In Vitro Antibacterial Activity Test of Jackfruit (Artocarpus

heterophyllus Lam.) Leaf Extract against Methicillin-Resistant

Staphylococcus aureus (MRSA)

Ika Ningsih*

, Dyah Ayu Rosalinda

, Ariyani Kiranasari

, Beti Ernawati Dewi

, Fithriyah Sjatha

Department of Microbiology, Faculty of Medicine, Universitas Indonesia,

Jl. Pegangsaan Timur no. 16 Jakarta 10320

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 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

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: 10.5220/0009845300002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

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

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.

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

C for 24 hours. MBC value was

determined as concentration of MHA plate whose

culture results do not show any growth of bacterial

colonies.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

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 researchers use

disc diffusion method.

In Vitro Antibacterial Activity Test of Jackfruit (Artocarpus heterophyllus Lam.) Leaf Extract against Methicillin-Resistant Staphylococcus

aureus (MRSA)

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 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.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

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

aureus (MRSA)