The Antioxidant Activity of Sterculia stipulata Korth Woods and Leaves by FRAP Method

The genus Sterculia rich of metabolite secondary flavonoids and phenolic compounds. The phenolic compounds such as phenolic acid, propanoic phenyl, coumarins, lignans, and lignin with much smaller amounts than flavonoids.1 The ironreducing antioxidant power (FRAP) test was a method used to determine antioxidant activity by reducing Fe (III) to Fe (II) by complexation with 2,4,6-Tripyridyl-s-Triazine (TPTZ). The test is carried out at acidic pH 3,6 to maintain iron solubility.2 FRAP value obtained with comparing changes in the absorption of the test mixture at 593 nm with the mixture, which contains Ferro ions whose concentration is known.3-5 The FRAP test based on electron transfer. FRAP cannot detect compounds that work by radical reduction (hydrogen transfer). Method FRAP testing is simple, fast, inexpensive, and requires no special equipment.2 In this study, the antioxidant activity of S. Stipulata determined. Antioxidants play an important role in the prevention and treatment of metabolic disorders caused by oxidative stress.6 Some sterculia genus plants are known to have antioxidant activity, including methanol extract Sterculia foetida stems with IC50 values of 20 μg/ ml.7 Sterculia villosa bark ethyl acetate fraction with IC50 23.99 μg/ml. 8


INTRODUCTION
The genus Sterculia rich of metabolite secondary flavonoids and phenolic compounds. The phenolic compounds such as phenolic acid, propanoic phenyl, coumarins, lignans, and lignin with much smaller amounts than flavonoids. 1 The ironreducing antioxidant power (FRAP) test was a method used to determine antioxidant activity by reducing Fe (III) to Fe (II) by complexation with 2,4,6-Tripyridyl-s-Triazine (TPTZ). The test is carried out at acidic pH 3,6 to maintain iron solubility. 2 FRAP value obtained with comparing changes in the absorption of the test mixture at 593 nm with the mixture, which contains Ferro ions whose concentration is known. [3][4][5] The FRAP test based on electron transfer. FRAP cannot detect compounds that work by radical reduction (hydrogen transfer). Method FRAP testing is simple, fast, inexpensive, and requires no special equipment. 2 In this study, the antioxidant activity of S. Stipulata determined. Antioxidants play an important role in the prevention and treatment of metabolic disorders caused by oxidative stress. 6 Some sterculia genus plants are known to have antioxidant activity, including methanol extract Sterculia foetida stems with IC 50 values of 20 µg/ ml. 7 Sterculia villosa bark ethyl acetate fraction with IC 50 23.99 µg/ml. 8

Extraction
Woods and leaves of Sterculia stipulata extracted by n-hexane, ethyl acetate, and the final solvent was methanol. Extraction by maceration method. The methanol extract identified the antioxidant activity and also the phenols total and flavonoids total.

Antioxidant activity with FRAP
The antioxidant activity with the FRAP method for sample preparation was carried out by five (5) mg sample (extract/quercetine) of 5 mg dissolve in 2 ml methanol p.a, (Concentration to 2500 μg/ml).
Based on the formula, FeEAC was the equality of ferric ions with antioxidant activity (µmol/g). ∆A = absorbance of samples that have been reduced by blank. GRAD (M -1 ) was the gradient of the AFS calibration graph. Av = total volume for the test (300 μl) , Spv = sample volume (30 μl), C ext = concentration of sample stock, weight (gram) in volume (g/l), D = dilution factor for sample before analysis (D = 1 if sample not diluted). GRAD (gradient) determined from the calibration curve on AFS.

Determination of total flavonoids content
The total flavonoids content determined by the method described by Farasat et al. 11 Twenty (20)

Determination of total phenols content
The total phenol content in the extract determined using the method from Farasat et al., with slight modifications. Twenty (20) µl of extract was added with 100 µL of Folin-C Reagent (1:10), shaking for 60 seconds and allowed to stand for 4 minutes. Added with 80 µl solution of Sodium carbonate (Na 2 CO 3 ) 7.5% in water, shaking for 60 s. This mixture was incubated at room temperature in a dark place for 2 hours. Furthermore, it read at 600 nm. The concentration of extract in the sample made at 100 µg/ml. The concentration of stock solutions was 1000 µg/ml. Blank was samples replaced with methanol. The treatment was the same as sample. In total phenols determination using gallic acid as the standard, total phenols calculated as equality of gallic acid (mg GAE/gram).

Total phenols compound
Determination of total phenols content in the methanol extract of wood and leaves of Sterculia stipulata showed that the total phenols content in wood was greater than that found in the leaves. The results showed in Table 1.

Flavonoids compound
Determination of total flavonoids levels in Sterculia stipulata results in greater flavonoids content in leaves compared to wood. The results showed in Table 2.

Antioxidant activity with FRAP (Ferric reducing antioxidant power)
In testing antioxidant activity using the FRAP method using the AFS comparison. The results of the regression curve for AFS standard chart results shown in the Figure 1.
The antioxidant activity with the FRAP method gives the results that wood extract has a greater activity compared leaves extract 41.17 ± 1.99 FeEAC (mol/g), while for woods it was 4.74 ± 1.03 FeEAC (mol/g). This value is still far compared to the positive control quercetine of 1201.61 ± 77.89 FeEAC (mol/g). The antioxidant activity showed on Table 3.

Phytochemical screening
Identification of the content of chemical compounds is carried out on terpenoids/steroids, alkaloids, tannins, flavonoids, saponins and anthraquinones. The results of their identification showed on Table 4.
For the result of chemical content showed that there is no differences between woods and leaves.
The FRAP test based on the ability of the phenol to reduce the yellow color of ferric tripyridyltriazine (Fe (III) -TPTZ) to the blue color of the Ferro (Fe (II) -TPTZ complex) by antioxidant activity that contributes to electrons. The blue color produced was measured spectrophotometrically at 593. Ferric salt used as an oxidant and its redox potential (<0.70 V). The FRAP test required an acidic condition (non-physiological, low pH value = 3.6) to maintain iron solubility. One FRAP unit defined as a reduction of 1 mol of Fe (III) to Fe (II)). 10,12 The advantages of FRAP Test are simple, fast, cheap, and strong and do not require special equipment. FRAP tests can done using automatic, semiautomatic, or manual methods. 2 Fe (II) was a prooxidant that can react with H 2 O 2 to produce hydroxyl radicals (OH -), dangerous free radicals found in vivo. The ability of compounds to produce Fe (II) from Fe (III) is defined as "antioxidant strength" in the FRAP test. The ability to reduce Fe (III) is possible reflecting the ability to reduce reactive species. However, not all reductants capable of reducing Fe (III) are antioxidant. [13][14][15] The total phenols, total flavonoids and antioxidant activity in leaves better than woods. Phenols and flavonoids have an antioxidant activity. 11  Note : + = presence, -= absence