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Gunawan et al. World Journal of Pharmacy and Pharmaceutical Sciences
POTENTIAL OF EUCHRESTA HORSFIELDII LESCH BENN LEAF
EXTRACT PREVENT OXIDATIVE STRESS THROUGH DECREASE
OF MALONDIALDEHYDE LEVELS AND PROFILE
HISTOPATHOLOGY PANCREATIC Β-CELLS IN DIABETIC RATS
1*I.W.G. Gunawan and 2K. Suastika, 1A. A. Bawa Putra
1*Laboratory of Organic Chemistry, Department of Chemistry, Udayana University.
2Faculty of Medicine, Udayana University, Bali-Indonesia.
ABSTRACT
Oxidative stress is damage to cells caused by chemical reactions
between free radicals and molecules in the body. Cell damage caused
by oxidative stress is believed to be the cause of various diseases such
as diabetic. As a result, the intensity of the oxidation process of normal
body cells becomes higher and cause more damage. The results
showed that the Euchresta horsfieldii lesch benn leaf extract can
prevent oxidative stress due to the production of compounds reactive
oxygen species (ROS), thereby protecting cells from damage functions.
Oxidative stress can be formed through decreased malondialdehyde
levels in the body, then carried out research with the aim to prove that
the n-hexane extract of leaves Euchresta horsfieldii lesch benn has the
ability as through decreased malondialdehyde levels of antioxidative and histopathological
profile improvement of pancreatic β-cells in diabetic rats. This study is a laboratory
experimental study design was randomized pre and post-test control group design. A total of
36 rats were divided into four groups: one control group and three groups treated with
different the Euchresta horsfieldii lesch benn leaf extract that each dose of 0.5 mg/kg bw, 2
mg/kg bw and 5 mg/kg bw. All groups induced by alloxan dose of 140 mg/kg bw in order to
get a diabetic condition. After treatment for 8 weeks, blood samples were taken for
examination malondialdehyde levels. The results showed that the Euchresta horsfieldii lesch
benn leaf extract can prevent oxidative stress by decreasing the average levels of MDA in the
positive control group (Glibenclamide) of (0.61 ± 0.07) µmol/L, the treatment groups
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 5.210
Volume 5, Issue 1, 1340-1352 Research Article ISSN 2278 – 4357
Article Received on
19 Oct 2015,
Revised on 10 Dec 2015,
Accepted on 30 Dec 2015
*Correspondence for
Author
I.W.G. Gunawan
Laboratory of Organic
Chemistry, Department of
Chemistry, Udayana
University.
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Gunawan et al. World Journal of Pharmacy and Pharmaceutical Sciences
respectively P1 = (0,96 ± 0.08) µmol/L; P2 = (0.72 ± 0.08) µmol/L; and P3 = (0.46 ± 0.06)
µmol/L of different statistic significant with p <0.05.
KEYWORDS: Euchresta horsfieldii lesch benn, Oxidative Stress, malondialdehyde,
Diabetic.
INTRODUCTION
Cardiovascular complications in diabetes mellitus is one of the factors causing oxidative
stress at the cellular level. Although the administration of insulin or oral antidiabetic drugs,
complications of diabetic is still difficult to overcome almost all over the world, including
Indonesia. Deaths due to these complications is estimated at 40% of patients with diabetes
mellitus. This situation triggered by failure of the pancreatic β-cells in the production of
insulin and glucagon, followed by high plasma (Srinivasan, 2007; Suastika, 2008).
Various complications can be caused by poor control of diabetes. The complications which
include systemic vascular disease (accelerated atherosclerosis), heart disease, microvascular
disease of the eye as a cause of blindness and retinal degeneration (diabetic retinopathy),
cataracts, kidney damage as a cause of kidney failure and peripheral nerve damage (diabetic
neuropathy). The extent of complications in diabetes seems to be correlated with blood
glucose concentration so that excess glucose is thought to be a major cause of tissue damage
(Rahbani et al, 1999; Halliwell et al, 1999). This phenomenon can be caused by the ability of
hyperglycemia in vivo in the oxidative modification of various substrates. In addition,
hyperglycemia is also involved in the formation of free radicals (Droge, 2002).
Hyperglycemia causes glucose autooksidasi, glycation of proteins and activation of metabolic
pathways polyols which further accelerates the formation of reactive oxygen species (Ueno,
2002). The formation of reactive oxygen compounds that can improve lipid modification,
DNA, and proteins in different tissues. Molecular modifications in various tissues has
resulted in an imbalance between antioxidant protective (antioxidant defense) and increased
production of free radicals. It was the beginning of oxidative damage known as oxidative
stress (Nuttal et al, 1999). Oxidative stress is a pathological condition caused by damage to
tissues in the body because of the increased number of free radicals that are not normal. The
existence of these free radicals can be known through MDA as lipid peroxidation products.
Diabetes mellitus is a multifactorial disorder associated with proinflammatory cytokines that
are characterized by the formation of compounds of reactive oxygen species (ROS) excess
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(Sahebari et al, 2011). ROS is part of the free radicals which are the product of normal cell
metabolism. Free radicals are one of the products of chemical reactions in the body in the
form of atoms or groups that have unpaired electrons on the outer orbital, so that these
compounds are highly reactive (unstable). In the body, free radicals can cause lipid
peroxidation process (Favier., 1995). Lipid peroxidation is the oxidative destruction of the
unsaturated fatty acid chain length (Polyunsaturated Fatty Acids) that produce compounds of
malondialdehyde (MDA). Thus, MDA can be used as an index measuring the activity of free
radicals in the body. High levels of MDA in the body can be caused by increased activity of
free radicals (Haliwell., 1999).
Malondialdehyde an end product of lipid peroxidation, which is usually used as a biological
biomarkers of lipid peroxidation and describes the degree of oxidative stress
(Hendromartono, 2000). According Suryohudoyo (2000), MDA is dialdehydes compounds or
three reactive carbon is the final product of lipid peroxidation in cell membranes. MDA in
biological material present in free form or form a complex bond with other elements in the
network.
MDA measurements carried out by the researchers as an indirect index of oxidative damage
caused by lipid peroxidation. According to a statement Tokur et al. (2006), MDA
measurement principle is the reaction of one molecule of MDA with two molecules
tiobarbiturat acid (TBA) form a complex compound of the MDA-TBA pink and quantity can
be read with a spectrophotometer. These conditions lead to oxidative stress, ie an imbalance
between free radicals with antioxidants in the body causes changes in pancreatic tissue
histology and increased levels of malondialdehyde. However, in anticipation of the buildup of
excess free radicals, it is necessary given the exogenous antioxidants from natural sources,
one of which is the n-hexane Euchresta horsfieldii lesch benn leaf extract.
Kloppenburgh, (2006), reported that one of the traditional medicinal plants that have the
potential to be developed as an antidiabetic drug leaves Euchresta horsfieldi lesch benn,
because traditionally leaves Euchresta horsfieldi lesch benn has been used to treat various
diseases, such as: diabetic, asthma, coughing up blood and a dry cough. Other uses are as an
antidote to snake venom, alfrodisiakum and induce vomiting due to food poisoning. The
results showed that the Euchresta horsfieldii lesch benn leaf extract contains several chemical
compounds, including; α-humulene, trans-caryophyllene, eugenol, 1,2-benzene dicarboxylic
acids, phenolic acids and a new compound with a molecular weight (m/z) 302. The
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compound α-humulene sesquiterpenoids included in the class, a compound known to have
activity as an antioxidant and repair function of pancreatic β-cell destruction through
decreased levels of blood glucose and AGEs in Wistar rats hyperglycemia (Gunawan, 2013).
According Astuti (2003) Euchresta javanica R Br seed extract in male rats can increase
superoxide dismutase and lower levels of malondialdehyde, so it can protect cells against
oxidative stress.
RESEARCH METHODS
Research design
This study was a laboratory experimental design with The Randomized Pre and posttest
control group design (Pocock, 2008). The sample in this study are male rats, aged 3 months
were obtained center study of animal diseases Faculty of Veterinary Medicine Udayana
University. The sample size in this study was 36 Wistar rats were classified into four groups:
one control group and three treatment groups.
The initial step is to create uniform conditions of rats then 36th rats fed a diet try ITB rich in
vitamin B12 for 4 weeks. Furthermore, to get the mice with diabetes are given alloxan dose
of 140 mg/kgbw for 2-3 days. Each group was given the Euchresta horsfieldi Lesch benn leaf
extract known antioxidant capacity with various doses of 0.5 mg/kg bw, 2 m/kg bw and 5
mg/kgbw for 8 weeks. Later examination malondialdehyde levels and see the profiles of
pancreatic β-cells histopathology rats.
Euchresta horsfieldii lesch benn Leaf Extraction
The Euchresta horsfieldii lesch benn leaf which has been cleaned and then dried in the open
air with open air circulation and is not exposed to direct sunlight. Subsequently milled to a
powder blender. Euchresta horsfieldii lesch benn leaf powder that was dried weighed 1 kg
and was extracted by maceration using n-hexane for 24 hours and then evaporated using a
rotary vacuum evaporator. While the residue obtained re-extracted using n-hexane. n-hexane
extract is evaporated to obtain a thick extract n-hexane and subsequently under taken
phytochemical test and analysis Gas Chromatography Mass Spectroscopy (GC-MS) to
determine the class of compounds in Euchresta horsfieldii lesch benn leaf extract.
Treatment with Rats
A total of 36 male rats aged 3 months were measured weight and given a standard diet
enriched formula of vitamin B12 for 1 month. All mice in cages adapted for 1 week. After all
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the mice in uniform conditions, the rats were made diabetic for 3 days by means of induced
alloxan dose of 140 mg/kg, then made four experimental groups: one control group and three
treatment groups were each given Euchresta horsfieldii lesch benn leaf extract dose of 0,5
mg/kg, 2 mg/kg and 5 mg/kg for 8 weeks. Euchresta horsfieldii lesch benn leaf extract is
done by dissolving the extract into 5 ml of distilled water, then homogenized and exposed to
mice orally in accordance treatments by disonde.
After the 8-week treatment period was terminated rats (euthanasi) with CO2 done quickly and
sterile. Pancreatic tissue is taken and washed with Phosphate Buffer Saline (PBS), drained
and weighed, then packed with alufo and stored in freeser (-20°C) is then performed
histopathological examination of the rat pancreatic tissue.
Blood Examination
Rats will have blood drawn fasted for 12 hours but drinking still be given as usual, then the
mice weighed. After anesthesia by inhalation of diethyl ether until reached the respiratory
rhythm regular blood taken 3 ml. Blood collected allowed to stand for 30 minutes at room
temperature, then mess around at 2700 rpm for 10 minutes. Serum was separated, extracted
and inserted into the bottle and then closed. the sample is then stored at a temperature of 40C.
Furthermore, the determination of MDA levels in the blood of mice is measured by the
amount of malondialdehyde reacts with the reagent acid tribarbiturat (mol/L) during the
oxidative stress.
Laboratory Testing
Laboratory tests performed at the time of treatment until there is diabetic, with testing
procedures as follows.
a. Determination of MDA levels in rat blood is done using Mansila Espinosa. Measurement
is based on the amount of malondialdehyde raects with acidic reagents tribarbiturat
b. Examination histopathological profile of pancreatic tissue rats performed using binocular
microscope with Gomori-Nuclear fast red staining, magnification 400x.
Data Analysis
The data collected from this research was statistically analyzed by the following procedures.
Statistical analysis was conducted using SPSS 13.0 application program for windows (Triton,
2006) for.
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1. Normal distribution using Shapiro-Wilk within α = 0,05.
2. Homogeneity of variance were analyzed using Levene's test to determine whether
variations in respective homogeneous group.
3. Analisis of differentiation on mean increase of MDA levels from each group were
analyzed using one-way ANOVA. Further analysis is one way anova Post Hoc Test;
assuming homogeneous variance is then selected Post Hoc Test was LSD at significance
level α = 0.05.
RESULTS AND DISCUSSION
Characteristics of Euchresta horsfieldii lesch benn Leaf Extract
Euchresta horsfieldii lesch benn leaf extract condensed n-hexane maceration results give as
much as 62.15% yield blackish green. While the results of antioxidant capacity against DPPH
(2,2-diphenyl-1-picrylhydrazyl) showed that the seed extract has Euchresta horsfieldii lesch
benn percentage reduction of 63.05% in 5 minutes and 82.90% within 60 minutes.
Antioxidant capacity in the blood of rats was calculated based on the ability of catching free
radical DPPH (2,2-diphenyl-1-picrylhydrazyl) with the capture reaction.
The results of the separation of active compounds by GC-MS analysis showed that the
Euchresta horsfieldii lesch benn leaf extract provides nine peaks with a retention time (tR),
peak area (%) and has a different molecular weight. The compounds were detected in the n-
hexane of Euchresta horsfieldii lesch benn leaf extract be presented in detail are presented in
Table 1.
Table 1. The compounds were detected in the Euchresta horsfieldii lesch benn leaf
extract.
Peak No
Retention time
(Minutes)
Wide
Area (%)
Molecular
Formula
Compound Name
1
2
3
4
5
6
7
8
9
14.822
15.998
16.495
21.947
23.747
26.539
26.716
27.408
27.891
17.00
17.55
1.67
1.30
3.66
10.06
8.45
38.02
2.30
C10 H12O2
C15 H24
C15 H24
C17 H34 O2
C19H34 O2
C22H42 O4
C15 H24 N2O
BM = 302
C24 H38 O4
Eugenol
Trans-Caryophyllene
α-humulene
Hexadecanoic acid
9,12-octadecadienoic acid
Hexanedioic acid
Matrine
New Compound
1,2-benzene dicarboxylic acid
Description: The results of spectroscopic analysis QP2010S Shimadzu GC-MS.
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Decrease in blood MDA Wistar rats Diabetic
Data mean MDA blood hyperglycemia rats both pre and posttest are presented in Table 2
MDA profiles before and after treatment with various doses of Euchresta horsfieldii lesch
benn leaf extract is presented in Figure 1.
Table 2. MDA Levels Before and After Treatment.
Treatment
Observations MDA Levels (μmol/L)
Pretest
The mean ± SD
Posttest
The mean ± SD
Difference of MDA
Levels (μmol/L)
Control group (K-) and (K+)
Euchresta L benn extract dose 0,5 mg
Euchresta L benn extract dose 2 mg
Euchresta L benn extract dose 5 mg
2,42 ± 0,13
2,49 ± 0,12
2,47 ± 0,09
2,47 ± 0,09
0,61 ± 0,07
0,96 ± 0,08
0,72 ± 0,08
0,46 ± 0,06
1,808 b
1,533 d
1,750 c
2,011 a
Note: Difference in average values followed by different letters in the same column, shows
the test results were significantly different (p<0.05) LSD test for posstest, K-(Negative
Control), K+ (Positive Control/Glibenclamide).
Figure 1: Profile of MDA Levels Before and After Treatment.
Test results with the Shapiro-Wilks normality and homogeneity test with Levene'stest shows
that the data mean levels of MDA Wistar rats before and after administration of various
Euchresta lesch benn extract showed dose throughout the data were normally distributed and
homogeneous variants (p>0,05).
Results of analysis and one way ANOVA followed by LSD test showed that there were
significant differences between the levels of MDA Wistar rat control group (K+) treatment
group after the administration of Euchresta horsfieldii lesch benn leaf extract dose of 0.5
mg/kg bw, 2 mg/kg bw and 5 mg/kg bw with a value of p<0.05. The results also showed that
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the control group (K+) gives the equivalent effect of the treatment group a dose of 5 mg/kg
bw.
Furthermore, the limit of significance with paired t-test showed a significant difference in the
mean decrease in blood MDA levels between the control group (K-) with control group (K+)
with p<0.05. In contrast, the treatment group a dose of 0.5 mg/kg bw dose group treated with
2 mg/kg bw bothwere significantly different (p<0.05), while the treatment group a dose of 5
mg/kg bw occur both significant differences with p<0.05.
Euchresta horsfieldii lesch benn extract with various doses can lower blood levels of MDA
wistar rats diabetic. MDA mean data are shown in Table 5. Mean blood MDA levels of
diabetic in rats negative control group (K-) and positive control group (K+) was 2.42 ± 0.13
μmol/L and 0.61 ± 0.07 μmol/L. From the same table can also be seen due to the extract
MDA mean dose 0.5 mg/kg bw/day, 2 mg/kg bw/day and 5 mg/kg bw/day in a row for the
pre is (2.49 ± 0.12), (2.47 ± 0.09) and (2.47 ± 0.09) μmol/L, whereas for the posttest was
(0.96 ± 0.08), (0.72 ± 0, 08) and (0.46 ± 0.06) μmol/L. Compared with the positive control
group (glibenclamide), the extract dose of 0.5 mg/kg bw/day decreased the levels of MDA,
but not enough to dampen ROS produced by the metabolism of the body, so that ROS rapidly
reacts with the double bond in the acid fatty acids to form lipid peroxidation is MDA on the
cell membrane. The results are supported by Tjokroprawiro (2005), found that diabetic
patients indicated a number of free radical formation such as H2O2, hydroxyl radicals that
facilitate the formation of lipid peroxide (MDA) on the cell membrane. In the meantime, the
results Wresdiyati et al (2003) states that α-tocopherol administration at a dose of 60
mg/kg/day for seven days can reduce the levels of MDA in rats with stress conditions.
The mean decrease in blood MDA levels were significantly diabetic rats already occurred in
the extract dose of 0.5 mg/kg bw/day, 2 mg/kg bw/day and 5 mg/kg bw/day with a value of
p<0.05. Overall the data presented in Table 6. From the table it appears that the highest levels
of MDA decreased blood wistar rats diabetic occurs in the extract dose of 5 mg/kg bw/day,
which amounted to 0.50 μmol/L. This means that Euchresta horsfieldii lesch benn extract
contains several classes of carboxylic acids such as Hexadecanoic acid, 9.12-hexadecadienoic
acid, hexanedioic acid and 1,2-benzenedicarboxylic acid can lower blood levels of MDA in
rats wistar due to lipid oxidation of unsaturated fatty acid chain length (unsaturated fatty
acids). Declining levels of MDA in the blood may also prevent decreased membrane fluidity
and cell damage. The results of animal studies indicate that consumption of saturated fatty
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acids cause a buildup chain unsaturated fatty acids in the body which can lead to increased
toxic products of unsaturated fatty acid synthesis (lipid peroxidation), namely
malondialdehyde that can cause damage to cell membranes. Kutlu et al (2009) stated that the
granting of apricot kernel oil which is rich in fatty acids such as acids, oleic and linoleic acid,
and the bioactive compounds such as thiamine. Riboflavin, vitamin C, α-tocopherol,
sitosterol and kaempesterol able to reduce levels of MDA in Wistar rats.
Profile of Histopathology Pancreatic β-cells Rats
Gomori-Nuclear fast red staining done to see qualitative changes in the structure of rat
pancreatic tissue treatment. Staining is composed of two color components and the Gomori-
Nuclear fast red. Gomori an alkaline dye in order to color the cell nucleus that are acidic
while Nuclear fast red is an acidic dye that can stain the cytoplasm is alkaline.
Histopathological changes in pancreatic tissue morphology Wistar rats with 400 times
magnification and staining Gomori-Nuclear fast red from the normal state to occur alloxan-
induced diabetic caused a dose 140 mg/kg bw can be seen in Figure 2,3,4, 5, 6 and 7
Figure 2: Normal Histopat Pancreas Wistar Rats (Magnification 400x).
Figure 3: Histopat Pancreas Wistar Rats K- (alloxan) (Magnification 400x).
Figure 4: Histopat Pancreas Wistar Rats dose of 0,5 mg/kg bw (Magnification 400x).
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Figure 5: Histopat Pancreas Wistar Rats Dose 2 mg/kg bw (Magnification 400x).
Figure 6: Histopat Pancreas Wistar Rats Dose 5 mg/kg bw (Magnification 400x).
Figure 7: Histopat Pancreas Wistar Rats K+ (Glibenclamide) (Magnification 400x).
Figure 2 shows that the number of cytoplasmic granules wistar rats normally looks still intact,
no visible presence of clinical symptoms and found no β cell nuclei and other cell
degeneration to necrosis around the islets of Langerhans in mikroskpis examination, both
qualitatively and quantitatively compared control group (K-and K+) and the treatment group.
In contrast to Figure 3 shows that the pancreatic β-cells were detected by staining with
Gomori-Nuclear fast red shown in the figure are colored purple cytoplasmic granules. The
loss of a number of cytoplasmic granules around the islets of Langerhans. Rupture of a
number of β-cell nuclei (karyoreksis), shrinking the cell nucleus and no visible piknosis clear
cell boundaries between β-cells and α-cells around the islands of Langerhans. Rats pancreatic
β-cell degeneration to necrosis caused by alloxan induced a dose 140 mg/kg bw more than
the Wistar rat pancreatic β-cells in the treatment group. This is because alloxan is selectively
destroy pancreatic β-cells through the formation of reactive oxygen species that begins by
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alloxan reduction and characterized by elevated blood glucose levels. Nowhere β-cells around
the islets of Langerhans beta cells than in normal rats. Stroma reduced density of Langerhans
on the island, there is edema, congestion, to undergo necrosis (cell death).
In Figure 4 above have been changes in pancreatic tissue morphology histopathology in rat
islets of Langerhans due Euchresta lesch benn extract dose of 2 mg/kg bw compared with the
negative control group (K-), although the amount of pancreatic β-cell degenerating to
necrosis rather reduced. This means Euchresta horsfieldii lesch benn extract dose of 2 mg/kg
bw can not help the process of pancreatic tissue repair damage caused by alloxan induced. In
contrast to Figure 5 shows that employment Euchresta horsfieldii lesch benn extract dose of 5
mg/kg bw to changes in pancreatic tissue morphology structure is to stimulate cell division.
There is an increasing amount of pancreatic β-cells means in accordance with the theory that
when the cells are injured due to something so potentially stimuli undergo reversible changes
that can be back to normal. Mechanisms of pancreas due to improved Euchresta horsfieldii
lesch benn extract dose of 5 mg/kg bw is likely Euchresta horsfieldii lesch benn extract dose
of 5 mg/kg bw contains large insulincompare treatment groups in a dose of 2 mg/kg bw
resulting in destruction of pancreatic β-cells quickly and normal. Figure nearing still visible
above the cells undergoing necrosis.
Figure 6 does not appear on any cell degeneration to necrosis of the rat pancreatic tissue
around the islands of Langerhans thus showing clear boundaries between β-cells by α-cells.
Similarly, the amount of cytoplasmic granules in the beta cell nucleus has increased to near-
normal conditions so that the pancreatic tissue repair process can take place quickly. In
contrast to Figure 7 morphological changes in the structure of the network in the rat
pancreatic islands of Langerhans due antidiabetic drug administration (Glibenclamide) as a
posttest control. No visible cytoplasmic granules and clear boundaries between β-cells by α-
cells, still seems the cells undergoing necrosis and pancreatic β-cell repair process is not
perfect.
Carried observation of β-cells quantitatively by calculating the number of β-cells in the rat
pancreatic tissue of each group and the control group both treatment groups. β-cells were
detected by staining with Gomori-Nuclear fast red and 400x magnification images of cells are
shown in blue on the islets of Langerhans cell while the other is red.
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CONCLUSION
1. The Euchresta horsfieldii lesch benn leaf extract at a dose of 0.5 mg/kg bw/day, 2 mg/kg
bw/day and 5 mg/kg bw/day can prevent oxidative stress through decrease of
malondialdehyde levels in diabetic rats
2. The Euchresta horsfieldii Lesch benn leaf extract at a dose of 5 mg/kg the best potential
to prevent oxidative stress decreasing of malondialdehyde levels in diabetic rats. Decrease
of malondialdehyde levels by (0,46 ± 0,06) µmol/L and differ statistically significant
with p<0.05
3. The Euchresta horsfieldii lesch benn leaf extract improve the profile of histopathological
tissue of wistar rat pancreatic β-cell damage caused by alloxan induced dose of 140
mg/kg bw.
ACKNOWLEDGMENTS
The author would like to thank all those who have helped to make this research can be done
well.
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