Effects of Administering Breadfruit Leaf Extract (Artocarpus altilis) towards SOD Activity

and Ileum Histopathology Rat Animal Model Inflammatory Bowel Disease Indomethacin
Induction

Nathania Aryani1 Chanif Mahdi2 Analis Wisnu Wardhana1

1
Veterinary Medicine Faculty Brawijaya University
2
Mathematic and Science Faculty Brawijaya University
1
Puncak Dieng Eksklusif, Kalisongo, Kec. Dau, Kab. Malang 65151
2
Jalan Veteran, Ketawanggede, Kec. Lowokwaru, Kota Malang 65145
Email: nathania0301@gmail.com

ABSTRACT
Inflammatory bowel disease is a disease associated with inflammation of the digestive
tract and can cause complications such as obstruction, perforation, and intestinal carcinoma. IBD
can be aggravated by the presence of oxidative stress from NSAID induction, especially
indomethacin. The presence of tissue damage can be seen from decreased levels of Superoxide
Dismutase (SOD) and changes in the ileal mucosa. Breadfruit leaves (Artocarpus altilis) contain
flavonoids, tannins triterpenoid, saponin, alcaloid, and polyphenols which can be used as anti-
inflammatory, antioxidant, and antidiarrheal. This study is aimed to examine the interaction of
leaf extracts against Superoxide Dismutase (SOD) activity and ileal histopathology in rats. This
study was divided into 5 groups of rats consisting of group A (negative control), group B
(positive control), group T1 (IBD rats with therapy dose 100 mg / kgBB), group T2 (IBD rats
with therapeutic doses of 200 mg / kgBB), and group T3 (IBD rats with therapy dose of 400 mg /
kgBB). After 14 days, the rats ileum was taken and then analyzed. The enzyme activity of
Superoxide Dismutase (SOD) was obtained using the spectrophotometric method. The
qualitative histopathological description of the ileum was updated using a microscope with 400x
magnification. Data analysis using One Way ANOVA (Variant Analysis) with a confidence level
of 95% and agreed with the BNJ test. The results showed that administration of breadfruit leaf
extract was able to increase SOD activity very significantly (P <0.01) to reach an average of
38.38 ± 3.47 with an increase of 69.9%. Histopathological features can cause inflammation
characterized by erosion, edema, and inflammatory cell infiltration. Giving breadfruit leaf extract
can overcome inflammation in the ileum.

Keywords : IBD, SOD, Artocarpus altilis, histopatology

PREFACE
Inflammatory Bowel Disease consists of (UC), which are diseases associated with
Chron's disease (CD) and Ulcerative colitis inflammation in the gastrointestinal tract and
can cause complications such as obstruction,
perforation, and intestinal carcinoma.
Ulcerative colitis is limited to the colon,
while Chron's disease covers the entire
digestive tract (Danastri, 2013). IBD can
occur simultaneously in different parts of the
digestion, the incidence of IBD can be
intermittent or prolonged (Mansfield, 2011).
Non Steroidal Anti-Inflammatory Drug
(NSAID) is a drug for various inflammatory
conditions, especially in cases
osteoarthritis or rheumatoid arthritis.
Indomethacin causes an increase in COX 2,
the gastrointestinal system becomes
obstructed due to barriers to COX 1
(Fokunang, 2018). In experimental animal
models, indomethacin-induced
gastrointestinal shows acute morphological
changes, such as smooth muscle contraction,
microvascular damage, and changes in
permeability. Provision of indomethacin can
also cause an increase in TNFα in mice
(Nandi, 2008).
Indomethacin can trigger the production of
ROS such as superoxide anions. If the
amount of ROS exceeds antioxidants, the
body needs antioxidants from outside such
as breadfruit leaves. Medicinal plants have a
significant contribution to drug development
and are abundant in number. Breadfruit
leaves contain saponins, flavonoids,
polyphenols, and tannins. Flavonoids
function as anticancer, antioxidant, anti-
inflammatory, antiplatelet, and anti-
atherosclerotic. Saponins act as antibacterial,
and tannins play a role in antidiarrhea
(Nazira, 2018). Based on the review, a study
was conducted on the therapy of breadfruit
leaf extract against IBD from indomethacin
induction seen from SOD activity and white
rat ileum histopathology
of RESEARCH METHOD
ANIMAL PREPARATION
The experimental animals used were 8-12
week male rats (Rattus norvegicus) with a
body weight of 150-200 grams. Mice were
acclimatized for 7 days for adaptation. Rats
are placed in a 20 cm x 16 cm x 15 cm cage
with 20 pieces with a base in the form of an
underpad, a tub cover made of wire. The
optimum temperature of the mouse room is
22-240C with air humidity of 50-60%. All
rats were fed BR Comfeed and drank ad
libitum (Subandi, 2018).
INDOMETHACIN PREPARATION
Indomethacin induction using the sonde
method. The indomethacin dose given is 15
mg / kgBB (Sholichah, 2012). The average
weight of mice used is around 200 grams, so
the indomethacin doses used are:
Indomethacin: 15mg / kgBW x 0.2 kg = 3
mg / rat According to Bures (2011) the
making of indomethacin solution every 45
ml was dissolved in 4 ml of corn oil solvent.
So the amount of corn oil needed is The
need for corn oil: 3 mg / 45 mg x 4 ml =
0.27 ml

BREADFRUIT LEAVES EXTRACT
PREPARATION
Breadfruit leaves are obtained from materia
medika Batu, Malang. Breadfruit leaves are
cleaned of sticking dirt, then washed and
dried with herbs dryer for 48 hours at 550C.
Breadfruit leaves that are dried are milled
and sifted so that 500 grams of breadfruit
leaves are produced. The leaf powder was
macerated with 96% ethanol for 3 days as
much as 3 liters. The extract was then
filtered to remove the residue, remaserated
with ethanol 96% solvent for 2 days. The
filtrate obtained was concentrated with a
rotary evaporator temperature of 600C and
evaporated at 600C to remove excess
solvents until thick extracts were obtained
(Tandi, 2017).
ADMINISTRATION OF
INDOMETHACIN AND BREADFRUIT
LEAVE EXTRACT
Indomethacin is given once on the 8th day at
a dose of 15 mg / kg. Breadfruit leaf extract
therapy (Artocarpus altilis) was given for 14
days with three doses, namely a dose of 100
mg / kgBB, 200 mg / kgBB, and 400 mg /
kgBB. Based on Nazira (2018) breadfruit
leaf extract can be used as an antidiarrheal.
RESEARCH PARAMETER
The parameters observed were SOD activity
and ileal histopathology. SOD activity was
measured based on absorbance using the
spectrophotometric method. While ileal
histopathology is observed for inflammatory
cell infiltration, villous erosion, goblet cell
hypertrophy, and other abnormalities. Iile
histopathology was observed from five
visual fields using a microscope with a
magnification of 40x and 400x (Abrianto,
2015)
DATA ANALYSIS
Analysis of the data in the form of
quantitative data to determine the SOD
activity analyzed by the One Way ANOVA
test using SPSS version 16. Furthermore, to
determine the level of significance by
comparing Fncount and F table 5% and 1%.
If there is a real difference followed by a
BNJ test with α = 0.05. The
histopathological description of the ileum
was analyzed qualitatively descriptive
RESULT AND DISCUSSION
The clinical manifestations of CD vary
depending on the species and severity of
IBD, including diarrhea accompanied by
blood or not, abdominal pain, and weight
loss. According to Zakaria et al (2011), 10%
of CD sufferers do not experience diarrhea.
Diarrhea on CD can be caused by excessive
secretion of fluid due to disruption of fluid
absorption due to inflammation,
malabsorption of bile salts due to
inflammation, and steatorrhea due to
malabsorption of bile salts. Other symptoms P3
besides diarrhea are fecal bleeding. In the K + group, black stools with soft
Inflammation of the intestine can cause consistency can be seen, which can be
bleeding that appears on the stool, the more caused by bleeding from indomethacin
proximal bleeding, the stool will be black. administration. In groups P1, P2, and P3,
Black or melena stools occur around 50% in stools are not hard or flabby and appear to
CD cases (Mazal, 2014). resemble negative controls. This is because
breadfruit leaf extract contains tannins as
antidiare and inhibits inflammation does not

K+ get worse (Ashok, 2012), triterpenoids as

K-
antibacterial, flavonoids that can inhibit
intestinal bleeding, and alkaloids as
antibacterial which can damage bacterial

P1 P2 walls (Yaqin, 2014).

SOD (Superoxide dismutase) Activity

Kelompok Rata-rata SOD Penurunan SOD Peningkatan SOD
(Unit/100mg) terhadap Kontrol terhadap Kontrol
Negatif (%) Positif (%)
c
Kontrol Negatif 43,92±4,05 -

Kontrol Positif 22,59±2,88a 48,56%

P1 (Dosis 100 29,10±5,72ab 28,82%

mg/kgBB)
P2 (Dosis 200 34,98±6,90bc 54,85%
mg/kgBB)
P3 (Dosis 400 38,38±3,47bc 69,90%
mg/kgBB)
Superoxide dismutase (SOD) is an as the body's first defense from ROS
endogenous antioxidant enzyme, which acts (Reactive Oxygen Species), stopping
mutations and converting superoxide
radicals into harmless molecules such as
oxygen (O2) and hydrogen peroxide
(H2O2). SOD measurements using EDTA,
NBT, xanthine, and xanthine oxidase.
Xanthine oxidase is an enzyme that converts
hypoxanthine to uric acid, and produces
superoxide radicals. The principle of
measuring SOD activity is inhibition of
xanthine and xanthine oxidase reduction in
NBT (Nitroblue tetrazolium) (Cekic, 1999).
One unit of SOD activity is the amount of
enzyme needed to inhibit the reduction of
NBT by 50% under certain conditions. In
group P1 (dose of 100 mg / kgBB), SOD
activity was the lowest among other
treatments due to the inability of SOD to
inhibit NBT reduction so that superoxide
radicals were formed. Based on the SOD
activity test in Table 1 shows there are
significant differences between the average
SOD activity of the negative control group
ILEUM HISTOPATHOLOGY
K-
and the positive control group. This is
evidenced by the average value of negative
control SOD activities and positive controls
with different notations. Negative control
has the highest average SOD of 43.92 units /
100 mg, this is due to the absence of
intestinal infections. In group P1 (dose of
100 mg / kgBB), P2 (dose of 200 mg /
kgBB), and P3 (dose of 400 mg / kgBB)
experienced increased SOD activity when
compared with positive controls, this was
due to the presence of antioxidants in
breadfruit leaf extract (Artocarpus altilis).
Flavonoids acting as oxygen scavenger in
antioxidants will inhibit the oxidation
process, in addition flavonoids containing
phenol compounds that induce antioxidant
enzyme genes such as Cu / Zn-SOD or Mn-
SOD, then induce antioxidant and DNA
receptor elements to increase the production
of antioxidant enzymes (Sugito, 2012).
K+
 P1 P2

P3

Keterangan: ( ) hipertropi sel goblet, ( ) sel radang, ( ) erosi vili, ( ) hemoragi,

The histological results of
negative control group showed a normal
ileal picture. The mucous layer consists of
villi that are elongated, intact, and have no
erosion. Epithelial cells predominate and at
100x magnification there is no goblet cell
hypertrophy. In addition, there
inflammatory cells such as macrophages
which are differentiations of monocytes.
Under normal circumstances, the ileum has
inflammatory cells such as macrophages.
The function of macrophages is not limited
to phagocytosis of foreign microorganisms,
( ) edema
the but also plays a role in maintaining organ
homeostasis and producing functioning
growth factors in reepithitelization and
angiogenesis (Price and Wilson, 2006).
Positive control histopathology induced by
indomethacin has damage in the form of
are inflammatory cell infiltration, hemorrhage,
edema, villous erosion, and goblet cell
hypertrophy. Goblet cell hypertrophy is
caused by increased goblet cell activity to
produce mucus for intestinal mucosal
defense. According to Carty (2002), IBD
conditions can increase the synthesis of
excess thromboxane which can induce an platelets which will gradually be replaced by
inflammatory state due to excessive epithelium. The damaged epithelium will be
neutrophil production. The inflammatory released from the basal cell and new
response can cause increased capillary epithelium will form from the mitotic
permeability resulting in edema. This is in process. CONCLUSION Giving breadfruit
accordance with the statement of Aini et al leaf extract (Artocarpus altilis) was able to
(2011) that edema can be caused by significantly increase the activity of SOD
NSAIDs. In P1, P2, and P3, the (Superoxide dismutase) and was able to
histopathological picture was improved. improve ileal histopathology in IBD
Breadfruit leaf extract has the potential to indomethacin induction.
improve ileal histopathology because it
BIBLIOGRAPHY
contains flavonoids, tannins, and
polyphenols which can be antioxidant and Abrianto, W., Aulanni’am, D. K. Wuragil.

anti-inflammatory. Antioxidants function in 2013. Therapeutic Study of

neutralizing free radicals by giving electrons Breadfruit Leaf Water Extract

(H +) so that oxidative stress does not occur, against Malondyaldehide Level and

in addition antioxidants can inhibit increased Jejunum Histopathology Overview

production of cytokines such as IL-6 in Hypercholesteromia White Mice.

(interleukin-6) or TNF-α (Tumor Necrosis [Essay]. Faculty of Veterinary

Factor) which are inflammatory cytokines Medicine. Brawijaya University.

( Parwata, 2016). Flavonoids also play a role Aini, Z., Jamaluddin, and D. Dama. 2011.

in anti-inflammation because it inhibits Swelling. Kendari: Haluoleo

prostaglandin which is an inflammatory University.

agent. In the P3 group there was Ashok, P. and K. Upadhyaya. 2012. Tannin

reepithelization, due to breadfruit leaf are Astringent. Journal of

extract containing flavonoids that function Pharmacognosy and

in inhibiting COX-2, iNOS, and NF-kB Phytochemistry Vol. 1 No. 3.

(Nuclear Factor kappa B) and preventing Carty, E., C. Nickols, R. Feakins, and D. S.

bleeding or platelet aggregation (Rathee, Rampton. 2002. Thromboxane

2009). Reepithitelization can occur because Synthase Immunohistochemistry in

the tissue is dominated by macrophages and Inflammatory Bowel Disease.

 Journal of Clinical Pathology. Issue Nazira. 2018. Antidiareous Activity of
55; 367-370. Ethanol Extract of Breadfruit
Cekic, O., Y. Bardak, Y. Totan, O. Akyol. Leaves (Artocarpus altilis) in Mice
1999. Superoxide Dismutase, [Thesis]. Faculty of Pharmacy.
Catalase, Glutathione Peroxidase, University of Northern Sumatra
and Xanthine Oxidase in Diabetic Parwata, I. 2016. Teaching Materials:
Rat Lenses. Opthalmic Res 31; Antioxidants. Bali: Udayana
346-350. University. Price and Wilson.2006.
Danastri, I. and Ida Bagus. 2013. Pathophysiology of the Clinical
Inflammatory Bowel Disease. Concept of the Disease Process.
Denpasar: Udayana University. Jakarta: EGC.
Fokunang, C. Overview of Non-Steroidal Rathee, P., H. Chaudhary, S. Rathee, V.
Anti-Inflammatory Drugs in Kumar, and K. Kohli. 2009.
Resource Limited Country. Toxical Mechanism of Action of
MOJ 2018; 4 (1): 5-13. Flavonoids as Inflammatory Anti-
Mansfield, C. 2011. Diagnosis and Agents: A Review. India: Botham
Management of Canine Science Publisher.
Inflammatory Bowel Disease. Subandi, I. 2018. Female Ovary Protein
Small Animal Veterinary Congress Profile (Rattus novergicus) Female
WSAVA. Jeju (Korea). after Giving Ethanol Extract of
Mazal, Jonathan. 2014. Crohn Disease: Dragon Scales Leaf (Pyrrosia
Pathophysiology, Diagnosis, and piloselloides). [Essay]. Medical
Treatment. Radiologic Technology. School. Maulana Malik Ibrahim
Volume 85, No. 3 State Islamic University.
Nandi, J, Bipin Saud. 2008. 5- Sugito. 2012. Biological Antioxidant
Aminosalicylic Improves Activity of Sorghum and Jewels
Indomethacin - Induced and Their Application to Prevention
Enteropathy by Inhibiting Inos of Degenerative Diseases. Human
Transcription in Rats. Springer Development Journal Vol.6 No. 1.
Science Business Media. Tandi, J., M. Rizky, R. Mariani, and F.
Alan. 2017. Effect Test of Ethanol
 Extract of Breadfruit Leaves
(Artocarpus altilis) on Decreasing
Blood Glucose Levels, Total
Cholesterol, and Histopathological
Overview of Male White Rat
Pancreatic Hypercholesterolemia
Diabetes. Journal of Science and
Health. Vol. 1. No. 8.
Yaqin, Ainnul. 2014. Antibacterial Potential
of Ethanol Extract, Water Ethanol
Fraction, and N-Hexan Fraction
Ethanol Extract of Grape Leaves
against Pseudomonas aeruginosa.
Surakarta: Muhammadiyah
University of Surakarta.
Zakaria, R., A. Fauzi, M. Abdullah, A.
Fahrial. 2011. Diagnostic Problems
in Crohn’s Disease. The Indonesian
Journal of Gastroenterology,
Hepatology, and Digestive
Endoscopy Volume 12, Number 3.