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EFFECTIVITY OF WATERMELON (CITRULLUS LANATUS) SEEDS EXTRACT ON INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) EXPRESSION DURING WOUND HEALING ON GUINEA PIG (CAVIA COBAYA)

Authors:

Abstract

Background : Watermelon seed contains high L-arginine concentration. If present in certain concentration, L-arginine theorytically induces the expression of Inducable-Nitric oxide synthase (i-NOS) enzyme which catalyzes the production of nitric oxide (NO) from L-arginine. In Addition, the higher the nitric oxide (NO) concentration on damaged tissue, the rapider the wound healing proceed. However, there is no research on the effect of watermelon seed extract on the expression of i-NOS. Purpose: The aim of this study is to prove the effect of watermelon seed extract on inducing the expression of i-NOS by immunoreactive cells. Methods: The back of 32 Guinea Pigs (Cavia cobaya) are incised and covered with gauze and then divided into 2 experimental groups and 2 control groups. The experimental groups are given watermelon seed extract per oral 1gr/day for 24 hours (group 1) and 72 hours (group 2). The control group was given distilled water. After 24 hours and 72 hours, the skin tissues of all guinea pigs were taken and prepared for Immunohistochemical-paraffin staining to observe the expression of iNOS enzyme. The number of poly-morpho-nuclear, fibroblasts, endothelial and macrophage cells expressing iNOS are counted in all groups using light microscopes. Results: Using Independent-Samples T-test, the results shows significant increase (μc<μe; p <0.05) on the number of immunoreactive fibroblasts cells on experimental group 2 compared to control group 2. Conclusion: Watermelon seeds extract significantly increases the number of fibroblasts immunoreactive cells expressing iNOS after 72 hours on Guinea pig.
EFFECTIVITY OF WATERMELON (CITRULLUS LANATUS) SEEDS EXTRACT ON
INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) EXPRESSION DURING WOUND
HEALING ON GUINEA PIG (CAVIA COBAYA)
Author: Gusti Citra Putra*, William Suryajaya*, Theresia Indah Budhy S**, Diana
Nurwati**
*Undergraduate Student
Faculty of Dentistry, Airlangga University
**Department of Oral Pathology
Faculty of Dentistry, Airlangga University
ABSTRACT
Background : Watermelon seed contains high L-arginine concentration. If present in certain
concentration, L-arginine theorytically induces the expression of Inducable-Nitric oxide synthase
(i-NOS) enzyme which catalyzes the production of nitric oxide (NO) from L-arginine. In Addition,
the higher the nitric oxide (NO) concentration on damaged tissue, the rapider the wound healing
proceed. However, there is no research on the effect of watermelon seed extract on the expression
of i-NOS. Purpose: The aim of this study is to prove the effect of watermelon seed extract on
inducing the expression of i-NOS by immunoreactive cells. Methods: The back of 32 Guinea Pigs
(Cavia cobaya) are incised and covered with gauze and then divided into 2 experimental groups
and 2 control groups. The experimental groups are given watermelon seed extract per oral 1gr/day
for 24 hours (group 1) and 72 hours (group 2). The control group was given distilled water. After
24 hours and 72 hours, the skin tissues of all guinea pigs were taken and prepared for
Immunohistochemical-paraffin staining to observe the expression of iNOS enzyme. The number of
poly-morpho-nuclear, fibroblasts, endothelial and macrophage cells expressing iNOS are counted
in all groups using light microscopes. Results: Using Independent-Samples T-test, the results shows
significant increase ce; p <0.05) on the number of immunoreactive fibroblasts cells on
experimental group 2 compared to control group 2. Conclusion: Watermelon seeds extract
significantly increases the number of fibroblasts immunoreactive cells expressing iNOS after 72
hours on Guinea pig.
Keywords : wound healing, arginine, watermelon seeds extract, inducible nitric oxide synthase.
Korespondensi (correspondence): Gusti Citra Putra, William Suryajaya, Theresia Indah Budhy S. (Lecturer of Oral
Pathology), Diana Nurwati (Lecturer of Oral Pathology), Fakultas Kedokteran Gigi Universitas Airlangga. Jl. Mayjend.
Prof. Dr. Moestopo 47 Surabaya 60132, Indonesia. E-mail: xilentikus @ live .com
BACKGROUND
In daily life, wound is inevitable. Wounds
require sufficient time and nutrition to heal.
Sometimes, wounds cannot heal completely and
causing scar.
Wound healing are divided into 4 phase,
which is hemostatis, inflammation, proliferative
and remodelling.1,2 Each phase plays a major role
in wound healing. During inflammation phase,
there is an increase on the amount of
polymorphonuclear (PMN) cells followed by the
production of free radical. Nitric oxide, one of the
free radicals, is produced from arginine by Nitric
Oxide Synthase (NOS) enzyme. There are 3 types
of NOS enzyme isoforms: neuronal, endothelial
and inducible. The neuronal and endothelial
1
isoform are constantly produced in small amount
notwithstanding any stimulation like tissue
damage or infection. On the other hand, the
inducible type, inducible nitric oxide synthase
(iNOS) is only produced during bacterial
infection.3 iNOS concentration reaches its peak 24
hours after tissue damage and gradually
decreases.4
Proliferative phase are marked by
proliferation of fibroblasts, macrophages and
endothelial cells. Production of Nitric Oxide
during inflammation phase gives rise to
fibroblasts, macrophages and endothelial cells
proliferation which in turn affects regeneration
time and quality of the damaged tissue.1
Arginine, one of amino acids, resides
inside tissue protein. It is used to synthesize other
amino acid, polyamine, and keratin. Watermelon,
especially its seed, exhibits substantial amount of
Arginine (100 gram of watermelon seeds contains
4897 mg of arginine)5,6,8.
Until recently, there are limited studies
about watermelon seed extract effect on wound
healing. This study aims on elucidating
watermelon seed extract effect on inflammation
process and proliferation of fibroblast,
macrophage and endothelial cells during wound
healing.
METHOD
Extraction Method
Watermelon seeds extract (Citrullus lanatus) is
acquired by percolation methods using ethanol to
obtain maximum amount of arginine. Afterwards,
the extract is stored in 4°C temperature.11
Full Thickness Incision Procedure
Experimental subjects are divided into 4 groups
(Control 1 (C1), Experimental 1(E1), Control
2(C2) & Experimental 2(E2)). Each group
consists of 8 guinea pigs (Cavia cobaya, Sp.).
Subjects are then anesthesized using inhalant
anesthesia (10% Ether). Antiseptic (10%
Povidone Iodine) is applied13 before making full
thickness incision (10 mm in length and 2 mm
deep)14 on the back of the subject using scalpel no.
11.15 Subsequently, 10% Povidone Iodine is
applied topically on the wound and covered with
gauze. 16
Dosage and Application
The effective arginine daily dosage to enhance
wound healing is 0.252 gram for Cavia cobaya,
Sp., converted from human dosage of 14 gram. In
each experimental group, 1 gram of watermelon
seeds extract(11 grams of watermelon seed)
mixed with sterile aquadest is given orally every
day. Every gram of watermelon sees extract
contains roughly 0,5 gram of arginine. Control
group was given aquades. Experimental group 1
was given extract one time (24 hours) and
experimental group 2 was given extract 3 times
(72 hours).
Results Examination
After 24 and 72 hours, excision of the wounded
skin are made and prepared for histologoy
examination. iNOS expression was examined
using anti-iNOS antibody NB 120-15324 Novus
Biologicals through paraffin-
immunohistochemistry staining.19 The number of
polymorphonuclear, fibroblast, macrophage, and
edothelial cells expressing iNOS are counted in 5
distinct predetermined coordinate in every slides
using light microscope and Pentax 230 (1000x
magnificaton).
RESULT
A total of 32 histologic preparations (8
from each group) are examined. As a general rule,
immunoreactive cells expressing iNOS are more
brownish that the non-immunoreactive cells.
(figure 1-4)
The mean of each immunoreactive cell type
expressing iNOS from 5 distinct predetermined
coordinates in the light microscope are
summarized in Graph 1.
Graph 1. Mean of immunoreactive cells in C1, E1, C2
and E2
2
Figure 1. Immunoreactive polymorphonuclear cells
(above: negative; below: positive)
Figure 2. Immunoreactive macrophage cells
(above: negative; below: positive)
Figure 3. Immunoreactive fibroblast
(above: negative; below: positive)
Figure 4. Immunoreactive endothelial cells
(above: negative; below: positive)
3
Table 1. Mean Number of Immunoreactive Cells
Group Polymorphonuclear Macrophage Fibroblast Endothelial Cells
Control Group 1 21.63 12.75 14.75 19.25
Experimental Group 1 7.75 12.88 4.63 18.38
Control Group 2 33.29 20.43 8.71 29.57
Experimental Group 2 12.38 35.13 25.38 34.00
Independent T-test shows that there is significant
difference on fibroblasts expressing iNOS
between Control Gorup 2 and Experimental
Group 2 (p < 0,05). Whlist, no significant
difference on the number of other immunoreactive
cells between control groups 1-experimental
groups 1 and control group 2-experimental group
2(p>0,05).
DISCUSSION
Result shows no significant difference of
immunoreactive polymorphonuclear cells between
control group 1 and experimental group 1 or
between control group 2 and experimental group
2; however, there is a significant difference on the
mean value of both experimental groups
compared to each control groups. In experimental
group 1, the expression of iNOS is lower; thus, the
production of NO was lower. This is attributed to
negative feedback mechanism commenced by
PMN cells to control the production of NO.
Studies by Ajuebor et al. shows iNOS could
regulate the function and accumulate PMN cells
during early phase of wound healing and suppress
the adhesion of PMN to endothelial cells around
the wound. 20 Another study shows that PMN
expressing iNOS and producing huge amount of
NO will caused tissue damage since NO is
cytotoxic.21 Hence, suppression of the number of
PMN expressing iNOS during wound healing is
important.
There are no significant difference of
immunoreactive macrophage between both
control group and experimental group. This might
be attributed by variance between each subject
either in genetic or in their physiologic condition.
The difference of mean in control group 2 and
experimental group 2 was considered high but not
significant. This is probably due to watermelon
seeds extract which is rich in arginine. It increases
the proliferation of macrophage in experimental
group 2. Experimental group 1 which is also
received watermelon seeds extract this increase
did not happen because during 24 hours of wound
healing macrophage has not migrating to the are
of wound healing. Slight higher mean of in
experimental group 1 (24 hours) compared to
control group 1 (24 hours) is might be on account
of acute inflammation phase.
As shown by Independent T-test, there is
a significant difference in the number of fibroblast
expressing iNOS on experimental group 2
compared to control group 2. This possibly due to
NO produced by PMN which inhibits NF-κB
inductin. Consequently, immunoreactive
fibroblasts increase significantly.
In addition, increased cytokine level
during inflammation amplifies the expression of
iNOS in fibroblasts. Hence, NO production that
has the ability to protect cells from free radical
produced by PMN and macrophage within
fibroblasts increases.25 This can lead to better
wound healing since fibroblast activity is
uninterrupted by free radicals products.
There is a slight decrease in
immunoreactive endothelial cells in experimental
group 1 compared to control group 1. This may be
due to uncontrollable variable in each subject,
such as different genetic and physiology
condition. In the second group, there are
insignificant difference between control group 2
and experimental group 2. This might be
attributed by lower amount of NF-κB in
immunoreactive PMN cells in experimental group
2.26 Decrease of NF-κB results in increase of
immunoreactive endothelial in experimental group
2.
Increase of immunoreactive fibroblasts
and immunoreactive endothelial cells in
experimental group 2 shows that the expression of
iNOS contributes considerably during
proliferative phase of wound healing. Significant
increase of immunoreactive fibroblasts shows that
iNOS expression has greater effect to the
proliferation of fibroblast than endothelial cells.
4
CONCLUSION
Watermelon seed extract enhances wound
healing on full thickness incision in guinea pig
indicated by significant increase in the number of
fibroblasts expressing iNOS.
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