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Journal of Microbial Systematics and Biotechnology
2021, 3(1): pp.41-48
ISSN (Online): 2685-4430
Copyright© 2019, Indonesian Culture Collection, LIPI
41
Received August 16, 2021 / Accepted August 31, 2021 / Published online August 31, 2021
DOI:10.37604/jmsb.v3i1.78
Corresponding Author: khusnul@stikes-bth.ac.id
Effect of clove essential oil (Syzygium aromaticum) against the
growth of dandruff scalps-causing fungal pathogen using Kirby-
Bauer method in vitro
Khusnul*1, Puput Putuh Anisa1, Dewi Peti Virgianti1
1School of Technology Laboratory Medic, Sekolah Tinggi Ilmu Kesehatan Bakti Tunas Husada Tasikmalaya,
Indonesia
Khusnul, Anisa PP, Virgianti DP. 2021. Effect of clove Flower Essential Oil (Syzygium
aromaticum) against the growth of several pathogenic fungi that cause dandruff in vitro.
Journal of Microbial Systematics and Biotechnology 3(1), 41-48.
Abstract
One of the strongest contributing factors in dandruff scalps is the presence of fungal
pathogen or called as Malassezia. Several studies have found a connection between
Malassezia and scalp health, one of which is caused by pathogenic fungal infections
including Pityrosporum ovale,Microsporum gypseum, and Candida albicans. Clove flowers
are known to contain eugenol and its derivative compounds which have antimicrobial,
antifungal, antiseptic, and local anesthetic activity. This research aims to determine the effect
of clove flower essential oil (Syzygium aromaticum) against some dandruff-causing fungi.
The research method was to test the inhibitory power of clove flower essential oil using the
diffusion method (Kirby Bauer technique). Samples of pathogenic fungi were cultured on
SDA media (Saboroud Dextrose Agar) and given discs that had been given several
concentrations of clove flower essential oil. The concentrations used were 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%, positive control using 2% ketoconazole and
negative control using virgin coconut oil (VCO). The results showed a significant effect
between the ability of essential oils to inhibit the growth of some fungi that cause dandruff.
The treatment of clove flower essential oil against M. gypseum at the concentration of 10%
has an inhibitory ability of 33,05 mm and at the concentration of 100% amounted to 63.95
mm. Meanwhile, the inhibition percentage in P. ovale with a concentration of 10% and
100% of extract were 14,75 mm and 46,15 mm, respectively. The inhibition rate in C.
albicans with the concentration of 10% extract and 100% of extract was 13,5 mm and 39,35
mm. The research shows that clove flower essential oil inhibits the growth of several
pathogenic fungal in vitro.
Keywords: dandruff scalps, fungal pathogen, in vitro, Kirby-Bauer, Syzygium aromaticum
Introduction
Indonesia is a tropical country with a perfect location for the development of diseases
caused by fungal pathogen. Most of fungi can live optimally in humid environmental
conditions. In addition, these climatic conditions allow the scalp to become more humid,
which has the potential for the growth of pathogenic fungi supported by air pollution, dust,
and sunlight that can cause damage to the hair and scalp.
42
In humans, the scalp is unlike any other skin. It has a thick layer of skin, a high
follicle density, and numerous sebaceous glands. The hair shaft has a pH of 3.67 and the
scalp has a pH of 5.5. The existence of these glands, together with the scalp's dark and warm
environment, renders it more prone to fungal infections like dandruff and seborrheic
dermatitis, as well as parasitic infections like Pityriasis capitis. Despite the fact that scalp
problems are not among the most common diseases that cause serious physical illness or
morbidity, they have a significant social impact. The state of one's scalp and hair has a
greater psychological impact on the general population. Even minor changes in hair such as
gray hair and dandruff, affect a person's self-confidence. Scalp disorders including fungus
and bacterial infestation causing problems such as Tinea capitis (Narshana and Ravikumar,
2017). Pityriasis capitis (dandruff) is a squamous scalp disorder that is practically
physiological and is defined by the formation of small scales without indications of irritation.
It is commonly thought to be a minor type of seborrheic dermatitis.
Herbal medications, in addition to synthetic drugs, can be employed as a drug that can
be used for our bodies. Antifungal chemicals have been found in a variety of herbal plants in
Indonesia, including clove flower or Syzygium aromaticum. However, most of people chose
alternative medicine first because of lower risk of medication side effects, more affordable
price, and easier application. Clove flowers are a type of spices in Indonesia which is
commonly used as an alternative. Clove flowers contain a variety of beneficial chemicals.
According to Prianto, et al. (2013), clove flowers contain eugenol, trans-caryophyllene,
alpha-humulene, eugenol acetate, caryophyllene oxide, and trimetoxyacetophenone. The
content of eugenol and its derivative compounds has pharmacological activity as an analgesic,
anti-inflammatory, antimicrobial, antiviral, antifungal, antiseptic, antispasmodic, antiemetic,
stimulant, and local anesthetic (Towaha, 2012).
The results of previous research conducted by Yuliana (2014) showed that clove
flower essential oil had antifungal activity on wood with an inhibition zone of 29 mm.
Research conducted by Febrianti and Riyanta (2012) also found that clove leaf essential oil
could effectively inhibit the growth of Candida albicans with a volume concentration of 0.3
ml extract. Subsequent research conducted by Alfauziah and Budiman (2016) has also
successfully found that clove essential oil with a concentration of 1% exhibiting antifungal
activity with an inhibition zone of 1.12 cm. Emulsion with 0.5% CMC emulsifier was the
best formula and showing inhibition activity against wood fungi of 2.9 cm.
Based on the previous relevant studies, no research has been conducted on the
inhibitory activity of clove flower essential oil against some dandruff fungus (P. ovale, C.
albicans, and M. gypseum). Many researchers conclude that clove flower essential oil can be
an alternative herbal medicine against antifungals but there is no specific study on the
inhibitory this so that it is important for the finding of new alternatives for the scalp treatment.
Materials and methods
Sample preparation
The materials used in this study are Sabouraud Dextrose Agar (oxoid), Physiological
NaCl, 1% BaCl2, 1% H2SO4, 2% ketoconazole, clove flower essential oil, stock culture of P.
ovale,M. gypseum,C. albicans, and aquadest sterile. The equipment used were autoclave
(Portable M300), light microscope (Olympus CX23), evaporator (IKA RV 10), dry sterilizer
(Corona ZTP8A-7 (IR Ray), incubator (Memmert UNB 4000, analytical balance (Excellent
Analytical), balance AB HZK-2104, oven (T100-200), stir bar, mattress thread, petri dish,
beaker, Erlenmeyer, measuring cup, spirit flask, round loop, hot plate, micropipette, tip,
spatula, test tube, gauze , umbrella paper. In this study, the inhibition activity of clove flower
essential oil against the growth of some dandruff scalp-causing fungal pathogen (P. ovale, C.
43
albicans, and M. gypseum) was tested in vitro using the Kirby-Bauer method with samples
taken from pure cultures that have been cultured on Saboraud Dextrose Agar (SDA). The
sample was then given clove flower essential oil followed by incubation at a temperature of
250-300C for 4 days.
Preparation of Mc.Farland's standard
The Mc.Farland's standard 0.5 was done by pipetting 0.05 ml of 1% H2SO4 solution
and adding 9.95 ml of 1% BaCl solution, the two solutions were mixed (Clinical And
Laboratory Standard Institute, 2012).
Preparation of suspension of pathogenic fungi
Three collected tubes containing physiological NaCl were prepared, followed by the
suspension of pure cultured strains (P. ovale,M. gypseum and C. albicans) was taken 1 oose
of each and put into a tube containing physiological NaCl. It was then homogenized properly
and the turbidity was calculated compared to the standard 0.5 Mc Farland's (Clinical and
Laboratory Standards Institute, 2012).
Inhibition activity testing of clove flower essential oil against the three pathogenic fungi
(P. ovale, M. gypseum, and C. albicans)
The fungal suspension was planted on the media using a scattering technique by
pipetting 100 μl of the fungal suspension into a petri dish that already contained Saboroud
Dextrose Agar and was then leveled with a stirring rod. The discs that have been pre-soaked
with a concentration of clove flower essential oil with concentrations of 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90% and 100% was then put into the agar media. The media
were wrapped in plastic and with umbrella paper which was then incubated for 2 x 24 h for
the growth of C. albicans and P. ovale and 3 x 24 hours for the growth of M. gypseum. The
results were observed by measuring the inhibition zone formed around the disc using a
caliper (Pratiwi et al., 2008).
Data analysis
The results of the diameter of the inhibition were analyzed using a parametric test
with the One way ANOVA method (one-way analysis of variance) with a 95% confidence
level or = 0.05, if the test results showed significant results, it is followed by Duncan's test or
DMRT.
Results and discussion
Based on the results of the study, it was shown that there was a clear zone between the
paper discs, at a concentration of 10% to 100% there was an inhibition zone (mm). The
treatment with the greatest inhibitory ability was the treatment on M. gypseum, with the
average inhibition at 10% concentration of 33.05 mm, and at 100% concentration of 63.95
mm with positive control of 24.7 mm. On the inhibition zone then compared to the fungus C.
albicans and the fungus P. ovale, this M. gypseum is larger.
Analysis of one-way variance data or one-way ANOVA showed a very significant
difference between treatments of several concentrations of clove flower essential oil on the
average diameter of the inhibition zones of several dandruff-causing fungi (M. gypseum, P.
ovale, and C. albicans), where p = 0.000 or p-value < 0.05. This matter shows the significant
results of the concentration variation of clove flower essential oil against the dandruff-
causing fungal growth so that a further test was carried out using Duncan's test.
44
Duncan's test results show the mean different diameters of resistance of any
concentration or type mold. Clove essential oil treatment results showed that almost all
concentration showed different effectiveness, but some concentrations has a same effective
inhibition. The test results are presented in Table 1, 2, 3 and Figure 1.
Table 1. The average diameter of the inhibition zone of clove flower essential oil
on the growth of Pityrosporum ovale after Duncan's test.
Treatment(s)
The average diameter of
inhibition zone (mm)
Interpretive Category
10%
14,75a± 0,25
Intermediate
20%
17,4b± 1
Intermediate
30%
21,05c± 0,35
Susceptible
40%
26,9d± 0,1
Susceptible
50%
28d,e ± 0,6
Susceptible
60%
29,1e± 0,3
Susceptible
70%
31,05f± 0,95
Susceptible
80%
35,35g± 0,65
Susceptible
90%
40h± 1
Susceptible
100%
46,15i± 1,05
Susceptible
Control (+)
45,7i± 0,1
Susceptible
Control (-)
0
Note(s):
-
Number followed by the same alphabet was identical based on Duncan test
-
Susceptible response signified ≥ 20 mm, I ntermediate 15 -19mm, and resistant < 14 mm. (CLSI 2018)
Table 2. The average diameter of the inhibition zone of clove flower essential oil
on the growth of Microsporum gypseum after Duncan's test.
Treatment(s)
The average diameter of
inhibition zone (mm)
Interpretive Category
10%
33,05b± 0,55
Susceptible
20%
37,85b,c ± 6,65
Susceptible
30%
39,45c± 5,65
Susceptible
40%
45,93d± 4,38
Susceptible
50%
49,05d± 0,05
Susceptible
60%
50,95d,e ± 0,75
Susceptible
70%
55,65e,f ± 1,75
Susceptible
80%
56,85e,f ± 2,55
Susceptible
90%
58,35f,g ± 3,65
Susceptible
100%
63,95h± 4,15
Susceptible
Control (+)
25,7a± 0,1
Intermediate
Control (-)
0
Note(s):
-
Number followed by the same alphabet was identical based on Duncan test
-
Susceptible respo nse signified ≥ 20 mm, Intermediate 15 -19mm, and resistant < 14 mm. (CLSI 2018)
45
Table 3. The average diameter of the inhibition zone of clove flower essential oil
on the growth of Candida albicans after Duncan's test.
Treatment(s)
The average diameter of
inhibition zone (mm)
Interpretive Category
10%
13,5a± 0,17
Susceptible
20%
19,23b± 0,30
Susceptible
30%
25,03c± 0,66
Susceptible
40%
27d± 0,2
Susceptible
50%
33,26e± 0,41
Susceptible
60%
35, 66g± 0,15
Susceptible
70%
36,4h± 0,55
Susceptible
80%
36,56h± 0,35
Susceptible
90%
37,23i± 0,55
Susceptible
100%
39,53j± 0,15
Susceptible
Control (+)
34,23f± 0,25
Susceptible
Control (-)
0
Note(s):
-
Number followed by the same alphabet was identical based on Duncan test
-
Susceptible respo nse signified ≥ 20 mm, Intermediate 15 -19mm, and resistant < 14 mm. (CLSI 2018)
Figure 1. The clear zone shows the inhibitory activity of clove essential oil against
Microsporum gypseum on saboraud dextrose agar medium at 30°C for 4 days. (a.
concentration 100%, b. positive control : ketoconazole 2%).
Table 4. Clove flower essential oil phytochemical screening test results
Phytochemical Test
Results
Saponin
Negative
Phenol and Tannin
Negative
Flavonoid
Positive
Alkaloid
Negative
Poli Fenolat
Positive
Kuinon
Steroid
Positive
Positive
Menoterpen
Positive
46
Based on the results of Duncan's test, it showed that the ability of each concentration
of clove flower extract to the growth of M. gypseum resulted in a significantly different
inhibition zone. The ability of the 10% concentration was significantly different and better
than the control treatment of 2% ketoconazole, namely the inhibitory power at a
concentration of 10 % was 33.05 mm while the ability of 2% ketoconazole was 25.7 mm.
Likewise, the ability of clove flower essential oil to have an inhibitory zone of C. albicans, at
a concentration of 60% was significantly different and better than the control, with inhibitory
abilities of 35.66 mm and 34.23 mm, respectively. The ability of clove flower essential oil to
the P. ovale inhibition zone, the inhibitory ability between 100% concentration and 2%
control was not significantly different, namely the inhibition diameters were 46.15 mm and
45.7 mm, respectively. The inhibition zone was showed as the clear zone around the disc. It
implied that the active compounds able to inhibit fungal growth.
The active compounds contained in clove flower essential oil has antimicrobial
activity because it contains eugenol and flavonoids (Table 4). Eugenol belongs to the
polyphenolic group which has bacteriostatic or bactericidal activity depending on the
concentration. Eugenol inhibits the biosynthesis of ergosterol - an important component in
fungal cell membranes so that fungal cell membranes are damaged and their function
decreases. Because eugenol is a lipophilic compound, eugenol can penetrate the lipid bi-layer
membrane which is composed of fatty acid chains, thereby changing the fluidity and
permeability (Alfauziah and Budiman, 2016). Flavonoids act by denaturing cell proteins,
which can inhibit the work of enzymes in cells, resulting in a faulty cell wall construction
process. Because of the inclusion of eugenol chemicals, which are phenol components, clove
flower extract contains antifungal substances that can suppress fungal growth. These phenol
components can harm cell microorganisms by producing protein coagulation and cell wall
membrane permeability, as well as inactivating enzymes involved in microorganism cell
metabolism (Sundari et al., 2001).
Eugenol compounds are other active chemicals discovered in clove flowers. Eugenol
is a widely utilized substance in the pharmaceutical industry due to its wide range of
pharmacological properties, including antiseptic, anti-inflammatory, antiviral, antibacterial,
antifungal, antispasmodic, stimulant, and local anesthetic properties. (Alisa et al., 2015).
Eugenol has been shown to inhibit gram-positive and gram-negative bacteria, as well as those
resistant to antibiotics. Because of their hydrophobic character, the chemicals will harm cell
structure by interacting with the lipopolysaccharide already present in cell membranes.
(Utami et al., 2019).
Several studies linked to this subject, like Andries et al (2014), reported that clove
flower extract (S. aromaticum) has antibacterial activity against Streptococcus mutans in vitro,
with 5 repetitions at concentrations of 40%, 60%, and 80%. It indicated the inhibitory zone at
40 percent 20.41 mm, 60 percent 21.0 mm, and 80 percent 25.81 mm concentrations, in that
order. Nurhayati (2017) discovered that the clove flower extract (Syzygium aromaticum)
inhibited the growth of bacteria. Paliling et al. (2016) found that the clove flower extract can
prevent the growth of Porphyromonas gingivalis using 96 percent ethanol solvent and a
diameter of 13.0 mm using the Kirby Bauer method.
Khusnul et al. (2017) did another research on the fungus Trichophyton rubrum, this
time testing the effects of an ethanol extract of galangal rhizome (Alpinia Galanga L) against
Trichophyton rubrum growth. There is no inhibition at concentrations of 10% to 20%, and
there is inhibition at concentrations of 30% inhibition zone 3 mm, 40% inhibition zone 6 mm,
50% inhibition zone 12 mm, 60% inhibition zone 12 mm, 70% inhibition zone 14 mm, 80%
inhibition zone 14 mm, 90% inhibition zone 16 mm, and 100% inhibition zone 18 mm.
Trichophyton rubrum growth is inhibited to a lesser extent by galangal rhizome extract,
whereas Trichophyton rubrum growth is inhibited to a greater extent by clove flower extract.
47
Lova et al. (2018) found that clove flower essential oil had the greatest antibacterial
activity, with an inhibition zone of 25.85 mm - 26.75 mm, followed by flower stalk essential
oil with an inhibition zone of 20.60 mm - 21, 20 mm, and clove essential oil with an
inhibition zone of 18.04 mm - 18.58 mm. When compared to the essential oils from the
flower stem and clove leaf, the essential oil from the clove flower exhibits the best activity
against P. acnes. When used as an anti-bacterial for P. acnes, the essential oil from the flower
stem and clove leaf is not similar to that from the clove flower.
Cloves can inhibit bacteria, fungus, protozoa, and viruses, giving them a broad
spectrum of antibacterial activity. Gram-positive and Gram-negative bacteria have different
MIC values. Clove flowers have been shown to destroy germs (Pathirana et al, 2019).
Although the inhibition against Gram-positive bacteria is larger than the resistance against
Gram-positive bacteria (Saikumari et al, 2016), cloves has a very low or very strong MIC
value against some Gram-negative bacteria, depending on the bacterium types (Moon et al.,
2011; Pandey and Singh, 2011).
Conclusion
Based on the results of this study, it can be concluded that the essential oil clove
flower (S. aromaticum) can affect fungal growth that causes dandruff (Microsporum gypseum,
Pityrosporum ovale, and Candida albicans).
Conflict of interest
The authors state no conflict of interest from this manuscript.
Acknowledgment
This research was supported by the Direktorat Jenderal Pendidikan Tinggi (DIKTI) or
DRPM through SIMLITABMAS as the research funder on Program Penelitian Kerjasama
antar Perguruan Tinggi (PKPT) with master contract number 065/SP2H/LT/DRPM/2021.
This research is also supported by the School of Technology Laboratory Medic, STIKes
Bakti Tunas Husada Tasikmalaya and to the mycology laboratory team who had helped to
carry out this research.
Author contributions
All authors have reviewed the final version of the manuscript and approved it for
publication. KK designed the study; PPA performed research and collected the data; KK
analysed the data; KK, DPV wrote and reviewed the paper. KK is the main contributor of this
manuscript.
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