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The 5th International Conference on Pharmaceutical Nanotechnology / Nanomedicine 2021 | 27
ANTIOXIDANT AND ANTI-DYSLIPIDEMIC ACTIVITY OF BROWN SEAWEED (SARGASSUM
POLYCYSTUM) EXTRACT IN RATS FEED WITH HIGH-FAT CONTENT
Original Article
SARAH ZAIDAN1*, SYAMSUDIN1,2, DIAH KARTIKA PRATAMI1, HANA AMARANI SASTRA1, FIORINTINA SUTIKNO1,
DESI NADYA AULENA1
1Faculty of Pharmacy, Universitas Pancasila, South Jakarta, 12640, Indonesia, 2Postgraduate Program, Faculty of Pharmacy, Universitas
Pancasila, South Jakarta, 12640, Indonesia
*Email: sarah.zaidan@univpancasila.ac.id
Received: 22 Dec 2021, Revised and Accepted: 15 Mar 2022
ABSTRACT
Objective: This research aimed to obtain the antioxidant and antidyslipidemic activity of brown seaweed (S. polycystum) extract in vivo.
Methods: Two tests, such as antioxidant and anti-dyslipidemia, were conducted on the sample animals. Also, the number of Wistar rats was divided
into 2 sections which consisted of 6 treatment groups, respectively. The test animals received high-fat feed for 35 d, after which there administered
brown seaweed extract for 14 d. An antioxidant test consisting of 6 treatment groups, such as normal, negative, positive control (vitamin E), doses of
50, 100, and 200 mg/kg BW, was conducted in the first group. Meanwhile, the anti-dyslipidemia test consisting of 6 treatment groups, including
normal, negative, positive (simvastatin), doses of 50, 100, and 200 mg/kg BW, was carried out in the second group. Hematological and statistical
analysis was measured and performed using a 300 micro lab photometer as well as ANOVA, respectively.
Results: The antioxidant test results showed that superoxide dismutase (SOD) activity obtained a percentage increase of 63.41%, 75.01%, and
177.11%, while the dyslipidemia test results showed that after the administration of brown seaweed (S. polycystum) extract at a dose of 200 mg/kg
BW, there was a significant difference between the negative controls, with p less than α (0.05), such as total cholesterol (0.000) and triglycerides
(0.000). The percentage decrease in total cholesterol levels in the dose group was 31.98%, 41.47%, and 49.45%, with triglyceride levels of 30.65%,
37.78%, and 47.96%, respectively.
Conclusion: Considering these results, it was concluded that brown seaweed extract has an antioxidant activity on SOD parameters as well as an
antidyslipidemic activity on total cholesterol and triglyceride parameters. Therefore, the most effective dose in improving the levels of total
cholesterol and triglycerides is 200 mg/kg BW.
Keywords: Brown seaweed (Sargassum polycystum), Antioxidant, SOD, High-fat feed, Dyslipidemia, Cholesterol
© 2022 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2022.v14s3.06 Journal homepage: https://innovareacademics.in/journals/index.php/ijap
INTRODUCTION
Degenerative diseases have recently increased due to morbidity and
mortality in developed countries. Furthermore, it is caused by high-
fat feed that raises cholesterol levels, resulting in cardiovascular
disorders such as atherosclerosis [1]. The high-fat feed also causes
Low-Density Lipoprotein (LDL), which is easily oxidized, produces
reactive oxygen species that leads to oxidative stress, and triggers an
increase in the lipid peroxidation process. The body produces
endogenous antioxidant compounds such as the enzyme superoxide
dismutase (SOD). However, this cannot control the oxidation in the
body without causing oxidative stress, which requires more
significant amounts of antioxidants. This is performed by providing
an external intake of exogenous antioxidants from natural and
synthetic sources into the body [2].
Dyslipidemia is a lipid metabolism disorder due to genetic and
environmental factors, which is indicated in the parameters of total
cholesterol, LDL cholesterol, and triglycerides [3]. Plasma lipid levels
increase due to high carbohydrate consumption, stored as fat.
Therefore, the consumption of high-fat foods and a high-
carbohydrate diet affects energy requirements and the formation of
triglycerides and plasma cholesterol, as well as reduces HDL
cholesterol in the body [4].
The decrease in plasma lipids helps in the treatment of dyslipidemia.
Meanwhile, brown seaweed can reduce lipid levels in the blood.
According to Akbarzadeh et al. (2018), brown seaweed (Sargassum
oligocystum) lowers triglyceride levels in Wistar rats [5]. Therefore,
an antidyslipidemic activity test is conducted on the total cholesterol
and triglyceride parameters of brown seaweed extract in rats fed
with a high-fat content feed. Furthermore, brown seaweed also acts
as a source of antioxidants. This plant has flavonoids, steroids,
triterpenoids, fucoidans, and components that act as a source of
antioxidants, as stated by Ganapathi and Lutfiyana [6].
This research aims to obtain the antioxidant and antidyslipidemic
activity of brown seaweed (S. polycystum) extract in vivo. During this
research, brown seaweed was developed into natural medicines
with antioxidant and antidyslipidemic activity.
MATERIALS AND METHODS
Materials
The materials used in this research included brown seaweed,
vitamin E, high-fat feed consisting of animal fat, vitamins, cellulose,
cholesterol, sucrose, cornflour, and casein, 20% EDTA, TEP, Aqudest,
20% TCA, 0.67% TBA, Carbonate buffer solution pH 10.2,
Epinephrine solution 0.01 M, Chloroform–Ethanol 96% with a ratio
of 3:5, H2O2 0.059 M, and Phosphate buffer 0.05 M pH 7.
Furthermore, test animals used were male and female Wistar rats.
Tools
The tools used in this research were rat cages with food and water,
rat scales, analytical scale, syringe, oral sonde, Eppendorf tube,
measuring cup, beaker, stirring rod, stove, pestle mortar,
micropipette, capillary tube, test tube, centrifugation devices, UV-Vis
spectrophotometers, refrigerators, and pH meters.
Plant determination
The brown seaweed (Sargassum polycistum) was determined by
Research Center for Oceonagraphy Indonesian Institute of Sciences,
Jakarta, Indonesia with No. B-2716/IPK.2/IF/X/2016.
Preparation of test animals
Male Wistar rats were acclimatized for 7 d. During adaptation, the
rats were fed and provided with water (ad libitum). After
adaptation, the test animals were divided into 2 sections consisting
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ISSN- 0975-7058 Vol 14, Special Issue 3, 2022
S. Zaidan et al.
Int J App Pharm, Vol 14, Special Issue 3, 2022, 27-30
The 5th International Conference on Pharmaceutical Nanotechnology / Nanomedicine 2021 | 28
of 6 groups, respectively. The test animals other than the normal
control group were administered high-fat feed for 35 d and then
received brown seaweed extract for 14 d. Two tests were carried
out, including antioxidant and anti-dyslipidemia tests [7].
Ethical approval
The pharmacodynamic test protocol on animals was ethically approved
by the Health Research Ethics Committee of the Jakarta Veterans
National Development University with No: 231/VI/2021/KEPK.
Animal treatment
A total of 24 healthy rats were divided into 6 groups, including
normal (rats fed with a standard feed), a negative control (rats fed
with a high-fat feed), test group I (brown seaweed extract at a dose
of 50 mg/kg BW), test group II (brown seaweed extract at a dose of
100 mg/kg BW), test group III (brown seaweed extract at a dose of
200 mg/kg BW), and positive control (vitamin E), which consisted of
4 rats, respectively.
Blood sample
Blood samples were taken on day 0, day 14, day 35 after providing
the high-fat feed. Blood was taken through the eyes of rats using±3
ml capillary tubes, it was collected in a test tube containing 20%
EDTA, after which it was left for a while, then centrifuged at 3000
rpm for 10 min. A clear portion of the blood (plasma) was used to
measure malondialdehyde levels.
Analysis of superoxide dismutase (SOD) activity
SOD activity was examined on red blood cells according to the
modified Misra and Fridovich method with modification [8]. A total
of 250 L red blood cell hemolysate was added to 400 L 96% (3:5) of
the chloroform-ethanol mixture. The mixture was mixed for 1 min,
then centrifuged at 3000 rpm for 10 min. The clear light-yellow
filtrate was collected, after which a 50 L distilled water, 2775 L
0.0518 M carbonate buffer pH 10.2, and 125 L 0.01 M epinephrine
solution were added. Afterward, it was mixed homogeneously and
placed in a cuvette. Furthermore, the absorption measurements
were performed after 1, 2, 3, and 4 min at a wavelength of 480 nm
and 30 °C. The same method was also used for distilled (empty)
water, with absorption readings taken after 1, 2, 3, and 4 min.
Antidyslipidemic activity test
The number of male Wistar rats was divided into 6 treatment
groups, including normal, negative (high-fat feed), positive
(simvastatin), and test groups with doses of 50, 100, and 200 mg/kg
BW. After being treated with high-fat feed and sample
administration, dyslipidemic rats were treated for 14 d. The lipid
levels were observed on days 0, 7, 14 after being given a high-fat diet
and 7, 14 d after being treated. In addition, the total cholesterol and
triglyceride levels were analyzed using the "CHOD PAP" and "GPO
PAP" methods, respectively [9].
In vivo toxicity test
In stage I, 40 male and female DDY rats weighing 20-35 g were
acclimatized (adapted) for approximately 7 d, then grouped
randomly. As a result, each sex's bodyweight was evenly spread
among four groups. Finally, in stage II, a total of 50 male and female
DDY rats weighing 20-35 g were acclimatized to experimental
animals for roughly 7 d before being randomly divided into 5 groups
for each sex [10].
Stage 1 was divided into 4 groups consisting of 5 experimental
animals, administered doses of 10, 50, 250, and 1250 mg/Kg BW.
Therefore, the probe dose was increased by using a more significant
concentration of the preparation when no death was recorded after
24 h.
Stage 2 was divided into 5 groups, each consisting of 5 experimental
animals, which received doses: 1000; 2000; 3000; 4000, and 5000
mg/kg BW. First, observations were made after 24 h, counting the
number of experimental animals that died in each group and sex.
Afterward, the LD50 value was calculated.
RESULTS
Plant determination result
The brown seaweed was identified by Research Center for
Oceonagraphy Indonesian Institute of Sciences, Jakarta, Indonesia as
macroalgae Sargassum polycystum with this classification:
Divisio: Ochraphyta
Class: Phaeophyceae
Ordo: Fucales
Famili: Sargassaceae
Genus: Sargassum
Species: Sargassum polycystum
Measurement of superoxide dismutase (SOD) levels
The results of the antioxidant activity test in sample animals were
used to determine the levels of superoxide dismutase (SOD), as
shown in fig. 1. The average SOD activity in the negative control
group was 20.84 U/ml, which was lower than the average SOD
activity in the normal, positive, and treatment group. Meanwhile, the
average result of SOD activity in the 50 mg/kg BW treatment group
was 29.17 U/ml, with an increase of 63.41%, which was lower than
the positive control group of 108.3 U/ml. The average result of SOD
activity in the treatment group at a dose of 100 mg/kg BW 50 U/ml
with an increase of 75.01% percent was lower than the positive
control group of 108.3 U/ml. The dose of 200 mg/kg BW of brown
seaweed extract with an increase of 177.11% increases the activity
of SOD in the body as well as the positive control group.
Fig. 1: The mean value of SOD before and after administration of brown seaweed extract, Data was given in mean+SD, n=5
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Measurement of total cholesterol levels
The results of antidyslipidemic testing activity in experimental
animals were measured for cholesterol levels, the results of which
are shown in fig. 2.
Measurement of triglyceride levels
The results of testing for antidyslipidemic activity in experimental
animals were measured for triglyceride levels, the results of which
can be seen in fig. 3.
Fig. 2: Total cholesterol concentration, data was given in mean+SD, n=4
Fig. 3: Triglyceride concentration, data was given in mean+SD, n=4
In vivo toxicity test
The 24h observations of stages I and II showed no death. Based on
observations, the LD50 results of the preparation were 0.64 ml/20g
BW or 320 mg/20g BW (16 g/Kg BW). LD50 70% ethanol extract of
brown seaweed>15g/kg BW. Therefore, this showed that brown
seaweed's 70% ethanolic extract was practically non-toxic.
DISCUSSION
Antioxidant activity test of brown seaweed extract against SOD
activity in rats
The measurement result of SOD activity for each treatment group
are presented in fig. 2. The average SOD activity in the negative
control group was 20.84 U/ml lower than the average SOD activity
in the normal, positive, and treatment groups. Superoxide dismutase
is an antioxidant produced by the body that neutralizes free radicals
and protects cells from damage. Furthermore, it catalyzes the
transformation of superoxide to hydrogen peroxide (H2O2) [11].
These results are supported by Yang RL's research, which stated
that human subjects with high fat feeds experience oxidative stress
conditions characterized by an increase in free radicals and a
decrease in the status of antioxidant enzyme capacity [12]. The
average increase in SOD activity in the treatment group was 50
mg/kg BW of 29.17 U/ml, which was lower than the positive control
group of 108.3 U/ml. Therefore, the brown seaweed extract was
unable to boost SOD activity and vitamin E, which was utilized as a
positive control.
The average results of SOD activity from the treatment group at a
dose of 100 mg/kg BW 50 U/ml showed an increase of 75.01%
percent, which is lower than the positive control group 108.3 U/ml.
Therefore, the brown seaweed extract did not increase the SOD
activity and vitamin E, which was a positive control.
The dose of 200 mg/kg BW of brown seaweed extract with a
percentage rise of 177.11% increases the activity of SOD in the body
as well as the positive control group. These results showed that a
high dose of administered seaweed extract leads to an increase in
SOD activity. Furthermore, Widiyantoro et al. (2010). Stated that the
administration of vitamin E as an exogenous antioxidant enhances
the maintenance of SOD activity compared to the group that was not
given vitamin E [13]. Also, another research discovered that
flavonoids help maintain SOD activity in the body; hence, the
antioxidant status is maintained [14].
Total cholesterol in antidyslipidemic activity testing of brown
seaweed extract in rats
High-fat feeding for 14 d showed an increase in blood cholesterol
levels. Oral administration for 14 d was conducted, and the lipid
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levels in the administration of brown seaweed extract decreased, as
shown in fig. 4. This was caused by the pharmacological activity of
fucoxanthin, which happens to be a phenolic compound found in
brown seaweed. Fucoxanthin is a pigment from the carotenoid
group found in brown seaweed [15]. Furthermore, it affects the
hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) and Acyl-CoA
cholesterol acyltransferase (ACAT), which is one of the actions of
synthetic dyslipidemia drugs, as well as a sterol-binding factor-
binding transcription factor (SREBP-1): SREBP-1a. The activity is
controlled by the sterol levels in cells, which regulates the genes
associated with lipid and cholesterol production [16]. The most
significant percentage decrease in total cholesterol levels at the III
dose of 200 mg/kg BW was 49.45%. Statistical analysis showed a
significant difference in doses I, II, and III with negative control
(p>0.05). Therefore, the decrease in lipid levels at dose III had no
significant difference in the positive group, while at doses I and II,
there was a significant difference in the positive control group,
implying that the effect of lowering total cholesterol at dose III is the
same as that of the positive control.
Triglyceride levels in antidyslipidemic activity testing of brown
seaweed extract in rats
The observation of triglyceride levels was performed 5 and 3 times
after administering high-fat feed and 2 times after administering an
extract of brown seaweed (Sargassum polycystum). The decrease in
triglyceride levels is shown in fig. 5. The presence of a secondary
component, such as fucoxanthin, which considerably reduces
plasmatic and hepatic triglyceride concentrations [16], causes a
reduction in triglyceride levels discovered in brown seaweed
(Sargassum polycystin). The highest percentage decrease in
triglyceride levels at the dose III of 200 mg/kg BW was 47.85%.
Triglyceride levels at doses I, II, and III were significantly different
(p>0.05) from the negative and positive controls, implying that
brown seaweed extract reduces the plasma triglyceride levels, but
this has not been able to match the positive control.
In vivo toxicity test
This aims to determine the toxicity of brown seaweed extract in
both sexes using 5 male and female rats each. Furthermore, the
female rat was first subjected to an acclimatization process for 7 d
before receiving treatment to adapt to their new environment and
avoid false-positive results.
The 5 male and female rats, each weighing between 20-25 grams,
were given a suspension at a dose of 0.64 ml/20g BW which was
derived from the stock suspension extract solution in 0.5% CMC Na
with a concentration of 500 mg/ml. Subsequently, from 24 h
monitoring, no one died.
Based on the above observations, the LD50 results of the
preparation are 0.64 ml/20g BW or 320 mg/20g BW (16 g/kg BW).
Hence, the LD50 of 70% brown seaweed ethanol extract is>15g/kg
BW, implying that brown seaweed's 70% ethanolic extract was
practically non-toxic [7].
CONCLUSION
The extract of brown seaweed (Sargassum polycystum) has an
antioxidant activity on SOD parameters and antidyslipidemic
activity on total cholesterol and triglyceride parameters. Therefore,
a 200 mg/kg BW dose is the most effective in improving total
cholesterol and triglyceride levels.
ACKNOWLEDGEMENT
The author is grateful to the Faculty of Pharmacy, Pancasila
University, which has provided Faculty Incentive Grants in carrying
out this research. Research reported in this publication was
supported by the Fogarty International Center of the National
Institutes of Health under Award Number D43TW009672. The
content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of
Health.
FUNDING
The financial support was supported by the Faculty of Pharmacy,
Universitas Pancasila
AUTHORS CONTRIBUTIONS
All the authors have contributed equally.
CONFLICTS OF INTERESTS
The author has no conflicts of interest to declare.
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