Study of the Temperature and Molarity Ratio Effects in Geraniol

Esterification and Testing Its Antibacterial Activity

Stefanie Sugiarto

, Novita Ariani

2 b

, Elvina Dhiaful Iftitah

and Galuh Widiyarti

Department of Chemistry, Brawijaya University, Veteran, Malang, Indonesia

Research Center for Chemistry, National Research and Innovation Agency (BRIN) Puspiptek, South Tangerang, Indonesia

Research Center for Pharmaceutical Ingredients and Traditional Medicine, BRIN, Puspiptek, South Tangerang, Indonesia

Keywords: Geraniol, Geranyl Isobutyrate, Disc Diffusion Method.

Abstract: This study modifies the molarity ratio of geraniol to isobutyric acid (1:1, 1:1.1, and 1:1.3) and temperature

(RT, 40°C, 60°C, and 80°C) in the synthesis of geranyl isobutyrate ester using 5% (w/w) NaOH as a base

catalyst. The antimicrobial activity was tested against both gram-positive and gram-negative bacteria. Ester

products were separated and purified using column chromatography, and identified using Fourier Transform

Infrared Spectroscopy (FTIR) and Gas Chromatography-Mass Spectrometry (GCMS). The antimicrobial

activity was assessed using the disk diffusion method. The results showed that the esterification product with

a 1:1.1 molar ratio at 80°C had the best separation based on thin-layer chromatography (TLC) and

antimicrobial properties. GCMS analysis of the purified product revealed five compound peaks with geranyl

isobutyrate at R

= 13.376 minutes (2.77%). FTIR confirmed the presence of C=O ester carbonyl groups at

1717.82 cm

-1

and C-O groups at 1080.37 cm

-1

. Antimicrobial tests showed inhibition zones on gram-positive

bacteria of 18.33±2.62 mm for Bacillus subtilis and 15.67±0.47 mm for Staphylococcus aureus, and against

gram-negative bacteria of 10.67±0.47 mm for Pseudomonas aeruginosa and 16.67±2.36 mm for Escherichia

coli.

1 INTRODUCTION

The development of essential oils in Indonesia is

progressing rapidly due to their diverse benefits in the

pharmaceutical and medicinal fields. Essential oils,

commonly derived from plants, include lemongrass

oil, which is currently popular with global

consumption reaching around 2,000-2,500 tons per

year (Direktorat Jendral Perkebunan, 2020). The

diverse benefits of lemongrass oil are its antiseptic

properties and medicinal uses, make it a highly

valuable commodity. Lemongrass oil is rich in

beneficial compounds such as citronellal, citronellol,

and geraniol.

In Indonesia, there is a significant demand for

geraniol derived from lemongrass essential oil,

especially in the pharmaceutical and perfume

industries. Furthermore, geraniol exhibits a variety of

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https://orcid.org/0000-0002-0235-596X

beneficial medical properties, including antioxidant,

anti-inflammatory, antimicrobial, antitumor,

hepatoprotective, cardioprotective, and

neuroprotective effects (Pavan et al., 2018). Some

researchs have shown that geraniol has strong

antimicrobial activity due to its lipophilic properties,

which allow it to bind to the lipid membranes of

microorganisms, demonstrating effectiveness against

various bacteria, including Candida and

Staphylococcus(Lira et al., 2020). Nevertheless,

geraniol exports from Indonesia decreased from

11,789.3 million USD in 2019 to 8,251.1 million

USD in 2020 (Badan Pusat Statsitik, 2021).

Therefore, efforts are needed to increase the market

value of geraniol by discovering its derivatives to

enhance bioactivity and application potential through

derivatization into geranyl esters.

Sugiarto, S., Ariani, N., Iftitah, E. D. and Widiyarti, G.

Study of the Temperature and Molarity Ratio Effects in Geraniol Esteriﬁcation and Testing Its Antibacterial Activity.

DOI: 10.5220/0013553900004612

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of BRIN’s 2nd International Conference for Health Research (ICHR 2024), pages 38-46

ISBN: 978-989-758-755-9

Numerous studies have shown that derivatives

of geraniol, such as geranyl formate, geranyl acetate,

geranyl butyrate, and geranyl isobutyrate, possess

antibacterial properties. For instance, a study

indicated that organophilic bentonite incorporated

with geranyl acetate exhibited antibacterial activity

against Staphylococcus aureus and Salmonella

typhimurium (Capelezzo et al., 2023). Additionally,

geraniol derivatives such as geranyl butanoate have

shown potential as anticancer agents (Widiyarti et al.,

2019). On the other hand, geranyl isobutyrate has

been proven to have superior antimicrobial properties

compared to other geraniol derivatives, as

demonstrated in studies testing antimicrobial activity

against Escherichia coli, Staphylococcus aureus,

Pseudomonas aeruginosa, and Staphylococcus

epidermidis. The antibacterial effect of geranyl

isobutyrate against E. coli was the strongest among

the 12 compounds tested (Zhaoshuang et al., 2016).

However, further research is needed due to the limited

studies examining the antimicrobial activity of

geranyl isobutyrate, and most existing studies have

not included B. subtilis as one of the bacteria.

Moreover, additional research is required regarding

the synthesis method of geranyl isobutyrate, as its

quantity derived from plants is usually limited, such

as in Conyza incana, which yields only about 2.5%

(Zabin, 2018). The synthesis of geranyl isobutyrate

generally involves an esterification reaction between

alcohol and carboxylic acid compounds to form the

desired ester.

Conceptually, the esterification reaction requires

high temperatures or heating above 55°C (Tolvanen

et al., 2014). Additionally, catalysts play a role in

accelerating the reaction. Acid catalysts are generally

used, but recent research has shown that the use of

base catalysts can be an effective alternative. Base

catalysts are known to increase reaction efficiency, be

environmentally friendly, and reduce water formation

(Khan et al., 2021). Although the use of base catalysts

for esterification is still in the research phase,

previous studies have successfully produced geraniol

derivatives using a base catalyst of NaOH. These

derivatives include geranyl butyrate, geranyl

caproate, and geranyl caprylate, with yields of

66.94%, 76.72%, and 54.92%, respectively

(Widiyarti et al., 2019). This finding indicates the

potential use of heterogeneous base catalysts in the

esterification process, which has not been widely

reported.

Based on the above issues, this research aims to

synthesize geranyl isobutyrate using sodium

hydroxide (NaOH) as a base catalyst to enhance the

antibacterial activity of geraniol derivatives. The

geraniol esters will be identified using TLC, FTIR,

and GCMS. The geranyl isobutyrate product is then

antibacterial activity tested against B. subtilis, S.

aureus, E. coli, P. aeruginosa. These four bacteria are

bacterial models that are usually used for antibacterial

activity tests for natural products screening and their

derivative compounds that have the potential to be

antibacterial.

2 RESEARCH METHODS

2.1 Synthesis of Geranyl Isobutyrate

(GI)

The esterification of GI based on 1:1 molarity ratio is

carried out by reacting 20 mmol (3.086 g) of geraniol,

which is placed into a 250 mL round-bottom flask and

mixed with NaOH catalyst at 5% of the weight of

geraniol. After 4 hours, 20 mmol (1.762 g) of

isobutyric acid is added. The solution mixture is

reacted at room temperature (RT) for 24 hours. The

same procedure is repeated for each molar variation

of isobutyric acid for 1:1.1 (22 mmol, 1.939 g) and

1:1.3 (26 mmol, 2.290 g).

Next, the synthesis product is extracted using a

100 mL separatory funnel with a solvent solution of

ethyl acetate and distilled water (1:1). The mixture is

shaken and allowed to stand until two phases form,

and the organic phase is collected. This process is

performed in triplicate. The collected organic phase

is evaporated using a rotary evaporator until crude GI

ester product is obtained. The yield of the crude ester

is calculated using the following formula:

% 𝑌𝑖𝑒𝑙𝑑 =

  

   ()

𝑥100% (1)

The product ester is then purified using column

chromatography with eluent n-hexane, ethyl acetate,

and methanol eluted gradually. The separated

compound is identified using TLC, FTIR, and

GCMS.

This procedure is repeated for each molar

variation at different temperatures of 40

C, 60°C and

80°C.

2.2 Purification and Characterization

of GI Ester

The purification of GI crude ester is carried out using

gradient column chromatography with n-hexane and

ethyl acetate as solvents. The purified product is

subsequently analyzed by GCMS Agilent

Study of the Temperature and Molarity Ratio Effects in Geraniol Esteriﬁcation and Testing Its Antibacterial Activity

Tecnologies 7890C and FTIR Shimadzu prestige 21

using KBr pellets.

2.3 Antibacterial Test Using Disc

Diffusion Method

The bacterial culture of B. subtilis ATCC 6633, S.

aureus ATCC 25923, E. coli ATCC 8739, and P.

aeruginosa ATCC 9027 was prepared by swiped of 1

ose of bacteria culture from stock culture on the

sterile Nutrient Agar (NA) agar slant. The culture is

stored in an incubator at 37°C for 24 hours. Bacterial

culture on rejuvenated agar slants was then added

with 10 mL of 0.9% NaCl solution, shaken until all

colonies on the surface are loose and suspended in

NaCl solution 0.9%. The bacteria as much as 100 μl

were put into the petri dish, and then an amount of 10

mL of NA is added, and allow it to solidify.

Afterwards, the discs (approximately 6 mm in

diameter) are placed on the NA. Finally, 5 μl of the

sample at a desired concentration is added on top of

each disc. For each sample, a triplicate test was

carried out. The petri dishes are then incubated under

suitable conditions at 37

C for 18-24 hours, then the

resulting diameters of inhibition zone was measured

(Yusmaniar et al., 2017; Hossain, 2024).

3 RESULTS AND DISCUSSION

3.1 Synthesis of GI

Esters are produced through the reaction of

carboxylic acids with alcohols, forming water as a by-

product and replacing the hydroxyl group (-OH) with

an alkoxy group (-OR) (Melvine et al., 2021). The

esterification of geraniol with isobutyric acid, aided

by the base catalyst NaOH, results in the formation of

geranyl isobutyrate (GI). The presence of NaOH is

used to increase the reaction rate without undergoing

chemical change and to lower the activation energy

(Ea) (Setyaningsih et al., 2017). The synthesis and

mechanism reaction of GI as shown in Figure 1 and

Figure 2.

Figure 1: Synthesis Reaction of GI.

Figure 2: Esterification Reaction Mechanism of GI.

The hydroxyl group (-OH) reacts with the catalyst,

resulting in proton abstraction and the formation of an

alkoxide ion. This nucleophilic alkoxide ion attacks

the carbon atom of the carbonyl group, leading to

resonance stabilization and the formation of GI ester.

The mechanism occurs because the alcohol acts as a

good nucleophile, making the reaction with

carboxylic acid more effective in forming the geranyl

ester. This aligns with previous research, which states

that heterolytic cleavage occurs in the presence of the

catalyst, followed by the alcohol attacking the

catalyst, enhancing its nucleophilicity and forming an

intermediate. Subsequently, this intermediate

decomposes, eliminating the catalyst and forming the

ester (Kohsaka et al., 2018). However, this reaction

may produce side products if the alkoxide ion reacts

with other compounds in the mixture.

The reaction product exhibits a pH of 5-6,

indicating that it is non-corrosive. Liquid-liquid

extraction is then performed to separate the organic

phase from the aqueous phase (Figure 3). Ethyl

acetate (EtOAc) is used as the solvent to ensure the

efficient distribution of the geranyl ester in the

organic phase. EtOAc is soluble in various

compounds and has relatively low viscosity,

facilitating separation (Schneider et al., 2021).

Figure 3: Liquid-liquid extraction separation of geranyl

ester.

Subsequently, the crude ester as a yellowish-brown

substance was produced by evaporating the extracted

material as shown in Figure 4.

ICHR 2024 - BRIN’s International Conference for Health Research (ICHR)

Figure 4: Crude GI product.

The percentage yields of GI crude esters were

calculated based on the percentage molarity of GI

divided by the molarity of starting material/reactan

from geraniol.

The relatively high yield is attributed to the

presence of impurities and geraniol that have not been

completey synthesized into GI ester. The crude ester

yield under various reaction conditions is presented in

Table 1.

Table 1: Crude ester yield.

Sample Weight (g) Yield (%)

GI (1:1)

–

RT 3.480 77.28

(

1:1,1

)

–

RT 4.011 89.11

(

1:1,3

)

–

RT 4.208 93.53

(

1:1

)

- 40°C 4.143 92.15

GI (1:1,1) - 40°C 3.472 77.10

GI (1:1,3) - 40°C 3.791 84.46

GI (1:1) - 60°C 3.433 76.43

(

1:1,1

)

- 60°C 3.522 78.29

(

1:1,3

)

- 60°C 4.035 89.46

(

1:1

)

- 80°C 4.104 90.98

GI (1:1,1) - 80°C 4.248 94.78

GI (1:1,3) - 80°C 4.173 92.95

The highest yield of 94.78% was produced in the

esterification of geraniol at a temperature of 80℃ and

a reactant molarity ratio of 1:1.1.

Afterthat, esterification results were analyzed by

TLC using silica gel plates (Kiesel gel 60F254 0.25

mm), with an eluent ratio of n-hexane to ethyl acetate

(9:1) at λ = 254 nm. The results showed that at RT,

the Retention factor (R

) value for GI matches the R

of geraniol of 0.73, indicating esterification has not

occurred yet. However, there is a gradual decrease in

the R

value from 0.65 to 0.6 and finally to 0.55 at

80°C, suggesting that 80°C is the optimal temperature

for forming the geranyl ester product.

Temperature is a crucial parameter that regulates

the rate and extent of esterification. An increase in

reaction temperature significantly impacts

esterification, where higher temperatures lead to

faster conversion. However, higher reaction

temperatures risk increasing product darkness. It has

been observed that even a small temperature

difference of 10-20°C can significantly affect the

reaction rate and product yield (Mazubert et al.,

2014).

In the esterification process, the addition of

excess reactants can drive the esterification reaction

toward higher product yield, consistent with Le

Chatelier's principle (Peris, 2021). Based on TLC also

showed that (GI) with a concentration ratio of 1:1.1

has the best R

value, while other concentrations show

no significant change. Although the 1:1.3

concentration is higher than 1:1.1, the R

value

decreases because the 1:1.1 concentration achieves

the optimal stoichiometric level. There is a threshold

where further increases do not significantly enhance

conversion and may even reduce it due to reaction

mixture saturation and catalyst deactivation (Lade et

al., 2014).

The rate of esterification is influenced by the

structure of the carboxylic acid. Linear chain acids

esterify more readily than branched acids due to steric

hindrance. The presence of branched chains in the

acid slows the reaction rate. Adding more chains to

the acid structure further reduces the reaction rate.

However, branched chain acids offer higher

conversion rates than linear chain acids. Additionally,

certain substituents can either accelerate or decelerate

the reaction rate (Jin et al., 2012).

value for synthesized compound of GI is 0.55,

lower than R

value of 0.73 from the starting material

geraniol. This is allegedly because larger size and

structure of GI than geraniol, less polar and causing

more interaction with the stationary phase of silica

gel.

Furthermore, antibacterial tests were conducted to

determine the efficacy of the crude ester with the best

results, as shown in Table 2.

Table 2: Antibacterial test results of crude GI extract.

Sample

Inhibition Zone

ubtilus

aureus

aeruginosa

E. coli

GI(1:1,1) -

80°C

16.67

±0.94

17.00

±1.63

16.33

±0.47

16.67±

1.25

GI(1:1) -

80°C

16.67

±0.47

15.33±0

.47

16.33

±1.70

16.00±

0.82

GI(1:1,3) -

80°C

16.33

±0.47

17.00±0

.82

15.33

±0.47

16.33±

1.25

GI(1:1,1) -

17.67

±2.05

16.33±0

.94

16.00

±0.82

15.33±

1.25

GI(1:1,1) -

40°C

16.33

±0.94

16.67±0

.47

16.33

±0.47

16.00±

1.63

GI(1:1,1) -

60°C

17.00

±0.82

16.33±1

.25

16.67

±1.70

16.00±

1.41

Geraniol

12.33

±1.25

11.67±1

.25

11.33

±0.47

10.00±

0.00

Study of the Temperature and Molarity Ratio Effects in Geraniol Esteriﬁcation and Testing Its Antibacterial Activity

Antibacterial test results indicate that the crude

ester with an isobutyric acid molar ratio of 1:1.1 at

80°C demonstrated the best antibacterial activity

against both gram-positive and gram-negative

bacteria. This condition proved to be more effective

compared to other molarity and temperature

variations. Therefore, the molar ratio of 1:1.1 at 80°C

is selected as the optimal synthesis condition. This

condition will be used for subsequent purification and

characterization stages. This is in accordance with the

yield produced, where the highest yield is produced

under these conditions.

3.2 Purification and Characterization

of GI by Using GCMS and FTIR

A total of 3500 mg of crude GI was purified using

gradient column chromatography, yielding 19

fractions. This method utilized silica gel 60 (230-400

mesh) and a gradient solvent mixture of n-hexane and

ethyl acetate. Fractions F6-F9 in n-hexane: ethyl

acetate (9:1), and F10-13 in n-hexane ; ethyl acetate

(8:2) were identified as geranyl isobutyrate, with

yields of 24 mg (0.682%), and 28 mg (0.795%)

respectively. The TLC of the purified ester as shown

in Figure 5.

Figure 5: The TLC of purified GI.

Tailing was observed in the TLC of the purified

product grom GI, likely due to the presence of

impurities or other by-products in the sample. To

confirm the presence of the compounds, further

analysis was conducted using GCMS and FTIR.

GCMS analysis was performed to identify the

components of the synthesis product based on mass

spectra, area and molecular weight. GC

chromatogram of pure GI showed 62 compound

peaks. It is due to geraniol as starting material itself

had 30 contaminant compounds with an area of

71.56% (Figure 6).

Figure 6: The chromatogram of geraniol

Table 3: Dominant peak in GCMS of GI ester.

No Compound

(minut1es)

Area

(%)

1 3-Buten-2-ol 17.976 33.89

Cyclohexanecarboxylic

Aci

18.177 5.75

3,7-dimethyl-2,6-

octadien

13.376 2.77

4 Geraniol 11.409 4.50

5 Oxalic Aci

7.326 3.28

Figure 7: GC chromatogram of synthesis product.

The five dominant peaks of GI ester chromatogram

presented in Table 3 and Figure 7. The retention time

) of 13.376 minutes corresponds to 3,7-dimethyl-

2,6-octadienyl or geranyl isobutyrate (GI) compound

with the area percentage was 2.77%, as shown in

Table 3 and Figure 7.

Based on the mass spectra in Figure 7, the geranyl

isobutyrate compound has a molecular mass (M) of

224 with a base peak at m/z = 69. Other characteristic

ICHR 2024 - BRIN’s International Conference for Health Research (ICHR)

peaks appear at m/z = 136 (M+-88), indicating the

loss of isobutyric acid. Another important peak is at

m/z = 93, formed from the loss of ethylene from m/z

= 121, and the peak at m/z = 154 is suspected to be a

fragment of the ester moiety.

FTIR analysis was conducted to determine the

functional groups present in the ester product. The

FTIR spectrum can be seen in Figure 8. The

interpretation of the wavenumbers for GI is provided

in Figure 8 and Table 4.

Figure 8: FTIR Spectra of GI.

Table 4: The IR Interpretation of GI.

Wavenumber (cm

-1

) Functional Group

3403,01 O-H

tretch

2925,40;2971,23;2879,78 C-H s

1717,83 C=O

1456,707 -C=C- bend

1080,37 C-O

The FTIR results show several characteristic

absorption bands. At a wavenumber of 1717.83 cm

-1

there is a significant absorption band corresponding

to the carbonyl (C=O) stretching vibration of the

ester. The presence of this group is further confirmed

by the absorption at 1080.37 cm

-1

, indicating the

presence of the C-O group of the ester. Additionally,

the absorption at 3403.01 cm

-1

shows the presence of

residu of unreacted geraniol. Wang et al. reported that

geranyl esters have an IR peak around 1735.87 cm

-1

indicating the presence of a carbonyl (C=O) group,

and a C- O absorption at 1175.32 cm

-1

(Wang et al.,

2019).

3.3 Antibacterial Test Using Diffusion

Method

The diffusion method is carried out using the paper

disk, with the bacterial count for sensitivity testing

ranges from 10

to 10

CFU/mL. Paper disks

containing antibiotics or samples are placed on a

medium containing the microbes, then incubated and

the results read based on the microbial inhibition

around the disk (Yusmaniar et al., 2017). The basic

principle of the method is the measurement of the

diameter of the clear zone, which indicates the

antibacterial compound's inhibition of bacterial

growth in the test sample (Bhargav et al., 2016.).

GI is a monoterpenoid with good antimicrobial

properties, and its results are optimized with proper

incubation time. The incubation process is conducted

for 18-24 hours as the bacteria are still in the

exponential phase. Interaction with the hydrophobic

structure of bacteria plays a key role in the

antimicrobial effects of hydrocarbons. Bacteria are

more sensitive during the exponential phase

compared to the stationary phase. Several

antimicrobial agents cause significant changes to the

plasma membrane, resulting in total cell lysis.

Although the activation of autolytic enzymes may be

responsible for this effect, lysis can also be caused by

the weakening of the cell wall and disruption of the

cell membrane due to osmotic pressure, rather than

specific action on the membrane (El Kolli et al.,

2016).

The antibacterial test conducted on crude ester

and pure GI produced at optimum esterification

process at a 1:1.1 molarity ratio and 80°C temperature

compared to reactant of geraniol (G). Tetracycline

(TS), streptomycin (MS), and dimethyl sulfoxide

(DMSO) were used as positive and negative controls,

respectively, at a concentration of 16,000 ppm. This

activity test was performed against gram-positive

bacteria, namely B. subtilis and S. aureus, as well as

gram-negative bacteria, P. aeruginosa and E. coli.

The results showed at Table 4. It can be seen that

DMSO, as a negative control, exhibited no

antimicrobial activity, with a consistent inhibition

zone of 6 mm. This confirms that the observed

antimicrobial activity is due to GI ester compound.

Tetracycline and streptomycin were used as positive

controls due to their broad-spectrum activity against

various gram-positive and gram-negative bacteria

and their use in both veterinary and human medical

treatments (Araby et al., 2020). Comparatively, GI

showed fairly good antibacterial activity, indicating

potential as a therapeutic agent. Visualization of the

inhibition zones in the antibacterial disk diffusion test

is shown in Figure 9.

Study of the Temperature and Molarity Ratio Effects in Geraniol Esteriﬁcation and Testing Its Antibacterial Activity

Table 5: The antibacterial activity of samples.

Sample

Inhibition Zone

(

)

ubtilus

aureus

aeruginosa

E. coli

23.67

±0.47

30.00

±0.00

18.00

±0.00

20.00

±0.00

21.33

±1.89

30.00

±0.00

23.33

±2.36

26.33

±0.94

DMSO

6.00

±0.00

6.00

±0.00

6.00

±0.00

6.00

±0.00

Pure GI

18.33

±2.62

15.67

±0.47

10.67

±0.47

16.67

±2.36

Crude

16.67

±0.94

17.00±

1.63

16.33

±0.47

16.67

±1.25

12.33

±1.25

11.67±

1.25

11.33

±0.47

10.00

±0.00

B. subtilus S. aureus

aeruginosa

E. coli

Figure 9: Inhibition zone of pure GI.

Additionally, Table 5 shows stronger antibacterial

activity against gram-positive bacteria, with

inhibition zones of 18.33 mm for B. subtilis and 15.67

mm for S. aureus. Based on these inhibition zones GI,

is categorized as a strong antibacterial (Ullah & Ali,

2017). Zabin declared that essential oils with geranyl

isobutyrate as the main product also exhibit good

activity against gram-positive bacteria (Zabin, 2018).

Previous studies have shown that the antibacterial

effect of monoterpenoids is weaker against gram-

negative bacteria due to their hydrophilic nature,

which prevents the contact of hydrophobic

monoterpenoid components with bacterial cells. In

contrast, gram-positive bacteria can directly damage

their cell membranes, leading to cell membrane

rupture, inhibition of enzyme systems, and increased

ion permeability (Lang, 2010). Pure GI showed a

significant increase in inhibition zones compared to

geraniol as starting material. However, there was a

decrease in against P. aeruginosa and S. aureus when

compared to crude GI (1:1.1). This may be due to the

some compounds present in GI were separated during

purification, which may contribute to antibacterial

activity against P. aeruginosa and S. aureus.

Additionally, P. aeruginosa is challenging to

eradicate due to its biofilm-forming ability

(Srivastava et al., 2021). Meanwhile, S. aureus is a

major concern due to its high resistance levels (Zabin,

2018). Consequently, pure GI showed decreased

antibacterial activity against these bacteria compared

to crude GI, which contains more compounds with

potential antibacterial activity against these bacteria.

5 CONCLUSIONS

The study indicate that both temperature and reactant

molarity ratio significantly influence the efficiency of

GI ester formation. The optimum reaction of

esterification was at a 1:1.1 of molarity ratio and a

temperature of 80°C which produces the largest crude

GI of 4.248 g (94.78%), nevertheless the pure GI

produced was only 52 mg (1,48%). One of the

dominant peaks on the GCMS chromatogram

revealed the presence of GI compound at R

13.376 minutes with an area of 2.77% as well as the

FTIR spectra displayed functional groups of carbonyl

(C=O) at 1717.82 cm

-1

from ester compound.

Antibacterial activity test demonstrated that GI

exhibited significant antibacterial activity against

gram-positive bacteria, with inhibition zones of

18.33±2.62 mm for B. subtilis and 15.67±0.47 mm for

S. aureus, and against gram-negative bacteria, with

inhibition zones for P. aeruginosa 10.67±0.47 mm

and for E. coli 16.67±2.36 mm. Therefore GI is

classified as an strong antibacterial agent against

gram-positive bacteria.

Further purification is needed to increase the pure

ester yield, as well as minimum inhibitory

concentration (MIC) and minimum bactericidal

concentration (MBC) analysis.

ACKNOWLEDGEMENTS

The authors would like to express their deepest

gratitude to Advanced Characterization Laboratories

Serpong, BRIN for their facilities support, and the

Research Organization of Health BRIN for

supporting this research with the funding number

5/III.9/HK/2024.

Galuh Widiyarti who had the research idea and

Stefanie Sugiarto who helped conduct the research by

doing synthesis and antibacterial analysis of GI are

the main contributors. Whilst Novita Ariani who

helped antibacterial test and Elvina Dhiaful Iftitah

who gave comments on the draft manuscript are co

contributors.

ICHR 2024 - BRIN’s International Conference for Health Research (ICHR)

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ICHR 2024 - BRIN’s International Conference for Health Research (ICHR)