Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2011, Article ID 419612, 6 pages
Carapaguianensis L.LeafforItsWoundHealing Activity
B. Shivananda Nayak,1Joel Kanhai,1DavidMalcolmMilne,1
LexleyPinto Pereira,2and WilliamH.Swanston3
1Department of Preclinical Sciences, The University of West Indies, Faculty of Medical Sciences,
Biochemistry Unit, EWMSC, Trinidad and Tobago
2Department of Para Clinical Sciences, Faculty of Medical Sciences, The University of the West Indies,
St. Augustine, Trinidad and Tobago
3Diagnostic Laboratory Services, North Central Regional Health Authority, Trinidad and Tobago
Correspondence should be addressed to B. Shivananda Nayak, firstname.lastname@example.org
Received 26 January 2009; Accepted 13 September 2009
Copyright © 2011 B. Shivananda Nayak et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
The leaves of Carapa guianensis have been used to treat ulcers, skin parasites, and skin problems. The ethanolic extract of C.
guianensis leaf was evaluated for its antibacterial and wound healing activity using excision, incision and dead space wound
models in rats. The animals were randomly divided into two groups (n = 6) in all the models. In the excision wound model test
group animals were treated topically with the leaf extract (250 mg kg−1body weight) whereas, control animals were treated with
petroleum jelly. Intheincisionanddeadspacewoundmodels,thetestgroupanimalswere treated withextract(250mgkg−1day−1)
orallyby mixingin drinkingwater andthe control group animalswere maintainedwith plain drinkingwater. Healingwasassessed
by the rate of wound contraction, period of epithelialization, skin breaking strength, granulation tissue weight and hydoxyproline
content. On Day 15 extract-treated animals exhibited 100% reduction in the wound area when compared to controls (95%) with
significant decrease in the epithelialization period. The extract failed to demonstrate antibacterial activity. Skin breaking strength
(P < .001), wet (P < .002) and dry (P < .02) granulation tissue and hydroxyproline content (P < .03) were significantly higher
in extract treated animals. The increased rate of wound contraction, skin breaking strength and hydroxyproline content supports
potential application of C. guianensis in wound healing.
Traditional herbal medicine practitioners have described the
healing properties of various wild plants [1, 2]. Various heal-
ing constituents in these plants have prompted researchers
to examine them with a view to determine their potential
wound healing activities.
Healing of skin wounds is a complex process which
cell lineage. The behavior of each of the contributing cell
types during the phases of proliferation, migration, matrix
synthesis and contraction, as well as the growth factor and
matrix signals present at a wound site are now well under-
a predictablefashion torepair the damage. In the subsequent
inflammatory response following an injury the cells below
the dermis (the deepest skin layer) begin to increase collagen
(connective tissue) production reaching the last stage of
regeneration of, epithelial tissue (the outer skin layer) .
Carapa guianensis L. (Meliaceae) is also known as
Andiroba. The leaves have been used for fever and the tea
made from this plant is applied externally for ulcers, skin
parasites and otherskin problems. Traditional forestdwellers
particularly those dwelling on the river bank in Brazil called
caboclos, make a medicinal soap using crude Andiroba oil,
wood ash and cocoa skin residue. This soap is especially
recommended for the treatment of skin diseases. Andiroba
oil is also applied directly on joints to relieve arthritic pain,
2 Evidence-Based Complementary and Alternative Medicine
and mixed with hot water and human milk it is used as
drops for ear infections. Many of these uses continue today
in the Brazilian herbal medicine systems either in pure form
ormixedwith otheroilsornatural products.Braziliansapply
sage oil and natural insect repellant, and employ it topically
for many skin diseases and conditions, including psoriasis.
All parts of the C. guianensis tree have a bitter taste
attributed to a group of terpene chemicals called meliacins,
which are very similar to the bitter antimalarial chemicals
found in tropical plants. One of these meliacins, named
gedunin, has recently been documented to have antiparasitic
properties and an antimalarial effect equal to that of
quinine. Chemical analysis of C. guianensis oil, leaves has
also identified the presence of another group of chemicals
calledlimonoids. Theanti-inflammatory andinsectrepellent
properties of andiroba oil are attributed to the presence
of these limonoids , including a novel one which has
been named andirobin. Another limonoid called epoxyazadi-
radione is found in C. guianensis oil. The three chemicals
present in Andiroba have been found to have antiparasitic
and insecticidal actions [6, 7]. Carapa guianensis oil is
well known in Brazil and widely employed to heal many
skin conditions and as a natural insect repellant. North
American practitioners and consumers are just beginning to
learn about the powerful healing properties of C. guianensis.
Andiroba oil can be applied topically several times daily to
rashes, muscle/joint aches and injuries, wounds  insect
bites, boils and ulcers.
However, there is not enough scientifically proven data
to support the wound healing and antimicrobial activities of
C. guianensis in literature. We undertook the present study
to explore the antimicrobial and wound healing effects of C.
guianensis leaf extract.
2.1. Plant Material and Extract Preparation. The C. guia-
nensis leaf (625g) was collected locally in March 2008 and
identified by the plant taxonomist and curator, National
Herbarium of Trinidad and Tobago, The University of the
West Indies, St. Augustine, Trinidad and a voucher specimen
was deposited at the herbarium (specimen number: TRIN
36521). The leaves were washed with tap water and finally
with deionized water. After shade drying they were ground
into a powder using an electric blender. The fine powder
(620g)was suspendedin 4000ml ofethanol for48 hat room
temperature. The mixture was filtered using a fine cloth and
the filtrate was placed in a water bath to dry at 40◦C. The
dried residue (60g) was used for the study . The extract
was subjected to preliminary phytochemical and microbial
2.2. PhytochemicalScreening Methods
2.2.1. Saponins. The extract (2g) was boiled with 20ml
water for 4 min; the mixture was cooled and mixed vigor-
ously and left for few minutes. The formation of frothing
indicates the presence of saponins .
Test for tannins: To an aliquot of the extract (dissolved
in water) 2ml of ferric chloride (1%) was added. Color
development from red brown to blue black indicates the
presence of tannins .
2.2.2. Triterpenes. The extract (1g) was mixed with 10ml
chloroform and warmed at 55◦C for 30min. Few drops (1-
2ml) of concentrated sulfuric acid were added and mixed
well. The appearance of a reddish brown color indicates the
presence of triterpenes .
2.2.3. Test for Sterols. The extract (1g) was mixed with 10ml
chloroform and warmed at 55◦C for 30 min. Few drops (1–
2ml) of concentrated sulfuric acid were added and mixed
well. The appearance of reddish brown color indicates the
presence of sterols .
2.2.4. Alkaloids. The extract (1g) was boiled with 50ml
methanol for 20 min in a water bath and the cooled filtrate
was tested separately with Mayer’s, Wagner’s Hager’s and
ammonium reineckate reagents. Cloudy precipitate of the
alcoholic layer indicates the presence of alkaloids .
2.2.5. Flavonoids. About 1g of extract was boiled with 10ml
ethylacetateoverasteambathfor3min. Thefiltrate ofabout
4ml was mixed with 1ml of dilute ammonia solution and a
yellow precipitate indicates the presence of flavonoids .
Thin layer chromatography of the aqueous extract on
silica gel was done using the medium chloroform: methanol
(9 : 1 v/v) and chloroform: acetone (1 : 1 v/v) as the mobile
2.3. Antimicrobial Activity. Pseudomonas aeruginosa (ATCC
27853), Klebsiella pneumonia (ATCC 700603), Enterococcus
fecalis (ATCC29212),Escherichia coli(ATCC25922),Staphy-
lococcus aureus (ATCC25923) and Methicilin-resistant S.
aureus (ATCC43300)were tested forantibacterial sensitivity.
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 and a
known volume of bacterial suspension was transferred to
each microplate well. Ten microliters of the ethanolic extract
(5mgml−1) ofC. guianensis leaf was added to the microplate
wells and incubated at 35–37◦C for 18–20 h. Results were
analyzed visually for inhibition zones.
180–200g were used for the study. They were individually
housed and maintained on normal food and water ad
libitum. Animals were periodically weighed before and after
the experiment. The rats were anesthetized prior to and
during infliction of the experimental wounds. The surgical
interventions were carried out under sterile conditions using
ketamine anesthesia (120mgkg−1body weight). Animals
were closely observed for any infection and if they showed
signs of infection were separated, excluded from the study
Evidence-Based Complementary and Alternative Medicine3
Figure 1: (a) Excision wound on Day 1 (test group animal). (b)
Excision wound on Day 15 treated with ethanolic extract of C.
guianensis leaf (test group animal).
2.5. Animal Ethical Committee Approval. The study was
approved by the ethics committee for animal experimenta-
tion (AHC06/07/1) by The Faculty of Medical Sciences, The
University of the West Indies, St. Augustine.
2.6. Excision Wound Model. The anesthetized rats were
inflicted with excision wounds as described by Morton and
Malone . 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 250mm2and 2mm
depth was created along the markings with a surgical blade.
The animals were randomly divided into two groups of six
jelly . Animals of Group 2 (experimental) applied with
the leaf extract mixed with petroleum jelly at a dose of
250mgkg−1daily until complete epithelialization. This was
The wound contraction rate was assessed by tracing
the wound on alternate days using transparency paper
and a permanent marker. The wound areas recorded were
measured using a graph paper. The point at which the eschar
fell off without any residual raw wound was considered
Figure 2: (a) Excision wound on Day 1 (control group animal).(b)
Excision wound on Day 15 without any treatment (control group
Wound area (mm2)
Figure 3: Effect of ethanolic extract of C. guianensis leaf on wound
contraction in excision wound model.
2.7.Incision WoundModel. Thedorsalfuroftheanesthetized
animals was shaved with an electric clipper. A longitudinal
paravertebral incision, 6cm in length was made through
the skin and cutaneous muscle on the back as described by
Ehrlich and Hunt et al. . Surgical sutures were applied
to the parted skin at intervals of 1cm. The wounds were
left undressed. The animals were randomly divided into two
groups of six each. The test group rats were given leaf extract
orally in their drinking water at a dose of 250mgkg−1daily.
The controls were given drinking water. As an average, rat
consumes 110ml of water kg−1day−1, 250mg of leaf extract
was dissolved in 100ml of drinking water. The sutures were
removed on the 8th post wound day and the treatment was
continued. The skin-breaking strength was measured on the
10th day by the method described by Lee .
4 Evidence-Based Complementary and Alternative Medicine
Figure 4: Effect of ethanolic extract of C. guianensis leaf on
Tensile strength (mg/100g)
Figure 5: Effect of C. guianensis leaf extract on skin breaking
2.8. Determination of Wound Breaking Strength. The anes-
on either side of the wound 3mm away from the suture
line. The line on either side of the suture was gripped with
a forceps one at each end opposed to each other. One end
of the forceps was supported firmly, whereas the other was
connected to a freely suspended lightweight measuring jar.
Water was slowly added continuously till the wound began
to gape. As soon as wound gaping appeared the addition
of water was stopped. The volume of water was determined
and noted as a measure of breaking strength in grams. An
average of three readings was recorded for a given incision
wound and the mean reading for the group was taken as an
individual value of breaking strength .
OH-proline Dry wtWet wt
Figure 6: Effect of C. guianensis leaf extract on biochemical
2.9. Dead Space Wound Model. Dead space wounds were
inflicted by implanting sterile cotton pellets (5mg each), one
on either side of the groin and axilla on the ventral surface of
each rat by the technique of D’Arcy et al. (1960) described by
Turner . The animals were divided into two groups (n =
6). The test group rats were given leaf extract orally in their
drinking water at a dose of 250mgkg−1daily. The controls
were given drinking water. On the 10th post-wounding day,
was carefully removed under anesthesia. The wet weight of
the granulation tissue was noted. These granulation tissues
were driedat60◦Cfor12h,weighed, andthedrygranulation
tissue weight was recorded. To the dried tissue 5ml 6N HCl
was added and kept at 110◦C for 24h. The neutralized acid
hydrolysate of the dry tissue was used for the determination
of hydroxyproline . Wet granulation tissue preserved in
10% formalin was used for histological studies.
2.10. Estimation of Hydroxyproline. Dry granulation tissue
from both the control and treated groups was used for
the estimation of hydroxyproline. Hydroxyproline present in
the neutralized acid hydrolysate was subsequently oxidized
by sodium peroxide in the presence of copper sulfate fol-
lowed by complexing with para-dimethylaminobezaldehyde
to develop a pink color and that was measured at 540 nm by
2.11. Statistical Analysis. The means of wound area mea-
surements were analyzed using one-way ANOVA descriptive
test. The epithelization period, tensile strength, wet and dry
weight and hydroxyproline content of the granulation tissue
between the test and control groups were compared using
independent t-test. Data were analyzed using SPSS (Version
12.0,Chicago,USA)and P-valuewas set<.05forall analyses.
3.1. Phytochemical Analysis. The phytochemical analysis of
the leaf extract by qualitativemethod showed the presence of
Evidence-Based Complementary and Alternative Medicine5
Phase 1: Inflammation
Tissue and immune cells
Phase 3: Remodeling
Collagen deposition and linkage
Scar tissue formation
Phase 2: Proliferation
Granulation tissue formation
Fibroblast migration and maturation
Epithelial cell production
Figure 7: Possible role of phytochemical constituents of C. guianensis on wound healing.
alkaloids, essential oils, saponins and tannins and absence of
triterpenoids and flavonoids.
3.2. Excision Wound Model. On Day 15 a significant increase
in the wound-healing activity was observed in the animals
treatedwith theC.guianensis extract (Figure 1(b))compared
with those who received the placebo control treatments
(Figure 2(b)). Figure 3 shows the effects of the C. guianensis
leaf extract administered topically on wound healing activity
in rats inflicted with the excision wound. In this model C.
guianensis treated animals were found to epithelize faster
(12.00 ± 0.44) when compared with controls (15.60 ± 0.80)
(P < .03) (Figure 4). The rate of reduction in the wound area
controls (95%) and it was statistically significant (P < .03).
3.3. Incision Wound Model. In the incision wound model, a
significant increase in the wound breaking strength (576.60
± 26.03) was observed when compared with the controls
(380.10 ± 21.40) (Figure 5).
3.4. Dead Space Wound Model. In the dead space wound
model, the extract-treated animals showed significantly
increased levelsofhydroxyproline content(58.00 ± 26.72) as
comparedwith the controlgroup(38.16 ± 14.33)ofanimals.
Asignificant increase wasobservedin thewet and dry weight
(P < .002) of the granulation tissue in the animals treated
with the extract (Figure 6).
3.5. Antimicrobial Activity. All microbial organisms tested
(P. aeruginosa, K. pneumonia, E. fecalis, E. coli, S. aureus
and Methicilin-resistant S. aureus) were resistant against C.
guianensis leaf extract.
Excision, dead space and incision wound models were used
to study wound contraction, skin braking strength which
are the parameters of tissue cell regeneration, collagenation
capacity and mechanical strength of the skin respectively
The ethanolic leaf extract of C. guianensis showed
increase in the rate of wound contraction, skin breaking
strength, the rate of epithelialization, weight and hydrox-
yproline content of granulation tissue. Granulation tissue
formed in the final part of the proliferative phase is
primarily composed of fibroblasts, collagen, edema and
new small blood vessels. The increased hydroxyproline
content of the granulation tissue indicates increased collagen
turnover. Collagen, the major component of granulation
tissue, strengthens and supports extracellular tissue, which is
composed of the amino acid hydroxyproline and it has been
used as a biochemical marker for tissue collagen .
Researchers have showed the anti-allergic and anti-
inflammatory properties of tetraterpenoids and limnoids
isolated respectively from C. guianensis . However, our
phytochemical analysis of the leaf extract by qualitative
analysis showed the presence of alkaloids, saponins, tannins
penoids and flavonoids. Possibly, constituents like tannins,
saponins and alkaloids may play a role in the process of
wound healing (Figure 7), however, further phytochemical
studies are needed to isolate the active compound(s) respon-
sible for these pharmacological activities.
Several studies demonstrated that the phytochemical
constituents present in medicinal plants promote the wound
healing process [22–25]. The beneficial response of C. guia-
nensis on wound healing may be due to its various reported
activities which include antiallergic and antiparasitic [21, 26,
27] effects among its medicinal activities.
While the anti-inflammatory effects of C. guianensis
are attributed to its possible antihistaminergic activity, the
antioxidant activity of C. guianensis cannot be discounted.
Like honey which is known to have anti-inflammatory,
wound-healing promoting action  and antibacterial
activity , C. guianensis may also have anti-inflammatory,
immunostimulant and pro-healing properties. As C. guia-
nensis did not inhibit the growth of microorganisms asso-
ciated with wound infections, its wound-healing promoting
activity is independent of its antimicrobial activity. Future
study using isolated active compounds from C. guianensis is
required to know the reason for negative role of leaf extract
in inhibiting the growth of microorganisms.
6 Evidence-Based Complementary and Alternative Medicine Download full-text
We have shown that the leaf extract of C. guianensis
facilitates wound healing in the experimental animal model.
Studies to isolate the active ingredients of the extract that
promote wound healing are recommended before proposing
its potential application for therapeutic use.
The authors sincerely thank Mrs Yasmin S. Baksh-Comeau,
Curator, National Herbarium, Trinidad and Tobago and Mr
Mathew Everlsley and Ms Sabana Mayers, for their excellent
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