This document summarizes a study on the synthesis of ferulic acid derivatives using peroxidase enzyme. Ferulic acid is known to have antioxidant properties due to its phenolic structure. The goal was to synthesize compounds with increased antioxidant activity by adding more hydroxyl groups. Initially, ferulic acid was esterified to ethyl ferulate. Then, peroxidase enzyme catalyzed the oxidation of ethyl ferulate into a suspected tetramer derivative. Testing found the tetramer had the strongest antioxidant effects against DPPH due to its multiple hydroxyl groups. Further analysis is recommended to fully characterize the synthesized tetramer compound.

1. SYNTHESIS OF FERULIC ACID DERIVATIVES USING PEROXIDASE
ENZYME
Raissa Melina, S.T., B. Eng., Hery Sutanto, M.Si., Yulia Anita M.Sc.
Department of Food Technology, Faculty of Life Sciences and Technology
Swiss German University
EduTown BSD City, Tangerang 15339, Indonesia
raissamelina85@gmail.com
Abstract
Ferulic acid is widely known as antioxidant due to the hydroxyl
group in its phenolic structure. The synthesis reaction was done in
order to increase the antioxidant activity. It was expected to
synthesize a new compound that contains more hydroxyl group.
Initially, ferulic acid was esterified to obtain maximum result of
synthesis product. Esterification product was tested using GCMS.
The synthesis reaction was then assisted by peroxidase enzyme as the
catalyst. Synthesis of ferulic acid derivatives using peroxidase
enzyme is still limited. Peroxidase enzyme catalyzed the oxidation
process by breaking down hydrogen peroxide and covert the substrate
into a radical molecule. The synthesis product was suspected as
tetramer of ethyl ferulate. The LCMS spectrum supports the facts
related to tetramer of ethyl ferulate formation. Tetramer of ethyl
ferulate was found to be strong antioxidant due to its structure. The
antioxidant of ferulic acid, ethyl ferulate and tetramer of ethyl
ferulate was tested against DPPH. The antioxidant activity of
tetramer of ethyl ferulate in this experiment was the highest
compared to the others. In the next research, the synthesis product is
recommended to be purified and analyzed further about the structure
using FTIR and NMR
Keywords: synthesis, Ferulic acid, ferulic acid derivatives, tetramer
of ethyl ferulate, antioxidant activity.
I. INTRODUCTION
Antioxidant demand is growing rapidly all over the world
lately [1]. The demand growth can be triggered by the
potential of antioxidant to give human health benefits [2].
Antioxidant is able to conquer the harmful effects caused by
free radicals [3].
Free radicals are compound with an unpaired electron. With
the lacking of electrons, free radicals are unstable. In order to
be more stable, free radicals become more active and react
quickly with other compounds so that it is able to obtain an
electron to fulfil the pair [4].
Ferulic acid as a phenolic compound has high antioxidant
activity [5]. In other word, ferulic acid has an ability to
scavenge free radicals. Ferulic acid can be found abundantly
in many varieties of plants and foods such as corn bran, navy
bean et cetera [6]. Moreover, ferulic acid has very high
potential usages in food and pharmaceutical industry due to its
ability to whiten skin, reduce melanin production, increase
shelf life stability during long storage time, retain color and
odor et cetera [7].
On the other hand, polymerization process is proven to be able
to increase the antioxidant value. A synthesis reaction of
polymerization may produce several derivatives products
which one of the simplest is dimeric compound. Ferulic acid
dimers are more effective inhibitors of lipid peroxidation than
ferulic acid [8].
The synthesis of ferulic acid dimers is using an enzyme as
catalyst. Enzyme as homogeneous catalyst used because it is
dissolved in the medium and able to make a good contact with
the reactants. Peroxidase enzyme used since it has already
been commonly utilized as a catalyst for the dimerization
process. High number of peroxidase can be taken from
Brassicaceae family [9].
II. RESEARCH METHOD
Esterification Based on Fischer Method
970 mg 5 mmol Ferulic acid was mixed with 5 ml ethanol and
then stirred using magnetic stirrer. After that, 0.027 ml H2SO4
was added. The mixture was refluxed for 8 hours at 88o
C.
After cooling to 25o
C, ethyl acetate was added along with
3.5% brine solution forming layer separation. The ethyl
acetate layer was taken and dried over by adding MgSO4. The
mixture was evaporated using rotary evaporator.
Synthesis Reaction
1 g ethyl ferulate was dissolved with heating at 60o
C into 1-
liter acetate buffer pH 4. After the solution cooled down to
40o
C, 0.38 ml hydrogen peroxide was added along with 5 mg
peroxide in 1 ml phosphate buffer pH 7.
TLC analysis
The most suitable ratio is 7 to 3 n-hexane and ethyl acetate
respectively.
DPPH Antioxidant Assay
110 ppm concentration of DPPH was made by dissolving 11
mg of DPPH in 100 ml of ethanol. Then, different
concentrations of sample were made. 0.1 μl of each sample
concentrations were mixed with 0.75 μl of ethanol and 0.15 μl
of DPPH 110 ppm. The samples were measured by using UV-
VIS spectrophotometer at wavelength 517 nm.

2. III. RESULTS AND DISCUSSION
Esterification
Figure 1. Mass Spectrum of GCMS at RT 51.310 minutes
The fragment shows that ethyl ferulate or Ethyl (2E)-3-(4-
hydroxy-3-methoxyphenyl)-2-propenoate with molecular
weight 222.1 is present in the sample. The amount is 57.57%
of the total.
Synthesis Reaction
Figure 2. Mass Spectrum of LCMS at RT 15.320 minutes
It is assumed that the product of synthesis reaction would be
the tetramer of ethyl ferulate. Some fragments that indicates
the presence of the tetramer of ethyl ferulate are 907.46
[M+2H+Na]+
, 908.47, 465.19, 466.08.
Figure 3. Proposed structure of tetramer of ethyl ferulate
DPPH Antioxidant Assay
Table 1. IC50 value comparison
Substrate IC50 value (ppm)
Ferulic Acid 166.846
Ethyl Ferulate 170.991
Tetramer of Ethyl Ferulate 140.122
Tetramer of ethyl ferulate as a derivative of ferulic acid shows
the lowest IC50. It was able to scavenge 50% DPPH radical
with 140.122 ppm concentration. Tetramer of ethyl ferulate has
more hydroxyl group compared to ferulic acid and ethyl
ferulate, make it a better antioxidant agent.
The higher amount and strength of electron donating
substituent in phenolic acid play an important role on
becoming better antioxidant. The antioxidant activity of
ferulic acid is better than its ester due to the structure of ferulic
acid. With having more OH group, ferulic acid is able to
stabilize more free radicals.
ACKNOWLEDGMENT
I want to thank the God Almighty, my family, friends and
everyone who always support me no matter what happens and
also for my advisor and co-advisor for their endless kindness
and help.
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