The correlation of in vitro antioxidant potentials with the various biochemical responses of salinized basil leaves

One of the environmental sustainability issues is salinity. Basil seedlings (Ocimum basilicum L.) were treated using NaCl solutions of three different concentrations prepared using irrigation (40, 80, and 130 mM), and various biochemical analyses were performed on basil leaves. The number of leaves, leaf area, moisture, weights, and MDA content of basil decreased significantly as salinity levels increased from 40 to 130 mM; however, dry matter increased. As well, the current study investigated a significant increase in osmolytes (including total soluble sugars and proline) and Na contents. The highest activities of CAT and SOD in the leaf tissues of basil were recorded after treatment with 130 mM NaCl, whereas the polyphenol and total flavonoid contents were negatively influenced. On the other hand, the highest ABTS scavenging activity was observed in the 40 mM-treated leaves at a concentration of 1000 μg/mL; however, the DPPH scavenging potential increased significantly in the 80 mM-treated leaves at 3000 μg/mL. Furthermore, the correlation between in vitro antioxidant potentials and biochemical responses was described. A strong correlation was identified between the in vitro antioxidant capacities of salinized O. basilicum leaves and SOD activity, total flavonoids, and the presence of phenolic acids, particularly p-hydroxybenzoic and o-coumaric acids at various concentrations. As a result, this is the first study to explain how basil may resist salinity by producing specific antioxidant compounds; therefore, our research recommends use of salinity issue to obtain a better plant material for producing dietary supplements or herbal drugs.


Introduction Introduction Introduction Introduction
Salinity is a main abiotic stress that noticeably disturbs crop production globally. Annually, approximately 1.5 million hectares of cropland are excluded owing to high salinity. In addition, irrigation water containing traces of NaCl increase the salinity levels of nearby arable soil (Tester and Davenport, 2003).
2 Even though salinity negatively affects plant growth by directly damaging and inhibiting it and its effect depends on the salinity of the environment and the salinity tolerance of the plant, saline groundwater is still widely used to irrigate plants that are grown in areas with poor water supply (Abbas et al., 2021;Osakabe et al., 2014;El-Beltagi et al., 2022a).
Salinity generally induces osmotic stress in plants by decreasing water availability to the roots and increasing Na + and Cl − ion accumulation, which consequently alters various physiological processes of the plant, including the transpiration rate, stomatal conductivity, photosynthesis, and relative growth rate (Caliskan et al., 2017;Negrao et al., 2017). Reactive oxygen species (ROS) is regenerated under salinity conditions; the plant overcomes the deterioration of ROS by increasing antioxidant defense including catalase (CAT) and superoxide dismutase (SOD) (Jabeen and Ahmad, 2013;Sevindik et al., 2017). Enhanced salinity tolerance in plants can be attributed, at least partially, to the activity of antioxidant enzymes present in the leaves, such as peroxidase and catalase (CAT), which are considered the primary salinity sensitive enzymes, and proline can induce an increase in these activities (Nawaz and Ashraf, 2010).
Furthermore, malondialdehyde (MDA) levels can be used as a marker of the degree of salinity-induced oxidative stress (Jakovljević et al., 2017); it plays a vital role in regulating the properties of membrane lipid compounds in response to the deleterious effects of osmotic stress. Since salinity particularly enhances ROS formation, it consequently affects the proportion of secondary compounds, such as phenolic compounds, essential oils, and photosynthetic pigments (Talebi et al., 2018;de Azevedo Neto et al., 2019;Scagel et al., 2019). Basil (Ocimum basilicum L.; Lamiaceae) grows abundantly throughout the Mediterranean, especially in warmer areas. Ocimum spp. are known to contain high quantities of bioactive compounds with several medicinal, nutritional, and pharmaceutical applications. For example, O. basilicum is one of the chief species from which essential oils are obtained. The essential oils obtained from O. basilicum vary in composition, and hence, are used for various applications, such as in condiments, as flavouring agents, and even in folk medicine for treating mental fatigue, dysentery, nausea, rhinitis, and cold (Tarchoune et al., 2012;Ramadan et al., 2022a).

Materials and Methods Materials and Methods Materials and Methods Materials and Methods
Authentication and experimental design O. basilicum seeds were authenticated at the Flora and Phyto-Taxonomy Research Department of the Agricultural Research Centre, Dokki-Cairo, Egypt. The study was conducted in 2020 and 2021 in a greenhouse at the Faculty of Agriculture, Cairo University, Egypt. The experiments were performed as described here using a completely randomized design and 30 replicates for each treatment. The soil and water used were first examined according to a method previously reported by Estefan et al. (2013). In April, 25-day-old seedlings were transferred into pots (diameter, 20 cm; height, 18 cm) packed with soil (pH = 7.86 and electric 3 conductivity (EC) = 2.11 ds/m). When the seedlings were 45 days old, three NaCl solutions with different concentrations (40, 80, and 130 mM) were prepared by dissolving NaCl in the irrigation water (pH = 7.54 and EC = 0.28 ds/m) and supplied to the plants. The pots were kept at a greenhouse (27 °C and 17 °C day and night air temperatures, respectively). Overall, eight irrigation treatments were applied, and the plants were harvested 25 days following NaCl treatment. In the current study, the treatments were designated as C (Control), S1 (40 mM), S2 (80 mM), and S3 (130 mM).

Chemicals and reagents
All solvents and kits used were of analytical grade and sourced from Sigma-Aldrich (St. Louis, MO, USA).

Determination of O. basilicum leaf area and weight
O. basilicum leaves were harvested and the leaf area was determined using Image J (National Institute of Health, USA) (Schneider et al., 2012). In addition, the fresh leaves were weighed (FW), after which the leaves were dried at 105 °C for 24 h before measuring their dry weight (DW).
The percentage (%) of moisture and dry matter (DM) contents of the leaves were calculated using the following formulas (Arunachalam and Parimelazhagan 2014; Mehdizadeh et al., 2019): Where DW is the dry weight and FW is the fresh weight.

Determination of proline content
Fresh leaves (0.5 g) were homogenized in 10 mL of aqueous sulfosalicylic acid (3%) and filtered through a Whatman #2 filter paper. Next, 2 mL of the filtrate was added to a mixture of 2 mL of ninhydrin and 2 mL of glacial acetic acid. The mixture was then incubated at 100 °C for 1 h before cooling. Next, 4 mL of toluene was added to the mixture and mixed. The toluene fraction was measured at 520 nm using a spectrophotometer, Jenway, England. The concentration of proline was determined by interpreting a standard curve (R 2 = 0.9916) (Bates et al., 1973).

Determination of total soluble sugar content
Fresh leaves weighing 0.2 g were soaked in methanol (80%) with refluxing for 6 h at 80 °C. Then, 2 mL of the solution was added to 0.05 mL phenol (80%), to which 5 mL of H2SO4 conc. was added. The tubes were incubated at 30 °C for 20 min. The absorbance of reaction was measured at 490 nm (for hexose) and 480 nm (for pentose and uronic acid) against a blank. The concentrations of the sugars were calculated using the standard curve (R 2 = 0.991 ) (Dubois et al., 1956).

Determination of Na + and K + ion concentrations
One gram of leaves was added to a mixture of 7 mL of H2SO4 and 2 mL of H2O2 in a digestion tube placed on a temperature-controlled hotplate. The samples were homogenized by gently swirling the solution, and the resulting homogenate was heated until clear. The solution was then cooled and transferred to a volumetric flask before filtration. The levels of Na + and K + in the solution were detected by iCE 3300 Atomic Absorption Spectrometer (Thermo Scientific, Germany)(Christian 1970).

Determination of antioxidant enzyme activities and MDA content
Preparation of enzyme extract We homogenized 500 mg of O. basilicum leaf tissue in a mixture comprising 3 mL sodium phosphate buffer (100 mM, pH 7, polyvinylpyrrolidone [1%], and EDTA [1 mM]). The centrifugation of resulting 4 homogenate was at 7000 rpm for 20 min under cooling. The supernatant was stored till further analyses at -80 °C.
Determination of CAT activity CAT activity was investigated as described Aebi (1984). CAT activity was expressed in U/ mg protein, where each unit of CAT reacts with H2O2 (1 µM, pH 7.0)/min at 25 °C (Aebi 1984).
The mixture was incubated for 10 min under fluorescent light. The absorbance was read at 560 nm. One unit of SOD was described as the extent of enzyme that inhibits 50% of the NBT reduction under the assessment condition (Giannopolitis and Ries, 1977).

Determination of MDA content
Equal volumes of the extract and a mixture of thiobarbituric acid (TBA) (0.5%) and trichloroacetic acid (TCA) (20%) were incubated at 95 °C for 30 min. The absorbance of reaction was measured at 532 nm (Stewart and Bewley, 1980).

Extraction of O. basilicum leaves
The leaves of O. basilicum were left at 37 °C for three days until dry, and then a fine powder of air-dried leaves was soaked in methanol overnight. The extracts were filtered through a Whatman filter paper and evaporated using a rotary dryer at 45 °C.

Estimation of total polyphenol content
Polyphenol content was determined as previously described by Prior et al. (2005) with some adjustments. Briefly, 125 µL of the methanolic extract was diluted to 400 µL of distilled water, and then 125 µL of Folin-Ciocalteu reagent (diluted 1:1) was added to solution. The resulting mixture was incubated for 3 min at room temperature; following which 250 µL of Na2CO3 (7%) was added. The reaction was stored for 30 min at room temperature in the dark. The absorbance was measured at 760 nm. The gallic acid standard was prepared (10-50 mg/L) (R 2 =0.9713). The total polyphenol content was calculated as mg of gallic equivalent [GAE]/g of dry leaves (Prior et al., 2005).

Estimation of total flavonoid content
Briefly, 0.5 mL of methanolic was added to 0.5 mL of AlCl3 (2%). An orange-yellowish color was obtained after 1 h and absorbance was measured at 420 nm. The quercetin standard (10-100 mg/L) was prepared (R 2 =0.9728). The total flavonoid content was calculated as mg of quercetin equivalent [QE]/g of dry leaves (Ordonez et al., 2006).

Statistical analyses
All data are described by mean (n = 3) ± standard error (SE). SE and correlation were calculated using Microsoft Excel. The statistical significance of the differences among the mean values was measured via oneway ANOVA by Web Agri Stat Package 2.0 software. (https://ccari.icar.gov.in/wasp/index.php). Different letters designate statistically significant differences between the means at (p < 0.05).

Appearance and biochemical analyses of basil leaves under salinity stress
The number, area, and moisture content of the leaves were 41.84, 61.60, and 4.64% lower, respectively, in S3-treated leaves compared with control leaves. However, the increasing levels of salinity also increased the DM of the leaves, with S1-treated leaves showing a greater increase (with 59% increase) compared with control leaves (Table 1). All values represented as mean ± SE (n=3) and comparison of treatment means with (p < 0.05).
6 Figure 1, A shows a decrease in MDA content in response to treatments with higher NaCl concentrations, with non-significance for S1 and S2 treated basil leaves. Conversely, the proline and total soluble sugars contents increased significantly at three NaCl treated basil leaves, compared to control leaves, with S3-treated leaves increasing by 2 folds in sugar content (Figure 1, B and C).  The Na + and K + ion concentrations exhibited significant differences between the NaCl-treated and control O. basilicum leaves, with S3-treated leaves increasing by 9.5 folds in Na + content, in response to NaCl treatments ( Figure 2).

Effect of salinity stress on antioxidant defense outlines of O. basilicum leaves
Although the highest polyphenol content was observed under moderate NaCl concentrations (S2), S1, S2, and S3-treated leaves showed decreased flavonoid content (by 8.33 and 12.01, and 23.02 %, respectively). However, the polyphenol content remained unaffected at 40 and 80 mM of NaCl concentrations, S3-treated leaves displayed a decrease by 21.95 %. Furthermore, the CAT and SOD activities were displayed an increase by 5.26 and 45%, respectively for S3-treated leaves (Table 2).

In vitro antioxidant potentials
The antioxidant capacity of O. basilicum leaves was measured using DPPH and ABTS assays. A substantial increase in DPPH scavenging activity was observed at concentrations of 1000 and 2000 µg/mL in the control and S1-treated leaves, respectively. When salinity was increased from 80 to 130 mM NaCl, DPPH scavenging potential increased significantly by 17.62% in S2-treated leaves at 3000 µg/mL, while S3 treated leaves investigated 1.8 folds of scavenging at the same concentration (Figure 3, A). However, the highest ABTS scavenging activity was observed in the S1-treated leaves, showing 84.23 and 7.77 % at 1000 and 2000 µg/mL, respectively, while S3-treated leaves increased by 21.31 % at 1000 µg/mL (Figure 3, B). The IC50 of control and NaCl-treated basil leaves for two antioxidant assays were compared and S1-treated leaves was displayed the lowest value in ABTS assay (1037.63 µg/mL).

Effect of salinity stress on phenolic acids and flavonoids abundance
The results of our HPLC analysis of the O. basilicum leaf methanolic extracts are shown in Table 3. Several phenolic acids and flavonoids were identified, including p-hydroxybenzoic acid, quercetin, benzoic acid, ferulic acid, o-coumaric acid, kaempferol, and syringic acid. Interestingly, myricetin was a major compound present in both control and treated O. basilicum leaf extracts, showing an increase in abundance by 70.20% in c S2-treated leaves. Although, o-coumaric acid was displayed a decrease with 90.84% in S3-treated basil leaves, rutin and resveratrol were observed to be highly abundant. Moreover, p-hydroxyenzoic acid content declined with increasing salinity. Concurrently, S1 was associated with decreases in the contents of benzoic acid and quercetin by 96.27 and 87.12%, respectively. Conversely, ferulic acid content increased by 178.50, 65.74, and 39.08% in S1, S2, and S3-treated leaves, respectively, compared with control leaves. Treatment of O. basilicum leaves with 80 mM NaCl increased the contents of phenolic acids and flavonoid compounds (Table 3). Table 3. Table 3. Table 3.

Correlation traits
The correlations recognized among the in vitro antioxidant of DPPH and ABTS and the various biochemical responses of untreated and NaCl treated basil leaves are described in Figure 4. In details, the antioxidant capacity (DPPH) was shown to have a strong positive correlation with total flavonoids and phydroxybenzoic acid contents (0.941 and 0.930) at 1000 µg/mL and (0.793 and 0.917) at 2000 µg/mL, respectively. Furthermore, total flavonoids and p-hydroxybenzoic acid levels were found to have a powerful correlation (0.993) and (0.960), respectively, with antioxidant capacity (ABTS) at 3000 µg/mL (Figure 4). On the one side, the amounts of quercetin and benzoic acid were investigated (0.705 and 0.663), respectively at 3000 µg/mL, the content of ferulic acid was displayed a correlation (0.712) with antioxidant capacity (ABTS) at 1000 µg/mL, and moreover, TPC was positively correlated with ABTS scavenging potential (0.659) at 3000 µg/mL, while the DPPH scavenging potential was displayed a low or negative correlation.
Furthermore, the o-coumaric acid content was positively correlated (0.635 and 0.534) with antioxidant capacity (DPPH) at 1000 and 2000 µg/mL, respectively, although the correlation was increased from -0.233 to 0.792 with the o-coumaric acid content in ABTS scavenging potential. The activity of SOD was strongly correlated with DPPH scavenging (> 0.9) at 1000 and 2000 µg/mL, in spite of the fact that the antioxidant capacity of ABTS gradually ranged from 0.2 to 0.9. Mutually, kaempferol content showed a positive correlation (0.407) with antioxidant capacities at 1000 and 2000 µg/mL for DPPH and ABTS, respectively. On the other hand, the amounts of total soluble sugars and proline showed a positive correlation (0.805 and 0.515, respectively), with antioxidant capacity (only DPPH) at 3000 µg/mL.

Discussion Discussion Discussion
The one of salinity effects is a stomata closure, which causes a decrease of moisture inside leaf tissues (Copolovici et al., 2021). Studies have also reported that the degree of salinity tolerance of crops is often inversely associated with its growth rate, since salt tension might directly constrain cell expansion and inhibit water absorption (Munns et al., 2006). Moreover, reductions in the biomass, area, and number of O. basilicum leaves have been reported in response to salinity stress (Munns and Tester, 2008;Menezes et al., 2017). Organic solutes or osmolytes such as sugars, proline, and glycine betaine help plants cope with high salinity stress by adjusting the osmotic pressure of the cytoplasm and maintaining the integrity of photosystem II (Heidari, 2011;Krasensky and Jonak, 2012). Moreover, the organic solutes play critical roles associated with reducing the levels of ROS by forming hydrophilic complexes (Krasensky and Jonak, 2012), scavenging ˙OH radicals, quenching singlet oxygen (Szabados and Savouré, 2010), and activating defensive pathways inside the plant cells (Fariduddin et al., 2013) MDA levels are therefore used to evaluate the extent of membrane lipid peroxidation. The correlation between proline and MDA contents was calculated as a negative correlation (-0.604) (Figure 1 A and B), in agreement with Wang et al. (2016) who investigated the converse relationship between proline and MDA contents in stressed -Arabidopsis thaliana leaves. The last relationship might be ascribed to the auxiliary roles of superior proline accumulation at salinity stress through ROS scavenging and the consequent defense of membrane integrity (Wang et al., 2016). Also, the increase in the concentration of total soluble sugars in NaCltreated O. basilicum leaves was dose-dependent and significant compared with control leaves (Figure 1, C), this finding disagrees with that reported by Heidari (2011) who found that treatment with 6 ds/m NaCl exhibited no significant effect on carbohydrate content (Heidari, 2011).
Moreover, the concentrations of Na + in NaCl-treated O. basilicum leaves displayed dose-dependent and significant increase compared with control leaves (Figure 2). Despite the comparatively high levels of Na + ion accumulation in O. basilicum leaves, the lowest levels of K + ions were observed in S1-and S3-treated leaves (Figure 2). Apse and Blumwald (2007) suggested that O. basilicum can accumulate and compartmentalize Na + ions inside its leaves without undergoing dehydration. Furthermore, the salinity tolerance of O. basilicum may be associated with its capacity to (1) sustain K + provision to its leaves and (2) maintain Na + accumulation in its leaves at levels compatible with normal cellular physiology. Several genes encode transporters responsible for the movement of Na + from the xylem of the root to the leaves (Apse and Blumwald, 2007).
Subsequently, to alleviate the ROS mediated oxidative stress, plants utilize an antioxidant defense scheme involving antioxidant enzymes, for instance CAT and SOD, as well as nonenzymatic antioxidants, such as phenolic acids, carotenoids, and flavonoids (Gengmao et al., 2015;Wei et al., 2015). In similar, the present study clarified the pathways of basil for tolerance the salinity including: elevating the antioxidant enzyme activities including (CAT and SOD) and accumulation of polyphenols and flavonoids ( Table 2). The enzyme SOD is obtained in various partitions of the cell and converts the superoxide radicals (O2 •− ) to hydrogen peroxide (H2O2) which removed by peroxidases and catalase. The increase in SOD activity was observed in basil shoots after NaCl treatment, while the decline in SOD activity may be due to extra plant growth; developed plants may improve a suitable enzyme scheme in addition to antioxidant developments.
Furthermore, this finding is in accordance with the data found in the extant study, which revealed that CAT activity was the highest in the S3-treated leaves (Table 2). Generally, the increases or decreases in CAT activity are caused by differences in the period, type, and intensity of stress. A previous study reported that the roots and shoots of O. basilicum displayed maximum CAT activity during their early growth phase (i.e., < 14 days) in response to salinity (Sharma et al., 2012;Jakovljević et al., 2017). The methanolic extracts of the control and salinized O. basilicum leaves exhibited higher polyphenol and flavonoid contents in the present study (Table 2) compared with those reported in previous studies. For example, one study reported phenol and flavonoid contents in ethanolic extract of basil leaves were 29.60 mg GAE/g and 19.58 mg QE/g, respectively (Nguyen et al., 2021). This increase was due to the use of methanol solvent that was previously showed a highest efficacy of phenolic compound extraction (Nguyen et al., 2020). On the other hand, the decrease in total polyphenol content at 130 mM NaCl was previously referred to inferior capability to adjust reactive oxygen species and the oxidative capacity in O. basilicum (Copolovici et al., 2021).
Our results showed significant reductions in DPPH scavenging activity in salinized O. basilicum leaf extracts (at concentrations of 1000 and 2000 µg/mL) in response to the 130 mM NaCl treatment (Figure 3, A). This finding is reliable with the those of Robatjazi et al. (2020) who reported a similar relationship in O.
The results of the current study correspond with those found in the relevant literature. For instance, Imen et al. (2012) observed a decline in the caffeic and rosmarinic acid contents of a fine cultivar of basil following 15 days of Na2SO4 stress (Imen et al., 2012). Furthermore, the previous studies have also reported a decrease in the p-coumaric acid content of wheat leaves in response to salinity , as well as a high abundance of phenolic acids and flavonoid compounds (including vanillic, chlorogenic, ferulic, and ellagic acids and rutin) in the leaves of Aegilops cylindrical Host and Amaranthus tricolor L. subjected to salinity stress (Kiani et al., 2021). As result, differences in the DPPH and ABTS scavenging potentials of control and salinized O. basilicum leaves may be due to the presence of kaempferol, myricetin, ferulic acid, and p-hydroxybenzoic acid.

12
The role of phenolic acids and flavonoids in the scavenging of free radicals were showed by Gould and Lister (2006). Generally, the increase in polyphenol contents of treated basil leaves at 80 mM NaCl (Table 2) may be because of their antioxidant properties, which allows them to inhibit lipid peroxidation in response to salinity stress (Sivaci et al., 2014).
The total flavonoid, quercetin, and phenolic acids involving (p-hydroxybenzoic, benzoic, ferulic, and ocoumaric acids) were displayed a positive correlation (Figure 4), in agreement with Isetkomolmat et al. (2023) who showed a positive correlation of quercetin with antioxidant capacities of rice husk (Isetkomolmat et al., 2023). As well, the latter study recognized a positive correlation between antioxidant capacity (DPPH) and total flavonoids of safflower (Golkar and Taghizadeh, 2018;El-Beltagi et al., 2019) and the positive role of hydroxybenzoic acid of silk in antioxidant activity was previously displayed (Ratha et al., 2023). Also, a previous report by Złotek et al. (2016) demonstrated a positive relationship between the antioxidant potential and total polyphenol and flavonoid contents of untreated O. basilicum leaves, which was validated by our experimental results (Złotek et al., 2016). Structurally, the presence of catechol group on ring B of flavonoids and 5-OH group of A ring are explained the positive correlation between antioxidant capacities (DPPH and ABTS) and total flavonoids content (TFC) and some of phenolic acids for basil leaves under NaCl stress. Moreover, the methoxyl group and number of hydroxyl groups in phenolic acids had a determinant role in their antioxidant activity (Moazzen et al., 2022). In addition, phenolics mixture showed dose de-pendent activity among concentrations and combinations of radical antioxidants such as phenolics, catechin, p-hydroxybenzoic, benzoic, ferulic, coumaric acid, caffeic acid, chrysin, quercetin, kaempferol…etc showed greater activity against TMV infection and their oxidative stress (Dhawi et al., 2021). The high contents of antioxidant compounds in these extracts, such as total phenolics and flavonoids, may contribute to their greater total antioxidant capacity. Due to their high redox properties, phenolic compounds are crucial for quenching singlet or triplet oxygen, as well as for the breakdown of peroxides. They also absorb and neutralize free radicals, which helps to prevent oxidative chain reactions El-Beltagi et al., 2022c).

Conclusions Conclusions Conclusions
This work suggests the pathway of basil to resist NaCl pressure by showing the correlation of in vitro antioxidant potentials with various biochemical responses. In conclusion, the correlation of antioxidant potential with the SOD activity and content of TFC, o-coumaric acid, and p-hydroxybenzoic acid was powerfully positive. While the CAT activity and the amounts of total soluble sugars, proline, quercetin, TPC, kaempferol, and phenolic acids involved (benzoic, ferulic, chlorogenic, and rosmarinic acids) displayed a moderate correlation with antioxidant potential.

17
 Maps and affiliations: The publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  Responsibilities: The editors, editorial board and publisher do not assume any responsibility for the article's contents and for the authors' views expressed in their contributions. The statements and opinions published represent the views of the authors or persons to whom they are credited. Publication of research information does not constitute a recommendation or endorsement of products involved.