Wound-healing Properties of the Oils of Vitis vinifera and Vaccinium macrocarpon

Abstract

Vitis vinifera (grape) and Vaccinium macrocarpon (cranberry) are well known medicinal plants; most of the pharmacologically active phytochemicals have been isolated from the skin, fruit juice, fermented extract and alcohol fractions of the plants above. Here, the pharmacological properties of the phytochemical constituents present in oils of cranberry and grape were investigated. The oil of grape and cranberry has been evaluated for their wound healing activity by using an excision wound model in rats. The animals were divided into four groups of six each (n = 6). The experimental group 1 and 2 animals were treated topically with the grape and cranberry oil (100 mg/kg body weight), respectively. The controls were treated with petroleum jelly. The standard group of animals were treated with mupirocin ointment (100 mg/kg body weight). The healing was assessed by the rate of wound contraction and hydroxyproline content. On day 13, animals treated with cranberry oil exhibited a (88.1%) reduction in the wound area compared with grape-oil treated (84.6%), controls (74.1%) and standard group animals (78.4%) (p < 0.001). The hydroxyproline content of the granulation tissue was significantly higher in the animals treated with cranberry and the grape-oil (p < 0.000). Comparative investigation of the curative properties of the oils of V. vinifera and V. macrocarpon revealed a significant result which suggests their wound-healing potential.

Full text

Wound‐healing Properties of the Oils of
Vitis vinifera and Vaccinium macrocarpon
B. Shivananda Nayak,
1
*D. Dan Ramdath,
1,2
Julien R. Marshall,
1
Godwin Isitor,
1
Sophia Xue
2
and John Shi
2
1
Department of Preclinical Sciences, Faculty of Medical Sciences, The University of the West Indies, St Augustine, Trinidad
2
Guelph Food Research Centre, Agriculture and Agri‐Food Canada, 93 Stone Road West, Guelph, Ontario N1G 5C9, Canada
Vitis vinifera (grape) and Vaccinium macrocarpon (cranberry) are well known medicinal plants; most of the
pharmacologically active phytochemicals have been isolated from the skin, fruit juice, fermented extract and
alcohol fractions of the plants above. Here, the pharmacological properties of the phytochemical constituents
present in oils of cranberry and grape were investigated. The oil of grape and cranberry has been evaluated for
their wound healing activity by using an excision wound model in rats. The animals were divided into four
groups of six each (n= 6). The experimental group 1 and 2 animals were treated topically with the grape and
cranberry oil (100 mg/kg body weight), respectively. The controls were treated with petroleum jelly. The
standard group of animals were treated with mupirocin ointment (100 mg/kg body weight). The healing was
assessed by the rate of wound contraction and hydroxyproline content. On day 13, animals treated with
cranberry oil exhibited a (88.1%) reduction in the wound area compared with grape‐oil treated (84.6%),
controls (74.1%) and standard group animals (78.4%) (p< 0.001). The hydroxyproline content of the granulation
tissue was significantly higher in the animals treated with cranberry and the grape‐oil (p< 0.000). Comparative
investigation of the curative properties of the oils of V. vinifera and V. macrocarpon revealed a significant result
which suggests their wound‐healing potential. Copyright © 2011 John Wiley & Sons, Ltd.
Keywords: hydroxyproline; excision wound; wound area.
INTRODUCTION
Ethnomedicinal research has searched arduously for
remedies that can be derived from the abundant
resources of nature. Recent discoveries into the wound
healing potential of Vitis vinifera,Morinda citrifolia and
Vinca rosea has prompted further work; which seeks to
increase the knowledge of the medicinal properties of
the wide variety of plants. Grape and cranberry are two
well known plants within ethnomedicine. Research has
highlighted the potential curative properties of antho-
cyanins, tannins and resveratrol found in grape extract.
Research has also discovered significant concentrations
of polyphenols such as catechins, cyanidin‐3‐glucoside
and peonindin‐3‐glucoside in cranberry fruit. Intense
research has been directed to the discovery of the
phytochemical constituents of the skin and fruit of those
plants.
The common grape V. v i n i f e r a is a historically
important plant. The fruit is a berry, known as a grape.
In the wild species, it is 6 mm diameter and ripens dark
purple to blackish with a pale wax bloom. In cultivated
plants, it is usually much larger, up to 3 cm long and can
be green, red, or purple. Red wines from several red
grape varieties have been reported to have health
benefits based on the significant amount of polyphenolic
derivatives present (Pastrana‐Bonilla et al., 2003).
Resveratol, a non‐flavanoid biomolecule is found in
significant quantities in red wine and has been reported
to possess antioxidant, antiinflammatory and anticancer
properties (Elliott and Jirousek, 2008; Asensi et al.,
2002).Additionally, the topical application of a high
resveratol from grape seed extract was shown to
accelerate wound healing in mice, which was attributed
to modulation of the redox‐sensitive processes that
drive dermal tissue repair (Khanna et al., 2002).
Cranberries are a group of evergreen creeping shrubs
or vines up to 2 m long and 5–20 cm in high. It is edible
with an acidic taste that can overwhelm its sweetness.
Cranberries and cranberry juice are abundant food
sources of the anthocyanidin, flavonoids, cyanidin,
peonidin and quercetin (Duthie et al., 2006). Cranberry
juice is used in the prevention and treatment of urinary
system infections (Hood et al., 2004; Jenkins et al.,
2005), as well as in the treatment of periodontitis
(Ratcliffe and Shachar‐Hill, 2005; Rios et al., 2002) and
other disorders.
It has been proposed that alkaloids and terpenoids
may provide astringent, antifungal or antimicrobial
properties that may be of benefit to the progression of
the wound healing cascade (Scortichini and Pia Rossi,
1991; Miot et al., 2004; Roy and Saraf, 2006). Antho-
cyanins, leucoanthocyanins and other polyphenols have
been demonstrated to have significant antioxidant
properties which also can be of benefit in the progression
of wound healing (Shetty etal.,2007; Nayak et al., 2009;
Parry et al., 2006; Shi et al., 2003). It is reasonable to
expect that the phytochemical constituents in oil
fractions of grape and cranberry would have significant
wound healing properties. However, there appear to be
* Correspondence to: B. Shivananda Nayak, Department of Preclinical
Sciences, Faculty of Medical Sciences, The University of the West Indies,
St Augustine. Trinidad and Tobago.
E-mail: shivananda.nayak@sta.uwi.edu
PHYTOTHERAPY RESEARCH
Phytother. Res. (2011)
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.3363
Copyright © 2011 John Wiley & Sons, Ltd.
Received 30 March 2010
Revised 22 October 2010
Accepted 26 October 2010

very few reports on the wound healing potential and
antimicrobial activities of Vitis vinifera and Vaccinium
macrocarpon in literature. Therefore, this study was the
first attempt to report the efficacy of Vitis vinifera and
Vaccinium macrocarpon for their possible role in the
treatment and management of wounds.
MATERIALS AND METHODS
Grape seed oil production. The grape seeds oil was
obtained by compressing grape seeds (supplied by
Joseph’s Natural Products Inc.) using a screw extrusion
press (Komet S 87 G, IBG Monforts, Mönchengladbach,
Germany). Grape seed pressing was performed without
heating (cold pressed).
Cranberry oil production. The cranberry oil was
obtained by compressing cranberry (supplied by Joseph’s
Natural Products Inc.) using a screw extrusion press
(Komet S 87 G, IBG Monforts, Monchengladbach,
Germany). Pressing was performed without heating (cold
pressed).
Lipid extraction, separation, methylation and GC
analysis. Grape seed oil (10 mL) was extracted with
140 mL chloroform: methanol (1:1, v/v) using an Ultra‐
Turrax homogenizer (T25, Ika Works Inc., USA). After
homogenization, it was stirred for 5 min to obtain a
uniform consistency in 63 mL water that was added to
the solution to avoid any miscellaneous phase (Bligh
and Dyer, 1959). The chloroform phase was collected
and partitioned again with some chloroform. The
combined extracts were taken to dryness by a vacuum
rotary evaporator (<30 °C) until constant weight and
then stored at −20 °C until analysis.
The total crude lipids obtained were dissolved in
15 mL chloroform. Fatty acid methyl esters (FAME)
analysis was done to determine the total fatty acids. In
this FAME analysis, 0.25 mL (10–12 mg) of each lipid
extract was methylated with 1.5 mL 5% anhydrous
HCl/methanol (w/v) in a 15 mL culture tube equipped
with a Teflon‐lined screw cap at 80 °C for 1 h. After
cooling to room temperature, a few drops of water and
2 mL hexane was added. The hexane layer was
collected and reduced in volume before the FAME
mixture was applied to TLC plates. The loaded TLC
plates were developed in the solvent mixture of hexane/
ethyl ester/acetic acid (85:15:1, v/v/v), sprayed with
2′,7′‐dichlorofluoroscein/methanol (0.1% w/v), and
viewed under UV light (254 nm). The corresponding
FAME band was removed and eluted with chloroform.
The filtrate was blown dry under nitrogen and the
FAME mixture was dissolved in 1.5 mL hexane and
analysed by GC.
For separating the lipid classes, 0.4 mL (at least
20 mg) of each crude extract was dried under a stream
of nitrogen, then added to chloroform and applied
onto TLC plates before methylation of the FA. TLC
plates were developed and viewed under UV light as
above. Major lipid class bands were scraped from the
plates. The first two bands near the bottom were
eluted with methanol: chloroform (1:1, v/v), and the
upper three bands were eluted with chloroform. After
removal of the chloroform with nitrogen, the lipid
classes were methylated for 1 h, and then water
(5 mL) and 2 mL hexane were added to collect the
FAME in hexane as above. Calibration and identifica-
tion of the different lipid classes were made by running
standards in parallel with the samples. The extractable
lipids were separated into five classes: polar lipids
(phospholipids and glycolipids, PL), diacylglycerols
(DAG), free fatty acids (FFA), triacylglycerols (TAG)
and steryl esters (SE).
A GC (model 5890; Hewlett‐Packard, Palo Alto, CA)
equipped with a flame ionization detector and an auto
sampler (model 7673, Hewlett‐Packard, Palo Alto, CA,
USA), a 100 m CP‐Sil 88 fused capillary column (Varian
Inc., Mississauga, ON, Canada) and ChemStation
software system (version A.09, Hewlett‐Packard,
Palo Alto, CA, USA) were used for analysing FAME.
The injector and detector temperatures were 250 °C.
The temperature programme for the column was: hold
at 45 °C for 4 min, increase by 13 °C/min to 175 °C,
hold again at 175 °C for 27 min, increase at 4 °C/min to
215 °C, and then finally hold at 35 °C. A FAME
standard (mixture 463) was used to identify the FAME
and the FA amount was expressed as the percent of
total fatty acids.
The above procedure was repeated for cranberry oil
for lipid extraction, separation, methylation and GC
analysis.
Determination of total phenols of cranberry oil and
green grape seed oil. Extraction of the phenols was
carried out according to a method presented by Parry et al.
(2005) with some modifications. For the extraction of oils,
15 mL of methanol (methanol: water, 90:10 ) was added to
5.0 g oil, followed by vortex for 3 min and centrifugation
for 10 min at 3500 rpm. The supernatant was collected for
phenolic content measurements.
The quantitative determination of total phenols (TP)
was analysed by using the Folin‐Ciocalteau colorimetric
method (Swain and Hillis, 1959). Five hundred micro-
litres of sample extract was placed in a test tube along
with 1.0 mL of Folin‐Ciocalteau reagent and 2 mL of
distilled water. After leaving for 5 min, 0.5 mL of
sodium carbonate (10%) was added, mixed and allowed
to stand for 2 h in a dark place for reaction. Absorption
at 765 nm was measured in a UV‐Vis spectrophotometer
against a blank sample. The TP content was expressed
as gallic acid equivalents in milligrams per gram of oil,
using a standard curve generated with 5 mg, 10 mg,
25 mg, 50 mg, 75 mg and 100 mg gallic acid per 100 mL
extraction solvent.
Phenolic compound analyses by HPLC. An HP 1100
HPLC system equipped with an alphaBond C
18
125A
column (4.6×250 mm, particle size 5 μm), coupled with
an Agilent 1100 series ChemStation software was used
for quantifying the individual phenolic compounds. The
mobile phases consisted of 2.0% acetic acid in distilled
water (A) and acetonitrile (B). The column was eluted
at 1.0 mL/min under a linear gradient from 5% mobile
phase B to 75% over 20 min, to 100% over 5 min,
isocratic 5 min at 100%, and then to 25% over 5 min
and to 5% over another 5 min. Sample injection
volumes were 20 μL. The compounds were detected
at 280 nm with an HP 1100 series ultraviolet (UV) diode
array detector. Standards of catechin and epicatechin
were injected for identification.
B. SHIVANANDA NAYAK ET AL.
Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. (2011)

Animals. The study was approved by the Ethics
Committee for animal experimentation (AHC06/07/1)
by The Faculty of Medical Sciences, The University of
the West Indies, St Augustine. Healthy inbred male
Sprague Dawley rats weighing 180–200 g were used for
the study. They were housed individually and main-
tained on normal food and water ad libitum. Animals
were periodically weighed before and after the experi-
ment. The rats were anaesthetized prior to and during
infliction of the experimental wounds. The surgical
interventions were carried out under sterile conditions
using ketamine anaesthesia (120 mg/kg body weight).
The animals were closely observed for any infection and
if they showed signs of infection were separated,
excluded from the study and replaced.
Wound‐healing activity. An excision wound model was
used to evaluate the wound‐healing activity of oils of
Vitis vinifera and Vaccinium macrocarpon.
Excision wound model. The animals were anaesthetized
prior to and during creation of the wounds. The rats
were inflicted with excision wounds as described by
other researchers (Morton and Malone, 1972). The
dorsal fur of the animals was shaved with an electric
clipper and the area of the wound to be created was
outlined on the back of the animals with methylene blue
using a circular stainless steel stencil. A full thickness of
the excision wound of circular area 250 mm
2
and 2 mm
depth was created along the markings using toothed
forceps, a surgical blade and pointed scissors. The
wound closure rate was assessed by tracing the wound
on alternate days (day 1, 3, 5, 7, 9 etc.) using
transparency paper and a permanent marker. The
wound areas recorded were measured using graph
paper. The point at which the eschar fell off without
any residual raw wound was considered epithelization.
Estimation of hydroxyproline. Dry granulation tissue
from both the control and treated group was used for
the estimation of hydroxyproline. The hydroxyproline
present in the neutralized acid hydrolysate was
subsequently oxidized by sodium peroxide in the
presence of copper sulphate followed by complexing
with para‐dimethylaminobenzaldehyde to develop a
pink colour and that was measured at 540 nm by a
spectrophotometer.
Histological study. The granulation tissue was obtained
on day 13 from the test and control group animals for
the histological study. Van Gieson’s and haematoxylin
and eosin stains were used to show the fibroblasts and
collagen deposition.
Antimicrobial activity. Pseudomonas aeruginosa
(ATCC 27853), Klebsiella pneumonia (ATCC 700603),
Enterococcus fecalis (ATCC 29212), Escherichia coli
(ATCC 25922), Staphylococcus aureus (ATCC25923)
and methicillin resistant Staphylococcus aureus
(MRSA) (ATCC 43300) were the organisms tested.
The bacterial strains were obtained from fresh colonies
grown on MacConkey and blood agar plates. The
sensitivity testing was done using Muller Hinton agar
plates. A known volume of bacterial suspension was
transferred to each microplate well. Ten microlitres of
(5 mg/mL) of V. v i n i f e r a and V. macrocarpon oils was
added to the microplate wells and incubated at 35–37 °C
for 18–20 h. The results were analysed visually.
Statistical analysis. The means of wound area measure-
ments between groups at different time intervals were
compared using one‐way ANOVA, descriptive test,
followed by Tukey’s post‐hoc test. One‐way ANOVA
was used to examine the mean difference in epitheliza-
tion period between the groups. Data were analysed
using the SPSS (Version 12.0, Chicago, USA) and a
value of p< 0.05 was used for all analyses.
RESULTS
Phytochemical analysis
Phytochemical analyses of grape and cranberry oils, by
quantitative methods have shown the presence of
significant amounts of polyphenolic derivatives,
leucoanthocyanins and fatty acids (Table 1). Analysis
of the oils revealed a significant amount of phenolic
derivatives, expressed as gallic‐acid equivalents and it
was 3.167 mg/g ± 0.124 and 3.330 mg/g ± 0.123 for
cranberry and grape oil, respectively (Table 2). A
quantitative evaluation of grape oil was done and the
total polyphenolic and leucoanthocyanin equivalents
were determined for grape seed. The seed contained
34.1 ± 5.3 mg of total polyphenolic equivalents. The
leucoanthocyanin equivalents were determined and it
was noted that the seed of the V. v i n i f e r a contained
368.6 mg/g ± 71.4 of cathechin and 276.3 mg/g ± 56.3 of
itsepimerepi‐cathechin. These results suggest that
Table 1. Fatty acid composition of grape seed and cranberry oil
Fatty acid (file) Grape (Niagara) Grape W (WI) Cranberry
Total 95.15 97.79 93.73
Saturated 10.99 11.64 6.84
Monounsaturated 15.22 16.56 18.77
Polyunsaturated 68.94 69.59 68.12
Total fat (as TG) 95.63 98.29 94.21
Omega‐30.35 0.40 31.26
Omega‐668.59 69.19 36.86
Omega‐914.86 16.23 18.70
Palmitic acid 7.10 7.36 5.63
Stearic acid 3.67 3.96 1.21
Samples from Joseph Estate Wines, 1811 Niagara Stone Road,RR#, Niagara‐on‐the‐lake, Ontario L0S 1 J0 Canada.
WOUND HEALING ACTIVITY OF GRAPE AND CRANBERRY OILS
Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. (2011)

leucoanthocyanin and other polyphenols may exist in
the oil extracts of grape and cranberry, since these oils
were obtained from cold‐pressing of the seeds of these
fruits.
Microbiological study
Microbial analysis to support the efficacy of grape and
cranberry oil revealed that concentrated oils of both
fruits exhibited some antibacterial effects. Concentrated
grape oil was proven to be effective against two strains
of Escherichia coli. Similarly concentrated cranberry oil
proved to be effective against methicillin resistant
Staphyloccocus aureus and two strains of Escherichia
coli and Klebsiella pneumonia (Table 3 and 4).
Biochemical and wound area measurements
Animal specimens treated with cranberry oil showed
faster rates ofwound area contraction of 88.1% by day 13,
whereas the grape oil treated animals had 84.6% of
wound area contraction (Figs. 1 and 2). Biochemical
analysis of hydroxyproline content of granulation tissue
suggested that both grape and cranberry oils are effective
potential wound healing agents based on the recorded
hydroxyproline content. The results of the hydroxyproline
content of cranberry oil (21.22 mg/g±0.02) was slightly
higher compared with grape oil (20.10 mg/g ± 0.02)
which suggests that the cranberry has better wound
healing potential than grape oil (Fig. 3). These results
suggest that grape and cranberry oil have strong wound
healing potential.
Histological study
The histological study of the granulation tissue obtained
on day 13 from the experimental animals (grape and
cranberry treated) showed increased well organized
bands of collagen (Figs. 4 and 5) compared with the
controls, which showed scanty collagen fibres (Fig. 6).
Mupirocin treated animals showed well organized
collagen fibres (Fig. 7). Numerous fibroblasts in H & E
staining of the tissues obtained from the experimental
and standard suggesting a significant amount of collagen
and fibronectin deposition.
DISCUSSION
Antibiotic therapy represents one mechanism involved
in the progression of the inflammatory pathway, in vivo
which is carried out by resident leucocytes. Exogenous
antibiotics are often given to augment the activity of
leucocytes. Initial antibiotics were derived from a
natural source, namely bacteria. Most antibiotics are
classified as bacteriostatic or bactericidal based on their
relative action on the bacterial organism. A bacterio-
static chemical usually arrests protein or carbohydrate
synthesis in the bacterium causing the organism to
remain in static growth, but does not kill the cell.
Whereas bactericides may inhibit the production of
essential material required for organism survival, e.g.
inhibition of peptidoglycan layer formation in Gram‐
positive bacteria, or via induction of DNA instability
(Walsh, 2003; Spratt, 1977; Kasten and Reski, 1997;
Widmer, 2008).
Possible role of phytochemical constituents on
wound healing
Both oils demonstrated significant antimicrobial activity
against Escherichia coli, which suggest the intrinsic
effect of polyphenols modulating the integrity of the
peptidoglycan layer of Gram‐positive bacteria. It has
been also suggested that they act synergistically with
Table 2. Phenolic content of cranberry and grape oil
Sample Gallic acid eq. (mg/g) % content/(g) of oil
Cranberry oil 3.167± 0.124 0.32
Green grape oil 3.330± 0.123 0.33
Total phenol content was analysed as gallic acid equivalent mg/g
of oil, values were taken as the average of triplicates.
Table 3. Comparative antimicrobial analysis of 1 in 20 dilutions of oils of cranberry and grape
Sample QCAQCB123456789
Grape NG NG GGGGGGGGG
Cranberry NG NG GGGGGGGGG
Table 4. Comparative antimicrobial analysis of concentrated oils of cranberry and grape
Sample QCAQCB123456789
Grape NG NG G G G NG NG G G G G
Cranberry NG NG GGNGNGNGNGGGG
1, Pseudomonas aeruginosa ATCC 27853; 2, Staphylococcus aureus ATCC 25923; 3, Staphylococcus aureus ATCC 43300 (MRSA); 4,
Escherichia coli ATCC 25922; 5, Escherichia coli ATCC 35218; 6, Klebsiella pneumonia ATCC 700608; 7, Enterococcus faecalis ATCC
29212; 8, Candida albicans (bench strain yeast); 9, Clostridium perfringens (bench strain anaerobe bacteria); QCA, Muller Hinton agar (no
extract/bacterial challenge) Control; QCB, direct culture of extract (no bacterial challenge) Control; G, growth of bacterial colonies; NG, no
growth of bacterial colonies.
B. SHIVANANDA NAYAK ET AL.
Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. (2011)

beta‐lactams to act as beta‐lactamase at a capacity
similar to clavulanic acid (Spratt, 1977; Gianfranco
et al., 2008). Phenolic compounds in products play
important roles for antioxidant, antiinflammatory and
antimicrobial effects (Shi et al., 2003). At this stage, it
can be stated that the antioxidant activity of both oils
might have contributed more value to the antimicrobial
activity against Escherichia coli.
The demonstrated antimicrobial activity of cranberry
oil against Staphyloccocus aureus and Klebsiella
pneumonia may be due to the presence of a phyto-
chemical constituent in the oil. Polyphenols have been
investigated previously for their curative properties and it
has been indicated that cathechin, a monomeric flavonoid
component of leucoanthocyanin; conjugates of cathechin
such as cathechin gallate, and its epimer epi‐cathechin
may have inhibitory effects against (MRSA) methicillin
resistant Staphyloccocus aureus and Streptoccocus mutans
(Ikigai et al., 1993; El‐Gammal and Mansour, 1986;
Muroi and Kudo, 1993).
Recent research has suggested that in addition to
antimicrobial activity, polyphenols may possess protease
inhibitor properties. Matrix proteases are required for
the degradation of the extracellular matrix proteins,
fibrin and fibronectin, in preparation for the deposition
of collagen by fibroblasts. This protease activity gradually
Figure 1. Wound‐healing activity of grape and cranberry oil,in comparison with standard and control (n=6). Each column represents mean ± SE.
This figure is available in colour online at http://wileyonlinelibrary.com/journal/ptr
Figure 2. (C) Excision wound on day 1 treated with petroleum jelly, (C13) excision wound on day 13 treated with petroleum jelly (74.2%
wound closure), (T) excision wound on day 1 treated with V. macrocarpon, (Cr13) excision wound on day 13 treated with V. macrocarpon
(88.1% wound closure), (T) excision wound on day 1 treated with V. vinifera, (Gr13) excision wound on day 13 treated with V. vinifera
(84.6% wound closure), (S) excision wound on day 1 treated with mupirocin ointment, (S13) excision wound on day 13 treated with
mupirocin (78.4% wound closure). This figure is available in colour online at http://wileyonlinelibrary.com/journal/ptr
WOUND HEALING ACTIVITY OF GRAPE AND CRANBERRY OILS
Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. (2011)

decreases as the more stable collagen type (III) fibre
is deposited. If the protease activity is not curtailed in a
systematic pattern extracellular matrix repair, granulation
tissue development and wound area closure is affected
(Sartor et al., 2002).
There is much information on the antioxidant
properties of polyphenols. The antioxidant activity
may be significant in accounting for the antioxidant
activity proposed by the varied literature that currently
exists. Research into the effect of free radicals on
wound healing has suggested that the presence of these
radicals inhibits the activity of fibroblasts and macro-
phages. Inhibition of these cells may prevent the
progression of the inflammatory pathway. One method
of controlling the presence of free radicals is to solicit
the activity of radical scavenger molecules. Fatty acids
such as oleic acid, linoleic acid were proven to have a
significant antioxidant profile when compared with
α‐tocopherol, and may be able adequately to scavenge
free radicals. Based on the quantitative results of grape
oil and cranberry, it is noted these oils contain
significant amounts of omega‐6 and omega‐9 fatty acids.
Therefore the wound‐healing activity could be attrib-
uted to the antioxidant activity of the fatty acids and
anthocyanins measured in the oil fractions (Gupta et al.,
2002; Al‐Naqeeb et al., 2009).
Pro‐inflammatory molecules are responsible for the
progression of the inflammatory phase in would
healing. Arachidonic acid is considered to be a
significant pro‐inflammatory molecule and it has been
suggested that its precursor, linoleic acid, may be
antiinflammatory as it has been observed in recent
studies to weakly inhibit cyclooxygenase (Henry et al.,
2002).
Figure 3. Hydroxyproline content of the granulation tissue in
comparison with standard (mupirocin), control (petroleum jelly),
grape and cranberry oil (n=6). Each column represents mean ± SE
(p<0.000) versus control and standard (one‐way ANOVA,
descriptive test). This figure is available in colour online at http://
wileyonlinelibrary.com/journal/ptr
Figure 4. (HE) H&E stain of granulation tissue of animal specimen treated with grape oil shows a matrix of collagen fibres, and fibroblast in the
vicinity. a, macrophages; b, fibroblast; c, collagen. (VG) Van Gieson stain of granulation tissue of animal specimen treated with grape oil shows
a dense matrix of wavy collagen fibres. a, collagen fibres. This figure is available in colour online at http://wileyonlinelibrary.com/journal/ptr
Figure 5. (HE) H&E stain of granulation tissue of animal specimen treated with cranberry oil shows a matrix of collagen fibres, and fibroblast
in the vicinity. a, macrophages; b, fibroblast; c, collagen. (VG) Van Gieson stain of granulation tissue of animal specimen treated with
cranberry oil, the stain shows a dense matrix of wavy collagen fibres. a, collagen fibres. This figure is available in colour online at http://
wileyonlinelibrary.com/journal/ptr
B. SHIVANANDA NAYAK ET AL.
Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. (2011)

The data demonstrate that grape and cranberry oil
facilitate significant wound healing. Its wound healing
promoting activity could be due to a combination of
antimicrobial, antiinflammatory and antioxidant activities,
by the respective constituents or it could be due to the
individual activity of active leucoanthocyanins, fatty acids
and other polyphenols. Further investigation is required to
isolate the active ingredients that promote wound healing,
before it can be considered for clinical application.
Acknowledgements
We extend our sincere thanks to Dr William Swanston and Mrs
Sabana Mayers for their excellent microbiological work.
Conflict of Interest
The authors state there was no conflict of interest.
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Figure 6. (HE) H&E stain of granulation tissue of animal specimen treated with petroleum jelly shows sparse collagen deposition, numerous
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a, collagen fibres. This figure is available in colour online at http://wileyonlinelibrary.com/journal/ptr
Figure 7. (HE) H&E stain of granulation tissue of animal specimen treated with mupirocin ointment shows a matrix of collagen fibres, and
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http://wileyonlinelibrary.com/journal/ptr
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