Potency of Andaliman (Zanthoxylum acanthopodium DC.) Extracts as

Quorum-sensing Inhibitor to Serratia marcescens

Adelya Irawan Manalu

, It Jamilah

and Sovia Lenny

Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia

Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia

Keywords: Andaliman, Quorum-sensing inhibitor, Serratia marcescens, Zanthoxylum acanthopodium DC.

Abstract: Quorum-sensing is a specific communication type among microbial species, exposing several virulence

factors to host or internal environment. The phenomenon is currently being studied as potential target for

drug discovery and development. Natural product derived from plant source may be evaluated as potential

quorum-sensing inhibitor (QSI). The study aimed to determine the optimum concentration of Andaliman

methanolic (MeOH) and ethyl acetate extract (EtOAc) against prodigiosin synthesis by Serratia marcescens

as one of phenotypes controlled by quorum sensing. Based on optical density (OD

600

), both extracts did not

interfere with the growth of microorganism. The optimum concentration of MeOH extract in inhibiting the

prodigiosin synthesis was at concentration of 0.05% (w/v) in the end of incubation period (30 h).

Meanwhile, the EtOAc extract inhibit the prodigiosin synthesis at concentration of 0.4% in the end of

incubation period. Our results showed that Andaliman possess natural products as QSI which needed

further evaluation in the future.

1 INTRODUCTION

Qourum sensing (QS) is a bacteria communication

type based on genetic expression in a population

which impact on development of biofilm, pigment

production, toxin synthesis and other virulence

factors. The communication used a molecule signal

called autoinducer. It produced when microbial

population density reached an optimum state

(Bassler, 1993; Hentzer & Givskov, 2003).

Autoinducer commonly synthesized by Gram

negative bacteria is N-acyl-homoserine lactone

(Blaschek, 2007; Bai & Vittal, 2011).

Serratia marcescens is an opportunistic pathogen

from gram negative group. The species is able to

produce N-hexanol homoserine lactone (C6-HSL),

form biofilm and synthesis prodigiosin, a red

pigmented compound. Strains of Serratia are known

to exhibit QS to control their genetic expression that

code for extracellular virulence factor (Morohoshi et

al., 2007). Previous study had revealed a decrease in

AHL level to less production of exoenzymes and

prodigiosin leading to an immature biofilm

formation (Kievit et al., 2000). Several strains are

known as nosocomial infection factor due to

antibiotic resistance (Traub, 2000). Therefore, a

strategy is needed to combat this infection without

dependence on antibiotics.

The alternative way to manage virulence factors

is by using Quorum-sensing inhibitor (QSI) from

various plant phytochemicals (Bai & Rai, 2011;

Packiavathy et al., 2012; Bai & Vittal, 2014). QSI

role to decrease the level of virulence factor without

producing any bacteriostatic and bacteriocidal effect

as usual factor to trigger antibiotic resistance

(Yarmolinsky et al., 2015; Chang et al.,2017).

One of commonly utilized plant commodity in

North Sumatera is Andaliman (Zanthoxylum

acanthopodium DC.). Andaliman is one of native

plants in North Sumatera, commonly used as spices

in Bataknese traditional dishes. Extract of

Andaliman fruits have been reported to contain α-

pinene, limonene, geraniol, citronella, gerany-aceate,

and monoterpeneoids (Wijaya et al., 2002).

Study of Andaliman has focused on antimicroial,

antioxidant, anti-inflammation, inhibition of

xanthine oxidase and cytotoxic properties (Kristanty

& Suriawati, 2015). However, information of their

ability as potential QSI is still limited. The ability of

Andaliman as QSI will be evaluated in this study as

inhibitor to prodigiosin synthesis by S. marcescens.

Manalu, A., Jamilah, I. and Lenny, S.

Potency of Andaliman (Zanthoxylum acanthopodium DC.) Extracts as Quorum-sensing Inhibitor to Serratia marcescens.

DOI: 10.5220/0008506300870090

In Proceedings of the International Conference on Natural Resources and Technology (ICONART 2019), pages 87-90

ISBN: 978-989-758-404-6

2 RESEARCH METHODOLOGY

2.1 Inoculum preparation

Isolate of S. marcescens was firstly sub-cultured in

Luria Bertani agar prior laboratory test. Isolate was

obtained from collection of Department of

Microbiology, Faculty of Mathematics and Natural

Sciences, USU.

2.2 Phytochemical extraction

Fresh fruits and seeds of Andaliman (Z.

acanthopodium DC.) were washed and dried under

open air environment. Dried Andaliman fruits and

seeds were crushed using blender to obtain simplisia

powder. Simplisia powder was extracted using ethyl

acetate solvent (w/v) (1:6) as semi-polar fraction and

macerated for 3 d under agitation. Macerates were

obtained by filtering and simplisia was further

extracted using methanol solvent as polar fraction.

Each macerates were concentrated using rortary-

evaporator at 45

C. Both concentrated extracts were

diluted in various concentrations using Dimethyl

Sulfoxide (DMSO) (Muzafri et al.,2018)

2.3 Determination of QSI activity

Measurement of inhibitory activity of extract to

prodigiosin synthesis is based on protocol by

Morohoshi et al. (2007). Overnight culture of S.

marcescens was inoculated into Luria Bertani broth

supplemented with various concentration of

Andaliman extracts 0.4, 0.2, 0.1, 0.05, 0% (control),

and incubated for 30 hr. Indirect measurement of

bacterial growth was obtained through OD

600

of 5-hr

interval sampling of aliquots. Prodigiosin was

extracted from bacterial cells using acidified

methanol solution (4% 1 M HCl in EtOH).

Inhibition of prodigiosin was measured by

quantifying absorbance value as A

534

using

following formula:

%inhibition=

Absorbance of control-absorbance of treatment

×100%:

Absorbance of control

3 RESULTS AND DISCUSSIONS

Potency of Andaliman extracts as QSI was

evaluated from decrease of pigment quantity

produced by reference strain. The first thing to

consider in QSI testing was that extract did not

inhibit bacterial growth.

Samples were taken at various times (up to 30 h)

and analyzed to determine bacterial growth. The

tested concentrations of both MeOH and EtOAc

extracts did not show growth inhibitory activities as

shown in Figure 1 and 2, as bacterial density

proceed to increase following incubation time. This

shows that the extract had condition as QSI.

Figure 1: Effect of Andaliman MeOH extract on the

growth of S.marcescens on 0 h to 30 h incubation.

Figure 2: Effect of Andaliman EtOAc extract on the

growth of S.marcescens on 0 h to 30 h incubation.

Prodigiosin production by Serratia marcescens

was also analyzed at various times (up to 30 h)

together with its growth. Intracellular production

was extracted every 5 h. In the MeOH extract, the

prodigiosin production had increased in the end

incubation, and showed control was superior to the

treatment shown in Figure 3.

ICONART 2019 - International Conference on Natural Resources and Technology

Figure 3: Effect of Andaliman MeOH extract on prodigiosin

production by S.marcescens extracted every 5 hours (up to

30 hours)

The same event is also shown in EtOAc extract,

where prodigiosin production increases over time. In

this analysis, prodigiosin production at the end of

incubation showed that control was also superior to

treatment shown in Figure 4.

Figure 4: Effect of Andaliman EtOAc extract on

prodigiosin production by S.marcescens extracted every 5

hours (up to 30 hours)

The highest inhibition was obtained in

concentraion of 0.05% for MeOH and 0.4% for

EtOAc with percentage of inhibition 43.4% and

72.0% respectively in the end of incubation period

(30h). Packiavathy et al (2014) reported that

Curcuma longa extract also exhibit potency in

inhibiting prodigiosin production.

Figure 5. Percentage of prodigiosin inhibition on MeOH

Andaliman extract in the end of incubation period.

Figure 6. Percentage of prodigiosin inhibition on EtOAc

Andaliman extract in the end of incubation period.

A decrease in red-color intensities were observed

from concentration treatments. In the figure below

shown that the intensity of red is more likely to be

seen in cultures without the addition of Andaliman

extract.

Figure 7. Red-pigmented colour intensity in LB medium

with the addition of concentration variants extract

andaliman.

In this study, the QSI potential of Andaliman

fruit extract (Zanthoxylum acanthopodium DC.) is

evaluated through synthesis of red pigments by S.

marcescens. Quantitative analysis showed that

Potency of Andaliman (Zanthoxylum acanthopodium DC.) Extracts as Quorum-sensing Inhibitor to Serratia marcescens

extracts reduced the prodigiosin production in

percentage of 43.4 and 72.0% for methanol and

ethyl acetate fraction, respectively. Prodigiosin

synthesized by S.marcescens was known as one of

essential virulence factors (Liu & Nizet, 2009). Two

signals of the N-butanoyl homoserine lactone and

HHL molecules are shownto regulate prodigiosin

production (Morohoshi et al., 2007). Therefore, any

interference with this QS system will leads to a

reduction in prodigiosin production. A study by

Packiavathy et al. (2014) reported that inhibition of

prodigiosin using Curcuma longa was 58%. Bai &

Vittal (2014) reported that inhibition of violacein

production of Chromobacterium violaceum by M.

koenigii essential oil is due to inhibition of CIRIR-

dependent QS signaling either by preventing the

reception of AHL signals or decreasing AHL gene

expression.

A QS can be inhibited by certain compounds,

with mechanical inhibition in the form of blocking

the function of regulating transcription signals or

receptors. Persson et al. (2005) dividing inhibitory

compounds into two main groups, namely (a)

molecules which are analogous structures of the

original signal molecules; (b) other molecules with

structures that are not similar to the original signal

molecules. In this study the mechanism of inhibition

of QS by Andaliman is still not known specifically.

4 CONCLUSIONS

Fruit extract of Andaliman from methanolic and

ethyl acetate fractions showed inhibitory activities to

prodigiosin production by S.marcescens. In this

study, we evaluated that both extracts did not inhibit

bacterial growth as concluded to become unique

feature of QSI in combating and preventing

microbial resistances.

ACKNOWLEDGEMENTS

The authors would like to express the highest

gratitudes to Ministry of Research, Technology, and

Higher Education of the Republic of Indonesia for

financially supporting us through research grant

scheme of Penelitian Tesis Magister 2019, Number:

11 / E1 / KP. PTNBH / 2019 to IJ, USU.

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