Phytomedicine 18 (2011) 976984

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Phytomedicine
journal homepage: www.elsevier.de/phymed

Hydroalcoholic extract based-ointment from Punica granatum L. peels with

enhanced in vivo healing potential on dermal wounds
E.A. Hayouni a,e, , K. Miled b , S. Boubaker c , Z. Bellasfar b , M. Abedrabba d , H. Iwaski e , H. Oku e , T. Matsui e ,
F. Limam a , M. Hamdi f
a

Laboratory of Bioactive Compounds at Biotechnology Center, Ecopark of Borj Cedria, BP-901, Hammam Lif 2050, Tunisia
Experimental Commodities for Animal Care, Institute of Pasteur, Tunis, Tunisia
Laboratory of Anatomo-pathology, Institute of Pasteur, Tunis, Tunisia
d
UR Molecular Physico-chemical, IPEST, La Marsa, Tunisia
e
Center of Molecular Biosciences, University of the Ryukuyus, Nishihara, Okinawa, Japan
f
Microbial Ecology and Technology Laboratory, INSAT, Tunis, Tunisia
b
c

a r t i c l e

i n f o

Keywords:
Punica granatum L. peels
Biological activities
Wound healing
Ointment
Biochemical
Histopathological

a b s t r a c t
The present study reports for the rst time, the in vivo wound healing potential of Punica granatum
L. peels. A 5% (w/w) methanolic extract based-ointment was formulated and evaluated for its wound
healing in guinea pigs. The ointment was applied in vivo on the paravertebral area of twelve excised
wounded models once a day for 10 consecutive days. The ointment signicantly enhanced the wound
contraction and the period of epithelialization as assessed by the mechanical (contraction rate, tensile
strength), the biochemical (increasing of collagen, DNA and proteins synthesis) and the histopathological
characteristics. Such investigation was encouraged by the efciency of the methanolic extract as antimicrobial and antioxidant. Indeed, the extract showed antioxidant activity as strong as natural and synthetic
compounds (Trolox, BHA, Quercetin). Furthermore, the extract exhibited signicant antibacterial and
antifungal activity against almost all tested bacteria: Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Klebsiella pneumoniae, Salmonella anatum, Salmonella
typhimurium, Streptococcus pneumoniae, and fungi Candida albicans, Candida glabrata, Trichopyton rubrum
and Aspergillus niger. The formulated ointment might well nd use as skin repair agent without hazard to
human health based on these results and on the fact that it has been well established that the extracts of
pomegranate used in conditions similar to those applied by traditional medicine, showed no toxic effects.
2011 Elsevier GmbH. All rights reserved.

Introduction
The concept of developing drugs from plants used in indigenous medical system is much older, while in some cases direct
links between a local and biomedical use exists, in other cases the
relationship is much more complex (Heinrich and Gibbons 2001).
Wounds and particularly chronic wounds are major concerns for
the patient and clinician alike, chronic wounds affect a large number of patients and seriously reduce their quality of life. Balick and
Cox (1996) reported that only 13% of drugs listed in Western pharmacopoeia are intended for use in the skin and for wounds, by
comparison, at least one third of herbal remedies are for such use.

Corresponding author at: Laboratory of Bioactive Compounds at Biotechnology

Center, Ecopark of Borj-Cedria, BP-901, Hammam-Lif 2050, Tunisia.
Tel.: +216 25600851.
E-mail addresses: a.hayouni@gmail.com, a hayouni@yahoo.com (E.A. Hayouni).
0944-7113/$ see front matter 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2011.02.011

Wound healing involves a chain of well orchestrated, biochemical and cellular events, leading to the growth and regeneration of
wounded tissue. In coetaneous wound healing, the inammation
stage begins immediately after injury, rst with vasoconstriction
that favours homeostasis and releases inammation mediators. The
proliferative phase is characterized by granulation tissue proliferation formed mainly by broblast and the angiogenesis process. The
remodelling stage is characterized by reformulations and improvement in the components of the collagen bers that increases the
tensile strength. Although the rate of collagen synthesis slow down
after about three weeks, collagen cross-linking and reorganisation
occur for months after injury in the remodelling phase of repair
(Beanes et al. 2003).
Due to the lack of side effects compared to synthetic drugs,
approximately 60% of the worlds population relies almost entirely
on plants for medication, and natural products have long been
recognized as an important source of therapeutically effective
medicines. Indeed, many plants have been shown to possess ther-

E.A. Hayouni et al. / Phytomedicine 18 (2011) 976984

apeutic potential as promoters of wound healing for example,

Jatropha curcas, Aloe barbadensis, Centella asiatica (Villegas et al.
1997; Shukla et al. 1999). These plants exhibited often antifungal,
antimicrobial, antioxidant, anti-inammatory activities (Turkoglu
et al. 2007). North African folklore and tribal medicines employ
a number of plants and animal products for treatment of cuts,
wounds and burns (Ahmed et al. 1995). Some of these plants
have been screened scientically for the evaluation of their wound
healing activity in different pharmacological models and human
subjects, but the potential of most of the plants remain unexplored.
Punica granatum L. (Punicaceae), commonly called
pomegranate, is a large deciduous shrub or small tree used
medicinally in Europe, Indo-China, the Philippine Islands, North
Africa, and South Africa. The plant is used in folklore medicine for
the treatment of various diseases, such as ulcer, hepatic damage,
snakebite, etc. The rind of the fruit is antihelminthic, useful in
dysentery and ulcer (Lansky and Newman 2007). The plant also
shows high antioxidant and antiartherogenic activity (Aviram et al.
2000). Modern uses of pomegranate derived products now include
treatment of acquired immune deciency syndrome (AIDS) in
addition to use for cosmetic beautication and enhancement
hormone replacement therapy, resolution of allergic symptoms,
cardiovascular protection, oral hygiene, ophthalmic ointment
weight loss soap and as an adjunct therapy to increase bioavailability of radioactive dyes during diagnostic imaging (Aviram et al.
2000; Lansky and Newman 2007).
There are only few prospective randomized controlled trials that
have proved the clinical efcacy of the traditional wound healing
agents. Accordingly, based on its ethnopharmacological prole and
reputed medicinal use in traditional practice, the present study was
undertaken to (i) evaluate, the in vitro, antioxidant and antimicrobial activities (against various wound pathogens) of the methanolic
extract obtained from the peels of P. granatum fruits (PgME) (ii) to
evaluate systematically the possible in vivo wound healing potential of a 5% ointment formulated using this extract.
Materials and methods
Chemicals
The anthocyanidins standards were obtained from Apin Chemicals Ltd. (Abingdon, England). Ellagic acid and gallic acid were from
Wako (Japan), all the standards and solvents were HPLC-grade.

977

ABTS, -carotene, were from SigmaAldrich (Steinheim, Germany).

All other chemicals used in this study were obtained either from
Merck Chemical Company (Darmstadt, Germany) or from Fluka
Biochemika (Buchs, Switzerland).
Plant material and preparation of the extract
Pomegranates (Romman as Tunisian vernacular name) were
purchased from a local market in Tunis (October 2008) and
identied at the Aromatic and Medicinal Plants Unit, Biotechnology Center at Borj-Cedria where a voucher specimen was
deposited. The peels were dried in shade and than pulverized
by a mechanical grinder. 100 g of the powder were extracted
with aqueous methanol (75%) using a soxhlet extraction apparatus, for 8 h. The obtained organic extract was concentrated by
rotary evaporation under vacuum at 45 C, using a HACH UVVis
spectrophotometer (Model DR/4000, Colorado, USA). The semisolid mass (18.7% 0.88, methanol free) obtained was used for
the in vitro biological activities and for the formulation of a 5%
(w/w) extract-based ointment, using soft white parafn, as a vehicle (Fig. 1D).
HPLC-ngerprint analysis of PgME
Sample preparation. The PgME was prepared to a concentration
of 12 mg/ml, with injection volume of 10 l. Before injection, PgME
was centrifuged (4 min at 5000 rpm) and the centrifuged supernatant was ltered through a 0.45 m PTFE lter (Waters), and
submitted to HPLCDAD and HPLCESIMS analysis.
Apparatus. Shimadzu controller (SLC-10A vp) with of a vacuum
degasser (DGU-14A), a binary pumps (LC-10AD vp), an oven (CTO10AC vp) and a Shimadzu Diode Array Detector (SPD-M10A vp). UV
spectra from 200 to 600 nm were recorded for peak characterization.
Separation. Column: Inertsil C18 ODS-3 column (5 m,
200 mm 4.6 mm) with a suitable guard column (C18, ODS, 5 m,
4 mm 3.0 mm). Temperature: 30 C.
Solvent: A: 0.1% (v/v) aqueous solution of formic acid and B:
methanol. Flow rate: 1 ml/min.
Gradient: 05 min, 99% B, 520 min, linear gradient 120% B;
2030 min, linear gradient 2040% B; 3035 min, linear gradient
4095% B and maintained constant at 95% B over the next 15 min.

Fig. 1. Main steps of the experimental protocol: (A) shaving; (B) wound creation under anaesthesia; (C) medication using cetrimide-based cream (left) and our ointment
(right) which is shown next and (D) the ointment formulated using PgME.

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E.A. Hayouni et al. / Phytomedicine 18 (2011) 976984

Detection: 254 nm, 520 nm (anthocyanidins), 366 nm (ellagitannins).

HPLCESIMS analysis
HPLC coupled to mass spectrometry was performed using a
Squire 4000 plus ion trap spectrometer tted with electrospray
ionization (Bruker Daltonics, Billerica, MA, USA). The capillary voltage was 3500 V. Nitrogen was used as nebulizer gas at 350 C at a
ow of 8.0 l min1 , nebulizer pressure was 27.5 psi. Spectra were
recorded between m/z 150 and 1400 in negative ion mode. Collision
induced dissociation (CID) spectra were obtained with a fragmentation amplitude of 1.00 V (MS/MS) using helium as the collision gas.
Punicalagins A and B were identied by comparison to their respective ions (1083.7 m/z mother ion) and MS/MS (601.0 m/z daughter
ion) ions as reported (Cedra et al. 2003).
Quantication of the antioxidant activity
ABTS assay
ABTS radical scavenging activity of the extract was determined
according to (Re et al. 1999) with some modications.
-Carotene bleaching test
The antioxidant activity of the extract was evaluated according
to a slightly modied version of the -carotene bleaching method
(Pratt 1980).
Antimicrobial activity
Microbial strains and culture conditions
The extract was tested against a panel of 11 wound pathogens
including 7 bacteria: Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Klebsiella
pneumoniae, Salmonella anatum, Salmonella typhimurium, Streptococcus pneumoniae; two yeasts: Candida albicans, Candida glabrata;
and two fungi: Trichopyton rubrum and Aspergillus niger.
Determination of minimum inhibitory concentration (MIC)
A broth microdilution susceptibility assay was used for bacteria, as recommended by NCCLS, for the determination of the
MIC (NCCLS 1999). All tests were performed in Nutrient Broth
(NB, Oxoid Ltd, Hampshire, UH). Bacterial strains were cultured
overnight at 37 C in NB. Bacterial strains were suspended in NB to
give a nal density of approximately 106 cfu/ml and these were conrmed by viable counts. Geometric dilutions ranging from 2 mg/ml
to 0.062 mg/ml of the methanolic extract from the peels of P. granatum fruits, were prepared in a 96-well microtitre plate, including
one growth control (NB) and one sterility control (NB + Tested
extract). Plates were incubated under normal atmospheric conditions at 37 C for 24 h. The bacterial growth was indicated by
the presence of a pellet on the well bottom. Amikacin and Clindamycine were used as positive controls.
A macrodilution broth method was used to determine the minimal inhibitory concentrations for yeasts and lamentous fungi
(NCCLS 1997). A serial dilution of the extract was prepared over the
range 0.0622 mg/ml. Recent cultures in Sabouraud Dextrose Broth
(SDB), in log phase, of each strain were used to prepare the cell suspension adjusted to (12) 103 cells/ml for yeasts and (12) 104
cells/ml for fungi. The test tubes were incubated aerobically at 37 C
for 48 h (yeasts) or at 30 C for 35 days (dermatophytes) and MICs
were determined. The fungal growth was indicated by the turbidity.
MIC was dened as the lowest concentration that reduced growth
in 80%, compared with antifungal-free controls. Herein, Amikacin
and Amphotricin B were used as positive controls and the negative
controls were used as above.

Animals and experimental protocol

This study was conducted in the Experimental Animal Commodities at the Institute of Pasteur, Tunis. Male and female guinea
pigs, weighing 250300 g, were used. All animals were housed, fed
and treated in accordance with the in-house guidelines for animal
protection. Animals were kept for 2 weeks to be acclimatized prior
to the investigation. Throughout experimentation period, animals
were given standard pellet diet and water ad libitum.
The animals were anesthetized with Ketamin chlorhydrate at
a dose of 1.5 mg/kg body weight intramuscularly (IM). Then a
local anaesthesia using Lidocan hydrate (2%) was applied on the
excision area. This type of anaesthesia prevents any pain and movement of the animals at least 2 h after the administration of the
anaesthetic solution. The dorsal back skins of the animals were
shaved and ethanol (70%) was used as antiseptic for the shaved
region before creating the wound (Fig. 1A). One excision wound
was inicted by cutting away a 400 mm2 full-thickness skin in the
dorsal inter-scapular. The wound was left undressed to the open
environment and no local or systemic anti-microbial agents were
used (Fig. 1B). After wound creation, experimental animals were
randomly divided into the following groups (n = 12):
- Group 1: untreated (control) animals
- Group 2: wounds treated topically with a simple ointment base
(placebo group)
- Group 3: wounds treated topically with reference standard a 2%
(w/w) cetrimide-based cream
- Group 4: wounds treated topically with the methanolic extract
based-ointment prepared from P. granatum peels.
The herbal ointment along with standard and vehicle were
applied (enough to cover the wound) once daily during 10 consecutive days (Fig. 1C).
Determination of wound contraction
For the determination of the wound contraction, excision
wounds were traced on a transparent paper having a scale, and
the change in wound size at 4 day-intervals was calculated as the
percentage of wound area that healed. The rate of wound contraction was expressed in terms of the percentage of wound area that
had healed: % wound contraction = (healed area/total area) 100.
Tensile strength
Tensile strength of wound represents the effectiveness of wound
healing. Dened as the force required opening the healing skin it
is used to measure the completeness of healing. Usually woundhealing agents promote the gaining of tensile strength. For this
purpose the newly repaired tissue including scar was excised to
measure the tensile strength by mean of a tensiometer. For the
quantication one of the edges of the skin was xed while applying a measurable force to the other one. The load (weight) in grams
required to disrupt the wound is determined 20 days after surgery.
Biochemical analyses of the newly formed skins
Animals were sacriced on day 8 after wound creation, and the
entire wound on each animal was cut out and stored at 70 C
until analyses. Hydroxyproline, the basic constituent of collagen,
was taken as a marker of collagen synthesis. Tissues were dried in
a hot air oven at 6070 C to constant weight and were hydrolysed
in 6 N HCl at 130 C for 4 h in sealed tubes. The hydrolyzed samples
were adjusted to pH 7.0 and were subjected to chloramines-T oxidation for 20 min. The reaction was terminated by addition of 0.4 M
perchloric acid and color was developed with the help of Ehrlich
reagent at 60 C (Woessner 1961), and measured at 557 nm using a
HACH spectrophotometer.

E.A. Hayouni et al. / Phytomedicine 18 (2011) 976984

979

Fig. 2. HPLCDAD chromatogram of PgME recorded at 254 nm (A) and 366 nm (B).

For estimations of total protein and DNA content, wet granulation tissues were rst extracted with TCA by the method of
Schneider (1957). Briey, the tissue was rst homogenized in 5%
TCA and centrifuged. The pellet was washed with 10% TCA, resuspended in 5% TCA, and kept for 15 min in a water bath maintained
at 90 C. The contents were centrifuged and the supernatant was
used for the determination of DNA content by the method of
Burton (1956). The precipitated proteins were suspended in 0.1 M
TrisHCl, pH 7.4, and the protein content was estimated by the
method of Lowry et al. (1951).
Histopathological studies
Animals were sacriced on the 4th, 8th, 12th, 16th and 20th days
after wound creation. Wound tissues were immediately preserved

in 10% buffered formalin and passed through different grades of

alcohol in order to ensure complete dehydration of tissues and
then embedded in parafn wax. Serial sections of parafn embedded tissues of 6 m thickness were cut using a microtome and
stained with haematoxylin and eosin (H&E). Additional sections
were stained with Masson Trichrome for the assessment of collagen content and maturation within the dermis. Micrographs were
obtained with an Axiophot Zeiss Light Microscope.
Tissues samples were evaluated for the following histological
criteria: the extent of re-epithelization, the maturation and organization of the epidermal squamous cells, the thickness of the
granular cell layer, the degree of granulation tissue formation, and
for collagenisation and scar formation in the dermis. For the epidermal changes, the adjacent non-traumatized skin served as the

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E.A. Hayouni et al. / Phytomedicine 18 (2011) 976984

internal control for the assessment of epidermal collagen bers,

prominent vascularity and inammation considered as an indication for immature granulation tissue. Contrary, the presence of
well formed and horizontally-oriented collagen bers, as evident
by Masson Trichrome stain, along with scarce inammation cells
and inconspicuous blood vessels were considered as an indication
for the maturity of the scar.

comparisons between variables were performed with Students

t-test. Signicances were calculated by comparing rates of healing
in group treated either with PgME ointment or with cetrimide
cream versus controls and in group treated with PgME ointment
versus cetrimide cream group. p-Values less than 0.05 were
considered to be signicant.
Results and discussion

Statistical analysis
For the in vitro tests, all data were expressed as
means standard errors of triplicate measurements. One-way
analysis of variance (ANOVA) was carried out to identify the
differences between treated groups and controls. The statistical

Fingerprint analysis of PgME

Fig. 2 shows the chemical pattern of PgME with ellagitanins (punicalagin A, punicalagin B, gallic acid and ellagic

Fig. 3. The antioxidant activities of the methanolic extract from P. granatum peels as determined by: (A) the ABTS Free radical scavenging activity and (B) the -carotene
bleaching test. Results are mean S.E of three parallel measurements. In (B) error bars are too small to be seen.

E.A. Hayouni et al. / Phytomedicine 18 (2011) 976984

acid) being the major compounds. Some anthocyanidins were

also detected namely delphinidin-3,5-diglucoside; quercitin3,4 -O-diglucoside; pelargonidin-3-glucoside and quercitin-4 -Oglucoside (Spiraeoside). It has been reported that pomegranate is
a rich source of anthocyanidins with delphinidin-3,5-diglucoside
being a major anthocyanin in pomegranate juice (Harborne
1967; Du et al. 1975). The seed coat of this fruit contains
delphinidin-3-glucoside, delphinidin-3,5-diglucoside, cyanidin-3glucoside, cyanidin-3,5-diglucoside, pelargonidin-3-glucoside, and
pelargonidin-3,5-diglucoside (Du et al. 1975). In addition, it is well
established that pomegranate peel contains substantial amounts of
hydrolyzable tannins punicalagins, punicalin, ellagic acid and gallic
acid (Nasr et al. 1996; Tanaka et al. 1986).
Antioxidant activity of PgME
Fig. 3A illustrates the decrease in absorbance of ABTS+ radical
due the scavenging ability of the PgME. At used concentrations, the
ABTS+ radical scavenging activity of PgME was found signicantly
higher than quercitin (p 0.05, at least). Furthermore, the extract
exhibited a scavenging potency, as strong as the positive control
BHA. It is well established that extracts obtained using high polarity solvents (such as methanol) were considerably more effective
radical scavengers than those using less polarity solvents (Zhou
and Yu 2004). The peel of this fruit had recently been reported to
have higher antioxidant activity than its pulp and seed (Li et al.
2006). Furthermore, Ricci et al. (2006) reported the antioxidant
capacities of some extracts from P. granatum arils, juice and rind
and correlated them to their polyphenols contents. In another work
undertaken by Okonogi et al. (2007) it was reported that the extract
of pomegranate peel showed the highest antioxidant activity, with
an IC50 of 0.003 mg/ml in a DPPH assay. This extract exhibited also
the highest TEAC value of 4.07 mM/mg.
In addition, according to the results presented in Fig. 3B, we
can notice that our extract, used at 500 ppm, showed more than
75% bleaching of the -carotene solution, which is as strong as the
synthetic antioxidants Trolox and BHA. Garca-Alonso et al. (2004)
using the TBARS method found markedly similar results when
investigating the antioxidant capacities of aqueous extracts from
pomegranate peels. Indeed, they pointed out that the fruits with
greater antioxidant activity are all rich in anthocynanidins, suggesting that these pigments could be contributing to this activity.
It is consented that reactive oxygen species (ROS) are deleterious to wound healing process due to the harmful effects on cells and
tissues (Aliyeva et al. 2004). The wound site is rich in both oxygen
and nitrogen reactive species along with their derivatives. The presence of these radicals will result in oxidative stress leading to lipid
peroxidation, DNA breakage, and enzyme inactivation, including
free-radical scavenger enzymes. Evidence for the role of oxidants
in the pathogenesis of many diseases suggests that antioxidants
may be of therapeutic use in these conditions.
In this context, topical applications of methanolic extractbased ointment, with free-radical-scavenging properties, could be
able to signicantly improve wound healing and protect tissues
from oxidative damage. It has been suggested that free radical
scavenging and antioxidant activities of extracts from various
parts of P. granatum play an important role in prevention of
free radical-related diseases, including aging, wounds and ulcers
(Harmam 2001). In addition, anthocyanidins prevented lipid peroxidation of cell or liposome membranes (Tsuda et al. 1996).
Ellagitanins, namely punicalagin, had shown remarkable pharmacological activities including anti-inammatory hepatoprotective
and antigenotoxic activities. One of the important factors responsible for all of the above activities of punicalagin could be ascribed
to its antioxidant activity which has been attributed to the presence
of 16 dissociable OH groups (Lin et al. 2001).

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Table 1
Minimum inhibitory concentrations of PgME on wound pathogens using micro- and
macrodilution methods.
MIC
P. granatum extract
Staphylococcus aureus ATCC 25923
Streptococcus pneumoniae
Escherichia coli ATCC 25922
Klebsiella pneumoniae
Pseudomonas aeruginosa ATCC 9027
Salmonella typhimurium
Salmonella anatum

>2
1
0.5
>2
0.5
0.25
0.25

Candida albicans
Candida glabrata
Trichopyton rubrum
Aspergillus niger

0.5
2
0.125
>2

Antibiotic
2a
0.125b
2a
1b
1a
1a
2a
0.5a
0.125a
15.62c
15.62c

MIC, minimum inhibitory concentration. Values are given as mg/ml for the methanolic extract and as g/ml for antibiotics: amikacin, clindamycine, amphotericin B.
a
Amikacin.
b
Clindamycine.
c
Amphotericin B.

Antimicrobial activity of the methanolic extract

The inhibitory effects of the crude-methanolic extract from
the peels of P. granatum against 11 pathogens are given in
Table 1. Among the test microorganisms, Trichopyton rubrum
was found to be the most sensitive with MIC = 0.125 mg/ml.
Salmonella typhimurium and Salmonella anatum came next with
MIC = 0.25 mg/ml, closely followed by Escherchia coli, Pseudomonas
aeruginosa, Candida albicans (MIC = 0.5 mg/ml). Streptococcus pneumoniae was the less sensitive strain (MIC = 1 mg/ml). Similar results
were reported in many other studies, where various extracts
obtained from different parts of P. grantum, were tested against
large panels of microorganisms. For example, Navarro et al. (1996),
recorded MICs values of 0.62 mg/ml (against Staphylococcus aureus
ATCC 6538) and 10 mg/ml (against Escherchia coli ATCC 8937, Pseudomonas aeruginosa ATCC 10231 and Candida albicans ATCC 10231)
when they tested a methanolic extract from the fruit shell. In previous research, ellagitanins inhibited the growth of the pathogenic
Gram-negative bacteria E. coli (IC50 = 9.2 M) and P. aeruginosa
(IC50 = 3.2 M). It is also likely that ellagitanins act synergistically when combined with anthocynanidins and other phenols to
enhance their action against bacteria (Reddy et al. 2007). Since MIC
values of P. granatum extract were substantially low namely against
Trichopyton rubrum (125 g/ml), and taking into accounts the high
antioxidant activity of the extract, it was proposed to undertake its
in vivo wound healing capacity.
Mechanical survey of the wound healing process
The restoration of a functional barrier during the healing process is dependent on the successful regeneration of new skin with
architecture that closely resembles the injured tissues. Wound contraction in different groups is shown in Fig. 4. It clearly appears that
wound contraction started immediately from day 4, in all groups.
However, on later days, the rate is much faster in the positive control group and the P. grantaum extract-based ointment group than
in the control groups (untreated and placebo groups). On day 8,
animals of both treated groups exhibited signicant increase in
the percentage of wound contraction as compared to untreated
and placebo groups. On day 16 post-surgery, It may be seen that
the positive control group showed 85% healing, whereas group
treated with P. granatum ointment showed 83.5% healing, when
compared to the controls (43% and 54% for untreated and placebo
group, respectively). On day 20, contrary to the control groups, no
scars were observed on animals treated with certimide cream or

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Fig. 4. Percentage reduction of wound size in control groups (untreated and placebo) and treated groups (with topical application of cetrimide cream or P. granatum ointment),
at 4-day intervals. Results are presented as means S.E (n = 46). Results were found signicant in ointment group versus untreated or placebo group (* p < 0.05 or ** p < 0.01)
and also in cetrimide group versus the same control groups (* p < 0.05 or ** p < 0.01). One-way ANOVA also supported t-test analysis and showed that both treatments have
similar potency and wound closure. The upper serial photos show the kinetics of wound closure in P. granatum ointment group.

P. granatum ointment. This may be considered as an indication for

complete healing and hence on this day measurements of tensile
strength were carried out.
Indeed, the results showed that the tensile strength of animals treated with cetrimide cream or the PgME-based ointment
was signicantly greater than those of the untreated group (control) and the placebo group. We recorded 506 g and 494 g (no
statistically signicant difference between treated groups: p > 0.05)
as breaking forces in certimide-cream group and PgME ointment
group, respectively. Meanwhile, in the untreated animals and the
placebo group, only 389 g and 412 g were recorded, respectively.
The increase in tensile strength of treated wounds may be due to
increase in collagen concentration and stabilization of the bers
(Udupa et al. 1995). The results obtained in this study were similar to those given by of Aloe vera on collagen characteristics
in healing dermal wounds in rats (Chithra et al. 1998). Also, a
similar effect has been observed with the ethanolic extract of
Centella asiatica on the rat dermal wound healing (Suguna et al.
1996).
Biochemical survey of the wound healing process
The collagen, DNA and total protein contents of granulation tissues on day 8 are given in Table 2. Wounds treated with P. granatum

extract-based ointment as well as the cetrimide cream exhibited a

very signicant increase in hydroxyproline levels as compared to
the untreated group (63% and 61.9% increase, respectively) and to
the vehicle group (74.2% folds and 72.5% rise, respectively). Collagen is the predominant extracellular protein in the granulation
tissue of a healing wound and there is a rapid increase in the synthesis of this protein in the wound area soon after an injury. In addition,
to provide strength and integrity to a tissue matrix collagen plays
an important role in haemostasis and subsequent epithelialization
requires also collagen.
Furthermore, the DNA levels were also increased by 77.5% in the
wound receiving topical applications of P. granatum extract-based
ointment as compared to the untreated animals. Similar results
were recorded in the certimide-treated group which showed a signicant DNA content increase (70.1% rise when compared to the
untreated animals). In parallel, similar behaviour was noticed, concerning the rates of the total proteins. Indeed, it was observed that P.
granatum and certimide groups had 74.9% and 73.6%, higher protein
contents, when compared to the vehicle, respectively. Such observations are corroborated by the collagen/DNA ratio in granulation
tissue. This parameter may be taken to be an index of the synthesis
of collagen per cell in the wound area. The order of the recorded
ratios was as follows: P. granatum ointment (29.9) > cetrimide
cream (27.7) > placebo (24.56) > untreated (24.45).

Table 2
Effects of topical treatment for 8 days on selected biochemical markers of wound healing in guinea pig excision models.

Hydroxyproline
DNA
Total proteins

Untreated

Placebo

Cetrimide cream

P. granatum ointment

62.6 1.22
2.56 0.03
52.3 0.51

73.2 1.1
2.98 0.09
65.5 0.33

101.1 0.87**
3.65 0.05**
89 0.53**

98.7 1.01**
3.3 0.08*
87.4 0.41**

Results are given in mg/g wet weight tissue. Values are mean S.D (n = 6 animals). There are no statistically signicant difference (p > 0.05) when comparing cetrimide group
to ointment group.
*
As compared to untreated or placebo group: p < 0.05.
**
As compared to untreated or placebo group: p < 0.01.

E.A. Hayouni et al. / Phytomedicine 18 (2011) 976984

983

Fig. 5. Histological evaluation of wound repair by Haematoxylin and Eosin (H&E 200) staining of granulation tissue on different day of healing. (A) Normal skin; on day 4:
(B) control and (C) P. granatum treated wounds; on day 12: (D) vehicle, (E) cetrimide cream treatment, and (F) P. granatum treatment; on day 20: (G) cetrimide treatment
and (H) P. granatum treatment.

The protein and DNA content of granulation tissues indicate the levels of protein synthesis and cellular proliferation.
Higher protein and DNA contents (compared to the untreated controls) of the treated wounds suggest that P. granatum ointment,
probably through an unknown mechanism, stimulates cellular
proliferation. Even though, it was recently established that an
aqueous extract of pomegranate peel had a potent dermal effect
by stimulating dermal broblast proliferation and collagen synthesis while inhibiting the major collagen-degrading enzyme in
skin (matrix metalloproteinase-1), but had no growth-supporting
effect on keratinocytes (Aslam et al. 2006). The collagen/DNA ratio
of the granulation tissues also suggests that P. granatum ointment may increase the synthesis of collagen per cell. The collagen
molecules synthesized are laid down at the wound site and become
crosslinked to form bers. Wound strength is acquired from both,
remodelling of collagen, and the formation of stable intra- and
inter-molecular crosslinks. P. granatum ointment-treated wounds
also showed an increased rate of wound contraction, leading to a
prompt healing as conrmed by decreased period of epithelialization when compared to untreated control wounds.
Histhopathalogical survey of the wound healing process
Haematoxylin and eosin (H&E) stained sections of granulations tissue collected on various days were examined for cellular
inltration, neo-vascularisation, epithelial regeneration and matrix
organization. Day 4 sections showed increased cellular inltration

in animals topically treated with the P. granatum ointment (Fig. 5C)

than control (Fig. 5B) and, no epidermal regeneration was observed.
On day 12, a well-advanced organization of granulation tissue and
on-going epithelialization was observed in treated groups, either
with certimide cream or P. granatum ointment (Fig. 5E and F,
respectively) than the vehicle (Fig. 5D) and the control. The histological examination showed that the original tissue regeneration
was much faster in the skin wound treated with extract ointment or
cetrimide cream without any oedema, congestion or inammatory
disorders (results not shown).
On day 20, animals treated with the extract-based ointment
(Fig. 5H) and the certimide-based cream (Fig. 5G) showed very close
proles when compared to one another and when both groups were
compared to the control and the vehicle. There was full thickness
epidermal regeneration which covered completely the wound area.
The epidermis was thick and disorganized, especially when compared with the adjacent normal skin. The keratin layer was thick
with focal parakeratosis. In both groups the granular layer was well
formed but 23 cells in thickness. The basal layer was well formed
in both groups as it was in the control and the vehicle groups. In
addition, in the dermis, maturation and organization of the collagen bers as judged by the Masson Trichrome stain was similar
(Fig. 6B and C). The dermis was cellular with proliferation of broblasts, laying down disorganized and poorly oriented collagen bers.
Prominent capillary-sized blood vessels were seen. Scattered collections of inammatory cells, consisting mainly of macrophages
and few neutrophils were also present throughout the whole thick-

Fig. 6. Histological analysis of wound-edge tissue obtained on day 20 of the experiment. Neutral buffered formalin-xed sections were stained with Masson Trichrome
procedure (400) which results in blue-black nuclei, blue collagen/cytoplasm, and keratin/muscle bers/intracellular bers all stained red. (A) Vehicle treated group showing
spongiosis of the epidermis with persistence of inammatory cells in the dermis. (B) Cetrimide cream treatment showing maturation of the epidermis and the dermis. (C) P.
granatum treatment showing thin well-formed epidermis with hair follicle formation in the dermis and no inammatory cells in a well organized dermis. (For interpretation
of the references to color in this gure legend, the reader is referred to the web version of the article.)

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E.A. Hayouni et al. / Phytomedicine 18 (2011) 976984

ness of the dermis. Whereas, in vehicle group (Fig. 6A) there was a
failure of re-epithelization of the wound. Indeed, there was persistence, within the dermis, of immature granulation with few
haphazardly-oriented collagen bers. Inammatory cells, predominantly neutrophils, were still detected within the granulation tissue
together with blood vessels which were prominent and dilated.
Conclusion
In addition to its value as a table fruit, pomegranate preparations have been used for ages in various folk medicines. In recent
years there is important focus on the bioactive moieties of its different parts which showed a large panel of activities: antioxidant,
anti-inammatory, angiogenic, anti-cancer, skin repair. . . these are
namely attributed to the polyphenolic and lipophilic fractions.
The different phases of the wound healing process overlap and
ideally a plant-based remedy should affect at least two different
processes before it can be said to have some scientic support for
its traditional use. Since there is a denite role of free radicals in the
pathogenesis of wound, the antioxidant activity was studied. The
results indicate that the PgME possesses potent antioxidant activity
by inhibiting lipid peroxidation and increasing the potency of free
radicals scavenging.
In the eld of wound healing, there are several unknowns,
this includes the wound itself. Mechanical and biochemical survey
together with the histological results showed that the methanolic extract-based ointment from Tunisian pomegranate exhibits
potent healing properties on excision wounds, in guinea pigs
model. This again validates the potent wound healing activity of
P. granatum extracts as claimed by the ethnopharmacological data.
However, the exact mechanism of the healing process of wound
is not clearly understood. Indeed, one has to remember that there
are a number of parameters which are involved in the healing of
wound including epithelization, antioxidant defense and biochemical changes (hydroxyproline). Therefore, further approaches are
needed to clearly elucidate the full mechanism of action of such
natural preparations.
Acknowledgments
We are very grateful to Dr. Nazek (Pasteur Institute, Tunis) for
providing us with microorganisms. The ointment formulated using
the crude methanolic extract from Punica granatum L. peels is protected by a Tunisian patent (N 7260).
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