European Journal of Scientific Research
ISSN 1450-216X Vol.22 No.4 (2008), pp.494-500
© EuroJournals Publishing, Inc. 2008
http://www.eurojournals.com/ejsr.htm
Acute Toxicological Studies on the Extract of Iraqi Peganum
Harmala in Rats
Zuhair Muhi-eldeen
School of Pharmacy / Petra University, Amman / Jordan
Kassim J. Al-Shamma
School of Pharmacy / Baghdad University, Iraq
Tawfik M. Al- Hussainy
School of Pharmacy / Petra University, Amman / Jordan
Elham N. Al- Kaissi
Department of Pharmaceutics, School of Pharmacy
Petra University, P.O. 961343 Amman, Jordan
School of Pharmacy / Petra University, Amman / Jordan
E-mail: kaielham@hotmail.com
Tel: 00962-605715546; Fax: 00962-6-5715551
Ali M. Al-Daraji
School of Veterinary / Baghdad University, Iraq
Hassan Ibrahim
School of Medicine / Baghdad University, Iraq
ِAbstract
Acute toxicological studies of the aqueous extract of Iraqi Peganum harmala in rats
were conducted. The LD
50
value of the extract in rats given intramuscularly was 420
mg/kg. Tremor and convulsion were observed in most of the treated rats specially those
given large dose. These toxic signs are probably due to the alkaloids content of the extracts
which have a central nervous system stimulant effects.
Histological examination of organs and tissues of treated rats were normal
indicating that the extract has a low level of toxicity: However, sever local inflammatory
reaction in the muscles at the site of injection was observed which caused a significant
increase in the counts of leucocytes and neutrophil. This indicates that the intramuscular
route is not a proper route of administration.
Keywords: Peganum harmala, Acute and subacute toxicity.
1. Introduction
Peganum is a small genus belonging to the family Zygophylaceae and mainly distributed in the
Mediterranean region. Pegenum harmala is the only species found growing wild in the middle and
Acute Toxicological Studies on the Extract of Iraqi Peganum Harmala in Rats
495
northern parts of Iraq. The plant is rich in alkaloids and contains up to 4% total alkaloids (Abdel-Fattah
et al, 1997; Chopra et al, 1958). The principle alkaloids present are harmaline, harmine, harmalol and
peganine (Abdel-Fattah et al, 1995). It also contains fixed oils. There are several reports which
indicated the great variety of pharmacological and biological activities of Peganum harmala such as
antibacterial, antifungal and MAO inhibition (Abdel-Fattah et al, 1997) and to be effective in the
treatment of dermatosis (Saad and Rifaie, 1980), hypothermic (Abdel-Fattah et al, 1995) and cancer
(Adams, 1983). Recently groups of workers reported that the aqueous extract of peganum harmala
possesses antinociceptive analgesic and anti-inflammatory properties (Monsef et al, 2004).
In order to assess the toxicity of the aqueous extract, acute and subacute toxicological studies
were carried out in rats. The aqueous extract was given by intramuscular route in order to exclude the
role of gastrointestinal tract in regard absorption and first pass effect.
2. Materials and Methods
2.1. Extract preparation
The dry seeds of Iraqi Peganum harmala (100 g) were grinded and then were extracted with purified
water for 24 hours in continuous (Soxhelt) apparatus. The extract was filtered, and water was removed
by evaporation on a rotator evaporator under vacuum at 60ºC to a small volume. A small amount of
diluted ammonia was added to make pH of 9. Subsequently, 100 ml of chloroform was added and
slowly shake for several minutes until the alkaloids enter to the chloroform phase. This was repeated
for three times and total chloroform phase was evaporated, yielding a total alkaloid extract.
2.2. Animals
Albino Wister rats of either sex, weighing 200-350 g were used throughout the study. They were fed a
commercial chow, water was given ad libitum.
The animals were divided into four groups designated as A, B, C, D. each group consists of 42
rats divided to 6 subgroups of 7 rats. Rats of group A treated with a single daily intramuscular (IM)
LD
50
dose of the aqueous extract. Rats of group B treated with a single daily (IM) 1/2 LD
50
dose of the
aqueous extract and group C treated with a single daily (IM) 1/4 LD
50
dose of the aqueous extract.
Group D taken as control treated with IM saline. Treatment was done for 6 weeks.
The experiment protocol was revised and approved by the ethical committee of school of
pharmacy, Petra University
2.3. Haematological methods
Total RBC count, WBC count and differential leucocytes count were performed visually according to
the methods described in (Lewis et al, 2006). Haematological studies were done weekly for 6 weeks.
Blood was withdrawn from the heart directly after anesthetizing the animals lightly with ether. Bone
marrow examination was done on all animal remained alive after the end of the experiment.
2.4. LD
50
determination
LD
50
(420 mg/kg) was calculated according to Litchifield and Wilcoxon (1949) method. The aqueous
extract was given intramuscularly. Groups of 6 rats were used for each dose given.
2.5. Biochemical tests
Biochemical tests include the measurement of GPT, GOT and ALP enzymes. Sodium, potassium,
sugar, urea and creatinine were estimated in the plasma of some rats. The enzymes were estimated by
autoanalyzer (Technicon AA2). Other biochemical tests were carried out by autoanalyzer (Technicon
SMA-600).
496
Zuhair Muhi-eldeen, Kassim J. Al-Shamma, Tawfik M. Al- Hussainy,
Elham N. Al- Kaissi, Ali M. Al-Daraji and Hassan Ibrahim
2.6. Pathological methods
Rat killed by exsanguinations. Tissue specimens (heart, lung, spleen, skin, testis, ovary, glands) were
cut transversely into 1 cm thick slices. These slices were fixed with 10% formalin for 3-4 days,
embedded in paraffin and cut at 4 thick. The sections were generally stained with haematoxylin and
counter stained with eosin.
3. Results
3.1. LD
50
value and signs of toxicity
Table (1) showed the mortality rate induced by different doses of the aqueous extract of harmala plant.
From this table and the dosage mortality curve, the LD
50
value of the extract by IM route in rats was
calculated to be 420 mg/kg.
Table 1:
Percent mortality in rats given IM injection of the aqueous extract of Peganum harmala
Dose (mg///kg)
No. of animals
No. of deaths
Percent mortality
200 6
0 0
300 12 2 16.6
350 12 3 25
400 12 4 33.7
450 6
5 83.3
500 18 14 77.7
550 6
6 100
There were no significant changes in the weight of rats given the extract compared to the
control rats. Tremor appeared in most of rats of group A and it was less evident in rats of group B and
C. treated rats appeared more quite and less reactive than control rats. Convulsions appeared in some
treated rats specially those die after treatment.
3.2. Biochemical data
Table (2) showed the activity of the enzymes ALP, GOT and GPT in blood of rats at different intervals
after treatment.
Table 2:
Effect of the aqueous extract of harmala plant on the GPT, GOT and ALP enzymes in blood of
treated rats.
Group
Time (weeks)
GPT (U/L)
GOT (U/L)
ALP (U/L)
A
–
125
± 21
242
± 57
201
± 25
1
91
± 11
289
± 17
208
± 54
2
125
± 18
295
± 11
266
±13
3
107
± 15
250
± 36
211
± 13
B
6
145
± 5
277
± 21
170
± 20
1
96
± 5
More than 300
194
± 10
2
108
± 10
287
± 12
171
± 24
3
111
± 15
276
± 31
175
± 15
C
6
179
± 13
292
± 13
144
± 11
1
106
± 14
187
± 6
235
± 18
2
108
± 18
215
± 35
211
± 74
3
85
± 10
170
± 30
224
± 46
D
6
165
± 30
183
± 6
136
± 40
Acute Toxicological Studies on the Extract of Iraqi Peganum Harmala in Rats
497
No significant changes in these enzymes were observed between treated and control rats.
3.3. Pathological data
3.3. 1. Gross Pathology
The animals were at poor nutritional status and the hair was rough. Lesions were limited to the
muscles, mainly at the site of inoculation. These muscles had haemorrhagic, necrotizing appearance
with serosanguinous fluid oozing from them. They had pale or yellowish-green discoloration, a softer
consistency and swollen appearance. This lesion was exaggerated in animals of group A and B, and
was seen to a lesser degree in group C. there was also haemorrhagic appearance and necrosis of the
subcutis. Occasionally seen between the muscle bundles are cyst-like formation filled with inspissated
material colored like that of the drug i.e. dark brown or chocolate color. Also there was sloughing of
the skin above the site of infection with scab formation.
On the next weeks, especially the 5
th
and 6
th
weeks, post-inoculation, lesions became more
focally extensive and some foci measured 2 X 1.2 cm. in six rats from two groups (B & C), the hind
legs had rounded, firm, well- delineated masses in the muscles with a necrotic, granular, pale green to
grayish centers and an outer whitish firm band of connective tissue. These masses were seen within the
thigh muscles and they were 1.5 cm at largest diameter.
There were no lesions in muscles or organs of the saline- injected rats.
3.3.2. Histopathology
In all experimental groups, the skeletal muscles showed swelling to shrinkage of muscle fibers with
loss of their differential staining but mostly there is increased eosinophilia of the sarcoplasma with loss
of boundaries of muscle cells, loss of their nuclei and loss of their cross striations. Also seen is
fragmentation of muscle fibers. In most other foci, this lesion progressed to focal areas of frank
necrosis. There is infiltration by mixed inflammatory cell types consisting of polymorphonuclears,
predominantly neutrophilic and mononuclear, predominantly macrophages. The cytoplasm of the latter
was loaded with yellowish-brown pigment. On special stains, some of this engulfed material appeared
to be haemosiderim. There is oedematous and haemorrhagic separation of muscle bundles together
with focal area of small, scattered irregular, basophilic foci of regenerating muscle fibers. Towards the
end of the experiment and mainly on the 5
th
and 6
th
weeks, there were fibroplasias, proliferation of
capillaries together with formation of numerous foreign body-type multinucleated giant cells. The
cytoplasm of the latter contained similar type substance to that seen in macrophages cytoplasm.
3.3.3. Haematological data
Table (3) showed the effect of the extract on the percent haematocrit and percent haemoglobin.
498
Zuhair Muhi-eldeen, Kassim J. Al-Shamma, Tawfik M. Al- Hussainy,
Elham N. Al- Kaissi, Ali M. Al-Daraji and Hassan Ibrahim
Table 3:
Effect of the aqueous extract of harmalas plant on percent haemaglobin and haematocrit in blood of
treated rats.
Group
Week
A B C D
% H
ct
37.1
± 2.7
40..8
± 0.9
35.3
± 1.6
40.3
± 3.0
1
Gm / 100H
b
12.3
± 0.8
13.8
± 0.4
12.0
± 0.5
13.5
± 1.1
% H
ct
−
38.5
± 1.8
35.1
± 3.5
37.0
± 1.0
2
Gm / 100H
b
−
12.9
± 0.5
11.9
± 0.5
12.7
± 0.4
% H
ct
−
37.8
± 2.1
36.3
± 1.5
40.5
± 1.3
3
Gm / 100H
b
−
12.9
± 0.6
12.9
± 0.9
13.4
± 0.3
% H
ct
−
37.8
± 2.1
40.3
± 1.3
40.6
± 1.7
4
Gm / 100H
b
−
12.6
±0.6 13.5
± 0.6
13.7
± 0.5
% H
ct
−
38.6
± 1.3
38.8
± 1.1
39.3
± 2.0
5
Gm / 100H
b
−
12.8
± 0.2
12.9
± 0.3
13.5
± 0.6
% H
ct
−
39.6
± 2.0
38.8
± 1.1
40.0
± 0.8
6
Gm / 100H
b
−
13.4
± 0.5
13.0
± 0.4
13.3
± 0.2
H
b
= Haemoglobin
H
ct
= haematocrit
No significant changes were observed in these value in treated rats compared to controls.
Similarly no significant changes in the RBC count were observed between treated and control rats
(Table 4).
Table (4) showed the effect of the extract on the number of reticulocytes.
Table 4:
Effect of the aqueous extract of harmala plant on RBC and WBC counts in blood of treated rats.
Group
Weeks
A B C D
No.X100 WBC
3.6
± 1.3
2.33
± 0.9
2.6
± 0.5
4.0
± 1.6
1
No.X100 RBC
1315.6
± 68.2
1315.6
± 151
1274.1
± 177
963.3
± 82
No.X100
−
4.0
± 1.6
3.35
± 1.03
2.0
± 0.2
2
No.X100 RBC
−
13002.3
± 108
1135
± 149
922
± 43
No.X100
−
4.4
± 1.1
7.0
± 1.8
4.8
± 2.0
3
No.X100 RBC
−
1135
± 41.7
1232
± 123
892
± 47
No.X100
−
5. *
± 1.4
5.1 **
± 0.7
1.58
± 0.2
4
No.X100 RBC
−
1136
± 122
1294
± 160
861
± 254
No.X100
−
5.28 *
±0.6 4.2
*
± 0.8
2.2
± 0.5
5
No.X100 RBC
−
1238
± 132
1293
± 132
935
± 94
No.X100
−
8.2**
± 2.9
6.45 *
± 2,9
1.8
± 0.4
6
No.X100 RBC
−
1155
± 139
1155
± 136
974
± 61
* P
< 0.01
** P
< 0.001
Value given are for the probability level (p) where significant differences occurs between the
treated groups and the control group
Significant increase in the number of these cells was observed after the first week of treatment,
in treated rats compared to control rats. Similarly significant increase in neutrophil counts was
observed in treated rats compared to the controls (Table 5). Bone marrow examination revealed no
significant change.
Acute Toxicological Studies on the Extract of Iraqi Peganum Harmala in Rats
499
Table 5:
Effect of the aqueous extract of harmala plant on the differential leucocytes count in blood of treated
rats
Group
Week
A B C D
N
46 38 42 26
L
49 57 52 66
E
3.1 2.0 1.8 3.0
1
st
M
1.6 2.3 3.5 3.3
N
57
49
28
L
35
45
59
E
4
4
1.3
2
nd
M
1.8
1.8
1.3
N
49
42
21
L
48
53
76
E
1.3
2.6
2
3
rd
M
1.6
1.3
1
N
55
65
21
L
41
30
74
E
2.3
2
1
4
th
M
1
1.5
3.3
N
32
51
32
L
55
44
61
E
5
3.6
1.1
5
th
M
6.8
2.2
4
N
28
41
11
L
67
52
83
E
2
3
2
6
th
M
2.6
3
3.2
N = Neutrophil
L = Lymphocytes
E = Eosinophil
M = Monocytes
4. Discussion
According to the method of Litchfield and Wilcoxon (1949), the LD50 value of the extract given IM to
rats was calculated and found to be 420 mg / kg. (Barnes and Eltherington 1973).
Tremors and convulsion which were observed in most of the treated rats, specially those given
high dose of (500-550)mg / kg since these alkaloids are known to have central nervous system
stimulant effects (Brunton et al, 2005). The significant increase in the number of leucocytes and
neutrophiles are due to the sever local inflammatory response in muscles at the site of injection which
was observed in most of the treated rats (table-5).
Histological examination of other tissues and organs were normal indicating that the extract
have low level of toxicity, this was supported by the biochemical data which were also normal. The
extract has no apparent toxic effects on the liver and kidney. The results indicated that the IM route is
not a proper route of administration, since it produces a serious local inflammatory reaction. Further
work is in progress to find out wether these toxicities are common to harmeline, harmine, harmala and
peganine or their might be some significant differences between the toxicities of these alkaloids.
500
Zuhair Muhi-eldeen, Kassim J. Al-Shamma, Tawfik M. Al- Hussainy,
Elham N. Al- Kaissi, Ali M. Al-Daraji and Hassan Ibrahim
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