Anti Oxidant Activity Test

3.1 Anti Oxidant Activity Test

The antioxidant activity of various foods can be determined accurately, conveniently, and rapidly using DPPH testing. The trend in antioxidant activity obtained by using the DPPH method is comparable to trends found using other methods reported in the literature. This method can be used successfully for solid samples without prior

extraction and concentration, which saves time [16] .

3.1.1 Quantitative Analysis of Antioxidant Activity using DPPH (2,2-diphenyl-1- picrylhydrazyl)

The quantitative measurement of the scavenging of DPPH radical allows one to determine exclusively the intrinsic ability of substance to donate hydrogen atom or electrons to this reactive species in a homogenous system. The method is based on the reduction of methanol-DPPH solution with the presence of antioxidant substances having hydrogen donating groups (RH) such as phenolics and flavonoids compounds due to the formation of non radical DPPH-H form [15] . The primary reaction, which takes place, is the formation of free radical R . and the reduced form of DPPH (Figure 1).

Figure 1. Structure of DPPH and its reduction form by the antioxidant RH (Rohman et al., 2010)

The parameter used to measure/evaluate the radical scavenging activity of extracts and fractions was IC 50 , defined as the concentration of antioxidant required for 50% scavenging of DPPH radicals in a specified time period. The smaller the IC 50 value,

the higher the antioxidant activity [12] . On the wavelength scan it was found that the maximum wavelength for these experiments was 521 nm, while the time scan was

30 min and 34 min for the conventionally dried and for the DIC fruits, respectively.

Table 1. Antioxidant activity (IC50 value) of Red Fruit Extracts and Red Fruit Oil by DPPH method

No DIC-assisted

Oil Ethanol Ext

Conv. Drying

DIC-assisted

Conv. Drying

Ethanol Ext

Hexane extr

Hexane extr

1 y= 0.1506x+1.6771 0.0729x+2.1058 0.0011x+0.5221 0.001x-2.4677 0.0099x+1.9138 IC50(ppm) 320.87

0.1326x+4.1219 0.0724x+3.485 0.0011x+0.4311 0.001x-2.7523 0.0098x+2.4401 IC50(ppm) 345.99

0.001x-2.4225 0.0101x+1.5917 IC50(ppm) 336.26

0.1412x+2.5194 0.0743x+3.2137

0.0011x-0.1239

4792.09 Average (ppm) 334±13

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Table 2. IC50 value of the extracts, oil, and positive control equal to the concentration in extracts

Extracts IC 50 Value ± SD (mg/L) Ethanol Extract of DIC red fruit powder

334.37 ± 12.66 Ethanol Extract of conventionally dried red fruit powder

643.05 ± 13.65 Hexane Extract of DIC red fruit powder

45203.24 ± 317.88 Hexane Extract of conventionally dried red fruit powder

52547.50 ± 178.80 Red Fruit Oil

4834.38 ± 35.98 Positive Control ( α-Tocopherol)

38.73 ± 0.66 Positive Control (Catechin monohydrate)

19.70 ± 0.18 Positive Control (Gallic Acid)

9.90 ± 0.03

The ethanol and the hexane extracts obtained from the DIC red fruit powder had antioxidant activities approximately better than the un-treated ones. From the data above, all of the red fruit extracts and red fruit oil were not as potent as the positive controls of antioxidant. The positive controls which were used were α-tocopherol, catechin monohydrate and Gallic acid, and the concentrations were taken equal to their concentrations in the extracts. The intensity of antioxidant activity of active substances using the DPPH method can be classified according to the values of IC 50

Table 3. The intensity of antioxidant activity with the DPPH method

Intensity

IC 50 Value

Very strong

< 50 μg/mL

Strong

50-100 μg/mL

Moderate

101-150 μg/mL

Weak

> 150 μg/mL

As seen from IC 50 value, red fruit extracts and red fruit oil were classified as weak antioxidants. All of the ethanol and hexane extracts and the red fruit oil cannot be considered as potent antioxidants. Besides, according to Molyneux et al., (2004) if

the IC 50 value of active substances at the concentrations of 200-1000 ppm, the substances is less active but still have an antioxidant activity. But, when observed from the active substances content of the red fruit extract, antioxidant activity from the substances in its extract can be classified as a potent antioxidant.

Table 4. IC 50 value of red fruit extracts and red fruit oil based on its

flavonoid content

No. Sample

Flavonoid

Extract in

Flavonoid in

IC 50 Value from

bulk

extract

flavonoid content (ppm)

13.81 ± 0.88 red fruit powder dried conventionally 2. Ethanol Extract of

1. Ethanol Extract of

7.99 ± 0.30 red fruit powder pre-dried by DIC 3. Red Fruit Oil

Sept em ber 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a

Table 5. IC 50 value of red fruit extracts and red fruit oil based on its

total phenol content

No. Sample

Total

Extract in Total phenol in IC 50 Value from

total phenol content (ppm)

24.88 ± 0.49 conventionally dried red fruit powder 2. Ethanol Extract of DIC

1. Ethanol Extract of

14.31 ± 0.54 red fruit powder 3. Red Fruit Oil

Table 6. IC50 value of red fruit extracts and red fruit oil based on its α -tocopherol content

No. Sample

α-tocopherol

extract

α-tocopherol

IC 50 Value from

in bulk

in extract

α-tocopherol content (ppm)

1. Hexane Extract of

339.02 ± 2.39 conventionally dried red fruit powder 2. Hexane Extract of

189.17 ± 0.64 DIC red fruit powder 3. Red Fruit Oil

All of the extracts cannot be considered as a potent antioxidant because the percentage of the active substances on the whole extracts are low. This condition may be due to the high percentage of Cab-O-Sil in the bulk, causing the active substances that act as antioxidant in the extracts cannot dissolve completely because the extracts is bound to a high percentage of Cab-O-Sil.

The solvents that are used to pull the active substance out were ethanol and hexane. Ethanol is a polar solvent due to its hydroxyl (OH) group, with the high electronegativity of oxygen allowing hydrogen bonding to take place with other molecules. While hexane is a non-polar solvent due to the bonds between carbon and hydrogen in hexane are uniform. Since in this research ethanol was used only as a polar solvent and hexane as a non polar solvent, a further study is needed to better study the impact of other solvents for extraction. Probably, a semi polar solvent can be used to dissolve not only polar, but also non polar active substances.

Another reason is may be due to the dynamic maceration as extraction method, because it should not allow optimally extracting all the active substances. Further studies need to be achieved using the same dynamic maceration as extraction method but with extraction of the red fruit powder repeatedly.

3.1.2 Data Analysis The results of antioxidant activity in the red fruit extracts and the red fruit oil were analyzed statistically by one-way ANOVA using MINITAB program. This aimed at establishing the effect of DIC treatments on the antioxidant activity of the red fruit extracts. In this experiment, the extract concentrations act as a factor (independent

variable), while IC 50 Value as a response (dependent variable).

Sept em ber 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a

Figure 2. Normal Probability Plot of Red Fruit Extracts and Red Fruit Oil

Minitab Output 1. One-way ANOVA DIC, Ethanol Conven, Hexane DIC, Hexane Conven, Oil

Source DF SS MS F P Factor 4 8050054747 2012513687 74661.83 0.000 Error 10 269551 26955 Total 14 8050324297

S = 164.2 R-Sq = 100.00% R-Sq(adj) = 100.00%

Individual 95% CIs For Mean Based on Pooled StDev

Level N Mean StDev +---------+---------+---------+--------- Ethanol DIC 3 334 13 * Ethanol Conven 3 645 17 *)

Minitab Output2. Hexane DIC 3 45203 318 * Hexane Conven 3 52548 179 * Results in Data Analysis Using Tukey’s Method

Oil 3 4834 36 * +---------+---------+---------+--------- 0 15000 30000 45000

Grouping Information Using Tukey Method

N Mean Grouping

Hexane Conven 3 52548 A Hexane DIC 3 45203 B Oil 3 4834 C Ethanol Conven 3 645 D Ethanol DIC 3 334 D Means that do not share a letter are significantly different.

Sept em ber 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a

The output from this analysis showed there were 4 groups of results (A to D), where the extracts in a same group do not have significant difference in antioxidant activity

measured with IC 50 value.

Group A consists of Hexane extract of Conventional dried fruits. Group B consists of Hexane extract of DIC fruits. Group C consists of Red Fruit Oil, and for the last group (group D) consists of Ethanol extract of DIC fruits and Ethanol extract of conventionally dried fruits.

Although the means or averages of IC 50 values from the first group (group A-C) are high, but the antioxidant activity is lower than that of the last group (group D), because the higher the IC 50 value, the lower the ability in scavenging the activity of free radicals the IC 50 value, the lower the antioxidant activity.

From the data above using one-way ANOVA, ethanol extract of DIC fruits and ethanol extract conventional dried fruits are at the same group. Even so, the ethanol extract of DIC fruits had the highest antioxidant activity because the value of the IC 50 was the smallest among the others.

Based on the experiment, the IC 50 values of the ethanol extract of the DIC fruits, the ethanol extract of the conventionally dried fruits, the red fruit oil, the hexane extract of the DIC fruits, and the hexane extract of the conventionally dried fruits were 334.37 ppm; 643.05 ppm; 4834.38 ppm; 45203.24 ppm; and 52547.50 ppm, respectively. The antioxidant activity of the red fruit extracts was much higher for DIC fruits than conventional dried fruits. From this study, the red fruit extracts and the red fruit oil are categorized as weak antioxidants. Also the hexane extract from the red fruit powder pre-dried by DIC has antioxidant activity better than that of dried conventionally.

In addition studies of antioxidant activity of the Red Fruit extracts were also carried out by Frap Method [4] .