Content uploaded by Daniel Gyamfi Amoako
Author content
All content in this area was uploaded by Daniel Gyamfi Amoako on Dec 19, 2023
Content may be subject to copyright.
Citation: Anokwah, D.;
Asante-Kwatia, E.; Asante, J.;
Obeng-Mensah, D.; Danquah, C.A.;
Amponsah, I.K.; Ameyaw, E.O.;
Biney, R.P.; Obese, E.; Oberer, L.; et al.
Antibacterial, Resistance Modulation,
Anti-Biofilm Formation, and Efflux
Pump Inhibition Properties of
Loeseneriella africana (Willd.) N. Halle
(Celastraceae) Stem Extract and Its
Constituents. Microorganisms 2024,12,
7. https://doi.org/10.3390/
microorganisms12010007
Academic Editor: Giuseppe
Mancuso
Received: 13 November 2023
Revised: 2 December 2023
Accepted: 13 December 2023
Published: 19 December 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
microorganisms
Article
Antibacterial, Resistance Modulation, Anti-Biofilm Formation,
and Efflux Pump Inhibition Properties of Loeseneriella africana
(Willd.) N. Halle (Celastraceae) Stem Extract and Its Constituents
Daniel Anokwah 1, * , Evelyn Asante-Kwatia 2, Jonathan Asante 1, Daniel Obeng-Mensah 1,
Cynthia Amaning Danquah 3, Isaac Kingsley Amponsah 2, Elvis Ofori Ameyaw 1, Robert Peter Biney 1,
Ernest Obese 1, Lukas Oberer 4, Daniel Gyamfi Amoako 5, Akebe Luther King Abia 5,6
and Abraham Yeboah Mensah 2
1School of Pharmacy and Pharmaceutical Sciences, College of Health and Allied Sciences,
University of Cape Coast, PMB, Cape Coast, Ghana; jonathan.asante@ucc.edu.gh (J.A.);
daniel.obeng-mensah@ucc.edu.gh (D.O.-M.); eameyaw@ucc.edu.gh (E.O.A.);
robert.biney@ucc.edu.gh (R.P.B.); ernest.obese@ucc.edu.gh (E.O.)
2Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah
University of Science and Technology, PMB, Kumasi, Ghana; eamireku@knust.edu.gh (E.A.-K.);
akila.amponsah@gmail.com (I.K.A.); aymensah@yahoo.com (A.Y.M.)
3Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah
University of Science and Technology, PMB, Kumasi, Ghana; cadanq@yahoo.com
4Novartis Institutes for BioMedical Research, CH-4056 Basel, Switzerland; lukas.oberer@bluewin.ch
5Antimicrobial Research Unit, College of Health Sciences, University of KwaZulu-Natal,
Durban 4001, South Africa; amoakodg@gmail.com (D.G.A.); lutherkinga@yahoo.fr (A.L.K.A.)
6Environmental Research Foundation, Westville 3630, South Africa
*Correspondence: daniel.anokwah@ucc.edu.gh
Abstract:
This study investigated the antibacterial, resistance modulation, biofilm inhibition, and
efflux pump inhibition potentials of Loeseneriella africana stem extract and its constituents. The antimi-
crobial activity was investigated by the high-throughput spot culture growth inhibition (HT-SPOTi)
and broth microdilution assays. The resistance modulation activity was investigated using the
anti-biofilm formation and efflux pump inhibition assays. Purification of the extract was carried
out by chromatographic methods, and the isolated compounds were characterized based on nuclear
magnetic resonance, Fourier transform infrared and mass spectrometry spectral data and comparison
with published literature. The whole extract, methanol, ethyl acetate, and pet-ether fractions of
L. africana all showed antibacterial activity against the test bacteria with MICs ranging from 62.5 to
500.0
µ
g/mL The whole extract demonstrated resistance modulation effect through strong biofilm
inhibition and efflux pump inhibition activities against S. aureus ATCC 25923, E. coli ATCC 25922
and P. aeruginosa ATCC 27853. Chromatographic fractionation of the ethyl acetate fraction resulted in
the isolation of a triterpenoid (4S,4
α
S,6
α
R,6
β
S,8
α
S,12
α
S,12
β
R,14
α
S,14
β
R)-4,4
α
,6
β
,8
α
,11,11,12
β
,14
α
-
Octamethyloctadecahydropicene-1,3(2H,4H)-dione) and a phytosterol (
β
-sitosterol). These com-
pounds showed antibacterial activity against susceptible bacteria at a MIC range of 31–125
µ
g/mL
and potentiated the antibacterial activity of amoxicillin (at
1
4
MIC of compounds) against E. coli and
P. aeruginosa with modulation factors of 32 and 10, respectively. These compounds also demonstrated
good anti-biofilm formation effect at a concentration range of 3–100
µ
g/mL, and bacterial efflux pump
inhibition activity at
1
2
MIC and
1
4
MIC against E. coli and P. aeruginosa.Loeseneriella africana stem bark
extracts and constituents elicit considerable antibacterial, resistance modulation, and biofilm and
efflux pump inhibition activities. The results justify the indigenous uses of L. africana for managing
microbial infections.
Keywords:
Loeseneriella africana; phytochemical screening; friedelane-1,3-dione;
β
-sitosterol; chromatography
Microorganisms 2024,12, 7. https://doi.org/10.3390/microorganisms12010007 https://www.mdpi.com/journal/microorganisms
Microorganisms 2024,12, 7 2 of 17
1. Introduction
The evolution of multidrug-resistant (MDR) bacteria remains a global health menace
due to the consequent increased morbidity and mortality [
1
]. Globally, the treatment of
infectious diseases with the currently available antibiotics is becoming increasingly difficult
due to the development of multidrug resistance mechanisms by bacteria [
2
]. Unfortu-
nately, this problem is exacerbated by the rapidly depleting pipeline of new antibiotics,
necessitating an urgent search for new antimicrobial agents [3].
Bacterial biofilm formation and the overexpression of efflux pumps have been shown
as major resistance mechanisms [
4
,
5
]. In comparison to their planktonic counterparts,
bacteria in biofilms are about 1000 times more resistant to antibiotics. The biofilm matrix
decreases permeability and allows type IV secretion systems (complex transmembrane
secretion proteins) of bacteria to mediate horizontal gene transfer, which facilitates antibiotic
resistance among bacteria through adaptation to environmental changes [
5
,
6
]. When
formed, the biofilm matrix facilitates the action of antibiotic-modifying enzymes, and
the expression of multidrug efflux pumps in the bacterial cells [
6
–
8
]. Overexpression
of efflux proteins by bacteria allows them to extrude several antibiotics to their exterior,
preventing antibiotics from reaching their therapeutic concentration, thereby rendering
them ineffective [
5
,
6
]. Therefore, developing medicines that prevent biofilm formation and
combine chemical efflux pump inhibitors with existing antibiotics is a potential strategy for
fighting antimicrobial resistance.
Medicinal plants have been demonstrated to be rich sources of secondary metabolites
with potential uses as therapeutic agents. These secondary metabolites possess diverse
chemical structures and mechanisms of action which may be valuable in developing new
treatment against resistant infections [
9
,
10
]. Furthermore, medicinal plants are widely used
in many communities due to their availability and favourable safety profile [11].
As part of a continuing effort to explore tropical medicinal plants as sources of an-
timicrobial agents, the antibacterial activity, anti-biofilm, efflux pump inhibitory and re-
sistance modulation potentials of the crude extract, fractions and some constituents of the
stem of Loeseneriella africana were investigated in this study. Loeseneriella africana (Willd.)
N. Halle is among 16 known species of the genus Loeseneriella of the family Celestraceae.Loe-
seneriella africana is a tough, flexible and durable liane or scandent shrub with widespread
distribution in West Africa, South Africa, Sri Lanka, India, Laos, and Myanmar [12,13].
Loeseneriella africana is used in African folklore medicine to treat inflammatory and
infectious diseases. In Ghana, the stem decoction is used for treating malaria, wounds,
oedema, menstrual pains, and in combination with other plants for infectious diseases and
hypertension [
14
]. Previous reports show that Loeseneriella africana and related species have
antidiabetic and hypolipidaemic, analgesic, anti-inflammatory, antipyretic, antioxidant,
antidiarrheal and antinuclear activities [
13
,
14
]. Previous phytochemical profiling of the
stem and leaves of L. africana has used HPLC-MS detected flavonoids including rutin,
p-coumaric acid, isoquercitrin, quercetin, quercitrin, ferulic acids and gentisic as bioactive
molecules, which could be responsible for the anti-inflammatory and antioxidant potentials
of the plant [
13
]. However, little is known about its antimicrobial properties, prompting
this study. Therefore, the objective of the study is to determine the antibacterial, resistance
modulation, biofilm inhibition, and efflux pump inhibition potential of L. africana stem and
its constituents as possible alternatives in mitigating microbial resistance through direct
antimicrobial activity or resistance modulation activity.
2. Materials and Methods
2.1. Drugs and Chemicals
Solvents were supplied by BDH Laboratory Supplies, London, UK. Silica gel
70–230 mesh and aluminum sheet TLC silica gel 60 F
254
were obtained from Merck KGaA
(Darmstadt, Germany), and amoxicillin was purchased from Phyto-Riker, Accra, Ghana.
Microorganisms 2024,12, 7 3 of 17
2.2. Bacterial Strains and Inoculum Standardization
Clinical bacterial strains (Streptococcus pyogenes,Vibrio cholerae,Salmonella typhi,Kleb-
siella pneumoniae) were obtained from the pharmaceutical microbiology laboratory, Kwame
Nkrumah University of Science and Technology (KNUST). Also, American type culture
collection (ATCC) bacteria strains (Enterococcus faecalis ATCC 29212, Escherichia coli ATCC
25922, Staphylococcus aureus ATCC 25923, Proteus mirabilis ATCC 12453 and Pseudomonas
aeruginosa ATCC 27853) were supplied by the Department of Pharmacology, KNUST,
Kumasi, Ghana.
Overnight broth cultures of test organisms were used to create a standardized bac-
teria culture. The organisms were standardized through serial dilution in sterile nor-
mal saline (0.9 percent NaCl, w/v) to achieve about 1.5
×
10
8
CFU/mL (equivalent to
0.5 McFarland standard).
2.3. General Experimental Procedures
Column chromatography (CC) was carried out using silica gel 60 (70
−
230 mesh).
Thin layer chromatography (TLC) was conducted using pre-coated silica gel 60 plates
(0.25 mm). Separated compounds were visualized by spraying with vanillin-sulphuric
acid spray reagent followed by heating. Mass spectrometry on isolated compounds was
carried out with high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).
The instrument used was Bruker MaXis 288882.20181 (LC-QTOF) supplied by the Bruker
Corporation, Billerica, MA, USA. Nuclear magnetic resonance (NMR) data were recorded
on a Bruker 500 MHz Advance III HD NMR spectrometer (Bruker Corporation, Billerica,
MA, USA), operating at 500 MHz (
1
H) and 125 MHz (
13
C) with deuterated methanol
(CD
3
OD) as a solvent. The chemical shifts were expressed as parts per million (ppm)
with tetramethylsilane (TMS) as the internal standard and the coupling constants (J) were
measured in hertz (Hz). Functional groups in the isolated compounds were identified using
the Fourier transform-infrared spectrometry (FT-IR) performed on a Perkin Elmer device
(UATR Spectrum Two Series) to obtain infrared (IR) spectra over a 4000–350 cm
−1
wave
number range.
2.4. Plant Material Collection
The stem of L. africana was harvested from the wild at Kwahu Asakraka village in
the eastern region of Ghana (06
◦
37.356
0
N/000
◦
41.396
0
W) in November 2017. The plant
material was authenticated by Mr. Clifford Asare of the Department of Herbal Medicine,
KNUST, Kumasi, Ghana, and a voucher specimen (KNUST/HM/2017/SB017) was reserved
in the department’s herbarium.
2.5. Preliminary Phytochemical Screening
Secondary metabolites such as tannins, flavonoids, triterpenoids, and glycosides were
detected in the L. africana stem powder using simple qualitative phytochemical screening
methods [15].
2.6. Preparation of Extracts and Fractions
After washing under running water, the stem was cut into smaller sizes suitable for
milling, and air-dried for 10 days. The dried plant material was powdered with a hammer
mill into coarse powder of about 3 kg and was extracted with methanol for 6 h using
a Soxhlet extractor. The mother liquor was concentrated on a rotavapor under reduced
pressure and further dried at 65
◦
C overnight to obtain a brown solid extract (6.4% yield)
referred to as LA or “the whole extract” in this report. About 120 g of LA was adsorbed
onto silica gel (70–230 mesh size) successively partitioned with petroleum ether (pet-ether),
ethyl acetate (EtOAc) and methanol (MeOH) to afford pet-ether (LAPE, 7.3 g), EtOAc (LAEt,
27.9 g) and MeOH (LAM, 82.6 g) fractions. A desiccator was used to store the extract and
fractions until required for use.
Microorganisms 2024,12, 7 4 of 17
2.7. Isolation and Characterization of Phytoconstituents
About 25 g of the ethyl acetate fraction (LAEt) was mounted and purified by column
chromatography (CC) [
16
]. Ethyl acetate was used to reconstitute the extract, adsorbed
onto silica gel 60, and dry-packed onto the stationary phase (silica gel, 70
−
230 mesh).
The solvents (pet-ether, EtOAc and MeOH) were used as the mobile phase by gradient
elution and the eluates were monitored using TLC. Two compounds were obtained and
characterized by comparing their
1
H,
13
C NMR and mass spectral data with published
data (Figure 1). Details of the isolation and characterization are presented in Figure S1
(Supplementary Materials).
Microorganisms 2023, 11, x FOR PEER REVIEW 4 of 17
Soxhlet extractor. The mother liquor was concentrated on a rotavapor under reduced pres-
sure and further dried at 65 °C overnight to obtain a brown solid extract (6.4% yield) re-
ferred to as LA or “the whole extract” in this report. About 120 g of LA was adsorbed onto
silica gel (70–230 mesh size) successively partitioned with petroleum ether (pet-ether),
ethyl acetate (EtOAc) and methanol (MeOH) to afford pet-ether (LAPE, 7.3 g), EtOAc
(LAEt, 27.9 g) and MeOH (LAM, 82.6 g) fractions. A desiccator was used to store the ex-
tract and fractions until required for use.
2.7. Isolation and Characterization of Phytoconstituents
About 25 g of the ethyl acetate fraction (LAEt) was mounted and purified by column
chromatography (CC) [16]. Ethyl acetate was used to reconstitute the extract, adsorbed
onto silica gel 60, and dry-packed onto the stationary phase (silica gel, 70−230 mesh). The
solvents (pet-ether, EtOAc and MeOH) were used as the mobile phase by gradient elution
and the eluates were monitored using TLC. Two compounds were obtained and charac-
terized by comparing their
1
H,
13
C NMR and mass spectral data with published data (Fig-
ure 1). Details of the isolation and characterization are presented in Figure S1 (Supple-
mentary Materials).
Figure 1. Isolated compounds from the stem bark of Loesenoriella africana.
2.8. Antimicrobial Testing
2.8.1. Evaluation of Antibacterial Activity of Crude Extracts and Fractions
The crude MeOH extract and major fractions were tested for antibacterial activity
using the high-throughput spot culture growth inhibition assay (HT-SPOTi) as previously
described [17]. Briefly, a twofold serial dilution of a stock solution of the extracts, prepared
in dimethyl sulphoxide (2% DMSO), was carried out in a PCR half-skirted plate to give a
concentration range of 0.49–500.00 µg/mL. Then, 2 µL of each dilution was alloed into
corresponding wells of a 96-well plate. After that, 200 µL of molten agar was added to
wells containing the test samples and swirled to mix thoroughly. After seing, the wells
were spoed with 2 µL of standardized suspension of the test microorganisms and al-
lowed to stand for 20 min to enable diffusion of the test sample into the agar. Wells con-
taining only media and bacteria were used as a negative control and wells containing me-
dia, bacteria and amoxicillin were used as a positive control. The plates were sealed and
incubated at 37 °C for 24 h. Visual examination for comparison with the control wells was
used to determine the presence or absence of growth.
Figure 1. Isolated compounds from the stem bark of Loesenoriella africana.
2.8. Antimicrobial Testing
2.8.1. Evaluation of Antibacterial Activity of Crude Extracts and Fractions
The crude MeOH extract and major fractions were tested for antibacterial activity
using the high-throughput spot culture growth inhibition assay (HT-SPOTi) as previously
described [
17
]. Briefly, a twofold serial dilution of a stock solution of the extracts, prepared
in dimethyl sulphoxide (2% DMSO), was carried out in a PCR half-skirted plate to give a
concentration range of 0.49–500.00
µ
g/mL. Then, 2
µ
L of each dilution was allotted into
corresponding wells of a 96-well plate. After that, 200
µ
L of molten agar was added to wells
containing the test samples and swirled to mix thoroughly. After setting, the wells were
spotted with 2
µ
L of standardized suspension of the test microorganisms and allowed to
stand for 20 min to enable diffusion of the test sample into the agar. Wells containing only
media and bacteria were used as a negative control and wells containing media, bacteria
and amoxicillin were used as a positive control. The plates were sealed and incubated
at 37
◦
C for 24 h. Visual examination for comparison with the control wells was used to
determine the presence or absence of growth.
2.8.2. Determination of the Antibacterial Activity and Antibiotic Modulation Effect of
Isolated Compounds
The isolated compounds were investigated for their antibacterial activity by the broth
microdilution method against selected Gram-negative (P. aeruginosa ATCC 27853 and E. coli
ATCC 25922) and Gram-positive (S. aureus ATCC 25923 and E. faecalis ATCC 29212) bacteria
according to the World Health Organization (WHO) priority [
18
]. Briefly, respective wells
of a 96-well microplate were filled with 100
µ
L of nutrient broth (NB) and corresponding
volumes of compounds reconstituted in DMSO (2%) to obtain a concentration range be-
tween 7.8 and 500.0
µ
g/mL. Then, 20
µ
L of test organisms (approximately 10
8
CFU/mL)
were inoculated and the plates incubated at 37
◦
C for 24 h. The presence or absence of
growth was determined using the visual colour change of 3-(4,5-dimethylthiazol-2-yl)-2,5-
Microorganisms 2024,12, 7 5 of 17
diphenyl-2H-tetrazolium bromide (MTT) (0.125% w/vthiazoyl blue tetrazolium bromide)
within 30 min of addition to the wells.
For the resistance modulating test, the MIC of the standard drug (amoxicillin) was
determined in the presence of 1/4 MICs of the phytoconstituents against E. coli and
P. aeruginosa. The resistance modulation activity of the phytoconstituent on the MIC
of the standard drug was determined by the modulation factor (MF). The estimation was
done using the formula: MF = MIC (antibiotic)/MIC (antibiotic + modulator) [19].
2.8.3. Biofilm Inhibition Assay
The whole extract (LA) and the two compounds were screened for their effect on the
biofilm formation by E. coli ATCC 25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 25923
using the crystal violet retention method as previously described [
16
,
20
]. Briefly, 180
µ
L of
microorganisms freshly cultured in tryptone soy broth (TSB) was pipetted into corresponding
wells of a flat-bottom 96-well polystyrene microtiter plate. Concurrently, 20
µ
L of test extract
(15–500
µ
g/mL) or compounds (3.1–100.0
µ
g/mL) prepared in TSB was added and the plates
incubated at 37
◦
C for 24 h (subsequently referred to as the test). Wells containing only media
and bacteria were used as control wells for the assay. The experiment was conducted in
quadruplicate. After 24 h of incubation, the supernatant was aspirated, and the planktonic
cells were washed off with phosphate buffer saline (PBS, pH 7.2). The adherent biofilms
were fixed by drying the plates in the incubator at 50
◦
C for 30 min. The biofilms were then
stained with 200
µ
L of 0.1% w/vcrystal violet
(aq)
(CV) dye for 10 min at 26
◦
C. Excess dye
was aspirated and the wells were gently washed three times with sterile water for injection.
Finally, the stain bound to the biofilms in each well was solubilized with 200
µ
L of 95%
ethanol and allowed to stand for 10 min. The differential staining absorbance was measured
at 600 nm using a microtiter plate reader (Biotek Synergy H1 Hybrid Multi-Mode Reader:
271230 supplied by Agilent Technologies, Santa Clara, CA, USA). The mean absorbance of
the samples was determined, and the percentage inhibition of biofilm was calculated as:
Percentage biofilm inhibition (%) = [(control OD600 −Test OD600)/control OD600]×100.
2.8.4. Efflux Pump Inhibition Assay
The extract (LA) and compounds were investigated for their effect on the efflux
pump activity of bacterial cells according to previously published protocols [
19
,
21
]. The
organisms (E. coli ATCC 25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 25923) were
grown to the mid-log phase in the nutrient broth (OD
600
adjusted to 0.4) and collected
by centrifugation (3000 rpm for 15 min at 4
◦
C). The supernatant was discarded and the
cells were re-suspended twice in sterile PBS at pH 7.4 and diluted to a final OD600 of 0.5.
Aliquots of the bacterial cell suspension in PBS were added to filter sterilized glucose
(mixed to a final concentration of 0.4% v/v) and pipetted into corresponding wells of a
96-well microtiter plate. The plates were vortexed at room temperature to distribute the cells
uniformly. Ethidium bromide (EtBr) was added to a final concentration of 0.5 mg/L and
different volumes of the extract/compound (1/2 or 1/4 MIC) were added to corresponding
wells and the fluorescence was measured over 60 min at 3 min intervals using excitation
and emission wavelengths of 530 nm and 585 nm, respectively. Chlorpromazine and
verapamil, known efflux pump inhibitors (EPI), were included as comparative probes and
a drug-free culture as the negative control. The ability of the test substance to enhance
the accumulation of EtBr (a substrate for efflux pumps) is considered as an efflux pump
inhibition effect [18].
3. Results
3.1. Preliminary Phytochemical Investigation of the Stem of L. africana
Preliminary phytochemical analyses of the dried powdered L. africana stem confirmed
the existence of many secondary metabolite groups, including triterpenoids and phytos-
terols (Table 1).
Microorganisms 2024,12, 7 6 of 17
Table 1. Phytochemical screening of the stem of L. africana.
Secondary Metabolite Result
Reducing sugars +
Tannins +
Flavonoids +
Coumarins +
Triterpenoids +
Phytosterols +
Saponins −
Alkaloids +
+: detected; −: not detected.
3.2. Isolated Compounds from the Stem of L. africana
Chromatographic fractionation and purification of the bioactive EtOAc fraction led to
the isolation of two compounds based on their
1
H and
13
C NMR, MS and FTIR data. The
white amorphous crystals (compound
LA1
) were identified to be friedelane-1,3-dione triter-
penoid, characterized as 4S,4
α
S,6
α
R,6
β
S,8
α
S,12
α
S,12
β
R,14
α
S,14
β
R-4,4
α
,6
β
,8
α
,11,11,12
β
,
14
α
Octamethyloctadecahydropicene-1,3(2H,4H)-dione. The white amorphous powder
(compound
LA2
) was identified as a phytosterol, characterized as
β
-sitosterol (Figure 1).
The
1
H NMR (600 MHz in CDCl
3
) and
13
C (125 MHz in CDCl
3
) NMR data are shown in
Table 2. Further spectral data and some physicochemical constants of the compounds are
available in the Supplementary Materials, Figures S2–S4.
Table 2. 1H and 13C NMR data for compounds LA1 and LA2.
Position LA1 LA2
δCδH (J in Hz)δCδH (J in Hz)
1 202.8 (C) - 37.2 (CH2) 1.81
2 60.6 (CH2)3.21, d(15.9 Hz)
3.43, d(15.9 Hz) 31.6 (CH2) 1.81
3 204.2 (C) - 71.8 (CH) 3.50
4 59.0 (CH) 2.55, q(6.7 Hz) 42.2 (CH2) 2.21, 2.27
5 37.8 (C) - 140.7 (C) -
6 40.5 (CH2)1.35, m
1.86 m(12. 5, 6.8, 3.2 Hz) 121.7 (CH) 5.33, d(4.7 Hz)
7 18.0 (CH2) 1.42, 1.51 31.9 (CH2) 1.55, 1.56
8 52.1 (CH) 1.22 31.9 (CH) 1.42
9 37.13 (C) - 50.1 (CH) 0.89
10 71.79 (CH) 2.35, s36.5 (C) -
11 34.5 (CH2)1.12, m
2.12, m(13.6, 6.8, 3.5 Hz) 21.0 (CH2) 1.44, 1.47
12 30.1 (CH2) 1.26, 1.38 39.7 (CH2) 1.13, 1.99
13 39.4 (C) - 42.2 (C) -
14 38.2 (C) - 56.7 (CH) 0.96
15 32.3 (CH2) 1.50, m24.3 (CH2) 1.03, 1.56
16 35.8 (CH2) 1.33, 1.52 28.2 (CH2) 1.26, 1.81
17 30.0 (C) - 56.0 (CH) 1.08
18 42.6 (CH) 1.54, m11.8 (CH3) 0.65 s
19 35.2 (CH2) 1.32, m19.4 (CH3) 0.98, s
20 28.1 (C) - 36.1 (CH) 1.32, m
21 32.7 (CH2) 1.24, m18.7 (CH3) 0.90, d(6.5 Hz)
22 39.2 (CH2) 0.90, 1.47 33.9 (CH2) 0.97, 1.30, dd (8.4, 15.0 Hz)
23 7.3 (CH3) 1.02, d(6.7 Hz) 26.0 (CH2) 1.13, dd (8.4, 15.0 Hz)
Microorganisms 2024,12, 7 7 of 17
Table 2. Cont.
Position LA1 LA2
δCδH (J in Hz)δCδH (J in Hz)
24 15.9 (CH3) 0.66, s45.8 (CH) 0.90
25 18.0 (CH3) 1.17, s29.1 (CH) 1.65
26 20.3 (CH3) 1.00, s19.0 (CH3) 0.81, d(2.5 Hz)
27 18.7 (CH3) 0.99, s19.8 (CH3) 0.79, d(6.8 Hz)
28 32.0 (CH3) 1.15, s23.0 (CH2) 1.21, 1.25
29 31.7 (CH3) 0.97, s12.0 (CH3) 0.82, t(6.0 Hz)
30 35.0 (CH3) 0.91, s22.3 (CH2) 0.84, m
3.3. Antibacterial Activity of L. africana Stem Extract, Major Fractions and Isolated Compounds
In the HT-SPOTi assay, the methanol-chloroform (4:1) whole extract, petroleum ether,
ethyl acetate, and methanol fractions inhibited growth of Gram-positive and Gram-negative
bacteria to varying degrees. The MIC for susceptible bacteria ranged between 125 and
500
µ
g/mL depending on the bacteria and solvent extract (Table 3). The MICs of the
extracted compounds from L. africana stem are shown in Table 4.
Table 3.
MICs of L. africana stem extract and fractions against clinically significant bacteria in HT-
SPOTi assay.
Microorganism Minimum Inhibitory Concentration (µg/mL)
LA LAPE LAEt LAM Amox
S. aureus 125 500 >500 250 3.91
S. pyogenes 500 >500 >500 >500 1.95
E. faecalis 125 >500 250 >500 0.49
P. aeruginosa 500 >500 >500 >500 500
P. mirabilis >500 >500 250 >500 31.25
K. pneumoniae 125 >500 250 >500 31.25
S. typhi 500 >500 500 >500 62.50
E. coli 500 >500 >500 >500 125
V. cholerae 125 500 250 125 125
Key: LA—whole extract, LAPE—Pet. ether fraction, LAEt—Ethyl acetate fraction, LAM—Methanol fraction, and
Amox—Amoxicillin.
Table 4. MICs of isolated compounds from L. africana stem extract.
Microorganism Minimum Inhibitory Concentration (µg/mL)
LA1 LA2 Amoxicillin
S. aureus 31.25 31.25 10
E. faecalis 31.25 31.25 10
E. coli 62.5 125 20
P. aeruginosa 62.5 125 >320
3.4. Antibiotic Modulation Effect of Isolated Compounds from L. africana Stem
The isolated compounds were tested against E. coli and P. aeruginosa for an antibacterial
resistance modulatory effect with amoxicillin at sub-inhibitory concentrations (1/4 MIC). In
the broth dilution assay (Table 5), the MIC values of amoxicillin against E. coli (20
µ
g/mL)
and P. aeruginosa (>320
µ
g/mL) were higher than the clinical breakpoint (8
µ
g/mL), indi-
cating possible resistance.
Microorganisms 2024,12, 7 8 of 17
Table 5.
Minimum inhibitory concentration (MIC) of amoxicillin in the absence or presence of
compounds at 1
4MIC concentration.
Microorganism MIC (µg/mL) MIC Combined (µg/mL) Modulation Factor
Amoxicillin Only LA1 LA2 LA1 LA2
P. aeruginosa >320 <31.25 <31.25 >10 >10
E. coli 20 <0.625 <0.625 >32 >32
3.5. Biofilm Inhibitory Effect of L. africana Stem Extract and Isolated Compounds
The extract inhibited biofilm formation in a concentration-dependent manner be-
tween 15.6 and 500.0
µ
g/mL. The strongest biofilm inhibition effect was observed against
E. coli, followed by S. aureus and P. aeruginosa (Figure 2). The percentage biofilm inhi-
bition ranged between 40 and 59% for S. aureus at 15.0–62.5
µ
g/mL, and 49–77% for
E. coli at 15–250
µ
g/mL. For P. aeruginosa, the percentage biofilm inhibition ranged between
15 and 56% at a sub-inhibitory concentration of 62.5–250
µ
g/mL whereas at concentrations
between 15 and 31 µg/mL, LA enhanced the biofilm-forming capacity of P. aeruginosa.
Microorganisms 2023, 11, x FOR PEER REVIEW 8 of 17
E. coli 62.5 125 20
P. aeruginosa 62.5 125 >320
3.4. Antibiotic Modulation Effect of Isolated Compounds from L. africana Stem
The isolated compounds were tested against E. coli and P. aeruginosa for an antibac-
terial resistance modulatory effect with amoxicillin at sub-inhibitory concentrations (1/4
MIC). In the broth dilution assay (Table 5), the MIC values of amoxicillin against E. coli (20
µg/mL) and P. aeruginosa (>320 µg/mL) were higher than the clinical breakpoint (8 µg/mL),
indicating possible resistance.
Table 5. Minimum inhibitory concentration (MIC) of amoxicillin in the absence or presence of com-
pounds at ¼ MIC concentration.
Microorganism MIC (µg/mL) MIC Combined (µg/mL) Modulation Factor
Amoxicillin Only LA1 LA2 LA1 LA2
P. aeruginosa >320 <31.25 <31.25 >10 >10
E. coli 20 <0.625 <0.625 >32 >32
3.5. Biofilm Inhibitory Effect of L. africana Stem Extract and Isolated Compounds
The extract inhibited biofilm formation in a concentration-dependent manner be-
tween 15.6 and 500.0 µg/mL. The strongest biofilm inhibition effect was observed against
E. coli, followed by S. aureus and P. aeruginosa (Figure 2). The percentage biofilm inhibition
ranged between 40 and 59% for S. aureus at 15.0–62.5 µg/mL, and 49–77% for E. coli at 15–
250 µg/mL. For P. aeruginosa, the percentage biofilm inhibition ranged between 15 and
56% at a sub-inhibitory concentration of 62.5–250 µg/mL whereas at concentrations be-
tween 15 and 31 µg/mL, LA enhanced the biofilm-forming capacity of P. aeruginosa.
Figure 2. Biofilm formation inhibitory effect of the stem extract of L. africana against S. aureus ATCC
25923, E. coli ATCC 29212, and P. aeruginosa ATCC 27853 expressed as percentage biofilm inhibition;
values recorded as mean ± SEM (n = 3); p < 0.05.
The effect of triterpenoid compounds LA1 and LA2 (3.1–100.0 µg/mL) on biofilm for-
mation in S. aureus, E. coli and P. aeruginosa is presented on Figure 3. LA1 showed good
antibiofilm activity against S. aureus (54–65%) and E. coli (47–74%) whereas it showed low
Figure 2.
Biofilm formation inhibitory effect of the stem extract of L. africana against S. aureus ATCC
25923, E. coli ATCC 29212, and P. aeruginosa ATCC 27853 expressed as percentage biofilm inhibition;
values recorded as mean ±SEM (n= 3); p< 0.05.
The effect of triterpenoid compounds
LA1
and
LA2
(3.1–100.0
µ
g/mL) on biofilm
formation in S. aureus,E. coli and P. aeruginosa is presented on Figure 3.
LA1
showed good
antibiofilm activity against S. aureus (54–65%) and E. coli (47–74%) whereas it showed low
to good activity against P. aeruginosa (42–57%).
LA2
demonstrated good antibiofilm activity
against S. aureus (56–72%), E. coli (60–65%) and P. aeruginosa (50–65%).
3.6. Effect of L. africana Stem Extract and Isolated Compounds on Ethidium Bacterial Efflux Pump
Figure 4depicts the accumulation behavior of the extract (LA) in S. aureus,E. coli,
and P. aeruginosa over 60 min compared to the two standard EPIs, verapamil (VP) and
chlorpromazine (CP). According to the results, the crude extract (LA) inhibited efflux
pumps, resulting in EtBr fluorescence, whereas both standard EPIs were more effective in
causing higher EtBr accumulation in the bacterial cells (S. aureus,E. coli, and P. aeruginosa)
than the test extract measured over 60 min (Figure 4A–C).
Microorganisms 2024,12, 7 9 of 17
Microorganisms 2023, 11, x FOR PEER REVIEW 9 of 17
to good activity against P. aeruginosa (42–57%). LA2 demonstrated good antibiofilm activ-
ity against S. aureus (56–72%), E. coli (60–65%) and P. aeruginosa (50–65%).
Figure 3. Biofilm formation inhibitory effect of isolated compounds LA1 and LA2 on bacteria biofilm
formation in S. aureus ATCC 25923, E. coli ATCC 29212, and P. aeruginosa ATCC 27853 expressed as
percentage biofilm inhibition; values recorded as mean ± SEM (n = 3); p < 0.05.
3.6. Effect of L. africana Stem Extract and Isolated Compounds on Ethidium Bacterial
Efflux Pump
Figure 4 depicts the accumulation behavior of the extract (LA) in S. aureus, E. coli, and
P. aeruginosa over 60 min compared to the two standard EPIs, verapamil (VP) and chlor-
promazine (CP). According to the results, the crude extract (LA) inhibited efflux pumps,
resulting in EtBr fluorescence, whereas both standard EPIs were more effective in causing
Figure 3.
Biofilm formation inhibitory effect of isolated compounds
LA1
and
LA2
on bacteria biofilm
formation in S. aureus ATCC 25923, E. coli ATCC 29212, and P. aeruginosa ATCC 27853 expressed as
percentage biofilm inhibition; values recorded as mean ±SEM (n= 3); p< 0.05.
Microorganisms 2024,12, 7 10 of 17
Microorganisms 2023, 11, x FOR PEER REVIEW 10 of 17
higher EtBr accumulation in the bacterial cells (S. aureus, E. coli, and P. aeruginosa) than the
test extract measured over 60 min (Figure 4A–C).
LA1 and LA2 acted on efflux pumps by increasing ethidium bromide accumulation
in E. coli (Figure 5A,B) and P. aeruginosa (Figure 6A,B).
Figure 4. Effect of L. africana whole extract (LA) at ½ MIC concentration on EtBr accumulation in S.
aureus (A), E. coli (B) and P. aeruginosa (C).
Figure 4.
Effect of L. africana whole extract (LA) at
1
2
MIC concentration on EtBr accumulation in
S. aureus (A), E. coli (B) and P. aeruginosa (C).
LA1
and
LA2
acted on efflux pumps by increasing ethidium bromide accumulation in
E. coli (Figure 5A,B) and P. aeruginosa (Figure 6A,B).
Microorganisms 2024,12, 7 11 of 17
Microorganisms 2023, 11, x FOR PEER REVIEW 11 of 17
Figure 5. The effect of compounds LA1 (A) and LA2 (B) at ½ and ¼ MICs on the intracellular accu-
mulation of EtBr in E. coli.
Figure 5.
The effect of compounds
LA1
(
A
) and
LA2
(
B
) at
1
2
and
1
4
MICs on the intracellular
accumulation of EtBr in E. coli.
The efflux pump inhibition activity of the compounds against E. coli (Table 6) and
P. aeruginosa (Table 7) was calculated every 15 min as a percentage inhibition over the
negative control and compared to the standard drugs (verapamil and chlorpromazine).
Values lower than 50% were considered low efflux pump inhibition activity (a), values
between 50 and 100% were considered good efflux pump inhibition activity (b), whereas
values above 100% were considered very good efflux pump inhibition activity (c). The
result revealed that in E. coli (at
1
4
MIC), compounds
LA1
and
LA2
had good to very
good efflux pump inhibition activity with percentage inhibition of 87–111% and 86–110%,
respectively (Table 6). In P. aeruginosa (both 1/4 and 1/2 MICs.), both compounds showed
low efflux pump inhibition activity with values less than 50% (Table 7).
Microorganisms 2024,12, 7 12 of 17
Microorganisms 2023, 11, x FOR PEER REVIEW 12 of 17
Figure 6. The effect of compounds LA1 (A) and LA2 (B) at ½ and 1/4 MICs on the intracellular
accumulation of EtBr in P. aeruginosa.
The efflux pump inhibition activity of the compounds against E. coli (Table 6) and P.
aeruginosa (Table 7) was calculated every 15 min as a percentage inhibition over the nega-
tive control and compared to the standard drugs (verapamil and chlorpromazine). Values
lower than 50% were considered low efflux pump inhibition activity (a), values between
50 and 100% were considered good efflux pump inhibition activity (b), whereas values
above 100% were considered very good efflux pump inhibition activity (c). The result re-
vealed that in E. coli (at ¼ MIC), compounds LA1 and LA2 had good to very good efflux
pump inhibition activity with percentage inhibition of 87–111% and 86–110%, respectively
(Table 6). In P. aeruginosa (both 1/4 and 1/2 MICs.), both compounds showed low efflux
pump inhibition activity with values less than 50% (Table 7).
Figure 6.
The effect of compounds
LA1
(
A
) and
LA2
(
B
) at
1
2
and 1/4 MICs on the intracellular
accumulation of EtBr in P. aeruginosa.
Table 6.
Effect of L. africana compounds on E. coli ATCC 25922 efflux pump activity expressed as
a percentage.
MIC Compound % Inhibition of Efflux Pump Activity
15 min 30 min 45 min 60 min
1/4 LA1 111.0 ±19.03 c108.4 ±15.43 c100.0 ±11.05 c87.1 ±12.47 b
LA2LA2 109.6 ±12.89 c105.4 ±12.17 c95.4 ±12.82 c86.3 ±9.34 b
1/2
LA1 59.5 ±11.35 b45.3 ±8.01 a34.6 ±5.00 a29.1 ±1.78 a
LA2 61.3 ±12.55 b50.8 ±6.35 b39.2 ±12.55 a30.7 ±7.10 a
Verapamil 60.3 ±5.41 b120.3 ±6.82 c125.8 ±7.23 c113.4 ±8.29 c
Chlorpromazine 95.6 ±7.91 b97.9 ±15.04 b78.64 ±5.78 b71.9 ±4.49 b
Key: superscripts (a, b, and c) represent low, good, and very good efflux pump inhibition activity, respectively,
expressed over the negative control. Data presented as mean ±SEM (n= 3); p< 0.0001.
Microorganisms 2024,12, 7 13 of 17
Table 7.
Effect of L. africana compounds on P. aeruginosa ATCC 27853 efflux pump activity expressed
as a percentage.
MIC Compound % Inhibition of Efflux Pump Activity
15 min 30 min 45 min 60 min
1/4 LA1 35.3 ±26.88 a18.1 ±10.87 a6.7 ±10.69 a14.5 ±14.21 a
LA2 6.7 ±5.34 a2.2 ±7.66 a0.5 ±8.55 a8.7 ±5.27 a
1/2
LA1 41.7 ±10.06 a19.3 ±11.08 a13.22 ±9.91 a13.4 ±0.28 a
LA2 30.6 ±11.04 a26.2 ±8.94 a29.2 ±11.20 a37.1 ±9.67 a
Verapamil 147.3 ±28.78 c145.5 ±17.35 c129.3 ±7.34 c128.4 ±10.89 c
Chlorpromazine 105.4 ±9.02 c107.6 ±9.03 c86.0 ±3.86 b85.9 ±9.91 b
Key: superscripts (a, b, and c) represent low, good, and very good efflux pump inhibition activity, respectively,
expressed over the negative control. Data presented as mean ±SEM (n= 3); p< 0.0001.
4. Discussion
The antimicrobial activity of the methanol-chloroform (4:1) extract of L. africana stem,
three solvent fractions (petroleum ether, ethyl acetate, and methanol fractions), and some
isolated phytoconstituents from the stem were investigated in this study. The effects of
the crude extract and isolated compounds on bacterial biofilm formation and efflux pump
activity were also studied. The resistance modulation effect of compounds on amoxicillin
against E. coli and P. aeruginosa was also investigated.
Secondary metabolites, including those detected in this study, have been shown to
have several biological actions, including antibacterial, anti-inflammatory, and antioxidant
properties [
17
]. Terpenoids, phytosterols, and alkaloids have been reported to have antimi-
crobial, resistance modulation, antibiofilm, and efflux pump inhibition activities [
17
,
22
].
Tannins, flavonoids, and terpenoids have demonstrated antimicrobial, anti-inflammatory
and antioxidant activities [
22
,
23
]. The antimicrobial effect of these secondary metabolites is
elicited through various mechanisms. Phytochemicals such as terpenoids and phytosterols
disrupt bacterial cell membrane through lipophilic action that leads to the disturbance of
membrane-embedded proteins including efflux proteins, an increase in membrane perme-
ability and fluidity and alteration of ion transport processes in both Gram-positive and
Gram-negative bacteria [
23
]. Polyphenols such as flavonoids and tannins inhibit DNA
and RNA synthesis in bacterial cells [
23
]. Therefore, the secondary metabolites found in
L. africana may explain the traditional applications of the plant for treating a variety of
ailments, including inflammatory disorders and infections.
The extracts and compounds demonstrated considerable antibacterial activities. Some
authors [
22
–
24
] have proposed criteria for classifying the level of antimicrobial activity of
plant extracts. Kuete et al. [
22
] classify the antimicrobial activity of plant extracts as significant
(MIC < 100
µ
g/mL), moderate (100
≤
MIC
≤
625
µ
g/mL) or weak (MIC > 625
µ
g/mL).
According to this criterion, the whole extract and fractions had moderate antibacterial activities.
Generally, the extract and fractions had moderate antibacterial activities (MIC
≤
500) against
most of the organisms tested (Table 2). At MICs of 125
µ
g/mL, the whole extract showed
activity against S. aureus ATCC 25923, E. faecalis ATCC 29212, K. pneumoniae (clinical strain),
and V. cholerae (clinical strain). At MICs of 500
µ
g/mL, the whole extract showed activity
against E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. typhi (clinical strain), and S. pyogenes
(clinical strain). The ethyl acetate fraction showed activity against E. faecalis ATCC 29212,
K. pneumoniae (clinical strain), V. cholerae (clinical strain), and P. mirabilis ATCC 12453 at MICs
of 250
µ
g/mL and against S. typhi (clinical strain) at 500
µ
g/mL. At MICs of 125
µ
g/mL and
250
µ
g/mL, the methanol fraction showed activity against V. cholerae (clinical strain) and
S. aureus ATCC 25923, respectively. At MICs of 500
µ
g/mL, the petroleum ether fraction
showed activity against V. cholerae (clinical strain) and S. aureus ATCC 25923. As a result,
among the L. africana extract fractions, the ethyl acetate fraction demonstrated the most
significant antimicrobial activity. However, the whole extract outperformed its fractions in
terms of antimicrobial activity.
Microorganisms 2024,12, 7 14 of 17
In the broth dilution assay, the compounds isolated from the stem of L. africana were tested
for antimicrobial activity against Gram-positives (S. aureus and E. faecalis) and Gram-negatives
(P. aeruginosa and E. coli). The compounds (
LA1
and
LA2
) showed antibacterial activity at
MICs ranging from 31 to 125 g/mL (Table 4). The lower MICs of the isolated compounds
compared with those of the parent fraction suggests that fractionation produced more active
samples. The antimicrobial activity for pure compounds may be classified as significant
(MIC < 10
µ
g/mL), moderate (10
≤
MIC
≤
100
µ
g/mL), or weak (MIC > 100
µ
g/mL) [
22
].
The friedelane triterpenoid (
LA1
) demonstrated moderate antibacterial activity whereas
the phytosterol,
β
-sitosterol (
LA2
) showed weak to moderate antibacterial activity against
the selected pathogens (Table 3). Triterpenoids and phytosterols have been reported
to elicit their antibacterial effect through disruption of the cell membrane due to their
lipophilic nature that leads to increased membrane fluidity, permeability, and disruption
of embedded proteins [
23
]. It could therefore be speculated that the bulkier lipophilic
nature of the friedelane triterpenoid (
LA1
) compared to the phytosterol,
β
-sitosterol (
LA2
)
contributes to its higher activity over the
β
-sitosterol. The presence of these compounds in
L. africana may thus contribute to its antibacterial activity.
According to the results of the resistance modulatory tests (Table 4), all compounds
notably potentiated amoxicillin’s antibacterial activity against E. coli and P. aeruginosa. In
the presence of the isolated compounds, the MIC of amoxicillin decreased from 320
µ
g/mL
to 31.25
µ
g/mL, indicating a modulation factor (MF) of about 10 for P. aeruginosa. The MIC
of amoxicillin in E. coli decreased from 20
µ
g/mL to 0.625
µ
g/mL, resulting in MF = 32.
This means that when the compounds are co-administered with amoxicillin, the MIC of
amoxicillin is about 10 times lower to elicit an antibacterial effect against P. aeruginosa and
32 times lower to elicit an antibacterial effect against E. coli. The findings indicate that the
isolated compounds can enhance amoxicillin’s antibacterial effect against these bacteria.
Plant extracts and plant-derived compounds have been extensively studied for their
resistance-modulatory effect on antibiotics [
25
–
30
]. According to some studies, the interac-
tion of plant-derived compounds and antibiotics modulates bacterial resistance through
bacterial membrane destruction, increased antibiotic influx into the bacterial cell, inhibition
of bacterial efflux pumps, inhibition of quorum sensing, and gene expression modula-
tion [
31
,
32
]. Triterpenoids have been reported to enhance the antibacterial activity of several
classes of antibiotics including
β
-lactams, fluoroquinolones, tetracyclines, macrolides and
glycopeptides [
32
]. Their mechanism of action is thought to be due to membrane disruption
that enhances the influx of the antibiotics into the bacterial cell to reach high concentrations
for maximum antibacterial action [
32
]. Amoxicillin is an example of a
β
-lactam antibiotic;
hence, the resistance modulation action of the friedelane triterpenoid and
β
-sitosterol could
be mediated by membrane destruction and the ability of the compounds to enhance the
influx of the antibiotic into bacterial cells.
The whole extract of L. africana stem (LA) was tested for its ability to inhibit biofilm
formation against S. aureus ATCC 25923, E. coli ATCC 29212, and P. aeruginosa ATCC 27853.
The microorganisms used are known biofilm-forming pathogens [
5
]. The extract inhibited
biofilm formation in a concentration-dependent manner between 15.6 and 500
µ
g/mL.
The strongest biofilm inhibition effect was observed against E. coli, followed by S. aureus
and P. aeruginosa (Figure 2). The percentage biofilm inhibition ranged between 40% and
59% for S. aureus at its sub-inhibitory concentration (15–62.5
µ
g/mL), and 49 and 77% for
E. coli at its sub-inhibitory concentration (15–250
µ
g/mL). For P. aeruginosa, the percentage
biofilm inhibition ranged between 15 and 56% at the sub-inhibitory concentration of
62.5–250
µ
g/mL whereas at concentrations between 15 and 31
µ
g/mL, LA enhanced the
biofilm-forming capacity of P. aeruginosa. The effect of the triterpenoid compounds
LA1
and
LA2
(3.1- 100
µ
g/mL) on biofilm formation in S. aureus,E. coli, and P. aeruginosa is
presented in Figure 3.
LA1
showed good antibiofilm activity against S. aureus (54–65%) and
E. coli (47–74%) whereas it showed low to good activity against P. aeruginosa (42–57%).
LA2
demonstrated good antibiofilm activity against S. aureus (56–72%), E. coli (60–65%) and
P. aeruginosa (50–65%).
Microorganisms 2024,12, 7 15 of 17
A variety of plant extracts and compounds produced from plants have been shown to
inhibit the formation of biofilms by blocking the adhesion or implantation of planktonic
bacterial cells on abiotic surfaces [
20
,
23
]. Although this is the first report on the ability of
L. africana and its friedelane-type triterpenoid (
LA1
) to inhibit biofilm formation, previous
research has described the biofilm-inhibitory activity of a number of pentacyclic triterpenes
and sterols, including
β
-sitosterol (
LA2
), which demonstrated remarkable anti-biofilm
activities [16,32,33]. By destroying microbial membrane structures, blocking the synthesis
of peptidoglycans, nucleic acids, quorum sensing, and anti-cell adhesion molecules, plant
extracts and plant-derived chemicals may prevent the formation of biofilms [34–37].
The EtBr accumulation assay was used to test the ability of the crude extract (LA)
and isolated compounds (
LA1
and
LA2
) to act as efflux pump inhibitors (EPIs). EtBr, a
multidrug efflux pump substrate, emits a strong fluorescent signal when bound to DNA
intracellularly but only a weak signal when present extracellularly. As a result of the
retention of fluorescence over time if the efflux is reduced, the activity of putative EPIs
can be measured fluorometrically [
38
]. The bacterial efflux pump phenomenon has been
observed in P. aeruginosa,E. coli, and S. aureus [
39
]. P. aeruginosa and E. coli are classified as
critical priority pathogens, whereas S. aureus is classified as a high-priority pathogen [
40
].
As generally known efflux pump expressors, P. aeruginosa,E. coli, and S. aureus were chosen
for the efflux pump inhibitory effects.
Plant secondary metabolites, such as triterpenoids and sterols, have been found to
block the action of bacterial efflux pumps [
16
].
LA1
and
LA2
acted on efflux pumps
as shown by the increased ethidium bromide accumulation in E. coli (Figure 5A,B) and
P. aeruginosa (Figure 6A,B). Although their mechanism of action on efflux proteins is not
clearly established, triterpenoids are reported to elicit efflux pump inhibition activity [
41
].
This suggests that the efflux pump inhibition effect demonstrated by
LA1
and
LA2
is due
to their triterpenoid nature and thus contributes to the overall antibacterial activity of
L. africana.
The antibacterial activity of L. africana and its constituents demonstrated in this study
confirms the plant as a potential source of novel antibacterial agents alone and as adjuvants
in combination with known antibiotics.
5. Conclusions
This work has established that Loeseneriella africana stem extract has antimicrobial
resistance modulation, anti-biofilm generation, and efflux pump inhibitory activities. This
lends scientific credence to the traditional usage of L. africana stem for treating infections.
Friedelane-1,3-dione and
β
-sitosterol isolated from L. africana demonstrated an antibacterial
resistance modulation effect, and antibiofilm and efflux pump inhibition activities in
Gram-negative and Gram-positive organisms. The promising antimicrobial activities
demonstrated by the constituents of L. africana further support the antimicrobial activities
of the plant.
Supplementary Materials:
The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/microorganisms12010007/s1, Figure S1: Schematic presen-
tation of isolation procedure; Figure S2: (A) Structure of
LA1
(B) 1H-1H COSY and crucial 1H-1H
NOE correlations of
LA1
(C) Crucial HMBC correlations of
LA1
; Figure S3: Structure of
LA1
(A)
rotated horizontally at 180
◦
(B), then further rotated in a plane counterclockwise at 60
◦
C (D) Structure
of
LA1
numbered according to the position presented in Table 1; Figure S4: (A) Structure of
LA2
(B)
1H-1H COSY and crucial 1H-1H NOE correlations of LA2 (C) Crucial HMBC correlations of LA2.
Author Contributions:
Conceptualization, D.A. and A.Y.M.; methodology, D.A., J.A., D.O.-M., L.O.
and I.K.A.; software, D.A., J.A., A.L.K.A., D.G.A. and L.O.; formal analysis, D.A., C.A.D., E.O.A.,
R.P.B. and E.O.; investigation, D.A., J.A., D.O.-M., C.A.D., E.A.-K., E.O.A., E.O., R.P.B., L.O. and I.K.A.;
resources, D.A., D.G.A. and A.L.K.A.; data curation, D.A.; writing—original draft preparation, D.A.
and E.A.-K.; writing—review and editing, J.A., D.O.-M., C.A.D., E.A.-K., E.O.A., E.O., R.P.B., L.O.,
I.K.A., D.G.A. and A.L.K.A.; visualization, R.P.B.; supervision, A.Y.M. All authors have read and
agreed to the published version of the manuscript.
Microorganisms 2024,12, 7 16 of 17
Funding: This research received no external funding.
Data Availability Statement:
The dataset supporting the conclusions of this article are included
within the article and its additional files. Raw data sets used and analysed during the study are
available from the corresponding author on reasonable request.
Acknowledgments:
The authors are grateful to Clifford Asare for his assistance in plant collection
and the technicians in the Departments of Pharmacognosy, Pharmacology and Microbiology, Fac-
ulty of Pharmacy and Pharmaceutical Sciences, KNUST, Kumasi. We are also grateful to Thomas
Allmendinger of Novartis Pharma AG, Switzerland, for efforts in accessing the NMR equipment.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Seukep, A.J.; Kuete, V.; Nahar, L.; Sarker, S.D.; Guo, M. Plant-derived secondary metabolites as the main source of efflux pump
inhibitors and methods for identification. J. Pharm. Anal. 2020,10, 277–290. [CrossRef]
2.
Laukkanen, M.O. Return of Communicable Diseases as a Result of Antibiotic Resistance? Antioxid. Redox Signal.
2021
,34, 439–441.
[CrossRef]
3. Igarashi, M. New natural products to meet the antibiotic crisis: A personal journey. J. Antibiot. 2019,72, 890–898. [CrossRef]
4.
Kvist, M.; Hancock, V.; Klemm, P. Inactivation of efflux pumps abolishes bacterial biofilm formation. Appl. Environ. Microbiol.
2008,74, 7376–7382. [CrossRef]
5.
Tamfu, A.N.; Ceylan, O.; Cârâc, G.; Talla, E.; Dinica, R.M. Antibiofilm and anti-quorum sensing potential of cycloartane-type
triterpene acids from cameroonian grassland propolis: Phenolic profile and antioxidant activity of crude extract. Molecules
2022
,
27, 4872. [CrossRef]
6.
Sánchez, E.; Morales, C.R.; Castillo, S.; Leos-Rivas, C.; García-Becerra, L.; Martínez, D.M.O. Antibacterial and antibiofilm activity
of methanolic plant extracts against nosocomial microorganisms. Evid. Based Complement. Altern. Med. eCAM
2016
,2016, 1572697.
[CrossRef]
7.
Touani, F.K.; Seukep, A.J.; Djeussi, D.E.; Fankam, A.G.; Noumedem, J.A.; Kuete, V. Antibiotic-potentiation activities of four
Cameroonian dietary plants against multidrug-resistant Gram-negative bacteria expressing efflux pumps. BMC Complement.
Altern. Med. 2014,14, 258. [CrossRef]
8.
Wolcott, R.; Costerton, J.; Raoult, D.; Cutler, S. The polymicrobial nature of biofilm infection. Clin. Microbiol. Infect.
2013
,19,
107–112. [CrossRef]
9.
Tamfu, A.N.; Ceylan, O.; Fru, G.C.; Ozturk, M.; Duru, M.E.; Shaheen, F. Antibiofilm, antiquorum sensing and antioxidant activity
of secondary metabolites from seeds of Annona senegalensis, Persoon. Microb. Pathog. 2020,144, 104191. [CrossRef] [PubMed]
10. Isah, T. Stress and defense responses in plant secondary metabolites production. Biol. Res. 2019,52, 39. [CrossRef] [PubMed]
11.
Van Wyk, A.S.; Prinsloo, G. Health, safety and quality concerns of plant-based traditional medicines and herbal remedies. S. Afr.
J. Bot. 2020,133, 54–62. [CrossRef]
12. Burkill, H. The useful plants of West Africa, families MR. R. Bot. Gard. Kew 1985,4, 359.
13.
Compaoré, M.; Meda, R.N.-T.; Bakasso, S.; Vlase, L.; Kiendrebeogo, M. Antioxidative, anti-inflammatory potentials and
phytochemical profile of Commiphora africana (A. Rich.) Engl. (Burseraceae) and Loeseneriella africana (Willd.) (Celastraceae) stem
leaves extracts. Asian Pac. J. Trop. Biomed. 2016,6, 665–670. [CrossRef]
14.
Anokwah, D.; Asante-Kwatia, E.; Jibira, Y.; Amponsah, I.K.; Kyei, S.; Cobbold, G.P.; Adzekey, B.; Obese, E.; Ameyaw, E.O.;
Biney, R.P. Pharmacognostic study, anti-inflammatory and antioxidant activities of Loeseneriella africana. S. Afr. J. Bot.
2023
,157,
174–188. [CrossRef]
15. Evans, W.C. Trease and Evans’ Pharmacognosy; Elsevier Health Sciences: Amsterdam, The Netherlands, 2009.
16.
Anokwah, D.; Asante-Kwatia, E.; Mensah, A.Y.; Danquah, C.A.; Harley, B.K.; Amponsah, I.K.; Oberer, L. Bioactive constituents
with antibacterial, resistance modulation, anti-biofilm formation and efflux pump inhibition properties from Aidia genipiflora stem
bark. Clin. Phytosci. 2021,7, 28. [CrossRef]
17.
Danquah, C.A.; Maitra, A.; Gibbons, S.; Faull, J.; Bhakta, S. HT-SPOTi: A rapid drug susceptibility test (DST) to evaluate antibiotic
resistance profiles and novel chemicals for anti-infective drug discovery. Curr. Protoc. Microbiol.
2016
,40, 17.8.1–17.8.12. [CrossRef]
18.
Cos, P.; Vlietinck, A.J.; Berghe, D.V.; Maes, L. Anti-infective potential of natural products: How to develop a stronger
in vitro
‘proof-of-concept’. J. Ethnopharmacol. 2006,106, 290–302. [CrossRef]
19.
Prasch, S.; Duran, A.G.; Chinchilla, N.; Molinillo, J.M.; Macías, F.A.; Bucar, F. Resistance modulatory and efflux-inhibitory
activities of capsaicinoids and capsinoids. Bioorg. Chem. 2019,82, 378–384. [CrossRef]
20.
Abidi, S.H.; Ahmed, K.; Sherwani, S.K.; Bibi, N.; Kazmi, S.U. Detection of Mycobacterium smegmatis biofilm and its control by
natural agents. Int. J. Curr. Microbiol. Appl. Sci. 2014,3, 801–812.
21.
Stavri, M.; Piddock, L.J.; Gibbons, S. Bacterial efflux pump inhibitors from natural sources. J. Antimicrob. Chemother.
2007
,59,
1247–1260. [CrossRef] [PubMed]
22.
Kuete, V. Potential of Cameroonian plants and derived products against microbial infections: A review. Planta Medica
2010
,76,
1479–1491. [CrossRef] [PubMed]
Microorganisms 2024,12, 7 17 of 17
23.
Simoes, M.; Bennett, R.N.; Rosa, E.A. Understanding antimicrobial activities of phytochemicals against multidrug resistant
bacteria and biofilms. Nat. Prod. Rep. 2009,26, 746–757. [CrossRef] [PubMed]
24.
Aligiannis, N.; Kalpoutzakis, E.; Mitaku, S.; Chinou, I.B. Composition and antimicrobial activity of the essential oils of two
Origanum species. J. Agric. Food Chem. 2001,49, 4168–4170. [CrossRef] [PubMed]
25.
Sharifi, M.S.; Hazell, S.L. Isolation, analysis and antimicrobial activity of the acidic fractions of Mastic, Kurdica, Mutica and
Cabolica gums from genus Pistacia. Glob. J. Health Sci. 2012,4, 217. [CrossRef] [PubMed]
26.
Sharifi, M.S. Antimicrobial activities of triterpenoids against Methicillin-resistant Staphylococcus aureus (MRSA). Worldw. Res.
Efforts Fight. Against Microb. Pathog. 2013,9, 51.
27.
Ododo, M.M.; Choudhury, M.K.; Dekebo, A.H. Structure elucidation of
β
-sitosterol with antibacterial activity from the root bark
of Malva parviflora. SpringerPlus 2016,5, 1210. [CrossRef] [PubMed]
28.
Do˘gan, A.; Otlu, S.; Çelebi, Ö.; Aksu, P.; Sa˘glam, A.G.; Do˘gan, A.N.C.; Mutlu, N. An investigation of antibacterial effects of
steroids. Turk. J. Vet. Anim. Sci. 2017,41, 302–305. [CrossRef]
29.
Sen, A.; Dhavan, P.; Shukla, K.K.; Singh, S.; Tejovathi, G. Analysis of IR, NMR and antimicrobial activity of
β
-sitosterol isolated
from Momordica charantia. Sci. Secur. J. Biotechnol. 2012,1, 9–13.
30.
Mailafiya, M.M.; Yusuf, A.J.; Abdullahi, M.I.; Aleku, G.A.; Ibrahim, I.A.; Yahaya, M.; Abubakar, H.; Sanusi, A.; Adamu, H.W.;
Alebiosu, C.O. Antimicrobial activity of stigmasterol from the stem bark of Neocarya macrophylla.J. Med. Plants Econ. Dev.
2018
,
2, a38.
31.
Anand, U.; Jacobo-Herrera, N.; Altemimi, A.; Lakhssassi, N. A comprehensive review on medicinal plants as antimicrobial
therapeutics: Potential avenues of biocompatible drug discovery. Metabolites 2019,9, 258. [CrossRef]
32.
Catteau, L.; Zhu, L.; Van Bambeke, F.; Quetin-Leclercq, J. Natural and hemi-synthetic pentacyclic triterpenes as antimicrobials
and resistance modifying agents against Staphylococcus aureus: A review. Phytochem. Rev. 2018,17, 1129–1163. [CrossRef]
33.
da Silva, G.N.S.; Primon-Barros, M.; Macedo, A.J.; Gnoatto, S.C.B. Triterpene derivatives as relevant scaffold for new antibiofilm
drugs. Biomolecules 2019,9, 58. [CrossRef] [PubMed]
34.
Sibanda, T.; Okoh, A. The challenges of overcoming antibiotic resistance: Plant extracts as potential sources of antimicrobial and
resistance modifying agents. Afr. J. Biotechnol. 2007,6, 2886–2896.
35.
Wro´nska, N.; Szlaur, M.; Zawadzka, K.; Lisowska, K. The synergistic effect of triterpenoids and flavonoids—New approaches for
treating bacterial infections? Molecules 2022,27, 847. [CrossRef] [PubMed]
36.
Romero, C.M.; Vivacqua, C.G.; Abdulhamid, M.B.; Baigori, M.D.; Slanis, A.C.; Allori, M.C.G.d.; Tereschuk, M.L. Biofilm inhibition
activity of traditional medicinal plants from Northwestern Argentina against native pathogen and environmental microorganisms.
Rev. Soc. Bras. Med. Trop. 2016,49, 703–712. [CrossRef] [PubMed]
37.
Sasirekha, B.; Megha, D.; Chandra, M.; Soujanya, R. Study on effect of different plant extracts on microbial biofilms. Asian J.
Biotechnol. 2015,7, 1–12. [CrossRef]
38.
Blair, J.M.; Piddock, L.J. How to measure export via bacterial multidrug resistance efflux pumps. mBio
2016
,7, e00840-16.
[CrossRef]
39.
Kumar, R.; Jandaik, S.; Patial, P. Screening of medicinal plants of Himachal Pradesh for efflux pump inhibitory activity against
Escherichia coli.J. Pharmacogn. Phytochem. 2016,5, 96–100.
40.
World Health Organization. 2019 Antibacterial Agents in Clinical Development: An Analysis of the Antibacterial Clinical Development
Pipeline; World Health Organization: Geneva, Switzerland, 2019.
41.
Shriram, V.; Khare, T.; Bhagwat, R.; Shukla, R.; Kumar, V. Inhibiting bacterial drug efflux pumps via phyto-therapeutics to combat
threatening antimicrobial resistance. Front. Microbiol. 2018,9, 2990. [CrossRef]
Disclaimer/Publisher’s Note:
The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
