ANTIOXIDANT ACTIVITY OF ETHANOL EXTRACT COMBINATIONS OF JERUK PURUT LEAF (CITRUS HYSTRIX DC.) AND JATI BELANDA LEAF (GUAZUMA ULMIFOLIA L.)

Ethanol extracts of Guazuma ulmifolia L. and Citrus hystrix Dc., have a strong antioxidant activity. The purpose of this research is to determine the antioxidant activity of ethanol extracts combination of Guazuma ulmifolia L. (jati belanda leaf) and Citrus hystrix Dc (jeruk purut leaf). Determination of antioxidant activity was done by DPPH method (1,1-diphenyl2picrylhydrazil) using UV-Vis spectrophotometer with maximum wavelength 516.5 nm. The antioxidant activity of jeruk purut leaf is smaller than jati belanda leaf (IC50 48.70 g/mL for jeruk purut leaf, IC50 35.92 g/mL for jati belanda leaf). Antioxidant activity of the two extracts combination was lower than the antioxidants of each extract (without combination). The low antioxidant activity of both extract combination due to the antagonist activity as the interaction of the content of each extract so that the extract combination showing a weaker antioxidant activity. At the concentration of each different comparison of each extract combination volume shown no difference with P-value > 0.05 (P-value: 0.99).


INTRODUCTION
Free radicals are relatively unstable molecules with the outermost orbits of atoms that have one or more unpaired electrons [1].Free radicals are one of the factors causing DNA damage other than virus.Cell division can be disrupted because of the breaking of DNA chains that cannot be repaired by the DNA repair system until it becomes a cancer [2].
Antioxidants are automatically produced by our body to prevent the free radicals spread through the body [3].Antioxidant is a compound that have an important role in maintaining health because it can capture free radical molecules thereby inhibiting oxidative reactions in the body which cause some various diseases [4].The body must obtain sufficient amount of antioxidants.Antioxidants can decrease their activity when excessive free radicals are forming so that our body needs an external antioxidant intake [5].Natural antioxidants are obtained from classes of phenol derivatives such as flavonoids, hydroxamic acid derivatives, coumarins, tocopherols and organic acids which is present in plants [3].
Jati belanda is one of Indonesia's tropical plants that are often used to support the health of the human body [6].Jati belanda leaf have antioxidant effects and phytochemical screening shows that it contains flavonoid compounds [7].Jeruk purut leaf has exogenous antioxidant effect that can be used as an alternative to inhibiting the aging process (antiaging) because it contains citronellal, a powerful antioxidant.Phytochemical screening shows the jeruk purut leaf contain phenol, flavonoids, and terpenoids [8].This study aims to determine the antioxidant activity of the combination of both plants because of the high antioxidant activity.

Sample Extraction and Preparation
The extraction of both samples was done by maceration.50 grams of Jeruk purut leaf simplicia and 200 grams of jati belanda leaf were soaked with 96% ethanol (1:10) and stored in a glass container, tightly closed, left for 3 days, protected from light to avoid oxidation or evaporation, and stirred it occasionally.After 3 days, the maserate was filtered with a Buchner funnel and remaserated the dregs with the same amount of solvent.Maserate is evaporated with a rotary evaporator and waterbath until it becomes a viscous extract.
Sample preparation for the DPPH assay was made from a total of 100 mg from each extract was dissolved with 10 mL of ethanol p.a and synthesized to ensure the homogeneity of the solution.

Antioxidant Assay
Antioxidant assay was done by DPPH method.The DPPH solution was prepared from 15.77 mg of DPPH powder dissolved with 100 mL ethanol p.a.The combination of extracts with a certain ratio was added with 1 mL of DPPH solution and ethanol p.a boundary marker of 5.0 mL flask.Incubation of the solution for 30 minutes and avoid it from the lights, read the absorbance of each ratio with UV-Vis spectrophotometer at maximum wavelength 516.5 nm.The blank solution was ethanol p.a and the DPPH control consist of 1 mL DPPH solution added with ethanol p.a up to 5 mL boundary marker on the flask.Sample control of each combination of 1 mL and added ethanol p.a to the 5 mL boundary marker on the flask.Table 1 shows the certain ratio of extract combination.
The antioxidant activity determined with the inhibitory concentration (IC50) and % antiradical from the extracts.Vitamin E used as the standard of high antioxidant activity.IC50 of vitamin E is 12.59 g/mL.

Statistical Analysis
Parameter used to find out the antioxidant activity of the extract combination was % antiradical.The formula to calculate % antiradical is as follows.Another parameter to find out the antioxidant activity of the sole extract of jeruk purut leaf and jati belanda leaf was IC50 value.IC50 value was calculated by linear regression of the % antiradical of sole extract.The result obtained from the experiments analyzed using anova test.

RESULT AND DISCUSSION
Extract rendemen was 5.42% (jati belanda leaf extract) and 12.44% (jeruk purut leaf extract) and its calculated from the extract mass divided by simplicia mass.
The method used to test the antioxidant activity of ethanol extract combination of jeruk purut leaf and jati belanda leaf is DPPH method (2,2diphenyl-1-picrylhydrazyl).The principle of the DPPH method is by reacting the sample with DPPH reagent to see the inhibitory ability of the sample to DPPH as a radical compound.The parameters used to determine the antioxidant activity of the sample are % antiradical and IC50 values.The results obtained in the study are presented as follows.The results obtained in the study are presented at table 1 and table 2.
From the result obtained, the greater concentration of extracts result in the greater activity of antioxidants.IC50 of jati belanda leaf extract is smaller than the extract of jeruk purut leaf, where the results indicate that the antioxidant activity of jati belanda leaf extract is higher than the jeruk purut leaf extract.Smaller value of IC50 means greater effect of antioxidant to resist the DPPH activity as a free radical [9].IC50 calculation results also prove that in the combination of the two extracts, which has contributed to the high antioxidant activity is jati belanda leaf extract which has the ability to inhibit DPPH radical activity higher.In addition, there were a compound as an antagonist to the combination result in lowering the antioxidant activity of the combination or lowering stability of the antioxidant.Stability of the antioxidant can be affected by temperature, changes of pH, light, oxygen, and metal ions [10].Positive controls used were vitamin E. Positive controls were used to compare IC50 or sample inhibitory concentrations of vitamin E that had high antioxidant activity.Vitamin E has an IC50 value of 12.59 g/mL [7].The data then analysed by anova test, and the result are tabulated in Table 3  Based on the results obtained, it is known that the antioxidant activity combination of jeruk purut leaf extract and jati belanda leaf extract is increasing when the amount of jati belanda leaf extract greater.The more the amount of jati belanda leaf extract in combination, it will further suppress the antioxidant activity of lime leaf extract.

Table 1 .
% Antiradical from certain ratio of extract combination

Table 2 .
IC50 value of the sole extract

Table 3 .
ANOVA test for the extract combinations % antiradical

Table 4 .
ANOVA test for IC50 of 2 samples and vitamin E and Table 4.There is an average difference in IC50 value of both extracts with the standard of vitamin E. Different IC50 values indicate different antioxidant activity.The smaller value of IC50, has the better antioxidant activity.CONCLUSIONSThe IC50 values of jeruk purut leaf extracts is 48.70 g/mL, while the jati belanda leaf extracts is 35.92 g/mL.