Anggung Praing et al. / The Effect.........

THE EFFECT OF ETHANOLIC EXTRACT OF FALOAK BARK (Sterculia

quadrifida R.Br) AGAINST FREE RADICAL DPPH (in vitro) AND ACTIVITY OF
GLUTATHIONE PEROXIDASE ENZYME IN DIABETIC MICE

Rambu Konda Anggung Praing, Titik Sunarni1, Sunarti1

1
Faculty of Pharmacy, Setia Budi University Surakarta.
Jln. Letjen Sutoyo, Mojosongo-Solo 57127
Tlpn: 0271 - 852 518, Website: www.setiabudi.ac.id

ABSTRACT
Faloak bark (Sterculia quadrifida R.Br) is the part of faloak tree which has antioxidant and
antidiabetic activity. The purpose of this study is to know the antioxidant effect of faloak bark
by in vitro and in vivo method.
Ethanolic extract of faloak bark is tested to the antioxidant activity by bind the free radical
DPPH method (in vitro) with IC50 parameter and using rutin as standard to compare with the
extract. Antioxidant activity test (in vivo) in this study using increased activity of glutathion
peroxidase enzyme in the tissue of mices liver which has inducted by aloxan, and then
analyze the datas using Kolmogorov-Smirnov test and continued by Mann-Whitney test.
The result of this study showed that the ethanolic extract of the faloak bark has antioxidant
activity. Antioxidant activity of ethanolic extract of faloak bark by in vitro test has IC50 value
4,86 ppm, this indicated that the ethanolic extract of faloak bark include of a strong
antioxidant. The result of in vivo test showed that glutathion peroxidase enzyme with the
effective dose 260 mg/Kg weight of mice and the value of increase level of glutathion
peroxidase enzyme is 56,10 U/mg and there is significant difference compared with the
diabetic control group.

Keywords: faloak bark, DPPH, gutathion peroxidase, antioxidant.

INTRODUCTION
Diabetes mellitus (DM) is a group of metabolic disease with hyperglycemic that occur
because abnormalities of insulin secreation and insulin activity or both. Symptoms that
appear because the blood glucose level is increase and decreased insulin secreation10. DM
causes autooksidation of glucose, protein glycation and activation of polyol metabolism
pathways which further accelerate the formation of reactive oxygen compounds12.
The formation of reactive oxygen compounds can increase lipid modification, DNA, and
protein in various tissues. Moleculer modification of these tissues cause imbalance between
protective antioxidant and increased free radical production, this is the beginning of oxidative
damage known as oxidative stress. Furthermore, oxidative stress also contributes to the
deterioration and development of the incidence of complications7.
Faloak bark contains natural antioxidant compounds namely flavonoid and phenolics that are
able to protect the body from free radical attack9. Flavonoid as one of the phenolic
compounds find in many plant tissues can be antioxidant. The antioxidant activity of
flavonoid in faloak bark can donate its hydrogen atom or through bind the metals 6.
Flavonoids can improve insulin receptor sensitivity11.

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RESEARCH METHODS
Making powder from faloak bark. Wash the faloak bark which obtained from Kupang
NTT. Then cut into small pieces and dry using oven with temperature 50C, then continue the
process to make powder from faloak bark using grinder.
Making ethanolic extract of faloak bark. Macerate 500 g powder of faloak bark using 5000
ml ethanol 70% for 5 days (protected from light). Then concentrated the filtrate using rotary
evaporator with temperature 50C.
Identification the ethanolic extract compounds of faloak bark. Identification of the
compounds using chemical reaction method, first preparing the test solution (ethanolic extract
of faloak bark) and reacted with the reactant. The color will change when the chemical
reaction happend.
Test of antioxidant activity (in vitro). The antioxidant activity test was done by mixing 2 ml
of test solution of each concentration series and 2 ml DPPH solution 80 ppm into vials and
leave in dark for 30 minutes and then observed its absorbance at = 517 nm (3 replication),
using blanco from 2 ml metanol p.a and 2 ml DPPH solution 80 ppm. After obtained the %
inhibition datas from all concentration series of extract and rutin, then calculate the value of
IC50 using regression linear equation based on Y = a + bx (% inhibisi vs concetration series of
extract and rutin solution).
Absorbance of blancoAbsorbance of sample
% Inhibition = x 100%
Absorbance of blanco

Test of antioxidant activity (in vivo). Testing was performed for 14 days where blood
sampling was done to measure the initial glucose level (T0). In the same day the mice were
given a solution of alloxan monohydrate 180 mg/Kg weight of mice intraperitoneally. After
5 days of alloxan induced, each test animals with blood glucose level >200 mg/dl (positive
diabetic) was then grouped for blood glucose measurent for the first day (T1). Taking the
blood from venous plexus mice. In the 15th day the mice were killed by dislocation the neck
and then taking the mice liver for measure the GPx activity. Measured of GPx activity using
modified Lawrence and Burk method (1976). 200 l liver supernatants werw added 200 l
phosphate buffers 0,1 mM EDTA, 200 l GSH 10 mM and 200 l GDR (2,4 unit). Then
incubated for 10 minutes at 37C, added 200 l NADPH 1,5 mM and incubated again for 3
minutes at the same temperature and continued addition of 200 l H2O2 1,5 mM. The change
of absorption rate during conversion of NADPH to NADP was measured
spectrophotometrically with = 340 nm for 3 minutes. GPx activity is expresed as mol
NADPH that is oxidized to NADP+ minute-1 mg-1 protein with extrinsic coefficient for
NADPH. The calcultion is
Abs x Vt x 2 x 1000 x 1/mg protein
M unit GSH-Px = 6,22 x Vs
Data analysis
Datas from test of antioxidant activity (in vitro) will analysis using independent samples t-test
to compare the average of IC50 rutin and extract and the function is show that the values are
differ significantly or not.
Datas from test of antioxidant activity (in vivo) will analysis using Kolmogorov-Smirnov test
and continued by Mann-Whitney test.

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RESULTS ANALYSIS
Making of ethanol extract of
faloak bark. 3,6 Kg faloak bark dried and
after drying the weight become 1,8 Kg so
the percentage of dry weight to wet weight
is 50%. The viscous extract obtained from
the maceration process of 500 g of faloak
bark powder is 67,68 g and the yeild of the
extract is 13,54 %. The results of the
calculations can be seen in tables 1 and 2.

Table 1. Drying yield of faloak bark

Sample Wet weight (Kg) Dry weight (Kg) Yield (%)

Faloak bark 3,6 1,8 50

Table 2. Percent yield of the ethanolic of extract faloak bark

Powder weight (g) Extract weight (g) Yeild (%)
500 67,68 13,54

Identification the compound of datas from all concentration series of

ethanol extract of faloak bark.
Identification the compounds using
chemical reaction method showed that the
ethanol extract of faloak bark contains
flavonoid, terpenoid, saponin, tanin and
alkaloid.
Test of antioxidant activity (in
vitro). The solution of extract and DPPH
were reacted for 30 minutes resulting a
stable reaction between the active
compound and DPPH and then, measured
its absorbance at wavelength 517 nm
accordance the maximum wavelength of
DPPH. After obtained the % inhibition
extract and rutin, then calculate the value of
IC50 using regression linear equation based
on Y = a + bx. The value of IC50 is more
effective as antioxidant if the value is
smaller. The definition of IC50 is the ability
of the compounds to inhibit 50% DPPH
activity. The value half maximal inhibitory
concentration (IC50) of ethanol extract of
faloak bark and rutin are 4,86 ppm and 4,23
ppm. They are included as powerful
antioxidant because it contains high
flavonoids and phenolic compounds which
can inhibit the activity of free radical
DPPH9;3.

Table 3. IC50 of ethanolic extract of faloak bark and rutin

Solutions Replication
Extract
Rutin
The result of statistic analysis using
Independent samples t-test to compare the
average of IC50 rutin and extract showed
IC50 (ppm) Mean IC50SD
1 4,86
2 4,86 4,860,01
3 4,87
1 4,23
2 4,24 4,230,01
3 4,23
that the values are differ significantly
(p<0,05).

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Test of antioxidant activity (in the glutathione reduction (GSH) become

vivo). The dose of extract (65, 130, 260 glutathione disulfide (GSSH), while
mg/Kb BB rat) are able to increase the reducing amount of hydrogen peroxide
activity of GPx enzyme in the homogenate (H2O2) converted into 2 water molecules
of rats liver during 14 days. GPx catalyze (H2O)1.

Figure 1. GPx activity (U/mg) of entire treatment groups

GPx activity
90 77.79
GPx activity (U/mg)

80
70 62.83
56.1
60 50.87
50 36.73
40
30
25.87
20
10
0
Normal Negative Compare Dose I Dose II Dose III
group group group group group group

Table 4. Significant difference analysis of variation extract dose groups to control

groups
Groups GPx
N activity (U/mg)SD % Increased the GPx activity
Normal group 5 77,793,28 -
Negative group 5 25,871,07 -
Compare group 5 62,831,44 58,82
Extract dose 65 mg/Kg weight
5 36,7abc0,66 29,57
of mice
Extract dose 130 mg/Kg weight
5 50,87abc1,90 49,14
of mice
Extract dose 260 mg/Kg weight
5 56,10abc0,94 53,89
of mice

Information :
a : differed significantly from the normal group
b : differed significantly from the negative group
c : differed significantly from the compare group

(U/mg)
% Increased the GPx activity = %
(U/mg)
Increased the GPx activity = 100 % - %

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Mean of GPx activity value in normal
group show the highest value (77,79 U/mg)
because various organ in normal condition
are able to work well, including liver that
has a natural defense mechanism of
intracellular antioxidant enzyme such as
GPx that serves to neutralize and accelerate
the degradation of free radical compounds
to prevent damage the macromolecular
components of cells7. In physiological
state, Reactive Oxygen Species (ROS)
maintain homeostasis. Under normal
conditions there is a balance between free
radicals and endogenous antioxidants so
that lipid peroxidation does not occur
which is a oxidation process of long chain
sarurated lipid acid in membranes cells13.
The negative control group showed the
lowest GPx activity value (25,87 U/mg)
and differed significantly when compared
with the normal group (p<0,05), the
comparison group, and the variation extract
dose group. This is because the negative
control group for 14 days was only given
0,5% NaCMC and hyperglycemia
conditions due to alloxan induction.
Alloxan as a free radical agent that causes
autooksidation of glucose, protein
glycation, and activation of polyol
metabolism pathways which further
accelerate the formation of ROS that can
cause moleculer modification of various
tissues, this is the beginning of oxidative
damage due to an imbalance between
protective antioxidants and increased free
radical production. The production of free
radicalcan not be neutralized by
group give glibenclamide showed a
significant difference (p<0,05). This
happened because in this research using
pure glibenclamide who is able to increase
endogenous antioxidant activity in GPx,
SOD, catalase with mechanisms causing
homeostasis in the pancreas so that
endogenous antioxidant (GPx). The
negative control group in this study is used
as a marker of oxidative damage caused by
free radicals. Oxidative damage in this
group can be seen from decrease in GPx
activity when compared with the normal
group because the normal group is not
induced by alloxan so that no oxidative
process occurs5.
Mean of GPx activity value in the
compared control group is 62,83 U/mg and
differed significantly when compared with
the negative control group (p<0,05) based
statistic analysis. This is because the
compared control group was given
glibenclamide. This drug works by binding
and inhibiting the ATP-sensitive potassium
channels (KATP) inhibitory regulatory
subunit sulfonylurea receptor 1 (SUR1) in
pancreatic beta cells. This inhibition causes
cell membrane depolarization, opening
voltage-dependent calcium channels. This
results an increase intrasellular calcium in
the beta cell and subsequent stimulation on
insulin release2.
Mean of GPx activity value in the groups
who give extract with doses 65, 130, and
260 U/mg are 36,73 U/mg ; 50,87 U/mg
dan 56,10 U/mg when compared with the
negative control group showed a significant
difference. These results prove that the
extract has ability to increase GPx activity
and stop the chain reaction of free radical
production because the extract contains
compounds that have antioxidant activity
and antidiabetic. In the test group with the
highest dose of extract compared with the
endogenous antioxidants can nuetralize or
bind the free radicals8.
Compounds that have antioxidant activity
on faloak bark are flavonoid and phenolic.
Flavonoid as one of the phenolic
compounds find in plant tissues can be
antioxidant. The antioxidant activity of
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flavonoid in faloak bark can donate its
hydrogen atom or through bind the metals6.
CONCLUSION
First, ethanolic extract of faloak bark has
antioxidant activity to bind free radical
DPPH with IC50 4,86 ppm. Second,
ethanolic extract of faloak bark can
increase glutathione peroxidase enzyme in
diabetic mice who induced using alloxan.
The effective dose 260 mg/Kg weight of
mice and the value of increase level of
glutathion peroxidase enzyme is 56,10
U/mg.
SUGGESTION
First, further research is needed to see what
compounds are contained in the bark of
faloak which is able to inhibit the free
radical of DPPH and increase the activity
of glutathione peroxidase enzyme.
Secondly, further research is needed by
using other methods and parameters related
to antioxidant effects on ethanol extract of
faloak stem bark.
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