In vitro evaluation of effects of two Ghanaian plants relevant to wound healing.

Abstract

Commelina diffusa and Spathodea campanulata are used as wound-healing agents in Ashanti traditional medicine in Ghana. The methanol extracts of Commelina diffusa herb and Spathodea campanulata bark showed some level of antimicrobial activity with C. diffusa exhibiting selective antifungal activity against Trichophyton species. The extracts reduced the peroxidation of bovine brain extract with an IC(50) value of 1.39 mg/mL and 0.24 mg/mL, respectively. In addition the extracts also exhibited significant antioxidant activity by protecting MRC-5 cells from hydrogen peroxide induced oxidant injury at concentrations between 1 microg/mL and 10 microg/mL. The extracts showed no inhibition of NF-kappaB at 100 microg/mL. The antioxidant activities and antimicrobial activities suggest that the use of the plants in wound healing may be based on antioxidant and antiseptic effects of its constituents.

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Copyright © 2006 John Wiley & Sons, Ltd.Phytother. Res. 20, 941–944 (2006)
Received 31 July 2005
DOI: 10.1002/ptr
Accepted 27 April 2006
Copyright © 2006 John Wiley & Sons, Ltd.
* Correspondence to: Peter J. Houghton, Pharmacognosy Research Labor-
atories, Department of Pharmacy, King’s College London, 150 Stamford
Street, London SE1 9NH, UK.
Contract/grant sponsor: Wellcome Trust Fund as an International
Research Development in Tropical Medicine Award.
Contract/grant sponsor: European Union, FP 6.
aqueous extracts obtained are applied to wounds to aid
their healing.
Wound healing is a complex process involving three
main overlapping stages; inflammation, cell prolifera-
tion and contraction of the collagen lattice formed
(Bodeker and Hughes, 1996). Inflammation is a funda-
mental physiological process common to all wounds and
begins immediately injury occurs. It involves the con-
striction and activation of the coagulation process pro-
ducing a platelet plug to limit blood loss. In addition to
this the local immune system is activated and soluble
biological active mediators are released which attract
leukocytes and monocytes to enter the wound and
attack contaminating foreign objects. The neutrophils
form the first line of defence against invading micro-
organisms. They phagocytose microorganisms then kill
the ingested microbes by the production of oxygen-free
metabolites such as hydroxyl radicals and superoxide
ions. Paradoxically, normal tissues may be damaged by
some of these reactive oxygen species in excess amount,
thus prolonging the inflammatory phase of wound heal-
ing. The inflammatory phase may also be prolonged
when the wound is colonized by bacteria (Cherry et al.,
1994; Morrison and Kindlen, 1997; Laupattarakasem
et al., 2003).
Some aspects of the inflammatory process exploited
for screening for antiinflammatory extracts in vitro
include evaluating the protection offered by extracts
against oxidant injury to cells and monitoring the inhibi-
tion of peroxidation of lipid membranes (Aruoma
et al., 1989; Mensah et al., 2001). Inhibition of NF-κB
activation is used as an indicator of possible anti-
inflammatory action (Karin et al., 2004).
The present study was undertaken to determine
whether there was any scientific justification for the
INTRODUCTION
Recent years have witnessed an increase in interest in
plants used in traditional medicine for healing wounds
(Bodeker and Hughes, 1996; Houghton et al., 2005).
Commelina diffusa Burn. F. (Commelinaceae) and
Spathodea campanulata Beav. (Bignoniaceae) are two
such species used in Ghana, which were identified as
being used by several healers of the Ashanti ethnic
group following a survey carried out amongst them. C.
diffusa, also known as ‘spreading dayflower’, is a versa-
tile perennial tropical herb with numerous traditional
uses (Akobundu and Agyakwa, 1987; Burkhill, 1985).
Its tenacious nature is adequately exemplified by its
local Ghanaian name which when translated literally
means ‘God will die before I die’. In Ghana the leafy
aerial parts are used to treat boils and sores (Ayensu,
1979; Burkhill, 1985). S. campanula is widely dis-
tributed in Africa and the bark is used traditionally
as an antimalarial, hypoglycaemic agent and also for
the treatment of ulcers (Irvine, 1961; Makinde et al.,
1988; Niyonzima et al., 1993). Phytochemical investiga-
tion of S. campanulata bark has shown the presence of
triterpenes and sterols (Ngouela et al., 1991). There is,
however, no documented information on the phyto-
chemistry of C. diffusa. In Ghana, decoctions of both
plants are made with water and the concentrated
Abraham Y. Mensah1, Peter J. Houghton2*, Rita A. Dickson1, T. C. Fleischer1,
M. Heinrich3 and P. Bremner3
1Department of Pharmacognosy, Faculty of Pharmacy, Kwame Nkrumah University of Science and Technology (KNUST),
Kumasi, Ghana
2Pharmacognosy Research Laboratories, Department of Pharmacy, King’s College London, 150 Stamford Street, London SE1
9NH, UK
3Centre for Pharmacognosy and Phytotherapy, School of Pharmacy, University of London, 29/39 Brunswick Square, London
WC1N 1AX, UK
Commelina diffusa and Spathodea campanulata are used as wound-healing agents in Ashanti traditional
medicine in Ghana. The methanol extracts of Commelina diffusa herb and Spathodea campanulata bark
showed some level of antimicrobial activity with C. diffusa exhibiting selective antifungal activity against
Trichophyton species. The extracts reduced the peroxidation of bovine brain extract with an IC50 value
of 1.39 mg/mL and 0.24 mg/mL, respectively. In addition the extracts also exhibited significant antioxidant
activity by protecting MRC-5 cells from hydrogen peroxide induced oxidant injury at concentrations between
1 µ µ µ µ µg/mL and 10 µ µ µ µ µg/mL. The extracts showed no inhibition of NF-κ κ κ κ κB at 100 µ µ µ µ µg/mL. The antioxidant activities
and antimicrobial activities suggest that the use of the plants in wound healing may be based on antioxidant
and antiseptic effects of its constituents. Copyright © 2006 John Wiley & Sons, Ltd.
Keywords: Commelina diffusa; Spathodea campanulata; ringworm fungi; bacteria; wound healing; lipid peroxidation.

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Copyright © 2006 John Wiley & Sons, Ltd.Phytother. Res. 20, 941–944 (2006)
DOI: 10.1002/ptr
campanulata as wound healing agents in Ghana, par-
ticularly since no such report has been documented in
the literature.
MATERIALS AND METHODS
The leafy young stems and leaves of Commelina
diffusa Burn. F. (Commelinaceae) and the stem bark
of Spathodea campanulata Beav. (Bignoniaceae) were
collected in April from the campus of Kwame Nkrumah
University of Science and Technology, Kumasi, Ghana
(KNUST) and authenticated by the curator of the her-
barium at the Department of Pharmacognosy. Voucher
specimens of the Commelina diffusa and Spathodea
campanulata (CD1 and SC1) are deposited at the
Faculty of Pharmacy, KNUST.
The plant materials were dried in sunlight and
powdered. 200 g of each powdered material was con-
tinuously extracted with MeOH in a Soxhlet apparatus
for 8 h to obtain 3.5 g of greenish syrupy extract of the
Commelina diffusa and 2.4 g of a dark brown extract of
the Spathodea campanulata, respectively.
In this investigation the inhibition of NF-κB was used
as an indicator of possible antiinflammatory action
(Karin et al., 2004). Inhibition of NF-κB activation was
measured utilizing luciferase in HeLa cells under the
control of an IL-6 promoter (a gene target for acti-
vated NF-κB). The cells were exposed to the extracts
(100 µg/mL) in 1 mL of growth media in replicates of
three and incubated at 37 °C for 1 h. At this point
phorbol myristicyl acetate (PMA) (50 ng/mL) was added
to stimulate NF-κB activation. PMA stimulated and
unstimulated cells acted as positive and negative con-
trols, respectively. The cells were then incubated for a
further 7 h before cell harvesting and luminometric
measurements in an automated 96-well format. Details
of the experimental protocol can be found in Bremner
et al. (2004).
Inflammation also involves the formation of excess
reactive oxygen species (ROS) so antioxidant proper-
ties in a plant extract can be antiinflammatory and also
protect the new cells formed during the proliferation
stage from oxidative damage. Two tests for antioxid-
ant activity was performed, one using liposomes and
the other for protection of cultured cells. The liposome
assay measured oxidative damage in terms of malon-
dialdehyde (MDA) through its reaction product with
thiobarbituric acid (Burits and Bucar, 2000). Serial
dilutions (10 mg/mL to 0.32 mg/mL) based on the pre-
vious protocol of each extract were tested, assayed
according to the method described by Aruoma et al.
(1989) and Guvenc et al. (2005) and propyl gallate was
used as the positive control.
The percentage inhibition (I) of lipid peroxidation
was calculated by the equation
I(%) = 100 × (A1/A0)
where A0 is the absorbance of the control reaction (full
reaction, containing no test compound) and A1 is the
absorbance in the presence of the inhibitor.
The other test for antioxidant protection was per-
formed using a confluent MRC-5 cell line (Sigma, UK)
challenged with H2O2 according to a method described
Catalase (250 IU/mL) was used as the positive anti-
oxidant control and the normal control consisted of the
cells alone (exposed only to the HBSS). Different doses
of the extracts were tested and 1 µg/mL, 5 µg/mL and
10 µg/mL doses of the extract were selected and used
since they gave reproducible and consistent results. The
neutral red assay was used to assess the protection
offered by the doses (Zhang et al., 1990). The cells were
also visually examined to check for any visible sign of
damage. Four separate experiments were carried out
and each experiment was done with seven replicates.
The data from both experiments were analysed by one-
way analysis of variance and Dunnet’s test using the
Microsoft Excel package. Differences at the 95% level
were considered to be significant.
As noted above, antimicrobial effects in the extract
would aid wound-healing so possible activity of the
extracts in this respect was also tested. The organisms
used were: Staphylococcus aureus (ATCC 2593);
Bacillus subtilis (ATCC 6633); Escherichia coli
(ATCC 25922); Pseudomonas aeruginosa (NCTC 5055);
Candida albicans (ATCC 10231); Saccharomyces
cerevisiae (NCTC 080178); Trichophyton interdigitale
(NCPF 654) Trichophyton tonsurans (NCPF 656) and
Microsporum gypseum (NCPF261). The bacteria were
maintained on nutrient agar at 37 °C and the yeast and
moulds on Sabouraud’s dextrose agar maintained at
30 °C and 26 °C, respectively. The agar serial dilution
method was used to determine the minimum inhibitory
concentration (MIC) for each extract with concentra-
tions of the extracts from 125 to 1000 µg/mL (Vanden
Berghe and Vlietinck, 1991; Mensah et al., 2000).
Chloramphenicol and clotrimazole were used as posi-
tive controls for the bacteria and fungi, respectively,
whilst the culture media used for the assay were used
as negative control and the assay was performed in
triplicate.
RESULTS AND DISCUSSION
In the tests for antioxidant effect using liposomes,
the methanol extract of C. diffusa gave an IC50
of 1.39 mg/mL and that of the S. campanulata was
0.24 mg/mL, propyl gallate having an IC50 of 1 × 10−4M
(21.2 mg/mL).
When the extracts were tested for protection of cul-
tured MRC-5 cells, the methanol extract of C. diffusa
at concentrations of 1.0, 5.0 and 10 µg/mL gave statist-
ically significant protection (p < 0.05) against hydrogen
peroxide-induced damage (Fig. 1), although the lowest
Figure 1. Neutral Red assay of the protective effect of
Commelina diffusa extract on MRC-5 cells (P16) against hydro-
gen peroxide induced oxidant injury.

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Copyright © 2006 John Wiley & Sons, Ltd. Phytother. Res. 20, 941–944 (2006)
DOI: 10.1002/ptr
showed some activity against E. coli (MIC 750 µg/mL).
The C. diffusa extract exhibited moderate antifungal
activity against the Trichophyton species with MIC
values of 500 µg/mL and 250 µg/mL against T.
interdigitale and T. tonsurans, respectively, but no such
activity was observed for the S campanulata extract.
Thus there is a weak level of antimicrobial activity
in the crude extracts of the assayed plants, although
the C. diffusa shows marked activity against the
Trichophyton species, so may be of use in preventing
fungal infection of wounds.
The antioxidant activities in particular show that there
is some scientific justification for the traditional uses
of the plants as wound healing agents in Ghana. The
antimicrobial activity is unlikely to be very effective
against infection of wounds at the concentration used,
and there was no indication of NFκB inhibition as an
indicator of antiinflammatory effects. Other tests for
properties such as fibroblast stimulation or other mech-
anisms associated with antiinflammatory action will be
of interest to investigate other activities related to
wound-healing properties.
Acknowledgements
Financial support was provided by the Wellcome Trust Fund as an
International Research Development in Tropical Medicine Award.
Research at ULSOP has been supported by the European Union,
FP 6.
Figure 2. Neutral Red assay of the protective effect of Spathodea
campanulata extract on MRC-5 cells (P18) against hydrogen
peroxide induced oxidant injury.
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