Vol 52 No 1 Jan-Mar 2019_New.indd


4545

Dental Journal
(Majalah Kedokteran Gigi)

2019 March; 52(1): 45–50

Research Report

The role of Kuniran (U. moluccensis) and Gurami (O. goramy) fish 
thorns and scales in increasing salivary leukocyte and monocyte 
cells viability against Streptococcus mutans 

I Dewa Ayu Ratna Dewanti,1 I Dewa Ayu Susilawati,1 P. Purwanto,1 Pujiana Endah Lestari,1 Roedy Budirahardjo,2 Dyah Setyorini,2 Ristya Widi 
Endah Yani,3 Erawati Wulandari,4 and Melok Aris Wahyukundari5
1Department of Biomedical Science
2Department of Pedodontics
3Department of Dental Public Health
4Department of Conservative Dentistry
5Department of Periodontics
Faculty of Dentistry, Universitas Jember, Jember – Indonesia

ABSTRACT

Background: Kuniran thorns and Gurami fi sh scales are rich in protein and minerals such as dentin believed to increase cell viability 
against Streptococcus mutans (S. mutans) that causes dental caries. These, in turn, can cause systemic diseases if left untreated. Purpose: 
This study aims to analyze the infl uence of Kuniran thorns and Gurami fi shes scales on the viability of monocytes and salivary leukocytes 
against S. mutans. Methods: Monocytes and leukocytes salivary cells were placed on a microtiter plate and treated according to the 
nature of each group. This study comprised the following groups: control group: untreated; S. mutans group: induced by S. Mutans; 
Gurami thorn group: thorns + S. Mutans; Gurami scales group: scales + S. Mutans; Kuniran thorn group: thorns + S. Mutans; Kuniran 
scales group: scales + S. mutans. Viability analysis involved staining with Tripan Blue. Furthermore, the number of viable cells (white) 
was calculated under an inverted microscope at 200 times magnifi cation from fi ve fi elds of view. Data was analyzed by means of an 
ANOVA test followed by LSD test. Results: The ANOVA and LSD tests confi rmed signifi cant diff erences (0.01<P). Kuniran thorns and 
Gurami fi sh scales increased the viability of monocytes and salivary leukocytes, but not signifi cantly. The content of fl avonoids, amino 
acids, omega 3, omega 6 and antioxidants increased leukocyte metabolism, thereby increasing cell resistance to S. mutans infection. 
Conclusion: Kuniran thorns (U. moluccensis) and Gurami (O. goramy) fi sh scales increase the viability of salivary leukocyte and 
monocyte cells against Streptococcus mutans.

Keywords: Osphronemus goramy; scales; Streptococcus mutans; thorns; Upeneus moluccensis; viability

Correspondence: I Dewa Ayu Ratna Dewanti, Faculty of Dentistry, Universitas Jember. Jl. Kalimantan 37, Jember, 68121, Indonesia. 
E-mail: idewadewanti@yahoo.com

INTRODUCTION

In 2018, the National Basic Health Research Department 
(Riskesdas) stated that the rate of dental and oral problems 
stood at 57.6%. Dental caries remain an intractable national 
problem in Indonesia which, according to Riskesdas figures 
for 2013 and 2018, had reached the level of 4-5 teeth 
per person, while the international standard stood at 2.5 
teeth.1 Streptococcus mutans (S. mutans) is a bacterium 
constituting the main cause of dental caries whose spread 

can lead to systemic diseases. Consequently, researchers 
are increasing their understanding of its presence and 
studying the pathogenesis of dental caries together with 
their systemic expansion. There is evidence that bacteria 
(S. mutans) originating in dental caries play a role in the 
occurrence of endocarditis 2 since they are found in the 
coronary atherosclerotic plaque of patients who suffer fatal 
heart attacks.3 Since monocytes and salivary leukocytes 
play a role in the spread of S. mutans from the mouth 
throughout the systems of the body, the importance of 

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v52.i1.p45–50

mailto:idewadewanti@yahoo.com
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46 Dewanti, et al./Dent. J. (Majalah Kedokteran Gigi) 2019 March; 52(1): 45–50

the role of leukocytes in saliva and monocytes in blood 
in the spread of S. mutans is evident. On the other hand, 
prevention and treatment of dental caries can be achieved 
by employing natural resources readily available in 
Indonesia. Two such resources are fish thorns and scales 
which, since they are regarded as waste, remain under-
utilised. However, such materials must be compatible.4 
In Jember, Kuniran (Upeneus moluccensis) and Gurami 
(Osphronemus goramy) are two of the five most common 
fish species5 with the latter representing an important 
national aquaculture commodity.6 Therefore, this study 
seeks to analyze the viability of Kuniran thorns and Gurami 
fish scales in monocytes and salivary leukocytes acting 
against S. mutans.

Researchers have previously proved that fish thorns 
and scales consist of proteins that supposedly act as 
immunomodulators because they contain omega 3, omega 6, 
flavonoid, various amino acids (alanine, leucine-isoleucine, 
valine, arginine, methionine, proline, glutamic acid, 
histidine, lysine, glycine, serine and tyrosine) which are 
believed to prevent dental caries. A number of researchers 
have confirmed that fish waste such as scales, thorns and 
skin represent a potential source of collagen that is crucial 
to tissue formation and organ function, while also being 
involved in various functional expressions of cells.4,7–9 
Scientists from Nanyang Technological University (NTU) 
in Singapore have demonstrated that collagen derived 
from fish scales is highly effective across a variety of 
biomedical applications such as wound healing.10 Research 
conducted by Zhou et al. (2013), strongly suggests that 
fish scales containing collagen can increase blood vessel 
and lymph formation, thereby promoting tissue repair and 
regeneration.11 However, the findings of these studies 
suggest that the viability of the scales and thorns of Gurami 
and Kuniran fish remains unproven. It is known that Gurami 
(fresh water) and Kuniran (salt water) are the types of fish 
most frequently consumed by the population of Jember, 
East Java, Indonesia.

Cell viability testing is central to drug discovery 
studies.12 Determination of cell viability is the most 
commonly employed method of assessing the impact of 
various types of stressors in both toxicity and industrial 
microbiology studies. Viability is defined as the percentage 
of living cells within the entire population.13 Therefore, 
this study was intended to analyze the effects of thorns 
and scales from Gurami and Kuniran fish on the viability 
of salivary leukocyte and monocyte cells after induction 
of S. mutans.

MATERIALS AND METHODS

The research, approved by the Ethical Committee, Faculty 
of Dentistry, University of Jember (079/UN.25.8/KEPK/
DL/2018), was conducted between April and September 
2018. The materials comprised the following: peripheral 

blood, S. mutans from the Microbiology Laboratory, 
Faculty of Dentistry, University of Jember; Ficoll-hypaque 
(Sigma); HBSS/Hank’s Balanced Salt Solution (Gibco); 
RPMI medium (Roswell Park Memorial Institute/Gibco); 
Immunostaining KIT (Daco); PBS/Phosphate Bufer Saline 
(Sigma); DAB/Diaminobenzidine (Daco); HRP/horseradish 
peroxidase (Daco) and fungizone (Gibco). 

The fish thorns/scales were placed in an alumunium pan 
once the water temperature had reached 80°C and boiled 
for 60 minutes, before being dehydrated in a drying oven 
for 48 hours at 65°C, mashed using a grinder mill and 
sieved through a 100 mesh filter. ±1g of the dry sample 
was subsequently weighed and dissolved using PBS at a 
ratio of 1:1.14 

Blood and saliva were collected from healthy individuals 
(males deemed to be free of systemic disease and caries who 
were teetotal non-smokers). Saliva was collected by means 
of the subject spitting onto a dish placed on ice. The blood 
and saliva was then carefully layered with histopaque and 
ficollpaque to avoid their mixing, before being centrifugated 
at 1,900 rpm for 30 minutes at 26oC. The monocyte and 
salivary leukocyte layers were added to HBSS (1:1) and 
pipetted. Monocytes and salivary leukocytes were filtered 
on each coverslip (@ 100 μL), placed in a microplate and 
incubated for 15 minutes. 1 cc of RPMI medium, 20 μL of 
stain and 5 μL of fungizone were added to each well and 
incubated for 20 minutes, washed twice to eradicate any 
contamination and, finally, treated according to the group.15 
 The monocyte group consisted of six groups: a control 
group; a monocyte isolates untreated S. mutans group; a 
monocyte isolate induced by S. mutans group; a Gurami 
thorn group: @ 100 μL of monocyte isolates were added to 
200 μL of Gurami thorns and 100 μL of S. mutans; a Gurami 
scales group: @ 100 μL of monocyte isolates were added to 
200 μL of Gurami scales and 100 μL of S. mutans. 

The salivary leukocytes group consisted of six groups: 
a control group: untreated salivary leukocytes isolates; 
S. mutans group: salivary leukocytes isolates induced by 
S. mutans; Gurami thorns group: @ 100 μL of salivary 
leukocytes isolates were added to 200 μL thorns of Gurami 
and 100 μL of S. mutans; Gurami scale group: @ 100 μL of 
salivary leukocytes isolates were added to 200 μL of scales 
Gurami and 100 μL of S. mutans; Kuniran thorn group: @ 
100 μL of salivary leukocytes isolates were added to 200 μL 
of Kuniran thorns and 100 μL of S. mutans; Kuniran scales 
group: @ 100 μL of salivary leukocytes isolates were added 
to 200 μL of Kuniran thorns and 100 μL of S. mutans. All 
groups were painted with Tripan Blue to facilitate analysis 
of cell viability. Treatment ensued with S. mutans (@ 100 
μL and thorns/scales @ 200 μL.

Cell viability was analyzed by counting the number of 
living cells (white) under the inverted microscope at 200 
times magnification from five fields of view. The data 
obtained was subsequently analyzed by means of ANOVA 
and LSD Tests.

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v52.i1.p45–50

http://e-journal.unair.ac.id/index.php/MKG
http://dx.doi.org/10.20473/j.djmkg.v52.i1.p45-50


47Dewanti, et al./Dent. J. (Majalah Kedokteran Gigi) 2019 March; 52(1): 45–50

RESULTS

The results of the viability of salivary monocyte and 
leukocyte cells exposed by thorns and scales of Kuniran 
and Gurami fish after being induced by S. mutans were 
as follows. The ANOVA analysis results were p=0.000 
(p<0.05), while the LSD test produced a significant 
difference between the control with Gurami thorns and 
scales and the control with Kuniran thorns and scales. 
However, there were no significant differences between 
Gurami thorns and Gurami scales p=0.86 (p>0.05), Gurami 
thorns with Kuniran scales p=0.14 (p> 0.05), Gurami thorns 
with Kuniran scales p=0.147 (p>0.05), Gurami scales with 
Kuniran scales p=0.765 (p>0.05). 

The ANOVA analysis results were p=0.000 (p<0.05), 
while the LSD Test indicated a significant difference 
between the control group with Gurami thorns and scales 
and the control group with Kuniran thorns and scales. 
However, there were no significant differences between 
Gurami thorns with Gurami scales p=0.86 (p>0.05), Gurami 
thorns with Kuniran thorns p=0.14 (p>0.05), Gurami thorns 
with Kuniran scales p=0.147 (p>0.05) and Gurami scales 
with Kuniran scales p=0.765 (p> 0.05). These results 
illustrated that Kuniran thorns and scales possess the same 
ability to increase the viability of salivary leukocytes, 
whereas Gurami scales increased the viability of salivary 
leukocytes compared to their scales. It could be said that 
the thorns and scales of Kuniran and Gurami fish usually 
increased both the viability of monocytes and salivary 
leukocytes (Tables 1-6 and Figures 1-4).

Table 1. Normality test of monocyte cells viability One-sample 
Kolmogorov-Smirnov test

Treatment Viability of monocyte cells

2424N

Normal Parametersa,b
92.41673.5000Mean

59.960071.74456Std. Deviation

Most Extreme Differences

.401.138Absolute

.235.138Positive

-.401-.138Negative

1.966.678Kolmogorov-Smirnov Z

.001.748Asymp. Sig. (2-tailed)

a. Test distribution is normal; b. Calculated from data.

Table 2. Test of homogeneity of monocyte cells viability

Levene 
Statistic

Sig.df2df1

5 18 .796.467

Table 3. One-way ANOVA of monocyte cell viability 

Sum of 
squares

df
Mean 
square

Sig.F

Between 
groups

.00012146.74316533.067582665.333

Within 
groups

1.3611824.500

Total 82689.833 23

Table 5. Test of salivary leucocyte homogeneity

Sig.df2df1Levene Statistic

.692185.612

Table 4. Normality test of salivary leucocyte viability One-sample 
Kolmogorov-Smirnov test

Treatment
Viability of 
monocyte 

cells

2424N

Normal 
Parametersa,,b

44.83333.5000Mean

Std. 
Deviation

27.641601.74456

Most 
Extreme 
Differences

.375.138Absolute

.222.138Positive

-.375-.138Negative

1.837.678Kolmogorov-Smirnov Z

Asymp. Sig. (2-tailed) .748 .002

a. Test distribution is Normal; b. Calculated from data.

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v52.i1.p45–50

http://e-journal.unair.ac.id/index.php/MKG
http://dx.doi.org/10.20473/j.djmkg.v52.i1.p45-50


48 Dewanti, et al./Dent. J. (Majalah Kedokteran Gigi) 2019 March; 52(1): 45–50

Table 6. One-way ANOVA of salivary leucocytes viability

Sig.FMean squaredfSum of squares

.0002938.9123510.367517551.833Between groups

1.1941821.500Within groups

2317573.333Total

10.5 
3 

63.75 65.25 61.5 
65 

0 

10 

20 

30 

40 

50 

60 

70 

80 

Control S. mutans Gurami 
Thorns 

Gurami 
Scales 

Kuniran 
Thorns 

Kuniran 
Scales 

Th
e 

nu
m

be
r 

of
 c

el
ls

 
Figure 3. Diagram of salivary leukocytes viability

13.5 
5.5 

133.75 135.25 131.5 135 

0

20

40

60

80

100

120

140

160

Control S. mutans Gurami
Thorns

Gurami
Scales

Kuniran
Thorns

Kuniran
Scales

Th
e 

nu
m

be
r 

of
 c

el
ls

 

Figure 1. Diagram of monocytes viability

 

Control 

S. mutans 

Kuniran Scales 

Kuniran Thorns 

Gurami Scales 

Gurami Thorns 

Figure 2 Microscopic description of monocytes viability of Kuniran and Gurami fish scales and thorns (arrow). Observations.
conducted with an inverted microscope at 200x magnification. 

Control 

S. mutans Gurami Thorns 

Kuniran Scales 

Kuniran Thorns 

Gurami Scales 

Figure 4. Microscopic description of salivary leucocyte viability of Kuniran and Gurami fish scales, thorns (arrow). Observations 
using a inverted microscope at 200x magnification. 

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v52.i1.p45–50

http://e-journal.unair.ac.id/index.php/MKG
http://dx.doi.org/10.20473/j.djmkg.v52.i1.p45-50


49Dewanti, et al./Dent. J. (Majalah Kedokteran Gigi) 2019 March; 52(1): 45–50

DISCUSSION

As caries-related bacteria penetrate deeply into dentin, 
coming into close proximity to the pulp, inflammatory cells 
(such as lymphocytes, macrophages and neutrophils) infiltrate 
the bacterium-invaded area resulting in the development of 
pulpitis. Many types of cytokines and adhesion molecules are 
responsible for the initiation and progression of pulpitis.16 
Bacteria (S. mutans) then spread systemically through the 
blood vessels in the oral cavity to other parts of the body. 
At this point, the role of immunocompetent cells such 
as monocytes, macrophages, neutrophils, lymphocytes 
is crucial in preventing the spread of S. mutans from 
the oral cavity to other parts of the body’s systems. 
Immunocompetent dental pulp cells have an important 
function in maintaining the structural integrity of connective 
tissue. These cells include odontoblasts which produce pro-
inflammatory cytokines and express adhesion molecules 
in response to pathogens such as pathogen-associated 
molecular patterns (PAMP) which are structures expressed 
by microorganisms. Generally, the initial recognition of 
microbial pathogens is mediated by pattern recognition 
receptors (PRRs), such as toll-like receptors (TLRs) and the 
nucleotide-binding oligomerization domain-like receptors 
(NLRs).17–19After recognition by receptors, the cells will 
initiate proinflammatory cytokines and oxidant-producing 
phagocytic processes, both of which cause damage to 
cells and tissues. Therefore, a material is required that 
can increase cell resistance to damage caused by bacterial 
infections, especially S. mutans. 

The research reported here has proved that the thorns and 
scales of Kuniran and Gurami fish can inhibit the growth 
of S. mutans by increasing the viability of monocytes and 
salivary leukocytes. Kuniran and Gurami fish thorns and 
scales increased the viability of monocytes and salivary 
leukocytes due to their content, including: amino acids, 
flavonoids and unsaturated fatty acids (omega 3, omega 6). 
Amino acids play a role in the vitamin B process (especially 
that of B5 and B6), produce leucine and isoleucine in 
protein synthesis, form antibodies, activate various types 
of hormones, energy providers, ketone and glucose 
makers. Arginine can strengthen the immune system, while 
methionine nourishes blood vessels, reduces inflammation 
and treats allergies. Glycine can be employed for wound 
healing and arginine for various metabolic urea synthesis, 
lymphocyte proliferation and wound healing. Glutamine is 
one of the three amino acids present in glutathione which are 
antioxidant compounds used as ingredients in leukocytes 
metabolism. Glutathione is one of the antioxidants with a 
role in protecting cells from damage caused by reactive 
oxygen. On the other hand, the main components of cell 
membranes are phospholipids, glycolipids and cholesterol. 
These components contain polyunsaturated fatty acids 
which are highly susceptible to oxidation that causes free 
radicals.20,21

Antioxidants can prevent cell damage caused by 
donating hydrogen electrons to free radicals. Antioxidants 

can give hydrogen atoms to lipid radicals (R •, ROO •) 
and transform them into more stable forms. In addition, 
antioxidants can also slow the rate of auto-oxidation 
resulting in the presence of amino acids, thought to 
positively affect body cell metabolism, thereby enhancing 
cell resistance to infection. The flavonoid content is thought 
to work through its inhibiting of the production of Nitric 
Oxide (NO) through the mechanism of the cytokine-induced 
NO synthase (iNOS) enzyme. It may also inhibit arginine 
transport through the mechanism of Cationic Amino 
Acid Transporter-2 mRNA (CAT-2 mRNA). It is said 
that flavonoids are potential cancer-reducing compounds 
which can inhibit oxidation reactions induced by enzymes 
or non-enzymes and act as a good source of hydroxyl and 
superoxide radicals that protect membrane lipids from 
reactions that can damage cells (monocytes and salivary 
leukocytes).22

Unsaturated fatty acids constitute the main component of 
phospholipids which act as a constituent of cell membranes, 
DNA and proteins. DNA represents a cell’s genetic device, 
while proteins play an important role as enzymes, receptors, 
antibodies, matrix formers and cytoskeleton. Research 
has demonstrated that omega-3 polyunsaturated fatty acid 
fish oil reduces stress-induced oxidative DNA in vascular 
endothelial cells.23 Therefore, all bioactive components of 
Kuniran and Gurami fish thorns and scales increase cell 
viability. The conclusion of this research is that the thorns 
and scales of Kuniran (U. moluccensis) and Gurami (O. 
goramy) fish can increase the viability cells of salivary 
leukocytes and monocytes against S. mutans. 

ACKNOWLEDGMENTS

The authors express their gratitude to the Indonesian 
Ministry of Research, Technology and Higher Education 
(RISTEKDIKTI) for its provision of funding for this 
research and to the Chairperson of the Institute for Research 
and Community Service (LP2M), Universitas Jember, and 
Dean of the Faculty of Dentistry, Universitas Jember for 
their recommendations relating to its conduct.

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Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
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Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. 
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v52.i1.p45–50

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