{Bhebhe et al., 2016}). This is supported by studies on Macadamia tetraphylla shells, herbal infusions of Limnophila aromatic and Withnaia somnifera (L.), where there is a strong influence of the solvent on the antioxidant activity of plant extracts (^{Do et al., 2014}; ^{Dailey and Vuong, 2015}). This variation in antioxidant activity may possibly be associated with the compounds of the plant, such as phenolic constituents, which are soluble based on the polarity of the solvents used for extraction or other secondary metabolites present in each of the varieties studied (^{Dowlath et al., 2020}).