Histological evaluation of Plantago lanceolata L. extract in accelerating wound healing

Abstract

This study was conducted to evaluate the effects of topical application of Plantago lanceolata L. (PL; Plantaginaceae) extract on the rate of wound healing closure and histology of healed wound. An area of uniform wound 7 mm in diameter using circular punch was excised from the nape of the dorsal neck of all rats. The animal groups were topically treated with 0.75 and 1.5% of PL and two groups were treated as control and placebo groups. Macroscopically, wound dressed with PL extract significantly healed earlier than those treated with placebo and control groups. Histological analysis of healed wounds dressed with PL extract showed comparatively less scar width at wound site and healed wound contained less inflammatory cells and more collagen with angiogenesis compared to wounds dressed with placebo. Results of wound enclosure assessment showed that PL was significantly effective in wound enclosure. The best results (100.0% healing) were seen in third group (0.75% of PL extract) on 14 th day. In conclusion, wounds dressed with PL extract significantly enhanced the acceleration of wound healing enclosure in rats.

Full text

Journal of Medicinal Plants Research Vol. 6(34), pp. 4844-4847, 5 September, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.516
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Histological evaluation of Plantago lanceolata L. extract
in accelerating wound healing
Hojjat Ismayilnajadteymurabadi1, Mohammad Reza Farahpour2* and Amir Amniattalab3
1Graduated Veterinary Medicine, Islamic Azad University, Department of Veterinary Medicine, Urmia Branch, Iran.
2Department of Clinical Sciences, Islamic Azad University, Urmia Branch, Urmia, Iran.
3Department of Basic Sciences, Pathology, Islamic Azad University, Urmia Branch, Urmia, Iran.
Accepted 4 January, 2012
This study was conducted to evaluate the effects of topical application of Plantago lanceolata L. (PL;
Plantaginaceae) extract on the rate of wound healing closure and histology of healed wound. An area of
uniform wound 7 mm in diameter using circular punch was excised from the nape of the dorsal neck of
all rats. The animal groups were topically treated with 0.75 and 1.5% of PL and two groups were treated
as control and placebo groups. Macroscopically, wound dressed with PL extract significantly healed
earlier than those treated with placebo and control groups. Histological analysis of healed wounds
dressed with PL extract showed comparatively less scar width at wound site and healed wound
contained less inflammatory cells and more collagen with angiogenesis compared to wounds dressed
with placebo. Results of wound enclosure assessment showed that PL was significantly effective in
wound enclosure. The best results (100.0% healing) were seen in third group (0.75% of PL extract) on
14th day. In conclusion, wounds dressed with PL extract significantly enhanced the acceleration of
wound healing enclosure in rats.
Key words: Plantago lanceolata, wound healing, rat, histological study.
INTRODUCTION
Normal wound healing response begins the moment the
tissue is injured. Wound healing is the process of repair
that follows injury to the skin and other soft tissues.
Following injury, an inflammatory response occurs and
the cells below the dermis begin to increase collagen
production. Later, the epithelial tissue is regenerated
(Souba and Wilmore, 1999). The objectives of the
pharmacology of wound healing are to study the
influence of various measures in wound management
programmers on healing and to screen drugs that
promote healing. Several materials have so far been
used and are reported to affect healing differently.
However, intensive research in wound healing has not
yielded, economic and efficacious pro-healing agent that
*Corresponding author. Email: m.farahpour@iaurmia.ac.ir. Tel:
+98(441) 4373676. Fax: +98(441) 3460980.
could obviate the long hospitalization of patients following
surgery and wound infliction (Shivananda et al., 2006).
Plantago lanceolata L. (PL; Plantaginaceae), is a
perennial plant species with a worldwide distribution and
large ecological amplitude. Iridoid Glycosides (IGs) are a
group of monoterpene-derived compounds that have
been recorded in over 50 plant families (Bowers, 1991).
The main IGs found in P. lanceolata are catalpol and its
precursor aucubin (Jensen, 1991).
Several therapeutic effects including: Therapeutic
effects on gastrointestinal, blood and respiratory (asthma
and dyspnea) disorders have been described for the
Plantago lanceolata in Iranian ancient medical books
(Zargary, 1990). PL is used internally to suppress coughs
associated bronchitis and upper respiratory inflammation,
to reduce skin inflammation, treatment of wounds and as
a laxative (Baytop, 1999).
In this study, we investigated wound healing activity of
PL under pathological review which was based on anti-

inflammatory characteristic of PL.
MATERIALS AND METHODS
Plant material preparation of the extract
Dried PL flowers were collected and Cold aqueous extract was
prepared. Dried flowers were steeped for 6 h at 4°C in 300 ml
distilled water, with constant stirring. The material was centrifuged
and the supernatant was filter-sterilized and then freeze-dried.
Acute toxicity studies
The acute toxic study was used to determine a safe dose for PL.
Thirty healthy Wistar rats (15 males and 15 females) were obtained
from the Experimental Animal House, Faculty of veterinary,
University of Urmia, and were divided equally into 3 groups labeled
as placebo, 5 and 10% of PL. respectively. The animals were
observed for 30 min and 2, 4, 24 and 48 h after the administration
for the onset of clinical or toxicological symptoms.
Mortality, if any, was observed over a period of two weeks. The
animals were sacrificed on the 15th day. Hematological, serum
biochemical and histological (liver and kidney) parameters were
determined following standard methods (Bergmeyer, 1980; Tietz et
al., 1983). Throughout the experiments, all animals received human
care according to the criteria outlined in the ―Guide for the Care and
Use of laboratory Animals―.
Experimental animals
32 Male Wister rats (190 to 210 g) of 10 weeks were obtained from
the Experimental Animal House, Faculty of Veterinary University of
Urmia and the rats were divided randomly into 4 groups of 8 rats
each. The animals were housed separately (one rat per cage). The
animals were maintained on standard pellet diet and tap water.
Animal houses were in standard environmental conditions of
temperature (22 ± 3°C), humidity (60 ± 5%), and a 12 h light/dark
cycle.
Experimental induced excision wounds
After anesthesia induction with Xylizaine 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. With this excisional wounding method,
epidermis, dermis, hypodermis and Panniculus Carnosus layers
were removed completely (Luisa and DiPietro, 2003). Wound
contraction was monitored by measuring wound area, on alternate
days till the wounds were completely healed. Wound contraction
was calculated as percentage reduction in wound area.
Topical wound application
After being made of surgical wound, all rats randomly was colored
with none toxic color and divided to three groups. In group A, 0.75%
PL extract was administered. Group B received 1.5% PL. group D
as placebo were administrated with Eucerin and Vaseline and
group C as control group did not received any administration. All
extracts were applied in topical route. All rats were followed 21 days
Ismayilnajadteymurabadi et al. 4845
later. Daily observation was performed and any wound fluid or any
evidence of infection or other abnormalities were noted.
Histopathological evaluation of healed wounds
The skin samples were obtained during days 3, 7, 14 and 21, from
all groups of animals and 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, epithelial growth,
hyperemia, collagen density 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
Acute toxicity
Acute toxicity of P. lanceolata was carried out on animal
at dose of 5 and 10% and animals were kept under
observation for 14 days. All animals remained alive and
did not show any significant differences between control
and treated groups. We concluded that P. lanceolata
orally administrated to rats was safe and no side effect
was seen even at highest doses.
Wound healing activity
Groups: A: 75%, B: 1.5%, C: Control, D: Placebo
On 3rd day angiogenesis in A group was more than B
group. But it was same between B and C groups. On 7th
day, both hyperemia and bleeding were same in all
groups. Leukocytes in A group were less than B group
but it was significantly high in C group compare to A and
B. There was no remarkable difference in angiogenesis
between groups. Collagenation was not noticeable. The
main difference between A group and other groups on 7th
day was epithelization which was macroscopically higher
than other groups. Scar formation in treated group was
also better than C or D.
During 14th day, hyperemia was equal both in A and C
but it was high in B. There was no bleeding in A but both
B and C showed a bit of bleeding. There was no sign of
inflammatory in A and B but leukocytes were seen in C
group. Angiogenesis was still observable in A and B.
Epithelization was remarkably up in A and B.
On 21st day there was no sign of hyperemia in C but it
was seen in A and B. There was no inflammatory cell in A
and B but in C group lymphocytes were still in the site

4846 J. Med. Plants Res.
Table 1. Effect of B. Plantago lanceolata extract on percentage (%) wound healing in experimental rats.
No.
Group
3 days
7 days
14 days
21 days
1
Control
26.04±0.49
45.25c±1.35
71.54d±0.74
95.13b±1.38
2
Placebo
26.14±1.42
46.42c±0.22
75.35c±0.81
100a±0.0
3
0.75%
26.30±0.37
71.47b±1.17
100a±0.00
100a±0.0
4
1.5%
26.44 ±1.66
75.29a±1.20
85b±0.73
100a±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).
and there was no difference between groups according to
epithelization and scar formation.
Wound enclosure
Percentages of wound healing have been documented in
Table 1. 3 days after drug application no statistical
significant differences were seen between groups (NS),
although treated groups showed better wound enclosure
percentage. Topical ointment demonstrated its effective-
ness and treated groups had significant difference
compare to control and placebo groups in 7th day. In
14th day, complete enclosure was seen in A group and B
group showed higher closure compare with placebo and
control groups. Consider the fact that during whole study
placebo showed a slight effect on better healing. As it can
be seen, 21st day of study all groups showed complete
closure except control group which was not completely
closed in healing site.
DISCUSSION
It is important to note that throughout the period of wound
treatment, the PL extract did not cause irritation or pain to
the animals as the rats neither show any signs of
restlessness nor scratching/biting of wound site when the
extract were applied. All the surgical interventions were
carried out under sterile conditions and animals were
closely observed for any infection. This is very important
that the control microbial infection is necessary for better
healing and its management (Muhammad and
Muhammad, 2005).
Wound healing is a complex and dynamic process of
restoring cellular structures and tissue layers in damaged
tissue as closely as possible to its normal state. Wound
contracture is a process that occurs throughout the
healing process, commencing in the fibroblastic stage
whereby the area of the wound undergoes shrinkage.
The proliferative phase is characterized by angiogenesis,
collagen deposition, granulation tissue formation,
epithelialization, and wound contraction resulting in a
smaller amount of apparent scar tissue (Midwood et al.,
2004). In the present study, topical application of PL
extract significantly accelerated the rate of wound
healing, and histology, healed wound contain
comparatively less inflammatory, more collagen and
angiogenesis. Topical application of PL extracts
demonstrated its effectiveness and treated groups
presented significant difference compare to control and
placebo groups in 7th and 14th days however, group A
showed complete wound closure (100%) sooner (14th)
than other groups (groups B, C and D). Wound healing
effects may be due to regulation of collagen expression
(Bonte et al., 1993).
Similarly, enhanced healing activity has been attributed
to collagen formation and angiogenesis (Shukla et al.,
1999; Trabucchi et al., 1986). Collagen played a central
role in the healing of wounds and it is a principal
component of connective tissue and provides a structural
framework for the regenerating tissue (Cohen et al.,
1992); although we did not completely measure collagen
rate in wound site, wound enclosure results show
remarkable collagenation in treated groups (Table 1).
Angiogenesis in granulation tissues improves circulation
to the wound site thus providing oxygen and nutrients
essential for the healing process (Szabo et al., 1995) that
includes re-epithelization (Habibipour et al., 2003) and
showed that histological analysis of the treated healed
wound group contained a large amount of fibroblast
proliferation, collagen synthesis, and neovascularization,
which resulted in an increased wound tensile strength
and accelerated healing of wound. It is proved that PL
has an anti-inflammatory activity (Herold et al., 2003;
Wegener and Kraft, 1999) which might be a reason in
accelerating wound healing.
According to this characteristic of PL, inflammatory
cells rate will decrease in the wound site which lead to
better wound healing. Extract from PL and Plantago
major are antiphlogistic in carrageenan and PGE1-
induced inflammations in rats (Shipochliev, 1981).
Aceteoside, the main phenylethanoid from PL inhibits
arachidonic acid induced mouse ear oedema (Murai et
al., 1995). Therefor PL can inhibit oedema in wound site
as it did in present study some wound healing process
could be accelerated under none-oedema condition in
wound site.

Compounds such as the iridoid glycosides aucubin and
catalpol, the aglycone aucubigenin, and caffeic acid
derivatives including plantamajoside and acteoside have
been isolated from Plantago spp. and have demonstrated
antimicrobial activity (Samuelsen, 2000; Blumenthal et
al., 2000) thus PL can decrease microbe volume in
wound site which is a useful trend to accelerate wound
healing.
In this study, we demonstrated that extract from PL was
beneficial in wound healing base on its anti-microbial and
anti-inflammatory which helped to faster wound
contraction and better histological properties of treated
wounds.
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