Biology, Medicine, & Natural Product Chemistry  ISSN 2089-6514 (paper) 

Volume 13, Number 2, October 2024 | Pages: 407-413 | DOI: 10.14421/biomedich.2024.132.407-413 ISSN 2540-9328 (online) 
 

 

 

 

Effectiveness of Ethanol Extract Clove Leaves (Syzygium aromaticum) 

In Inhibiting Biofilm of Candida Albicans ATCC 14053 

 
Firdausi Nuzula1, Masfufatun2,* 

1Faculty of Medicine, University of Wijaya Kusuma Surabaya, Indonesia. 
2Department of Biochemistry, Faculty of Medicine, University of Wijaya Kusuma Surabaya, Indonesia 

Jl. Dukuh Kupang XXV No. 54, Dukuh Kupang, Dukuh Pakis, Surabaya, Indonesia. 

 

 

Corresponding author* 
masfufatun@uwks.ac.id 

 

 
Manuscript received: 15 July, 2024. Revision accepted: 07 October, 2024. Published: 09 October, 2024. 

 

 

Abstract 

 

Candida albicans is the most common type of Candida. It is a normal microbiota in the healthy human body that can become pathogenic 

when its balance is disturbed, causing an infection referred to as candidiasis. Antifungal resistance to biofilms is estimated to be 10,000 

times that in planktonic form. Clove leaves (Syzygium aromatium) can treat infectious diseases, including candidiasis. This study aimed to 

analyze whether the ethanol extract of clove leaves (S. aromaticum) destroys the biofilm of C. albicans ATCC 14053. The research 

design was pure experimental with a post test control group only design approach. The extraction method used was maceration. Test 

antibiofilm activity of ethanol extract of clove leaves using microtiter plate biofilm assay method. Measurement of results with a 

microplate reader was carried out using a wavelength of 595nm in accordance by the wavelength of crystal violet used as a colouring 

agent. The results showed that ethanol extract of clove leaves (S. aromaticum) has significant effectiveness against the maturation of C. 

albicans biofilm (p<0.05) with the highest percentage of activity at 20% concentration. The MBEC50 was determined by probit analysis, 

so the concentration of clove leaf ethanol extract (S. aromaticum) that can eradicate 50% of C. albicans biofilm is located at a 

concentration of 0.45%. The clinical benefits that can be developed from the results of this study are the potential use of clove leaf ethanol 

extract as an alternative therapy for C. albicans infection in the form of soap or ointment. 

 

Keywords: Biofilm; Candida albicans; Candidiasis; Clove leaf. 

 

Abbreviations: MBEC Minimum Biofilm Eradication Concentration; ATCC American Type Culture Collection 

 

 

INTRODUCTION 
 

Candida is a genus of fungi often found in the 

environment, especially in tropical climates (Mbatu et 
al., 2018). Candida albicans is the most commonly found 

type of Candida and is a normal microbiota in the 

healthy human body, for example, on the skin, mouth, 

intestines, genital tract, and under the nails (Khafidhoh et 

al., 2015). C. albicans can become pathogenic when its 

balance is disturbed, which will accelerate its 

proliferation and cause infection. Fungal infections 

caused by Candida are referred to as candidiasis 

(Masfufatun et al., 2014).  

In Indonesia, candidiasis is around 20-25% 

(Puspitasari et al., 2019). The occurrence of candidiasis 

recurrence is related to the pathogenicity of C. albicans, 

one of which is supported by the ability to form biofilms. 

The stages of biofilm formation include attachment, 

microcolony formation, biofilm formation, maturation, 

and cell dispersion (Purbowati, 2016). Biofilms are 

composed of various types of cells (yeast-shaped, having 

round buds, oval pseudohyphal cells, and long hyphal 

cells), attached to biotic and abiotic surfaces and 

embedded in a layer called the exopolysaccharide matrix 

(Gulati et al., 2016). The presence of this matrix causes 

resistance to antifungals and hinders phagocytosis by 

host cells (Tsui et al., 2016). 

Currently used antifungals include azoles, polyenes, 

echinocadin, allylamine, and fluoropyrimidine (Kabir & 

Ahmad, 2013). Although azoles have long provided 

effective treatment, recent epidemiological studies have 

shown azole resistance in some cases of candidiasis 

(Whaley et al., 2017). Antifungal resistance to biofilms is 

estimated to be 10,000 times that in planktonic form 

(Rabin et al., 2015). Antifungal resistance occurs due to 

complex biofilm-forming components, making it more 

difficult for antifungals to work (Sharma et al., 2016). In 

addition to causing resistance, prolonged use of synthetic 

drugs can result in respiratory depression, uritaccharia, 

hepatotoxicity, decreased platelet levels, and 

gastrointestinal disorders (Lestari et al., 2019). 

An effort is needed to inhibit growth in both 

planktonic and biofilm forms. One of them is by using 

https://doi.org/10.14421/biomedich.2024.132.407-413
mailto:masfufatun@uwks.ac.id


 

 
 

408 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 407-413 

 

 

natural materials. According to research that has been 

done, there is an antibiofilm effect of clove (S. 
aromaticum) essential oil nanoemulsion against 

Staphylococcus aureus with an MIC of 0.62 mg/mL, 

while against Cryptococcus neoformans with an MIC of 

3.12 mg/mL, and against with an MIC of 50 mg/mL 

(Shehabeldine et al., 2023). 

Clove is a spice plant native to Indonesia. Almost all 

parts of cloves can be utilized, starting from the leaves, 

flowers, and twigs. Clove leaves (Syzygium aromatium) 
can be used to treat infectious diseases, including 

candidiasis. In the phytochemistry of clove leaf extract, 

both crude and purified extracts contain flavonoids, 

tannins, saponins, terpenoids, and alkaloids (Novema et 

al., 2022). 

 

 

MATERIALS AND METHODS 
 

Research design 

The research design used was pure experimental with a 

post test control group only design approach. Test the 

antibiofilm activity of clove leaf ethanol extract using the 

microtiter plate biofilm assay method.  

 

Extraction of clove leaves 

The method used to perform the extraction is maceration. 

Prepare 995 grams of simplistic powder and then put it in 

a macerator. Adding 96% ethanol solvent into the 

macerator. After that, let stand until all the simplistic 

powder is submerged for 3x24 hours while stirring 

periodically. Then the mixture is filtered and the pulp is 

soaked again using new 96% ethanol. The further 

filtering process will be carried out twice using 96% 

ethanol. Liquid extracts are put together and concentrated 

using a vacuum rotary evaporator at 78oC and 

concentrated again using a water bath at 40oC to produce 

thick extracts (Wahyulianingsih et al., 2016). 

 

Concentrations of extract 

Each treatment requires 4 repetitions in 8 test groups, so 

the concentrations used in each treatment are 20%, 10%, 

5%, 2.5%, 1.25%, 0.63%, 0.31%, 0.16%. The finished 

thick extract of clove leaves (S. aromaticum) was 

weighed by starting the largest concentration of 20%. 

Then put into a vial bottle. The thick clove leaf extract 

solution was diluted using dimethyl sulfoxide (DMSO) 

solvent according to the desired concentration using the 

following dilution formula: 

 

𝑀1 𝑥 𝑉1 =  𝑀2 𝑥 𝑉2 

 

𝑀1 : Concentration of solution before dilution 

𝑉1 : Volume of solution before dilution 

𝑀2 : Concentration of solution after dilution 

𝑉2 : Volume of solution after dilution (Iqhasari R, 

2017). 

Regeneration and preparation of C. albicans 

inoculum 

C. albicans isolates were inoculated as much as 1 ose by 

streak plate method or scraping the colony. Furthermore, 

the new SDA media is incubated using an incubator for 

2-3 days so that a single colony of C. albicans will form. 

Inoculation of C. albicans on SDB media for 18-20 hours 

at room temperature using a shaker at 120 rpm produces 

inoculum (Naitullah et al., 2014). 

 

Harvesting of inoculum and preparation of C. 

albicans suspension 

Single colonies of C. albicans grown on SDA for 2-3 

days were put into Erlenmeyer flasks containing 10 ml of 

SDB. SDB media was used as a dilution solution because 

it supports biofilm growth. Next, the flask was shaken for 

24 hours at 120 rpm in an Erlenmeyer flask. Performed 

centrifugation by moving the C. albicans inoculum into a 

centrifuge tube for 15 minutes until a pallet was 

produced. The pallet formed was separated from the 

filtrate using PBS and resuspended to 1 x 106 CFU/ml 

with RPMI media. The Optical Density (OD) of the C. 

albicans suspension will be calculated using a microplate 

reader. If the OD > 0.5, continue diluting until OD = 0.5 

is obtained. C. albicans OD 0.5 suspension is ready for 

an antibiofilm test (Fauzan et al., 2023). 

 

Mature biofilm formation  

100 μl of C.albicans suspension was put into the wells 

starting from rows A-D, columns 1-6, 8, 10, and 12. 

Then it was incubated for 2 hours at 37o C for the 

C.albicans attachment stage. Next, 100 μl RPMI media 

was added to all wells and incubated for 48 hours at 37o 

C for the growth stage of C. albicans. Then, added 

extracts in concentrations of 20%, 10%, 5%, 2.5%, 

1.25%, 0.63%, 0.31%, 0.16%, RPMI (for positive, 

negative, and blank media controls), and fluconazole (for 

positive control). For blank samples, 50 μl of C. albicans 

suspension was also placed into the wells in rows G and 

H. Incubated at 37°C for 24 hours for the C.albicans 

maturation stage (Zhu et al., 2023).  

 

Mature biofilm eradication test 

After incubation at 37°C for 24 hours for the C.albicans 
maturation stage. Each well was washed twice using 

PBS. The unattached planktonic cells of C. albicans were 

gently removed by inverting and pouring on a tissue to 

obtain C. albicans attached to the microplate. The 

biofilm was fixed using methanol for 15 minutes and 

then dried at room temperature. Next, 0.1% crystal violet 

solution was added to each well for staining for 30 

minutes at room temperature. Next, wash 3 times using 

PBS. Adding 96% ethanol to each well then incubated 

for 1 hour at room temperature and measured using a 

microplate reader (Zhu et al., 2023). 
 



 

 
 

 Nuzula & Masfufatun – Effectiveness of Ethanol Extract Clove Leaves (Syzygium aromaticum) … 409 
 

 

Minimum biofilm eradication concentration 

(MBEC50) 

Minimum Biofilm Eradication Concentration (MBEC50) 

is the lowest clove leaf ethanol extract concentration that 

can eradicate 50% of C. albicans biofilm. The MBEC50 

value was determined using OD maturation data 

converted to % eradication and analyzed SPSS probit 

(Gupta et al., 2016). 

 

Data analysis 

The data analysis method used SPSS (Statistical Product 

of Service Solution) statistical analysis for Windows. 

The normality test used if the data is less than 50 is the 

Shapiro-wilk test. Furthermore, a homogeneity test was 

carried out using the Levene test if it was proven that the 

data was typically distributed or close to normal. Data 

that are normally distributed and homogeneous (P>0.05) 

will be analyzed using the One-Way Analysis of 

Variance (Anova) test, which is a parametric test to 

compare differences in the means of data from more than 

two groups. The significance level was set at P<0.05. 

After the One-Way ANOVA test was conducted, the Post 

Hoc test was followed. The Post Hoc test is an advanced 

test that analyzes which test group has a significant 

difference. Furthermore, the MBEC50, it was determined 

using SPSS probit analysis.  

 

 

RESULT AND DISCUSSION 
 

RESULT 

Characteristics of Clove Leaf  
This study aimed to test the antibiofilm effect of ethanol 

extract of clove leaves (S. aromaticum) at the maturation 

stage. Clove leaves were obtained from Jatirejo District, 

Mojokerto. The clove leaves were picked, washed, and 

aerated for one week. After drying, the clove leaves were 

pulverized using a blender until they became powder 

(simplistic). Clove leaf simply obtained as much as 995 

grams. The clove leaf ethanol extract was prepared by 

maceration method, namely by soaking the fine 

simplistic using 96% ethanol solvent and filtering using 

filter paper for 3x24 hours while stirring periodically. 

The liquid extract was combined and concentrated using 

a vacuum rotary evaporator at a temperature of 78o C 

and concentrated again using a waterbath with a 

temperature of 40o C to produce 156 grams of clove leaf 

ethanol extract with a greenish brown color. 

 

Biofilm Maturation Detection Test 

To see the biofilm maturation, C. albicans was incubated 

at 37°C for 2 hours for the attachment stage. 

Furthermore, another incubation was carried out for 48 

hours at 37o C for the growth stage of C. albicans. Then, 

the ethanol extract of clove leaves (S. aromaticum) was 

added. The wells that have been treated are incubated for 

24 hours at 37°C.  The results of the C. albicans biofilm 

maturation test can be seen in Figure 1 

 

 

 
 

Figure 1. Mature Biofilm Eradication Test Results with Crystal Violet 

Staining. 
Column 1A-E = concentration 20% 

Column 2A-E = concentration 10% 

Column 3A-E = concentration 5%  
Column 4A-E = concentration 2.5% 

Column 5A-E = concentration 1.25% 

Column 6A-E = concentration 0.63% 
Column 7A-E = concentration 0.31% 

Column 8A-E = concentration 0.16% 

Column 9A-E = positive control 
Column 10A-E = negative control 

Column 12A-E = media blank 

 

 

Based on Figure 1., the results show that clove leaf 

extract at a concentration of 20% after giving crystal 

violet has a faded purple color and the smaller the 

concentration, the more intense the purple color 

produced, meaning the higher the density of biofilm 

biomass.  

The most intense purple color was found in the 

negative control group. Furthermore, the 

absorbance/Optical Density (OD) value was measured 

which can be seen in Table 1. 

 

 
 

Table 1. OD Mature Biofilm Eradication Test Results with Clove Leaf Extract. 
 

Replication 
Clove Leaf Extract Concentration Control 

20% 10% 5% 2.5% 1.25% 0.63% 0.31% 0.16% K (+) K (-) 

1 0.29 0.60 0.66 0.76 0.84 0.92 1.56 3.36 1.92 3.27 

2 0.13 0.46 0.59 0.85 0.68 0.96 1.24 3.22 2.06 3.16 

3 0.20 0.61 0.54 0.68 0.73 0.62 1.28 3.15 2.08 3.06 

4 0.19 0.50 0.41 0.63 0.60 0.64 0.84 3.07 1.80 3.32 

Average 0.20 0.54 0.55 0.73 0.71 0.79 1.23 3.20 1.97 3.20 

Std 0.06 0.07 0.09 0.08 0.09 0.16 0.26 0.10 0.11 0.10 

 



 

 
 

410 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 407-413 

 

 

In Table 1, the C. albicans mature biofilm test results 

were replicated four times, with the highest average OD 

value obtained at a concentration of 0.16%, namely 3.20, 

and the weakest at a concentration of 20%, namely 0.20. 

At a concentration of 0.16%, the highest OD value is 

obtained, meaning that the density of the biofilm biomass 

is the highest so that the ability of clove leaf extract to 

eradicate C. albicans biofilm is lower and at a 

concentration of 20% has the lowest OD, meaning that 

the density of the biofilm biomass is the lowest so that 

the ability of clove leaf extract to eradicate C. albicans 
biofilm is higher. Meanwhile, for the control group, the 

highest OD value was found in the negative control (K-) 

because the negative control group was not given any 

treatment for C. albicans cells that matured on the 

microplate. Table 1 shows a decrease in the average 

maturation of C. albicans biofilm and an increase in 

concentration. 

 

 
Figure 2. Post Hoc Analysis Results of Mature Biofilm Eradication with Clove Leaf Extract. 
Superscript abc, if it contains the same letters means there is no significant difference.  

If it contains different letters, it means there is a significant difference between the treatment groups based on the Post hoc test (P value <0.05 shows a 

significant difference) 

 

 

Based on the Post hoc test results in Figure 2, 

significantly different groups were at a concentrations of 

20%, 0.31% and positive control while groups that did 

not have significant differences were at concentrations of 

10%, 5%, 2.5%, 1.25%, 0.63%, 0.16% and negative 

control. 

MBEC50 Value Determination Results 

The Optical Density of the Table was used to determine 

the percent of C. albicans biofilm eradication testing 

using the formula below: 

 

 

% Biofilm maturation rate =
(OD negative control − OD sample)

(OD negative control)
x 100% 

 

 
 

Table 2. Eradikasi Biofilm. 

 

% Eradikasi 

Clove Leaf Extract Concentration 

20% 10% 5% 2.5% 1.25% 0.63% 0.31% 0.16% 

93.67 83.10 82.82 77.21 77.78 75.43 61.58 0.01 

 

 

From table 2, the highest eradication results are in the 

clove leaf ethanol extract group with a concentration of 

20% having the highest eradication ability, meaning that 

the growth of C. albicans biofilm is getting lower and 

vice versa at a concentration of 0.16% has the lowest 

eradication ability, meaning that the growth of C. 

albicans biofilm is getting higher.  
Determination of MBEC50 using probit analysis 

obtained the results of the concentration of clove leaf 

extract that can eradicate 50% of C. albicans biofilm is 

located at a concentration of 0.45%, meaning that clove 

leaf ethanol extract can eradicate 50% of C. albicans 
biofilm at a concentration of 0.45%. 

 

 

DISCUSSION 

Clove leaf extract yield 

The clove leaves used in this study are about 6-8 months 

old, green , and fresh. Clove leaves that have been picked 

must be washed first to remove dirt, then aerated for one 

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

20% 10% 5% 2.5% 1.25% 0.63% 0.31% 0.16% K (+) K (-)

A
v
er

an
g
e 

O
p
ti

ca
l 

D
en

si
ty

 (
5
9
5
n
m

)

Clove Leaf Extract Concentration

Maturation Stage Curve

b
a

b b b b
c

d d

e



 

 
 

 Nuzula & Masfufatun – Effectiveness of Ethanol Extract Clove Leaves (Syzygium aromaticum) … 411 
 

 

week to reduce the water content so that it can inhibit 

microbial growth. After drying, the clove leaves are 

mashed using a blender until they become powder 

(simplisia) (Warnis, et al., 2020). The particle size of the 

sample is related to the surface area that will contact the 

extraction solvent. The finer the simple, the easier the 

compounds in the leaves move into the solvent (Aji, 

2018). 

The extraction method used in this research is 

maceration using 96% ethanol solvent. The 96% ethanol 

solvent was chosen because it is a universal solvent that 

can dissolve organic compounds in samples of both polar 

and non-polar compounds (Munte et al., 2015). The use 

of 96% ethanol as a solvent because ethanol is more 

accessible to enter the cell membrane so that it can 

extract secondary metabolite compounds such as 

flavonoids, tannins, saponins, terpenoids, and phenols 

(Dewi et al., 2021). From the maceration results of 995 

grams of clove leaf simplisia, 156 grams of thick extract 

were obtained with a percentage yield of 16%. This 

research is in line with that conducted by Suhendar and 

Sogandi (2019) who used the maceration method extract 

clove leaves. However, the solvent in the study used 70% 

methanol. The results of this method from 1 kg of clove 

leaf simplistic obtained a thick extract of 100.08 grams 

with a percent yield of 10%. Where good yield results 

have a value of> 10% (Novema et al., 2022). 96% 

ethanol solvent has a lower polarity than 70% methanol. 

Methanol 70% also contains 30% water, which can 

reduce the extraction efficiency of some lipophilic 

compounds. Water can cause hydration or precipitation 

of specific components that should be extracted. Thus, 

compounds in clove leaves with lipophilic properties 

(like fat) will dissolve more easily in 96% ethanol. 

Organoleptic results show that the methanol extract of 

clove leaves has a thick form, reddish brown color, bitter 

taste, and smells typical of cloves. Meanwhile, the 

ethanol extract of clove leaves also has a thick texture, 

greenish brown color, and smells typical of cloves. 

Dimethyl sulfoxide (DMSO) is used to dissolve 

organic compounds that are less soluble when using 

distilled water. DMSO is a solvent that can dissolve 

almost all compounds both polar and non-polar. DMSO 

has low toxicity, has anti-inflammatory, and analgesic 

effects. In research conducted by Rahmi & Putri (2020), 

it was found that the use of DMSO as a solvent had no 

effect on the test results carried out using the disc paper 

method against C. albicans. In this study, DMSO solvent 

was used because clove leaf ethanol extract does not 

have an aqueous phase. 

 

Effect of Clove leaf extract on mature biofilm  

Based on the results of the mature biofilm eradication 

test in Table 2. the OD value of the test group of Clove 

Leaf Extract is lower than the OD of the positive control 

except at a concentration of 0.16%. This indicates that at 

concentrations of 0.31% to 20%, clove leaf ethanol 

extract has a better mature biofilm effect than 

fluconazole antifungal. The lowest OD value was found 

at 20% concentration. The OD value from concentrations 

of 0.16% to 20% at the stage of mature biofilm of C. 
albicans is decreasing, which shows that with the 

increasing concentration of extracts used, the ability to 

eradicate biofilms is increasing. Based on the results of 

anova analysis, indicate that there is an effect of giving 

clove leaf ethanol extract (S. aromaticum) on the 

maturation stage of C. albicans biofilm significantly 

(p<0.05). In Figure 2, Groups that have significant 

differences are at concentrations of 20%, 0.31%, and 

positive control, while groups that do not have 

substantial differences are at concentrations of 10%, 5%, 

2.5%, 1.25%, 0.63%, 0.16%, and negative control. 

Clove leaf can eradicate mature biofilm because it 

contain active compounds such as saponins, alkaloids, 

tannins, flavonoids, and terpenoids. Tannins will inhibit 

the icaA and icaD genes. These genes will synthesize 

PIA, which plays a role in EPS formation and cell 

aggregation. The action of saponins is to increase cell 

wall permeability by reducing surface tension, so that 

cells leak and intracellular components come out. The 

role of saponins as antibiofilm monospecies of C. 

albicans is at the stage of biofilm adherence and 

maturation (Hamzah et al., 2021). 

From Table 2. the most effective test group was at 

20% concentration because the percentage was the 

highest at 93.67%. Thus, the antibiofilm activity of clove 

leaf ethanol extract to eradicate C. albicans biofilm is 

high because the percentage of antibiofilm activity is 

≥50% (Famuyide et al., 2019). 

At the maturation stage, research that supports this 

research is research conducted by Mirzaei et al., (2022), 

where melittin, vancomycin, and rifampicin against 

Staphylococcus epidermidis biofilms produced MBEC50 

for melittin, rifampin, and vancomycin, are 0.002%, 

0.0512%, and 0.1024%. 50 According to Abidah's 

research, (2020), black mulberry leaf extract (Morus 

nigra L.) against Escherichia coli biofilm has an 

MBEC50 of 0.016%. Meanwhile, in this study the 

MBEC50 is 0.45%. 

As the mature biofilm, the synergy and 

communication between the fungi in the biofilm becomes 

more complex. The EPS matrix layer that is formed is 

more numerous and thicker. The EPS matrix can increase 

the defense of fungi compared to still in planktonic form. 

The eradication ability of an extract is related to the 

ability of the compounds contained in the extract to 

penetrate the EPS matrix layer that envelops the bacteria 

and destroys the EPS matrix in the biofilm. 

Microcolonies that have formed will develop and 

undergo maturation. maturation is influenced by 

electrolyte concentration, carbon source, pH, surface 

type, temperature, and osmolarity. Maturation is 

characterized by an increase in biofilm density and 

complexity (Kumar et al., 2017). 

 

 



 

 
 

412 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 407-413 

 

 

CONCLUSIONS 
 

Based on the results of the research that has been done, it 

can be concluded that the ethanol extract of clove leaves 

(S. aromaticum) has an antibiofilm effect on the 

maturation stage of C. albicans significantly. At the 

highest concentration, 20% was able to eradicate 

93.67%. the Minimum Biofilm Eradication 

Concentration (MBEC50) in eradicating of 50% C. 

albicans biofilm is 0.45%. Thus, clove leaf ethanol 

extract has the potential to be an alternative therapy for 

C. albicans infection. 

 

 

Acknowledgments: Special gratitude goes to the 

Department of Biochemistry, University of Wijaya 

Kusuma Surabaya and Infectious Hospital University of 

Airlangga. 

 

Authors’ Contributions: Firdausi Nuzula laboratory 

work and wrote the manuscript. Masfufatun designed the 

study and final coordinator of the manuscript. All authors 

read and approved the final version of the manuscript.  

 

Competing Interests: The authors declare that there are 

no competing interests. 

 

 

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