Antibacterial Activity of Synthesized Silver Nanoparticle using

Langsat Leaf Extract (Lansium domesticum var. pubescen Kooders et

Valeton) as Bioreductor against Escherichia coli and

Staphylococcus aureus

Khairunnida Rahma

, Agung Dwi Wahyu Widodo

, Rebekah Juniati Setiabudi

Retno Indrawati Roestamadji

, Maftuchah Rochmanti

, Pudji Lestari

Basic Medical Science Study Program, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia

Department of Medical Microbiology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia

Department of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia

Department of Pharmacology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia

Department of Public Health-Preventive Medicine, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia

Keywords:

Antibacterial Activity, Escherichia coli, Green Synthesis, Lansium domesticum, Silver Nanoparticles,

Staphylococcus aureus

Abstract: The emerging of antimicrobial resistance has caused the urgency to find new alternative agents. The

application of silver in the form of silver nanoparticles (AgNP) began to be studied again. The less toxic and

ecofriendly method to synthesize AgNP is through the green synthesis approach using plant extract. Langsat

is one of the endemic plants of South Kalimantan and its leaf provides secondary metabolites substances to

help the synthesize of AgNP. The synthesis of AgNP was prepared and AgNO

using Langsat Leaf (LL)

extract as the bioreductor. The synthesized LL-AgNP was then characterized by UV-Vis spectrophotometer

on 575 nm. The MIC study was done using broth dilution method with 6 different concentrations ranged from

3.125% to 100% and 2 controls, followed by the MBC study on MHA plates. The LL-AgNP successfully

inhibited the growth of Escherichia coli and Staphylococcus aureus in concentration of 6.25% and 25%,

respectively. The LL-AgNP also showed bactericidal activity against Escherichia coli in concentration of

25% but showed no activity on Staphylococcus aureus. This result indicates that LL-AgNP has potential as

an antibacterial agent against Escherichia coli and Staphylococcus aureus.

1 INTRODUCTION

Infection caused by microorganisms is one of the

main causes of chronic infection and even death

(Linlin, Chen, & Longquan, 2017). There are more

than 200 known diseases that can be transmitted from

bacteria, fungi, viruses, and other microbes to human

(Ganesan et al., 2017). It increases from time to time

and becomes a real threat for the community.

Antibiotics are currently used as preferred method for

https://orcid.org/0000-0002-6089-3619

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https://orcid.org/0000-0002-9222-9376

https://orcid.org/0000-0003-4725-4676

bacterial infection treatment because they are

considered to be more effective in cost and have

proven to give strong and clear results.

However, nowadays, the use of antibiotics has

also started to cause other dangerous phenomena. The

emergence of bacteria causing antibacterial resistance

has increased quite rapidly throughout the world

(Ventola, 2015). The limitation of new antibiotics in

nature to replace antibiotic agents that are no longer

effective raises the urgency to develop new

298

Rahma, K., Wahyu Widodo, A., Setiabudi, R., Roestamadji, R., Rochmanti, M. and Lestari, P.

Antibacterial Activity of Synthesized Silver Nanoparticle using Langsat Leaf Extract (Lansium domesticum var. pubescen Kooders et Valeton) as Bioreductor against Escherichia coli and

Staphylococcus aureus.

DOI: 10.5220/0010491702980304

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 298-304

ISBN: 978-989-758-499-2

antibiotics to strengthen the effectiveness of existing

antibiotic agents (World Health Organization, 2014).

There are many types of precious metals that are

used in the medical world, especially in antimicrobial

research. One of them is silver metal (Ag). Ag has

been used to demonstrate antibacterial effects and has

been frequently used in medicine including

orthopedics (Castiglioni, Cazzaniga, Locatelli, &

Maier, 2017). The advantage of Ag utilization is that

it can be formed into silver nanoparticles (AgNP).

AgNP gives promising potential as antibiotic

alternatives because AgNP has better

physicochemical and biological properties than

whole silver (Qing et al., 2018). One of the methods

to create AgNP is the green synthesis approach. This

approach is seen from the biology perspective,

especially the utilization of natural organisms has

offered a method that is reliable, simple, non-toxic,

and environmentally friendly (Velusamy, Kumar,

Jeyanthi, Das, & Pachaiappan, 2016). In green

synthesis, many sources from nature such as plants,

bacteria, and fungi can be used in the process.

Indonesia is a country which has rich biodiversity.

This provides a good opportunity and potential to

develop the synthesis of nanoparticles that is more

environmentally friendly using plant extracts from

various plants in Indonesia. One of the endemic plants

found in South Kalimantan, especially Tabalong, is

Langsat. Langsat (Lansium domesticum var.

Pubescen Kooders et Valeton) is a plant that comes

from Meliaceae family. Every fruiting season,

Langsat produces a lot of fruits but people in South

Kalimantan only eat the fruits, despite the results of

previous research proving that other parts such as

bark, fruit skin, and leaf have potential as traditional

medicine.

To the best of our knowledge, the synthesis of

AgNP using Langsat leaf has been hardly explored

until now. This fact supports the research of

antibacterial activity using silver nanoparticles

synthesized by biological methods using a

bioreductor from Langsat leaf extract (Lansium

domesticum var. Pubescen Kooders et Valeton) in

vitro as one of the endeavor to find the potential of

new antibacterial agents in the future.

2 MATERIALS AND METHODS

This research is a true experiment and was done with

randomized post-test only control group design.

There were 6 different LL-AgNP concentrations and

2 controls (positive and negative). Silver Nitrate

(AgNO

) 99% from Merck was used for the synthesis

of silver nanoparticle. Langsat (Lansium domesticum

var. pubescen Kooders et Valeton) leaf were collected

from a private plantation in Tabalong District, South

Kalimantan, Indonesia. Bacterial strain of Gram-

negative Escherichia coli ATCC 25922 and Gram-

positive bacteria Staphylococcus aureus ATCC

25923 were purchased from Center of Health

Laboratory (BBLK) Surabaya, East Java, Indonesia.

The research was conducted in Microbiology

Laboratory Faculty of Medicine, Universitas

Airlangga Surabaya, East Java Indonesia.

2.1 Preparation of Leaf Extract

The fresh Langsat leaves were collected from

Langsat trees and wiped gently using dry paper towel

to remove dust on surface. The Langsat leaves were

then washed thoroughly with running water and were

tapped gently with paper towels afterward. The leaves

were dried using a microwave at medium temperature

for 6 minutes. The dried leaves were chopped using

blender and were sifted with 60 mesh strainer. The

extraction process was following the maceration

steps. First, 100 grams of fine dried leaves were

weighed and added by ethanol 96% until the volume

reached 1000 ml. The mixture was left for 24 hours

and was strained after that. The process was repeated

for 3 times and was evaporated using vacuum rotary

evaporator until a thick extract was obtained.

2.2 Silver Nanoparticles Synthesis

Synthesis of silver nanoparticles using Langsat Leaf

extract (LL-AgNP) was done using the green

synthesis method. The ratio of AgNO

solution and

the extract was 1:10. Around 15 ml of the extract was

mixed with 150 ml AgNO

solution inside a beaker

glass that has been covered with aluminum foil to

prevent light intrusion. The solution was then warmed

inside a water bath at 60

C and was stirred once in a

while. The discoloration is one of the indications that

the synthesis of LL-AgNp has been done.

2.3 Silver Nanoparticles

Characterization

The characterization of synthesized LL-AgNP was

done using UV-Vis Spectrophotometer. As much as

1 ml of LL-AgNP solution was scanned between 300

- 650 nm for determining the wavelength peak. The

scan was done 4 times; right after the synthesis

process (D+0) and one month after watching the

stability of LL-AgNP (D+37, D+44, D+51, and

D+58) with the same steps as mentioned above. The

Antibacterial Activity of Synthesized Silver Nanoparticle using Langsat Leaf Extract (Lansium domesticum var. pubescen Kooders et

Valeton) as Bioreductor against Escherichia coli and Staphylococcus aureus

299

result from LL-AgNP characterization was then used

to calculate its size using a formula described by

Amirjani, Firouzi, & Haghshenas, (2020).

Nanoparticle size  0,78 𝜆



266

(1)

2.4 Minimum Inhibitory Concentration

Assay

The Minimum Inhibitory Concentration (MIC) assay

was done by the broth dilution method. First, 5 ml

MHB sterile was pipetted to 8 different test tubes and

were given labels; tube 1-5, Control (+), and Control

(

–

). Then, 2 ml of LL-AgNP was added to the first

tube and homogenized. After being homogenized, 1

ml of the solution from tube 1 was pipetted and was

moved to the second tube and was vortexed. This step

was done until the fifth tube and from tube 5, 1 ml of

the solution was discharged and became the lowest

concentration.

Aquadest was used as negative control, while

Meropenem and Vancomycin were used as the

positive control for Escherichia coli and

Staphylococcus aureus, respectively. The tubes were

vortexed well and were incubated on an incubator for

24 hours at 35

C. The color of the solution on each

tube was observed after the incubation. Tubes with

color turbidity imminent with turbidity on positive

control were then noted and the lowest concentration

was decided as MIC. The tubes were proceeded to

MBC assay. This assay was done in triplicate. The

result was noted and analyzed descriptively.

2.5 Minimum Bactericidal

Concentration Assay

One loop of the solution from tubes that has been

selected from previous MIC assay was taken and was

streaked to the Mueller-Hinton Agar plates. The

plates were then incubated on an incubator for 24

hours at 35

C. The colony growth was observed after

the incubation period. Plates without any colony

growth observed were then noted and the lowest

concentration of it was decided as MBC. The result

was noted and analyzed descriptively.

3 RESULTS

3.1 Silver Nanoparticles Synthesis

The Langsat leaf (LL) extract had a dark brown color

after the extraction process was done. Meanwhile,

AgNO

had no color with a clear solution just like the

solvent used. After both of the components were

mixed with ratio 1:10 of AgNO

1 mM solution and

LL extract, discoloration was observed. The mixed

solution showed a yellowish color and slightly turbid.

Based on the color changes, it could indicate the

formation of silver nanoparticles. However, a further

test was done in order to make sure the silver

nanoparticles were successfully synthesized.

Figure 1: AgNO

solution before the synthesis process (left)

and LL-AgNP after the synthesis process (right).

3.2 Silver Nanoparticles

Characterization

The solution was then run through the

spectrophotometer UV-Vis reading to get the

wavelength peak in order to confirm the formation of

LL-AgNP. The reading was done at λ 300 - 650 nm.

Based on the reading process, the peak of LL-AgNP

was observed at λ 398 nm with an absorbance value

of 0.92. This result confirmed that LL-AgNP has been

synthesized and reflected on Figure 1.

Figure 2: Characterization of LL-AgNP.

The recorded wavelength of LL-AgNP was then

calculated with the formula to roughly estimate its

size. Based on the calculation, a 44 nm LL-AgNP was

synthesized. As stated in Khodashenas & Ghorbani

(2019), AgNP shape with the size similar to the

synthesized AgNP would be spherical. Biological

methods for AgNP synthesis are still under

development. Nanoparticles with shapes other than

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

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300

spherical and cubic could only be synthesized through

physical or chemical methods.

One month after the synthesis, another reading on

the spectrophotometer was done to measure the

current absorbance value and the stability of LL-

AgNP. The LL-AgNP absorbance value shifted from

0.92 (D+0) to 1.06 (D+37), 2.66 (D+44), 3.25

(D+51), and 3.57 (D+58) (Figure 3). It could indicate

the LL-AgNP has agglomerated and alteration of size

and shape has occurred.

Figure 3: The LL-AgNP stability observation on several

days after the initial synthesys.

3.3 Minimum Inhibitory Concentration

Assay

The Minimum Inhibitory Concentration (MIC) result

of various LL-AgNP concentrations presented on

table 1.

Table 1: MIC of LL-AgNP.

Isolate

LL-AgNP Concentration (%)

100

12.5

6.25

3.125

–

E. coli 1

–

aureus

–

+ + ++

–

R (replication), + (Meropenem for Escherichia coli and

Vancomycin for Staphylococcus aureus), – (Aquadest)

Based on the data in table 1, LL-AgNP could

inhibit the growth of Escherichia coli at concentration

6.25% and Escherichia coli ESBL at 12.5%.

Meanwhile, on the Gram-positive bacteria, the

growth of Staphylococcus aureus and MRSA

inhibited by LL-AgNP at concentration 25%.

3.4 Minimum Bactericidal

Concentration Assay

The Minimum Bactericidal Concentration (MBC)

result of previously obtained from MIC assay

presented on tablr 2.

Tabel 2: MBC of LL-AgNP.

Isolates R

LL-A

NP Concentration (%)

+ 100 50 25 12.5 6.25

–

E. coli 1

–

+ ++

–

aureus

–

++ + + ++

–

++ + + ++

–

++ + + ++

R (replication), + (Meropenem for Escherichia coli and

Vancomycin for Staphylococcus aureus), – (Aquadest)

Based on the data in table 2, LL-AgNP showed

bactericidal activity against Escherichia coli at

concentration 25% and Escherichia coli ESBL at

12.5%. Discrepant with the result of Gram-negative

bacteria, there was no bactericidal activity observed

against Staphylococcus aureus and MRSA.

4 DISCUSSION

Langsat leaf extract was proven can be used as

bioreductor on silver nanoparticle synthesis. This

could happen because Langsat leaf extract contains

secondary metabolites that act as a reduction agent.

Phytochemical screening of Langsat leaf was done

previously by Mayanti et al. (2015), Yunus, Boddhi,

& Queljoe (2018), and Matsumoto et al. (2019). The

results showed Langsat leaf posses phenolic

compounds, saponin, and triterpenoid/steroid. The

phenolic compound on Langsat leaf has the potential

as metal salt reducer and agent to stabilize the

nanoparticles from agglomeration.

Pal, Rai, & Pandey (2019) explained reduction

and stabilization of silver ions by biomolecule

combination like protein, amino acid, polysaccharide,

secondary metabolites, and vitamin that plant has,

will serve as the easiest and cheapest way to

synthesize NP. Generally, plant leaf has high

polyphenol level. The phenolic compound has

hydroxyl and ketonic group that has the ability to

bond with metal and reduces metal salt and gives

stability from agglomeration. Kawas (2016)

described the process of secondary metabolites as a

reducer of silver nitrate is AgNO

will detach into

Ag+ and NO

- form and greatly influenced by

0,92

1,06

2,66

3,25

3,57

Day 0 Day 37 Day 44 Day 51 Day 58

Absorbance (AU)

Antibacterial Activity of Synthesized Silver Nanoparticle using Langsat Leaf Extract (Lansium domesticum var. pubescen Kooders et

Valeton) as Bioreductor against Escherichia coli and Staphylococcus aureus

301

temperature and light. During the process, it is

necessary to seal the beaker glass with a cover like

aluminum foil to make the beaker dark. The plant

extract also gives proteins and enzymes to AgNO

solution in which the Ag+ ion will combine with

enzymes to make enzyme-substrate complex. The

enzymes released from plant extract work on silver

ion and release nanoparticles as the product (Prasad,

2014). This formation is not just through covalent

bond, but also because of the existence of protein

attraction through hydrogen bond, electrostatic, or

other supramolecular interactions (Ballottin et al.,

2016).

Characterization of synthesized nanoparticles is

one of the important steps. This has to be done in

order to ensure the formation of nanoparticles. Noah

(2019) explained that AgNP shows strong absorbance

band and specific colors on its solution. The

synthesized LL-AgNP color is yellowish and slightly

turbid. The color variation occured because of the

variety of phytochemical compounds on plant extract

that used for the AgNP synthesize process (Ovais,

2016). Moreover, the variety of shape and size of the

formed AgNP also will contribute to the AgNP

solution color diverseness (González, Noguez,

Beránek, & Barnard, 2014). Beside color observation,

a more quantitative test needs to be done as part of NP

characterization. One of the methods us

spectrophotometer Uv-Vis. The LL-AgNP shows a

wavelength peak at λ 398 nm. According to Seifipour,

Nozari, & Pishkar (2020), AgNP that successfully

synthesized has a peak observed at around 370 nm -

500 nm. With this result, it can be considered that LL-

AgNP has formed.

Almost all AgNP is prone to agglomeration and it

is a commonly found phenomenon. Agglomeration is

a process when nanoparticles lose their nano

characteristic (Bae, Lee, Kim, Choi, & Yi, 2013).

Based on the finding result, one month after the LL-

AgNP was formed, agglomeration was observed.

AgNP stability can be monitored from time to time.

The wavelength will shift and it indicates the change

of absorbance spectrum on the UV-Vis area. Badiah,

Seedeh, Supriyanto, & Zaidan (2019) explained that

large surface tension force causes greater cohesion

force. This causes the interaction between AgNPs to

become greater as well. Over time, the particles will

become larger in size due to the formation of groups

amongst AgNPs.

Silver nanoparticles are stabler, more consistent in

size, and not toxic to human tissues compared to

silver in metal form. It affects the effectiveness of

AgNP as antibacterial (Nolan, 2018). AgNP can

penetrate to microbe cell wall easier because of its

smaller size than the microorganism (Siddiqi, Husen,

& Rao, 2018). Based on the obtained result, it is

known that synthesized LL-AgNP has potential as an

antibacterial agent against Escherichia coli and

Staphylococcus aureus. The LL-AgNP could inhibit

the growth of said bacteria.

The MIC and MBC assay showed that LL-AgNP

works better against Escherichia coli than

Staphylococcus aureus. In line with the result,

according to Qing et al. (2018), AgNP gives a

stronger effect on Gram-negative bacteria. One of the

theories that support this finding is because Gram-

negative bacteria have thinner peptidoglycan cell

walls, while Gram-positive bacteria have thicker cell

walls (Sizar & Unakal, 2020). Kailasa, Park, Rohit, &

Koduru (2019) also mentioned the amount of Ag+

ions that successfully penetrate into the Gram-

positive bacteria is fewer. It shows that there is a

strong interaction between AgNP and Gram-negative

bacteria.

The specific mechanism of AgNP for each

bacteria is still under investigation. Generally, AgNP

mechanisms as antibacterial are similar, both on

Gram-negative and Gram-positive (Baptista et al.,

2018). The observed mechanisms are inhibition of

cell wall synthesis, protein synthesis, and nucleic acid

synthesis. Furthermore, damage on the cell surface

and respiration chin are also known.

There are several mechanisms of synthesized

AgNP using plant extract as the reducer that has been

explained by experts. The AgNP will release silver

ions (Ag+) and it will directly penetrate into the

bacteria cell wall (Rajeshkumar & Bharath, 2017).

Wong and Liu (2010) in Durán et al. (2016) also

explained that AgNP has wide surface area to come

into contact with bacteria. This makes AgNP possible

to adhere to the cell membrane and easily get into the

bacteria. The released Ag+ ion has strong

antibacterial properties. The Ag+ ion will interact

with the bacteria cell membrane and cell wall

components. This is one of the crucial mechanisms of

AgNP toxicity towards bacteria (Liu et al., 2020).

Silver nanoparticles have positive charge. It will

cause the electrostatic attraction between AgNP and

bacteria cell membrane that has negative charge. The

bacteria cell wall has negative charge because of

electron release that is caused by catalysis activity on

cell respiration. This charge interaction will help

AgNP to attach to the cell membrane (Abbaszadegan

et al., 2015). Hence, the antibacterial effect could be

enhanced by altering the surface charge of AgNP so

that stronger obstruction will be obtained (Mandal et

al., 2016).

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

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5 CONCLUSIONS

Nanoparticles were successfully synthesized using

AgNO

and Langsat leaf extract with the recorded

wavelength peak at 398 nm. The approximate size of

the LL-AgNP is 44 nm. Based on the results that are

obtained, we conclude that the LL-AgNP shows

antibacterial activity against Gram-negative bacteria

Escherichia coli and Gram-positive bacteria

Staphylococcus aureus. The LL-AgNP MIC is

observed at 6.25% on Escherichia coli and 25% on

Staphylococcus aureus. The MBC is observed at 25%

on Escherichia coli but no bactericidal activity is

observed on Staphylococcus aureus. Further tests

with different strains, concentrations, and methods

are suggested to add more diversity from the findings

of this study.

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