**1. Introduction**

*Centella asiatica* L. Urban (centella) as a medicinal plant of the Apiaceae family that has been used to treat a number of diseases, including varicose veins, certain eczemas, hypertonic scars, and keloids [1]. Centella is also considered a valuable plant in the cosmetics and pharmaceutical industries. As a leafy vegetable, this species is consumed as a juice blend in many Asian countries including Vietnam and Malaysia. The medicinal properties of centella are determined by a variety of phytochemicals such as phenolics, flavonoids, and terpenes [2]. The concentration of these compounds is influenced by several environmental stress factors, including salinity [3].

Salinity as an environmental stress factor affects arable lands worldwide, causing an annual monetary loss of approximately \$27.3 billion to the agricultural industry [4]. Excessive salt concentration reduces the water potential, resulting in osmotic stress and an increase in the toxic accumulation of sodium and chloride in plant cells. Ionic toxicity and osmotic stress disrupt photosynthetic functions and reduce growth of plants [5]. This results in the accumulation of reactive oxygen species (ROS), including hydrogen peroxide (H2O2), superoxide anions (O2•−), singlet oxygen (1O2), and hydroxyl radicals (OH•), resulting in protein, DNA, and lipid damages [6]. The extent of the damage depends on the type, variety, and growth stage of the plant [7].

Plants have evolved antioxidant defense systems to reduce oxidative damage from salinity stress. Phenolic compounds play a major role in scavenging free radicals [8] by acting as hydrogen or electron donors that stabilize and delocalize unpaired electrons or chelate metal ions, preventing the generation of ROS [9]. Plants with higher antioxidant levels have an increased tolerance to damages by ROS [10]. Recent studies have shown various changes in antioxidant compounds when plants are subjected to salinity.

**Citation:** Hoang, H.L.; Rehman, H. Unravelling the Morphological, Physiological, and Phytochemical Responses in *Centella asiatica* L. Urban to Incremental Salinity Stress. *Life* **2023**, *13*, 61. https://doi.org/ 10.3390/life13010061

Academic Editors: Hakim Manghwar and Wajid Zaman

Received: 6 December 2022 Revised: 20 December 2022 Accepted: 21 December 2022 Published: 25 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Salinity increased the total phenolic content in *Thymus vulgaris* L. [11] and *Brassica oleracea var. acephala* [12]. However, higher salt levels reduced the phenolic content of *Ocimum basilicum* L. [13] and *Nigella sativa* [14]. The significant correlation among phenolic content, antioxidant activity, and salt tolerance is well documented in literature [15]. For example, Sarker et al. [16] reported that salinity stress enhanced total phenolic, total flavonoid, and antioxidant activity of amaranth. Razieh et al. [17] also observed that phenolic content and antioxidant activity were significantly increased by salt stress in wheat. Santander et al. [18] determined that the greatest total phenolic content and antioxidant activity in lettuce prevailed at 50 mM NaCl treatment.

Despite numerous studies reporting the responses of plants to saline stress, there is limited research available on responses of centella to incremental salinity. The present study, therefore, determined the growth and biomass production of centella under saline conditions and their effects on ionic uptake, phytochemical content, and antioxidant activity.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Experimental Design*

Seeds of *Centella asiatica* L. were collected from the Lucky Seed Company, Vietnam. The seeds were sown in trays containing a mixture of coconut fiber and sand. At the third leaf stage, seedlings were transplanted into garden soil-filled, plastic pots (20 × 18 × 10 cm) containing 2.5 kg soil. The soil had a pH of 5.5, 1.3 ECe, 35% organic matter, 0.5 mg L−<sup>1</sup> Na+, 0.88 mg L−<sup>1</sup> Cl−, 0.65% N, 0.71% K2O, and 0.62% P2O5. The study was conducted in a greenhouse in Huong Tra Town, Thua Thien Hue Province, Vietnam, from February to November 2021.

The NaCl was applied as: 0, 25, 50, 75 and 100 mM. The experiment design was a completely randomized block (CRB) design with three replicates, and each replicate included 15 plants. One seedling was transplanted per pot. The soil was drenched with 25, 50, 75, and 100 mM NaCl after transplanting. NaCl was stepped up in daily increments of 25 mM until reaching the final concentration of each treatment. No additional nutrients or fertilizers were added. The experiment was terminated 45 days after transplantation. The plants were evaluated for plant growth, as well as phytochemical and ionic analyses.

#### *2.2. Growth Measurement*

Plant phenotypes, including the number of leaves, rosette diameter, petiole length, total leaf area, and specific leaf area were recorded. The millimeter graph paper method was used to measure total leaf area per plant [19]. At the end of experiment, shoots and roots were sampled, oven-dried at 60 ◦C for 60 h, and weighted.

#### *2.3. Total Chlorophyll Content Measurement*

Two hundred mg of fresh centella leaves were chopped and ground into fine powder in 5 mL 60% acetone (*v/v*). The extractant was filtered, and then the diluted acetone was added to make up the 20 mL final volume. The supernatant was recorded spectrophotometrically at 663 and 645 nm, and the formula given by Lichtenthaler (1987) was used to calculate the total chlorophyll content:

#### Total chlorophyll = 7.15A663 + 18.71A645

#### *2.4. Electrolyte Leakage (EC) Measurement*

The method described by Lutt et al. [20] was used to determine electrolyte leakage. The top 4th leaf was collected and thoroughly rinsed with distilled water to remove contamination. The samples were put into stoppered vials containing 10 mL of distilled water and then incubated at 25 ◦C on a shaker at 100 rpm for 24 h. After incubation, the electrical conductivity of the bathing solution (EC1) was immediately measured. After this, the same leaf samples were placed in an autoclaved at 120 ◦C for 20 min, and again, a reading EC2 was measured at room temperature using the portable meter HI993310 (Hanna

Instrument Company, Woonsocket, RI, USA). The electrolyte leakage was measured as a ratio of EC1/EC2 and expressed as a percentage.
