Qualitative test the presence of phenolic compounds

3.2 Qualitative test the presence of phenolic compounds

Callus generated from callus induction process M. oleifera seeds were then used to process the cell suspension cultures. After the callus was inoculated on the liquid medium and agitated it will form crumbs cells dispersed in a medium. Cell suspension cultures can be used for the process of improvement of secondary metabolites with the addition of elicitor [20] and the addition of precursor feeding [11].

Results of cell suspension cultures that had been treated salicylic acid and phenylalanine are then extracted using ethanol and generate total phenol extract compounds that are colorless (clear). Callus extracts of varying concentrations of precursor and elicitor treatment after being given the Folin-Ciocalteureagent produces blue chromogen. Chromogen blue color is the result of a reaction between the reagentFolin - Ciocalteu oxidizing the hydroxyl group (OH-) in the alkaline condition. In Figure 2 describes the mechanism of the reaction between phenol compounds can be reduced by the Folin-Ciocalteu reagent to form molybdenum blue complex [28].

Figure 2. The reactionbetween phenol compounds and the Folin-Ciocalteu reagent

3.3. Effect of salicylic acid in cell suspension cultures of M. oleifera Lam.

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One Way ANOVA test results p 0.092 (p 0.05) shows that the variation of the concentration of salicylic acid did not affect the total concentration of phenolic acids. This can be due to several factors, salicylic acid concentration range used is too low at 0.5 to 1.5 ppm. In addition, optimization of elicitor on cell suspension cultures depend on specificity, concentration, time elicitation, and plant species so that products which are accumulated by each plant is different elicitation results [30]. Calculation of the total concentration of phenol acid using a regression equation tannic acid standard curve y = 4,091x-0,001. Based on Table 1 is known that salicylic acid elicitor treatment of 0.5-1.5 ppm concentration showed an increase in total acid content of phenolic compounds and a reduction in dry weight compared to control cells. The highest concentration of total phenolic acids contained in elicitor treatment salicylic acid concentration of 1 ppm was 1.6067 ± 0.458 ppm and the lowest concentration found in the control 0 ppm was 0.6304 ± 0.148 ppm. Dry weight is highest in the control treatment was 25.1 ± 0 ppm was 2.42 mg dry weight while the lowest for the treatment of 1.5 ppm was 7.7 ± 1.35 mg.

Table 1. Total Acid Phenol concentration and cell dry weight of M. oleifera at elicitor treatment Salicylic Acid

Treatment

Total phenolic acid (ppm)

Cell dry weight (mg)

25,1 ± 2,42 0,5 ppm

12,5 ± 1,85 1,5 ppm

1 ppm

1,6067± 0,458

7,7 ± 1,35 The total phenolic acidincreased in1 ppm concentration of salicylic acid was 1.6067 ±

1,2399 ± 0,211

0.458 ppm but a decline in cell dry weight of 12.5 ± 1.85 mg compared to control, 0.6304 ± 0.148 for total phenol acid and 25.1 ± 2, 42 mg dry weight of cells. Increased total compound total phenolic acid and dry weight of this happening is one response when the plant is in stress condition to defense itself, so the plants produce more secondary metabolites than growth. This is consistent with the statement [31] that one of the compounds that play a role in plant defense mechanisms are phenols compounds. Increased phenol in salicylic acid elicitor treatment allegedly due to an increase in ROS (Reactive oxigen species) which is the initial response elicitor giving salicylic acid [31]. In addition, salicylic acid also affects the formation of the enzyme PAL (Phenylalanine ammonia lyase), which acts in the phenylpropanoid pathway and several enzymes that act to stabilize the ROS such as peroxidase and SOD (superoxide dismutase) [32].

An increase in ROS (Reactive Oxygen Species) which is in line with the increase in the concentration of salicylic acid may cause decreasingcell growth. According to [31] increased levels of ROS in the cells can cause direct binding of the enzyme catalase or other macromolecules such as proteins and lipids. So that the excessive accumulation of ROS can cause oxidative damage and cell death. In addition, salicylic acid also affects the increase in the hormone ABA (abscisic acid) that function in the process of defense against abiotic stress and dormancy. It can be presumed that the increase in ABA can cause a decrease in cell growth because more cells produce compounds such as phenols defense [33]. Results of the study

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[31] showed that the concentration of 3.125 to 12.5 mg/L showed a decrease in dry weight and an increase in phenolic compounds on cell cultures of S. Miltiorrhiza . At the Vitisvinifera plants giving 50 M salicylic acid methyl decreased dry weight of

16.4 ± 0.5 g/l compared control 17.6 ± 0.9 g/l and the content of phenolic compounds as much as 790 ± 30 mg / l compared to 760 control ± 60 mg/l after a ten-day incubation period [20]. Whereas in plants Chickpea ( Cicerarietinum L.) addition of 1.5 mM salicylic acid can increase the phenol content <60 mg after 96 hours of incubation [32].