3rd International Conferences and Workshops on Basic and Applied Sciences 2010

ISBN: 978-979-19096-1-7

Ball Sea Cucumber Phyllophorus sp. Improved

Interleukin-12 Response Against Mycobacterium
Tuberculosis
Dwi Winarni1, Dian Lia Palupi*, Erlix R. Purnama*, Moch. Affandi*,
Endang Dewi Masithah**, Alfinda Novi Kristanti***
*Department of Biology
Airlangga University, Surabaya, Indonesia
-mail: dwiwien711@yahoo.com
** Faculty of Fisheries and Marine
Airlangga University, Surabaya, Indonesia
***Department of Chemistry
Airlangga University, Surabaya, Indonesia

Abstract
1

The aim of this research was to know the potency

of Phyllophorus sp. as a immunostimulator for
enhance immune response against Mycobacterium
tuberculosis (Mtb). It was relied on the interleukin12 level of mice. For that, 24 male BALB/C strain
mice, 13-15 weeks old, weighed 25-30g, were
divided into 6 groups. They were given crude
extract, non polar fraction, semi polar fraction, and
polar fraction of Phyllophorus sp. respectively.
Control groups (negative and positive control
group) received no Phyllophorus sp. The doses of
Phyllophorus sp. was equal to 0,0462g dry
weight/kg BW/day for 14 days. A suspension
contained 106 Mtb infected intraperitoneally into
mice of all groups at day 15 except negative control
group. The blood samples were collected at day 18.
The level of serum IL-12 was measured by ELISA
and then the IL-12 data were analyzed by ANOVA
and Duncan test at = 0,05. The result suggested
that the ball sea cucumber Phyllophorus sp. has
immunostimulatory effect by improving IL-12
response against Mycobacterium tuberculosis and
that immunostimulatory effect was contributed
mainly by its non polar fraction

Introduction

Mycobacterium tuberculosis (Mtb) is the causative

agent of tuberculosis, which leads to an estimated
2-3 million deaths worldwide each year (Pathak et
al., 2005). The hallmark of pulmonary TB is
recruitment to the long tissue and activation of
inflammatory cells, resulting in granuloma
formation. This response restricts mycobacterial
dissemination and provides defense of the lung
against TB. However, it can lead to extensive tissue
damage, resulting both in the reduced lung function
and the aerosol transmission of mycobacteria
released
from
disintegrating
granulomas
(Eruslanov, et al., 2005). Indonesia is the 3rd
position of tuberculosis cases after India and China
(Departemen Kesehatan Republik Indonesia, 2006).
Mycobacterium tuberculosis able to evades the
immune response of the host by manipulating host
signaling pathways. One of the important
mechanism is the suppression of interleukin-12 (IL12) production. Interleukin-12 is a heterodimeric
cytokine composed of two disulfide-linked subunits
of p-35 and p-40. It is produced by macrophages
and dendritic cells in reponse to bacterial infection
or exposure to bacterial constituents such as
lypopolysaccharide. (Pathak et al., 2005). Beside
that, IL-12 promotes interferon- (IFN-)
production, proliferation, and cytolytic activity of
natural killer and Th cells (Vecchio, et al., 2007).
Interferon- is the predominant activator of
microbicidal function of macrophages, is detectable
at sides of Mtb infection and is essensial for
prevention of uncontrolled progression of infection.

Keywords: Ball sea cucumber, Phyllophorus sp,

immune response, Mycobacterium
tuberculosis, interleukin-12

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Dwi Winarni, Ball Sea Cucumber Phyllophorus sp. Improved Interleukin-12 Response Against Mycobacterium
tuberculosis

In addition, MTb infected macrophages synthesize

and secrete one or more polypeptides that inhibit
the response of uninfected macrophages to IFN-
(Nagabhushanam et al., 2003).

Methodology

2.1 Ball sea cucumber.

The ball sea cucumbers Phyllophorus sp were
caught from east coast of Surabaya, Indonesia. To
produce dry specimens, the fresh body walls were
dried by freeze dryer (-40oC, 50 mTorr) and then
powdered.

Indonesia is the largest single-country archipelago

in the world (8.3 million km2). Seventy per cent
(5.8 million km2) of this area is ocean with 17 508
islands and 81 000 km of coastline. Indonesian
coastal and marine areas are habitats for numerous
renewable marine resources, such as fish and
shrimp. Moreover, Indonesian coastal and marine
areas are a favourable habitat for many sea
cucumber species. There are at least 56 species of
sea cucumber found in these areas. Some
commercial species that are heavily exploited in
Indonesia and bring Indonesia to be the largest sea
cucumber exporter in the world. The main export
destination is Hong Kong SAR (China) and indeed
40-80 % of Hong Kong's import of sea cucumber
comes from Indonesia. Other export markets for
Indonesian sea cucumber are Japan, Korea Rep.,
Singapore, Taiwan PC, Malaysia and Australia
(Tuwo, 2004).

2.2 Extract preparation.

Dried specimens were extracted by ethanol
followed by n-hexane, ethyl acetate, and ethanol.
Evaporation at 50oC by vacuum evaporator for each
was done to obtain non polar, semi polar and non
polar fraction. While, the crude extract was yielded
from extraction of the other dried specimens by
ethanol. Then, all extract were subjected to
Lieberman-Burchard test semiquantitatively to
confirm their triterpene contain.
2.3 Mycobacterium tuberculosis (MTb)
The suspension of MTb 109 bacteria/ml has
obtained and prepared in Microbiology Laboratory
of Dr. Sutomo Hospital, Surabaya, Indonesia.
2.4 Animals
This study was carried out by using 24 male
BALB/C mice (3-4 moths, 30-40 g). Mice were
obtained from UPHP Gadjah Mada University,
Yogyakarta, Indonesia. The animals were grouped
and maintained under biosafety laboratory level 3
(BSL-3) conditions with dark and light cycle 12/12
h, and allowed free access to standart pellet feed
and water ad libitum. The mice were acclimatized
for 1 week before commencement of experiment.
All procedures conducted in accordance with
guideline from Animal Care dan Use
Committee/ACUC, Faculty of Veterinary Medicine,
Airlangga University.

Winarni et al. (2010) reported that at least seven

species of sea cucumbers which were found along
the east coast of Surabaya, East Java, Indonesia.
The one of them is ball-like shaped sea cucumber
Phyllophorus quadrangularis. Fishermen catch it
especially for local demand for traditionally food
use and it have not listed as a commercial species
for global market (Tuwo, 2004).
The body wall of sea cucumbers are known to
contain a variety of triterpene glycosides (Dang et
al., 2007; Hua et al., 2009; Dong et al., 2008). The
triterpene glycosides are secondary metabolites of
sea cucumber that are found in polar fraction of its
extract. Some of sea cucumbers triterpene
glycosides
have
antimicrobial,
antitumour,
anticancer, cytotoxic and immunomodulator
activities (Hua et al., 2009; Abraham et al., 2001;
Farouk et al., 2007). Immunomodulator able to
weaken or modulate the activity of the immune
system.The body wall of Phyllophorus sp. have
reported to contain triterpene qualitatively.
Treatment with body wall exctract of Phyllophorus
sp. decreased the number of E.coli in liver of mice
infected by E.coli 3 days before suggesting that it
could enhance the performance of immune system
(Winarni, et al., 2010). An enhancing immune
system of host was needed to overcome suppression
of IL-12 production and inhibition of uninfected
macrophages response in mycobacterial infection.
Beside that, exploration of the local ball sea
cucumber bioactivities will increase its commercial
value.

2.5 Treatment
Animals were divided into 6 groups. They were
given crude extract, non polar fraction, semi polar
fraction, and polar fraction of Phyllophorus sp.
respectively. Control groups (negative and positive
control group) received no Phyllophorus sp. The
doses of Phyllophorus sp. was equal to 0,0462g dry
weight/kg BW/day for 14 days. A suspension
contained 106 Mtb infected intraperitoneally into
mice of all groups at day 15 except negative control
group. The blood samples were collected
intracardiacly at day 18. To obtain their serum, the
coagulated blood samples were centrifuged at 4oC,
3000 rpm for 10 minutes after .
2.6 Measurement of Interleukin-12 level
To measure the level of IL-12, all samples of serum
were subjected to quantification total IL-12 level by
ELISA (enzyme-linked immmunosorbent assay)
using mouse IL-12 total ELISA BenderMed
system.
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3rd International Conferences and Workshops on Basic and Applied Sciences 2010

al., 2005). Doz et al. (2007) also stated that the

capacity of macrophages in IL-12 production
depend on binding of Man-LAM and LAM to TLR
and C-type lectin mannose receptors balance.
Macrophages continue to produce IL-12 if more
TLR-Mtb binding occurred, but it will be
suppressed to produce IL-12 if there were more Ctype lectin mannose receptors-Mtb binding.

3 Result
Macrophages are the main effector cells involved in
killing of MTb (van Crevel et al., 2002). Initial
response
of
macrophages
started
when
macrophages-bearing receptors recognize to certain
parts of MTb. These main receptors are Toll-like
receptor (TLR) and C-type lectin mannose
receptors that which able to recognize mannosecapped
lipoarabino-mannan
(Man-LAM),
lipoarabinoma-nnan (LAM), and other ligands that
are found in cell wall of MTb (Doz et al, 2007).
Generally, bacterial infections stimulate IL-12
production by macrophages (Vecchio et al., 2007).
But, the level of IL-12 in control group with MTb
infection didnt increase significantly compared
with control group without MTb (figure 1.)
indicating that there was suppression of IL-12
production in response to mycobacterial infection
(Pathak et al., 2005)

Treatment with the non polar fraction of body wall

of Phyllophorus sp into mice equal to 0,0462 g dry
weight/kg BW/day for 14 days (P2) could enhance
IL-2 response significantly after MTb infection (Fig
1) comparing with control with or without Mtb
infection. Increase in IL-12 level showed that active
ingredients in non polar fraction of ball sea
cucumber abled to upregulate IL-12 production.
This regulation might through enhancing TLR
expression so that more TLR could bind to Mtb,
increase ratio TLR-MTb binding to C-type lectin
mannose receptors-Mtb binding, and
further,
macrophages could produce more IL-12.
Increase in IL-12 level in mice which given by
crude extract (P1), that not significantly different
with control (K1 and K2)(figure 1.) suggesting that
it was needed further study to determine the range
of the dosage of extract which have
immunostimulatory effect, and that constituents in
semi polar and polar fraction might lowered the
imunostimulatory effect of the active ingredients of
non polar fraction. Beside that, because the highest
effect on IL-12 response showed by non polar
fraction, we conclude that it wasnt a triterpene
glycoside effect because triterpene glycosides were
found in polar fraction.

c
a,b

ISBN: 978-979-19096-1-7

b,c
a,b

a,b
a

4 Conclusions
Figure 1: Interleukin-12 level of all group of
treatments. The same script above each diagram
represent not significantly different after
Duncan test at =0.05). K1= control group
without Mtb infection; K2 = control group with
Mtb infection; P1, P2 and P3 were group that
given by crude extract, non polar fraction, semi
polar fraction and polar fraction of ball sea
cucumber Phyllophorus sp. respectively.

This research revealed that the ball sea cucumber

Phyllophorus sp. has immunostimulatory effect by
improving IL-12 response against Mycobacterium
tuberculosis and that immunostimulatory effect was
contributed mainly by its non polar fraction
References

The supression of IL-12 production was attributed

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able to induce IL-12 production in a TLRdependent manner and may also inhibit IL-12 in a
TLR-independent manner, so that the ratio of LAM
to Man-LAM may govern the capacity of the
organism to circumvent IL-12 production (Pathak et
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tuberculosis

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