In Vitro Antibacterial Activity Test of Jackfruit
(Artocarpus heterophyllus Lam.) Leaf Extract against Methicillin-
Resistant Staphylococcus aureus (MRSA)
Ika Ningsih*
1
, Dyah Ayu Rosalinda
2
, Ariyani Kiranasari
1
,
Beti Ernawati Dewi
1
, Fithriyah Sjatha
1
1
Department of Microbiology, Faculty of Medicine, Universitas Indonesia,
Jl. Pegangsaan Timur no. 16 Jakarta 10320
2
Undergraduate Program, Faculty of Medicine, Universitas Indonesia
Keywords: Antibacterial activity, jackfruit (Artocarpusheterophyllus Lam.) leaf extract, Methicillin-Resistant
Staphylococcus aureus (MRSA)
Abstract: Infectious diseases are still a public health problem in Indonesia. Methicillin Resistant Staphylococcus
aureus (MRSA) is one of bacteria causing infections that is a concern because of the nature of resistance to
various beta-lactam class of antibiotics. Therefore, it is necessary to find an alternative antibiotic for the
treatment of MRSA infections. This study aims to determine the antibacterial activity of jackfruit
(Artocarpus heterophyllus Lam.) leaf extract against MRSA by minimum inhibitory concentration (MIC)
and minimum bactericidal concentration (MBC) conducted using in-vitro test with broth macrodilution
method. Jackfruit (Artocarpus heterophyllus Lam.) leaf extract were used in half serial concentration of 1280
μg/ml to 0,625 μg/ml. MIC of jackfruit leaf extract against MRSA was found at concentration of 320 μg/ml
which showed by a clear solution in Brain Heart Infusion media. Meanwhile, MBC value of jackfruit leaf
extract against MRSA was found at concentration of 1280 μg/ml showed by the absence of MRSA colony
growth Mueller-Hinton agar. From this study showed that jackfruit (Artocarpus heterophyllus Lam.) leaf
extract has anti-bacterial activity against MRSA in-vitro.
1 INTRODUCTION
Until now infectious diseases are still a problem in
Indonesia (Ministry of Health, 2011; Mardiastuti et
al, 2007). One approach to manage infectious
disease is a rational antibiotic use which in
Indonesia misuse and overuse of antibiotics is
commonly occur (Mardiastuti et al, 2007). This
phenomena can lead to new problems of emerging
antibiotic-resistant microbial strains (Mardiastuti et
al, 2007; National Institute of Allergy and Infectious
Diseases, 2015).
Methicillin-Resistant Staphylococcus aureus
(MRSA) is a strain of beta-lactam-resistant
Staphylococcus aureus, including penicillin,
oxacillin, methicillin, and so on (National Institute
of Allergy and Infectious Diseases, 2015) MRSA
resistance to various antibiotics is caused the
existence of genetic changes caused by the use of
antiobiotics irrationally (Nurkusuma D, 2009).
MRSA bacterial transmission is divided into 2
categories, namely Hospital-Acquired (through
medical measures such as catheterization or surgery)
and Community-Acquired (via direct contact with
patients or due to low levels of hygiene) (National
Institute of Allergy and Infectious Diseases, 2008).
MRSA bacterial infections may cause skin diseases
such as eczema, furuncle, impetigo, pyoderma, or
clinical manifestations of skin abscesses, venous
thrombosis, osteomyelitis or even dissemination
resulting in meningitis, pneumonia, endocarditis or
sepsis (Brooks GF et al, 2016) .
Data on the prevalence of MRSA infections in
Indonesia is limited. In 2003 the prevalence of
MRSA infections at Atmajaya Hospital Jakarta was
recorded at 47%, whereas in 2010 the incidence of
MRSA infection in Dr. Moh. Hoesin Palembang
reached 46% (Yuwono, 2010). In 2013, research to
find the prevalence of MRSA infection was done in
ICU and surgical treatment room of RSUD Abdul
Ningsih, I., Rosalinda, D., Kiranasari, A., Dewi, B. and Sjatha, F.
In Vitro Antibacterial Activity Test of Jackfruit (Artocarpus heterophyllus Lam.) Leaf Extract against Methicillin-Resistant Staphylococcus aureus (MRSA).
DOI: 10.5220/0008360702450249
In Proceedings of BROMO Conference (BROMO 2018), pages 245-249
ISBN: 978-989-758-347-6
Copyright
c
2018 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
245
Moeloek Lampung and got prevalence rate 38,24%
(Mahmudah R, 2013). This relatively high
prevalence of multiresistant bacterial conditions is
one of particular concern as the choice of antibiotics
therapy becomes more difficult. The drug of choice
for the treatment of MRSA infections is
vancomycin, but since 1996 MRSA has found a
decreased sensitivity to vancomycin (Yuwono,
2010). Even in 2002, clinicians in the United States
found a strain of MRSA that was resistant to
vancomycin (although the numbers were few)
(National Institute of Allergy and Infectious
Diseases, 2008).
Jackfruit (Artocarpus heterophyllus Lam.) is a
dicotyl plant with woody stems that are native to
India, Malaysia and Bangladesh, but can also grow
well in other tropical-subtropical regions including
Indonesia (Manuel NV et al, 2012; Orwa et al,
2009). In addition to fruit, seeds, or young flowers
are useful as a source of food, other jackfruit plant
parts are also empirically often used as drugs.
Jackfruit root is used to treat skin diseases, asthma,
fever, and diarrhea. Jackfruit leaf is believed to cure
ulcers. Jackfruit sap is believed to cure abscesses,
snake bites, and swollen glands. While the seeds,
often used to cure gall disease (National Tropical
Botanical Garden, 2015).
Several studies have been conducted to prove the
usefulness of jackfruit plants in the medical field.
Jha and Srivastava in his study found that jackfruit
seed oil could inhibit the growth of Escherichia coli,
Pseudomonas aeruginosa and Staphylococcus
aureus bacteria with minimum inhibitory
concentrations (MIC) between 1.55-5.20 mg / ml
(Jha S et al, 2013). Other studies have also shown
the presence of antibacterial activity from jackfruit
extracts to MSSA and MRSA played by the
substance artocarpesin (Manuel NV et al., 2012).
Silver nanoparticles synthesized from jackfruit leaf
extracts have antibacterial activity against
Escherichia coli, Staphylococcus aureus, Bacillus
subtilis, Aspergillus niger, and Pichia pastoris
(Thombre R et al, 2012). Testing of jackfruit leaf
extracts on MRSA has not been found.
In this study we investigate the ability of jackfruit
leaf extracts to inhibit MRSA growth by
determining its MIC (minimal inhibitory
concentration) and MBC (minimal bactericidal
concentration).
2 MATERIAL AND METHODS
2.1 Preparation of Extracts and MRSA
Bacteria
The extract was obtained from Indonesian Institute
of Sciences (LIPI) diluted in DMSO (Dimethyl
sulfoxide). MRSA bacteria originating from the
Microbiology Department Universitas Indonesia
culture collection which is stock in cryotube beads
and re-growth on blood agar followed by incubation
at 37
o
C for 18-24 hours. The growing bacteria were
identified with Gram staining and biochemical tests
using commercial products (Vitex).
2.2. Preparation of Antibiotic and
Jackfruit Leaf Extract Stock
Solutions
The vancomycin in powder form was diluted in
(Brain Heart Infusion) media to reach concentration
of 256 μg/ml. Meanwhile, Jackfruit leaf extract was
also diluted 10 times higher than vancomycin
concentration (2560 μg/ml).
2.2 Dilution of Antibiotic and Jackfruit
Leaf Extract
Serial dilution of stock solution of antibiotics and
Jackfruit leaf extract were done using BHI media
until concentration of 0,25 μg/ml antbiotics and 2,5
μg/ml Jackfruit leaf extract were reached.
2.3 Determination of MIC (Minimum
Inhibitory Concentration)
Suspension of 0,5 McFarland value of MRSA
bacteria is inserted into each tube of BHI media
containing serial antibiotic and Jackfruit leaf extract
solution followed by incubation at 35°C-37°C for 24
hours. After that, an inspection was done on each
tube to assess the growth of bacteria characterized
by turbidity in the fluid in the tube. MIC value
defiened as the smallest extract concentration that
inhibits MRSA growth in BHI media.
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
246
2.4 Determination of MBC (Minimum
Bactericidal Concentration)
After MIC is known, 2 tubes at the lower and 2
tubes at the higher concentration of MIC were
subcultured into Mueller-Hinton Agar (MHA) media
followed by incubation at 37
o
C for 24 hours. MBC
value was determined as concentration of MHA
plate whose culture results do not show any growth
of bacterial colonies.
Table 1. Result of MIC test of Leaf Jackfruit Leaf Extract (Artocarpus heterophyllus Lam.)
on MRSA bacteria.
Treatment
Concentration
Result
(Turbidity [+]; Clear [-])
Positive control
-
+
Negative control
-
-
A. heterophyllus
1280 µg/ml
-
A. heterophyllus
640 µg/ml
-
A. heterophyllus
320 µg/ml
-
A. heterophyllus
160 µg/ml
+
A. heterophyllus
80 µg/ml
+
A. heterophyllus
40 µg/ml
+
A. heterophyllus
20 µg/ml
+
A. heterophyllus
10 µg/ml
+
A. heterophyllus
5 µg/ml
+
A. heterophyllus
2,5 µg/ml
+
A. heterophyllus
1,25 µg/ml
+
A. heterophyllus
0,625 µg/ml
+
Positive control = sterile BHI and MRSA bacterial inoculum; Negative control = BHI sterile
3 RESULT AND DISCUSSION
The experiments performed by finding MIC and
MBC value of jackfruit leaf extract (Artocarpus
heterophyllus Lam.) against MRSA bacteria in vitro.
The research was conducted by broth macrodilution
method . The extract used was a crude extract in a
DMSO solvent with a potential of 100,000 μg / mg
obtained from LIPI, a total volume of ± 235 μL
extract. Based on research that has been done,
obtained MIC jackfruit leaf extract to MRSA
bacteria of 320 μg/ml and MBC of 1280 μg/ml. In
this experiment we found different turbidity levels
on test tubes after incubation for 1x24 hours.
Basically, the addition of stock extract on sterile
BHI will produce a turbid liquid. Nevertheless, the
researchers compared the turbidity of each tube with
positive control and negative control. The tubes
contain a jackfruit leaf extract in a serial
concentration of 0.625 μg/ml to 160 μg/ml were
having similar turbidity with positive controls (BHI
and MRSA bacteria without extract). Meanwhile,
tubes containing jackfruit leaf extract with
concentrations of above than 320 μg/ml resulting
clearer turbidity. The results obtained can be seen in
the table 1.
Cultures on MHA medium were carried out to
confirm the results of MIC and determine the MBC.
In this experiment, culture of MHA medium was
performed for all extract concentrations. The results
of culture can be seen in the figure 1.
MBC is the lowest concentration in which no
bacterial colony growth is found in culture. Thus,
the MBC for jackfruit leaf extract (Artocarpus
heterophyllus Lam.) is 1280 μg/ml.
In addition, tubular macrodilution testing was also
performed to determine MIC and MBC vancomycin
against MRSA bacteria. In this study vancomycin
antibiotics were used as a comparison. From the
results of MIC and MBC vancomycin tests on
MRSA, 2 μg/ml result was obtained for both. Based
on the Clinical Laboratory Standards Institute
(CLSI), there are three classifications of S. aureus
based on their susceptibility to vancomycin, ie
sensitive (<2 μg/ml), intermediates (4-8 μg/ml), and
resistant (> 16 μg/ml) . Thus it can be concluded that
the MRSA used in this study is sensitive to
vancomycin.
Test method of jackfruit leaf extract in
macrodilution was chosen because the result
obtained is quantitative, that is by finding MIC and
MBC. The macrodilution method is less used than
the disc diffusion method. However, with the
consideration that the extract is a crude extract that
still contains solids material and easily settles, it is
feared the extracts are not spread evenly if the
In Vitro Antibacterial Activity Test of Jackfruit (Artocarpus heterophyllus Lam.) Leaf Extract against Methicillin-Resistant Staphylococcus
aureus (MRSA)
247
researchers use disc diffusion method. However, the
weakness of the macrodilution method is manual
preparation that often occurs errors, requiring more
places and reagents, and generally MIC or MBC is
found to be the range between the lowest
concentrations with no bacterial colonies and the
nearest concentration below (Jorgensen JH et al,
2009).
In this study, we used dimethylsulfoxide (DMSO) as
a solvent of jackfruit leaf extract. DMSO is a very
polar liquid that is generally used as a solvent.
DMSO was chosen because it does not affect the
antibacterial activity of the extract. Another study
show that DMSO has bacterial inhibition but is
weaker than methanol or ethanol (the solvent
commonly used to dissolve the extract), so the use of
DMSO is more recommended (Wadhwani T et al,
2008).
Jackfruit (Artocarpus heterophyllus Lam.) Has
active ingredients, ie phytosterol and terpenoids.
Jackfruit leaves contain flavonoids, terpenoids,
steroids, phenols, glycosides, and saponins
(Sivagnanasundaram P et al, 2015). Jackfruit leaf
extraction with silver nitrate can also produce silver
nanoparticles (Thombre R et al, 2012). These active
ingredients are believed to have antibacterial and
antioxidant effects. The content of the active
ingredient in jackfruit leaves is consistent with the
results of a study conducted by Thombre et al. which
shows that the use of jackfruit leaf extract can
inhibit the growth of Escherichia coli, Bacillus
subtilis and Staphylococcus aureus with inhibition
zones of 20 mm, 20 mm, and 17 mm, respectively.
The zone of inhibition was obtained by adding 20
μL of extract with concentration of 40000 μg/ml into
each diffusion well.
In this study the obtained concentration of jackfruit
leaf extract is smaller to inhibit bacteria compared
with the concentrations obtained from research
Thombre, et al. MIC jackfruit leaf extract was
obtained at concentration of 320 μg/ml and MBC at
a concentration of 1280 μg/ml. This difference may
be due to Thombre, et al in his research specifies the
active substance used, ie only using silver
nanoparticles. While in this study the extract used is
a whole extract without extraction or separation of
further compound fractions. There may be
contributions from other antibacterial substances
other than silver nanoparticles in jackfruit leaf
extract used by researchers, so the use of the
required concentration becomes smaller to obtain
MBC.
Until now, vancomycin is still the preferred
antibiotic for MRSA infections. Based MIC and
MBC values, there is a considerable concentration
difference between vancomycin and jackfruit leaf
extract against MRSA bacteria. Vancomycin has
MIC and MBC = 2 μg/ml, while jackfruit leaf
extract has MIC = 320 μg/ml and MBC 1280 μg/ml.
This may be due to differences in the mechanism of
vancomycin or jackfruit leaf extract in inhibiting
growth or killing MRSA bacteria.
In this study, we use crude extract of jackfruit leaf
which not yet purified for its active compound
towards MRSA or other bacteria, yet may become
one of the reason of high value of MIC and MBC.
Other active compound of jackfruit against MRSA
can be further analyzed as Manuel NV et al, 2012
found substance artocarpesin is the main compound
against MRSA, Furthermore, our MIC and MBC is
relatively low compare to study performed by Jha S
et al, 2013 which obtain 1.55-5.20 mg/ml of MIC
towards Escherichia coli, Pseudomonas aeruginosa
and Staphylococcus aureus bacteria.
However, with increasing MRSA resistance to
various antibiotics including vancomycin (Tenover
FC et al 2006), the use of jackfruit leaf extract as an
alternative antibiotic for MRSA infections may be
considered.
Figure 1. The results of the culture of jackfruit leaf extract (Artocarpus heterophyllus Lam.) With MRSA Bacteria on
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
248
Mueller-Hinton Agar medium. At concentrations of 0.625 μg/ml extract, 1.25 μg/ml, and so on up to 320 μg/ml there is a
solid colony growth that can not be counted. In extracts with concentrations of 640 μg/ml there is still a growth of 22
colonies of bacteria. While in the extract with a concentration of 1280 μg/ml no bacterial growth of MRSA was found.
4 CONCLUSION
Based on our study, jackfruit leaf extract is having
anti MRSA properties with MIC and MBC value
were 320 μg/ml and 1280 μg/ml respectively.
ACKNOWLEDGEMENTS
Publication of this was study supported by Hibah
PITTA UI 2018/2019.
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In Vitro Antibacterial Activity Test of Jackfruit (Artocarpus heterophyllus Lam.) Leaf Extract against Methicillin-Resistant Staphylococcus
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