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Bioscience Research
Print ISSN: 1811-9506 Online ISSN: 2218-3973
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RESEARCH ARTICLE BIOSCIENCE RESEARCH,201815(3):1487-1493. OPEN ACCESS

Iron chelation ability and hematological effect of

sappan wood (Caesalpinia sappan, L.) Extract tablet
on iron overload condition of rats
Ratu Safitri1 and Ani Melani Maskoen2,3,4*
1Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran.
Jl. Raya Bandung - Sumedang Km-21, 45363, Jatinangor, Sumedang West Java, Indonesia
2Faculty of Dentististry, Universitas Padjadjaran.Jl. Raya Bandung -Sumedang KM 21, Jatinangor 45363. Sumedang

West Java, Indonesia

3Laboratory of Molecular Genetics, Faculty of Medicine, Universitas Padjadjaran, Jatinangor 45363. Sumedang West

Java, Indonesia
4Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Padjadjaran, Jatinangor 45363.

Sumedang West Java, Indonesia

*Correspondence: amelani@yahoo.com, ratusafitrie@yahoo.com Accepted: 07 June 2018 Published online:27 July 2018

Research of iron chelation ability on sappan wood (Caesalpinia sappan, L) extract tablet of rats (Rattus
norvegicus,L.) on iron overload condition aims to obtain optimum dose sappan wood extract (CSE)
tablet effective as iron chelator herbal. The research was performed using a completely randomized
design (CRD) to 27 rats female Wistar 8 weeks old with an average weight of 200 grams. Research is
divided into 9 treatments with 3 repetitions.Negative control (KN1), the control formula (KN2), Tablet of
sappan wood extract (CSE) dose of 100 mg/kg BW/d (P1), dose of 200 mg/kg BW/d (P2), dose of 300
mg/kg BW/d (P3), dose of 400 mg/kg BW/d (P4), a satellite group (P5), deferiprone dose of 75 mg/kg
BW/d (P6). Data were statistically analyzed using analysis of variance (ANOVA) level of 95% (α = 0.05),
if there is a significant difference, then followed by the Duncan multiple comparison tests. The results
showed that the sappan wood extract tablet is in the normal values range at each measurement of the
blood parameter. It indicates that administered of sappan wood extract tablet only binds the excess of
iron body and does not destabilize of the eritrosit.
Keywords: Caesalpinia sappan, L, iron chelation, tablet, thalassemia

INTRODUCTION Iron overload in thalassemia is the side effect

Thalassemia is a hereditary disease
associated with a proportion of globin chain
synthesis (Vanichsetakul, 2011). Hemolysis and
erythropoiesis ineffective is the main cause of
anemia in thalassemia disease (Kotze et al,
2011). Blood transfusion therapy for thalassemia
should maintain hemoglobin in the normal range,
in order to prevent deleterious effects of anemia. It
is also important to restrict iron overload by
adequate chelation to prevent organ damage due
to the free radical formation (Sharaf et al., 2014).
of ineffective erythropoiesis, increased
gastrointestinal absorption of iron, lack of the
physiological mechanism for excreting the excess
of iron and multiple blood transfusions resulting in
hemochromatosis (Jeon and Sin, 2013).
Complication by iron overload is common and its
toxic effect progressively damage heart, liver and
endocrine glands at later age (Coates et al.,
2014).
Iron chelation therapy is the only method of
iron overload. Iron chelation therapy involves the
Safitri and Maskoen
use of lighting drugs which is able to coordinate
with iron forming complex to reduce exaggerated
levels of iron (Maskoen et al,, 2016). Currently,
desferrioxamine, deferiprone, and deferasirox
clinically used for the treatment of iron overload
(Hoffbrand, 2012). Due to its side effect like
agranulocytosis and neutropenia (Hoffbrand,
2012), as well as its high cost, the third chelator is
considered.
Phenolic and flavonoid compound is
commonly used as the chelating agent (Gupta et
al., 2011; Ebrahemzadeh, 2009). The heartwood
of Caesalpinia sappan, L (CS) contains various
types of phenolic components, such as flavones,
chalcones, xanthones a brazilin (Nirmal et al.,
2014). Brazilin in C. sappan, L has chelation
activity due to hydroxyl functional (Nirmal et al,
2014) and catechol group in its structure (Safitri et
al., 2016).
Dosages form to play the important role of
thalassemia patient. According to most of the
patient is in childhood age, a tablet can be
administered due to its advantages. Tablet can be
given for systemic effect, patient compliance, and
less expensive to manufacture. Tablet can provide
high precision dosing. Tablet form is the most
widely used dosage form because of self-
administration and ease to manufacture (Jaimi
and Rawat, 2013).
In this study, extract of CS heartwood was
made in tablet that consist in different doses 0
mg/kg BW/d, 100 mg/kg BW/d, 200 mg/kg
BW/day, 300 mg/kg BW/d, and 400 mg/kg BW/d
and satellite dose 400 mg/kg BW (P5). The state
of an iron body known by measurement red blood
cells parameter (hematocrit level, hemoglobin,
total erythrocytes, and Erythrocytes index) on rats
(Rattusnovergicus L.) with iron overload.
Determining blood parameter is helpful in
assessing the thalassemia Iron status. This study
aims to get an effective dose of CSE tablet as iron
chelation in rats with iron overload condition by
observing red blood cells parameters
MATERIALS AND METHODS
Material
The material used in this study were distilled
water, deferiprone (Ferriprox ®), ethanol 96%,
iron dextran, dried powder of CS heartwood,
chloroform, rats feed type of CV 151 (Laboratory
standard), 8 weeks old female rats (weight
between 150 – 200 g). Tablet excipient consist,
citric acid, Avicel PH 102, lemon oil. Na CMC,
sodium cyclamate, and Lactose.
Bioscience Research, 2018 volume 15(3):1487-1493
Hematological features of CSE tablet
Tablet Preparations
About 2.5 kg dried powder of CS heartwood
placed into macerator, macerated using ethanol
96% for 3 x 24 hours. Collect all macerate, and
dried using rotary evaporator at 400C to form the
crystalline powder. Sappan wood extract tablet
made by wet granulation method, in which citric
acid, sodium cyclamate, lactose, Avicel PH 102
and powder extract mixing with Na-CMC
mucilage. The mixture was homogenized and
granulated (mesh 12), then dried in the oven
(temperature ± 400C) until constant weight (2
days). The granules were mixed with Mg Stearate,
Avicel PH 102 and lemon oil, until homogeny, and
compressed using single punch machine.
Animal Test and Treatment
The research was performed using a
completely randomized design (CRD) to 27 rats
female Wistar (Rattus norvegicus, L.) 8 weeks old
with an average weight of 200 grams. Animal
initially acclimated to the laboratory environment
for a week. During this acclimatization, rats only
get rat feed type CV 151 and tap water ad libitum.
All treatment was administered orally, therefore
any given substance was made in solution. CSE
Tablet extracts, deferiprone, and iron dextran
dissolve in distilled water beforehand. Treatments
given for 28 days were performed with oral
gavage needle. Iron dextran was given 60 mg/kg
BW in every 3 days, whereas deferiprone and
tablet extract was daily given. Treatments given
are presented in table 1.
Measurement of Parameters
Blood samples were carried on day 29.
Hemoglobin (Hb) measured use Sahli method,
whereas Hb concentration can be determined by
reading the scale on dilution tube. Hematocrit or
pack cell volume (PCV) measured by the
microhematocrit method and the value of
hematocrit expressed in percentage. The
erythrocyte number measured use
hemocytometer while red blood cells number
performed under the microscope with
magnification 45 x 10. Erythrocyte number can
calculate using the formula.
Erythrocyte number = number of erythrocyte in 5 block R x
dilution x 50
RESULTS
Hemoglobin Level
Hemoglobin is the main substance of the
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Safitri and Maskoen Hematological features of CSE tablet

erythrocyte. It’s consist of protein part (globin) and
the non-protein part (heme). Hemoglobin level is
one of the parameters to determine anemia.
Hemoglobin is a compound containing iron, so the
availability of iron will affect hemoglobin levels in
the blood. Based on the results of analysis of
variance (ANOVA) test, showed that there is a
significant difference from each treatment to
hemoglobin levels of rats with iron excess. Then
proceed with Duncan’s multiple range test to know
the significant differences in each treatment. The
measurement results can be seen in figure 1.
Hematocrit Level
Hematocrit (Hct), the proportion of blood
volume occupied by red blood cells, is a major
determinant of blood viscosity. Hematocrit levels
also serve as an indicator of health conditions.
Based on the result analysis of variance
(ANOVA), it showed that CSE tablet in each
treatment had no effect or significantly different on
rats hematocrit value (p>0,05). The Duncan
distance test was not performed due to the result
of ANOVA. Graph of hematocrit values of rats with
iron excess given CSE tablet can be shown in
Figure 2.
Erythrocyte Number
Erythrocytes have been considered as cells
with a transport of oxygen (O2) and to modulate
the microcirculation. Based on the results of
analysis of variance (ANOVA) showed that the
CSE tablet in each treatment gave the significant
effect on the rat's erythrocyte number (p> 0.05),
so the Duncan distance test is performed. Graph
rats erythrocyte number with excess iron given by
CSE tablet can be shown in Figure 3.

18.00 16.33 14.67

Hemoglobin level (gr/dl)

16.00 13.33 c bc
12.00 abc 12.00 11.67
14.00 11.33 11.00
ab ab ab 10.67
ab a
12.00 a
10.00
8.00
6.00
4.00
2.00
0.00
KN1 KN2 KP P1 P2 P3 P4 P5 P6

Figure.1 Hemoglobin level of rats (Rattus norvegicus L) with iron overload condition, given CSE
tabletb.
Note : Based on Duncan test different letters in the same column showed a significant difference in the level of
confidence 95%
Table 1. Treatment of animal test
Iron CSE Tablet
Deferiprone Formula
Dextran Satelit
Ex 100mg/ 200mg/ 300mg/ 400mg/ 75mg/ Without
60 mg/ 400mg/kg
kg BW/d kgBW/d kgBW/d kgBW/d kg BW/d Extract
kg BW/d BW/d
KN1 - - - - - - - -
KN2 - - - - - - - √
KP √ - - - - - - -
P1 √ √ - - - - - √
P2 √ - √ - - - - √
P3 √ - - √ - - - √
P4 √ - - - √ - - √
P5 √ - - - - √ - √
P6 √ - - - - - √ -

Bioscience Research, 2018 volume 15(3):1487-1493 1489

Safitri and Maskoen Hematological features of CSE tablet

Note : KN1 = Rats were given distilled water (negative control); KN2 = Rats were given formulla
without extract / dose 0; KP = Rats were giveniron dextran dose of 60 mg/kgbw (Positive control); P1
= Rats were given iron dextran dose of 60 mg/kg BW and tablet sappan wood extract dose of 100
mg/kgbw/d; P2 = Rats were given iron dextran dose of 60 mg/kg BW and tablet sappan wood extract
dose of 200 mg/kg BW/d; P3 = Rats were given iron dextran dose of 60 mg/kg BW and tablet sappan
wood extract dose of 300 mg/kg BW/d; P4 = Rats were given iron dextran dose of 60 mg/kg BW and
tablet sappan wood extract dose of 400 mg/kg BW/d; P5 = Rats were given iron dextran dose of 60
mg/kg BW and tablet sappan wood extract dose of 100 mg/kg BW/d; Then allowed to be left for 14 days
post-treatment; P6 = Rats were given iron dextran dose of 60 mg/kg BW and deferiprone 75 mg/kg
BW/d

54.00 52.67
51.00
Hematocrite Level (%)

a
50.00 50.33 50.00 50.00
52.00 a
a a a a
50.00 47.67 47.33
a a
48.00 45.33
46.00 a

44.00
42.00
40.00
KN1 KN2 KP P1 P2 P3 P4 P5 P6

Figure.2 Hematocrit level of rats (Rattus norvegicus L) with iron overload condition, given CSE
tablet.
Note : Based on Duncan test different letters in the same column showed a significant difference in the level of confidence 95%
14.00 12.13 11.70 11.23
c 10.60
Number of Erythrosit (Juta/µl)

c bc
12.00 9.53 b 10.63b
9.07 9.23 9.17
a a a a
10.00
8.00
6.00
4.00
2.00
0.00
KN1 KN2 KP P1 P2 P3 P4 P5 P6

Figure.3 Erythrocyte number (Rattus norvegicus L) with iron excesses condition, given CSE
tablet.
Note: Based on Duncan test different letters in the same column showed a significant difference in the
level of confidence 95%

Bioscience Research, 2018 volume 15(3):1487-1493 1490

Safitri and Maskoen
DISCUSSION
Duncan test results showed that rats
hemoglobin levels, which were given iron dextran
(16.33 gr / dl) had higher levels and significantly
different than the negative control group which
was only given distilled water (12 g / dl), but not
significantly different from the group of rats who
were given CSE tablet at the dose of 100 mg /kg
BW/d and in group of rats given only tablet
formula. Hemoglobin levels of rats which were
given Fe addition in the feed were higher than the
group without Fe addition. Despite having lower
hemoglobin levels than iron dextran group, but the
rat's hemoglobin level which was given the tablet
formula was slightly higher than the negative
controls group.This is presumably because the
content of lemon oil in the tablet formula helped
the rats body in absorbing iron contained in the
feed. It is known that vitamin C in lemon oil can
help the body in improving iron absorption.
Meanwhile, in the administration of CSE tablet at
doses of 200, 300 and 400 mg/kg BW/d and
satellite groups showed values of hemoglobin not
significantly different than deferiprone and control
groups. This indicates that administration of CSE
has chelation effect as well as deferiprone.
The administered of CSE from low to high
doses shows the decreased of hemoglobin levels.
The Low level of hemoglobin is given by CSE
dose 400 mg/kg BW/d that is 11 gr/dl or decrease
about 48,45% compared to iron dextran group.
The decrease of Hemoglobin level which was
given of CSE shown the ability of CSE as iron
chelation, its mean that iron-induced into rats
body cannot be used for hemoglobin formation.
Winter, 2014 mentions that the iron absorbed
from the intestinal lumen will be bound directly to
apotransferrin which will bring Fe to the liver and
be used for hemoglobin formation (Winter and
William, 2014). However, in all treatments in rats,
it still showed a normal range of hemoglobin
levels because it was still in the normal state,
which is about 11-18g /dl. Murphy, W.G (2014),, it
is not the same in thalassemia patients who
received blood transfusions due to hemoglobin
formation disorders (Murphy, 2014).
The percentage of hematocrit is the ratio of
erythrocytes to total blood volume. Hematocrit is a
measure representing of erythrocytes in 100 ml of
blood, so the results are expressed in
percentages (Andrew et al., 2007). Based on
Figure 2, it is a known that the rat's hematocrit
level in general in each group showed no
significant difference. However, the iron dextrans
Bioscience Research, 2018 volume 15(3):1487-1493
Hematological features of CSE tablet
group had the highest hematocrit level which is
52.67%. The administration of iron dextran can
increased rats Hematocrit level 10.49% compared
with control rats group which is given distilled
water.
According to Murphy, W.G (2014), the
greater amount of iron in the body, the higher of
hematocrit level. The main reason is iron play
important role in the red blood cells (Erythrocytes)
formation (Murphy, 2014). Figure 2 showed that in
administered of CSE tablet with doses of 100,
100, 200, 300 and 400 mg/kg BW/d and
deferiprone 75 mg/kg BW/d have lower hematocrit
levels than dextran iron. The normal level of rat
hematocrit is between 36-48%, which means the
administered of CSE tablet did not decrease the
red blood cells number. This shows that CSE
tablet only bind the excess of iron body and not
disrupt the stability of the erythrocytes
In figure 3, it is known that erythrocyte number
is given iron dextran (12,13 x 106 / μl) showed the
higher amount of erythrocytes and significantly
different from control group (9.07 x 106 / μl). This
can explain that the excess iron from iron dextran
use to forming red blood cell in rat body, so it can
increase the erythrocyte number. While in the
administration of deferiprone cause decreased of
erythrocytes number lower than rats given iron
dextran. This may occur because some of the iron
ion excesses from iron dextran have bound to
deferiprone molecule, so only a few iron ions are
used for forming erythrocytes.
CSE tablet administration at dosage 100 and
200 mg/kg BW/d was not different from the
control treatment. Meanwhile, at the higher dose
which is 400 mg /kg BW/d give significant effect
compared with control group. Although not
significantly different from the iron dextran group,
the rat has received orally CSE tablet at 400 mg
/kg BW showed higher amounts of erythrocytes
than control rats treated only with distilled
water.This may be due to low doses (100 and 200
mg/kg bb) have been able to bind iron excess in
rat with iron overload condition, So it can’t be able
to form red blood cells. While at higher doses (400
mg/kg bb), there is also an ability to stimulate the
formation of erythrocytes with characterized by
the increasing of higher erythrocytes number than
the control group.
In this study, the total amount of erythrocytes
ranged between 9.07 to 12.13 106/μl, higher than
the normal erythrocyte range. According to
Luciana et al., (2015), normal erythrocytes
number in rats ranged from 7.2 to 9.6 x 106 /μl (da
SilveiraCavalcante, 2015). The amount of
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Safitri and Maskoen
erythrocytes is affected by several factors
including age, sex, hormones, weight and other
factors (Jeon et al., 2013; Galanello and Origa,
2010) add that nutritional factors, blood volume
also affects the number of erythrocytes.
The results showed that the sappan wood
extract tablet is in the normal values range at
each measurement of the blood parameter. It
indicates that administered of sappan wood
extract tablet only binds the excess of iron body
and does not destabilize of the erythrocyte.
CONCLUSION
Based on the research, it can be concluded that
iron content through iron dextran administration
can increase hemoglobin and hematocrit levels
however CSE at various doses can decrease
hemoblobin, hematocrit and erythrocytes to the
same level as normal rat condition. Administation
of CSE at various doses binds excess iron, but
does not disrupt the stability of the amount of
erythrocytes, even CSE can stimulate the
formation of erythrocytes. The results showed that
CSE able to bind iron excess and does not disrupt
the stability of hemoblobin formation, hematocrit
and erythrocytes. CSE produces just as safe as
depheriphrone.
CONFLICT OF INTEREST
The authors declared that present study was
performed in absence of any conflict of interest.
ACKNOWLEGEMENT
The team of research would like to expressed
appreciation to Ministry of Research, Technology
and Higher Education Republic of Indonesia that
funded the research through the Scheme of
Applied Research of excelence of higher
education (PTUPT) in 2018 with theme of
“Chronic Toxicity Studies, Assay for
Antituberculosis and Antibacterial Causes of
Respiratory Tract Infection Of Sappanwood
(Caesalpinia sappan L.) and Brazilin Compound.
Also thank to our student who helped this
reseach in animal experimental, and bio assay :
Kenti Prahmanti and Ahmad Sazali.
AUTHOR CONTRIBUTIONS
RS and AMM, designed the study and carried out
the collecting sample. The RS carried out the
experimental in-animal research, biochemical
testing. AMM interpreted the results and statistical
analysis, helped complete the draft manuscript. all
authors read and approved the final version. RS
Bioscience Research, 2018 volume 15(3):1487-1493
Hematological features of CSE tablet
Copyrights: © 2017 @ author (s).
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(CC BY 4.0), which permits unrestricted use,
distribution, and reproduction in any medium,
provided the original author(s) and source are
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journal is cited, in accordance with accepted
academic practice. No use, distribution or
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with these terms.
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