Title


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

Research Paper

Spirulina platensis Extract Reduces Serum TNF-α, Neutrophils, and Increases

Macrophage Count in Skin Incisional Mice Model

Riski Dwi Utami1, Tri Nur Kristina2*, Renni Yuniati3

1Department of Biomedicine, School of medicine, Medical Faculty of Diponegoro University, Semarang, Central Java, Indonesia
2Department of Microbiology, Medical Faculty of Diponegoro University, Semarang, Central Java, Indonesia
3Department of Dermatology-Venereology, Medical Faculty of Diponegoro University, Semarang, Central Java, Indonesia

*Corresponding author e-mail: renniyuniati@yahoo.com

Abstract
Spirulina sp. is an alga that has antioxidant and anti-inflammatory potential. Phycocyanin contained in Spirulina platensis can
reduce tumor necrosis factor-α levels, neutrophils, and M2 macrophages in inflammation. This research aims to investigate
the anti-inflammatory activity of S. platensis in a mice wound model. Thirty-two male Wistar rats were incised and divided
into 4 groups. The first therapeutic group (X1) received S. platensis extract at a dose of 500 mg/kgBW/day, and the
second therapeutic group received S. platensis extract at a dose of 750 mg/kgBW/day. The negative control group (C1)
received a saline solution and the positive control group (C2) received diclofenac sodium 20mg/kgBW/day. Serum TNF-α
levels examined by the Enzyme-Linked Immunosorbent Assay method. Neutrophils and M2 macrophages were calculated
by tissue biopsy and hematoxylin-eosin (HE) staining. Data analysis was performed with ANOVA test and LSD Post-Hoc
Test. The average levels of serum TNF-α on the 14-day were 394.50; 180.33; 2,980.33; and 607.42 pg/ml for X1, X2, C1
and C2, respectively. The average neutrophils number on the 14-day were 8.00; 6.83; 14.67; and 11.17 for X1, X2, C1 and
C2, respectively. The average macrophage number in the 14-day was 15.50; 19.17; 6.33; and 11.17 for X1, X2, C1 and
C2, respectively. We found significant differences between TNF-α levels, the number of neutrophils, and the number of
M2 macrophages. The administration of S. platensis extract at a dose of 750 mg/ kgBW/day reduces serum TNF-α levels,
neutrophil count, and increases M2 macrophages in skin incisional mice model.

Keywords
Spirulina platensis, anti-inflammatory, TNF-α, neutrophils, macrophages.

Received: 3 June 2020, Accepted: 12 June 2020

https://doi.org/10.26554/ijems.2020.4.2.34-38

1. INTRODUCTION

Skin is the largest organ of the body that works as a first-
line barrier to toxins, trauma, and pathogens. Any breach
on the skin surface disrupts the skin’s protective barrier
function and may result in the formation of scars when
the wound heals. There are several steps of wound healing,
such as hemostasis, inflammation, proliferation, and mat-
uration.(Rohl et al., 2015; Martin and Nunan, 2015) The
inflammatory phase in the wound healing process cleans
damaged cells, extracellular matrix and helps to inhibit the
growth of pathogenic organisms. This phase lasts about
5-7 days. Excessive inflammation phase can cause chronic
inflammation, inhibits re-epithelialization and increases the
occurrence of fibrosis.(Rohl et al., 2015).

The inflammatory phase of the wound in the skin denotes
the entry of mast cells, monocytes, neutrophils, and T cells
that originate from capillaries into the tissues. Neutrophils

and macrophages enter the wound area in the initial phase
of inflammation at different times. Neutrophils arrive at
the wound area approximately in the first 6-12 hours, and
they reach a peak one day after injury. (Rohl et al., 2015;
Gonzalez et al., 2016). Neutrophils migrate through the
process of diapedesis in capillary endothelial cells and ac-
tivated by proinflammatory cytokines such as interleukin
(IL)-1β, Tumor Necrosis Factor (TNF)-α and interferon
(IFN-γ) .(Karnen, 2012).

Macrophages enter the wound area in large numbers up
to 48 hours after injury and the number is stable on days
4-5; furthermore, they continue to decline until the 14-day.
Macrophages will differentiate into various kinds of cells
with different functions. At the initial phase of inflamma-
tion, macrophages will differentiate into proinflammatory M1
macrophages. M1 macrophages are classified as phagocytos-
ing bacteria, which can be produced TNF-α, IL-6, and IL-23.

https://doi.org/10.26554/ijems.2020.4.2.34-38


Utami et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 34-38

M1 Macrophages can be differentiated into M2 macrophages
after the phagocytic process of apoptosis in response to the
cytokines IL10 and IL4. M2 macrophages possess wound
repairing abilities and they release growth factors such as
TGF-β, Vascular Endothelial Growth Factor (VEGF), and
Platelet-Derived Growth Factor (PDGF)-β which recruit
endothelial (pro-angiogenic) cells and fibroblasts, promote
myofibroblast differentiation and secrete extracellular matrix
components.(Rohl et al., 2015).

Pharmacological therapies are useful to reduce symptoms
and to speed up the healing process of the wounds. One
of the therapeutic modalities that can be used is Phyto
therapeutics (Levine, 2017)., which has various potentials,
one of them is phytotherapeutic potential.(BPS, 2014; Nur,
2014; Triyono, 2013). Spirulina sp. is a type of cyanobacteria
that grows in water, freshwater, brackish water or salt-
water. Growth and nutrient content of Spirulina sp. are
influenced by factors such as light intensity, temperature,
growth media, pH and salinity. Phycocyanin plays a big role
in anti-inflammatory activities.(Nur, 2014; Wu et al., 2016).

Phycocyanin and β-carotene of Spirulina sp. play an
important role in the anti-inflammatory effects.(Wu et al.,
2016). The blue pigment phycocyanin in S. platensis works
as an anti-inflammatory agent, by acting as a selective
inhibitor of the cyclooxygenase-2 (COX-2) enzyme, inhibit-
ing the production of nitric oxide (NO) and prostaglandin
E2.(Wollina et al., 2018). Besides, phycocyanin is also
able to inhibit the expression of TNF-α, and IL-6.9 β-
carotene also has antioxidant and anti-inflammatory effects
by inhibiting the production of inducible nitric oxide syn-
thase (iNOS), COX-2, TNF-α, and IL-1β.(Sorg et al., 2017).
The previous study had concluded that S. platensis has an
anti-inflammatory effect on acute and chronic inflamma-
tion.(Quader et al., 2013). Besides that, S. platensis extract
significantly influences wound healing through granulation
tissue formation and neovascular enhancement in the wound
area.

Wound healing, especially chronic wounds, is not easy to
heal due to various disturbances and chronic inflammation
in the wound bed. Therefore, we aimed to evaluate the
potency of S. platensis extract as a novel wound-healing
enhancement agent. We hypothesized that S. platensis
could reduce inflammation that happened in the wound bed,
which can be measured by TNF-α, macrophage count, and
neutrophil count as inflammation indicators.

2. EXPERIMENTAL SECTION

2.1 Materials
The extraction of S. platensis requires 95% ethanol. The
materials needed for the maintenance of experimental an-
imals and incision procedures are AD-shaped pellet type
II, reverse osmosis drinking water, 70% alcohol and 10%
ketamine. For the examination of serum TNF-α levels, we
used the rat TNF-α ELISA kit (catalog no. E-EL-R0019:
Elab Science Biotechnology, Texas, USA). For examination

of neutrophil and macrophage counts, we used formalin as a
natural buffer, alcohol with multilevel concentration (10%,
70%, 80%, 90%, 95% and 100%), paraffin, xylol, and Mayer’s
hematoxylin stain.

2.2 Collection and Extraction of S. platensis
The S. platensis used in this study were obtained as S.platensis
powder with US FDA registration number of 15594742028
and CERES number of 50OGA1200043 (9241). The pow-
dered S.platensis microalgae were macerated in 95% ethanol
solution with 1:10 concentration (one part of S.platensis
powder macerated in 10 parts of 95% ethanol solution). The
maceration process done for five days in a glass container.
The glass container was stirred every day to make sure
the uniformity of the maceration process. After five days,
the solution was filtered through Whatman and was evap-
orated using a rotary evaporator machine at the ethanol
boiling point temperature until a thick extract was obtained.
This extract of S.platensis was used as the material in the
subsequent tests, further explained below.

2.3 Experiment subjects
Thirty-two male pure-breed laboratory-grade Wistar rats,
2-3 months obtained from a local laboratory rat breeder with
a lineage certificate of with a bodyweight of 100-200 grams,
were acclimatized for 7 days. The animal was fed with water
and rat pellets ad libitum at the cage. The animals were
randomly divided into 4 groups and were incised to make
a wound on the skin of their backs. The first therapeutic
group (X1) received S. platensis extract at a dose of 500 mg/
kgBW/ day (X1) orally, the second therapeutic group (X2)
received S. platensis extract at a dose of 750 mg/ kgBW/
day orally, the negative control group (C1) received saline
solution 0.9 % 0.5 mL and the positive control group (C2)
received diclofenac sodium 20mg/ kgBW/ day orally using
oral dosing (gavage) method. This study has been approved
by Health Research Ethics Committee, Faculty of Medicine,
Sultan Agung Islamic University with Ethical Exemption
certificate number 241/V/2019/Komisi Bioetik.

2.4 Procedure of incision
The mice were anesthetized using an intramuscular injection
of 60 mg/kgBW ketamine. The hair of the rats was shaved
about 1.5 cm to the right of the centerline on the back of the
head and cleaned with 70% alcohol. The skin was incised
using a 4 cm long scalpel parallel to the paravertebral lines
until reaching the muscle layer. The wound was sutured with
a simple interrupted suture technique at 0.5 cm intervals,
and the suture was removed on the 7th day.

2.5 Collection of blood and tissue samples
One milliliter of the blood sample was obtained from the
retroorbital blood vessels of the experimental rats on 7 and
14-days. We harvested the skin tissue nearest to the wound
incision site, put it into a paraffin block, sliced and was
processed for further histopathological analysis.

© 2020 The Authors. Page 35 of 38



Utami et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 34-38

Table 1. Serum TNF-α levels of experimental groups

Post-injury day Groups Mean ± SD (pg/mL) Min-Max (pg/mL) p Values

7th Day X1 377.83 ± 145.98 109.50-522.00 p < 0.001
X2 232.42 ± 40.54 164.50-277.00
C1 4634.08 ± 1956.02 2094.50-6707.00
C2 658.67 ± 35.38 612.00-719.50

14th day X1 394.50 ± 24.70 364.50-429.50 p < 0.001
X2 180.33 ± 39.10 134.50-229.50
C1 2980.33 ± 892.50 1707.00-4207.00
C2 607.42 ± 80.89 502.00-744.50

2.6 Measurement of serum TNF-α levels
Measurement of serum TNF-α levels from blood samples
was done using the Elabscience Rat TNF-α ELISA kit,
(Catalog number E-EL-R0019, Elabscience Biotechnology,
Houston, TX, USA). Blood was left to clot for 2 hours at
room temperature and centrifugated for 15 minutes. The
supernatant was collected and was stored in a clean container.
One hundred microliters of sample was put into the wells
and was incubated for 90 minutes at 37 C. One hundred
microliters of Biotinylated detection antibody were added to
the solution, and the plate was aspirated and washed three
times. Afterward, 100 µL of HRP conjugate working solution
and 90 µL of substrate reagent was added and incubated
according to the factory provided procedure. Finally, 50 µL
of stop solution was added and the plate was read using
microplate reader at 450nm.

2.7 Histopathology
Tissue samples were obtained through tissue biopsy on the
14th day. Subsequently, the histological preparations and
hematoxylin-eosin (H&E) staining were made. Histologi-
cal slides were analyzed by a pathologist by using a 40x
magnification binocular light microscope (Olympus CX23,
Shinjuku, Japan).

2.8 Statistical analysis
The statistical analysis was done using SPSS for Windows
version 21. We used One-Way ANOVA and Post Hoc LSD
Test for further analysis. The statistical significance used in
this study was set at p < 0.05.

3. RESULTS AND DISCUSSION

All of the mice used in this research were kept alive until the
end of the measurement period. No mice were dropped out
of the study. All of the mice were terminated after samples
were taken following ethical termination rules.

The mean baseline value of serum TNF-α levels on
the pre-wounded mice was 34.89 ± 22.47 pg/mL. One-way
ANOVA analysis showed significant differences (p < 0,001)
in serum TNF-α levels between study groups on both 7
and 14-days. On the 7th day, LSD post hoc tests showed

significant differences were found between the X1 group and
C1 group and between X2 group and the C1 and C2 groups.
There were also significant differences between C1 and C2
groups. We also found significant differences on the 14th
day between X1 group and X2, C1, and C2 group, between
X2 group and both control groups, and between C1 and C2.
(Table 1)

Table 2. The changes of the number of neutrophils per field
of view in experimental groups

Groups Mean ± SD Min-Max p Values

X1 8.00 ± 2.366 4-11 p < 0.001
X2 6.83 ± 1.722 4-9
C1 14.67 ± 1.862 12-17
C2 11.17 ± 2.483 8-15

The results from Table 2 showed that there was a signif-
icant difference between the negative control group and the
first therapeutic group, which received S. platensis extract
group with a dose of 500 mg/kg of body weight/day. More-
over, significant differences were also found between the
positive control group and both of the therapeutic groups.
Table 3 also shown the results of the changes in the number
of macrophages in experimental groups.

Table 3. The changes in the number of macrophages in
experimental groups

Groups Mean ± SD Min-Max p Values

X1 15.50 ± 2.429 12-19 p < 0.001
X2 19.17 ± 2.317 16-22
C1 6.33 ± 1.862 4-9
C2 11.17 ± 1.329 9-13

Analysis test showed that there was a significant in-
crease in the number of M2 macrophages in the both of
the therapeutic groups which received S. platensis extract
compared to the negative and positive control groups. A
significant difference was also found between the two ther-

© 2020 The Authors. Page 36 of 38



Utami et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 34-38

apeutic groups, where the second therapeutic group had a
significantly higher M2 macrophage compared to the first
therapeutic groups.

Analysis test showed that there was a significant in-
crease in the number of M2 macrophages in the both of
the therapeutic groups which received S. platensis extract
compared to the negative and positive control groups. A
significant difference was also found between the two ther-
apeutic groups, where the second therapeutic group had a
significantly higher M2 macrophage compared to the first
therapeutic groups.

In this study, we found a significant difference in TNF-α
levels between the experimental groups. Normal levels of
TNF-alpha in non-wounded rats were around 32 pg/mL,
and on wounded rats was around 124.6 pg/mL.(Rohl et al.,
2015; Martin and Nunan, 2015) These results are in accor-
dance with the previous study found that the administration
of S. platensis extract orally at a dose of 500mg/ kgBW/
day was able to suppress acute inflammation induced by
carrageenan and the administration duration of 7 days has
the potential to inhibit chronic inflammation which was
induced by granuloma. Another study also shows that the
administration of S. platensis could reduce TNF-α levels
under the inflammatory process condition (Wu et al., 2016).
Another study shows that the administration S. platensis
extract at a dose of 20 mg/kg of body weight and 30 mg/kg
of body weight in diabetic-induced mice decreases serum
TNF-α levels (Liu et al., 2016).

Cytokines play an important role in the inflammatory
process through the production of proinflammatory cy-
tokines such as TNF-α, IL-1β, and IL-6, which initiates
the immune system in the inflammatory process.(Liu et al.,
2016). TNF-α is mainly produced by macrophages, en-
dothelial cells, and mast cells. TNF-α increases adhesion
molecules in inflammatory cells (neutrophils, monocytes)
and endothelial cells.(Owen et al., 2013; Abbas et al., 2016).
Tumor Necrosis Factor-α is needed in the wound healing,
especially in the inflammatory phase, because the inflam-
matory process is a physiological process in wound healing.
However, if the inflammation in the wound healing process
occurs too long, the wound may become a chronic wound.
On the 7th day, TNF-α was still needed in the inflammatory
phase of the wound healing process. However, the increase
of TNF-α levels which continued for a prolonged period than
normal indicated an extension of the inflammatory phase.

Anti-inflammatory agents produced by S. platensis are
phycocyanin and β-carotene.(Wu et al., 2016) As an anti-
inflammatory agent, phycocyanin contained in S. platensis
works a selective inhibitor of the cyclooxygenase-2 (COX-2)
enzyme which is regulated during the inflammatory process
and has the ability to induce apoptosis in macrophages.
Other studies also concluded that phycocyanin was able to
inhibit the expression of iNOS, COX-2, TNF-α, and IL-6
while β-carotene was able to inhibit the production of iNOS,
COX-2, TNF-α, and IL-1β. (Wu et al., 2016; Sorg et al.,

2017).
In this study, we found that the mean TNF-α level of the

control group was negative on the 7th day compared to the
treatment group, which was given S. platensis extract. This
result shows that there was a possibility that the administra-
tion of S. platensis extract may disrupt the wound healing
process. This might happen because the average TNF-α
levels in the treatment group were much lower than the
negative control group. Therefore, the timing of S. platensis
administration is important to prevent the disruption of the
wound healing process.

The measurement of the number of neutrophils in wound
tissue was done to analyze how the development of the
inflammatory phase in the wound healing process. The pres-
ence of neutrophils at the wound inflammation area usually
peaks within one day after the initiation of tissue injury,
because the neutrophils are needed in the wound healing
process to phagocytose bacteria and to secrete proteinase
which works as an antimicrobial agent.(Rohl et al., 2015;
Gonzalez et al., 2016) In acute wounds, the inflammatory
phase lasts for less than 7 days. If the number of neutrophils
continues to increase or persist until the 14th day, the in-
flammatory phase might be prolonged and the wound might
become chronic wounds.(Rohl et al., 2015).

The results of this study indicate that the administra-
tion of S. platensis extract has the potential to reduce the
number of neutrophil inflammatory cells compared to the
control group. These results are consistent with a previous
study that found that the phycocyanin content possessed
by S. platensis has an anti-inflammatory effect. One of
the anti-inflammatory effects possessed by S. platensis ex-
tract is the inhibition of neutrophil migration to the area of
inflammation. (Liu et al., 2016).

Macrophages are involved in the inflammatory and prolif-
eration phase of the wound-healing phase. Macrophages en-
tered the area of injury in large numbers up to 48 hours after
injury and the number was stable on days 4-5. Furthermore,
they continued to decline until the 14th day. Macrophage
assessment in this study was conducted on the 14th day
of wound healing, which was purposely done to let the
macrophages differentiate into M2-type anti-inflammatory
macrophages. The therapeutic groups which received S.
platensis extract show a significant effect on increasing M2
macrophages compared to the control group. Therefore, the
administration of S. platensis extract helped the wound heal-
ing process by increasing the amount of M2 macrophages.

The results of this study differ from a previous study,
which might be attributed to the difference in data collection
time. Their previous research showed that the number of
macrophages in the group which received S. platensis extract
increased significantly on the 3rd day. However, it started to
decrease in the 7, 12 and 13-days. Therefore, an immunohis-
tochemical examination should be carried out to ascertain
the type of macrophage. Besides, It is necessary to examine
proinflammatory, other anti-inflammatory cytokines, and

© 2020 The Authors. Page 37 of 38



Utami et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 34-38

the growth factors to see the effectiveness of S. platensis in
the healing of proliferative and remodeling phases. There
are several limitations in this study that can be studied
in the future, such as the measurement of wound healing
markers, preferably should have been done together with
the microscopic (histopathological) analysis to accurately
measure the wound healing process and its correlation with
the wound healing markers.

4. CONCLUSIONS

S. platensis extract was found to have the capability to re-
duce serum TNF-α levels, reduce the number of neutrophils,
and significantly increase the amount of anti-inflammatory
M2 macrophages compared with the control groups. The ad-
ministration of S. platensis extracts at a dose of 750 mg/kg
body weight/day on the second experimental group has bet-
ter potential to reduce serum TNF-α levels and increases
the number of M2 macrophages when compared to the first
experimental group which was given with S. platensis extract
at a dose of 500 mg/kg body weight/ day.

5. ACKNOWLEDGMENT

This study was supported by Department of Biomedical,
Medical Faculty of Diponegoro University and supported by
Abdurrab University.

REFERENCES

Abbas, A., Lichtman, and P. Andrew (2016). Basic Im-
munology. 6th ed. Elsevier

BPS (2014). Badan Pusat Statistik. Statistik Sumber Daya
Laut dan Pesisir. Technical report

Gonzalez, A. d. O., T. Costa, Z. d. A. Andrade, and
A. Medrado (2016). Wound healing - A literature re-
view. An Bras Dermatol, 91(5); 614–20

Karnen, B. (2012). Imunologi Dasar. Jakarta: Badan
Penerbit Fakultas Kedokteran Universitas Indonesia; 132

Levine, J. (2017). The Effect of Oral Medication on Wound
Healing. Adv Skin Wound Care, 30(3); 137–42

Liu, Q., Y. Huang, R. Zhang, T. Cai, and Y. Cai (2016).
Review Article Medical application of Spirulina platensis
derived C-Phycocyanin. Evidence-Based Complementary
and Alternative Medicine; 1–14

Martin, P. and R. Nunan (2015). Cellular and molecular
mechanisms of repair in acute and chronic wound healing.
Br J Dermatol, 173(2); 370–378

Nur, M. (2014). Potensi Mikroalga sebagai Sumber Pangan
Fungsional di Indonesia. Eksergi, 9(2); 1–6

Owen, J., J. Punt, J. Stanford, Sharon, and K. Patricia
(2013). Immunology Kuby. 7th ed. New York: W.H
Freeman

Quader, S., S. Islam, A. Saifullah, and F. Majumder (2013).
The Parma Innovation-Journal In-vivo studies of the in-
flammatory effects of Spirulina platensis. Pharma Innov,
2013(4); 70–80

Rohl, J., A. Zaharia, M. Rudolph, M. Rz, R. Murray, and
J. Röhl (2015). The role of inflammation in cutaneous
repair. Inst Heal Biomed Innov, 23; 8–15

Sorg, H., D. Tilkorn, S. Hager, J. Hauser, and U. Mirastschi-
jski (2017). Skin Wound Healing: An Update on the
Current Knowledge and Concepts. Eur Surg Res, 58(1);
81–94

Triyono, K. (2013). Keanekaragaman hayati dalam menun-
jang ketahanan pangan. Innofarm J Inov Pertan, 11(1);
12–22

Wollina, U., C. Voicu, S. Gianfaldoni, T. Lotti, K. França,
and G. Tchernev (2018). Arthrospira Platensis – Potential
in dermatology and beyond antioxidant activity. Maced J
of Med Sci, 6(1); 176–80

Wu, Q., L. Liu, A. Miron, B. Kĺımová, D. Wan, and K. Kuča
(2016). The antioxidant, immunomodulatory, and anti-
inflammatory activities of Spirulina: an overview. Arch
Toxicol, 90(8); 1817–40

© 2020 The Authors. Page 38 of 38


	INTRODUCTION
	EXPERIMENTAL SECTION
	Materials
	Collection and Extraction of S. platensis
	Experiment subjects
	Procedure of incision
	Collection of blood and tissue samples
	Measurement of serum TNF- levels
	Histopathology
	Statistical analysis

	RESULTS AND DISCUSSION
	CONCLUSIONS
	ACKNOWLEDGMENT