Effect of Tapioca Flour Therapy on Malondialdehyde Levels and Features of Stomach

Histopathology in Rats (Rattus norvegicus) Model IBD Induction of Indomethacin

1
Melita Nono Lebang, 2Chanif Mahdi, 3Fajar Shodiq Permata
1,3
Faculty of Veterinary Medicine, Universitas Brawijaya, Puncak Dieng Exclusive ,
Kalisongo, Kec. Dau , Kab. Malang 65151
2
Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Jalan Veteran, Malang
65145
Email: melitanlebang@gmail.com, chanifmahdi@gmail.com,
drhfajarshodiqpermata@ub.ac.id

ABSTRACT
IBD is an inflammatory condition in the digestive tract that can be caused by
side effects of non steroidal anti-inflammatory drugs, such as indomethacin. Resistant starch
in tapioca flour acts as a mucoprotectant in the gastric mucosa. This study aims to determine
the effect of tapioca flour therapy on decreasing levels of MDA Malondialdehyde and
histopathological features of rats (Rattus novergicus) IBD indometacin induction model. This
research is experimental by using a completely randomized design (CRD). Experimental
animals use male rats (Rattus norvegicus) wistar strains aged 8-12 weeks weighing 150-200
grams. Rats were divided into 5 treatment groups; the control group, the IBD group, and the
therapeutic group were induced by indomethacin and tapioca flour therapy with a dose of 0.9
mg / kg body weight, 1.8 mg / kg body weight, and 3.6 mg / kg body weight respectively.
Gastric MDA levels were analyzed by oneway ANOVA and gastric histopathology was
analyzed descriptively. The results of data analysis showed that there were no significant
differences between the treatment groups for the reduction in MDA levels (P> 0.05). The
conclusion of this study was administration of tapioca flour therapy can not reduce MDA
levels, although there was a reduction trend and also in the improvement of gastric tissue in
rat model of Inflammatory Bowel Disease.

Keywoards : Inflammatory Bowel Disease, Indomethacin, mucoprotector, Tapioca Flour,

MDA and Gastric Histopathology.

1. INTRODUCTION
Inflamatory Bowel Disease (IBD)
is a chronic inflammatory condition in the
digestive tract. This disease is generally
caused by a distorted immune response
resulting in failure of regulation of the
immune system and stimulation of
microbial in the digestive tract. The cause
of the pathogenesis of IBD is still unclear
to date, but it involves complex
interactions between genetic factors and
environmental factors (Yi-Zhen and Yang,
2014).
A common symptom in animals
with IBD is continuous weight loss and
diarrhea. IBD can also be caused by side
effects of using non-steroidal anti-
inflammatory drugs (NSAIDs), such as
indomethacin. NSAID's drugs such as
indomethacin will inhibit the COX-1 and
COX-2 enzymes which play a role in the
formation of gastric prostaglandins so that
the defense system of the gastric mucosa
weakens (Sparkes, 2010).
Damage to the digestive tract due to
indomethacin will induce the release of
inflammatory cells such as neutrophils,
monocytes, macrophages and cytokines
that will trigger gastric damage.
Inflammatory cells will produce Reactive
Oxygen Species (ROS) which can produce
free radicals and proteolytic enzymes. Free
radicals cause lipid peroxidation. Lipid
peroxidation is the oxidation process of
long-chain unsaturated fats
(Polyunsaturated fatty acids or PUFA) on
cell membranes that produce aldehyde
products such as Malondialdehyde (MDA).
Malondialdehyde and proteolytic enzymes
produced by ROS have toxic properties to
gastric cells that can cause gastric cell
death and cause gastric cell histology
changes (Khennouf et.al., 2010; Segal,
2005).
Indonesia is a tropical country that is
rich in biodiversity, including food crops,
one of which is cassava (Mannihot
esculenta crantz). Cassava plants can be
processed into various types of food, one
of which is processed into starch or tapioca
flour. Tapioca is pure starch obtained from
cassava milling extraction (Indrianti et al,
2013). Tapioca flour is a food ingredient
which if modified to its starch will add
functional value. One of the modified
cassava flour products is resistant starch.
Starch can act as an energy source (ATP)
in the stomach to reduce inflammation and
stop the tissue necrosis by increasing
COX-1 production to aggregate platelets
for blood clotting and stop bleeding.
Resistant starch in tapioca flour will act as
a mucoprotectant to increase the mucosal
defense mechanism by stimulating
prostaglandin production by forming a
cytoprotective layer of the gastric mucosa.
Besides tapioca flour also contains
bioactive compounds such as phenolic
compounds which act as antioxidants to
reduce levels of MDA in the stomach
under conditions of Inflammatory bowel
disease.
The white rat (Rattus novergicus)
used in this study was divided into three
major research groups namely the negative
control group, the IBD group, and the
tapioca flour therapy group. The average
normal level of MDA in white rats (Rattus
novergicus), which is 1.599 µg / mL
(Agnes et al., 2013).
2. RESEARCH METHODS
2.1 Equipment
The equipment used in this study
included, mouse cages, rat drinking
containers, 1 cc syringes, Erlenmeyer,
sonde tools, glass objects, glass covers,
incubators, refrigerators, dissecting sets,
surgical boards, glove, water baths, feed
containers , Erlenmeyer tubes, object glass,
glass cover, microtome, light microscope
(Olympus BX51), digital camera, drop
pipette, vortex, centrifuge
(Thermoscientific Sorvall Biofud Primo R
Centrifuge), appendorf micropipette size
10-100 µl, UV-VIS spectrophotometer ,
rubber bulb, knives, scissors, bunsen,
organ pots, filter paper, autoclaves, timers,
incubators, refrigerators, microtome
machines, microtome knives, glass objects,
glass covers, and light microscopes.
2.2 Material
Materials used in this study include
white rats ( Rattus norvegicus ) male
Wistar strain, pellet feed, drinking water,
disinfectant , goat milk, brown rice bran
flour, starter, aluminumfoil , cotton, pricic
acid, formalin 10%, physiological NaCl 0 ,
9%, 1% acid alcohol, 70%, 80%, and 96%
alcohol, xylol, paraffin and Hematoxylin-
Eosin dyes.
2.3 Preparation of Animal Model
This study used 20 male white rats
(Rattus norvegicus) strain of Wistar, aged
8-12 weeks with a body weight of 150-200
grams. The use of experimental animals
has received ethical approval with No:
1065-KEP-UB. The mouse was divided
into 5 treatment groups and acclimatized
for 7 days by feeding and drinking ad-
libitum.
2.4 Procedures for Making IBD Animal
Models
Indomethacin dose used is 15 mg /
kg body weight (Aulani'am, 2012). The
average weight of mice used is ± 200
grams. Indomethacin was dissolved with
corn oil and given 0.27 mL / rat orally
(Bures et al., 2011).
2.5 Making and Giving Therapy of
Tapioca Flour
Tapioca flour used is obtained from
traditional markets in Malang, tapioca
flour therapy preparations are made by
providing 0.07 grams of tapioca flour, then
100 ml of stillet water is provided into a
glass baker and then mixed. Therapy is
given with gastric round, given for 14 days
as much as two times in one day. The
therapeutic doses of each treatment group
were 0.9 mg / 200g BW, 1.8 mg / 200g
BW, and 3.6 mg / 200g BW.
2.6 Euthanasia and Gastric Organ
uptake
Euthanation of white rats (Rattus
norvegicus) was done on day 15. The
animals were deuthanated by dislocating
the neck and then the rats were placed on a
tray for surgery with the ventral position
above. Next opened the abdomen, then
taken the stomach and wrapped using
aluminum foil washed with physiological
NaCl, then stored in formalin 10%, storing
organs in formalin to prevent changes in
components in the organs.
2.7 Measurement of Gastric
Malondialdehyde (MDA) Levels by
Thiobarbituric Acid (TBA) Test
Analysis of MDA done by grind the gastric
organ with a weight of 200 grams in a cold
mortar until smooth. Then 500 μL NaCl
0.9% was added and homogenated.
Homogenates are taken and transferred to
the ependorf tube. Next, centrifugation was
carried out at 8000 rpm for 20 minutes and
supernatant was taken. 100 μL supernatant
was put into a test tube, plus 550 μL
distilled water, 100 μL TCA, 100 μL HCl 1
N, 100 μL NaThio 1% and homogenized
again. After that, centrifuged 500 rpm for
10 minutes, heated in a water bath 100 ⁰ C
for 30 minutes. The sample is then
absorbed using a spectrophotometer at the
maximum wave length (max λ = 532 nm) .
2.8 Manufacture and observation of
gastric histopathology preparations
Preparation of gastric
histopathology preparations consists of
several stages, namely fixation of a gastric
sample into a 4% paraformaldehyde
sample pot for 24 hours, then a gastric
organ sample is trimmed 1 x 1 cm into a
tissue cassette. The tissue in the tissue
cassette is put into distilled water for 1
hour, then dehydrated using 70%, 80%,
90%, 95% alcohol, and absolute ethanol I,
II, III for 1 hour respectively. After that the
tissue in the tissue cassette is put into the
xylol I solution for 1 hour, II, and III for 30
minutes each. Then the sample in the tissue
cassette is put into liquid paraffin I, II, III
at a temperature of 56℃ for 2 hours, tissue
cassette of paraffin III is placed on an
electric stove and the gastric tissue is
removed from the tissue cassette. Liquid
paraffin IV is poured into paraffin molds,
then gastric tissue is planted in paraffin
molds and covered with a tissue cassette to
form paraffin blocks. Paraffin block
containing gastric tissue is mounted on the
microtome device. The paraffin block is
cut to a thickness of 5 microns. The piece
is placed on the glass object. The pieces
are placed on a hot plate 38-40℃ until dry
and then stored at 37℃ and ready to be
colored. The coloring process uses
hematoxylin and eosin dyes. Begin with a
hematoxylin solution for 10 minutes, then
wash with running water for 15 minutes,
and rinse with distilled water. After that
the preparation was stained with Eosin for
five minutes and washed with running
water for 10 minutes, after which it was
observed under a light microscope.
2.9 Data Analysis
Data analysis was carried out
quantitatively and qualitatively.
Quantitative data were used that checks the
levels of MDA of the gastric using
ANOVA test with a level of α = 0.05 and
in continuation with the test of Tukey . The
qualitative data used is the
histopathological description of the gastric
which will be analyzed and presented
descriptively.
3. RESULT AND DISCUSSION
3.1 The Effect of Tapioca Flour Therapy
on Normal Malondialdehyde (MDA)
Level of Stomach White Rat (Rattus
novergicus) IBD Model by Indometacin
Induction
MDA levels were analyzed with a
variety of ANOVA and further analysis
was done with the Tukey test (p> 0.05)
that the administration of tapioca flour
therapy had no significant effect (p> 0.05)
on the level of gastric MDA levels in
animal models of hypercholesterolemia. be seen in Table 3.1 and the results of the
The results of measuring MDA levels can tukey test can be seen in Table 3.2.

Table 3.1 ANOVA (Analysis of Varience) MDA Levels

S.V dfx SS MSxxx Fxxx Cale F5%
Treatment 4 10177.520 2544.380 0.794 3,06
Error 15 48063.574 3204.238

Total 19 58241.095

Table 3.2 Table Average MDA Levels in White Rats ( Rattus novergicus ) of All Groups
MDA Levels
Average mg / dL ± Improvement to Improvement to
Treatment Group
SD (K-) (K+)

Negative Control (K +) 284.67±58.55 - -

Positive Control (K +) 332.72±101.48
Rat Treatment (P1) 296.33±30.97
Rat Treatment (P2) 282.16±40.33
Rat Treatment (P3) 265.22±16.74
Note: The results of the average levels are not significantly different as indicated by the f
count <ftabel (p> 0.05) so that between treatments showed the same
notation.
Based on Table 3.1 shows that the
group with the highest average MDA level,
namely the IBD (K +) group of 332.72 ±
101.48 ng / mL. In the IBD group it
increased by 0.17% when compared to the
negative control group (K-) which had an
average of 284.67 ± 58.55 ng / mL.
Whereas the P1 group had an average of
296.33 ± 30.97 ng / mL and an increase of
0.04%. For the P2 group had an average of
282.16 ± 40.33 ng / mL with a percentage
decrease of 0.01%. Meanwhile, the P3
group had an average of 265.22 ± 16.74 ng
/ mL with a percentage decrease of 0.07%.
MDA levels were analyzed using the One
Way ANOVA statistical variance, which
was calculated at 0.794 which is smaller
than ftabel, which is 3.06. These results
prove that the average MDA level between
treatments did not differ significantly (p>
0.05).
The decrease in MDA levels which
was not significantly different in this study
proved that the three volumes of therapy
given for 14 days twice a day showed the
same results. Malondialdehyde (MDA) is
the final product of membrane lipid
0.17%
0.04% -
- 0.01%
0.07%
peroxidation by ROS, the reaction between
free radicals (hydroxy radicals) with Poly
Unsaturated Fatty Acid (PUFA) so that
measurement of MDA levels can indirectly
indicate the presence of free radicals.
Malondialdehyde is a free radical marker
in the body. Increased free radicals will
also cause an increase in MDA levels in
the body (Zaetun et al, 2018; Aulanni'am et
al, 2011). So we need exogenous
antioxidant compounds that are expected to
be able to neutralize excess free radicals in
the body.
The decrease in MDA levels in the treated
group of rats showed that the phenol
compound in tapioca flour as an exogenous
antioxidant had worked to reduce MDA
levels even though the level reduction was
not significant. Antioxidants are
compounds that can absorb or neutralize
free radicals. In general, phenol
compounds as antioxidants have an active
group - OH and a double bond> C = C
<which functions as a catcher and inhibitor
of free radical reactions, these groups can
provide one hydrogen molecule so that the
ROS becomes stable and forms new free
radicals that are less reactive. In the
condition of free radicals will damage cell
membranes, the radicals that play a role are
hydroxyl radicals (ᵒO2), so antioxidants
will work by changing H2O2 to H2O and
ᵒO2, so that the release of hydroxyl
radicals can be reduced (Parwata, 2015).
Based on the results of this study,
of the three therapeutic preparations given,
showed the results of an improvement in
the form of decreased MDA levels from
groups of mice given therapeutic dosages
with a dose of 1.8 ml / 200g BW (P.2) and
3.6 ml / 200g BW (P.3), and from the three
given therapeutic preparations, 3.6 ml /
200g BW (P.3) therapeutic preparations
are effective enough to reduce MDA levels
of rats in IBD conditions with the highest
levels of decreases of 0.07% although in
each treatment group did not show any
significant difference, meanwhile in the
group of mice P.1 with a dose of 0.9 mg /
200g BW did not show any improvement
in the form of decreased MDA levels and
an increase in MDA levels of 0.04%.
Figure 3.1 Histopathology of White Mouse Gastric with a Magnification of 10x & 40x
Note : epitel normal mukosa lambung;
Infiltrasi sel radang;
Histological picture of gastric mice
with HE staining (Figure 3.1) in the
3.2 The Effect of Tapioca Flour Therapy
on Histopathology of Gastric White Rats
(Rattus novergicus) Model IBD by
Indometacin Induction
Histopathological observation of
IBD rat model was carried out with the aid
of a microscope with magnification of 10x
and 40x (Figure 3.1) in all treatment
groups namely the control group, IBD
group, tapioca flour therapy group 0.9 mg /
200g BW, tapioca flour therapy group 1.8
mg / 200g BW and tapioca flour therapy
group at a dose of 3.6 mg / 200g BW.
Observations were made between
treatment groups (Figure 3.1) and analyzed
descriptively. Stomach is a digestive organ
that functions to store and process food
before it is transmitted to the duodenum. In
IBD conditions, the function of the
stomach will be disrupted so that the
process of food transport is disrupted as
well.
nekrosis sel; ulserasi;
erosi epitel; hemoragi.
healthy rat group (K-) shows the state of
gastric mucosal epithelium which is
composed of columnar simplex epithelium
in intact condition. Histological
observations can be used as a reference for
changes in the gastric mucosa tissue of
other treatment groups because in the
control group (K-) this is a group without
treatment. In the control group (K +)
histopathological damage in the form of
ulceration of the gastric mucosal
epithelium and found necrosis and
infiltration of inflammatory cells. The
results of observations from indomethacin-
induced groups indicate inflammation that
is marked by changes that occur, where
this parameter data can be used as a
reference supporting the measurement
results of increased MDA levels. Damage
that occurs in gastric epithelial tissue
Inflammatory Bowel Disease model
(Figure 3.1) is caused by indomethacin
induction of 15 mg / kg body weight.
Indomethacin induction in a short period of
time causes necrosis of gastric mucosal
cells, begins to lose the integrity of the cell
membrane, then will result in the
emergence of necrotic cells which will
activate neutrophil cells and macrophages
and cause an inflammatory process
(Tamisato et.al., 2001 ). The inflammatory
state of the gastric mucosal tissue will
affect the increase in proinflammatory
cytokines that cause neutrophil-endothelial
activation. The attachment of neutrophils is
related to the pathogenesis of gastric
mucosal damage through two main
mechanisms, namely occlus; causing
decreased gastric and ischemic blood flow
to cells and increased release of free
radical molecules; ROS (Reactive Oxide
Species). These free radicals react with
unsaturated fatty acids in the stomach
mucous membrane and cause fat
peroxidation and tissue damage to the
stomach (Amrulloh and Utami, 2016).
All treatment groups (P1, P2, and
P3) showed improvement in gastric
mucosa damage compared to the positive
control group (K +) although the results
were not significant. Tissue repair that
occurs due to starch as an energy source
will increase the formation of ATP and
increase COX-1 as a platelet for blood
clotting, besides that resistant starch
produced by tapioca flour can act as a
mucoprotectant. Resistant starch will
initiate prostaglandin production so that it
will increase the formation of mucus in the
gastric mucosa. This will help protect the
mucosa from damage due to gastric acid
secretion and suppress the invasion of
pathogenic bacteria which can play a role
in making inflammation worse; so
inflammation can decrease and oxidative
stress decreases. This situation will reduce
the production of free radicals and tissue
damage will be repaired.
Tapioca starch therapy in all
treatment groups (P1, P2, P3) showed a
slight improvement in gastric mucosal
damage compared to the K + group
although the changes that occurred were
not significant. This shows that the
inflammatory state still occurs in each
treatment group and is synchronous with
the results of MDA levels that are not
significant which indicates MDA levels are
still high. MDA levels are measured to
measure excess free radicals in the body
(Parwata, 2015), where the cause of gastric
tissue damage can be caused by the
production of excess free radicals
(Ancoferiawan et al, 2015). These results
indicate that resistant starch contained in
tapioca flour has not been quite effective in
its role as mucoprotectant in gastric
mucosa damaged by IBD conditions.
4. CONCLUSION
1) The administration of tapioca
starch therapy has no significant
effect on reducing MDA levels and
changes in gastric tissue of the
animal model Inflammatory Bowel
Disease.
2) The administration of tapioca flour
therapy group giving a dose of 1.8
mg / kgBB and 3.6 mg / kgBB
showed a decrease in MDA levels
in animal models of Inflammatory
Bowel Disease.
5. SUGGESTIOM
1) Further research needs to be done
on the use of improved tapioca
flour doses to see its effect on
reducing MDA levels and changes
 in the gastric tissue IBD induction
model.
2) In subsequent studies, testing can
be done by combining tapioca flour
with additional antioxidant
compounds to see its effect on
decreasing MDA levels and
changes in the gastric tissue image
of the IBD induction model.
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