The Effect of Elicitors on Total Flavonoid Content
3.3. The Effect of Elicitors on Total Flavonoid Content
The total flavonoid content of the samples was measured using quercetin as standard. Standard curve of quercetin is presented in Figure 4.
Figure 4. Standard Curve of Quercetin on Wavelength 510 nm
Regression equation y = 0,0003x + 0.0112 shows a linear relationship between absorbance and concentration measurement with a correlation coefficient 0.9857 (Figure 4). Figure 5 presents the results of the average concentration of total flavonoid in each treatment after elicitation treatment.
Figure 5. Average concentration of total flavonoid
The highest average concentration of total flavonoid (71.56±2.24 mg/L) was 0.5 mM salicylic acid treatment whereas the lowest total flavonoid concentration (23.78±1.89 mg/L) was the control. The total flavonoid concentrations in each replicate are shown in Table 1. Data were analyzed using ANOVA One Way with the acquisition of P<(0.05) which means that the concentration of elicitor (salicylic acid and NaCl) significantly influenced the total flavonoid content. Furthermore, Tukey test was conducted to determine the significance of the treatments.
Sept em ber 8 th
9 – th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a
Table 1. Total Flavonoid Concentration
Total Flavonoid Concentration (mg/L) Treatment
Average±SE Control b 19.33 16 36 23.78±1.89
Salicylic acid 0.5 mM a 56 72.67 86 71.56±2.24 Salicylic acid 1 mM ab 42.67 32.67 52.67 42.67±1.83 Salicylic acid 1.5 mM ab 32.67 59.33 76 56.00±2.70 NaCl 0.5 mM ab 49.33 56 36 47.11±1.84 NaCl 1 mM ab 29.33 56 56 47.11±2.27 NaCl 1.5 mM ab 46 62.67 49.33 52.67±1.71
The same letter in the column shows no significant difference by Tukey's test with a confidence level of 95%. R1: Repetition 1; R2: Repetition 2; R3: Repetition 3; SE: Standard Error.
The total flavonoid concentration of 0.5 mM salicylic acid treatment was significantly different from control. Salicylic acid triggers the increase of genes expression involved in flavonoid biosynthesis, thus accumulating the total flavonoid (e.g. CHS , CHI , F3H and ANS ) (Tounekti et al ., 2013). Furthermore, salicylic acid may trigger a signaling pathway that regulates the expression of genes encoding enzymes associated with phenylpropanoid pathway, e.g. chalcone synthase, the first enzyme for producing flavonoid compounds in the phenylpropanoid pathway (Ghasemzadeh et al ., 2012).
The total flavonoid concentration of salicylic acid treatment decreased at concentration of 1 mM and increased at concentration of 1.5 mM. Secondary metabolites of most plants are produced during the stationary phase (Malik et al .,, 2013). Plant cells treated by 1.5 mM salicylic acid were presumed to have longer stationary phase than plant cells treated by 1 mM salicylic acid. Therefore, the production of total flavonoid was also higher in the former. High concentration of salicylic acid may also activate pathways of other secondary metabolites, such as terpenoids (Tounekti et al ., 2013).
The total flavonoid concentration treated by NaCl 1.5 mM was higher than NaCl 0.5 mM and 1 mM, although the results of these treatments were not significantly different. The result obtained was in accordance with the study conducted by Hussein and Aqlan (2011) in which the addition of NaCl increased the total flavonoid production of Trigonella foenum-graecum L. NaCl causes perturbation of ionic steady state not only for Na + and Cl - but also for K + and Ca 2+ in the plant cells. External Na + negatively impacts intracellular K + influx, attenuating acquisition of this essential nutrient by cells. High NaCl causes cytosolic accumulation of Ca 2+ , a signal stress