*2.8. Determination of Total Flavonoid Content*

The flavonoid content was quantified following the method of Zhishen et al. [23]. The 0.5 mL of plant extract solution was added to 1.0 mL methanol, 0.5 mL of aluminum chloride, and 0.5 mL of 1 M potassium acetate and allowed to stand for 30 min. The absorbance of the reaction mixture was detected at 415 nm with a UV/Vis spectrophotometer (Shimazdu UV-2600, Kyoto, Japan). The total flavonoid content was calculated as quercetin from a calibration curve prepared by using quercetin solutions of different concentrations from 12.5 to 100 mg mL−<sup>1</sup> in methanol.

### *2.9. Determination of Total Antioxidant Activity*

The 1,1-Diphenyl -2-picryl-hydrazyl (DPPH) radical degradation method was used to estimate antioxidant activity [24]. The plant extracts (1 mL) were added at different concentrations with volumes equal to the methanolic solution of 10 mL DPPH (100 μM) in a test tube. The mixture was shaken vigorously and was then allowed to stand in the dark. After 15 min, the absorbance was detected at 517 nm as a lower IC50 value corresponding to its higher antioxidant activity. This measurement was repeated three times. The IC50 values indicate the concentration of the sample.

#### *2.10. Statistical Analysis*

The data was subjected to analysis of variance (ANOVA) using the Statistical Package for the Social Science (SPSS) software version 12. If the F-test was found significant, mean comparison was performed using the least significant difference (LSD) test at 5% level.

#### **3. Results**

#### *3.1. Plant Growth and Biomass Production*

Salinity significantly reduced the centella growth at all NaCl concentrations except 25 mM (Table 1). Plant growth was detrimentally reduced at 100 mM NaCl. High salinity level (100 mM) reduced the number of leaves, leaf area, and specific leaf area by 45%, 38.4% and 35%, respectively. Low and moderate salinity levels (25 and 50 mM NaCl) had no significant effect on the rosette diameter and petiole length of centella, while high NaCl concentration (100 mM) reduced these up to 18.9% and 33.6%, respectively.

**Table 1.** Morphological parameters of *Centella asiatica* L. as affected by different salinity levels. The experiment was carried out in triplicate. The data are presented as treatment mean ± SD. Different letters in the same column represent significant differences at the *p* < 0.05.


Plant dry weight also decreased with incremental salinity, except at 25 mM NaCl (Table 2). The decrease in dry weight ranged from 5.9 to 13.9% with highest reduction of 19.7% at 100 mM NaCl compared with the control.

**Table 2.** Biomass production of *Centella asiatica* L. as affected by different salinity levels. The experiment was carried out in triplicate. The data are presented as treatment mean ± SD. Different letters in the same column represent significant differences at the *p* < 0.05.

