*3.6. Alterations in the Compositions of Volatile Compounds under Salt Stress*

The compositions of the leaf volatile compounds of *H. cannabinus* and their relative percentages (%) at different NaCl levels are shown in Table 2. Seventeen compounds were identified in the control. Phytol (18.64%) and 1-heptacosanol (18.23%) were the major components; other notable components were oleamide (9.36%), 12-methyl-E, E-2,13 octadecadien-1-ol (8.27%), alterungsschutzmittel BKF (7.30%), cis-vaccenic acid (6.80%), phthalic acid, hep-tyl undecyl ester (6.70%), and methyl linolelaidate (4.36%). When subjected to salt stress, the relative proportions of these components changed significantly. The application of NaCl reduced the phytol levels by 69.26%, 87.55%, and 88.79% at 150, 50, and 100 mM, respectively, compared with the control. This compound disappeared with 200 and 250 mM of NaCl. Compared to the control, the concentration of 1-heptacosanol increased significantly by 6.80% with 200 mM of NaCl; decreased substantially with 50, 100, and 250 mM of NaCl; and was not detected with 150 MM of NaCl. The biosynthesis of phthalic acid and heptyl undecyl ester was enhanced considerably with 100, 200, and 250 mM of NaCl, and it emerged as the first and most abundant compound. Also, the relative content of 3-(octadecyloxy) propyl ester increased at 50, 100, 150, and 200 mM NaCl concentrations and eventually became the second most abundant compound. The salt stress induced the biosynthesis of new compounds, including heptacosane, 1-octadecanesulphonyl chloride, and tetratetracontane. The biosynthesis of terpenes and alcohols was suppressed under salt

stress. However, a high salt concentration stimulated the production of alkane. In addition, under all levels of salinity stress, the esters increased significantly and eventually became the most abundant chemical class in salt-treated plants.

**Table 2.** Changes in the compositions and relative percentages (%) of volatile compounds in the leaves of *H. cannabinus* under different NaCl concentrations (0, 50, 100, 150, 200, and 250 mM). The results, reported as percentages, are means ± SD (*n* = 3). According to the Duncan test (*p* < 0.05), different letters within a row differ significantly; nd, not detected.


#### *3.7. In Vitro Antioxidant Activities under Salt Stress*

The antioxidant activity of *H. cannabinus* extracts was determined via DPPH, ABTS, FRAP, and ferrozine assays. Under salt stress, the antioxidant activity was significantly influenced (Figure 6). The plants treated with 150 mM of NaCl showed the highest antioxidant activity levels in DPPH, ABTS, and ferrozine assays, with values of 83.60%, 91.08%, and 63.68%, respectively. The FRAP results revealed the maximum antioxidant activity in the group treated with 100 mM of NaCl, with a value of 8.49 mmol Fe2+/g.

The relative antioxidant capacity index (RACI) values were calculated by the merging antioxidant capacity values from different chemical methods to rank the samples' antioxidant capacities. The RACI values were calculated using the method described by Mari´c et al. [29] previously. The RACI is the mean value of transformed standard scores derived from initial data without unit or method restrictions. As shown in Figure 7, the group treated with 200 mM of NaCl exhibited the highest RACI value (0.36), followed by the group treated with 250 mM of NaCl (0.35). The lowest RACI value was observed at 150 mM of NaCl.

**Figure 6.** Changes in (**a**) DPPH inhibition, (**b**) ABTS inhibition, (**c**) ferrous-ion-chelating activity, and (**d**) FRAP antioxidant capacity levels in *H. cannabinus* leaves under different salt concentrations (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).

**Figure 7.** The relative antioxidant capacity index (RACI) was applied to combine the antioxidant capacity values from the various methods.

#### *3.8. Correlation Analysis of Physiological and Biochemical Characteristics*

The Pearson's correlations between physiological and biochemical character traits are illustrated in Figure 8. The DPPH and ABTS scavenging capacity levels correlated positively with the proline, total phenolic, and total flavonoid contents. In addition, soluble sugar was positively correlated with the ABTS scavenging capacity. MDA showed a significant positive correlation with the reactive oxygen species (O2 •− and H2O2), proline, and total flavonoid contents. However, MDA was negatively correlated with the plant height and fresh and dry weight of the leaves.

**Figure 8.** A correlation analysis of physiological, biochemical, and antioxidant activities of *H. cannabinus* seedlings under salt stress. \* Indicates significance at the 5% level, while color depth denotes the correlation coefficient. The strength of the correlation is represented by the size of the circles.

#### **4. Discussion**

Soil salinization is now a major environmental threat to the long-term growth of global agriculture. It induces alterations in many physiological and metabolic processes, eventually reducing the crop yield, depending on the severity and duration of the stress [64,65]. Plants can tolerate or avoid saline conditions [65]. This study examined the growth parameters, physiology characteristics, bioactive constituents, and antioxidant capacity of *H. cannabinus* to assess its ability to deal with salinity.

The salt stress inhibited the *H. cannabinus* growth regarding the plant height and the fresh and dry weights of the leaves. Several authors have reported that various levels of salinity stress reduce plant growth in other medicinal plants [43,66,67]. In saline soils, the inhibition of plant growth is primarily caused by osmotic stress, which reduces the absorption of essential macro- and micronutrients [66]. In the current study, the salt stress decreased the concentrations of N, K, Ca, Mg, and P. However, there were no noticeable changes in the N and Mg contents with 50 mM of NaCl and only a slight change in the K content with 100 and 150 mM of NaCl. As previously demonstrated, the decreases in these minerals may be directly related to increased Na uptake by the roots [66]. Moreover, it has been found that NaCl treatment reduces Ca and Mg concentrations in plants [6,43]. However, adequate K, Ca, and Mg are needed to perform fundamental metabolic functions such as cellular K homeostasis, which is necessary for efficient photosynthetic system functioning and stomatal opening regulation [67]. Potassium plays a significant role in plant salinity resistance. Therefore, large quantities are required to reduce osmotic stress in a saline environment [68].

In response to salt stress, it is well established that osmolytes such as organic and inorganic solutes regulate the cellular osmotic potential of plants. The presence of more of these compounds aids in the selection of stress-tolerant cultivars [69,70]. The increased proline and soluble sugar contents protect cells from salt stress by maintaining the osmotic potential and ionic balance in the cytosol and outside of the cell, resulting in increased water and mineral absorption and cell membrane stability [71]. These increases are also commonly used to protect and stabilize enzyme structures against ROS [5,7]. Under salt stress, the proline content increased significantly in the current study. The present findings are consistent with previous research on *Brassica species* [44], *Phaseolus vulgaris* [72], and *Xanthoceras sorbifoliu* [65], which revealed an increase in proline content under salt stress. The enhancement of the proline content might be due to increased activity of the pyrroline-5-carboxylate synthase (P5CS) of the proline biosynthetic pathway in *Hibiscus cannabinus* under salt stress. Higher enzymatic activities, which aid in regulating cellular

structures and functions via interactions with macromolecules, could explain the increased total soluble sugars [73,74]. The current study found a significant increase in the total soluble sugar when exposed to salinity stress, consistent with [75,76]. Furthermore, soluble proteins act as osmotin, and their accumulation may play a role in the development of salt tolerance [6]. The current study revealed that under salt stress, the protein content increased significantly. The present findings are consistent with previous studies [6,7].

Salt stress may disrupt the proper balance between the induced ROS production and elimination, resulting in oxidative stress. Excessive levels of radical species, such as H2O2, O2 •−, and OH, damage plant cell components, resulting in cell death [77,78]. This study investigated the redox state of *Hibiscus cannabinus* seedlings by measuring the H2O2 and O2 •− levels. The levels of hydrogen peroxide and superoxide anion increased significantly as the NaCl concentration increased. Abiotic-stress-induced increases in ROS generation often peroxidize cellular and organelle membrane lipids, resulting in membrane integrity losses [79]. MDA is commonly used to detect lipid peroxidation. It is a marker of oxidative damage induced by salinity stress, and the higher the level of MDA under stress, the greater the degree of membrane damage [69]. In this study, the MDA content under stress was significantly higher compared to the control. Similarly, a previous study found that increasing the NaCl concentration increased the MDA level in *H. cannabinus* [79]. Plants have a variety of defense systems against the harmful effects of oxygen radicals, including osmolytes and antioxidants [7,44].

Plants produce antioxidants such as phenolic and flavonoid compounds to scavenge or detoxify ROS [80]. In salt-exposed plants, the biosynthesis of such compounds is generally stimulated [81]. In this study, the phenolics and flavonoids increased significantly in salt-exposed seedlings compared to the control. Salinity alters the biosynthesis of primary and secondary metabolites in plants, as previously demonstrated in *Carthamus tinctorius* [82] and maize [83]. The increased phytochemicals with antioxidant properties in salt-stressed *H. cannabinus* improved the defense systems necessary to detoxify or prevent the detrimental effect of the increased production of ROS that occurs with stress conditions.

Alterations in the saponin content are reported in many plants subjected to salinity stress [84]. In this study, the saponin content was highest in plants treated with 200 mM of NaCl compared to the control, which then declined significantly with 250 mM of NaCl. Similarly, Mar and colleagues [85] found that the saponins level in *C. quinoa* treated with 200 mM of NaCl increased. Furthermore, the saponin content of cucumber increased with low and moderate salinity levels but decreased significantly with the highest concentration of salt [84]. The total saponin content showed changes under salt treatment, revealing the possible involvement of these compounds in the response of *Hibiscus cannabinus* to salt stress.

Previously, a GC-MS analysis of kenaf leaf hexane extract revealed 13 phytoconstituents [32]. The GC-MS analysis of the kenaf leaves revealed 19 compounds in our study. However, when exposed to salt stress, the relative percentages of the compounds changed significantly. The proportions of phytol decreased as the salt concentrations increased and then disappeared at higher levels of salinity stress. Phytol has antioxidant, antibacterial, anti-inflammatory, neuroprotective, analgesic, and anticancer properties [86]. The production of phthalic acid, heptyl undecyl ester, and β-monoolein disappeared under salt stress. The production of 1-heptacosanol was stimulated at low salinity but disappeared after the severe salt treatment. There have been several reports on the antibacterial and antioxidant activity of 1-heptacosanol [87,88]. Furthermore, a low level of salinity stress stimulated the production of oleic acid, 3-(octadecyloxy) propyl ester. However, a high level of salinity stress did not notably change the level of its content. Oleic acid, 3-(octadecyloxy) propyl ester has potent antifungal activity [89]. Some new compounds appeared under the salt stress, such as β-viscol, diisooctyl phthalate, and 3,7,11,15-tetramethyl-2E,6E,10E,14 hexadecatetraenyl acetate. Furthermore, alkanes such as heptacosane and tetratetracontane appeared at the highest salt concentration. These compounds may be responsible for antibacterial, anticancer, antiviral, and antifungal activities [90]. The biosynthesis of terpenes

was inhibited and then disappeared with 150 and 200 mM of NaCl. These findings are consistent with a previous study in which salt inhibited terpene biosynthesis. Salt treatment can have an impact on the medicinal properties of *Hibiscus cannabinus*.

The present study revealed significant increases in the DPPH, ABTS, FRAP, and ferrozine antioxidant activity levels of *H. cannabinus* under NaCl stress. A previous study found that salt treatment increased the antioxidant capacity of sea lavender in terms of DPPH, ABTS, and FRAP [91]. Muscolo et al. [63] found that the DPPH, ABTS, FRAP, and ferrozine antioxidant activities in lentils increased significantly under NaCl stress. These antioxidant capacities could be associated with phenolic compound levels [92]. Similarly, the current study found that the antioxidant activity of *H. cannabinus* extracts was strongly related to the total phenolic content. The RACI and chemical assay results correlated, suggesting that the RACI could be used to measure food antioxidant power levels [93]. The salinity stress, thus, had a significant impact on the antioxidant capacity of the plant extracts. It could be indicated as a suitable strategy to increase the antioxidant activities of medicinal plants.
