Abeer Temraz et al.

/ Journal of Pharmacy Research 2009, 2(5),798-802

Research Article
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Treatment of Experimental Hemolytic Anemia by Some Antioxidant Drugs

Walid Hamdy El-Tantawy and Abeer Temraz *
National Organization for Drug Control& Research, P.O. 29 Dokki, Cairo, Egypt
*Pharmacognosy Department, Faculty of Pharmacy for Girls, Al-Azhar University, Nasr City, Cairo, Egypt
Received on:30-09-2008; Accepted on: 25-03-2009
ABSTRACT
Hemolytic anemia is caused by abnormal breakdown of red blood cells (RBCs) either in the blood vessels (intravascular hemolysis) or
elsewhere in the body (extravascular). The aim of this work was to investigate the efficacy of treatment with four antioxidant drugs containing
Silymarin available in Egyptian market, in experimental anemia model of rat induced by single dose of phenylhydrazine (80 mg/kg) ip. The
induction of anemia led to a significant decrease in hemoglobin level, red blood cells count, and haematocrit percent, and there was an
increase in serum iron concentration, total iron binding capacity and percent of transferrin saturation when compared to their corresponding
control. Oral administration of four preparations containing antioxidants to rats for 2 weeks resulted in a significant increase of hemoglobin
level, red blood cells count, and haematocrit percent. There was also a decrease in serum iron concentration, total iron binding capacity and
percent of transferrin saturation, when compared to the group treated with phenylhydrazine (anemic group) to be within the normal values.These
results suggest the use of antioxidants in cases of diseases resulting in hemolytic anemia.

Keywords: , Anemia, PHZ, antioxidants

INTRODUCTION
Oxidative stress is involved in the pathogenesis of many
cardiovascular diseases, including hypercholesterolemia, atheroscle-
rosis, diabetes, hypertension, hypoxia, ischemia reperfusion injury
and heart failure (1, 2). The hemolytic anemia that can accompany
therapy with arylamine drugs, such as dapsone, and certain natural
products, such as the fava bean pyrimidine aglycone, divicine, has
been well documented (3).The mechanism underlying hemolytic ac-
tions of several agents on red cells has long been studied, and it has
been established that hemolytic injury is associated with oxidative
stress within erythrocytes. This concept is supported by the fact that
hemolytic damage is accompanied by the generation of reactive oxy-
gen species (ROS), glutathione depletion, haemoglobin (Hb) oxida-
tion, and Heinz body formation in red cells. It has also been reported
that hemolytic agents caused membrane lipid peroxidation and/or
denaturation of cytoskeletal protein (4).Silymarin refers to the extract
from the seeds of the plant Silybum marianum, also called “milk
thistle”. It has been used for over 2,000 years. During the Middle
Ages the seed of the milk thistle was commonly used to treat liver
diseases. The active ingredients of milk thistle are chemicals called
flavonoids. The flavonoids in milk thistle are silybin, silydianin, and
silychristin. Together, they are called silymarin (5, 6). Silymarin ap-
pears to promote the growth of some types of cells in the liver. Milk
thistle can help prevent or reverse liver damage caused by alcohol,
recreational drugs, pesticides, some poisons, or hepatitis.Rather, milk
thistle is used with the hope that it would minimize the damage to the
liver that HCV can cause. Studies suggest that silymarin can block
various types of toxins from entering and injuring liver cells (7).
*Corresponding author. Tele.: 002024018031
Telefax: +002024018033; E-mail: abeertemraz@yahoo.com
Silymarin may be an effective “antioxidant,” which means
milk thistle may help fight a destructive chemical process in the body
known as “oxidation.” In oxidation, harmful substances produced in
the body (called free radicals) can damage cells. Some studies suggest
that milk thistle silymarin can prevent these substances from damaging
liver cells. Milk thistle’s Silymarin is thought to prevent inflammation
(swelling) of the liver; this may be described as displaying anti-
inflammatory properties (8).
Glutathione (GSH) is a tripeptide consisting of glutamic acid,
cysteine and glycine (9). Glutathione in its reduced state has a nucleo-
philic sulfhydryl group which bind covalently with electrophilic sites
on the reactive metabolites. GSH is therefore an important defense
mechanism against certain toxic compounds such as some drugs and
carcinogens (10).GSH is likely to be involved in the detoxification of
free radicals. GSH play a role in protection against damage caused by
oxidizing environments such as hyperoxia, hyperbolic oxygen and oth-
ers (11, 12).GSH promotes the antioxidant properties of vitamins C and
E by maintaining them in the reduced state (13, 14).Vitamins are ideal
antioxidants to increase tissue protection from oxidative process due
to their easy, effective and safe dietary administration in a wide range
of concentrations without harmful side effects (15). Furthermore, sele-
nium (Se) is an essential trace element for mammalian cells. It has
regulatory functions in cell growth, cellular death and modulates sig-
nal transduction in various cells (16).The aim of this work was to in-
vestigate the efficacy of treatment with some antioxidants of phenyl-
hydrazine (PHZ) induced- hemolytic anemia in rats.

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540 nm and red blood count was determined using the method de-
MATIERALS & METHODS
scribed by (18). Hematocrit percent was measured using the
Biological procedure
microhaematocrit reader after centrifuging blood containing
heparinised capillary tubes at 6.7×g for 5 min (19).
Anaemia was induced by intraperioneal injection of PHZ at 80 mg/
kg once, as described previously by Swann and Contrera (17). PHZ
Biochemical analysis
was dissolved in saline and administered ip.
Serum iron and total iron binding capacity (TIBC) were determined
Animals and experimental protocol
according to (20) and (21). Transferrin saturation was calculated by
the serum iron/TIBC ratio.
Healthy male Swiss albino rats weighing between 120–150 g each
were used for the investigation. The animals were housed at a tem-
Statistical analysis
perature, humidity controlled room and a 12-h light-dark cycle (lights
on at 0600 h). Rats were supplied with a standard pellet diet and tap
Results are expressed as the mean ± SEM, The data were statistically
water was freely available. Six normal control rats treated with distilled
analyzed using a one-way analysis of variance (ANOVA).Statistical
water (2 ml/kg p.o.) they served as a control (Group 1).
significance was determined at a level of p < 0.05.
Thirty rats were injected ip with PHZ at 80 mg/kg once, following the
RESULTS
injections of PHZ, rats were divided in five groups each of six.
Hemoglobin level
Group (2): 6 rats treated with distilled water (2 ml/kg p.o.) and served
as untreated anemic group.
Fig (1) illustrates the mean hemoglobin level in different groups.
Group (3): 6 rats treated with 100 mg/kg Silymarin (Legalon 70 mg,
The level in anemic rats (Group 2) was 6.01 ± 0.46 g/dl which signifi-
CID, Giza, Egypt).
cantly decreased as compared to their corresponding control (Group
Group (4): 6 rats treated with 100 mg/kg Silymarin, 140 mg/kg 1) 8.79± 0.29 g/dl, P< 0.05. After treatment, the hemoglobin level in
Acetylcysteine, 70 mg/kg Vitamin E, 100 mg/kg Vitamin group (3) treated with Legalon was 8.06 ± 0.2, group (4) treated with
C , 18 µg/kg Selenium, 7 mg/kg Zinc and 200 IU Vitamin Silymarin Plus was 9.1± 0.15, group (5) treated with Selenium ACE was
A (Silymarin Plus, SEDICO pharmaceutical Co. 6 Octo- 8.83 ± 0.63 g/dl which was significantly increased when compared
ber City, Egypt). with that of anemic rats (group 2), P< 0.05 and no significant differ-
Group (5): 6 rats treated with 4.2 IU Vitamin A, 80 mg/kg Vitamin E, 250 ence as compared to that of normal control, P> 0.05.
mg/kg Vitamin C and 270 µg/kg Selenium (Selenium ACE,
Interpharma, Egypt) On the other hand, the hemoglobin level in group (6) treated with
Group (6): 6 rats treated with 200 mg/kg Glutathione, 250 mg/kg Vita- Hipamax Plus was (11.01 ± 0.46), revealed significant increase when
min C, 120 mg/kg Vitamin E, 200 µg/kg Selenium, 60 mg/ compared with that of normal control (Group 1), anemic rats (group 2),
kg Zinc, 8 mg/kg Nicotinamide, 8 mg/kg Vit B1, 8 mg/kg and other treated groups, P< 0.05.
Vit B2, 10 mg/kg Vit B6, 50 mg/kg Vit B12 and 580 mg/kg
Silymarin (Hipamax Plus,Marcyrl Pharmaceutical Indus- Red blood cells count (RBCs) x 10 6/µl
tries, El-Obour City, Cairo, Egypt)
In normal control (Group 1) the mean value of RBCs count was 3.03
Treatment and blood samples ± 0.12. In anemic rats (Group 2) the mean value (2.11± 0.12) which was
significantly decreased as compared to that of normal control (Group
The animals were administered the antioxidants orally by orogastric 1), P< 0.05. After treatment, the mean value of RBCs count in group
catheter. Treatment started after twenty four hours from injection of (3) treated with Legalon was 3.51 ± 0.23, group (4) treated with Silymarin
PHZ and once daily for two weeks. Blood samples were collected after Plus was 3.48 ± 0.19, and group (5) treated with Selenium ACE was 3.39
24 hours from PHZ injection (for testing of induction of anemia), and ± 0.2 which showed significant increase when compared with that of
two weeks after treatment from the tail vein into a microcentrifuge anemic rats (group 2), P< 0.05 and no significant difference as com-
tube containing 50 mM ethylenediamine tetraacetic acid (EDTA) for pared to that of normal control, P> 0.05.
the determinations of hematocrit and hemoglobin and red blood cells
count. Serum was separated for the determination of iron and total On the other hand, the mean value of RBCs count in group (6)
iron binding capacity (TIBC). treated with Hipamax Plus was 5.53 ± 0.33, revealed significant in-
crease when compared with that of normal control (Group 1), anemic
Hematological measurements rats (group 2), and other treated groups, P< 0.05, Fig (2).

The hemoglobin concentration was determined colorimetrically at

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Hematocrit %

In normal control (Group 1) the mean value of hematocrit % was

27.18 ± 0.98. In anemic rats (Group 2) the mean value (17.76± 1.15)
which was significantly decreased as compared to that of normal con-
trol (Group 1), P< 0.05. After treatment, the mean value of hematocrit
% in group (3) treated with Legalon was 24.23 ± 0.55, group (4) treated
with Silymarin Plus was 27.1 ± 0.49, and group (5) treated with Sele-
nium ACE was 26.89 ± 1.71 which revealed significant increase when
compared with that of anemic rats (group 2), P< 0.05 and no significant
difference as compared to that of normal control, P> 0.05.

On the other hand, the mean value of hematocrit % in group (6)

treated with Hipamax Plus was (32.46 ± 1.03), revealed significant in-
crease when compared with that of normal control (Group 1), anemic
rats (group 2), and other treated groups, P< 0.05, Fig (3).

Serum iron concentration (µg/dl)

Fig (4) describes the mean value of serum iron concentration among
control, PHZ, antioxidant-treated groups. The mean value in control
group (Group 1) was 119.5 ± 5.43 µg/dl. In anemic rats (Group 2) the
value was 501 ± 12.66 µg/dl which significantly increased as
compared to normal control (Group 1), P< 0.05. After treatment, the
mean value ofserum iron concentration in group (3) treated with Legalon
was 122 ± 5.03, group (4) treated with Silymarin Plus was 121.6 ± 5.1,
and group (5) treated with Selenium ACE was 120 ± 5 µg/dl which
revealed significant decrease when compared with that of anemic rats
(group 2), P< 0.05 and no significant difference as compared to that of
normal control, P> 0.05. The mean value in (Group 6) treated with
Hipamax Plus (95.5 ± 3.67) µg/dl revealed significant decrease when
compared with that of normal control (Group 1), anemic rats (group 2),
and other treated groups, P< 0.05.

Serum total iron binding capacity (TIBC µg/dl)

Fig (5) describes the mean value of serum total iron binding capacity

among control, PHZ, antioxidant-treated groups. The mean value in

control group (Group 1) was 461 ± 7.47 µg/dl. In anemic rats (Group 2)
the value was 850 ± 12.24 µg/dl which significantly increased as com-
pared to normal control (Group 1), P< 0.05.

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 Abeer Temraz et al. / Journal of Pharmacy Research 2009, 2(5),798-802
group (Group 1) was 25.7 ± 1.07. In anemic rats (Group 2) the value was
58 ± 0.55 which significantly increased as compared to normal control
(Group 1), P< 0.05. After treatment, the mean value in group (3) treated
with Legalon was 27.3 ± 1, group (4) treated with Silymarin Plus was
26 ± 1.29, and group (5) treated with Selenium ACE was 25.7 ± 0.93
which revealed significant decrease when compared with that of ane-
mic rats (group 2), P< 0.05 and no significant difference as compared
to that of normal control, P> 0.05. The mean value in (Group 6) treated
with Hipamax Plus 22.5 ± 0.91 revealed significant decrease when com-
pared with that of normal control (Group 1), anemic rats (group 2),and
other treated groups P< 0.05.

DISCUSSION

This study aimed to evaluate the effect of treatment with antioxi-

dants on the hemolytic anemia induced by phenylhydrazine in rat.

Data from this study showed that PHZ causes a significant re-
duction in hemoglobin level, red blood cells count, hematocrit per-
cent. On the other hand there was a significant increase in serum iron
70
concentration, total iron binding capacity and transferrin saturation.
*
Treatment with antioxidants led to restoring normal levels of these
60 hematological and biochemical parameters. The most pronounced ef-
tra fect in case of treatment with Hipamax Plus which is composed of
ns 50
fer
Glutathione, Vit. C, Vit. E, Selenium, Zinc, Nicotinamide, Vit. B1, Vit. B2,
rin 40 Vit. B6, Vit. B12 and Silymarin. This may be due to synergistic effect.
Sa
tur 30 * Phenylhydrazine (PHZ) is a strong oxidant agent, which is exten-
ati
on 20 sively used in industry, laboratory and therapeutic settings. A variety
of toxic effects of PHZ have been described, including hemolytic ane-
10 mia, hypoxia, inflammation, alterations in the liver, kidney, central ner-
0
vous system, autoimmune disturbances and cancer (22-26).PHZ is
known to shorten life-span of red blood cells (RBCs) resulting in se-
Control PHZ
Legalon vere hemolytic anemia, increased iron absorption and tissue iron over-
SilymarinSPlus Hipamax Plus
elenium ACE load (27).The hemolytic states are unusual in that increased absorp-
tion continues in the face of an iron-replete body store without accu-
Fig ( 6):Percent of transferrin saturation among mulating iron in massive quantities (28).
control,PHZ and antioxidants treated rats .
The auto-oxidation of PHZ leads to generation of reactive oxygen
Significantly different from control.
species (ROS) and a complex array of PHZ-derived radicals, such as
phenylhydrazyl radical, phenyldiazene and benzenediazonium ions
After treatment, the mean value in group (3) treated with Legalon was (Misra and Fridovich, 1976).Among reactive oxygen and nitrogen
445 ± 6.94, group (4) treated with Silymarin Plus was 470 ± 10.6, and species, superoxide anion (O.-2), hydrogen peroxide (H2O2) and nitric
group (5) treated with Selenium ACE was 471 ± 4.49 µg/dl which re- oxide (NO) appear most important in involving vascular pathology
vealed significant decrease when compared with that of anemic rats (29-31).
(group 2), P< 0.05 and no significant difference as compared to that of
normal control, P> 0.05. The mean value in (Group 6) treated with It has been demonstrated that the flavonoid (antioxidant), neutral-
Hipamax Plus (421 ± 8.16) µg/dl revealed significant decrease when ized ROS by directly reacting with (O.- ), NO and peroxynitrite (32-34).
2
compared with that of normal control (Group 1), anemic rats (group 2), Therefore, antioxidant may preserve vascular function and protect
and other treated groups, P< 0.05. vascular injuries from ROS and perhaps from other oxidant species,
including PHZ radicals (35). Another possible mechanism is that the
Transferrin Saturation: antioxidant could stimulate erythropoisis process (36).

Fig (6) describes the mean value of transferrin saturation among In a previous work, treatment a model of hemolytic anemia induced
control, PHZ, antioxidant-treated groups. The mean value in control by injection of PHZ with some Ayurvedic drugs. The author reported

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that the Ayurvedic drugs prevented the action of PHZ by maintaining
RBC count, and Hb content. These drugs might have counteracted
the action of PHZ by preventing lipid peroxidation and free radical
generation. Flavonoid silybin, protects RBCs from haemolysis by pre-
venting the generation of free radicals and maintaining the membrane
integrity of the RBCs thus preventing the haemolysis by counteract-
ing PHZ induced damage (37).
These mechanisms explain the antianaemic properties of the formu-
lations in the current study. In summary, the present study provide
the evidence that the antioxidant could reduce oxidative stress in the
PHZ model, and this associates with a prevention of hemolytic anemia
by restoration of normal hemoglobin level, red blood cells count and
hematocrit percent.
Further investigation regarding the mechanism of action of antioxi-
dant in the management of hemolytic anemia is still required.
CONCLUSION
The present study has lent credence to the efficacy of antioxdants
as anti-anemic agent in the management of hemolytic anemia.
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