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JOURNAL OF WOUND CARE VOL 14, NO X, XXXXXXXXX 2005 1
Wound healing activity of Persea
Americana (avocado) fruit: a
preclinical study on rats
B.S. Nayak, MSc, PhD,
FAGE, NRCC-CC, FACB,
job title? Department of
Preclinical Sciences,
Biochemistry Unit,
Faculty of Medical
Sciences, The University
of the West Indies, St.
Augustine, Trinidad;
S.S. Raju, MBBS, MD, job
title? Department of
Pharmacology, School of
Medical Sciences, RAK
Medical and Health
Sciences University, Ras-
al-khaimah, United Arab
Emirates;
A.V. Chalapathi Rao,
MBBS, MD, Department
of Para clinical sciences,
Pathology unit, Faculty of
Medical Sciences, The
University of the West
Indies, St. Augustine,
Trinidad
Email: shiv25@gmail.com
W
ound healing comprises a
complex but well-coordinat-
ed cascade of biochemical
events that follow injury to
the skin and other soft tis-
sues. It is categorised into three phases: inflamma-
tion, proliferation and remodelling. The initial
inflammatory reaction is followed by the prolifera-
tive phase, which is characterised by angiogenesis,
collagen deposition, granulation tissue formation,
epithelialisation and wound contraction.1-4 (Sta-
delmann et al 1998; Iba et al 2004; Quinn
1998; Midwood et al 2004) The extracellular
matrix (okay?) and collagen are remodelled in the
final phase to provide integrity and strength to the
healed tissue (reference?). Factors that impair
wound healing include poor blood supply, lack of
nutrients, systemic disease, infections and the pres-
ence of foreign bodies. The ability to identify and
address these factors will help facilitate healing. Var-
ious nutritional factors and botanical influences,
including carbohydrate, protein, lipid sources (such
as? I assume you mean natural oils, waxes
etc?), vitamins, minerals and other trace elements,
play an important role during the different phases
of wound healing (references?). Adequate dietary
protein is vital for wound healing, and tissue levels
of the amino acids arginine and glutamine may
influence wound repair and immune function.5
(Agrawal et al 2003) Botanical extracts from
sources including Aloe vera, Centella asiatica and the
enzyme bromelain from pineapple have also been
shown to improve healing times and wound out-
comes.6,7 (MacKay and Miller 2003; Douglas et
al 2003 - author, we need the full reference
details for Douglas, seems to be the same as
MacKay and Miller at the moment - thank
you)
Avocado oil (Persea americana) is rich in unsapon-
ifiables (sterolins - please define briefly) com-
prised of biologically active compounds, such as
sterols, tocopherols, squalene and lipidic furans and
also contains vitamins A, C, D and E, proteins, beta-
carotene, lecithin, fatty acids and potassium.8
(Swisher 1988)
The use of avocado oil is considered to be benefi-
cial (by who? how widely accepted is this? or
should we say it has been found to reduce
l Objective: Avocado (Persea americana) oil is rich in nutrient waxes, proteins and minerals, as well as
vitamins A, D and E. It is an excellent source of enrichment for dry, damaged or chapped skin. This study
aimed to evaluate the wound-healing activity of fruit extract of Persea americana in rats.
l Method: The effect of topical and oral administration of Persea americana fruit extract (300mg/kg/day)
on excision and dead space wound models was evaluated. The rats for excision wound model were
divided into two groups, five each for topical and oral treatment. The rats for dead space wound models
were treated orally. Healing was assessed by the rate of wound contraction, period of epithelialisation,
granulation tissue weight and hydoxyproline content.
l Results: Complete healing (full epithelialisation) was observed on average on day 14 in both oral and
topical extract-treated animals. Whereas, controls wound took around 17 days. The extract treated
wounds were found to epithelialise faster as compared with controls (p<0.001). Wet and dry granulation
tissue weight and hydroxyproline contents of the tissue obtained from extract treated animals were
significantly higher (p<0.05) as compared with controls.
l Conclusion: Rate of wound contraction, epithelization time together with the hydroxyproline
content and histological observations support the use of P.americana in the management of wound
healing
l Declaration of interest: None.
Persea americana; hydroxyproline; epithelialisation
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JOURNAL OF WOUND CARE VOL 14, NO X, XXXXXXXXX 20052
age spots etc? but was this in a clinical
study?) in reducing age spots, healing scars and
moisturising the upper layers of the skin.7 (Douglas
et al 2003) A mixture of avocado and soybean
unsaponifiables has also been found to aid wound
healing, possibly by stimulating the deposition and
repair of extracellular matrix components.9,10 (Mar-
garet et al 1982; Boumediene et al 1999)
However, there is no scientifically proven data to
support either the collagen-enhancing or wound-
healing activities of Persea americana. This study
therefore set out to explore the effects of Persea
americana fruit on wound-healing parameters
including collagen synthesis.
Method
Fruit paste preparation
The Persea americana fruit (avocado) of 600g in
weight was taken and washed thoroughly with
deionised water, and then ground into a paste
(extract) using an electric blender. Special care was
taken during the procedure, and no water was added
to the extract to avoid contamination. The extract
was stored at a temperature of 4°C. What concen-
tration/amount of avocado was in the final
paste per animal? How was this standard-
ised?
Animals
The ethics committee for animal experimentation
(AHC06/07/1) at the University of the West Indies
approved the study. Healthy, in-house bred, gender-
matched, Sprague Dawley rats weighing 200–250g
were used for the study. They were individually
housed in an animal house and maintained on nor-
mal food and water ad libitum. The rats were anaes-
thetised before and during (to check, is this cor-
rect - both before and during?) infliction of the
experimental wounds. Surgical interventions were
carried out under sterile conditions using ketamine
anaesthesia (120mg/kg body weight). Animals with
signs of infection were excluded from the study and
replaced.
Dose selection
The male rats were treated with increasing doses (1,
2, 4 and 8g/kg body weight) of the extract for 14
days. Doses of up to 4g/kg body weight did not pro-
duce any sign of toxicity, while the animals remained
physically active and consumed food and water in a
regular way. As 1g/kg body weight showed better
healing (do you mean resulted in faster heal-
ing times?), the dose of 300mg/kg body weight
was selected for our experimental study. Author, I
presume the dose response study was sepa-
rate from the main study? this needs to be
made clear. At the moment, this looks like
the same animals were used in both studies,
which would invalidate the study
Wound-healing activity
Excision and dead space wound models were used
to evaluate the wound-healing activity of Persea
americana. Author, you also need to state the
study duration for the two models - thanks
l Excision wound model Excision wounds were
created on the rats, as described by Morton and
Malone.11 The dorsal fur was shaved with an electric
clipper and the anticipated wound area was drawn
on the back of the animals in methylene blue using
a circular stainless steel stencil. A full-thickness
excision wound measuring 250mm2 and 2mm deep
was created along the markings using toothed
forceps, a surgical blade and pointed scissors. The
wounds were kept open. The animals were divided
into four groups of five each. Author, to check
were these 4 groups used in the excision
wound model only, or were groups 1 and 2
used for the excision wound model and
groups 3 and 4 for the dead space wound
model?
l Group 1: the fruit paste (300mg/kg/day) was
applied topically using a sterile stainless steel appli-
cator on a daily basis (how did you ensure the
correct dosage of paste was applied? And
how was this standardised between animals?
How was this kept place? More detail is
required to ensure validity of the study -
thank you ).
l Group 2 (control): 1% carboxymethyl cellulose
dissolved in deionsied water was applied topically
- also on a daily basis? (Author, why add CMC
to the control wounds? CMC on its own has
been demonstrated to affect wound healing.
Therefore, the rationale for this needs
explaining and referencing. Also, as with
the fruit paste, you need to make clear why
the carboxymethyl cellulose was used, and
how it was made up, applied and kept in
place - thank you).
l Group 3: 300mg of fruit paste (do you mean 1g
of fruit paste was dissolved in 100ml of
drinking water?) was dissolved in the animal’s
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JOURNAL OF WOUND CARE VOL 14, NO X, XXXXXXXXX 2005 3
on which the eschar fell off, without leaving any
residual raw wound, was considered the period of
epithelialisation. The rats were euthansised after the
study (A table or diagram summarising the
experimental protocol would be helpful, as
you can see it’s not quite clear which groups
were used for which model. Thank you).
l Dead space wound model Dead space wounds
were inflicted by implanting sterile cotton pellets
(10mg each), one on either side of the groin and
axilla, on the ventral surface of each rat using the
technique of D’Arcy et al., as described by Turner.12
(Again, you need to make clear which groups
were used for this model) This is a well-
established standard method of obtaining collagen
for histology and hydroxyproline determination
(reference?).
Animals in the test group were given the fruit
extract orally (you mean these animals were
all in group 3?). On the 10th post-wounding day,
granulation tissue that had formed on the implant-
ed cotton pellets was carefully removed under
anaesthesia and its wet weight noted. The tissue was
then dried at 60°C for 12 hours and its weight
recorded; 5ml 6N hydrochloric acid was added to
the dried tissue, which was stored at 110°C for 24
hours. The acid hydrolysate (author, please
check this reads correctly - thanks) was neu-
tralised using 10N sodium hydroxide, and the vol-
ume made up to 10ml. This neutralised hydrolysate
was used to determine hydroxyproline,13 (Neuman
and Logan 1950) which is an index of collagen
content. An additional piece of wet granulation tis-
sue was preserved in 10% formalin for histological
studies.
Estimation of hydroxyproline
Dry granulation tissue from both control and treat-
ed group (are these groups 3 and 4?) was used to
estimate the hydroxyproline content. Hydroxypro-
line present in the neutralised acid hydrolysate was
subsequently oxidised by sodium peroxide in the
presence of copper sulphate. Following this, it was
complexed with paradimethylaminobenzaldehyde
to turn it pink, and measured at 540nm by spectro-
photometer.13 (Neuman and Logan 1950)
Histological study
Granulation tissues (from dead space wound model)
were obtained on day 10 from the test and control
group animals (groups 3 and 4?) for the histo-
logical study. For the better appreciation of collagen
deposition Masson’s trichrome stain, which stains
the fibres green, was used. This technique is used for
quantitative determination and to observe cross-
linking of collagen (reference?).
Statistical analysis
The means of wound area measurements between
groups at different time intervals were compared
using a one-way ANOVA, descriptive tests. One-way
ANOVA was used to examine the mean differences
in epithelialisation time, weight of the tissue and
hydroxyproline content between the groups in dead
excision and dead space wound models. Data were
analysed using the SPSS (version 12) and p value
was set <0.05 for all analyses.
Results
In the excision wound model the topically treated
animal wounds were found to fully have epithelial-
ised faster (13.8 ± 0.48 days) than the controls (17 ±
0.00 days). This difference was statistically signifi-
cant (p<0.001).
Animals given the oral extract also achieved full
epithelialisation significantly faster (13.0 ± 0.54
days) than the controls, who received plain drink-
ing water (16.6 ± 1.1 days). Again, this was sta-
tistically significant correct? (p<0.001).
In the dead space wound model, the tissue
obtained from the extract-treated animals (which
group?) showed significantly increased levels of
hydroxyproline content (68.0 ± 12.1mg/g tissue)
compared with the controls (30.0 ± 16.2mg/g tis-
sue). The wet (120.0 ± 25.84mg/100g rat) and dry
(28.40 ± 6.20mg/100g rat) weight of the granulation
tissue of animals treated with the fruit extract was
higher when compared with wet (57.6 ± 9.70mg/100g
rat) and dry (11.80 ± 0.9 mg/100g rat) weights of
tissue from animals received placebo control (what
placebo? you state plain drinking water).
This was statistically significant in both cases
(p<0.001).
The histological study of the granulation tissue
obtained on 10th day from the experimental ani-
mals (which group?) showed increased well-
organised collagen and more fibroblasts (Fig 1) when
compared with a section of granulation tissue from
the controls which showed inflammatory cells,
scanty collagen fibres and fibroblasts (Fig 2).
Discussion
These results show that avocado extract significant-
ly increased the rate of wound contraction/epitheli-
alisation, and the weight of the granulation tissue.
The latter is predominantly composed of fibroblasts,
collagen, oedema and new small blood vessels. The
increased rate of wound contraction is indicative of
increased collagen synthesis. The heavier weight of
the dry granulation tissue from excision wounds of
the extract-treated animals suggests it had a higher
collagen content. It is possible that the proinflam-
matory activity of the constituents of avocado fruit
could attract macrophages to the wound site. Mac-
rophages stimulate the chemotaxis and prolifera-
13 Neuman, R.E., Logan, M.
A. The determination of
hydroxyproline. J Biol
Chem 1950; 184: 1,
299-306.
14 Lu, Q.Y., Arteaga, J.R.,
Zhang, Q. et al. Inhibition of
prostate cancer cell growth
by an avocado extract: role
of lipid-soluble bioactive
substances. J Nutr Biochem
2005; 16: 1, 23-30.
15 Pacetti, D., Boselli, E.,
Lucci, P., Frega, N.G.
Simultaneous analysis of
glycolipids and phospholids
molecular species in
avocado (Persea Americana
Mill) fruit. J Chromatogr A
2007; 1150: 1-2, 241-251.
16 Pereira, L.M., Hatanaka,
E., Martins, E.F. et al. Effect
of oleic and linoleic acids
on the inflammatory phase
of wound healing in rats.
Cell Biochem Funct 2007;
26: 2, 197-204.
17 Balkan, M., Beyzadeoglu,
M., Oysul, K. et al. Retinoic
acid and intestinal wound
healing in intra-operatively
irradiated rat. Acta Chir
Belg 2006; 106: 1, 73-76.
18 Tom, W.L., Peng, D.H.,
Allaei, A. et al. The effect of
short-contact topical
tretinoin therapy for foot
ulcers in patients with
diabetes. Arch Dermatol
2005; 141: 11, 1373-1377.
19 Muehlberger, T., Moresi,
J.M., Schwarze, H. et al. The
effect of topical tretinoin
on tissue strength and skin
components in a murine
incisional wound model. J
Am Acad Dermatol 2005;
52: 4, 583-588.
20 Lateef, H., Abatan, O.I.,
Aslam, M.N. et al. Topical
pretreatment of diabetic
rats with all-trans retinoic
acid improves healing of
subsequently induced
abrasion wounds. Diabetes
2005; 54: 3, 855-861.
21 Musalmah, M., Nizrana,
M.Y., Fairuz, A.H. et al.
Comparative effects of
palm vitamin E and alpha-
tocopherol on healing and
wound tissue antioxidant
enzyme levels in diabetic
rats. Lipids 2005; 40: 6, 575-
580.
22 Alleva, R., Nasole, E., Di
Donato, F. et al. alpha-Lipoic
acid supplementation
inhibits oxidative damage,
accelerating chronic wound
healing in patients
undergoing hyperbaric
oxygen therapy. Biochem
Biophys Res Commun
2005; 333: 2, 404-410.
23 Stipcevic, T., Piljac, A.,
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JOURNAL OF WOUND CARE VOL 14, NO X, XXXXXXXXX 20054
tion of fibroblasts, and attract endothelial cells to
the wound and stimulate their proliferation to pro-
mote angiogenesis (reference?).
Avocado fruit is a rich source of monounsaturated
fatty acids and contains the highest content of
lutein (70% of the measured carotenoids) of com-
monly eaten fruits, as well as significant amounts of
related carotenoids and (should we mention
vitamin A as well?) vitamin E,14 (Lu et al 2005)
various phospholipids and glycolipids.15 (Pacetti
et al 2007) Because the avocado also contains a
significant amount of monounsaturated fat, these
bioactive carotenoids are likely to be better absorbed
into the bloodstream.15 (Lu et al 2005) Vitamin A
is required for epithelial formation, cellular differ-
entiation and immune function, and vitamin E is
the major lipid-soluble antioxidant in the skin.
Monounsaturated fatty acids,16 (Pereira et al
2007) topical and systemic carotenoids17-20 (Bal-
kan et al 2006; Tom et al 2005; Muehlberget
et al 2005; Lateef et al 2005) and vitamin E21
(Musalmah et al 2005) promote wound healing.
Derivatives of phospholipids and glycolipids also
have wound-healing properties.22-25 (Alleva et al
2005; Stipcevic et al 2006; Xu et al 2007; Linet
al 2006)
We hypothesise that carotenoids and, to a certain
extent, monounsaturated fatty acids might have
accelerated (okay?) the epithelialisation rate in the
excision wound model. Phospholipids are essential
co-factors in the blood coagulation cascade and
platelet activation, and it is possible that the phos-
pholipids of avocado fruit might have facilitated the
initial phase of cutaneous repair by initiating hae-
mostasis and the development of (okay?) a provi-
sional matrix for cell migration.
One known, (2R)-(12Z,15Z)-2-hydroxy-4-oxohe-
neicosa-12,15-dien-1-yl acetate (please double
check this is written correctly - thanks), and
two novel compounds, persenone A and B, isolated
from avocado fruit have been identified as inhibi-
tors of superoxide (O2-) and nitric oxide (NO) gen-
eration in cell culture systems.26 (Kim et al 2000)
Avocado has good hypochlorous acid, but not
hydrogen peroxide, scavenging ability.27 (Murcia
et al 2001) While there has been controversy over
the role of free radicals in wound healing,28
(Juranek and Bezek 2005) the efficacy of honey
is well correlated with its antioxidant properties.29
(Henriques et al 2006)
Avocados also have 60% more potassium than
bananas and are rich in vitamins B and K.14 (Lu et
al 2005) Vitamin B promotes wound healing.30
(Neiva et al 2005) However, the role of potassium
and vitamins K and D on wound healing is not
known (according to our reviewer, this is not
true. There is evidence that vitamin D is
involved in regulation of SAMs (fibronec-
tin).
Author, you need to fully acknowledge the
study limitations
Conclusion
Avocado fruit promotes wound healing and the
constituents present in the avocado fruit extract
may be responsible for promoting the collagen for-
mation at the proliferative stage of wound healing.
Its wound healing promoting activity could be due
to its antioxidant, antimicrobial, vitamins or some
unknown activities. However, it needs to be studied
further to isolate the active ingredients that pro-
mote wound healing, before considering it for clini-
cal use.
Fig 1: Experimental group granulation tissue with
more collagen (Masson’s trichrome stain) a =
inflammatory cells, b= fibroblast, c= collagen fibres
Fig 2. Control group granulation tissue with less
collagen (Masson’s trichrome stain) a = inflammatory
cells, b= fibroblast, c= collagen fibres
Piljac, G. Enhanced healing
of full-thickness burn
wounds using di-
rhamnolipid. Burns 2006;
32: 1, 24-34.
24 Xu, K.P., Yin, J., Yu, F.S.
Lysophosphatidic acid
promoting corneal
epithelial wound healing by
transactivation of
epidermal growth factor
receptor. Invest
Ophthalmol Vis Sci 2007;
48: 2, 636-643.
25 Lin, C.I., Chen, C.N.,
Chen, J.H., Lee, H.
Lysophospholipids increase
IL-8 and MCP-1
expressions in human
umbilical cord vein
endothelial cells through an
IL-1-dependent mechanism.
J Cell Biochem 2006; 99: 4,
1216-1232.
26 Kim, O.K., Murakami, A.,
Nakamura, Y. et al. Novel
nitric oxide and superoxide
generation inhibitors,
persenone A and B, from
avocado fruit. J Agric Food
Chem 2000; 48: 5, 1557-
1563.
27 Murcia, M.A., Jiménez,
A.M., Martínez-Tomé, M.
Evaluation of the
antioxidant properties of
Mediterranean and tropical
fruits compared with
common food additives. J
Food Prot 2001; 64: 12,
2037-2046.
28 Juránek, I., Bezek, S.
Controversy of free radical
hypothesis: reactive oxygen
species: cause or
consequence of tissue
injury? Gen Physiol Biophys
2005; 24: 3, 263-278.
29 Henriques, A., Jackson,
S., Cooper, R., Burton, N.
Free radical production and
quenching in honeys with
wound healing potential. J
Antimicrob Chemother
2006; 58: 4, 773-777.
30 Neiva, R.F., Al-Shammari,
K., Nociti, F.H. Jr et al.
Effects of vitamin-B
complex supplementation
on periodontal wound
healing. J Periodontol 2005;
76: 7, 1084-1091.
