Title


Indonesian Journal of Environmental
Management and Sustainability
e-ISSN:2598-6279 p-ISSN:2598-6260

Research Paper

Effects of Spirulina platensis extract on serum Malondialdehyde (MDA)

levels and differential leukocyte count on motor vehicle exhaust emissions

exposure (in vivo study on Wistar rats)

Dea May Fitry 1*, Nani Maharani 2, Renni Yuniati 3

1Graduate School of Biomedicine, Faculty of Medicine, University of Diponegoro, Semarang, Indonesia
2Department of Pharmacology and Therapy, Faculty of Medicine, Diponegoro University, Semarang, Indonesia
3Department of Dermatovenerology, Faculty of Medicine, Diponegoro University , Semarang, Indonesia

*Corresponding author e-mail: deamfitry@gmail.com

Abstract
The particulate matter in vehicle emission has been shown to cause an increase in free radical levels which can increase serum
MDA levels and leukocyte count. Spirulina platensis is an algae that has antioxidant effects that can scavenge free radicals.
In this research, twenty-four of wistar mice are divided into 4 groups. Groups X1 and X2 received vehicle emission smoke
exposure treatment and were given oral Spirulina platensis extract at a dose of 500 and 750 mg / kgBW / day for 14 days,
the negative control group (C2) was given 0.9% NaCl orally and the normal control group did not get vehicle emission smoke
exposure and Spirulina platensis extract. Serum MDA levels were checked by ELISA method and leukocyte count by giemsa
staining. Data analysis was performed by using the one-way ANOVA test followed by Post Hoc Bonferroni. The results
showed the mean serum MDA levels there was a significant difference between group C2 and group X2. In the mean absolute
value of neutrophils and lymphocytes, there were significant differences between the C2 group with the administration of
X1 and X2. In the mean absolute value of eosinophils and monocytes, there were significant differences between groups
C1 and C2 and group X2 and between groups X1 and C1. Furthermore, the given doses of Spirulina platensis extract at a
dose of 750mg / kgBW had an effect on serum MDA levels as well as the number of neutrophils and lymphocytes and the
administration of doses of 500 and 750mg / kgBW affected the number of eosinophils and monocytes.

Keywords
Spirulina platensis, PM2.5, MDA, Leukosit

Received: 8 September 2020, Accepted: 28 September 2020

https://doi.org/10.26554/ijems.2020.4.3.85-89

1. INTRODUCTION

Ambient air pollution (outdoor air) is the biggest world
problem of environmental health. According to WHO, there
were an estimated 4.2 million deaths in 2016 due to ambient
air pollution. Adverse health effects can occur as a result of
short or long-term exposure. The main effect of ambient air
pollutants is particulate matter (PM), ozone (O3), nitrogen
dioxide (NO2) and sulfur dioxide (SO2). One of the main
sources of PM is vehicle emission from diesel and gasoline
(WHO, 2016). The presents of free radicals and the inflam-
matory response of ambient air pollution can cause several
health problems (Lu et al., 2018; Manzetti and Andersen,
2016; Gong et al., 2013; Lee et al., 2018). Free radicals
will cause cell damage, lipid peroxidation and protein oxi-
dation. The result of lipid peroxidation is malondialdehyde
(MDA)(Birben et al., 2012). The inflammation that occurs

in the short term can increase the risk of exacerbation of
several diseases such as COPD, asthma and autoimmune
diseases and in the long term can cause various cardiorespi-
ratory diseases. One marker of inflammation is leukocytes
(Lee et al., 2018).

As similarly reported by Gong et al. (2013) in china,
showed the increasing MDA urine percentage and exhaled
breath condensate (EBC) during the Beijing Olympics in
which air pollution was increased dramatically. According
to Yan et al. (2017) showed an increase in MDA d levels in
mice with were given exposure to PM 2.5 (particles <2.52µm
in size) compared to control rats. Lee et al. (2018) also
reported that in 2010 to 2016 showed an increase in the
number of leukocytes due to long-term exposure to ambient
air pollution in South Korea. However, Indonesian marine
biota has a huge opportunity to be explored as a source of
natural antioxidant compounds. One of the microorganisms

https://doi.org/10.26554/ijems.2020.4.3.85-89


Fitry et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 85-89

that have the potential as a source of natural antioxidants
is Spirulina platensis (Yudiati et al., 2011).

Spirulina platensis is a filamentous and multicellular
blue-green alga capable of reducing inflammation and also
manifesting antioxidant effects. Spirulina platesis is a natu-
ral source of protein, vitamins, macronutrients and micronu-
trients such as amino acids, gamma linoleic acid, chlorophyll
and phycocyanin (Abd El-Hakim et al., 2018) Previous
studies have shown the antioxidant potential of Spirulina
platensis. El-Baz et al. (2013)conducted a study which
showed that Spirulina platensis extract had a beneficial
effect in controlling hyperglycemia and reducing MDA levels
in streptozoticin-induced DM type 2 rats (STZ). In addi-
tion, research conducted by Al-Qahtani and Binobead (2019)
Spirulina platensis is produced in different areas. In this
research, the antioxidant potential possessed by Spirulina
platensis is expected to inhibit lipid peroxidation and in-
flammation which can be measured by seeing a decrease in
MDA levels.

2. EXPERIMENTAL SECTION

2.1 Spirulina Platensis extract
This study used Spirulina platensis powder with the reg-
istration number FDA 15594742028 and CERES number
50OGA1200043 (9241). Spirulina platensis powder was ex-
tracted by maceration (immersion), with a ratio of 1:10,
namely 1 part of the Spirulina platensis microalgae sample
powder soaked in 10 parts of 95% ethanol solution. Macera-
tion is carried out for 5 days in a glass container. Shake the
glass container every day for a few minutes. After 5 days
the solution was filtered with Whatman filter paper. The
next step is evaporation using a rotary evaporator at the
boiling temperature of ethanol until a concentrated extract
is obtained.

2.2 Vehicle emission and exposure setting system
The vehicle emission carried out by comes from a 125 cc
automatic motorcycle was product in 2017 and fueled by
pertalite. The exposure was carried out by setting the
system i.e., the exhaust is connected to the sucking pump,
where only particles less than 2.5µm in diameter can pass
(using Whatman filter paper no 42). The sucking pump
will flow the smoke into the exposure chamber. Then the
motorcycle engine is operated for 80 seconds. The PM2.5
concentration in the exposure chamber was measured using
a kanomax (Figure 1). The inhalation time of PM2.5 per
day per 80 seconds of fumigation is 100 seconds where the
PM2.5 dose exposed to mice is 0.625 + 0.069mg/m3.

2.3 Procedure of wistar mice exposure
Twenty-four male Wistar mice aged 2-3 months with a
weight of 100-200gr were acclimatized for 7 days with room
temperatures ranging from 25-28oC and room humidity
levels ranging from 70-75%. A light reception is set to 12
hours bright and 12 hours dark. Rats were given feed in the

Figure 1. Procedure for exposure to vehicle emission

(Note: 1 = motorcycle exhaust; 2 = sucking pump + filter
paper; 3 = exposure chamber; 4 = kanomax; 5 =

connecting pipe)

form of AD II pellets and reverse osmosis drinking water
which was given ad libitum. On the 8th day, Twenty-four
male Wistar rats that had met the inclusion criteria were
then divided into 4 groups randomly (randomization) where
each group consisted of 6 Wistar rats. Groups X1 and X2
received vehicle emission exposure treatment and were given
oral Spirulina platensis extract at a dose of 500 and 750
mg/kgBW/day, the negative control group (C2) was given
exposure to smoke and NaCl 0.9% orally and the normal
control group did not get vehicle emission exposure and
Spirulina platensis extract.

After fourteen-day treatment, the wistar rats were anes-
thetized by giving ketamine injection at a dose of 60 mg/kgBW
(i.m). Then two separate blood samples were taken from
the retroorbital veins of each mouse. The first sample was
used for examination of serum MDA levels by the ELISA
(Enzyme linked immunosorbent assay) method according
to the protocol specified in the commercial kit used (Cat-
alog no: E-EL-0060: Elab Science Biotechnology, Texas,
USA). The second sample was used for manual examination
of leucocyte count with Giemsa staining according to the
procedure. This research has been approved by the Med-
ical / Health Research Bioethics Commission, Faculty of
Medicine, Sultan Agung Islamic University, Semarang with
the Ethical Clearance number 521 / VIII / 2019 / Bioethics
Commission.

2.4 Statistical analysis using ANOVA
The statistical analysis of this study used the SPSS 25 com-
puter program. The test performed was One-Way ANOVA
and was further analyzed by Post Hoc Bonferroni. The
significance value in this study was p<0.05.

3. RESULTS AND DISCUSSION

3.1 Serum MDA levels
The results after measuring serum MDA levels using the
ELISA method, the measurement results are obtained as
presented in Table 1.

The results of the one-way ANOVA analysis on serum
MDA levels showed significant differences between the study
groups and then further analyzed using the Bonferroni Post

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Fitry et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 85-89

Table 1. Results of measurement of serum MDA levels (ng
/ mL)

Groups Mean+SD Min-Max p*

C1 195.0+22.15 163.56 – 220.22 p<0.001
C2 376.1+55.57 293.56 – 441.33
X1 326.7+44.51 280.22 – 392.44
X2 178.6+25.22 155.78 – 209.11

Hoc test. The results of the Post Hoc test showed that there
was a significant difference between the negative control
group (C2) and the group giving Spirulina platensis extract
750mg/kgBW (X2) and between the two control groups (C1
and C2).

3.2 Differential Leukocyte counts
The value of absolute neutrophil and lymphocyte count,
the results are shown in Table 2 and Table 3. The results

Table 2. The value of absolute neutrophil (x103/ µL)

Groups Mean+SD Min-Max p*

C1 4.7+2.07 3-8 p<0.001
C2 12.8+3.49 9-18
X1 4.0+1.52 2-6
X2 4.7+2.13 2-8

Table 3. The value of absolute lymphocyte (x103/ µL)

Groups Mean+SD Min-Max p*

C1 4.3+2.07 2-7 p<0.001
C2 11.8+5.27 3-19
X1 5.3+2.50 2-9
X2 3.8+1.94 1-6

of the one-way ANOVA analysis on the value of absolute
neutrophil and lymphocyte count showed that there were
significant differences between the study groups and then
further analyzed using the Post Hoc Bonferroni test. The
results of the Post Hoc test showed that there was a signifi-
cant difference between the negative control group (C2) and
the group giving Spirulina platensis extract 500mg/kgBB
(X1) and 750mg/kgBB (X2) and between the two control
groups (C1 and C2).

Thus, the results of the one-way ANOVA analysis on the
value of absolute eosinophils (Table 4) and monocytes (Table
5) showed that there were significant differences between the
study groups and were further analyzed using the Post Hoc
Bonferroni test. The results of the Post Hoc test showed that
there was a significant difference between the two control
groups (C1 and C2) with the group giving Spirulina platensis

extract 750mg / kgBB (X2) and between the group giving
Spirulina platensis extract 500mg / kgBB (X1) and the
normal control (C1).

Table 4. The value of absolute eosinophils (x103/ µL)

Groups Mean+SD Min-Max p*

C1 1.8+1.17 0-3 p<0.001
C2 8.5+1.87 6-11
X1 7.4+2.12 5-10
X2 5.1+0.98 4-6

Table 5. The value of absolute monocytes (x103/ µL)

Groups Mean+SD Min-Max p*

C1 1.7+1.21 0-3 p<0.001
C2 10.3+2.16 8-14
X1 10.4+2.26 7-13
X2 5.1+1.91 3-8

The Spirulina platens is has various health benefits such
as antioxidants, immunomodulators, anti-inflammatory, an-
ticancer, antiviral and antibacterial and has a positive effect
on hyperlipidemia, malnutrition, obesity, metal poisoning
and anemia (N’Djamena, 2012). Spirulina platensis antiox-
idants have activity against several types of free radicals
such as hydroxyl and peroxyl radicals and preventing the
occurrence of DNA damage(Soni et al., 2015). Hydroxyl rad-
icals play an important role in the lipid peroxidation process
where the final product is MDA(Ayala et al., 2014). This
study provides exposure to vehicle emission in experimental
animals for 14 days. PM2.5 contained in vehicle emission
caused the presents of free radicals which will cause oxida-
tive stress. Oxidative stress will trigger lipid peroxidation
which in turn will increase serum MDA levels (Manzetti and
Andersen, 2016; Gong et al., 2013).

El-Baz et al. (2013) was reported that Spirulina platensis
extract had a beneficial effect in controlling hyperglycemia
and significantly lowering MDA levels of type 2 DM rats
compared to a control group. Another study was conducted
on mice given furan, a food contaminant and pollutant.
Commonly, Spirulina platensis supplementation at a dose
of 300mg / kgBW can significantly reduce MDA levels com-
pared to the control group (Yudiati et al., 2011). In line
with these studies, this study can prove that giving Spirulina
platensis extract can improve lipid peroxidation conditions
that occur in rats with given exposure to vehicle emission.
This can be seen from the finding of significant differences
in the mean MDA levels between the group given the 750mg
/ kgBW. Spirulina platensis extract and the control group
that was given vehicle emission smoke exposure. However,
there were no significant differences between the groups
given Spirulina platensis extract 500mg / kgBW, this is

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Fitry et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 85-89

probably due to the insufficient dose given to prevent lipid
peroxidation. In addition, oxidative stress was increased
levels of ROS because of the inflammation (Xu et al., 2013;
Cho et al., 2018).

Inflammation is a non-specific immune response to clear
pathological agents. The first response to inflammation
is the recruitment of neutrophils that kill foreign bodies.
Once recruited, neutrophils release cytokines responsible
for monocyte recruitment (Xu et al., 2013). In vitro data
suggest that the organic and transition metal constituents
(e.g., Fe, Cu, Ni, and Zn) contained in PM are capable of
producing ROS directly or as a result of its ability to acti-
vate neutrophils or other leucocytes (Delfino et al., 2011).
According to Steenhof et al. (2014), a positive association
between PM and neutrophil count after 2 hours of exposure
and monocytes after 18 hours of exposure in populations
exposed to PM in underground areas. Another hands by
(Al-Homaidan et al., 2016) showed that PM2.5 can signifi-
cantly increase the number of neutrophils, eosinophils and
lymphocytes in bronchoalveolar lavage fluid (BALF ) (Yan
et al., 2017).

This study shows that giving Spirulina platensis extract
can affect inflammatory conditions that occur in experimen-
tal animals. This can be seen from the finding of significant
differences in the mean absolute value of neutrophils and
lymphocytes in the group given Spirulina platensis extract
at doses of 500 and 750 mg / kgBW with the control group
given smoke exposure. Significant differences were also found
in the mean absolute values of eosinophils and monocytes
between the group given Spirulina platensis extract at a
dose of 750mg / kgBW and the control group, but there was
no significant difference between the group given Spirulina
platensis extract 500mg/kgBB and the control group.

4. CONCLUSIONS

Based on results of this study, it was found that there was
an effect of giving Spirulina platensis extract on serum MDA
levels and Wistar rats leukocyte counts exposed to vehicle
emission. The administration of Spirulina platensis extract
at a dose of 750mg/kgBW had an effect on serum MDA
levels and the absolute value of eosinophils and monocytes,
and doses of 500 and 750mg/kgBW on the absolute values
of neutrophils and lymphocytes.

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© 2020 The Authors. Page 89 of 89


	INTRODUCTION
	EXPERIMENTAL SECTION
	Spirulina Platensis extract
	Vehicle emission and exposure setting system
	Procedure of wistar mice exposure
	Statistical analysis using ANOVA

	RESULTS AND DISCUSSION
	Serum MDA levels
	Differential Leukocyte counts

	CONCLUSIONS