Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
(2019) 19:315
https://doi.org/10.1186/s12906-019-2713-3
RESEARCH ARTICLE
Open Access
Antibacterial activities, proposed mode of
action and cytotoxicity of leaf extracts from
Triumfetta welwitschii against Pseudomonas
aeruginosa
Molly Mombeshora and Stanley Mukanganyama*
Abstract
Background: Pseudomonas aeruginosa has become a main cause of Gram-negative infection, particularly in
patients with compromised immunity. High rates of resistance to antibiotics are associated with nosocomial
infections caused by P. aeruginosa strains. The search for novel antimicrobials has been necessitated by the
emergence of antimicrobial resistance in some bacteria Plant-based antimicrobials has great potential to combat
microbial infections using a variety of mechanisms. Triumfetta welwitschii plant roots are traditionally used to treat
symptoms of diarrhoea and fever, suggesting that it possess antimicrobial and immunomodulatory effects. Since
research investigating antimicrobial properties of the roots of Triumfetta welwitschii has been explored, there is
need to investigate the antimicrobial activity of its leaf extracts in order to probe their prospective use
as new antimicrobial agents that can be used to combat nosocomial infections. The objective of this
study was to evaluate the antibacterial activities, the mode of action and cytotoxicity of T. welwitschii leaf extracts.
Method: Extracts of T. welwitschii leaves were obtained using eight different solvents, the serial exhaustive
extraction method and the cold maceration technique. In vitro antibacterial activity evaluation of the
extracts was done on eight bacterial isolates using the broth microdilution method. The mode of action
for the most potent extracts was investigated using the rhodamine 6G efflux assay and the propidium iodidebased membrane damage assay. Toxicity of the extracts was evaluated using the haemolytic and MTT (3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays.
Results: The results showed that acetone, ethanol and dichlorometane: methanol extracts had the most potent
antibacterial activities against Pseudomonas aeruginosa (ATCC 27853). All three extracts caused membrane
disruption of P. aeruginosa as shown by nucleic acid leakage. All three extracts were unable to inhibit
efflux pumps.
Conclusion: The presence of antibacterial activities and low toxicity shown by the extracts indicates
that the plant may be a source of effective antibacterial against some bacterial infections caused by
P. aeruginosa. The disruption of membrane integrity is one possible mode of action of antibacterial
activity of the potent extracts.
Keywords: Triumfetta welwitschii, Antibacterial, Toxicity, Haemolytic
* Correspondence: smukanganyama@medic.uz.ac.zw;
smukanganyama01@gmail.com
Biomolecular Interactions Analyses Group, Department of Biochemistry,
University of Zimbabwe, P.O. Box MP 167, Mount Pleasant, Harare, Zimbabwe
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
Background
Treatment of infectious diseases is becoming more challenging due to the development of resistance to multiple
classes of antibiotics by bacteria. This is especially true
for infections caused by Pseudomonas aeruginosa. P. aeruginosa is a frequent causative pathogen in nosocomial
infections. The Gram-negative bacterium is associated
with nosocomial pneumonia, and is frequently implicated in hospital-acquired bloodstream and urinary tract
infections [1]. In an attempt to counteract resistance to
antibiotics, a number of studies now focus on the search
for new antimicrobials. Plants are one of the main targeted sources in the search for novel antimicrobials.
Constituents of plant origin provide a good source of
antimicrobial compounds [2, 3], as plants have evolved a
variety of diverse chemical strategies to combat attack
from pathogens. The secondary metabolites of medicinal
importance include alkaloids, flavonoids, tannins, terpenes, and phenolic compounds. These active constituents
possess effective pharmacological activity [4]. Triumfetta
welwitschii Mast. belonging to the Tilicea family is an
important medicinal plant largely used in the Southern
African countries as traditional medicine. Its roots are
crushed and used in the form of decoction to treat symptoms of diarrhoea [5]. A mixture of milk and roots of T.
welwitschii is used as an oral antipyretic agent [6]. Root
extracts of T. welwitschii has been reported to possess
antiplasmodial activity [7] and antiproliferative activity
against Jurkat cells [8]. Antibacterial activity against
Escherichia coli, Bacillus cerus [9] and antimycobacterial
activity against Mycobaterium aurum and Myocobacterium smegmatis has been reported from root extracts of T.
welwitschii [10]. The current study shifts from investigating antimicrobial activity of the roots and focuses on the
leaves of T. welwitschii. The leaves from the same family
(Tilicea) of plants have been reported to possess analgesic
and antimicrobial activity [11–13], indicating the potential
for antimicrobial activity in leaves of T. welwitschii. The
main classes of secondary metabolites found in T. welwitschii are flavonoids, phenols and coumarins (unpublished data from BIA laboratory). The primary objective of
the current study was to investigate the antibacterial properties of the leaf extracts of T. welwitschii against six of
some of the common nosocomial pathogens [14]. The secondary objectives were to evaluate the possible mode of
action and cytotoxicity of the crude extracts.
(2019) 19:315
Page 2 of 12
the plant sample was done by Mr. Christopher Chapano of
the National Botanical and Herbarium Garden (Harare,
Zimbabwe). Permission to use the plant samples was
granted by the Faculty of Higher Degrees Committee,
Harare, Zimbabwe (HD/71/16). The leaves were washed
with tap water several times to remove any soil or dust particles. Drying of the leaves was carried out under shade for
21 days.
Preparation of extracts
All solvents used for extraction were of analytical grade and
were obtained from Sigma Aldrich (Steinheim, Germany).
The leaves were ground to a fine homogenous powder
using a pestle and mortar. A total of 384 g of powder was
obtained and stored. A mass of 50 g powder was placed in
a plastic beaker and 500 ml of 50: 50 v/v dichloromethane
(DCM): methanol added to the powder. The cold maceration method with modifications was used to extract phytochemicals from the powdered leaves [15]. Maceration
involved soaking plant materials with a solvent in a beaker
covered with foil paper. The mixture was allowed to stand
at room temperature for a period 2 days with frequent agitation. Solvents of different polarities namely: hexane,
DCM, acetone, ethyl acetate, methanol, ethanol and water
were used to serially extract phytochemicals from a new
powder sample. Serial exhaustive extraction [16] involved
extracting sequentially with a non-polar solvent (hexane) to
a moderately polar and finally polar solvent (water). The
slurry obtained was filtered through a No. 1 Whatman filter
paper. The filtrate obtained was concentrated under a vacuum using a rotary evaporator RII (BUCHI, LabortechnikAG, Switzerland). The extracts were dried to a constant
mass under a fan in a fume hood cabinet. All extracts were
stored in sterile tubes at − 4 °C until use.
Chemicals used in assays
Chemicals used in the study included; ampicillin (A9518),
levofloxacin (28266), dimethyl sulphoxide (DMSO) (D5879),
thiazolyl blue (M2128), reserpine (R0875), sodium citrate
(1613859), potassium ferricyanide (702587), sodium carbonate (1613757), glucose (G8270) and rhodamine 6G (R6G)
(252433) were purchased from Sigma Aldrich (Germany).
Tryptic soy broth (TSB) (22092), tryptic soy agar (TSA)
(22091) and Roswell Park Memorial Institute media (RPMI)
(8758) were also from Sigma Aldrich (Germany).
Microbial strains and culture media
Methods
Collection of plant material
T. welwitschii leaves voucher number C16 E7 were procured between January and April of 2017 from the communal lands of Centenary (16.8oS, 31.1167°E, and 1156
m above sea level), in the Mashonaland Central Province
of Zimbabwe. The identification and authentication of
Six of some of the common nosocomial pathogens [14]
were chosen for this study. Isolates of Bacillus subtilis,
Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus pyogenes and Klebsiella
pneumoniae isolated from patients were supplied by Parirenyatwa Group of Hospitals (Department of Medical Microbiology, College of Health Sciences, Harare, Zimbabwe). S.
Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
aureus was isolated from an ear infection and P. aeruginosa
from a urinary tract infection. Types of infection from
which the isolation of B. subtilis, S. pneumoniae, S. pyogenes
and K. pneumoniae were not specified. P. aeruginosa ATCC
27853 and S. aureus ATCC 9144 were acquired from the
Microbiological Section in the Department of Biological
Sciences at the University of Botswana (Gaborone,
Botswana). Bacteria were kept as glycerol stocks at − 35 °C.
For each assay bacteria were grown on tryptic soy agar
(TSA) for 24 h at 37 °C, followed by inoculation in tryptic
soy broth (TSB). Inoculum concentration was adjusted
to106 c.f.u/ml by diluting the inoculum using TSB using 0.5
McFarland standard.
Determination of antibacterial activities of leaf extracts
isolated from Triumfetta welwitschii
Antibacterial activities of the DCM: methanol, hexane,
DCM, ethyl acetate, acetone, ethanol, methanol and
water extracts were determined by reconstituting each
extract in dimethyl sulfoxide (DMSO). Required concentrations (12.5, 25, 50 and 100 μg/ml) of the extracts were
obtained by diluting using TSB. The broth microdilution
method [17] with minor modifications was used to determine the effects of the extracts against ATCC strains
of P. aeruginosa and S. aureus. Susceptibility of the
clinical strains of P. aeruginosa and S. aureus, K. pneumoniae, S. pneumoniae, S. pyogenes and B. subtilis were
also determined. Liquid cultures of each bacterium were
grown in TSB media. These were diluted in fresh TSB
and 100 μL was applied to the wells of a 96-well plate. In
each case, approximately 2 × 106 cfu/ml of exponentially
growing cells was inoculated for each strain. The extracts or antibiotics (100 μL) were added to these wells
in decreasing concentrations and mixed by pipetting.
Cells in tryptic soy broth were used as the positive control. While cells exposed to the standard antibiotic were
used as the negative control. Cell density of the plate
was measured at 590 nm using a microplate reader
(Tecan Genios-Pro microplate reader, Grödig, Austria)
before incubation. Plate was incubated at 37 °C for 24 h,
and cell density was measured. Growth of cells was determined by finding the difference of the pre-incubation
value from the post-incubation value. Data are presented
as percentage inhibition of inoculum. Percentage inhibition was obtained using the equation:
Percentage inhibition ¼
ðpositive control value sample valueÞ 100
positive control value
ð1Þ
Ampicillin (0 to100 μg/ml) was used as the standard
antibiotic used against P. aeruginosa and S. aureus. Ciprofloxacin (0 to1 μg/ml) against S. pyogenes and B.
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subtilis. Levofloxacin (0 to 1 μg/ml) was used against K.
pneumoniae, S. pneumoniae.
Determination of the possible mode of action of
antibacterial
Membrane damage potential
The cell membrane damage potential of the DCM/
methanol, acetone and ethanol extracts from leaves of T.
welwitschii against the ATCC strain of P. aeruginosa was
determined using propidium iodide as described by
Moyo and Mukanganyama [9], with modifications. Propidium iodide is a dye capable of binding to nucleic
acids of non-viable cells with damaged membranes only
[18]. The dye is unable to enter viable cells, thus, it is
useful for determining the effects of plant extracts on
bacterial membranes. P. aeruginosa cells were grown by
pipetting 200 μl of overnight inoculum into 200 ml TSB
and incubating overnight at 37 °C with shaking in an incubator. The optical density of the cells was adjusted to
an OD600 = 1.5 equivalent to 2 × 109 c.f.u/ml using PBS.
Cell suspensions were exposed to different concentrations of the extracts of a final concentration of 50 μg/ml,
100 μg/ml and 200 μg/ml for 30 min at 37 °C with shaking in an incubator. The negative control contained cells
with no extract added. All test samples were prepared in
triplicate. After incubation, 1 ml of each test sample was
centrifuged at 11000 rpm. The pellet was washed with
saline solution, resuspended in PBS and propidium iodide of a final concentration of 10 μg/ml added to the suspension. The mixture was kept in the dark for 10 min
after which 200 μl of test samples were transferred to a
96-well plate. Fluorescence was measured at 544 nm Excitation and 612 Emission using an fmax spectrofluorometer (Molecular Devices, Sunnyvale, USA).
Determination of the extracts on drug transport activity
The transport of R6G dye out of cells as described by Chitemerere and Mukanganyam [19] was used to evaluate the
effects of the acetone, ethanol and DCM/methanol leaf extracts as potential efflux pump inhibitors. Duplicate standards of R6G (0 μM to 3 μM) were prepared in PBS and
their absorbance values determined at 527 nm using a
Shimadzu UV/VIS UV-1601spectrophotometer (Shimadzu,
Kyoto, Japan). A calibration curve was generated from
values of absorbance obtained as a function of concentration using Graphpad™ version 5 for Windows (Graphpad™
Software Inc., San Diego, California, USA).
A sub-inhibitory concentration (25 μg/ml) of each extract was used in the R6G efflux assay using the laboratory strain of P. aeruginosa cells. The R6G efflux assay
was carried out by growing 200 μL of an overnight culture of cells in three 200 ml nutrient broth in 2 L flasks
and incubated overnight at 37 °C with shaking (120
r.p.m). Cells were centrifuged using a Rotafix 32A
Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
centrifuge (Hettich, Benin, Germany) and washed using
phosphate buffer solution (PBS pH 7.4). Cells were resuspended in PBS containing sodium azide to a final
concentration of 40 mg/ml. A final concentration of
10 μM R6G was added and the mixture incubated at
37 °C for an hour with shaking (120 r.p.m). Cells were
collected by centrifuging at 4000 r.p.m. for 15 mins and
cells exposed to the following reagents in six separate
tubes containing: glucose, no glucose, glucose + reserpine, glucose + acetone leaf extract, glucose + ethanol
leaf extract, glucose + DCM/methanol leaf extract. The
final concentration of reserpine used was 80 μg/ml.
All samples were incubated at 37 °C for 1 h. Cells
were collected by centrifugation at 4000 r.p.m. for 15
min and the supernatant was used for R6G efflux
quantification. Optical density values of the R6G
pumped out of the cells was determined using a
Shimadzu UV/VIS UV1601 spectrophotometer (Shimadzu
Corporation, Kyoto, Japan) at a wavelength of 527 nm.
The calibration curve was used to interpolate concentrations of R6G in samples in the efflux assay based on their
absorbance values.
Evaluation of the toxicity of the leaf extracts
Determination of toxicity using sheep erythrocytes
The cytotoxicity effects of the DCM/methanol, acetone
and ethanol extracts from leaves of T. welwitschii
against erythrocytes from sheep was determined using
the haemolysis assay as described by Malagoli [20],
with modifications. A volume of 50 ml sheep blood
was collected and added to an equal volume of Alsever
solution. Blood was centrifuged at 3000 r.p.m. for 10
min and the supernatant was discarded. The residue
was washed three times with a 1:5 volume of PBS. The
resulting cells were diluted four-fold using PBS to give
an erythrocyte suspension. Extracts were prepared in
PBS and final concentrations of 50 μg/ml (1/2MIC),
100 μg/ml (MIC) and 200 μg/ml (2MIC) were used in
the assay. The erythrocyte suspension (500 μl) was
mixed with 500 μl test sample extract and incubated
for 90 min at 37 °C. All test samples were prepared in
triplicate. After incubation, the tubes were spurn at
3000 r.p.m. for 1 min in a microcentrifuge (Geratebau
Eppendorf GmbH, Engelsdorf, Germany). The positive
control with 100% haemolysis was obtained by mixing
200 μl erythrocyte suspension with 1.5 ml Drabkin’s reagent; the negative control was a mixture of 500 μl
erythrocyte suspension and 500 μl PBS. Aliquots of
200 μl of supernatant were transferred into 96-well
plates. The absorbance (Abs) of haemoglobin released
was measured at 590 nm using a Tecan Genios microplate reader (Grödig, Austria). The percentage haemolysis for each sample was calculated using the
equation [21]:
(2019) 19:315
Percentage haemolysis ¼
Page 4 of 12
Abs:of sample Abs:of control 100
Abs:of maximal lysis Abs:of control
ð2Þ
Determination of toxicity using mouse peritoneal cell
This work on animals was conducted in accordance with
the internationally accepted principles for the protection
of animals used for scientific purposes [22]. Six weeks old
male laboratory-bred strain of the house mice (BALB/c) of
20–25 g weight were collected from the Animal House at
the University of Zimbabwe (Harare, Zimbabwe) and
used. The research was carried out according to the rules
governing the use of laboratory animals and the experimental protocol was approved by the Faculty of Higher
Degrees Committee, Harare, Zimbabwe (HD/71/16). To
increase the number of peritoneal cells within the mice,
20% sterile starch solution was intraperitoneally introduced into the mice using a syringe with a 27 g needle.
The mice were left for 48 h in plastic cages with unlimited
access to food and water in order to allow peritoneal cell
yield increase. Total peritoneal cells were isolated as described by Ray and Dittle [23]. Each mouse was euthanized by cervical dislocation. Then sprayed with 70%
ethanol and mounted on a styrofoam block on its back.
Scissors and forceps were used to cut the outer skin of the
peritoneum to expose the inner skin lining the peritoneal
cavity. A volume of 5 ml of ice cold PBS with 3% FCS was
introduced into the peritoneal cavity using a 27 g needle.
Due care was taken to avoid puncturing of organs. After
injection, the peritoneum was gently massaged to remove
any attached cells into the PBS solution. A 25 g needle, attached to a 5 ml syringe was used to collect the fluid from
the peritoneum into tubes kept on ice after removing the
needle from the syringe. The cell suspension collected was
spurn at 1500 r.p.m. for 10 min in a Rotofix 32A centrifuge. The supernatant was discarded and cells resuspended the cells in RPMI. Cells were cultured in RPMI
medium supplemented with 10% Fetal bovine serum
(FBS) and 1% PNS (penicillin, neomycin and streptomycin) and incubated in a Shellab incubator (CO2 series
Sheldon Mfg. Inc., Cornelius, USA) at 37 °C in a controlled atmosphere with 5% CO2 for 24 h. Cells were
stained with 0.4% trypan blue and viable cells counted
using a haemocytometer counting chamber under a Celestron digital light microscope (Celestron, Los-Angeles,
USA) using the × 10 objective lens. Toxicity was determined using the MTT assay as described by Mapfunde
et al., [24]. Extracts were dissolved in DMSO. Each of the
three extracts was double diluted to give concentrations of
12.5, 25, 50 and 100 μg/ml. The final concentration of
DMSO in each well was 1%. A typical plate set up is
as shown in Fig. 1. The cells were incubated in 96well plates in the presence of extracts for 24 h at
Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
37 °C in a 5% CO2 Shel lab incubator. Each well contained
100 μl of the test substance and 100 μl of 0.5 × 105 cells/
ml in RPMI. Cells exposed to the standard anticancer
drug daunorubicin (10 μg/ml) were used as the positive
control. Cells in RPMI were used as the negative control.
After the 24 h incubation, 25 μl of MTT (3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)
was added to each well and plates were incubated for 4 h.
A volume of 50 μl of DMSO was added and the absorbance of the contents in wells was measured at 590 nm
using a Tecan Genios-Pro microplate reader (Tecan
Group Ltd. M nnedorf, Switzerland).
Statistical analyses
One-way analysis of variance test (ANOVA) with Dunnett’s Multiple Comparison Post Test was used to analyse
the results. All sets of data were compared to the control.
The values with a p-value < 0.05 were considered statistically significant. Graphical and Statistical analyses were
carried out using GraphPad Prism 5® Software (Version
5.0, GraphPad Software Inc., San Diego, USA).
Results
Yield of extracts
The percentage yield for the various extracts was calculated using the formula:
Percentage yield ð%Þ ¼
Mass of extract obtained ðgÞ 100
Mass of plant powder used ðgÞ
The results of the percentage yields of the extracts are
shown in Table 1.
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The solvent mixture of DCM: methanol extracted
the highest percentage of extracts (8.06%) while water
extracted the least percentage of extracts (0.52%). The
polar solvents which included acetone, methanol and
ethanol yielded greater than 2% extract with water being an exception. The non-polar solvents which included hexane, DCM and ethyl acetate gave yields of
less than 2%.
Antibacterial activities of extracts
The percentage inhibition of bacterial growth caused by
leaf extracts of a 100 μg/ml concentration is as shown in
Fig. 2. All leaf extracts showed varied antibacterial activities against test bacteria. The most significant growth inhibition by the extracts was against P. aeruginosa ATCC
compared to the other seven bacterial isolates. The growth
inhibition of the clinical strain of P. aeruginosa by most of
the extracts was lower than that in the ATCC strain P.
aeruginosa. The extracts exhibited the least growth inhibition against the clinical strain of K. pneumoniae compared to the rest of the test isolates. Of the eight extracts,
the acetone, ethanol and DCM/methanol leaf extracts
showed growth inhibitory activities of 96, 81 and 99% respectively against P. aeruginosa ATCC. Polar extracts
(acetone, ethanol and methanol) with the exception of the
water extract showed growth inhibition of greater than
60% against P. aeruginosa ATCC. The non-polar extracts;
ethyl acetate, DCM and hexane showed less than 60%
inhibition of the growth of P. aeruginosa ATCC. The ethanol, acetone and DCM/ methanol leaf extracts were used
in subsequent biochemical and toxicity tests since they
had shown higher growth inhibition of P. aeruginosa
Fig. 1 Plate set up for the MTT assay using mouse peritoneal cells, exposed to different solvent extracts from T. welwitschii leaves. Cells in RPMI
row was the negative control, while the cells daunorubicin row was the positive controls
Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
Table 1 Yield of extracts from leaves of T. welwitschii
Solvent used for extraction
Yield (%)
DCM: methanol
8.06
Hexane
1.66
DCM
0.98
Ethyl acetate
0.90
Acetone
2.53
Methanol
2.53
Ethanol
3.42
Water
0.52
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the effects of the extracts on efflux pump activities in P.
aeruginosa (Fig. 3). The presence of the ethanol, acetone
and DCM/methanol leaf extracts stimulated the extrusion of R6G from the bacterial cells. The cells exposed
to glucose showed higher efflux of R6G when compared
to cells exposed to the efflux pump inhibitor (EPI) reserpine. The presence of the acetone, ethanol and DCM/
methanol extracts resulted in an increased extrusion of
R6G from P. aeruginosa compared with the extrusion in
the presence of reserpine or glucose.
Membrane damage potential of extracts
ATCC in comparison to the other extracts. Having noted
that growth inhibition by the extracts was greatest against
the ATCC strain of P. aeruginosa compared to the rest of
the test isolates, subsequent biochemical and toxicity tests
were performed using this strain. Total inhibition of
bacterial growth by the standard antibiotics were at concentrations of: 50 μg/ml for the ATCC strain of P. aeruginosa; 25 μg/ml for the clinical strain of P. aeruginosa;
0.4 μg/ml for the ATCC and clinical strain of S. aureus;
0.5 μg/ml for S. pyogenes and B. subtilis; 0.25 μg/ml for S.
pneumoniae.
Effects of extracts on efflux activity
The amount of R6G extruded in the presence acetone,
ethanol or DCM/methanol extracts was used to assess
Effects of the DCM/methanol, acetone and ethanol leaf
extracts on bacterial membrane integrity was determined
by exposing P. aeruginosa to varying concentrations of
the extracts followed by staining with propidium iodide.
The exposure to leaf extracts resulted in bacterial cell
membrane disruption. Membrane disruption was evidenced by an increased uptake of propidium iodide by
the exposed cells in comparison to the unexposed cells
(P < 0.05) (Fig. 4). All three extracts were able to cause
significant membrane permeability resulting in nucleic
acid leakage from P. aeruginosa cells when compared to
the control. The highest amount of nucleic acid leakage
was observed in cells exposed to 200 μg/ml ethanol leaf
extract while the acetone leaf extract caused the least
nucleic acid leakage at the same concentration.
Fig. 2 Percentage inhibition of bacterial cells upon treatment with 100 μg/ml extract from leaves of T. welwitschii. a K. pneumoniae, b S.
pneumoniae, c S. pyogenes, d B. subtilis, e ATCC strain of S. aureus, f clinical strain of S. aureus, g ATCC strain of P. aeruginosa, and h clinical strain
of P. aeruginosa. Values are for mean ± standard deviation (error bar) for n = 4. The asterisks indicate a significant difference from the control with
*p < 0.05, ***p < 0.001 and ns mean no significant difference
Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
(2019) 19:315
Page 7 of 12
Fig. 3 The effects the acetone, ethanol and DCM/methanol leaf extracts from T. welwitschii on efflux pump activity of the ATCC strain of P.
aeruginosa. Cells exposed to glucose served as the positive control where active efflux occurred maximally. The error bars show the standard
deviation from the mean of two samples read twice. The asterisks indicate a significant difference from the control with *p < 0.05, **p < 0.01
and ***p < 0.001
Effects of extracts on sheep erythrocytes
The haemolysis of sheep erythrocytes induced by the
acetone, ethanol and DCM/methanol leaf extracts from
T. welwitschii expressed as a percentage is as shown in
Fig. 5. At a concentration of 100 μg/ml, the DCM/
methanol leaf extract showed the highest haemolytic effect when compared to the ethanol and acetone extracts.
All three extracts showed a dose-dependent haemolytic
effect against the sheep erythrocytes. The DCM/methanol leaf extracts showed haemolytic activity of 16%. The
Fig. 4 Fluorescence of propidium iodide bound to nucleic acids of P. aeruginosa cells after exposure to the acetone, ethanol and DCM/methanol
leaf extracts from Triumfetta welwitschii. Cells with no extract were used as the control. Values are for mean ± standard deviation (error bar) for
n = 3. The asterisks indicate a significant difference from the control with ***p < 0.001
Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
(2019) 19:315
Page 8 of 12
Fig. 5 The percentage haemolysis of sheep erythrocytes induced by exposure to different concentrations of the DCM/methanol, acetone and
ethanol leaf extracts from T. welwitschii. Cells with no extract were used as the control. Values are for mean ± standard deviation (error bar) for
n = 3. The asterisks indicate a significant difference from the control with ***p < 0.001
acetone and ethanol leaf extracts at a concentration of
100 μg/ml had haemolytic activity of 10 and 11%
respectively.
Effects of leaf extracts on mouse peritoneal cells
Toxicity of the acetone, ethanol and DCM/methanol leaf
extracts from T. welwitschii was tested on mouse peritoneal cells. The effects of the extracts on the growth of
mouse peritoneal cells are as shown in Fig. 6. All test samples were non-toxic to the mouse peritoneal cells. The
three extracts showed a dose-dependent increase in
mouse peritoneal cells proliferation. The DCM/methanol
leaf extract and the ethanol leaf extracts had the highest
and least proliferation stimulatory properties respectively.
Discussion
The search for new antimicrobials is frequently based on
ethnobotany and ethnopharmacology [25]. T. welwitschii
was selected based on its ethnomedicinal use in the
Southern parts of Africa [5, 6]. Since work had already
been done on the roots [7–9] this study focused on the
leaves of the plant as there is a knowledge gap pertaining
the pharmacological value of the leaves of the plant. Solvents of varying polarities were used to prepare extracts
from leaves of T. welwitschii. Different solvents extract
different phytochemical groups; therefore, serial exhaustive extraction was used to enhance the isolation of
phytochemicals from the complex crude mixture [26].
The DCM: methanol solvent mixture gave the highest
percentage yield (8.06%). The solvent mixture constitute
of a polar and non-polar solvent which must have facilitated the extraction of both polar and non-polar phytochemicals. Polar solvents with the exception of water
gave yields of more than 2% while non-polar solvents
gave yields of less than 2%. Martini and Eloff [27]
showed that the polar solvents have higher extracting
potential than the non-polar solvents.
Leaf extracts from T. welwitschii possessed varying
potential of antibacterial activity against P. aeruginosa, S.
aureus, K. pneumoniae, S. pneumoniae, S. pyogenes and
B. subtilis (Fig. 2). Of the eight test isolate, P. aeruginosa
ATCC was the most inhibited by the majority of extracts. It is worth noting that the Gram-negative P. aeruginosa was inhibited by most of the extracts more than
the Gram-positives S. aureus, B. subtilis, S. pneumoniae
and S. pyogenes. Gram negatives possess two cellular
membranes, with the outer membrane covered with
lipopolysaccharides, making it a formidable barrier for
molecules to penetrate [28] which deviates from the expected results. In this study, the disruption of membrane
integrity was shown to be the mode of action of the
three extracts. The penetration of the outer membrane
of the Gram-negative P. aeruginosa by the extracts may
have been achieved through the pre-disruption of the
membrane.
The acetone, ethanol and DCM: methanol leaf extracts
from T. welwitschii were the most active extracts against
the ATCC strain of P. aeruginosa. Acetone, ethanol and
methanol (in the DCM: methanol mixture) are polar solvents known to extract a wide range of phytochemicals
[27]. Antibacterial activities shown by these extracts may
be attributed to phenols, flavonoids [29] and coumarins
[30] the common secondary metabolites in T. welwitschii. A total of six and two extracts showed more
than 50% growth inhibition against the ATCC and clinical strains of P. aeruginosa respectively. The inhibition
of growth of the clinical strain of P. aeruginosa by most
of the extracts was lower compared to that of the ATCC
strain. Laboratory strains have been sub-cultured for
years since they were first isolated. A diversity of genotypes subsequently changes over time [31] hence the
different responses noted for the clinical and laboratory
strains. These findings on the antibacterial activity of
extracts from T. welwitschii plant make the plant a
Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
(2019) 19:315
Page 9 of 12
Fig. 6 The effects of the a acetone, b ethanol and c DCM/methanol
leaf extracts of T. welwitschii on mouse peritoneal cells. Cells with
daunorubicin a standard antibiotic were used as the control. Values
are for mean ± standard deviation (error bar) for n = 3. The asterisks
indicate a significant difference from the control with **p < 0.01
and ***p < 0.001
possible source of compounds to explore for novel lead
compounds for drug development against P. aeruginosa.
A wide range of mechanisms provide bacteria with
resistance to antibiotics; these include target-site modification and antibiotic inactivation among others. The
expression of efflux pumps by some human pathogenic
bacteria confers multidrug resistance (MDR). A single
pump may provide bacteria with resistance to an extensive range of chemically and structurally different compounds. Natural products are a possible source of efflux
pump inhibitors [32–34]. The R6G efflux assay was carried out to determine the potential use of the acetone,
ethanol and DCM/methanol leaf extracts from T. welwitschii as efflux pump inhibitors. The R6G assay involves preloading the cell with a fluorescent substrate
(R6G) prior to the efflux assay. After the loading step,
R6G accumulates within the cells. Cells are then washed
to remove R6G on the outer surface of cells. Subsequently, glucose is added to the culture as a source of
energy, and the efflux of R6G is measured by fluorimetry
[35]. A known EPI (e.g reserpine) is included as a positive control for inhibition of the efflux of R6G. Results
from the R6G efflux (Fig. 3) showed that there was increased efflux of R6G in the presence of plant extracts
compared to cells in glucose. The plant extracts stimulated efflux. Thus, the extracts used in this study lacked
efflux pump inhibitory activity. While inhibition of efflux
pumps seems to be a worthy approach for improving the
efficacy of antibiotics which are substrates of such
pumps, it is important to identify antibiotics and target
bacteria for which this approach would be the most applicable [36].
Antibacterial agents, usually act on the membranes of
bacteria by causing disruption and permeabilisation [37].
The antibacterial mode of action of the acetone, ethanol
and DCM: methanol leaf extracts from T. welwitschii on
the membrane integrity of P. aeruginosa was determined
using propidium iodide a fluorescent nucleic acid stain.
Live bacterial cells are impermeable to propidium iodide,
but upon membrane disruption or permeabilisation, propidium iodide can enter the cells [18]. The exposure of
P. aeruginosa to the three leaf extracts resulted in bacterial cell membrane disruption as evident from the increased uptake of propidium iodide in comparison to
the unexposed cells (Fig. 4). The increased fluorescence
of propidium iodide by cells showed that there was disruption of the cell membrane since propidium iodide
Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
exclusively bind to nucleic acids of dead cells with damaged membranes only and not live cells. It has been
reported in other studies that some extracts cause membrane damage leading to nucleic acid leakage [38], and
induce cell damage [39]. Among extracts that cause membrane damage causing leakage of cell materials can be
found also the Plumbago zeylanica root [37], Trianthema
portulacastrum leaf [40], and Ocimum basilicum [41].
For a plant extract to be useful, it has to possess bioactive properties and exhibit non-cytotoxic profile. Some
plants possessing bio-active components may show toxicity thus it is important to investigate the primary toxicity
of plant extracts. Several researchers have used erythrocytes as a model system for determining the interaction of
drugs with mammalian membranes [42–45]. The erythrocyte model has been commonly used in toxicity profiling
as it provides a direct indication of toxicity of injectable
preparations in addition to a general indication of membrane toxicity [46]. Haemolysis is a result of the destruction of the erythrocyte caused by the lysis of the
membrane lipid bilayer. The lysis of erythrocytes can
cause anaemia, an increase in plasma haemoglobin leading
to nephrotoxicity and vasomotor instability [47]. In the
haemolytic assay, when the erythrocyte suspension was
diluted in Drabkin’s, the reagent haemolysed the erythrocytes. The haemolysis released haemoglobin into the
solution. The Fe2+ of the haemoglobin molecules were
oxidised by potassium ferricyanide to Fe3+. This oxidation
resulted in the formation of methaemoglobin which combined with the cyanide ions to form cyanmethemoglobin,
a stable compound colour pigment read calorimetrically
at 590 nm [48]. The acetone, ethanol and DCM: methanol
leaf extracts from T. welwitschii showed haemolytic
activity of 10–16% (Fig. 5). According to Vidhya and
Udayakumar [49], a 10–49% haemolytic activity is rated as
slightly toxic. Therefore, the 10–16% haemolytic activity
obtained for the three leaf extracts from T. welwistchii is
an indicator of non-significant toxicity to erythrocyte
membrane, consequently favouring further study of the
plant species.
Macrophages are highly phagocytic and considered to
be essential immune effector cells that participate in
innate and adaptive immune responses. Since the functioning of macrophages can be altered depending on
their surrounding environment and the stimuli they are
exposed to [50], they were used as a typical model to
study the cytotoxicity of plant extracts. The potential of
plant extracts to inhibit the growth or viability of murine
macrophages can, therefore, be used as an indication of
toxicity. Viability of mouse peritoneal cell was determined using the MTT assay. The yellow tetrazolium
MTT salt was reduced by metabolically active cells by
the action of dehydrogenase enzymes giving a purple
colour. The intensity of the purple colour was used to
(2019) 19:315
Page 10 of 12
calorimetrically measure viable cells [51]. The results of
the mouse peritoneal cells exposed to the acetone, ethanol and DCM: methanol extracts from T. welwistchii
(Fig. 6) showed that cell survival increased with increasing extract concentration. The proliferative effect of the
three extracts on the mouse peritoneal cells was an indication that the leaf extracts were not toxic towards the
mouse peritoneal cells. Similar results were reported by
Ragupathi., et al [52], saponins isolated from Quillaja
saponaria tree bark stimulated the production of
immune cells. Sun et al., [53], showed that most plant
polypeptides promote the proliferation of macrophages
among other immune cells. Therefore, the results of this
study provide evidence that the acetone, ethanol and
DCM/methanol leaf extracts are not toxic to mouse
peritoneal cells but may stimulate their growth. The
extracts may boost growth of the immune cells which
are vital in fighting some bacterial infections.
Conclusion
The acetone, ethanol and total leaf extracts from T. welwistchii showed antibacterial activity against P. aeruginosa ATCC. Membrane disruption was the mode of
action against the bacteria for the three extracts. The
three leaf extracts showed low toxicity, thus, they could
be potential sources of alternative antimicrobials against
infections caused by P. aeruginosa. Studies will be
conducted on the extracts in order to isolate and characterise the specific compounds responsible for these antibacterial activities.
Abbreviations
ANOVA: One way analysis of variance; ATCC: American type control culture;
CFU: Colony forming units; DCM: Dichloromethane; DMSO: Dimethyl
sulfoxide; MTT: 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium;
PBS: Phosphate buffered saline; R6G: Rhodamine 6G; RPMI: Roswell Park
Memorial Institute; TSA: Tryptic soy agar; TSB: Tryptic soy broth
Acknowledgements
The authors acknowledge the assistance of Biochemistry staff and
technicians with technical assistance on this project.
Authors’ contributions
MM conducted the experimental studies and data analyses. SM
conceptualised, designed and directed the study. MM and SM finalised the
manuscript. Both authors read and approved the final version of the
manuscript.
Authors’ information
Prof Stanley Mukanganyama is an associate professor of Biochemistry at the
University of Zimbabwe. He holds a PhD in Biochemistry and specialised in
drug metabolism, particularly the contribution of Phase II enzyme systems in
xenobiochemistry. He is a member of the Biochemistry and Molecular
Biology Society of Zimbabwe (BMBSZ), is the Head of the Department of
Biochemistry and the In-country president of the Natural Products Research
of Eastern and Central Africa (NAPRECA-Zimbabwe. Ms. Molly Mombeshora
(BSc. Hons Applied Biology and Biochemistry, MSc Biotechnology) is a Temporary
lecturer and postgraduate student in the Department of Biochemistry. She is the
treasurer of the BMBSZ and a committee member of NAPRECA.
Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
Funding
This work was supported by the International Science Programmes (ISP)
through the IPICSZIM01 Project (International Program in the Chemical
Sciences (IPICS), Uppsala University, Sweden), Centre for Emerging and
Neglected Diseases (CEND), University of California, Berkeley and the German
Academic Exchange Service (DAAD). ISP through IPICS provided funds for
the purchase of chemicals and consumables. ISP and CEND provided
funding for the equipment used in the study. DAAD paid for the tuition of
MM during the study period. Funding bodies played no role in the design of
the study; collection, analysis, and interpretation of data; and in writing the
manuscript.
Availability of data and materials
The data sets generated during and/ analysed during the current study are
available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was approved by the Faculty of Higher Degrees Committee (HD/
71/16) Harare, Zimbabwe.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Received: 23 May 2019 Accepted: 11 October 2019
References
1. Nathwani D, Raman G, Sulham K, Gavaghan M, Menon V. Clinical and
economic consequences of hospital-acquired resistant and multidrugresistant Pseudomonas aeruginosa infections: a systematic review and metaanalysis. Antimicrob Resist Infect Control. 2014;3:1–16.
2. Ngameni B, Fotso GW, Kamga J, Ambassa P, Abdou T, Fankam AG, et al.
Flavonoids and related compounds from the medicinal plants of Africa.
Med Plant Res Afr. 2013;Chapter 9.
3. Adonizio A, Leal SM Jr, Ausubel FM, Mathee K. Attenuation of Pseudomonas
aeruginosa virulence by medicinal plants in a Caenorhabditis elegans model
system. J Med Microbiol. 2008;57:809–13.
4. Raja R, Sreenivasulu RM. Medicinal plants secondary metabolites used in
pharmaceutical importance - an overview. World J Pharm Sci. 2015;4:
436–47.
5. Chinemana F, Drummond RB, Mavi S, De Zoysa I. Indigenous plant
remedies of Zimbabwe. J Ethnopharmacol. 1985;14:159–72.
6. Gelfand M, Mavi S, Drummond RB, Ndemera B. The traditional medical
practitioner in Zimbabwe. Gweru: Mambo Press; 1985. p. 79.
7. Clarkson C, Maharaj VJ, Crouch NR, Grace OM, Pillay P, Matsabisa MG, et al.
Antiplasmodial activity of medicinal plants native to or naturalised in South
Africa. J Ethnopharmacol. 2004;92:177–91.
8. Moyo B, Mukanganyama S. Antiproliferative activity of T. welwitschii extract
on Jurkat T cells in vitro. BioMed Res Int. 2015;2015:817624.
9. Moyo B, Mukanganyama S. Antibacterial effects of Cissus welwitschii and
Triumfetta welwitschii extracts against Escherichia coli and Bacillus cereus. Int
J Bacteriol. 2015;2015:162028.
10. Marime L, Chimponda T, Chirisa E, Mukanganyama S. Antimycobacterial
effects of Triumfetta welwitschii extracts on Mycobacterium aurum and
Mycobacterium smegmatis. J Antimicrobiol Photon. 2014;129:319–32.
11. Tropical Plants Database, Ken Fern. tropical.theferns.info. 2019. http://
tropical.theferns.info/viewtropical.php?id=Triumfetta+rhomboidea.
Accessed 2 Sept 2019.
12. Devmurari VP, Ghodasara TJ, Jivani NP. Antibacterial activity and
phytochemical study of ethanolic extract of Triumfetta rhomboidea Jacq.
IJPSDR. 2010;2:40–2.
13. Ahmed SS, Ibrahim ME, Khalid AK, El-Sawi SA. Phytochemicals, volatile
oil and biological activities of Triumfetta flavescens (Hochst). IFRJ. 2017;
24:2102–6.
14. Haque M, Sartelli M, McKimm J, Bakar MA. Health care-associated infections
– an overview. Infec Drug Res. 2018;11:2321–33.
(2019) 19:315
Page 11 of 12
15. Mbahi MA, Mbahi AM, Umar IA, Ameh DA, Joseph I, Amos PI.
Phytochemical screening and antimicrobial activity of the pulp extract and
fractions of Ziziphus mauritiana. Biochem Anal Biochem. 2018;7:1–6.
16. Pandey A, Tripathi S. Concept of standardization, extraction and pre
phytochemical screening strategies for herbal drug. JPP. 2014;2:115–9.
17. EUCAST (European Committee for Antimicrobial Susceptibility Testing).
Determination of minimum inhibitory concentrations (MICs) of antibacterial
agents by broth dilution. Clin Microbiol Infect Dis. 2003;9:1–7.
18. Crowley LC, Scott AP, Marfell BJ, Boughaba JA, Chojnowski G, Waterhouse
NJ. Measuring cell death by propidium iodide uptake and flow cytometry:
Cold Spring Laboratory Press; 2016. p. 647–52.
19. Chitemerere TA, Mukanganyama S. In vitro activity of selected medicinal
plants from Zimbabwe. Afric J Plant Sci Biotechnol. 2011;5:1–7.
20. Malagoli D. A full-length protocol to test hemolytic activity of palytoxin on
human erythrocytes. ISJ. 2007;4:92–4.
21. Lee CC, Tsai WS, Hsieh H, Hwang DF. Hemolytic activity of venom from
crown-of-thorns starfish Acanthaster planci spines. J Venom Anim Toxins incl
Trop Dis. 2013;19:170–7.
22. Directive 2010/63/EU of the European Parliament and of the Council of 22
September 2010 on the protection of animals used for scientific purposes.
Off J Eur Union 276, 33–79.
23. Ray A, Dittel BN. Isolation of mouse peritoneal cavity cells. J Vis Exp. 2010;35:
e1488. https://doi.org/10.3791/1488.
24. Mapfunde S, Sithole S, Mukanganyama S. In vitro toxicity determination of
antifungal constituents from Combretum zeyheri. BMC Complement Altern
Med. 2016;16:1–11.
25. Mahmoudvant H, Mousavi SAA, Sepahvand A, Sharififar F, Ezatpour B,
Gorohi F, Dezaki ES, Jahanbakhsh S. Antifungal, antileishmanial, and
cytotoxicity activities of various extracts of Berberis vulgaris (Berberidaceae)
and its active principle berberine. ISRN Pharmacol. 2014;2014:602436.
26. Tiwari P, Kumar B, Kaur M, Kaur G, Kaur H. Phytochemical screening and
extraction: a review. Inter Pharm Sci. 2011;1:98–106.
27. Martini N, Eloff JN. The preliminary isolation of several antibacterial
compounds from Combretum erythrophyllum (Combretaceae). J
Ethnopharmacol. 1998;62:255–63.
28. Nikaido H. Molecular basis of bacterial outer membrane permeability
revisited. Microbiol Mol Biol Rev. 2003;67:593–656.
29. Onivogui G, Letsididi R, Mohamed D, Wang L, Song Y. Influence of extraction
solvents on antioxidant and antimicrobial activities of the pulp and seed of
Anisophyllea laurina R. Br. Ex Sabine fruits. Asian Pac J Trop Biomed. 2016;6:20–5.
30. Bourgaud F, Poutaraud A, Guckert A. Extraction of coumarins from plant
material (Leguminosae). Phytochem Anal. 1994;5:127–32.
31. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, et al.
Complete genome sequence of Pseudomonas aeruginosa PA01, an
opportunistic pathogen. Nat. 2000;406:959–64.
32. Garvey MI, Rahman M, Gibbons S, Piddock LJV. Medicinal plant extracts with
efflux inhibitory activity against gram-negative bacteria. Int J Antimicrob
Agents. 2011;37:145–51.
33. 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.
34. Lu W, Lin H, Hsu P, Lai M, Chiu J, Lin HV. Brown and red seaweeds serve as
potential efflux pump inhibitors for drug-resistant Escherichia coli. Evid
Based Complement Alternat Med. 2019;2019:1836982.
35. Gbelska Y, Hervay NT, Dzugasova V, Konecna A. Measurement of energydependent rhodamine 6G efflux in yeast species. Bio-protocol. 2017;7:1–6.
36. Sonnet P, Izard D, Mullie C. Prevalence of efflux-mediated ciprofloxacin and
levofloxacin resistance in recent clinical isolates of Pseudomonas aeruginosa and
its reversal by the efflux pump inhibitors 1-(1-naphthylmethyl)-piperazine and
phenylalanine-arginine-β-naphthylamide. Int J Antimicrob Agents. 2012;39:77–80.
37. Saritha K, Rajesh A, Manjulatha K, Setty OH, Yenugu S. Mechanism of
antibacterial action of the alcoholic extracts of Hemidesmus indicus (L.) R. Br.
Ex Schult, Leucas aspera (wild.), Plumbago zeylanica L., and Tridax
procumbens (L.) RR. Br. Ex Schult. Front Microbiol. 2015;6:1–9.
38. Mautsa R, Mukanganyama S. Vernonia adoensis leaf extracts cause cellular
membrane disruption and nucleic acid leakage in Mycobacterium
smegmatis. JBAPN. 2017;7:140–56.
39. Chovanová R, Mikulášová M, Vaverková S. In vitro antibacterial and antibiotic
resistance modifying effect of bioactive plant extracts on methicillin
resistant Staphylococcus epidermidis. Int J Microbiol. 2013;2013:e760969.
https://doi.org/10.1155/2013/760969.
Mombeshora and Mukanganyama BMC Complementary and Alternative Medicine
40. Kavitha D, Vidhya S, Padma PR. Investigation on the mechanism of action of
the leaves of Trianthema portulacastrum on human pathogens. Asian J
Pharm Clin Res. 2016;9:135–40.
41. Sa’nchez E, García S, Heredia N. Extracts of edible and medicinal plants
damage membranes of Vibrio cholera. Appl Environ Microbiol. 2010;76:
6888–94.
42. Kumar G, Karthik L, Rao KVB. Haemolytic activity of Indian medicinal
plants toward human erythrocytes: an in vitro study. Elixir Appl Botany.
2011;40:5534–7.
43. Sulaiman TC, Gopalakrishnan KV. Radical scavenging and in-vitro hemolytic
activity of aqueous extracts of selected Acacia species. J App Pharm Sci.
2013;3:109–11.
44. Ishnava K. Anticariogenic and hemolytic activity of selected seed. J
Dentistry. 2014;11:576–86.
45. Khalili M, Ebrahimzadeh MA, Safdari Y. Antihaemolytic activity of thirty
herbal extracts in mouse red blood cells. Arch Ind Hyg Toxicol. 2014;
65(4):399–406.
46. Zohra M, Fawzia A. Haemolytic activity of different herbal extracts used in
Algeria. IJPSR. 2014;5:495–500.
47. Kalegari M, Miguel MD, Dias JFG, Lordello ALL, Peitz de Lima C, CMS M,
et al. Phytochemical constituents and preliminary toxicity evaluation of
leaves from Rourea induta planch. (Connaraceae). Braz J Pharm Sci. 2011;
47(3):635–42.
48. Acker JP, Croteau MI, Yi QL. An analysis of the bias in red blood cell
hemolysis measurement using several analytical approaches. Clin Chim
Acta. 2012;413:1746–52.
49. Vidhya R, Udayakumar R. Phytochemical screening and evaluation of in vitro
haemolytic, thrombolytic and antiinflammatory activities of Aerva lanata (l.).
IAJPS. 2016;6:6–7.
50. Zhang X, Gonçalves R, Mosser DM. The isolation and characterization of
murine macrophages. Curr Protoc Immunol. 2015; Chapter:Unit 14.1.
51. Vinjamuri S, Shanker D, Ramesh RS, Nagarajan S. In vitro evaluation of
haemolytic activity and cell viability Assa of hexanoic extracts of Bridellia.
WJPPS. 2015;4:1263–8.
52. Ragupathi G, Gardner JR, Livingston PO, Gin DY. Natural and synthetic
saponin adjuvant QS-21 for vaccines against cancer. Expert Rev Vaccines.
2011;10:463–70.
53. Sun Y, Hu X, Li W. Antioxidant, antitumor and immunostimulatory activities
of the polypeptide from Pleurotus eryngii mycelium. Int J Biol Macromol.
2017;97:323–30.
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