Jurnal Ilmu Farmasi dan Farmasi Klinik
(Journal of Pharmaceutical Science and Clinical Pharmacy)
ISSN 1693-7899 (print) - 2716-3814 (online)
DOI: 10.31942/jiffk.v21i1.9529

In Vitro Evaluation of Cholesterol-Lowering Activity of
Acetyleugenol Synthesized via Esterification of Eugenol
Rahmawati Salsa Dinurrosifa1*, Dewi Fitriani Puspitasari2
1 Pharmaceutical Science Department, Faculty of Medicine, Universitas Negeri Semarang, Semarang, Central

Java, Indonesia
2 Pharmaceutical Science Department, Faculty of Pharmacy, Stifar Yayasan Pharmasi Semarang, Semarang,

Central Java, Indonesia

ABSTRACT: Hypercholesterolaemia is a significant risk factor for cardiovascular disease that
necessitates alternative therapies instead of statins due to their adverse effects. Eugenol, a
phenolic molecule that has the ability to lower cholesterol levels, is found in clove oil, which is
derived from Syzygium aromaticum. The chemical stability and biological activity of its
derivative, acetyleugenol, are both significantly higher. The purpose of this research was to
determine whether or not acetyleugenol, which was produced by esterifying eugenol with
acetic anhydride, have the ability to be effective in decreasing cholesterol levels. In order to
determine the anticholesterol activity, UV-Vis spectrophotometry with the LiebermannBurchard method was utilised at a range of concentrations (50, 75, 100, 125, and 150 ppm).
The EC50 value was equal to 94.413 ppm, and the maximum cholesterol decrease was 27.97%
when the concentration was 150 ppm. Considering these data, it appears that acetyleugenol
possesses a considerable amount of potential as a natural cholesterol-lowering medication.
Keywords: cholesterol ; acetyleugenol; esterification; eugenol; EC50

*Corresponding author:
Name
:Rahmawati Salsa Dinurrosifa
Email
: salsadinurrosifa@mail.unnes.ac.id
Address :Faculty of medicine, Universitas Negeri Semarang, Semarang, Central Java, Indonesia

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Submitted: 11-04-2025; Received in Revised Form: 14-05-2025; Accepted:23-06-2025

Dinurrosifa and Puspitasari

INTRODUCTION
Hyperlipidemia, especially hypercholesterolemia, is one of the main risk factors for
cardiovascular disease, which is the leading cause of death globally. Death can occur at a
young age, which is contrary to the health development target to be achieved in 2025 in the
form of increasing life expectancy, which has started from 2005 to 2025 (Pashar et al.,
2025). Efforts to treat hypercholesterolemia generally involve the use of statin drugs, but
long-term side effects such as myopathy and hepatic disorders encourage the search for
safer alternative therapies, especially from natural ingredients (Khatiwada & Hong, 2024).
Clove is a fragrant spice made from the dried flowers of the clove tree. Cloves are used in
cosmetics, medicine, gastronomy, and agriculture due to the presence of many bioactive
components such as gallic acid, flavonoids, eugenol acetate, and eugenol (Hema Krishna,
2024)
Clove oil (Syzygium aromaticum) contains eugenol, a phenolic compound with
established anticholesterol properties (Djamaluddinet al., 2024). Clove leaf infusion at a
dose of 0.18 g/head/day can lower blood cholesterol in mice (Siauta & Pentury, 2021). Early
studies have shown that eugenol and its derivatives can interact with key enzymes in lipid
metabolism, such as HMG-CoA reductase, which is the main target in hypercholesterolemia
therapy (Harb et al., 2019). Eugenol derivative compounds, such as acetyleugenol, show
increased chemical stability and higher bioactivity potential than eugenol itself (Ardiansah
et al., 2024). Acetyl eugenol shows anti-inflammatory effects in vitro, as evidenced by its
ability to inhibit protein denaturation by 32.20% (Dinurrosifa & Indriyanti, 2022).
Additionally, the study suggests acetyl eugenol may act as an HIV-1 protease inhibitor.
While further research is needed, these findings suggest the potential of acetyl eugenol in
a variety of biological applications, including its potential to affect cholesterol levels (Sururi
et al., 2023).
Cholesterol levels in pharmaceutical preparations can be tested using the
Liebermann-Burchard method using UV-Vis spectrophotometry (Novyanti et al., 2025).
This method is specific in measuring cholesterol compounds, which are included in one of
the steroid groups (Luhurningtyas et al., 2019). The direct spectrophotometric method is
used in the analysis of strongly absorbing analytes, while the indirect approach, involving
derivatization, is used for weakly absorbing compounds. Cholesterol (cholesterol-5-en-3βol) has a weakly absorbing chromophore, which can be derivatized through the
Liebermann-Burchard reaction to obtain a chromophoric group that can absorb strongly at
the maximum wavelength (Anggraini & Nabillah, 2018). However, its derivative,
acetyleugenol, is reported to have enhanced stability and bioactivity. This study aims to
investigate the in vitro cholesterol-lowering potential of acetyleugenol.
METHODS
This research was conducted in the pharmaceutical chemistry laboratory of Sekolah
Tinggi Ilmu Farmasi Yayasan Pharmasi Semarang. The equipment and laboratory tools
used in this study are as follows: beakers, Erlenmeyer flasks, droppers, measuring pipettes,
volume pipettes, analytical balances, watch glasses, ovens, sonicators, water baths, 50 mL
measuring flasks, 10 mL measuring flasks, test tubes, filter paper, and Buchner pumps. The
materials used in this study were obtained from Nitra Kimia, Yogyakarta, as follows:
eugenol (for synthesis sigma aldrich), NaOH (analytical grade), Acetic Acid Anhydride
(analytical grade), ethanol (analytical grade Smartlab), chloroform (analytical grade
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Smartlab), H2SO4 (analytical grade Merck), cholesterol standard (analytical grade Sigma
Aldrich), and acetic acid (analytical grade Merck).
Synthesis of acetyl eugenol
Eugenol 5 mL mixed with 13 mL of 10% NaOH. Sonicate for 15 minutes at 70-80°C.
Add 9.2 mL of Acetic Anhydride. Sonicate for 150 minutes at 70-80°C. Extraction with 20
mL of chloroform twice. Two phases are formed, chloroform and water phases. The
chloroform phase is stored overnight in the refrigerator until the temperature is below .
Anticholesterol Test
Preparation of standard solution
Weigh the cholesterol standard as much as 100 mg, dissolve it in ethanol p.a. up to
100 mL, and heat it in a water bath at 45°C.
Determination of maximum wavelength
An aliquot of 0.5 mL from the standard solution was pipetted into a test tube and
diluted with ethanol to a final volume of 5.0 mL. Subsequently, 2.0 mL of acetic anhydride
and 0.1 mL of concentrated sulfuric acid (H₂SO₄) were added. The reaction mixture was
allowed to stand for 15 minutes at room temperature. After the incubation period, the
absorbance was measured within the wavelength range of 400–700 nm using a UV-Vis
spectrophotometer.
Determination of Operating Time
To evaluate the optimal reaction time, 0.5 mL of the standard solution was diluted
with ethanol (p.a.) to a final volume of 5.0 mL. After the addition of 2.0 mL acetic anhydride
and 0.1 mL of concentrated H₂SO₄, absorbance measurements were taken at 2-minute
intervals from 10 to 30 minutes. The reaction was maintained at ambient temperature
during this period.
Preparation of cholesterol standard curve
To construct the calibration curve, varying volumes of the standard solution (0.2,
0.25, 0.3, 0.35, 0.4, 0.45, and 0.5 mL) were each diluted with ethanol to a total volume of 5.0
mL, resulting in final concentrations of 40, 50, 60, 70, 80, 90, and 100 ppm, respectively.
Each solution was then treated with 2.0 mL of acetic anhydride and 0.1 mL of concentrated
H₂SO₄. After incubation for the optimized reaction time, the absorbance was measured at
the predetermined wavelength.
Preparation of the sample standard curve
The synthesized acetyl eugenol standard solution was prepared at a concentration of
1000 ppm each 0.5, 0.75, 1, 1.25, and 1.5 mL, then ethanol up to 10 mL. taken 5 mL each
and added 5 mL of 200 ppm cholesterol standard and mixed. Each of the solutions was
taken 5 mL, then added with 2 mL of acetic anhydride and 1 mL of H 2SO4. Measure the
absorbance according to the wavelength obtained.
Preparation of negative control
Measured 5 mL of 100 ppm cholesterol standard, added 2 mL of acetic acid, and 0.1
mL of H2SO4.
Data Analysis
The results obtained from the sample measurement are absorbance, which is then
compared with a standard cholesterol solution to determine the level of cholesterol
reduction in percentage form. The formula used is as follows:

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% reduction = 𝑎𝑏𝑠 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 −𝑎𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 𝑥 100%
𝑎𝑏𝑠 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑

Information:
% reduction = reduction in cholesterol levels (%)
Standard abs = initial cholesterol standard absorbance
Sample abs = cholesterol absorbance after treatment

RESULT AND DISCUSSION
The method used in this study is the esterification method between alcohol and
carboxylic acid derivatives. The structure of eugenol contains an -OH group bound to the
benzene ring (Maurya et al., 2020). Acetyl eugenol synthesis is carried out by reacting
eugenol and acetic anhydride. The esterification process is very slow without a catalyst
(Dinurrosifa & Indriyanti, 2022), so the addition of a catalyst aims to accelerate the
acetylation reaction. The catalyst used is NaOH because its basic properties can increase
the reactivity of nucleophiles through nucleophilic acyl substitution reactions, so that the
reaction takes place faster and the yield obtained is higher (Tursiloadi et al., 2015).

Figure 1. Reaction Mechanism of Acetyleugenol ((Dinurrosifa & Indriyanti, 2022)

This process is also assisted by ultrasonic waves. The sonochemical method is now
one of the approaches in green chemistry because it is easy to do, efficient, produces high
quantities of products, takes a short time, and does not damage the environment (Patel et
al., 2014) and can prevent the use of volatile and toxic solvents by using harmless chemicals
(M. Draye, 2020). This method is very important in the synthesis of organic compounds and
in the pharmaceutical industry. The use of alkali catalyst sodium hydroxide will cause the
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formation of nucleophiles, namely eugenolate ions, so that the reaction can run faster and
more effectively. This results in an increase in nucleophilicity, which causes the attack of
the carbonyl atom C on acetic acid anhydride to take place more easily. Thus, it is expected
that the resulting yield will be greater.
In the synthesis process of acetyleugenol, 5.5 grams were produced using the
ultrasonic wave method, where the use of ultrasonic radiation produces a faster reaction
rate, energy conversion, and minimizes waste compared to conventional methods.
Ultrasonic waves occur at frequencies of 20 kHz to 100 MHz. Ultrasonic waves are known
to accelerate various types of organic reactions and are believed to be an important
technique in organic synthesis. Next, the sample measurement to see the decrease in
cholesterol levels uses the Lieberman-Burchard method. The Lieberman-Burchard method
is a very specific method for measuring steroid compounds, one of which is cholesterol.
Standard cholesterol is dissolved in chloroform because 1 part of non-polar cholesterol
dissolves in a non-polar solvent, namely 4.5 parts of chloroform. The reaction carried out
in this method must be free of water because the reaction will be very sensitive and
unstable to water. In this method, it is necessary to add anhydrous acetic acid and
concentrated sulfuric acid. The addition of anhydrous acetic acid aims to extract
cholesterol, ensure that the media is free of water, and form steroid acetyl derivatives
which are then dripped with concentrated sulfuric acid through the wall to produce a green
color for steroid compounds including cholesterol. In this study, the standard cholesterol
concentration used was 200 ppm. Before getting the concentration results used for the
study, the results obtained were the actual concentration of 1007 ppm. Then the
concentration was diluted to a concentration of 40,50,60,70,80,90, and 100 ppm. Each
concentration is put into a test tube and added with a standard cholesterol concentration
of 200 ppm. The resulting mixture is added with anhydrous acetic acid solution and also
from H2SO4. The resulting solution is then left for 15 minutes in a dark place. The goal is for
the solution to form a green complex and then read the absorbance value at a maximum
wavelength of 668 nm at 15 minutes, in addition the cholesterol solution is
photodegradable, unstable to light and will turn into cholestenone (Figure 2).

Light

Figure 2. Photodegradation Reaction of Cholesterol to Cholestenone (Dinurrosifa et al., 2022)

The use of anhydrous acetic acid aims to ensure that the media is free of water
because in the Lieberman-Burchad method the reaction is very sensitive and unstable to
water. If there is water content, then the anhydrous acetic acid in the system can change
into hydrated acetic acid and the reaction cannot occur with cholesterol or with
concentrated sulfuric acid. In addition, anhydrous acetic acid also plays a role in the
formation of acetyl derivative compounds from steroids which when added with
concentrated sulfuric acid can produce a green complex, as shown in Figure 3.
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Figure 3. Green Complex Reaction between Cholesterol and Lieberman-Burchard reagent
(Anggraini & Nabillah, 2018)

To determine the wavelength of acetyleugenol, UV-Vis spectrophotometry is used.
Ultraviolet and visible spectrophotometry methods are used to determine low levels of a
substance, usually in ppm (parts per million) or ppb (parts per billion). The working
principle is based on the absorption of light or radiation energy by a solution. The amount
of light or radiation energy absorbed allows quantitative measurement of the amount of
absorbent in the solution. In this study, the maximum wavelength obtained was 534.60 nm.
The Lieberman-Burchard method obtained two maximum absorption wavelengths and
stated that the lowest absorption at the maximum wavelength is more stable.
In the process of reducing cholesterol levels, absorbance values and % reduction in
cholesterol levels were obtained (Table 1).
Table 1. Effect of Acetyleugenol Concentration on Cholesterol Reduction
% reduction
Standard
acetyl eugenol
sample
average
in
absorbance
concentration
absorbance
(%)
cholesterol
0,659
3,65
0,684
50 ppm
0,667
2,49
2,83
0,668
2,34
0,629
8,04
0,684
75 ppm
0,632
7,6
7,50
0,637
6,87
0,582
14,91
0,684
100 ppm
0,583
14,77
15,21
0,575
15,94
0,533
22,08
0,684
125 ppm
0,522
23,68
22,47
0,536
21,64
0,488
28,65
0,684
150 ppm
0,499
27,05
27,97
0,491
28,22

EC50

94,413 ppm

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Based on the table, it can be seen that the higher the concentration used, the higher
the % decrease in cholesterol levels and the lower the absorbance value. This means that
high concentrations have an effect on reducing cholesterol levels, where the higher
concentration provides the highest cholesterol reduction, so that the absorbance value is
smaller than the percentage of large anticholesterol activity. Measurements were carried
out in triplicate, and the average percentage reduction in cholesterol levels is depicted in
the curve in Figure 4.

% reduction in cholesterol

30.00
25.00
20.00
15.00
10.00
5.00
0.00
50 ppm

75 ppm

100 ppm

125 ppm

150 ppm

acetyl eugenol concentration
Figure 4. Relationship Curve Between Acetyl Eugenol Concentration and Cholesterol Decrease

The effects of acetyl eugenol on cholesterol are influenced by its chemical structure,
primarily through the formation of ester bonds and steric effects. Acetyl eugenol, with an
acetyl group (-COCH3) in its phenol group, can form ester bonds with cholesterol,
modifying the physical and chemical properties of cholesterol. The ester group in the
context of cholesterol lowering refers to cholesterol esters. Cholesterol esters are forms of
cholesterol bound to fatty acids. Cholesterol esterification, the process of forming
cholesterol esters, aids in the storage and transport of cholesterol in the body. Cholesterol
esters are more hydrophobic than free cholesterol, so they can be packaged more efficiently
in lipoproteins, facilitating the transport of cholesterol through the bloodstream. In
addition, stanol esters, which are esterified forms of stanols, may also help lower
cholesterol by competing with cholesterol during intestinal absorption. In addition, the
structure of acetyl eugenol, specifically the methyl group (-CH3) and aromatic groups, may
also cause steric effects, influencing how acetyl eugenol interacts with cholesterol in
biological systems.
The effective concentration (EC50) value in this study was 94.413 ppm. This EC 50
value aims to see the concentration that can reduce total cholesterol levels by 50%. So, from
these results, it means that to reduce 50% cholesterol, 94.413 ppm of acetyleugenol is
needed.

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CONCLUSION
From this study, it can be concluded that acetyleugenol has cholesterol-lowering
activity with an EC50 value of 94.413 ppm. Additionally, investigations into its mechanism
of action at the molecular level, pharmacokinetic properties, and potential for structural
optimization are recommended to support its development as a novel hypocholesterolemic
agent. Comparative studies with standard lipid-lowering drugs and toxicity assessments
will also be essential to validate its therapeutic applicability.
AUTHOR CONTRIBUTION
RSD: design; definition of intellectual content; literature search; experimental studies; data
analysis; manuscript preparation.
DFP: Concepts or ideas; design; definition of intellectual content; literature search;
experimental studies; data analysis; Manuscript editing; manuscript review.
CONFLICT OF INTEREST
None to declare
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