Ethnopharmacological survey and in vitro evaluation of wound-healing plants used in South-western Nigeria

Abstract

Traditional healers in Nigeria employ a range of plant preparations as wound healing agents. Despite the use of local plants in wound healing, there is only scant literature on the wound healing properties of these plants to support the continued therapeutic application of these herbal remedies.
To document plants commonly used to treat wounds in South-western Nigeria and to test the scientific basis of such claims using relevant in vitro tests.
Structured questionnaires were used to determine which plant preparations are in common use, via interviews with Yoruba traditional healers. Aqueous and ethanolic extracts of the nine most common plants cited by the healers were collected, identified and tested using relevant in vitro wound healing assays. Minimum inhibitory concentrations (MIC) were determined against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis. Antioxidant activity was measured by DPPH assay and fibroblast proliferation determined by neutral red assay.
A total of 20 traditional healers from South-western Nigeria were involved in the study. Thirty-six plant species were recorded with their local names and parts used in the traditional wound healing preparations. Ethanolic extracts of nine species most frequently cited by the healers exhibited strong antioxidant activities (3.8-31.3 μg/ml) comparable to ascorbic acid (7.3 μg/ml). Crude extracts of the selected plants also inhibited the growth of bacteria with MIC values 0.3-7.6 mg/ml. Ethanol extracts of Bridelia ferruginea Benth. (1-30 μg/ml) and Parkia biglobosa Jacq. (15-30 μg/ml) influenced the proliferation of dermal fibroblasts significantly (p<0.05). Extracts from the remaining seven plants either had no effect on fibroblast proliferation or were cytotoxic.
Traditional use of many wound-healing plants from Nigeria can be rationalised by activity determined in relevant in vitro investigations of ethanol and aqueous extracts. These results support the traditional selection of these plants in South-western Nigeria for wound healing.

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Journal
of
Ethnopharmacology
137 (2011) 50–
56
Contents
lists
available
at
ScienceDirect
Journal
of
Ethnopharmacology
j
o
ur
nal
homep
age
:
www.elsevier.com/locate/jethpharm
Ethnopharmacological
survey
and
in
vitro
evaluation
of
wound-healing
plants
used
in
South-western
Nigeria
Adewale
Adetutua,b,
Winston
A.
Morgana,
Olivia
Corcorana,∗
aMedicines
Research
Group,
School
of
Health
and
Bioscience,
University
of
East
London,
Water
Lane,
London
E15
4LZ,
UK
bDepartment
of
Biochemistry,
Ladoke
Akintola
University
of
Technology,
Ogbomosho,
Nigeria
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
26
December
2010
Received
in
revised
form
28
March
2011
Accepted
31
March
2011
Available online 8 April 2011
Keywords:
Wound-healing
Nigeria
Medicinal
plants
Fibroblast
proliferation
Antioxidant
Antibacterial
a
b
s
t
r
a
c
t
Ethnopharmacological
relevance:
Traditional
healers
in
Nigeria
employ
a
range
of
plant
preparations
as
wound
healing
agents.
Despite
the
use
of
local
plants
in
wound
healing,
there
is
only
scant
literature
on
the
wound
healing
properties
of
these
plants
to
support
the
continued
therapeutic
application
of
these
herbal
remedies.
Aim
of
the
study:
To
document
plants
commonly
used
to
treat
wounds
in
South-western
Nigeria
and
to
test
the
scientific
basis
of
such
claims
using
relevant
in
vitro
tests.
Materials
and
methods:
Structured
questionnaires
were
used
to
determine
which
plant
preparations
are
in
common
use,
via
interviews
with
Yoruba
traditional
healers.
Aqueous
and
ethanolic
extracts
of
the
nine
most
common
plants
cited
by
the
healers
were
collected,
identified
and
tested
using
relevant
in
vitro
wound
healing
assays.
Minimum
inhibitory
concentrations
(MIC)
were
determined
against
Staphylo-
coccus
aureus,
Escherichia
coli,
Pseudomonas
aeruginosa
and
Bacillus
subtilis.
Antioxidant
activity
was
measured
by
DPPH
assay
and
fibroblast
proliferation
determined
by
neutral
red
assay.
Results:
A
total
of
20
traditional
healers
from
South-western
Nigeria
were
involved
in
the
study.
Thirty-
six
plant
species
were
recorded
with
their
local
names
and
parts
used
in
the
traditional
wound
healing
preparations.
Ethanolic
extracts
of
nine
species
most
frequently
cited
by
the
healers
exhibited
strong
antioxidant
activities
(3.8–31.3
␮g/ml)
comparable
to
ascorbic
acid
(7.3
␮g/ml).
Crude
extracts
of
the
selected
plants
also
inhibited
the
growth
of
bacteria
with
MIC
values
0.3–7.6
mg/ml.
Ethanol
extracts
of
Bridelia
ferruginea
Benth.
(1–30
␮g/ml)
and
Parkia
biglobosa
Jacq.
(15–30
␮g/ml)
influenced
the
prolifer-
ation
of
dermal
fibroblasts
significantly
(p
<
0.05).
Extracts
from
the
remaining
seven
plants
either
had
no
effect
on
fibroblast
proliferation
or
were
cytotoxic.
Conclusion:
Traditional
use
of
many
wound-healing
plants
from
Nigeria
can
be
rationalised
by
activity
determined
in
relevant
in
vitro
investigations
of
ethanol
and
aqueous
extracts.
These
results
support
the
traditional
selection
of
these
plants
in
South-western
Nigeria
for
wound
healing.
© 2011 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
In
most
of
the
developing
world
plants
or
herbal
products
play
an
important
role
in
the
treatment
of
wounds
(Phillipson,
2001;
Mensah
et
al.,
2006).
The
choice
of
herbal
products
for
the
treat-
ment
of
wounds
varies
between
regions
and
cultures
(Sofowora,
1993).
In
South-western
Nigeria,
traditional
healers
provide
crude
extracts
from
a
range
of
plants
to
treat
skin
afflictions
including
wounds
such
as
sores,
bites,
burns
and
lacerations.
This
form
of
traditional
herbal
medicine
makes
a
significant
contribution
to
the
healthcare
provision
for
rural
communities
(Okeke
et
al.,
2006).
Despite
the
Nigerian
Government
providing
funding
for
develop-
∗Corresponding
author.
Tel.:
+44
208
223
4034.
E-mail
address:
o.corcoran@uel.ac.uk
(O.
Corcoran).
ment
and
integration
of
traditional
medicine
into
Nigeria’s
primary
healthcare
system,
the
scientific
basis
for
the
use
of
many
of
these
plant
extracts
for
wound
healing
remains
poorly
understood.
The
present
study
investigates
selected
wound-healing
plants
com-
monly
employed
by
traditional
healers
in
Oyo
State
using
three
key
in
vitro
assays
of
antibacterial
activity,
antioxidant
activity
and
fibroblast
growth
stimulation.
2.
Materials
and
methods
2.1.
Study
area
and
survey
An
ethnopharmacological
survey
of
traditional
healers
was
car-
ried
out
in
Oyo
State
in
South-western
Nigeria.
Data
collection
was
based
on
the
strategy
of
mutual
trust
suggested
by
Sofowora
(1993)
and
Heinrich
(2000).
Information
was
compiled
through
0378-8741/$
–
see
front
matter ©
2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2011.03.073

Author's personal copy
A.
Adetutu
et
al.
/
Journal
of
Ethnopharmacology
137 (2011) 50–
56 51
general
conversations
and
using
standard
questionnaires
(which
is
a
modification
of
the
format
recommended
by
Joly
et
al.
(1987)
and
Sofowora
(1993).
The
language
used
for
interview
was
Yoruba
since
most
traditional
herbal
practitioners
were
illiterate.
The
question-
naire
was
written
in
English
language,
but
was
translated
to
Yoruba
language
as
the
question
was
posed
and
answer
written
down
in
English
except
for
the
local
names
of
plants.
Prior
to
the
interviews,
the
healers
were
given
information
about
participation
in
the
project.
The
conversations
were
built
on
mutual
trust
with
the
common
goal
to
improve
the
health
sit-
uation
in
the
country
and
increase
the
knowledge
on
Nigerian
medicinal
plants.
More
than
thirty
traditional
healers
from
various
suburbs
and
villages
in
South-western
Nigeria
were
approached,
with
twenty
volunteering
to
complete
the
survey.
The
information
collected
included
local
names
of
the
plants
used
for
wound
heal-
ing,
methods
of
preparation
and
details
of
administration
(Table
1).
The
plants
used
by
the
healers
are
listed
alphabetically
by
Latin
names,
along
with
the
respective
families
and
vernacular
(Yoruba)
names.
The
number
of
times
each
plant
was
cited
was
recorded.
Nine
of
these
plants
are
most
frequently
cited
for
treatment
of
wounds
including
burns
and
ulcers
(Fig.
1).
Specimens
of
the
sam-
ple
were
collected
and
these
were
useful
in
the
translation
of
the
Yoruba
names
to
botanical
names.
2.2.
Plant
materials
Of
the
36
species
recorded
in
the
survey
(Table
1),
only
the
nine
with
most
frequent
occurrence
(Fig.
1)
were
collected.
Rel-
evant
plant
parts
were
collected
from
Oyo
State
in
South-western
Nigeria
during
the
rainy
season
of
August
to
September
2006.
This
corresponded
to
the
time
when
most
of
the
plant
species
were
available
and
growing
well.
The
plant
materials
were
identified
by
a
qualified
botanist
(Mr.
T.K.
Odewo)
at
the
Forestry
Research
Institute
of
Nigeria
(FRIN),
Ibadan,
Nigeria,
and
voucher
specimens
were
deposited
in
the
herbarium
at
FRIN.
All
plants
collected
were
air-dried
in
a
moisture-regulated
room
(temperature
25–28 ◦C).
2.3.
Preparation
of
plant
extracts
The
leaves
or
stems
of
the
nine
most
frequently
cited
plants
were
collected
for
screening.
The
protocol
used
for
extraction
in
this
work
reflects
the
preparation
used
by
traditional
healers
and
so
simple
aqueous
and
alcoholic
extracts
were
prepared.
The
plant
materials
were
ground
into
fine
powder
and
then
extracted
(100
g
each
in
500
ml
of
solvent)
by
maceration
for
three
days
in
distilled
water
or
ethanol
at
room
temperature.
Extracts
were
filtered
through
a
Whatman
No.
1
filter
paper
and
the
filtrates
collected.
The
aqueous
filtrates
were
freeze-dried
and
the
organic
filtrates
reduced
using
a
rotary
evaporator
at
37 ◦C.
The
yield
corresponding
to
each
plant
material
is
shown
in
Table
2.
The
reduced
extracts
were
stored
at
4◦C
until
required
for
analysis.
2.4.
In
vitro
assays
relevant
to
wound
healing
2.4.1.
Antibacterial
assay
The
extracts
were
tested
against
the
selected
bacteria
strains
for
their
inhibitory
activity,
using
a
common
broth
microdilu-
tion
method
(Eloff,
1998)
in
96-well
plates.
The
anti-bacterial
activity
of
the
extracts
was
tested
against
four
reference
bac-
terial
strains:
Escherichia
coli
UEL57,
Pseudomonas
aeruginosa
NCTC10701,
Staphylococcus
aureus
NCTC
7447
and
Bacillus
subtilis
NCTC3610.
One
hundred
microliter
of
Mueller
Hinton
broth
were
distributed
from
the
first
to
the
12th
test
wells.
One
hundred
micro-
liter
of
ethanolic
or
water
extract
(20
mg/ml)
in
1%
DMSO
were
added
to
the
first
test
well
of
each
microtiter
line,
and
then
100
␮l
of
scalar
dilution
were
transferred
from
the
second
to
the
ninth
well.
The
10th
well
was
considered
as
growth
control,
since
no
extract
solutions
were
added.
Then,
100
␮l
of
a
microbial
suspen-
sion
(0.5
McFarland
standard),
obtained
from
an
overnight
growth
at
37 ◦C,
were
added
to
each
well
and
incubated
overnight
at
37 ◦C.
The
final
concentration
of
the
extracts
adopted
to
evaluate
the
anti-
bacterial
activity
ranged
from
10
mg/ml
(first
well)
to
0.019
mg/ml
(11th
well).
A
blank
control
was
taken
using
1%
DMSO
alone
(100
␮l)
added
to
the
96-well
plate
and
the
MIC
was
evaluated
as
described
above.
The
determination
of
the
MICs
for
the
positive
control
chlo-
ramphenicol
against
all
the
reference
strains
was
simultaneously
carried
out.
After
the
incubation
period,
40
␮l
of
2
mg/ml
of
p-
iodonitrotetrazolium
violet
(Sigma)
(INT)
dissolved
in
water
was
added
to
each
well.
The
extracts
were
then
incubated
for
a
fur-
ther
30
min
and
bacterial
growth
was
indicated
by
the
red
colour
of
the
INT
formazan
produced.
The
MIC
was
determined
as
the
low-
est
sample
concentration
at
which
no
red
colour
(i.e.
no
bacterial
growth)
appeared.
2.4.2.
Cell
proliferation
and
viability
assay
of
crude
extracts
The
method
described
by
Mensah
et
al.
(2001)
was
used.
Human
foreskin
fibroblast
(FS5)
cells
were
seeded
at
a
density
of
1
×
103cells
per
well
in
96-well
plates
maintained
at
37 ◦C
in
a
humidified
incubator
of
5%
CO2:
95%
air
atmosphere.
The
medium
was
replaced
after
48
h
with
100
␮l
of
DMEM
containing
0.5%
FCS.
The
ethanolic
extract
residues
from
the
plants
were
initially
dis-
solved
in
1
ml
of
DMSO
and
filtered
to
give
the
sterile
stock
solution
(20
mg/ml)
and
further
diluted
to
give
a
final
concentration
of
1–60
␮g/ml
in
the
wells.
The
final
volume
of
the
medium
was
200
␮l
per
well.
Two
well
plate
columns
were
maintained
on
DMEM/0.5%
FCS
and
DMEM/10%
FCS
as
maintenance
(negative)
and
positive
growth
stimulation
controls,
respectively.
The
cells
were
incubated
for
48
h
and
cell
growth
determined
using
neutral
red
uptake
assay.
After
incubation
the
cells
were
washed
with
phosphate
buffered
saline
(PBS),
100
␮l
freshly
prepared
neutral
red
solution
was
added
to
each
well
and
the
cells
were
incubated
at
37 ◦C
for
4
h.
The
neu-
tral
red
was
then
removed
by
washing
with
1%
HCHO/1%
CaCl2
and
neutral
red
in
the
lysosomes
was
eluted
with
100
␮l
of
1%
acetic
acid/50%
ethanol
over
30
min
in
an
orbital
shaker
and
the
optical
density
measured
at
540
nm
using
a
96-well
plate
reader.
Three
independent
experiments
were
conducted
and
the
results
obtained
are
expressed
as
mean
±
standard
error
of
the
mean
of
the
absorbance.
The
data
from
the
experiments
were
compared
with
the
control
(0.5%
FCS)
by
one-way
analysis
of
variance
and
Dunnet’s
test.
Differences
at
p
<
0.05
were
considered
to
be
significant.
2.4.3.
Free
radical
scavenging
activity
of
selected
plants
The
modified
methods
of
Brand-Williams
et
al.
(1995)
and
Chen
et
al.
(1999)
were
used
in
this
study.
Scavenging
activity
was
deter-
mined
by
dissolving
the
extracts
to
obtain
a
final
concentration
of
500–1.9
␮g/ml
in
methanol.
For
a
typical
reaction,
200
␮l
of
6.5
×
10−5M
DPPH
solution
in
methanol
was
mixed
with
20
␮l
of
extracts,
in
96-well
plates.
The
diluted
solutions
were
allowed
to
stand
in
the
dark
for
30
min
and
thereafter
the
optical
density
was
recorded
at
517
nm
using
96-well
plate
reader.
For
the
con-
trol,
200
␮l
of
DPPH•solution
in
methanol
was
mixed
with
20
␮l
of
methanol
and
the
optical
density
of
the
solution
was
recorded
after
30
min.
The
assay
was
carried
out
in
triplicate
and
3
indepen-
dent
experiments
were
conducted.
The
decrease
in
optical
density
of
DPPH
on
addition
of
test
samples
in
relation
to
the
control
was
used
to
calculate
the
antioxidant
activity,
as
percentage
inhibition
(%IP)
of
DPPH•.
Percentage
inhibition
(%IP)
=A0−
A1
A0
×
100

Author's personal copy
52 A.
Adetutu
et
al.
/
Journal
of
Ethnopharmacology
137 (2011) 50–
56
Table
1
Medicinal
plants
used
in
the
treatment
of
wounds
in
South-western
Nigeria
as
identified
by
the
ethnopharmacological
survey.
Latin
names
Family
Local
name(s)
(Yoruba)
Part
used
Mode
of
application
No
of
times
cited
Aframomum
melegueta
(Roskoe)
K.
Schum.
Zingiberaceae
Ata-ire
Fruits
The
fruit
is
ground
and
mixed
with
palm
oil
and
used
as
paste
to
treat
wound
4
Acalypha
wilkesiana
Müll.Arg.
Euphorbiaceae
Aworoso
Leaves
The
poultice
of
the
leaves
are
applied
to
wounds,
burns,
rashes
and
itches
6
Ageratum
conyzoides
L.
Asteraceae
Imin-esu
Leaves
The
leaves
are
ground
and
applied
to
wounds
and
sores
6
Annona
senegalensis
Pers. Annonaceae Abo Leaves A
decoction
of
the
leaves
is
used
to
clean
wounds
2
Anogeissus
leiocarpa
(DC.)
Guill.
&
Perr.
Combretaceae
Ayin
Stem-bark
The
stem-bark
is
ground,
powdered
and
applied
to
wound
and
sores
2
Azadirachta
indica
A.
Juss
Meliaceae
Afoforo
oyimbo
Leaves
The
leaves
are
used
as
a
paste
for
wound
dressing
3
Bridelia
ferruginea
Benth. Euphorbiaceae Ira Leaves The
poultice
of
the
leaves
are
applied
to
wounds,
open
cuts,
and
sores
10
Capsicum
frutescence
L.
Solanaceae
Atarodo
Leaves
Ground
leaves
are
mixed
with
palm
oil
and
are
applied
to
cuts
and
wounds
1
Carica
papaya
L. Caricaceae Ibepe Leaves
The
roasted
leaf
pulp
is
placed
on
sores
or
wounds
for
healing
1
Chromolaena
odorata
(L.)
King
&
H.
Rob
Asteraceae
Awolowo
Akintola
Taku
Leaves
An
infusion
is
used
as
an
external
wash
for
wounds
2
Citrullus
lanatus
(Thunb.)
Matsum.
&
Nakai
Cucurbitaceae
Osan
Leaves
The
softened
warm
leaf
is
applied
to
sores
and
wounds
2
Cocos
nucifera
L.
Arecaceae
Igi
agbon
Fruits
The
fruit
is
ground
and
applied
to
the
wound
1
Crassocephalum
crepidioides
(Benth.)
S.
Moore
Asteraceae Ebolo
Leaves
Chopped
leaves
are
placed
on
the
sore
1
Datura
stramonium
L.
Solanaceae
Apikan
Leaves
and
seeds
The
crushed
leaves
and
seeds
are
mixed
with
palm
oil
and
applied
to
wounds
and
burns.
4
Dichrostachys
glomerata
Chiov.
Fabaceae
Kara
Leaves
An
infusion
of
the
leaves
is
used
as
wash
for
wounds
3
Dioscorea
hirtiflora
Benth.and
Hook.
Dioscoreaceae
Isanyinahun
Leaves
The
fresh
leaves
are
made
as
paste
and
applied
to
the
wound
2
Diospyros
canaliculata
De
Wild
Ebenaceae
Orile
ijebu
Leaves
The
leaves
are
used
as
paste
for
wound
dressing
3
Euphorbia
heterophylla
L.
Euphorbiaceae
Oro
Leaves
Hot
ash
in
a
cloth
is
dipped
in
the
oil
from
the
leaves
and
applied
to
the
wound
1
Euphorbia
poissonii
L.
Euphorbiaceae
Oro-adete
Leaves
and
sap
The
fresh
leaves
or
leaf
sap
are
applied
as
a
wound
closure
2
Ficus
asperifolia
Miq.
Moraceae
Eripin
Leaves
The
leaves
are
used
as
paste
for
wound
dressing
3
Flabellaria
paniculata
Cav.
Malpighiaecea
Lagbolagbo
Leaves
A
decoction
of
the
leaf
is
used
for
cleaning
of
wounds
3
Hibiscus
sabdariffa
L.
Malvaceae
Amukan-zobo
Fruits
The
dried
fruit
is
finely
powdered
and
applied
to
sores
and
wounds
1
Jatropha
curcas
L.
Euphorbiaceae
Botuje,
Lapalapa
Leaves
The
juice
from
the
leaves
is
applied
to
wounds
1
Lawsonia
inermis
L. Lythraceae
Lali
Leaves
An
infusion
of
the
leaves
is
used
as
wash
for
wounds
10
Lycopersicon
esculentum
L.
Solanaceae
Igi
Tomato
Leaves
Fresh
leaves
are
used
for
wound
dressing
1
Morinda
lucida
Benth
Rubiaceae
Oruwo
Leaves
The
leaves
are
used
as
paste
for
wound
dressing
2
Nymphaea
lotus
L.
Nymphaeaceae
Ira
Leaves
The
poultice
of
the
leaves
are
applied
to
wounds
and
burns
2
Ocimum
gratissimum
L.
Lamiaceae
Effirin
Leaves
An
infusion
of
the
leaves
is
used
as
a
wash
for
wounds
10
Olax
subscorpioides
Oliv.
Olacaceae
Ifon
Leaves
The
fresh
leaves
are
applied
as
paste
on
wounds
2
Parkia
biglobosa
(Jacq.)
R.Br.
ex
G.Don
Leguminosae
Igi-Igba
Bark
The
ground
bark
is
used
as
a
paste
used
for
wound
dressing
7
Piliostigma
thonningii
(Schumach.)
Milne-Redh.
Leguminosae
Abafe
Leaves
The
apical
part
of
young
leaves
are
macerated
in
water
for
topical
application
1
Ricinus
communis
L.
Euphorbiaceae
Ewé
ogohoun,
ilara
Leaves
The
fresh
leaves
are
made
as
paste
and
applied
to
the
wound
2
Sida
acuta
Burm.f
Malvaceae
Isekete
Leaves
A
decoction
of
the
leaves
is
used
to
clean
wound
6
Tridax
procumbens
L.
Asteraceae
Kodele
yiri
Leaves
The
fresh
leaves
are
applied
as
a
wound
closure
5
Vernonia
amygdalina
L.
Asteraceae
Ewuro
Leaves
A
decoction
is
used
as
a
wash
and
applied
on
wounds
8
Xylopia
aethiopica
(Dunal)
A.
Rich.
Annonaceae
Eeru
Leaves
The
fresh
leaves
are
applied
as
paste
on
wounds
4

Author's personal copy
A.
Adetutu
et
al.
/
Journal
of
Ethnopharmacology
137 (2011) 50–
56 53
0
2
4
6
8
10
12
A.w
ilkesiana
A.c
onyzoides
B. ferruginea
L. inermis
O.gr
atissimum
P. biglobosa
S. acuta
T. procumbens
V. am ydgalina
Other species
Plant species
Species frequency
Fig.
1.
Species
citation
frequency
in
the
ethnopharmacological
survey
of
plants
commonly
used
for
wound-healing
in
South-western
Nigeria.
where
A0is
the
absorbance
of
the
control
and
A1is
the
absorbance
of
the
test
sample.
The
concentration
required
to
inhibit
DPPH
radical
formation
by
50%
was
calculated
as
the
IC50 of
each
sample.
3.
Results
and
discussion
3.1.
Survey
on
wound
healing
plants
In
the
present
study
36
species
belonging
to
22
plant
families
have
been
identified
as
wound-healing
remedies
used
by
healers
in
Oyo
State
Nigeria
(Table
1).
The
survey
has
shown
the
range
and
extent
of
medicinal
plants
employed
in
the
traditional
treat-
ment
of
wounds
in
South-western
Nigeria.
The
most
common
plant
families
are
Euphorbiaceae
(six
cites)
and
Asteraceae
(five
cites).
Solanaceae
has
three
cites
and
those
families
with
two
cites
include
Annonaceae,
Leguminosae
and
Malvaceae.
Leaves
are
the
most
fre-
quent
plant
parts
used,
constituting
about
86%
of
the
preparations
followed
by
stem
bark,
sap,
seeds
and
fruits
(approximately
2%
each).
The
plants
are
used
as
first
aid,
in
the
cleaning
of
wounds,
as
well
as
for
wound
dressing.
The
most
common
treatment
seems
to
either
be
using
a
decoction
as
a
wash
followed
by
application
of
a
plant
paste
or
applying
a
plant
dressing
directly
on
the
wound.
The
treatments
are
usually
repeated
every
day
until
the
wounds
Table
2
The
percentage
yield
of
crude
extracts.
Plant
species
Plant
part
used
%
Yield
of
plant
extract
residues
Ethanol
Water
Ageratum
conyzoides
Leaves
12.5
9.5
Acalypha
wilkesiana
Leaves
9.0
8.2
Bridelia
ferruginea
Leaves
7.1
6.3
Lawsonia
inermis
Leaves
10.1
11.2
Ocimum
gratissimum
Leaves
8.6
6.5
Parkia
biglobosa
Stem
bark
9.5
8.0
Sida
acuta Leaves
7.0
6.0
Tridax
procumbens
Leaves
5.6
4.6
Vernonia
amygdalina Leaves
7.2
6.6
are
healed.
Wound
dressing
most
often
consists
of
whole
or
fresh
leaves
used
in
form
of
paste.
The
present
inventory
of
wound
heal-
ing
plants
used
by
the
Yorubas
in
South-western
Nigeria
opens
new
avenues
to
scrutinize
such
a
rich
natural
plant
resource
for
further
pharmacological
screening
towards
the
development
of
potential
phytotherapy
and
to
validate
the
traditional
knowledge
as
an
adjunct
to
Nigeria’s
primary
healthcare
system.
Therefore,
the
plants
most
frequently
cited
by
the
healers
were
subjected
to
in
vitro
pharmacological
investigations
relevant
to
wound-healing.
The
ultimate
goal
is
providing
efficient
and
non-toxic
medicines
to
the
rural
populations
of
Nigeria,
where
medicinal
plants
play
a
vital
role
in
the
primary
health
care.
The
demonstration
of
pharma-
cological
activity
by
plant
extracts
that
correspond
to
traditional
formulations
may
support
the
traditional
use
of
these
medicinal
plants.
3.2.
In
vitro
wound
healing
assays
3.2.1.
Antibacterial
assay
Wounds
provide
an
environment
for
the
growth
of
micro-
organisms
(Somboonwong
et
al.,
2003).
An
infected
wound
is
less
likely
to
heal,
thus
removal
and
prevention
of
further
infection
is
a
key
to
rapid
and
effective
would
healing.
Many
authors
have
pro-
posed
is
in
vitro
and
in
vivo
evidence
to
support
the
use
of
plant
materials
as
topical
anti-microbial
agents
to
enhance
wound
heal-
ing
(Chah
et
al.,
2006;
Muthusamy
et
al.,
2006;
Perumal
et
al.,
2006).
The
results
of
our
antibacterial
screening
of
nine
plants
against
four
bacteria
species
are
summarised
in
Table
3
(MIC
values).
Staphylo-
coccus
aureus,
Escherichia
coli,
Pseudomonas
aeruginosa
and
Bacillus
subtilis
were
selected
for
this
study
because
they
are
common
to
infected
wounds
(Mertz
and
Ovington,
1993;
Bowler
et
al.,
2001).
Amongst
the
test
organisms
used,
Staphylococcus
aureus
was
found
to
be
most
sensitive,
followed
by
Bacillus
subtilis,
Escherichia
coli,
and
Pseudomonas
aeruginosa.
Initially
water
extracts
of
the
plants
were
used
in
this
study
in
order
to
reflect
the
traditional
heal-
ers’
formulations.
These
were
not
as
active
as
ethanolic
extracts
against
bacteria
(Table
3).
This
is
in
accordance
with
work
by
other
researchers
who
have
reported
water
extracts
showing
low
activ-
ities
especially
towards
bacteria,
as
compared
to
organic
extracts
(Shale
et
al.,
1999;
Lall
and
Meyer,
2000).
An
initial
observation
is
that
the
healer’s
extraction
techniques
may
be
inefficient
and
vari-
able,
however
given
that
there
are
some
activities
in
the
aqueous
extracts,
there
may
still
be
some
efficacy
if
used
in
very
high
doses.
In
this
study
all
the
plant
extracts
were
able
to
inhibit
the
growth
of
at
least
two
of
the
tested
standard
strains
(Table
3).
The
highest
activity
was
shown
against
the
Gram-positive
bacteria
Staphylo-
coccus
aureus
and
Bacillus
subtilis.
The
findings
of
our
study
agreed
with,
or
showed
greater
antimicrobial
activity,
to
that
of
earlier
research
for
Lawsonia
inermis
L.
(Malekzadeh,
1968),
Bridelia
ferrug-
inea
Benth.
(Talla
et
al.,
2002)
and
Vernonia
amygdalina
L.
(Iwalokun
and
Bamiro,
2003).
In
other
cases
however
the
results
of
this
study
showed
less
activity.
Such
variation
may
be
due
to
the
different
assays
used
by
different
researchers,
the
different
extraction
meth-
ods
employed,
the
sources
of
plant
materials
and
the
different
strains
of
bacteria
tested.
In
the
field
such
variation
in
plant
for-
mulations
must
have
a
significant
impact
on
the
efficacy
of
the
treatment
used
by
traditional
healers.
One
of
the
most
active
plants
Parkia
biglobosa
(Jacq.)
showed
similar
activity
for
both
aqueous
and
ethanolic
extracts.
The
bark
extracts
showed
anti-bacterial
activity
against
Staphylococcus
aureus
with
MIC
=
0.31
±
0.01
mg/ml
for
both
the
aqueous
extract
and
the
ethanolic
extract.
Previous
screening
for
antibacterial
agents
from
the
leaves
and
seeds
of
this
plant
showed
that
gram
positive
bacteria
were
less
susceptible
to
the
leaf
extracts
than
the
gram
negative
bacteria
(Ajaiyeoba,
2002).
It
has
previously
been
suggested
that
the
outer
membrane
surrounding
the
cell
wall

Author's personal copy
54 A.
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56
Table
3
Minimum
inhibitory
concentrations
(MICs)
of
the
aqueous
and
ethanolic
extracts
of
the
selected
plants.
Plant
species
Extract
MIC
(mg/ml)
Staphylococcus
aureus
Escherichia
coli
Bacillus
subtilis
Pseudomonas
aeruginosa
Ageratum
conyzoides
Aqueous
0.95
±
0.02
1.86
±
0.07
0.95
±
0.02
1.25
±
0.05
Ethanolic
0.47
±
0.01
1.25
±
0.05
0.31
±
0.01
0.63
±
0.01
Acalypha
wilkesiana Aqueous 0.94 ±
0.02
0.95 ±
0.01
0.95 ±
0.01
1.56 ±
0.05
Ethanolic 0.31 ±
0.01
0.47
±
0.01
0.47
±
0.02
0.95
±
0.02
Bridelia
ferruginea Aqueous
0.95
±
0.02
1.25
±
0.05
0.95
±
0.03
1.25
±
0.06
Ethanolic
0.47
±
0.03
0.95
±
0.03
0.47
±
0.01
0.63
±
0.02
Lawsonia
inermis
Aqueous
0.31
±
0.02
0.95
±
0.01
0.31
±
0.01
0.63
±
0.01
Ethanolic
0.47
±
0.03
0.78
±
0.02
0.31
±
0.02
0.78
±
0.02
Ocimum
gratissimum Aqueous 2.50 ±
0.09
5.00 ±
0.25
3.75 ±
0.10
5.00 ±
0.20
Ethanolic 1.25 ±
0.05
3.75
±
0.10
3.75
±
0.09
2.50
±
0.12
Parkia
biglobosa
Aqueous
0.31
±
0.01
0.63
±
0.02
0.31
±
0.01
0.95
±
0.04
Ethanolic
0.31
±
0.01
0.63
±
0.02
0.47
±
0.02
0.63
±
0.02
Sida
acuta
Aqueous
5.00
±
0.24
5.00
±
0.12
2.50
±
0.14
5.00
±
0.15
Ethanolic
3.75
±
0.20
5.00
±
0.25
2.50
±
0.10
5.00
±
0.25
Tridax
procumbens
Aqueous
2.50
±
0.30
5.00
±
0.20
3.75
±
0.03
7.50
±
0.50
Ethanolic
1.86
±
0.10
3.75
±
0.10
2.50
±
0.15
5.00
±
0.25
Vernonia
amygdalina Aqueous 0.63 ±
0.02
1.25 ±
0.02
0.47 ±
0.01
1.25 ±
0.01
Ethanolic
0.47
±
0.01
0.94
±
0.02
0.47
±
0.02
0.63
±
0.01
Chloramphenicol
0.001
±
0.00
0.001
±
0.00
0.08
±
0.01
0.09
±
0.01
Values
represent
means
±
SEM
of
three
independent
experiments.
of
Gram-negative
bacteria
may
restrict
diffusion
of
hydrophobic
compounds
through
its
lipopolysaccharide
covering
(Burt,
2004;
Davidson
et
al.,
2005).
In
our
study,
both
ethanol
and
aqueous
extracts
of
the
leaves
of
Bridelia
ferruginea
Benth.
inhibited
the
growth
of
all
the
tested
bacteria;
the
greatest
activity
was
from
the
ethanolic
extracts.
This
result
confirms
previous
studies
by
others
on
antibacterial
activity
for
this
plant.
Talla
et
al.
(2002)
demonstrated
that
the
methanol,
ethylacetate
and
hexane
extracts
of
Bridelia
ferruginea
Benth.
leaves
exhibited
significant
antimicrobial
activity
against
Pseudomonas
frutescens,
Bacillus
subtilis,
Escherichia
coli,
Staphylococcus
aureus
and
S.
faecalis.
Akinpelu
and
Olorunmola
(2000)
reported
that
60%
methanolic
extract
of
Bridelia
ferruginea
fruit
exhibited
antimicro-
bial
activity
against
seven
bacterial
isolates.
Muanza
et
al.
(1995)
also
reported
that
methanol
extract
of
the
leaves
showed
a
marked
antibacterial
activity
against
the
microorganisms
Klebsiella
pneu-
moniae,
Staphylococcus
aureus
and
Streptococcus
mutans.
The
use
of
decoctions
of
Bridelia
ferruginea
Benth.
is
widespread
in
Africa
and
such
extracts
are
reported
to
contain
antimicrobial
constituents
and
activity
(Muanza
et
al.,
1995;
Akinpelu
and
Olorunmola,
2000).
Vernonia
amygdalina
L.
was
active
against
most
of
the
strains
tested
in
this
study.
Few
reports
exist
on
the
antimicrobial
prop-
erty
of
compounds
isolated
from
this
plant.
Previous
reports
indicate
antibacterial
activity
of
extracts
of
this
plant
and
the
two
constituent
sesquiterpene
lactones,
vernolide
and
vernodalol
(Akinpelu,
1999;
Iwalokun
and
Bamiro,
2003;
Erasto
et
al.,
2006).
The
crude
extracts
of
Ocimum
gratissimum
L.
inhibited
the
growth
of
all
strains
of
tested
bacteria
with
an
MIC
range
of
5.00
±
0.20
to
1.25
±
0.05
mg/ml.
The
data
obtained
here
varied
from
what
was
reported
by
other
researchers
who
reported
antibacterial
activ-
ity
of
Ocimum
gratissimum
L.
against
Gram
positive
and
Gram
negative
bacteria
(Ngassoum
et
al.,
2003;
Ijeh
et
al.,
2005).
The
difference
in
extraction
procedure,
plant
part
and
geographical
origin
may
contribute
to
different
results.
The
ethanol
but
not
the
water
extracts
of
Acalypha
wilkesiana
Müll.
Arg.
inhibited
the
growth
of
both
the
Gram-positive
and
Gram-negative
bacteria.
Similar
observations
were
reported
by
Alade
and
Irobi
(1993)
on
water
and
methanol
extracts
of
the
plant,
who
found
that
most
of
the
antibacterial
activity
observed
was
in
the
methanol
extracts.
Results
obtained
here
on
antibacterial
activity
of
Lawsonia
inermis
L.
leaves
(Table
3),
are
similar
to
those
obtained
by
Malekzadeh
(1968)
who
reported
that
aqueous
extracts
showed
antimicrobial
activity
against
Escherichia
coli,
and
Staphylococcus
aureus.
In
addi-
tion,
Awadh
et
al.
(2001)
reported
that
the
ethanolic
and
ethyl
acetate
extract
of
Lawsonia
inermis
L.
were
active
against
Staphy-
lococcus
aureus,
Escherichia
coli,
and
Pseudomonas
aeruginosa.
Our
results
were
comparable
to
Awadh’s
study
with
aqueous
extracts
(Table
3)
producing
similar
activity
to
the
ethanol
extracts
by
Table
4
Influence
of
ethanolic
extracts
at
1–60
␮g/ml
for
48
h
on
human
dermal
skin
fibroblasts
using
neutral
red
uptake
(NRU)-assay.
Data
and
SEM-values
are
from
3
independent
experiments
with
n
=
6
replicates.
Negative
control:
untreated
cells;
positive
control:
10%FCS.
*p
<
0.05,
**p
<
0.01
compared
to
the
untreated
control
group
(ANOVA).
Plant
species
Percentage
proliferation
1
␮g/ml
5
␮g/ml
15
␮g/ml
30
␮g/ml
60
␮g/ml
Catalase
250
IU/ml
Ageratum
conyzoides
101.4
±
9
106.8
±
8
109.5
±
9
94.6
±
6
97.3
±
5
168.9**
±
12
Acalypha
wilkesiana
102.7
±
12
101.8
±
13
101.8
±
10
111.1
±
12
107.4
±
9
185.4**
±
14
Bridelia
ferruginea
123.3*
±
8
133.3**
±
9
123.3*
±
7
125.0*
±
8
70.0
±
6
181.7**
±
12
Lawsonia
inermis
100.3
±
9
95.6
±
7
91.2
±
8
88.2
±
6
80.9
±
5
161.0**
±
12
Ocimum
gratissimum
103.3
±
9
101.6
±
9
104.9
±
10
105.7
±
10
93.4
±
9
174.0**
±
13
Parkia
biglobosa 111.9
±
9
110.4
±
8
114.9*
±
11
131.3**
±
12
104.4
±
11
186.6**
±
15
Sida
acuta
98.27
±
8
100.9
±
9
94.8
±
9
87.2
±
7
92.2
±
8
170.1**
±
14
Tridax
procumbens 102.7
±
9
103.1
±
10
100.3
±
9
105.4
±
8
105.5
±
11
187.2**
±
15
Vernonia
amygdalina
104.2
±
9
101.3
±
9
100.5
±
10
98.6
±
9
98.5
±
8
147.1**
±
14

Author's personal copy
A.
Adetutu
et
al.
/
Journal
of
Ethnopharmacology
137 (2011) 50–
56 55
Malekzadeh
(1968)
cited
above.
The
slight
variation
in
the
val-
ues
of
MICs
(Table
3)
obtained
from
this
study
and
that
of
Awadh
et
al.
(2001)
could
be
explained
partly
because
of
different
con-
centrations
of
plant
extracts
used
and
method
of
plant
extraction
employed.
By
comparison
to
the
plants
described
above,
the
antibacterial
activity
Ageratum
conyzoides
L.
has
well
been
studied
(Pari
et
al.,
2000).
The
essential
oil
showed
weak
activity
against
Staphylo-
coccus
aureus,
Staphylococcus
epidermidis
and
was
inactive
against
Escherichia
coli
(Pari
et
al.,
2000).
Our
data
demonstrates
that
the
leaf
extracts
also
showed
some
activity
against
all
the
tested
bac-
teria.
In
contrast
to
the
generally
broad-spectrum
activity
from
the
plant
extracts
highlighted
above,
the
extracts
from
the
leaves
of
Sida
acuta
Burm.
F
and
Tridax
procumbens
L.
were
found
to
exhibit
low
antibacterial
properties
mainly
against
Gram
positive
species.
This
is
in
accordance
with
previous
work
reporting
that
Gram-negative
bacteria
are
resistant
to
most
plant
extracts
(Lall
and
Meyer,
2000;
Cos
et
al.,
2006).
The
antibacterial
activity
of
Sida
acuta
Burm.
F
com-
ponents
have
been
reported
by
Damintoti
et
al.,
2006.
Cryptolepine
was
shown
to
contribute,
at
least
in
part,
to
the
antibacterial
effects
by
causing
cell
lysis
and
morphological
changes
of
Staphylococcus
aureus
(Sawer
et
al.,
2005).
3.2.2.
Influence
of
extracts
on
human
fibroblast
skin
cells
in
vitro
Skin
fibroblast
proliferation
is
important
in
tissue
repair
as
fibroblasts
are
involved
in
migration,
proliferations,
contractions
and
collagen
production
(Woodley
et
al.,
1985;
Mimura
et
al.,
2004).
The
ability
to
stimulate
fibroblast
cell
growth
is
a
useful
model
for
testing
wound
healing
activity
in
vitro
(Graham
et
al.,
1984;
Mensah
et
al.,
2001).
Parkia
biglobosa
(Jacq.)
and
Bridelia
ferruginea
Benth.
stimulated
the
growth
of
fibroblasts
at
15–30
and
1–30
␮g/ml,
respectively
(Table
4).
The
findings
obtained
indicate
that
these
two
plants
may
promote
wound
healing
by
the
stimulation
of
fibrob-
lasts,
although
it
is
difficult
to
relate
these
concentrations
to
those
achievable
by
the
healers
in
practice.
Flavonoids,
tannins,
saponins
and
terpenoids
have
been
found
in
these
plants
(Cimanga
et
al.,
2001;
Tringali
et
al.,
2000).
Previous
reports
have
associated
these
classes
of
compounds
with
wound
healing
activities
by
stimulat-
ing
the
growth
of
fibroblasts
(Kim
et
al.,
1998;
Stevenson
et
al.,
2002).
Conversely,
Vernonia
amygdalina
L.,
Tridax
procumbens
L.,
Acalypha
wilkesiana
Müll.
Arg.
and
Sida
acuta
Burm.
F
(Table
4),
at
various
concentrations
tested
did
not
affect
human
skin
fibroblasts
proliferation.
Ethnopharmacological
uses
of
these
plants
however
suggest
that
they
might
have
wound
healing
properties.
There-
fore,
the
wound
healing
properties
of
these
plants
may
be
achieved
through
other
properties
such
as
anti-inflammatory,
antioxidant
or
antibacterial
action
and
not
directly
by
fibroblast
growth
stimula-
tion.
The
data
obtained
in
this
study
indicates
stimulation
of
fibro-
blast
growth
with
the
lower
concentrations
(1–15
␮g/ml)
of
extracts
of
some
of
the
selected
plants.
However,
at
higher
con-
centrations
(30–60
␮g/ml)
for
some
of
the
plants
there
is
evidence
of
cytotoxicity
or
inhibition
since
the
absorbance
was
less
than
the
control
(Table
4).
Most
traditional
healers
apply
these
extracts
with
little
understanding
of
the
concentration
being
applied.
The
indirect
evidence
of
cytotoxicity
of
some
of
these
extracts
at
high
concentration
implies
that
caution
must
be
taken
when
using
infu-
sion
of
these
plant
species
in
treating
wounds.
3.2.3.
Free
radical
scavenging
activity
of
selected
plants
Infected
wounds
attract
high
levels
of
phagocytic
cells
which
release
reactive
oxygen
species
in
an
attempt
to
fight
infection,
however
these
molecules
can
damage
the
host
cells
and
delay
the
healing
process
(Altomare
et
al.,
1995).
As
wound
healing
can
be
positively
influenced
by
antioxidant
agents,
the
radical
scaveng-
ing
activity
of
the
ethanolic
test
extracts
was
determined
by
DPPH
Table
5
DPPH
scavenging
activity
for
the
selected
plants
estimated
by
means
of
IC50.
Ascorbic
acid
was
used
as
a
positive
control.
IC50 values
(mean
±
SD,
n
=
3).
Plant
(ethanolic
extract)
IC50 (␮g/ml)
Ageratum
conyzoides 31.25 ±
0.26
Acalypha
wilkesiana 15.25
±
0.25
Bridelia
ferruginea
12.50
±
0.31
Lawsonia
inermis 3.80
±
0.02
Ocimum
gratissimum
18.0
±
0.25
Parkia
biglobosa
15.65
±
0.20
Sida
acuta
500.0
±
2.95
Tridax
procumbens 62.50 ±
0.31
Vernonia
amygdalina
31.25
±
0.20
Ascorbic
acid 7.26
±
0.12
assay
and
IC50 values
were
calculated
(Table
5).
The
extracts
which
showed
strong
DPPH
radical
scavenging
activity
(IC50 <
16.0
␮g/ml)
were
those
of
Lawsonia
inermis
L,
Bridelia
ferruginea
L.,
Acalypha
wilkesiana
Müll.
Arg.
and
Parkia
biglobosa
(Jacq.).
The
observed
antioxidant
effects
offered
by
some
of
the
extracts
compare
well
with
antioxidant
effects
demonstrated
by
plant
species
which
find
their
use
in
the
treatment
of
wounds
in
Vietnam
(Hien
et
al.,
1997),
Oman
(Marwah
et
al.,
2007),
Ghana
(Mensah
et
al.,
2006)
and
in
similar
studies
involving
the
wound-healing
properties
of
Bud-
dleja
globosa
(Mensah
et
al.,
2001).
Thousands
of
compounds
occur
in
the
medicinal
herbs
which
are
believed
to
be
responsible
for
antioxidant
activities
(Xiao
et
al.,
2000).
Over
4000
flavonoids
and
hundreds
of
coumarins
and
lignans
have
been
reported
as
natu-
rally
occurring
antioxidant
compounds
(Iwashina,
2000
and
Xiao
et
al.,
2000).
The
antioxidant
activity
observed
in
this
study
could
be
one
possible
mechanism
which
contributes
to
the
selected
plants
potential
for
enhanced
wound
healing.
4.
Conclusion
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
scientific
support
for
its
traditional
use
(Houghton
et
al.,
2005).
Most
of
the
plant
extracts
reported
here
displayed
both
antioxidant
and
antibacte-
rial
activities
thereby
suggesting
all
have
some
potential
for
wound
healing.
By
contrast
only
two
plants
were
effective
at
stimulat-
ing
fibroblast
growth
with
Bridelia
ferruginea
L.
showing
activity
at
low
concentrations.
From
the
above
results
there
is
strong
indi-
cation
that
the
traditional
use
of
plant
materials
for
wound
healing
by
South-western
Nigerian
healers
in
many
cases
can
be
rational-
ized
by
in
vitro
investigations.
In
conclusion,
these
findings
support
the
use
of
the
nine
plants
selected
by
citation
frequency.
However
further
studies
to
isolate
the
pharmacologically
active
compounds
contributing
to
the
wound
healing
properties
of
these
plants
are
needed.
Acknowledgements
The
authors
are
grateful
to
the
traditional
healers
for
volunteer-
ing
vital
information
about
their
rich
heritage
and
Mr.
T.K.
Odewo
of
the
Forestry
Herbarium
Ibadan
also
helped
in
the
preparation
of
voucher
specimens.
A.A.
acknowledges
study
leave
from
Ladoke
Akintola
University
of
Technology,
Nigeria
to
complete
his
doctoral
studies.
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