Comp Clin Pathol (2010) 19:169–172

DOI 10.1007/s00580-009-0848-1

ORIGINAL ARTICLE

Study on the effect of Peganum harmala extract

on experimental skin wound healing in rat: pathological
and biomechanical findings
A. Derakhshanfar & M. M. Oloumi & M. Mirzaie

Received: 13 October 2008 / Accepted: 30 March 2009 / Published online: 29 April 2009
# Springer-Verlag London Limited 2009

Abstract This study was designed to evaluate the role of esteem throughout Asia Minor as a medicinal plant (Mirzaie
Peganum harmala extract on experimental skin wound et al. 2007). The smoke of P. harmala seeds is traditionally
healing. The investigation was carried out on 30 male used in Iran as a disinfecting agent (Shahverdi et al. 2005).
Sprague-Dawley rats in the same conditions. Two uniform Its seeds were known to possess hypothermic, hallucinogen-
7-mm diameter skin defect were created on the back of each ic, antibacterial, antifungal, antiviral, and antitumoral prop-
animal by skin punch (total of 60 wounds). The extract was erties (Lamchouri et al. 1999). Although, various authors
then applied once daily on half of the wounds. The animals have undertaken studies on the effect of P. harmala in
were sacrificed on day 10 for histopathologic and biome- treatment of certain dermatoses (El-Saad El-Rifaie 1980),
chanical examinations. P. harmala extract significantly documented studies on the effect of this extract in skin
increased the number of fibroblasts and capillary buds and wound healing are lacking in the literature. So, in this study
also decreased the epithelial gap which showed the better P. harmala extract has been used for exaggerating experi-
healing in treatment group. On the other hand, the mental skin wound healing in rats.
improvement of biomechanical indices in treatment group
revealed a significant increase in tensile strength of the
wounds. It can be concluded that P. harmala is an effective Materials and methods
herbal remedy in wound healing
Preparation of the extract The aerial parts of P. harmala
Keywords Peganum harmala . Skin . Wound healing were collected in Isfahan Province, Iran. The plant was
positively identified by botanists in the Department of
Biology, Shiraz University, Shiraz, Iran. A concentrated
Introduction extract of P. harmala was prepared from the seeds of the
plant according to the method for alkaloid extraction
Peganum harmala is a plant known from the first century A. described by Manske and Holmes (1952). Briefly, the
D. and currently used for therapeutic purposes (Lamchouri et crushed seeds of P. harmala were covered with three times
al. 2002). It originated in Central Asia, and is held in high their mass of water containing 30 g of acetic acid per liter
of water. The seeds swell as they absorb the liquid and form
a thick dough which was pressed after 3 days. The pressed
A. Derakhshanfar (*) : M. Mirzaie seeds were once again treated as above with twice their
Department of Pathobiology, Faculty of Veterinary Medicine,
mass of dilute acetic acid and after maceration the liquid
Shahid Bahonar University of Kerman,
Kerman 7616914111, Iran was again pressed out. The concentrated extract was
e-mail: [email protected] subsequently sterilized in ultraviolet light for 12 h then
dried below 70°C in an oven.
M. M. Oloumi
Department of Clinical Sciences, Faculty of Veterinary Medicine,
Shahid Bahonar University of Kerman, Experimental design and sampling Thirty male Sprague-
Kerman, Iran Dawley rats, weighing 180–200 g were housed under
170 Comp Clin Pathol (2010) 19:169–172

Fig. 1 Schematic drawing showing the new method used for

quantitative tissue characterization. ep. epithelium, ep.g. epithelial
gap, f the field in which the number of fibroblasts and capillary buds
were counted, gt. granulation tissue, l the depth of granulation tissue

controlled condition (12 h light–dark cycle, 22°C, 60%

Fig. 2 The epithelial gap in treatment group (H&E, ×100)
humidity). They were fed a rodent chow and had tap water
ad lib. All the procedures were conducted in accordance with
the European community guidelines for laboratory animals.
The back of animals in the thoraco-lumbar region was Ringer’s soaked gauze, aluminum foils, and plastic bags
surgically prepared for aseptic surgery. A circular wound and kept in −20°C freezer until tensile testing.
was created on each side by a 7-mm biopsy punch (total of 60 The wounds were digitally photographed during the
wounds). The surgical procedures were carried out under study and the wound area was measured by a digital
general anesthesia (90 mg/kg ketamine+10 mg/kg xyalzine). scanning software (Sigma Scans, Pro 5.0, SPSS Science,
All the wounds (treatment and control) were rinsed daily Chicago, IL, USA).
by 10-ml sterile normal saline. In treatment wounds (30
wounds) following rinsing, the extract was applied topically Histopathological examination Following routine prepara-
on each wound by sterile cotton tip swabs once daily for tion of tissues, serial sections of paraffin embedded tissues
10 days, whereas the control wounds (30 wounds) were left of 5 μm thickness were cut with a microtome and stained
untreated. Wounds remained uncovered in both groups with hemotoxylin and eosin and studied under light
throughout the experiment. microscope for fibroblastic proliferation, angiogenesis, and
After 10 days the animals were sacrificed by
intracardiac injection of 20 mg/kg thiopental sodium.
For histopathological studies (15 wounds from each
group), regenerated tissues were cut in the form of
square pieces along with normal skin on either side of
the wound and preserved in 10% buffered formalin. For
biomechanical studies (15 wounds from each group), a
strip of skin, 7 cm long, with the same widths of
wound diameter, in the manner that the wound was
located at the middle of the strip, was removed by a
double-blade scalpel. The skin was then wrapped in

Table 1 Histopathological parameters evaluated in treatment and

control groups

Histopathological parameters*

Cappillary buds Fibroblast Epithelial

(No./mm2) (No./mm2) gap (µ)

Treatment 1,121.55±146.28 1,171.67±125.27 143.71±20.83

Control 399.12±32.85 666.91±93.21 944.47±64.52
Fig. 3 The epithelial gap in control group (H&E, ×40). Compare the
*p≤0.05 (significant difference between the two groups) gap with the Fig. 2
Comp Clin Pathol (2010) 19:169–172 171

Table 2 Biomechanical parameters evaluated in treatment and control treatment group (p≤0.05). Also the epithelial gap in
groups
treatment group (Fig. 2) was significantly less than the
Biomechanical parameters* control group (Fig. 3), showing better re-epithelialization in
this group.
Y.P. (Kg) U.S. (Kg) Stif. M.E.S.
(Kg cm) (Kg cm)
Biomechanic All biomechanical parameters measured in
Treatment 1.11±0.18 1.21±0.24 0.88±0.17 1.16±0.26 this study were significantly higher in treatment group
Control 0.63±0.14 0.71±0.13 0.42±0.15 0.61±0.18 compared with control group (Table 2), which shows better
biomechanical properties of the treated tissues.
Y.P. yield point, U.S. ultimate strength, Stif. stiffness, M.E.S. Wound area in two groups is demonstrated in Table 3.
maximum stored energy These data show that in days 4, 6, 8, and 10 the wound area
*p≤0.05 (significant difference between the two groups) in treatment group is significantly less than the control
group.

re-epithelialization. A new method was used to quantify the

histopathological data. For this purpose, the depth of
granulation tissue in each slide was measured by objective Discussion
micrometry lens under ×40 magnification. A field at the
middle of this length was then considered and the number Therapeutic evaluations for medicinal plants are essential
of fibroblasts and capillary buds were counted under ×100 because of the growing interest in alternative therapies and
and ×400 magnifications, respectively and presented in the therapeutic use of natural products. This interest in
number per mm2. To evaluate re-epithelialization, the drugs of plant origin is due to several reasons, for example,
epithelial gap was measured under ×200 magnification conventional medicine can be ineffective, synthetic drugs
(Fig. 1). The values were averaged for each group. may have side effects, or folk medicine suggests that
natural products are harmless. P. harmala has pharmaco-
Biomechanical evaluation The samples were defrosted by logically active compounds which are several alkaloids that
immersing in 20°C Ringer's solution. They were then can be found especially in the seeds and the roots. Its active
mounted in a Strograph mechanical test frame (Toyoseiky alkaloids include β-carbolines such as harmine, harmaline,
Tensile Testing Unit, model R3, Japan) fitted with appro- harmalol, and harman and quinazoline derivatives vasicine
priate clamps, the distance between the clamps at the start and vasicinone (Derakhshanfar and Mirzaei 2008; Mirzaie
of testing being 4 cm. The strips were loaded with 0–50 kg et al. 2007). The extract of P. harmala was used topically to
load cell, with strain rate of 1 cm/min and the load- treat certain dermatoses of inflammatory nature. Results
elongation curves were drawn. The following parameters were encouraging and proved the antibacterial, antifungal,
were measured from the load-elongation curves: yield and antipruritic effects of the extract (El-Saad El-Rifaie
strength (yield point) (kg), ultimate strength (kg), maximum 1980). Harmala alkaloids are found endogenously in mam-
stored energy (kg cm), and stiffness (kg cm). malian tissue. These compounds also have antioxidative,
Statistical analysis: Student's t-test was used to compare platelet aggregation inhibitory and immunomodulatory
two means. A value of p≤0.05 was considered as significant. effects. The vasorelaxant effect of harmine and harmaline
are attributed to their actions on the endothelial cells to release
nitric oxide (NO) and on the vascular smooth muscles to
inhibit the contractions induced by the activation of receptor-
Results linked and voltage-dependent Ca2+ channels (Shi et al. 2000,
2001). However, the results of other investigation revealed
Histopathology As shown in Table 1, the number of that P. harmala extract exerts a vasodilatory effect not related
fibroblasts and capillary buds were significantly higher in to the presence of endothelium and the main mechanism may

Table 3 Wound area (mm2) in

two groups during the study Days

0 2 4a 6a 8a 10a

Treatment 38.45±3.58 34.21±3.98 25.36±2.86 20.12±2.93 18.49±2.33 14.32±1.91

a
Significant difference between Control 36.25±3.34 33.95±3.14 32.14±3.23 27.65±2.87 26.14±2.79 25.29±2.52
the two groups
172 Comp Clin Pathol (2010) 19:169–172

be related to the inhibition of cyclic AMP phosphodiesterase lance system for damaged tissue in Drosophila larvae. Proc Natl
Acad Sci USA
(Berrougui et al. 2002). It seems that inhibition of
Berrougui H, Herrera-Gonzalez MD, Marhuenda E, Ettaib A,
phosphodiesterase resulting in an increase of the second Hmamouchi M (2002) Relaxant activity of methanolic extract
messenger levels and finally reduces the levels of free from seeds of Peganum harmala on isolated rat aorta. Therapie
radicals in tissues (Berrougui et al. 2006). The results of 57:236–241
Berrougui H, Martin-Cordero C, Khalil A, Hmamouchi M, Ettaib A,
some investigations demonstrate that circulating cells function
Marhuenda E, Herrera MD (2006) Vasorelaxant effects of
as a surveillance system that monitors body tissues for harmine and harmaline extracted from Peganum harmala L.
damage. After epidermal wounding, circulating cells are seeds in isolated rat aorta. Pharmacol Res 54:150–157.
rapidly recruited to the site of damage by adhesive capture. doi:10.1016/j.phrs.2006.04.001
Derakhshanfar A, Mirzaei M (2008) Effect of Peganum harmala (wild
After capture, these cells distributed across the wound, appear rue) extract on experimental ovine malignant theileriosis:
phagocytically active, and are subsequently released back into pathological and parasitological findings. Onderstepoort J Vet
circulation by the healing epidermis (Babcock et al. 2008). Res 75:67–72
Increasing tissue blood supply, and therefore number of El-Saad El-Rifaie M (1980) Peganum harmala: its use in certain
dermatoses. Int J Dermatol 19:221–222. doi:10.1111/j.1365-
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in capillary buds results in the formation of granulation tissue Atif N, Hassar M (1999) Antitumour principles from Peganum
in a shorter time. Biomechanical results confirm the histo- harmala seeds. Therapie 54:753–758
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Hassar M (2002) Experimental toxicity of Peganum harmala
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Mirzaie M, Nosratabadi SJ, Derakhshanfar A, Sharif I (2007)
process in skin wound; however, more studies are needed to Antileishmanial activity of Peganum harmala extract on the in
clarify the exact mechanism of action of this herbal remedy. vitro growth of Leishmania major promastigotes in comparison
to a trivalent antimony drug. VETERINARSKI ARHIV 77:365
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for his kind cooperation in revising the final version of the manuscript. main chemical composition of two smoke condensates from
Peganum harmala seeds. Z Naturforsch 60(C):707–710
Shi CC, Chen SY, Wang GJ, Liao JF, Chen CF (2000) Vasorelaxant
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