Scientific African 14 (2021) e00980

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Scientific African
journal homepage: www.elsevier.com/locate/sciaf

Antimicrobial, antioxidant and wound healing activities of

methanol leaf extract of Bridelia micrantha (Hochst.) Baill.
Philip Asumang a, Yaw Duah Boakye b,∗, Theresa Appiah Agana b, Jibira Yakubu c,
Philomena Entsie d, William Gariba Akanwariwiak a, Francis Adu b,
Christian Agyare a
a
Department of Theoretical and Applied Biology, College of Science, Kwame Nkrumah University of Science and Technology, Ghana
b
Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and
Technology, Ghana
c
Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and
Technology, Ghana
d
Department of Herbal Medicine, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and
Technology, Ghana

a r t i c l e i n f o a b s t r a c t

Article history: Bridelia micrantha is a medicinal plant used traditionally for the treatment of wounds.
Received 30 April 2021 However, there are no documented scientific reports on the wound healing activities of
Revised 27 July 2021
this plant. This study was conducted to investigate the antimicrobial, antioxidant and
Accepted 23 September 2021
wound healing activities of methanol leaf extract of B. micrantha (BME). The antimicro-
bial activity was determined using the agar well diffusion and broth dilution methods.
Editor: DR B Gyampoh Antioxidant activity was performed in vitro using the DPPH scavenging antioxidant activity
followed by in vivo wound healing activities using the excision wound model. Preliminary
Keywords:
phytochemical screening of BME showed the presence of saponins, tannins, flavonoids,
Bridelia micrantha
alkaloids, triterpenes, glycosides, steroids and coumarins. BME exhibited dose-dependent
Phytochemicals
Wounds antimicrobial activities. The most susceptible organism to BME was N. gonorrhoeae with
Antimicrobial mean zones of inhibition ranging from 20.50 ± 1.00 to 28.50 ± 1.00 mm. The least sus-
Wound healing ceptible organism was S. typhi with mean zones of inhibition in the range of 15.50 ±
Antioxidant 0.00 to 21.50 ± 0.00 mm. The MICs of BME against the test organisms were in the range
of 1.25 to 2.5 mg/mL. Sub-inhibitory concentrations of BME potentiated the activities of
ciprofloxacin and ketoconazole. BME showed a dose-dependent increase in antioxidant ac-
tivity with IC50 of 0.2626 μg/mL. The BME formulated creams at concentrations of 0.625,
2.5, and 10% w/w showed a significant (p<0.0 0 01) increase in rate of wound closure
compared to the control group. Histological studies revealed marked angiogenesis, colla-
genation and re-epithelization in BME-treated wound tissues compared to the untreated
group. The methanol leaf extract of B. micrantha possess antimicrobial, antioxidant and
wound healing activities, thereby proving the folkloric use of the leaves of B. micrantha as
a wound-healing agent.
© 2021 The Authors. Published by Elsevier B.V. on behalf of African Institute of
Mathematical Sciences / Next Einstein Initiative.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

∗
Corresponding author at: Kwame Nkrumah University of Science and Technology, Department Of Pharmaceutics,Knust,…, Kumasi, Ashanti, Ghana
E-mail address: ydboakye.pharm@knust.edu.gh (Y.D. Boakye).

https://doi.org/10.1016/j.sciaf.2021.e00980
2468-2276/© 2021 The Authors. Published by Elsevier B.V. on behalf of African Institute of Mathematical Sciences / Next Einstein Initiative. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
P. Asumang, Y.D. Boakye, T.A. Agana et al. Scientific African 14 (2021) e00980

Introduction

Pathogenic microorganisms cause several diseases especially when conditions are favourable for their growth. In numer-
ous states around the world, particularly in developing countries, infectious diseases remains one of the foremost cause of
illnesses and deaths [1]. However, some pathogenic microorganisms have developed resistance to current antimicrobials.
Therefore, the need for alternative treatment and efficient therapeutic materials for the management of infectious diseases.
These treatments, however, should be anodyne, economical and efficient in the management of diseases. The discovery of
new and effective antimicrobials and/or resistance modulators is necessary to tackle the spread of resistance or to reverse
multi-drug resistance [2]. Other conditions including wounds and its related infections also pose serious threats to the ex-
istence of humans [3].
Wounds present a considerable economic burden to healthcare systems. The economic burden of wounds cannot be
underestimated [4]. For instance, in the UK the treatment and care of chronic wounds account for 3% of total healthcare
expenditure. Again in the USA wounds affect 5.7 million with an annual cost of US$20 billion [5]. Although there is no
proper documentation on the cost of treatment of chronic wounds in Africa, it is reported that in Ghana 16% of households
borrow money and 27% sell their assets and use the proceeds as medical cost for the treatment of chronic wounds such as
Buruli ulcer [6]. Chronic wounds thereby do not only impose an immense financial burden on the society, and health care
systems but also cause a substantial reduction in productivity and quality of life [7].
Throughout history, plants have been used as a source of medicine, for treating and managing various diseases [8]. Medic-
inal plants have proven effective in treating several ailments [3] including the treatment of wounds. Some medicinal plants
used as wound healing agents in folkloric medicine in Ghana have been investigated and found to possess wound healing
properties. These include Physalis angulate [9], Phyllanthus muellerianus [10], Spathodea campunulata [11] and Clerodendron
splendens [12]. Some of these plants have been reported to possess antimicrobial and antioxidant properties which also
contribute to the wound healing process [13].
Bridelia micrantha (Hochst.) Baill. belongs to the family Phyllanthaceae (formerly Euphorbiaceae). This semi-deciduous or
deciduous tree is commonly known as mitzeerie or coastal golden leaf. It grows up to a height of 20 m with thick rounded
crown and stem [14]. Some parts of the plant are used to treat diverse human ailment by traditional healers. In Ivory Coast,
it is used as a powerful purgative in cases of obstinate constipation and poisoning [15]. In South Africa, B. micrantha stem
bark is used in folkloric medicine to treat paralysis, gastrointestinal ailments and painful joints [16]. In Nigeria, stem bark is
used for the management of diabetes [17]. In Central Uganda, a decoction of the leaves and bark is used for treating syphilis
and the bark for pre-hepatic jaundice [18]. Some biological activities of B. micrantha including anthelmintic, anticonvulsant,
antidiabetic, antinociceptive, antiviral and hepatoprotective properties have been reported [19]. This study, therefore, aims
to investigate the antimicrobial, antioxidant and wound healing properties of methanol leaves extract of B. micrantha.

Methods

Sample collection and identification

The fresh matured leaves of Bridelia micrantha were collected from Kwahu Asakraka in the Kwahu South District of the
Eastern Region of Ghana (latitude: N 6°37 45.9048 ’ and longitude: W 0°41 11.30253 ’) with Mpraeso as the Capital between
24th and 26th September, 2019. Authentication of the plant sample was done by Mr. Clifford Osafo Asare, a botanist at
the Department of Herbal Medicine, Faculty of Pharmacy and Pharmaceutical Sciences (FPPS), KNUST, Kumasi. A specimen
sample (specimen number: KNUST/HM1/2019/L008) was deposited at the Department’s herbarium.

Preparation and extraction of plant sample

The leaves of B. micrantha were washed thoroughly under running water to remove debris and allowed to drain before
being air-dried at 28°C for 5 days. The dried leaves were then milled into a fine powder using an ultra-fine grinding mill
(Christy and Norris, Chelmsford, UK). The powdered leaves of B. micrantha (500 g) were cold macerated in 2.5 litres of 70%
v/v methanol at regular intervals of shaking for 72 h at 28°C. The extract was filtered with Whatman filter paper (No. 1)
(Sigma- Aldrich, Michigan, USA). The filtrate was concentrated in a rotary evaporator (Rotavapor BUCHI R- 200 with heating
bath B-490, Buchi, Konstanz, Germany) at 40◦ C under reduced pressure and then lyophilized (CHRIST® ALPHA 1-4 LSC)
and stored in a fridge at 4°C. The dried methanol leaf extract of BME was weighed, kept in Falcon tubes (Sigma- Aldrich,
Michigan, USA), and stored in a desiccator for use. The yield of the extract, relative to B. micrantha powdered plant material
was 4.26% w/w.

Test organisms
Pure cultures of Streptococcus pyogenes ATCC 19615, Salmonella typhi ATCC 19430, Neisseria gonorrhoeae (Clinical strain),
Candida albicans ATCC 10231 and E. coli ATCC 25922 were obtained from the Microbiology Section, Department of Pharma-
ceutics, FPPS, KNUST, Kumasi. The inoculum size of 1.0 × 106 CFU/mL was used in all the antimicrobial assays.

Phytochemical screening of methanol leaf extract of B. micrantha

Phytochemical screening of BME was carried out using standard protocols [20,21]. This was done to ascertain the pres-
ence or otherwise of secondary metabolites such as saponins, coumarins alkaloids, flavonoids, tannins and triterpenes.

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P. Asumang, Y.D. Boakye, T.A. Agana et al. Scientific African 14 (2021) e00980

Determination of the antimicrobial activity of BME using the agar well diffusion method

The preliminary antimicrobial activity of BME was evaluated using the agar well diffusion method [22]. Muller Hinton
(MH) agar plates were lawn cultured with standardize organisms (1 × 106 CFU/mL). With the aid of a sterile cork borer No.
5, holes (10 mm in diameter) were punched in the media. The holes were filled aseptically with respective concentrations
of BME (80, 40, 20 and 10 mg/mL). Ciprofloxacin (50 μg/mL) (Sigma-Aldrich, Michigan, USA) and ketoconazole (50 μg/mL)
(Sigma-Aldrich, Michigan, USA) served as positive controls for bacteria and fungi, respectively. The suspension was allowed
to diffuse for 1 h and incubated at 37°C for 24 h. Plates were observed for zones of growth inhibition (clear zones surround-
ing the wells). Zones were measured with the aid of a millimeter ruler. The procedure was carried out in triplicate and the
average zone of inhibition was calculated [23].

Determination of minimum inhibitory concentrations (MIC) of BME

The minimum inhibitory concentrations of BME was determined using the broth micro-dilution procedure in 96-well
microtitre plate [23]. A Stock solution of BME was prepared and serially diluted to obtain two fold dilutions in respective
wells containing MH broth. Twenty microliters (20 μL) of each organism was introduced into the respective well to obtain
final concentrations in the range of 10 to 0.08 mg/mL of BME. Ciprofloxacin (50 to 0.39 μg/mL) and ketoconazole (100 to
0.78 μg/mL) served as positive controls for bacteria and fungi, respectively. The plate was incubated at 37°C for 24 h. Twenty
(20) microlitres of 3-(4, 5-dimethylthiazol-2-1)-2, 5-diphenyltetrazolium bromide (MTT) was added to each well, incubated
for 30 mins at 37°C and observed for colour change. The least concentration at which there was no visible colour change
from yellow to dark purple after incubation was recorded as the minimum inhibitory concentration [23]. The procedure was
repeated in triplicate.

Determination of the effect of sub-inhibitory concentrations of BME on ciprofloxacin and ketoconazole

The MICs of ciprofloxacin and ketoconazole were determined as described in section 2.2.3. To determine the effect of
sub-inhibitory concentrations of BME on the antibiotics, stock solutions of the antibiotics were serially diluted to achieve
two fold dilutions in respective wells. BME was added to appropriate wells to produce sub-inhibitory concentrations against
the various test organisms. Twenty microlitres (20 μL) of suspension containing 1.0 × 106 CFU/mL of the test organisms
were finally added to the appropriate wells. The plates were incubated for 24 h at 37°C, after which 20 μL of MTT was
added to the wells, incubated for 30 mins at 37°C and the MIC’s recorded.

In vitro total antioxidant activity of BME

The antioxidant activity of BME was determined using the 2,2-diphenyl-1- picryl hydrazyl (DPPH) assay [24]. Fifty mi-
croliters (50 μL) of BME (250 to 1.95 μg/mL) prepared by two-fold serial dilutions were added to 150 μL of 2 mM DPPH
and incubated in the dark for 30 mins. Ascorbic acid was used as standard. The absorbances of the mixtures were then
measured at 517 nm after incubation [25]. A blank solution of DPPH was also measured and used as control absorbance.
The percentage inhibition of both BME and ascorbic acid were calculated using the formula below:
A control − A sample
%inhibition = × 100 %
A control
Where A control is the absorbance of control while A sample is the absorbance of the sample for either the extract or ascorbic
acid. The percentage inhibitions obtained for BME and ascorbic acid were plotted against concentration [24].

Wound healing studies

Research approval
The study was approved by the Department of Pharmacology, FPPS, KNUST, Kumasi, Ghana; with reference number FPPS-
AEC/CA017/19 (December, 2019), but no consent was needed. All protocols used in experiments were executed and observed
following internationally recognized principles for laboratory animal practice and care laid down by the National Institute
of Health (NIH, Department of Health and Human Services publication No 5, revised 1985).

Formulation of extract into cream

The aqueous cream used for the wound healing experiment was prepared using the method described in the British
Pharmacopoeia [26]. BME was incorporated into the aqueous cream base to produce concentrations of 0.625, 2.5, and 10%
w/w. The various concentrations of the formulated creams were stored in the refrigerator at 4°C.

Experimental animals
Male Sprague-Dawley rats (160 to 180 g) were used for the studies. They were obtained from the Department of Phar-
macology, FPPS, KNUST, Kumasi. The rats were housed in stainless steel cages with wood shavings as beddings, and under
standard conditions (12/12 h light cycle; temperature of 25 ± 2°C and relative humidity of 40 ± 10%). They were fed with
standard laboratory dietary pellets (produced by Ghana Agro Food Company Limited) and water ad libitum.

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Table 1
Antimicrobial activity of BME.

Concentration (mg/mL) S. pyogenes N. gonorrhoeae E. coli S. typhi C. albicans

80 22.00 ±0.50 28.50 ±1.00 23.50 ±0.00 21.50 ±0.00 25.75 ±0.75
40 19.75 ±0.25 24.75 ±0.25 21.25 ±0.25 19.75 ±0.25 25.00 ±0.50
20 18.75 ±0.75 23.00 ±1.50 19.00 ±0.50 18.25 ±0.75 23.75 ±0.75
10 15.50 ±1.00 20.50 ±1.00 17.00 ±0.50 15.50 ±0.00 21.75 ±0.25

Excision wound model

The wound healing property of BME was determined in vivo using the excision wound model [10]. The dorsal fur of the
rats were shaved and the area cleaned with 70% methanol. Full thickness wounds (approx. 20 mm in diameter) were excised
after rats were anaesthetized with ketamine (50 mg/kg body weight im). Rats were then randomly placed into five groups
(five rats per group) and their wounds treated topically daily for twelve days as follows:

Group 1: received 1% w/w silver sulphadiazine cream used as a reference drug

Group 2: received aqueous cream only (vehicle)
Group 3: received 10% w/w BME aqueous cream
Group 4: received 2.5% w/w BME aqueous cream
Group 5: received 0.625% w/w BME aqueous cream

Thw wound diameter was measured on days 1, 3, 5, 7, 9, 11 and 13 post injury with the aid of a pair of dividers and
a millimetre ruler to assess the rate of wound closure. The equation below was used to calculate the percentage wound
closure:
Diameter of wound (Initial ) − Diameter of wound (Final )
% wound closure = x 100
Diameter of wound (Initial )

Histological studies
Skin specimens were taken on day 13, fixed immediately in 10% w/v buffered formalin and transferred to the histology
laboratory. The fixed tissues were dehydrated sequentially in ethanol (50, 70, 95 and 100%) and rinsed thoroughly in xylene
to remove ethanol. The tissues were then embedded in paraffin blocks at 40 to 60°C, deparaffinized, sectioned (5 to 6 μm
thick sections) and stained with hematoxylin and eosin (HE). The stained sections were examined qualitatively under a light
microscope for collagen deposition, fibroblast proliferation, angiogenesis, and epithelialization [27].

Statistical analysis

All data were presented as the mean ± standard error mean (SEM). Two-way ANOVA followed by Bonferroni’s post hoc
test was used to analyze the wound healing time course curve. One-way ANOVA followed by Dunnett’s post hoc test for
the area under the curve (AUC) graph. These were performed using Graph Pad Prism Version 8.0 for Windows (Graph Pad
Software Inc, San Diego, CA, USA).

Results

Phytochemicals composition of methanol leaf extract of B. micrantha

Preliminary phytochemical screening of BME revealed the presence of saponins, tannins, flavonoids, alkaloids, triter-
penoids, glycosides, steroids and coumarins.

Antimicrobial activity of methanol leaves extract of B. micrantha (BME)

The methanol leaf extract of B. micrantha exhibited dose-dependent antimicrobial activity. BME exhibited the highest and
lowest antimicrobial activities at test concentrations of 80 and 10 mg/mL, respectively, against all test organisms. The most
susceptible organism to BME was N. gonorrhoeae with mean diameter zones of inhibition ranging from 20.50 ±1.00 to 28.50
±1.00 mm. The least susceptible organism was S. typhi with mean diameter zones of inhibition in the range of 15.50 ±0.00
to 21.50 ±0.00 (Table 1).

Minimum inhibitory concentration (MIC) of methanol leaves extract of B. micrantha (BME)

The MICs of BME against the test organisms were in the range of 1.25 to 2.5 mg/mL. BME exhibited antimicrobial activity
against S. pyogenes, S. typhi and C. albicans with MICs of 2.5, 1.25 and 2.5 mg/mL, respectively. The MICs of BME for E. coli
and N. gonorrhoea were greater than the highest test concentrations (Table 2).

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Table 2
Minimum inhibitory concentration of BME.

Test Minimum inhibitory concentration

Organism
BME (mg/mL) Ciprofloxacin (μg/mL) Ketoconazole (μg/mL)

E. coli >10 12.5 ND

S. pyogenes 2.5 12.5 ND
N. gonorrhoeae >10 25 ND
S. typhi 1.25 25 ND
C. albicans 2.5 ND 25

ND: not determined; BME: methanol leaf extract of Bridelia micrantha.

Table 3
MIC of test antibiotics alone and in the presence of sub-inhibitory concentrations of BME.

Test organisms MIC of ciprofloxacin/ MIC of antibiotic in the presence of Modulation factor
ketoconazole alone (μg/mL) sub-inhibitory concentrations of BME (μg/mL)
- ∗
E. coli 12.5
S. pyogenes 12.5 6.25 2
- ∗
N. gonorrhoeae 25
S. typhi 25 3.13 8
C. albicans (Ketoconazole) 25 12.5 2
-
means No activity.
∗
means modulation factor cannot be calculated.

Modulatory effects of of methanol leaf extract of B. micrantha (BME)

The MIC of ciprofloxacin alone against S. pyogenes was 12.5 μg/mL. However, when ciprofloxacin was combined with a
sub-inhibitory concentration of BME, the MIC of ciprofloxacin reduced to 6.25 μg/mL which represents a 2-fold increase in
antibacterial activity.
The MIC of ciprofloxacin alone against S. typhi was 25 μg/mL. However, ciprofloxacin in the presence of the sub-inhibitory
concentration of BME, had its MIC reduced to 3.13 μg/mL which represents an 8-fold increase in antibacterial activity. The
MIC of ketoconazole alone against C. albicans was 25 μg/mL. However, when ketoconazole was combined with a sub-
inhibitory concentration of BME, the MIC of ketoconazole reduced to 12.5 μg/mL which represents a 2-fold increase in
antifungal activity (Table 3).

In vitro total antioxidant activity of methanol leaf extract of B. micrantha (BME)

Fig. 1 indicates the DPPH free radical scavenging activity of BME at different concentrations. BME showed a dose-
dependent increase in antioxidant activity. The IC50 value of BME was 0.2626 μg/mL whiles that of ascorbic acid was deter-
mined to be 0.2531 μg/mL.

Influnece of BME on rate of wound closure

The BME formulated creams at concentrations of 0.625, 2.5 and 10% w/w showed an increase in wound contractions
from the 3rd day of application to the 12th day (Fig. 2 A). The AUC of the various concentrations of the BME formulated
creams showed a significant reduction (P < 0.0 0 01) in wound size when compared to the vehicle-treated group (Fig. 2 B).

Histopathology of BME treated-wounds tissues

Histological investigations of the vehicle-treated wound tissues showed persistent inflammation and little proliferation
at wound edges indicative of poor rate of wound healing. Appreciable fibroblast activities with the indication of substantial
collagen deposition were observed in wounds treated with 0.625% w/w. An increase in collagenation and re-epithelialisation
showed a high rate of wound healing in the 2.5% w/w BME-treated wound tissues. Marked collagenation, re-epithelialisation
and angiogenesis was observd in the 10% w/w BME treated wound tissue and 1% w/w silver sulphadiazine- treated wound
tissues which indicated high wound healing rate.

Discussion

Various studies have indicated that medicinal plants are the backbone of traditional medicine [8,9,24,28]. Plants are
beneficial to humans in exhibiting activities such as antioxidant, antimicrobial, wound healing and anthelmintic activities.
This study investigated the methanol extract of the leaves of B. micrantha (BME) for its antimicrobial, antioxidants and
wound healing potential. Phytochemical screening shows that BME contains several secondary metabolites such as tannins,

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Fig. 1. DPPH free radical scavenging activity of BME and ascorbic acid.

Fig. 2. Effect of B. micrantha leaf extract on wound contraction. A: time-course curve; B: Area under the curve of the time-course curve; Ctrl: Control
[Vehicle (aqueous cream only) -treated group]; SS: silver sulfadiazine-trea (1 % w/w); Values are mean ± Standard error mean (SEM), n= 5; ∗ ∗ ∗ ∗ P < 0.0 0 01
(One – Way ANOVA followed by Dunnett’s post hoc test).

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Fig. 3. Histological images (x400) showing the influence of B. micrantha leaf extract on excised wound tissues. A - vehicle-treated (aqueous cream
only) wound tissues, B - 0.625% w/w treated wound tissues; D - 2.5% w/w treated wound tissues, F - 10% w/w treated wound tissues, G - 1 % w/w silver
sulphadiazine-treated wound tissues. CO: collagen strands, BV: blood vessels; FB: fibroblast.

saponins, glycosides, alkaloids, flavonoids, triterpenoids, steroids and coumarins. These phytochemicals could be responsible
for some of the biological activities of BME. For instance, flavonoids are known to decrease lipid peroxidation by stopping
or decelerating the onset of cell necrosis and improving vascularity. Flavonoids, and terpenoids are also reported to promote
wound healing primarily due to their astringent and antimicrobial property, which could be responsible for wound con-
traction and improved rate of epithelialization [29]. Tannins, the main components of many plant extracts, also act as free
radical scavengers [30]. The phytochemicals identified in B. micrantha could contribute to its antimicrobial, antioxidant and
wound healing activities.
The antimicrobial properties displayed by the BME may be associated with the synergistic activities or the actions of in-
dividual phytochemicals present in the leaves [31]. The antimicrobial activity of BME was concentration-dependent (Table 1).
This agrees with a study that found out that the methanol and ethyl acetate extracts of B. micrantha showed concentration-
dependent activity against Staphylococcus aureus and Salmonella typhi [32]. Other studies also report the antimicrobial activ-
ities of B. micrantha [33]. BME potentiated the antimicrobial activity of ciprofloxacin and ketoconazole against S. pyogenes, S.
typhi and C. albicans (Table 3). This finding is in agreement with a study that observed the drug-resistant modulation action
of B. micrantha [34]. The absence of antibacterial activity of ciprofloxacin and ketoconazole against E. coli and S. typhi in
presence of BME could be due to the fact that the extract antagonized the activity of the standard antibiotics.
It is reported that plants with good antimicrobial activity are likely to exhibit antioxidant activity as well [35]. The DPPH
free radical scavenging was used to determine the IC50 of BME because it is robust and gives highly reproducible results
[24]. BME demonstrated significant antioxidant activity in a concentration dependent manner (Fig. 1). The lower IC50 of

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BME shows that BME has better antioxidant property. The antioxidant potential of BME could be due to the presence of
flavonoids, and tannins present in it. Shelembe et al. [33] report that the higher antioxidant activity of the B. micrantha
could be attributed to the presence of the flavonoid quercetin-3-O-glucoside. Quercetin glycosides have been found to ex-
hibit excellent antioxidant activities against reactive oxygen species (ROS)-induced oxidative damage [36,37] . Therefore B.
micrantha could be beneficial to humans in minimizing oxidative stress.
Since BME showed good antimicrobial and antioxidant activity, the study further investigated its ability to heal wounds.
The wound healing activity of BME was investigated using the excision wound model in rats. The excised wounds were
treated with topical application of aqueous creams of BME for 12 days. Results obtained indicate that BME significantly (p
< 0.0 0 01) increased rate of wound closure compared to the vehichle-treated group (Fig. 2). The wound contraction process
produces a healed segment that is made up mostly of the dermis. This produces contractile force using sustained myosin AT-
Pase, thus causing myofibroblast cell contraction, generating thicker collagen fibres that condense granulation tissue, thereby
pulling surrounding skin into the segment [38].
There was faster wound healing activity, especially in the 10 % w/w BME-treated wounds. The accelerated wound healing
exhibited by BME confirms the report that methanol leaves extract of B. micrantha caused increased collagen synthesis and
cellular proliferation which was demonstrated by the increase in collagen content in tissues, enhanced rates of epithelializa-
tion and contraction of wounds [39]. Again, it is known that secondary metabolites such as flavonoids, tannins and saponins
exert their action by resembling endogenous metabolites, signal transduction molecules, or neurotransmitters, and lead to
useful effects on the wound healing process [40].
The marked reduction in wound area observed in BME-treated wounds was confirmed by histological studies (Fig. 3). The
histological examination of BME-treated wound tissues showed an increase in collagen content compared to the vehicle-
treated wound tissues. collagen, an extracellular protein compound plays a critical role in wound healing due to its move-
ment of cells such as fibroblasts which promotes tissue growth. Hence the presence of collagen greatly improves wound
healing [41]. Again, plant extracts are known to contain free radicals that protect the skin and stimulate the production of
collagen [42]. The low collagen content of in the excised wound tissues of the untreated group could be attributed to the
longer period of the inflammatory phase during which the breakdown of collagen increases as compared to the build-up of
collagen [41].

Conclusion

Methanol leaf extract of Bridelia micrantha (BME) showed a broad-spectrum antimicrobial activity. Sub-inhibitory con-
centrations of BME potentiated the activities of ciprofloxacin and ketoconazole. The BME at concentrations of 0.625, 2.5 and
10% w/w showed wound healing activity with marked angiogenesis, collagenation and re-epithelization. The phytochemicals
present in BME could be responsible for its antimicrobial, antioxidant and wound healing property. These findings support
the folkloric use of BME as a wound-healing agent.

Authors’ Contributions

William Gariba Akanwariwiak, Francis Adu, Yaw Duah Boakye and Christian Agyare participated in the conceptualization,
the design and supervision of the research study. Philip Asumang conducted all laboratory experiments with assistant from
Philomina Entsie. Theresah Appiah Agana and Yakubu Jibira assisted in the analysis and interpretation of experimental re-
sults. Philip Asumang, Theresa Appiah Agana, Yaw Duah Boakye and Francis Adu wrote the first draft of the manuscript. All
authors read and approved the final draft of the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

Acknowledgment

Authors are grateful to the Technicians at the Departments of Pharmaceutics and Pharmacology, FPPS, KNUST, Kumasi,
Ghana for their support.

Funding

This research did not receive any grant from any funding agencies.

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