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African Journal of Pharmacy and Pharmacology Vol. 5(21), pp. 2386-2389, 8 December, 2011
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.258
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Wound healing activity of flaxseed Linum
usitatissimum L. in rats
Mohammad Reza Farahpour
1
*, Human Taghikhani
2
, Mostafa Habibi
3
and Mohammad Amin
Zandieh
3
1
Department of Veterinary Surgery, Islamic Azad University, Urmia Branch,Urmia, Iran.
2
Graduate of Veterinary Medicine, Islamic Azad University, Urmia Branch, Urmia, Iran.
3
Young Researchers Club, Faculty of Veterinary Medicine, Islamic Azad University, Urmia Branch, Urmia, Iran.
Accepted 16 November, 2011
Wound healing is a complex process which requires special factors and condition. Drugs which
accelerate wound healing are expanded, particularly herbal drugs which are mainly safe and effective.
Hence, the present study was conducted to evaluate wound healing activity of flaxseed oil on
experimentally induced incision wound. The therapeutic ointments were prepared according to two
concentrations, 0.75 and 1.5%, and were applied to wound. 32 male rats were divided into 4 groups of 8
rats. Two circle-shapes, full thickness wounds with 7 mm
2
in diameters were made in both side of
backbone. Tissue samples were obtained at the end of 3, 7, 14 and 21 days from all groups and were
stained with hematoxylin and eosin, then were reviewed under light microscope. Treated animals
showed significant reduction of inflammatory cells in the period of re-epithelization. Flaxseed oil
significantly accelerates wound healing process and suggested flaxseed as an effective herbal drug for
wound in skin.
Key words: Wound healing, inflammatory cell, flaxseed oil, epithelization.
INTRODUCTION
Wounds are inevitable events of life which are made due
to physical, chemical or microbiological infections. Nor-
mal wound healing response begins immediately after the
tissue is injured. Following injury, an inflammatory re-
sponse occurs and the cells below the dermis begin to
increase collagen production, later the epithelial tissue is
regenerated (Souba and Wilmore, 1999). Research on
drugs which accelerate wound healing is developing and
it is a crucial subject in biomedical science. Several
plants and herbs have been used experimentally to treat
skin disorders including wound healing in traditional
medicine (Nayak et al., 2009).
Flaxseed (Linum usitatissimum) is one of the oldest cul-
tivated plants in the world and is cultivated for its fiber
and oil. Flaxseed and its derivatives, flaxseed oil and in-
seed oil, are rich source of the essential fatty acid, alpha-
linolenic acid, which is a biological precursor to omega-3
*Corresponding author. E-mail: mrf78s@gmail.com. Tel:
+98(441)4373676. Fax: +98(441)3460980.
fatty acids, such as eicosapentaenoic.
Several animal studies suggested that omega-3 fatty
acids of this plant may have potential anti-cardiovascular
disease activity, anti-renal disease, anti-inflammation and
some autoimmune disease, such as inflammatory bowel
disease and rheumatoid arthritis. The lignan found in flax-
seed is called secoisolariciresinol diglucoside (SDG)
which is a type of carbohydrate classed as a phenolic
compound (polyphenol) and it is a powerful antioxidant
and has been shown to enhance the immune system
functioning and being effective against many different
diseases, such as cancers. The present study has been
conducted to evaluate probable wound healing charac-
terization of flaxseed on experimentally included wounds
in rats.
MATERIALS AND METHODS
Oil material and preparation of the extract
The flaxseed seed found in Iran is L. usitatissimum L., in this study,
flaxseed oil was extracted by cold pressure method, then it was
blended with eucerin-vaselin to prepare treatment ointments with
different doses (0.75 and 1.5%).
Experimental animals
32 Male Wister rats (190 to 210 g) of approximately 3 months were
used as experimental animals and were divided into four groups of
eight rats. The animals were housed in standard environmental
conditions of temperature (22 ± 3°C), humidity (60 ± 5% ) and a 12 h
light/dark cycle. During experimental time, rats were given standard
pellet diet (Pastor Institute, Iran) and water ad libitum.
Surgical procedures
After anesthesia induction with xylazine 2% and ketamine 10% (I.M.
60 mg/kg) rats were fixed in ventral posture on surgery table. Then,
the dorsal area from scapula to ilium were scrubbed and prepared
to surgery. Two circle-shapes, full thickness surgical wounds with 7
mm diameters in both side of the backbone, 1 cm away from
backbone and 5 cm away from each other were made with biopsy
punch 7 mm. With this excisional wounding method, epidermis,
dermis, hypodermis and panniculus carnosus layers were removed
completely (Luisa and DiPietro, 2003).
Treatment
After surgical wounds were treated, all rats randomly were colored
with none toxic color and divided into three groups. In group 1,
ointment with 0.75% flaxseed oil and in group 2 with 1.5% flaxseed
oil were administered. Group 3 as placebo was administrated with
eucerin-vaseline and group 4 as control group did not receive any
substance. All rats were treated for 21 days. Daily observation was
performed and any wound fluid or any evidence of infection or other
abnormalities were noted.
Histopathological study
The healing tissues samples obtained during days 3, 7, 14 and 21,
from all four groups of animals were processed for histological
study. The samples fixed in formalin and installed on slides, stained
with hematoxylin and eosin, and were reviewed under light
microscope. Recorded factors were scar, inflammatory cells, kind of
inflammatory cells, angiogenesis, fibroplasia, congestion, collagen
density fibroblast, fibrin and fibroblastic aggregation.
Statistical analysis
All values are reported as mean ± S.D, the statistical differences
among groups were assessed using Duncan multiple range test
and analysis of variance (ANOVA). A value of p < 0.05 was
considered significant. Statistical analysis was performed using
SAS 9.1 for Windows.
RESULTS
Groups A (1.5%), B (0.75%), C (control groups) and
placebo groups
In this study results of control groups and placebo groups
were almost same; in fact some insignificant differences
were seen.
Farahpour et al. 2387
3 days post-surgery
In group C, epithelization was very low. Whole tissue was
swollen inflammatory cells, especially, neutrophils were
highly seen but there was no sign of macrophages, neo-
angiogenesis collagen and fibroblast.
In group B, epithelization and inflammatory cell rate
were the same as C group but neo-angiogenesis was
high and wound was highly congested. Presence of new
collagen was acceptable and it was similar to group A.
Fibroblasts were slightly in the place as well.
In group A, epithelization was more than both B and
control groups. Macrophages were seen deep in the
wound and the number of inflammatory cells was de-
creased. Neo-angiogenesis was expanded and conges-
tion was lower than group B. Fibroblast rate increased as
compare to group B.
7 days post-surgery
Presence of neutrophils in superficial layer was
decreased in C group. Macrophages and fibroblasts were
remarkably seen in the beneath layer. Collagenation was
more than before.
In group B, the number of inflammatory cells decreased
and was only seen in beneath layer. Neo-angiogenesis
and collagenation is suitable. Epithelization was started
slightly and fibroblasts were seen.
Wound healing activity was much better in group A as
compared to groups B and C. Neo-angiogenesis in
superficial layer was good and epithelial layer were about
to form, even keratinied layer was being formed. Colla-
gen filaments had started forming cross junctions and
inflammatory cells were just under epithelial layer.
14 days post-surgery
Results on 14th day showed complete epithelization and
thin keratinized layers. Mononuclear inflammatory cells
were all over the wound. Collagen rate was acceptable,
but collagen maturation was not complete. In the super-
ficial area, neo-angiogenesis was not yet finished. Fibro-
blasts were more than fibrocytes.
There was no sign of scar tissue in group B and
inflammatory cell rate was very low. Neo-angiogenesis
which was just in superficial layer was lower than group
C. Collagen formation in superficial layers was better as
compared to beneath layers. Keratinized layers was visi-
ble and hair follicles were about to form, also, upper
layers have a bit granulation tissue. Maturation collagen
rate was much as compared to two mentioned groups.
Granulation was better; neo-angiogenesis was slightly in
upper layers. Collagen maturation was not completed
only in the upper layer.
2388 Afr. J. Pharm. Pharmacol.
Table 1. Effect of flaxseed L. usitatissimum L. extract on percentage (%) wound healing in experimental rats.
S/N Group 3 days 7 days 14 days 21 days
1 control 29.28
a
± 0.80 43.85
c
± 0.57 69.58
d
± 0.63 97.86
b
± 1.01
2 placebo 29.32
a
± 0.95 44.67
c
± 0.92 76.43
c
± 0.67 100
a
± 0.0
3 0.75% 29.82
a
± 0.29 54.35
b
± 1.09 88.81
b
± 1.91 100
a
± 0.0
4 1.5% 30.38
a
± 0.96 75.54
a
± 1.52 100
a
± 0.0 100
a
± 0.0
Significance - NS ** * **
*All expressed as mean and standard deviation (S.D). Mean in columns with different letters were significantly
different (NS, Not Significant; *p < 0.05; *p < 0.01).
21 days post-surgery
Wound healing trend was almost finished in group C, col-
lagens were in maturation phase but maturation was not
ended.
Granulation
tissue
also
was
formed. Fibrocytes
were outnumbering as compared to fibroblasts which
shows collagenation process. Thick layer of keratinized
tissue had been made, but low rate of neo-angiogenesis
showed that angiogenesis was not yet finished.
There was no sign of scar tissue in B group and neo-
angiogenesis was rarely seen in middle and beneath
layers. Collagen rate was much as compared to C group.
Epithelization quality was lower than A group.
Keratinization was acceptable and granulation tissue was
low in upper layer.
In group A, collagenation had no significant difference
with groups B and C, but the quality of epithelization and
epithelium thickness were much more than the aforemen-
tioned groups. Keratinized layer was thicker than the
other
groups,
but
granulation
differences
were
insignificant.
Wound enclosure
Percentages of wound healing have been documented in
Table 1. 3 days after drug application, no statistical sig-
nificant differences were seen between groups (ns),
although treated groups showed better wound enclosure
percentage. Significant results were seen from day 7 in
which treated groups showed higher closure of wound as
compared to control and placebo, but there was no
difference between control and placebo groups. Treated
groups showed statistically significant difference as
compared to each other as well. All groups demonstrated
differences to each other, while group B had best wound
closure percentage (100%) in 14th day. Complete closure
of wounds was observed 21 days after the experiment.
DISCUSSION
The results of the present study showed that the extract
of L. usitatissimum accelerate the progression of wound
healing activity. Wound healing consists of event roughly
divided into three overlapping phases, such as inflama-
mation,
granulation
tissue
formation
and remodeling of the
extra cellular matrix (Smith and Equist, 1967; Luisa and
DiPietro, 2003). Different studies show that wound hea-
ling therapeutic agents which have anti-inflammatory
antibacterial, antimycotic, insecticidal, antiseptic and anti-
maggot properties should be externally applicable.
L. usitatissimum (flax) is an annual plant widely
distributed in mediterranean and temperature climate
zone. It is among the oldest crop plants cultivated for the
purpose of oil and fiber (Millam et al., 2005).
Furthermore, there is a report that flax products are also
recommended for treating skin disease (e.g healing of
chronic skin ulceration) (De Spirt et al., 2008). Flax oil is
one of the richest sources of α-linoleic acid (α-LA), which
includes about 44 to 57% of all fatty acids and it also
contains 15 to 29% linolenic acid and 13 to 29% oleic
acid (Muir and Westcott, 2003). Between them, α-
linolenic acid and linolenic acid are both required for cell
membrane for the structural integrity (Flaxseed oil, 2003),
so it can be effective in wound integrity as it was in the
present study. Also, flaxseeds and oil (some extent) are a
valuable source of secoisolariciresinol diglucoside (SDG)
and other anti oxidative compounds, such as tocoferol,
carotenoids, phenolic acids and anthocyanins. SDG also
has anti-viral, anti-bacterial and anti-fungal properties
(Muir and Westcott, 2003; Bozan and Temelli, 2008)
which were proved to be major criteria to accelerate
wound healing process in the present study. There is a
report that flax fibers with high antioxidant level would be
the perfect material for linen wound dressing. The high
level of antioxidants (e.g. phenolic acids) can stimulate
natural process of wound clarity by macrophages (Sen et
al., 2002), and this bandage can assure the perfect milieu
for effective healing by helping the natural process to
progress. This can prevent fibroma formation, and it can
also keep optimal humidity that facilitates epithelial cells
migration (Dyson, 1988). It should be simultaneously
used with fibers, transgenic oil emulsion and transgenic
seedcake extract to promote healing of chronic skin
ulceration (Magdalena et al., 2003).
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