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: [e-mail]

Tel: [phone]; Fax: [phone]

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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