*2.9. Determination of Total Phenolic and Flavonoid Contents*

The phenolic content was determined using a Folin–Ciocalteau colorimetric assay [41]. Briefly, 1.5 mL of 20% (*v*/*v*) Folin–Ciocalteau reagent was mixed thoroughly into 0.2 mL of sample extract and allowed to stand for 5 min. The total volume was then built up to 10 mL with distilled water before being incubated in the dark for 90 min at ambient temperature. The absorbance was measured at 760 nm against a prepared blank. The calibration curve of gallic acid, which ranged from 20 to 800 mg/L, was used to determine the phenolic content. The results are presented in milligrams of equivalent gallic acid per gram of sample dry weight (mg GAE/g DW).

The flavonoid content of each extract was determined using the aluminum chloride technique [32]. First, 0.2 mL of sample extract was mixed with 4 mL of distilled water, followed by 0.3 mL of NaNO2 (5%), and reacted for 5 min. After this, 0.3 mL of AlCl3 (10%) was added and left to react for 6 min. Then, after adding 2 mL of NaOH (4%) and filling it with up to 10 mL of distilled water, the absorbance of the solution was measured at 510 nm. The flavonoid content was determined using a rutin standard curve ranging from 50 to 400 mg/L. The results are given as milligrams of rutin equivalents (mg RE/g DW) for each gram of dry weight of the samples.

#### *2.10. Determination of Total Saponin Content*

The saponin content of the plant extract was determined using the vanillin–sulfuric acid colorimetric method [59]. In brief, 0.1 mL of sample extract was mixed with 0.5 mL of ethanol (50%), 0.5 mL of vanillin solution (8%), and 4.0 mL of sulfuric acid (77.5%). The solution was then cooled to room temperature after 15 min incubation in a water bath at 60 ◦C. Then, the absorbance was measured using a spectrophotometer at 545 nm. The results were expressed as milligrams of tea saponin equivalents per gram of dry weight of the sample (TSE/g DW) using a calibration curve ranging from 50 to 400 mg/L.

#### *2.11. Gas Chromatography–Mass Spectrometry (GC–MS) Identification of Volatile Compounds*

The plant extraction for the GC-MS analysis was performed as previously described [32]. Briefly, 2.5 g of the powdered leaf was extracted in 50 mL of hexane using ultrasonicassisted extraction for 30 min for each treatment. The extracts were purified (Whatman no. 4), evaporated at reduced pressure and temperature using a rotary evaporator, weighed, and dissolved in hexane at 10 mg/mL. The samples were analyzed via GC-MS equipped with an HP-5ms capillary column (30 m × 0.25 mm × 0.25 μm). The carrier gas was pure helium with a purity greater than 99.99%, flowing at a rate of 1.2 mL/min. The sample was diluted with n-hexane at a 10% (*v*/*v*) concentration. The injection volume was 1 μL and the diversion ratio was 1:5. The temperatures for injection and detection were set to 250 and 280 ◦C, respectively. The chromatographic heating procedure was as follows: the temperature was initially set to 60 ◦C for 2 min, then raised to 280 ◦C at a rate of 5 ◦C/min for 9 min. The electron ionization mode of the mass spectrometry involved an electron energy of 70 eV, a scanning range of 40–400 (*m*/*z*), a scanning rate of 3.99 scans/s, and a solvent delay of 3 min. The retention time (RT) values and NIST05 mass spectral library were used to identify compounds. The relative peak area of each compound in the chromatogram was used to calculate the percentage of each compound.

#### *2.12. Detection of Antioxidant Activity*

The DPPH• scavenging assay was performed with minor modifications to the Brand–Williams method [60]. A DPPH solution in methanol (6 × <sup>10</sup>−<sup>5</sup> M) was prepared and mixed with 100 μL of each sample (3 mL). The sample absorbance (A1) was measured at 515 nm after the mixtures were incubated in the dark for 15 min at room temperature. The absorbance of a blank sample (A0) containing 100 μL of methanol was also measured. The scavenging ability of the triplicate experiments was estimated using the following equation:

$$\text{Inhibtition } (\%) = \left[ (\text{A0} - \text{A1})/\text{A0} \right] \times 100 \tag{2}$$

where A0 is the absorbance of the blank and A1 is the absorbance of the sample extract.

The ABTS assay was measured following the procedure described by Nisca et al. [61]. Furthermore, 100 μL of sample extract was mixed with 100 μL of ABTS reagent and left to react in the dark for 6 min. The absorbance of the sample was measured at 734 nm. The inhibition percentage was calculated using the above formula described in the DPPH method.

The FRAP was determined using a slightly modified Benzie and Strain [62] method. The fresh FRAP reagent working solution was prepared by mixing 20 mL of acetate buffer (300 mM, pH 3.6), 2 mL of TPTZ (10 mM) in 40 mM HCl, and 2 mL of FeCl3, 6H2O (20 mM). The mixture was then incubated in a water bath at 37 ◦C for 30 min. The samples (75 μL) were then vigorously mixed with 75 mL of FRAP reagent. The sample absorption was measured at 593 nm after 4 min. A ferrous sulfate solution (0.5–10 mg/mL) was used to create the standard curve. The results were expressed in millimoles of ferrous ion equivalent per gram dry weight of the sample (mmol Fe2+/g DW).

The chelation ability of the ethanol extract was determined using a previously described ferrozine-based colorimetric assay [63]. The ethanol extract (50 μL) was mixed with 200 μL FeSO4 (0.2 mM) and 200 μL ferrozine (0.5 mM). The mixture was shaken and left at room temperature for 10 min. Finally, the absorbance was measured at 562 nm. The inhibition percentage of the ferrozine–Fe2+ complex was calculated using the following equation:

$$\text{Inhibtitious (\%)}=[\text{(Ac}-\text{As)/Ac}] \times 100\tag{3}$$

where Ac is the absorbance of the control and As is the absorbance of the sample.

#### *2.13. Statistical Analysis*

The data for all parameters were subjected to a one-way ANOVA followed by Duncan's multiple comparisons (*p* < 0.05) test using SAS version 9.4 (SAS Inc., Cary, NC, USA). The results are presented as mean values ± standard deviations (SD). For graphical representations, OriginPro® version 9.8.0.200 software (Northampton, MA, USA) was used.

### **3. Results**

#### *3.1. Effect of Salt Stress on Growth Parameters*

The effect of NaCl on the *H. cannabinus* plant growth was assessed by measuring the plant height as well as the fresh and dry weights of the leaf. The results revealed that the salinity significantly decreased the plant growth, as depicted in Figure 1. In detail, the treatments with 100, 150, 200, and 200 mM of NaCl significantly reduced the plant height by 14.80%, 22.81%, 29.78%, and 44.81%, respectively, compared to the control (Figure 2a). At 200 and 250 mM NaCl concentrations, the fresh leaf weight was reduced by 42.46 and 62.51%, respectively (Figure 2b). The leaf dry weight was also affected by the stressor, decreasing by 8.96 (150 mM), 30.45 (200 mM), and 50.11% (250 mM) (Figure 2c).

#### *3.2. Impact of Salt Stress on Minerals in Leaves*

Salinity stress significantly impacted the mineral concentration in the *H. cannabinus* leaves (Table 1). The salinity stress (100, 150, 200, and 250 mM) decreased the N (up to 8.49, 17.76, 22.63 and 24.85%, respectively) compared with the control. However, the N content reduction was not statistically significant at 50 mM of NaCl. The concentration of K significantly decreased at 50 mM, 200 mM, and 250 mM of NaCl by 6.52%, 5.74%, and 3.87%, respectively, as compared to the control, whereas the changes were slight at 100 and 150 mM of NaCl. Moreover, the concentrations of Ca and Mg decreased as the salinity intensified. However, the Mg content decreased slightly at 50 mM of NaCl. The concentration of *p* significantly decreased under salt stress. The low saline concentration (50 mM) significantly the increased Fe concentrations, whereas the medium and high saline concentrations significantly reduced the concentrations.

**Figure 1.** Changes in morphology of *H. cannabinus* seedlings grown under different salt stress conditions (0 mM of NaCl, 50 mM of NaCl, 100 mM of NaCl, 150 mM of NaCl, 200 mM of NaCl, and 250 mM of NaCl).

**Figure 2.** Effects of different levels of salinity stress (0, 50, 100, 150, 200, and 250 mM of NaCl) on the plant height (**a**) and fresh weight (FW) (**b**) and dry weight (DW) (**c**) of *H. cannabinus* leaves. The results are expressed in cm or g plant-1, as the means ± SD of different measurements (*n* = 15). Different letters (a–f) above the bars indicate a significant difference between treatments according to the Duncan test (*p* < 0.05).

**Table 1.** Compositions of nitrogen, potassium, calcium, magnesium, phosphorous, and iron in the leaves of *H. cannabinus* subjected to different levels of salt stress (0, 50, 100, 150, 200, and 250 mM of NaCl). The results are expressed in mg/g dry weight (DW), as means ± SD (*n* = 3). According to the Duncan test (*p* < 0.05), different letters within a column indicate significant differences.


*3.3. Alterations of Proline, Total Soluble Sugar, and Soluble Protein Contents under Salt Stress*

Proline is one of the most common osmotic adjustment substances. The results indicated that the plant proline content increased significantly with the salt concentration (Figure 3a). The proline levels increased by 432.76 and 527.38% under 150 and 200 mM NaCl concentrations, respectively, as compared with the control.

**Figure 3.** Effects of salt stress on the proline (**a**), total soluble sugar (**b**), and total soluble protein (**c**) contents in leaves of *H. cannabinus* seedlings subjected to 0, 50, 100, 150, 200, and 250 mM of NaCl. The values shown are means ± SD (*n* = 3). The different letters (a–f) above the bars indicate a significant difference according to the Duncan test (*p* < 0.05).

The results revealed that the NaCl concentrations increased the total soluble sugar content but to varying levels (Figure 3b). The total soluble sugar increased continuously from 50 to 150 mM of NaCl treatment. Treatments with 200 and 150 mM of NaCl increased the total soluble sugar by 101.63 and 222.59%, respectively, as compared to the control. Likewise, the application of 250 mM increased the total soluble sugar content by 24.38%, as compared to the control.

The content of soluble proteins was affected by salinity stress. The protein content increased significantly with the increasing salt concentration compared to the control (Figure 3c). The soluble protein levels were highest with 150 and 200 mM of NaCl. The soluble protein content was enhanced by 185.77 and 211.37% at 200 and 150 mM NaCl concentrations, respectively, as compared to the control treatment.

#### *3.4. ROS Detection and Lipid Peroxidation*

The concentration of O2 •− in the leaves of *H. cannabinus* was altered by the salinity. In detail, the O2 •− content significantly increased with the increasing salt concentrations, as shown in Figure 4a. The levels of O2 •− were higher in seedlings treated with 200 and 250 mM of NaCl, with the values exceeding 136.07 and 291.80% of those found in the control plants, respectively. Likewise, in seedlings exposed to 50 mM of NaCl, the O2 •− production increased by 19.67%. The H2O2 production rate increased progressively with the increase in salt concentration (Figure 4b). The H2O2 levels rose by 191.23 and 290.34% at 200 and 250 mM of NaCl, respectively, as compared to the control treatment. Moreover, 50 mM of NaCl increased the H2O2 concentration by 43.83% compared to the control.

**Figure 4.** O2 •− contents (**a**), H2O2 contents (**b**), and MDA contents (**c**) in leaves of *H. cannabinus* under various salt stress levels (0, 50, 100, 150, 200, and 250 mM of NaCl). The values presented are means ± SD (*n* = 3). Different letters (a–f) above the bars indicate a significant difference between treatments according to the Duncan test (*p* < 0.05).

As illustrated in Figure 4c, the MDA content is significantly influenced by the different salt concentrations. The results indicated that the MDA content increased as the salinity intensified. Compared to the control treatment, the MDA levels increased by 29.27 and 178.71% in response to 50 and 250 mM of NaCl, respectively.

#### *3.5. Total Contents of Phenolics, Flavonoids, and Saponins*

According to the findings, the salinity stress affected the total phenolic, flavonoid, and saponin contents. Under salt stress, the total phenolic content was found to be increased (Figure 5a). In detail, the total phenolic contents increased by 68.84 and 51.55% with 150 and 200 mM of NaCl, respectively, compared to the control. Similarly, the total flavonoid contents increased by 96.85 and 105.8% with 150 and 200 mM of NaCl, respectively, compared to the control (Figure 5b). There was no significant effect on the total flavonoid content with the 50 mM NaCl treatment. There was a considerable difference in saponin contents with increasing salt concentrations compared to the control (Figure 5c). The content of saponins increased steadily from the 50 to 200 mM NaCl treatments. Compared to the control, the saponin accumulation increased by 91.61% with 200 mM of NaCl but was significantly reduced by 47.81% with 250 mM of NaCl.

**Figure 5.** Changes in (**a**) the total phenolic contents, (**b**) total flavonoid contents, and (**c**) total saponin contents under different levels of salt stress (0, 50, 100, 150, 200, and 250 mM of NaCl). The values presented are means ± SD (*n* = 3). Different letters (a–e) above the bars indicate a significant difference between treatments according to the Duncan test (*p* < 0.05).
