*3.5. Shoot and Root Na<sup>+</sup> and Cl*<sup>−</sup> *Content*

The presence of NaCl in the soil medium resulted in the accumulation of Na<sup>+</sup> and Cl<sup>−</sup> in the roots and shoots of centella, with higher accumulation found in the roots than in the shoots (Figures 4 and 5). Incremental salinity increased the accumulation of highest Na<sup>+</sup> content with the highest observed in the roots at 100 mM NaCl, which was 5.7 times greater, followed by 3 and 4.4 times at 50 mM and 75 mM NaCl salinity, respectively. A similar trend in Na<sup>+</sup> content was observed in the shoots. The highest Na+ in the shoot 3.8 times higher was found at 100 mM NaCl than the control.

**Figure 4.** (**a**) Shoot and (**b**) root Na+ content of *Centella asiatica* L. as affected by different salinity levels. The experiment was carried out in triplicate. Different letters represent significant differences at the *p* < 0.05.

**Figure 5.** (**a**) Shoot and (**b**) root Cl− content of *Centella asiatica* L. as affected by different salinity levels. The experiment was carried out in triplicate. Different letters represent significant differences at the *p* < 0.05.

#### *3.6. Phytochemical Content*

Salinity stress significantly increased the total phenolic and total flavonoid contents of the centella (Figure 6). The highest total phenolic and flavonoid contents were found in the plants at 75 mM NaCl. The increase in total phenolic and total flavonoid contents augmented the antioxidant activity up to 34% when compared to the control (Figure 7). At a high salt concentration (100 mM NaCl) their accumulation was reduced; however, it was not significantly different from 75 mM NaCl salinity.

**Figure 6.** (**a**) Total phenolic, (**b**) total flavonoid (**b**) of *Centella asiatica* L. as affected by different salinity levels. The experiment was carried out in triplicate. Different letters represent significant differences at the *p* < 0.05. (GAE: gallic acid; QE: quercetin; DW: dry weight).

**Figure 7.** Antioxidant activity of *Centella asiatica* L. as affected by different salinity levels. The experiment was carried out in triplicate. Different letters represent significant differences at the *p* < 0.05.

#### **4. Discussion**

The results of the present study showed that centella growth was decreased by incremental salinity. A similar response in plant growth was reported in *Portulaca oleracea* L. [25], *Prosopis strombulifera* [26], and *Tetragonia decumbens* [27] due to salt stress. High saline concentrations reduced growth by decreasing the uptake of water and nutrients by the plants [28], accumulating toxic ions in the plant cells, and disrupting the metabolic pathways [29]. In this study, specific leaf area decreased with an increase in salinity. Burslem et al. [30] showed that a higher leaf thickness is associated with an increase in the ratio of mesophyll area available for the absorption of CO2 per unit leaf area, thereby enhancing CO2 assimilation and biomass production. However, Omami et al. [31] found that CO2 assimilation decreased with increasing salinity in amaranth. They suggested that the lower specific leaf area in salt stressed plants overloaded the leaves with inorganic and organic solutes, thereby permitting osmotic flow but limiting the efficient use of carbon. Increase in the leaf thickness could be an adaptation of the plant to increase intercellular space and to counteract the decrease of transpiration [32].

The current study indicated that the root biomass decreased under high salt concentration treatments. According to Banaka et al. [33], the main reasons for reduced plant growth and biomass under high salinity were ion toxicity and nutrient imbalance. Moreover, the increase of soluble salts in the soil leads to an increase of osmotic pressure and a reduction of water potential, thus reducing the water uptake by the root [34]. In this study, although salt stress inhibited plant growth and decreased biomass production, the root/shoot dry weight increased. This indicated that salinity affected the aboveground part more severely than the underground part and the plant had the ability to change biomass allocation. It means that the plants had the ability to maintain the root system while salt stress inhibited shoot growth. This response is one of the most popular strategies of plants to adapt to abiotic stress.

Chlorophyll content is an important factor in assessing photosynthetic activity in plants [35]. The results showed a decrease in the total chlorophyll content of the centella under saline conditions. Previous studies showed that the depletion of photosynthetic pigments reducing plant growth and crop yield under saline stress was also evident from a significant relationship between total chlorophyll content and biomass production in the present study (r = 0.9, *p* < 0.001) (Table 3). This was observed in *Amaranthus tricolor* [36], *Typha domingensis* [37], and *Lactuca sativa* L. [38].

There was also a negative correlation between the total chlorophyll content and the shoot Na+ content (r = −0.67, *<sup>p</sup>* < 0.001), showing degradation of photosynthetic pigments under the incremental salinity (Table 3). This leads to a reduction in biomass production as indicated by the negative correlation between fresh weight/dry weight with Na+ concentration (Table 3). Depletion of chlorophyll under saline conditions may be caused by the accumulation of toxic ions, such as Na+ and Cl<sup>−</sup> inhibiting the enzymes function responsible for chlorophyll synthesis [39]. Zahra et al. [40] also reported that salt stress could reduce the CO2 supplement through hydrostatic stomata closure or by changing the mesophyll conductance. According to Farhat et al. [41], a high salt concentration may damage the thylakoid membranes and protein modulation by inhibiting photosynthesis. Recent studies showed that the formation of ROS disrupted the chloroplasts and ultimately reduced the total population of *Brassica napus* [42], *Chenopodium quinoa* [43] and *Solanum lycopersicum* [44].

In this study, the centella was able to maintain membrane stability under slight salt stress (Figure 2) as evident from the electrolyte leakage which increased when the plants were subjected to a high salt concentration. A similar response was observed by ElYacoubi et al. [45] in ryegrass and by Behdad et al. [46] in licorice. This was mainly due to the efflux of K<sup>+</sup> and the flow of counter ions (Cl–, HPO4 2–, NO3 –, citrate3–, and malate2–) counterbalancing the efflux of K<sup>+</sup> [47]. According to Tavakkoli et al. [48], the distribution of Na<sup>+</sup> within cells and organs may subsequently cause toxic effects on membrane permeability and increased electrolyte leakage.

In this study, the increase of Na<sup>+</sup> and Cl<sup>−</sup> concentrations in the tissues was accompanied by salinity stress. High accumulations of Na+ and Cl<sup>−</sup> reduced plant growth. High Na+ concentrations interfered with the absorption of K<sup>+</sup> and Ca2+ ions and disturbed stomatal regulation, thereby inhibiting photosynthesis and growth. High Cl− concentrations caused the degradation of chlorophyll, leading to a reduction in the photosynthesis rate [48]. However, plants have different coping mechanisms for dealing with Na<sup>+</sup> toxicity. Some plants transport Na+ from the roots to the leaves where it is retained in the vacuoles, whereas others store Na<sup>+</sup> in the roots [49]. Salt tolerance is associated with the ability to limit the uptake and/or to transport Na+ from the root zone to aerial parts [50]. Based on the distribution of Na+ and Cl<sup>−</sup> between shoots and roots, a similar mechanism could occur in centella. The accumulation of Na+ and Cl<sup>−</sup> in the roots provided a mechanism for centella to cope with salinity in the rooting medium. This mechanism reduced the transport of Na+ and Cl<sup>−</sup> to the leaves, thereby reducing the impact of the toxic ions to the aboveground parts of the plant. The leaves of centella are usually harvested, which is advantageous for growing this plant in saline environments. This mechanism has also been reported in amaranth [36] and rapeseed [51].

One of the effects of salt stress on plants is the overproduction of ROS, which leads to oxidative stress. However, plants have evolved mechanisms to counteract the effects of this process by producing compatible metabolites and different antioxidants [10]. Phenolic compounds are the most abundant secondary metabolites in the plant kingdom which have a pivotal effect in scavenging the excessive ROS. Flavonoids as a group belong to phenolic compounds and are known to have antioxidant properties [10]. The presence of phenolics and flavonoids in plants contributed to the prevention of cell damage by abiotic stress, as demonstrated by several studies on peas [52] and kale [53]. These compounds neutralize the radicals accumulated in lipids or prevent their breakdown into free radicals. Furthermore, they can inhibit lipoxygenase activity, thus preventing lipid peroxidation [54,55]. The result showed that there was a significant increase in the phenolic and flavonoid content in response to salt stress. The increase in phenolic and flavonoid content indicates that they play a significant role in the adaptation of centella to salinity as evident from a positive correlation between total phenolic content and antioxidant activity in the present study (Table 3). The increase in these compounds is related to their function as a non-enzyme antioxidant to counteract the increase of ROS and hence contribute to the plant's health under salt stress. In the present study, antioxidant activity of the centella leaf increased with the salt treatments, and the highest antioxidant activity was observed at 100 mM NaCl. This finding is consistent with the important relationship that exists between antioxidant activity and the total phenolic content in the leaves of *Leucojum aestivum* and *Lactuca sativa* under salt stress conditions [19,56]. Although the centella was also negatively affected by salt stress, as demonstrated by yield decline and increased accumulation of Na+ and

Cl− ions, the study results showed an increase in phytochemicals content and antioxidant activity in centellas. This opens the way to cultivating this plant in saline soils to boost the production of bioactive compounds used in the pharmaceutical and cosmetics industries. However, studies on extraction techniques for specific bioactive compounds should be carried out to ensure the exclusion of ions and impurities.

#### **5. Conclusions**

Salinity stress caused a reduction in biomass yield and induced some physiological and phytochemical modification in centella. The results indicated that *Centella asiatica* showed moderate tolerance to severe salt stress, which was attributed to the exclusion of Na+ and Cl<sup>−</sup> in the root to protect the aboveground plant tissues from salt toxicity and to increase the total phenolic and flavonoid content of the centella. The centella is an herb with a rich source of phytochemical content. Thus, the response of the centella under salt conditions may be used to improve the production of bioactive compounds to be used in the manufacture of pharmaceuticals, supplements, food, and cosmetics.

**Author Contributions:** Conceptualization, H.L.H.; Methodology, H.L.H.; Software, H.R.; Validation, H.R.; Formal Analysis, H.L.H.; Investigation, H.L.H.; Resources, H.L.H.; Data Curation, H.L.H.; Writing—Original Draft Preparation, H.L.H.; Writing—Review & Editing, H.L.H. and H.R.; Visualization, H.L.H.; Supervision, H.R.; Project Administration, H.L.H. and H.R.; Funding Acquisition, H.L.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Education and Training (Grant numbers B2020-DHH-03).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data recorded in the current study are available in all tables and figures of the manuscript.

**Acknowledgments:** We are grateful to University of Agriculture and Forestry for the support to conduct this research.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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