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Aqueous extracts of Flacourtia indica,Swartzia madagascariensis and
Ximenia caffra are strong antibacterial agents against Shigella spp.,
Salmonella typhi and Escherichia coli O157
Constance Chingwaru
a
, Tanja Bagar
b
, Walter Chingwaru
a,
*
a
Biological Sciences Department, Faculty of Science & Engineering, Bindura University of Science Education, Zimbabwe
b
ICANNAInternational Institute for Cannabinoids, Kodeljevo Castle, Ulica Carla Benza 16. SI-1000 Ljubljana Slovenia
ARTICLE INFO
Article History:
Received 4 May 2019
Revised 4 September 2019
Accepted 27 October 2019
Available online 13 November 2019
Edited by JJM Meyer
ABSTRACT
Aqueous extraction methods, particularly steeping in hot water, are frequently adopted as methods of choice
in traditional medicine (TM). Moreover, other more modern extraction methods tend to utilise sophisticated
tools and a number of organic solvents. Unfortunately, the comparative value of the aqueous extraction
methods as used in traditional medicine in relation to the other methods has remained largely undocu-
mented. This study sought to establish the comparative antimicrobial activities and composition of Flacourtia
indica bark, Swartzia madagascariensis leaf and Ximenia caffra leaf extracts obtained using (i) hot water
extraction (THWE), (ii) cold water extraction (CWEC) and (iii) ethanolic extraction (EEC) methods against
putative isolates of Shigella spp., Salmonella typhi and Escherichia coli O157. Bacteriostatic and bactericidal
effects of the extracts were determined using the standard well diffusion assay. The hot water extracts
(THWE) and the cold water extracts (desiccated) (CWEC) were shown to have significantly greater inhibitory
activities then the ethanolic extracts (EEC) (desiccated) against Shigella spp. and S. typhi (p<0.05). The chem-
ical composition of the extracts obtained from the THWE method was shown to be comparable to that of
extracts from the other two methods (CWEC and EEC) albeit the former having excluded desiccation as per-
formed with the latter two. This study therefore validates the widespread use of steeping (THWE) as a
method of choice in traditional medicine as it yields extracts with high antimicrobial activities and chemical
composition that are comparable to those from the other two methods. We recommend the use of the THWE
method in preparation of medicinal products from Flacourtia indica bark, Swartzia madagascariensis leaves,
and Ximenia caffra leaves for the management of diarrhoea that is caused by S. typhi or Shigella spp, but not
by E. coli. Conservation practices are also recommended to ensure sustenance of the biodiversity of the
medicinal flora.
© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
Keywords:
Extraction method
Antidiarrhoea
Medicinal
Phytochemicals
1. Introduction
The use of plants or their products as sources of traditional medi-
cines (TM) has, since historic times, remained significant in the man-
agement of various ailments including diarrhoea (Maroyi, 2016).
Notably, there is a renewed public interest in the use of TM owing to
the high costs of orthodox medicines and their associated side effects,
especially antimicrobial resistance (Patwardhan and Patwardhan,
2006). In African societies, 80% of the population reportedly rely on
TM for economic and cultural reasons (Maroyi, 2016). As such, efforts
are underway in many African countries to ensure proper regulation
and monitoring of TM (Patwardhan and Patwardhan, 2006).
Diarrhoeal diseases remain one of the leading global public health
threats with high morbidity and mortality especially in children
under the age of 5 years (Maroyi, 2016). Approximately 1 to 9 million
deaths are reported globally among children aged 5 years of younger,
with the highest rates occurring in low income countries (LIC) espe-
cially in Sub-Saharan Africa and Asia (Troeger et al., 2017;Njume
et al., 2011). Most communities in LIC tend to rely on phytomedicines
to manage various forms of diarrhoea including cholera, typhoid and
various forms of gastroenteritis (Bekoe et al., 2017;Dhama et al.,
2014).
Anti-diarrhoeal properties of medicinal plants continue to be
reported or validated by scientists around the globe. Medicinal plants
are largely seen as a favoured alternative to orthodox medicines
(Maroyi, 2016), owing to their greater health promoting properties
and fewer side effects than the latter (Cornelio Favarin et al., 2013;
Karimi et al., 2015;Moyo and Van Staden, 2014;Ramalingum and
Mahomoodally, 2014).
*Corresponding author.
E-mail addresses: wchingwaru@yahoo.co.uk,wchingwaru@buse.ac.zw
(W. Chingwaru).
https://doi.org/10.1016/j.sajb.2019.10.022
0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
South African Journal of Botany 128 (2020) 119127
Contents lists available at ScienceDirect
South African Journal of Botany
journal homepage: www.elsevier.com/locate/sajb
Plants have maintained their place in the traditional management
of diseases due to their rich composition of healthful bioactive chemi-
cal compounds/metabolites (Crozier et al., 2006). Bioactive com-
pounds are accumulated as secondary metabolites within plant
tissues (Crozier et al., 2006). These metabolites are often accessed
through the use of extraction techniques. However some plant mate-
rials are consumed whole to achieve the desired treatment. The com-
position of an extract depends on its inherent chemical composition,
the solvent used and the protocol followed during the extraction pro-
cess (Muhamad et al., 2017).
Traditional medicine has largely relied on the use of organic based
solvents, primarily water, in the preparation of the respective medi-
cines. However, the utilisation of these traditional extraction meth-
ods is often limited by a low extraction yield (Teo et al., 2010). As a
result, scientists have adopted protocols that use a number of organic
solvents in combination to maximize the extraction yield (Liu, 2008).
Well funded and equipped laboratories in the developed world can,
on the other hand, access highly efficient protocols that include vari-
ous high quality solvents and pieces of equipment to aid the accumu-
lation of the desired bioactive compounds. Scientists have developed
protocols that rely on a number of solvents with differing polarities
to ensure efficient extraction of compounds (Wong and Kitts, 2006).
Many laboratories in the LIC rely on the use of rudimentary extrac-
tion techniques that normally involve the use of solvents to dissolve
compounds and evaporation techniques to dry / enrich the extracts.
Traditional African medicine (TAM) tends to rely on the use of sim-
ple hot water extraction (steeping) and cold water extraction methods
(Mothibe and Sibanda, 2019). In TAM, aqueous extracts (hot or cold
water based) are often obtained from fresh or dried plant materials
and administered within hours following extraction (De Wet and Ngu-
bane, 2014;Odunmbaku et al., 2018). The hot water extraction method
is essentially a steeping method which allows users to administer
extracts within a short period of time after extraction (Hajiaghaalipour
et al., 2016). Sample preparation and extractive strategies are impor-
tant in traditional medicine since they affect both the quality and
quantity of bioactive compounds resulting in differences in clinical
efficacy (De Monte et al., 2014; Azwanida, 2015). Pharmacological
properties of medicinal plants are attributed to the presence of plants’
secondary metabolites which include alkaloids, saponins, tannins,
anthocyanins, flavonoids and phenolic acid (Chikezie et al., 2015;Win-
tola and Afolayan, 2015). The efficiency of the extraction procedures
that are followed reportedly affect the bioactivity of the resultant
extracts (Pilon et al., 2016).
More sophisticated / modernmethods in TAMtend to rely on water
and/or commercial solvents as well as simple to sophisticated pieces of
apparatus and equipment as compared to the traditional methods
(Oosthuizen et al., 2018). Additionally, more modern methods often
incorporate a concentration / desiccation step - where phytomedicines
are exposed to processes such as granulation, extrusion, spheronisa-
tion and freeze drying; with products available as powdered products,
capsules or tablets (de Araujo-Junior et al., 2013;Muley et al., 2016;
Qusaj et al., 2012;Zhang et al., 2018). Different extractive strategies
reportedly yield products with different clinical efficacies (Azwanida,
2015). The modern extraction methods that rely on a number of sol-
vents have major drawbacks since many such solvents are not friendly
to the environment (Vardanega et al., 2014). Greener extraction meth-
ods that avoid the use of organic solvents and hence are environmen-
tally friendly such as pressurised hot water extraction have been
adopted (Ong and Len, 2003; Teo et al., 2008).
Methods are available, however, to reduce the toxicity of extracts
obtained with organic solvent, including the evaporation of solvents
in equipments such as the Soxhlet apparatus or simple unaided evap-
oration (Sicaire et al., 2015). Evaporation is seen as an essential step
in order to increase bioactivity of extracts (Adenusi and Odaibo,
2009). Evaporation is often achieved through the use of apparatus
such as Soxhlet (Sicaire et al., 2015). Unfortunately, exposure of
extracts to agents of the weather or high temperatures may drasti-
cally affect composition and bioactivity of the resultant extracts/
products (Leung et al., 2018;Ngadze et al., 2018). Besides, the tradi-
tional extraction methods are often done over long periods of time,
further compromising products’bioactivity and composition (Pan
et al., 2013). As such processes such as oxidation and the natural
decay of materials exposed to heat or other elements of weather are
known to reduce the efficacies of the resultant phytomedicinal prod-
ucts (Ngadze et al., 2018;Pan et al., 2013).
Plant sample composition analysis can be accomplished using
technologies that range from simple qualitative tests including sim-
ple biochemical tests to the use of sophisticated pieces of equipment
such as liquid chromatography (LC), hyphenated to photo diode array
(PDA) and/or mass spectrometry (MS) detectors (de Vos et al., 2007).
However, the fact that plants produce a plethora of metabolites with
varying biochemical profiles, the selection of a phytochemical analyt-
ical tool poses undisputed analytical challenges (Ong, 2004). Qualita-
tive methods such as biochemical tests tend to give approximations
of components only; hence such methods are deemed unreliable
(Ezeonu and Ejikeme, 2016). However, qualitative composition ana-
lytical methods tend to be adopted as methods of choice in resource
limited settings (Gul et al., 2017) including a number of countries in
Africa and Asia.
Despite the use of the traditional aqueous methods side by side
with newer extraction and packaging methods, no study has com-
pared efficacies of the resultant extracts / medicinal products. As
such, there is an urgent need to close the existing knowledge gap.
The current study sought to compare the efficacies of the traditional
aqueous extraction method (without desiccation / concentration)
and the aqueous and ethanolic extraction methods with evaporation
or concentration. This study is expected to inform both the traditional
and orthodox medicinal practices on the relative utilities of the avail-
able methods. The current study was designed in order to assess the
validity of the traditional method which is a method of choice by tra-
ditional healers and members of the public in LIC.
2. Materials and methods
2.1. Collection and processing of plant materials
Fresh samples of, Flacourtia indica (Burm. f.) Merr. bark, Swartzia
madagascariensis (Desv.) J.H. Kirkbr. & Wiersama leaves, and Ximenia
caffra Sond. var. caffra leaves were collected in Chiraswa Village
under Chief Mangwende in Murehwa, Mashonaland East Province of
Zimbabwe (around -17°.69071.55}S, 31°.96048.90}E) during the months
from October to December 2018. Collection was done with informed
consent of the owner of the plot where the plants were found. Spe-
cies identification was done by qualified botanists at the National
Herbarium and Botanic Garden, Harare. Voucher specimens were
deposited in the Biological Sciences laboratory for future reference.
The collected plant materials were air-dried under shade at room
temperature for 72 h and ground using an electric grinder into fine
powders which were stored into dark airtight containers at room
temperature. Remaining fresh samples to be used in the traditional
extraction method were frozen in airtight plastics for future use.
2.2. Extraction
2.2.1. Cold water (CWEC) and ethanolic extraction (EEC)
The powdered samples were extracted using 150 mL of cold dis-
tilled water and 70% ethanol respectively. The extracts were continu-
ously swirled at 150 rpm on a rotary shaker for 72 h. The extracts were
filtered using a Whatman filter paper number 1 and the collected fil-
trates were concentrated and stored at 20 °C until further analysis.
120 C. Chingwaru et al. / South African Journal of Botany 128 (2020) 119127
2.2.2. Hot water extraction (THWE)
Fresh plant samples (10 g) were added to 100 mL of hot boiled
water and steeped for 30 min. The samples were filtered under asep-
tic conditions and were stored at 20 °C for further tests.
2.2.3. Qualitative phytochemical analysis
Phytochemical analysis was done on stored crude extracts
obtained using the CWEC, THWE and EEC methods. Phytochemical
analysis included tests for tannins, flavonoids, reducing sugars, sapo-
nins and alkaloids. Qualitative analysis of phytochemicals was taken
as the method of choice due to limited resources in the Biological Sci-
ences laboratory at Bindura University of Science Education. More
advanced methods could have resulted in the detection and isolation
of specific compounds.
2.2.4. Test for tannins (Ferric chloride test)
A few drops of 0.1 ferric chloride were added to 2 ml of aqueous
extracts (CWEC, EEC and THWE). A blue coloration indicated the
presence of tannins (Oseni et al., 2011).
2.2.5. Test for flavonoids
Dilute ammonia (5 ml) solution was added to each plant extract
(1 ml). Concentrated sulphuric acid was added and a yellow colora-
tion in each plant extract indicated the presence of flavonoids
(Edeoga et al., 2005).
2.2.6. Test for alkaloids
Aqueous hydrochloric acid (5 ml, 1%) was added to extracts and
heated in steam bath for 10 min. The aqueous extract solution (1 ml)
was treated with 610 drops of Dragendoff’s reagent. A creamish
precipitate indicated the presence of alkaloids (Oseni et al., 2011)
2.2.7. Test for saponins
Aqueous extracts was mixed with distilled water (5 ml) and
shaken vigorously for stable persistence froth. The froth was mixed
with 3 drops of olive oil and was shaken vigorously. Emulsion indi-
cated the presence of saponins (Oseni et al., 2011).
2.2.8. Test for flavonoids
Dilute ammonia solution (5 ml) was added to 1 ml of each plant
extract. Concentrated hydrochloric acid was added to the test tubes.
A yellow coloration indicated the presence of flavonoids (Edeoga
et al., 2005).
2.2.9. Test for reducing sugars
To 1 ml of the plant extract, a few drops of Benedict’s reagent
(alkaline solution containing cupric citrate solution) was added and
boiled in a water bath. A reddish brown precipitate indicated the
presence of reducing sugars (De et al., 2010).
2.3. Bacterial strains
The microorganisms used in determination of the antibacterial
activity of the plant extracts’were as follows: presumptive E. coli
O157, Shigella spp. and S. typhi. All bacterial strains were obtained
from our internal stock of strains isolated from various environmen-
tal sources. The different strains were maintained on nutrient agar
and subcultures were freshly prepared before use. The bacterial cul-
tures were prepared by transferring two colonies of the bacteria into
a universal bottle containing 10 ml of nutrient broth and allowed to
grow overnight at 37 °C.
2.4. Antibacterial screening
Antibacterial tests were performed using the agar well diffusion
assay. Agar plates were prepared using sterile HiCrome O157: H7
agar and XLD agar for E. coli O157 and Shigella spp. spp./S. typhi,
respectively. The turbidity of the bacterial cultures was standardised
to 0.2 using a Biobase Microplate Reader Biobase-EL10B at 620 nm.
The standardised cultures were evenly spread onto the surface of the
agar plates using sterile swab sticks. Wells (10 mm in diameter) were
made in each plate with sterile auger. Sixty (60) microlitres of ethanol
and water extracts (100 mg/ml) were added in each well were strep-
tomycin was used as the positive control. Each extract and antibiotic
was tested in triplicate. The diffusion of the extracts was allowed at
room temperature for 1 h in a laminar flow cabinet. The plates were
then incubated at 37 °C for 24 h.
Antibacterial activity was shown in terms of the diameter of zones
of inhibition of each treatment. The absence of the zone of inhibition
around the wells was interpreted as the absence of activity. The zones
of inhibition were measured in millimetres.
2.5. Determination of minimal inhibitory concentration (MIC)
The minimal inhibitory concentration (MIC) of the plant extracts /
control was determined using the classic well diffusion assay. Briefly,
sterile HiCrome 0157:H7 media (for E. coli 0157) and XLD media (for
Salmonella and Shigella spp.) were prepared following manufacturer’s
instructions. The turbidity of the bacterial cultures was standardised to
an optical density of 0.2 using a Biobase Microplate Reader Biobase-
EL10B (Jinan, China) at 620 nm. Optical density of 0.2 at 620 nm was
shown to yield a bacterial concentration of 1 £10
9
CFU/mL based on
an internal growth kinetic curve. The standardised cultures were
evenly spread on the surface of the agar plates using sterile swabs
under sterile conditions. Wells (5 mm diameter) were made in each
plate with a sterile auger. 60 microlitres of plants extracts of known
concentrations ranging from 0100 mg/ml were added in each well.
Streptomycin was the positive control. The diffusion of the extracts
was allowed at room temperature for 1 h in a sterile laminar flow cabi-
net and the plates were incubated at 37 °C for 24 h. The plates were
observed for antimicrobial activity and the diameters of zones of inhibi-
tion (mm) were measured using a ruler to indicate the minimum con-
centration at which the extracts inhibited the growth of the test
microorganisms. The concentration at which there was no zones of
inhibition were recorded as the minimum inhibition concentration.
2.6. Determination of minimal bactericidal concentration (MBC)
A modified assay was used to determine the minimum bacteri-
cidal concentration. The agar plates that were used in obtaining MIC
results were used in this assay. The plates were opened under sterile
conditions. A sterile inoculating loop was used to touch the zone of
inhibition of different concentrations of extracts where there was
invisible growth. The loops were used to streak labelled and prepared
agar plates. The plates were incubated for 24 h and observed for
growth at different concentrations.
2.7. Statistical analysis
Statistical analysis was done by two way ANOVA for significance
at p<0.05 between the extraction method and antimicrobial activity
(ZOI at 100 mg / ml)
3. Results
3.1. Extract yield (per unit weight of plant material)
Table 1 shows the yield of extraction methods on selected parts of
F. indica bark,S.madagascariensis leaves and X. caffra leaves. Extract
yield was not determined in THWE extracts because the concentra-
tion by drying was not included in obtaining the extracts. Generally,
45 g of material from each plant yielded roughly between 300 and
C. Chingwaru et al. / South African Journal of Botany 128 (2020) 119127 121
700 mg of desiccated extract, except for CWEC extract of X. caffra
(42.20 g) that yielded 2 160 mg. This translates to: 1kg of plant mate-
rial would yield between 66 and 150 g of desiccated extracts.
3.2. Phytochemical composition of extracts
Table 2 shows the phytochemical composition of extracts
obtained using different extraction methods (CWEC, THWE and EEC).
The results also revealed the presence and absence of phytochemicals
in different extracts.
3.2.1. Summary
The following hot water extracts (THWE) showed greater concen-
trations than the other two types: (i) saponins in F. indica bark
extract, (ii) flavonoids in S. madagascariensis leaf extract and (iii) X.
caffra leaf extract. The cold water extracts (CWEC) of X. caffra leaves
showed higher concentration of tannins than the other two extracts.
3.3. The effect of extract concentrations on inhibition of E. coli O157:H7
in vitro
Fig. 1 shows the effect of F. indica bark-, S. madagascariensis leaf-
and X. caffra leaf extract concentrations on inhibition of E. coli O157:
H7 in vitro. The graphs also shows the minimum inhibition concen-
trations (MIC) of the extracts where the line graphs touch the Xaxis.
3.3.1. Summary
Ethanolic extracts (EEC) of F. indica (Fig. 1a) and X. caffra (Fig. 3c)
had greater inhibition compared to CWEC extracts, in terms of; (i) the
EEC inhibition graphs were higher over the concentrations used, (ii)
EEC attained lower MIC values [F. indica; 3.125 mg/ml (EEC) vs 25 mg/
ml (CWEC) and X. caffra; 0.8 mg/ml (EEC) vs 1.6 mg/ml (CWEC)]. How-
ever, CWEC extracts of S. madagascariensis (Fig. 1b) had greater inhibi-
tion compared to EEC extracts, in terms of; (i) the CWEC inhibition
graphs were higher over the concentrations used, (ii) CWEC attained
lower MIC values [0.8 mg/ml (CWEC) vs 12.5 mg/ml (EEC)]. All THWE
extracts had no inhibition activity against E. coli O157.
3.4. The effect of extract concentrations on inhibition of S. typhi in vitro
Fig. 2 shows the effect of F. indica bark-, S. madagascariensis leaf-
and X. caffra leaf extract concentrations on inhibition of S. typhi in
vitro. The graphs also shows the minimum inhibition concentrations
(MIC) of the extracts where the line graphs touch the Xaxis.
3.4.1. Summary
Hot water extracts (THWE) of all plants (Fig. 2a, 2b and 2c) had
greater inhibition compared to CWEC and EEC extracts. The THWE
inhibition graphs were higher over the concentrations 3.1 mg/ml to
100 mg/ml (Fig. 2a and 2b) and concentrations 3.1 mg/ml to 30 mg/
ml (Fig. 2c). However, MIC values for THWE extracts were generally
not lower than those for EEC and CWEC.
3.5. The effect of extract concentrations on inhibition of Shigella spp. in
vitro
Fig. 3 shows the effect of F. indica bark-, S. madagascariensis leaf-
and X. caffra leaf extract concentrations on inhibition of Shigella spp.
in vitro. The graphs also show the minimum inhibition concentrations
(MIC) of the extracts where the line graphs touch the Xaxis.
3.5.1. Summary
Based on results in Fig. 3, the following outcomes were noted:
THWE extracts of F. indica bark and S. madagascariensis leaves had
the lowest minimum inhibition concentration (1.562 mg/ml) against
Shigella spp.. CWEC and EEC extracts had an MIC of 3.125 mg/ml in
F. indica bark and S. madagascariensis leaves. Ethanolic extracts of
X. caffra leaves had the lowest MIC (0.765 mg/ml) as compared to
THWE (1.562 mg/ml) and CWEC (3.125 mg/ml) extracts.
Table 1
Extract yield per unit weight of plant material used and extraction method (hot water
extraction - THWE, cold water extraction with concentration - CWEC and ethanolic
extraction with concentration - EEC).
Plant species and extract Weight of plant
material used (g)
Yield (mg)
F. indica bark CWEC 45.16 700
EEC 45.16 390
THWE 10 N.D
S. madagascariensis leaves CWEC 45.17 320
EEC 45.17 720
THWE 10 N.D
X. caffra leaves CWEC 42.20 2 160
EEC 42.20 510
THWE 10 N.D
Key.
N.D Not determined. Extract was used without evaporation; hence its dry weight
was not determined.
Table 2
Qualitative phytochemical composition of extracts obtained using hot water extraction (THWE), cold water extraction with concentration (CWEC) and
ethanolic extraction with concentration (EEC).
Plant species and extract Saponins Alkaloids Flavonoids Tannins Reducing sugars
F. indica bark CWEC
++ ++ +++
EEC
++ +++
THWE
+++ + +++
S. madagascariensis leaves CWEC
++ ++++
EEC
+++
THWE
++ +++ +++
X. caffra leaves CWEC
++ +++ +++
EEC
+++++
THWE
++ +++ +++
Key.
CWEC = aqueous extract.
EEC = ethanol extract`.
THWE = aqueous extract obtained by the traditional extraction method.
+
presence of phytochemical in trace amounts.
absence of phytochemical.
++
moderate amount of phytochemical.
+++
appreciable amounts of phytochemicals.
122 C. Chingwaru et al. / South African Journal of Botany 128 (2020) 119127
3.6. Analysis of variance of zones of inhibition (ZOI) for extracts at
100 mg/ml
For comparative analysis of antimicrobial activities of the extrac-
tion methods, One way Analysis of Variance was performed at the
highest achievable concentration of each extract (100 mg/ml)
(p<0.05) (Vassarstats Software).
Pairwise Analysis of Variance of zones of inhibition (ZOI) for
extracts at 100 mg/ml against E. coli O157:H7, S. typhi and Shigella spp.
3.7. Analysis
Hot water extracts (THWE) of F. indica yielded similar antimicro-
bial activities against Shigella spp. compared to EEC and CWEC
(p= 0.132). Hot water extracts (THWE) of S. madagascariensis leaves
were shown to have significantly greater inhibition than CWEC
extracts (F= 150, p= 0.0003) and EEC extracts (F= 294, p= 0.0001).
However, CWEC extracts of S. madagascariensis leaves were shown to
have significantly greater inhibition than EEC (F= 24, p= 0.008). Etha-
nolic extracts (EEC) of X. caffra leaves were shown to have signifi-
cantly greater inhibition than CWEC (F= 42.25, p= 0.003) and THWE
(F= 42.25, p= 0.003).
Hot water extracts (THWE) of F. indica yielded significantly
greater antimicrobial activities against S. typhi compared to EEC
(F= 25, p= 0.008) and CWEC (F= 306, p=<0.0001). Ethanolic (EEC) of
F. indica yielded significantly greater antimicrobial activities against
S. typhi compared to CWEC (F= 132, p= 0.003).
Hot water extracts (THWE) of S. madagascariensis yielded signifi-
cantly greater antimicrobial activities against S. typhi compared to
CWEC (F= 544, p<0.0001) and EEC (F= 32, p=0.005). Ethanolic
Fig. 1. Inhibition of E. coli O157:H7 isolate by varying concentrations of F. indica bark (3a), S. madagascariensis leaf (3b) and X. caffra leaf (3c) extracts.
Fig. 2. Inhibition of S. typhi isolate by varying concentrations of F. indica bark (2a), S. madagascariensis leaf (2b) and X. caffra leaf (2c) extracts.
C. Chingwaru et al. / South African Journal of Botany 128 (2020) 119127 123
(EEC) of S. madagascariensis yielded significantly greater antimicro-
bial activities against S. typhi compared to CWEC (F= 312, p<0.0001)
(Table 3).
Ethanolic (EEC) of X. caffra yielded significantly greater antimicro-
bial activities against S. typhi compared to THWE (F= 32, p= 0.02).
Similarly, cold water extracts (CWEC) of X. caffra yielded significantly
greater antimicrobial activities against S. typhi compared to THWE
(F= 32, p= 0.02). Ethanolic (EEC) of X. caffra yielded significantly
greater antimicrobial activities against S. typhi compared to THWE
(F= 1.5, p=0.290) (Table 3).
Cold water extract (CWEC) of S. madagascariensis (Table 3)hadsignifi-
cantly greater inhibition compared to EEC extracts (F= 661, p<0.0001).
3.8. Minimum inhibition concentration (MIC) of extracts
Table 4 below shows minimum inhibition concentration (MIC) of
extracts obtained using hot water extraction (THWE), cold water
extraction with concentration (CWEC) and ethanolic extraction with
concentration (EEC).
3.9. Comparative analysis of inhibitory effects of extracts (ZOI) (100 mg/
ml) and streptomycin (300 mg/ml) against E. coli =157:H7, S. typhi and
Shigella spp
The ZOI for Streptomycin against E. coli O157 was 25 mm. Gener-
ally, CWEC and EEC extracts of the 3 plants yielded greater ZOI than
that of Streptomycin (Table 5).
The ZOI for Streptomycin against Shigella spp. was 20 mm. Gener-
ally, CWEC and EEC extracts of the 3 plants yielded greater ZOI than
that of Streptomycin (Table 5).
The ZOI for Streptomycin against Salmonella spp. was 23 mm.
CWEC and EEC extracts of the 3 plants yielded greater ZOI than that
of Streptomycin. THWE extracts of F. indica bark and X. caffra leaves
yielded greater ZOI than that of Streptomycin against S. typhi. Addi-
Fig. 3. Inhibition of Shigella spp. spp. isolate by varying concentrations of F. indica bark (1a), S. madagascariensis leaf (1b) and X. caffra leaf (1c) extracts. .
Table 3
Pairwise Analysis of Variance of zones of inhibition (ZOI) for extracts at 100 mg/ml against Salmonella typhi and Shigella spp.
Plant species Extracts Fvalue pvalue Fvalue pvalue Fvalue pvalue
Shigella spp. S. typhi E. coli O157:H7
F. indica THWE vs CWEC 3.56 0.132 306 <0.0001
THWE vs EEC 3.56 0.132 24 0.008
CWEC vs EEC 132 0.0003 1.5 0.29
S. madagascariensis THWE vs CWEC 150 0.0003 544 <0.0001
THWE vs EEC 294 <0.0001 32 0.005
CWEC vs EEC 24 0.008 312 <0.0001 661 <0.0001
X. caffra THWE vs CWEC 13.5 0.02
THWE vs EEC 42.25 0.003 13.5 0.02
CWEC vs EEC 42.25 0.003 1.5 0.290
124 C. Chingwaru et al. / South African Journal of Botany 128 (2020) 119127
tionally, CWEC and EEC extracts of X. caffra leaves yielded greater ZOI
than that of Streptomycin against S. typhi (Table 5).
3.10. Relationship between extraction method and antimicrobial
activities of extracts (Two-way ANOVA)
Table 6 shows the level of association between extraction method
and their antimicrobial activities (zone of inhibition at 100 mg/ml)
against E. coli,S. typhi and Shigella spp.
3.10.1. Summary
There was no significant association between extraction method
used and level of inhibition at 100 mg/ml of each extract (F>0.31:
p>0.31) (Table 6).
4. Discussion
Phytomedicines have the potential to be used in the treatment of
diarrhoea and other ailments because of their low cost, safety and
ease of access. Extraction methods reportedly affect the clinical effi-
cacy of phytomedicines. Unfortunately the extraction methods that
are used in traditional medicine have remained largely unvalidated
(Azwanida et al., 2015). In the present study, hot water (THWE), cold
water (CWEC) and ethanolic (EEC) extracts of X. caffra leaves, F. indica
bark and S. madagascariensis leaves were analysed for their antimi-
crobial activities against presumptive E. coli O157, Shigella spp. spp.
and S. typhi, as well as their phytochemical composition.
The extraction methods gave yields (dry mass of desiccated
extracts) that lie between 300 and 2 200mg of the plant material used
(Table 1). This means for one to obtain between 6 and 15g of desic-
cated product, approximately 1 kg of dried plant material should be
used. This means that, should the plants not be domesticated, harvest
for widespread treatment of diseases in LIC would not be sustainable.
Qualitative phytochemical analysis revealed the presence of sapo-
nins, flavonoids, tannins and reducing sugars in the plant extracts
obtained using the different extraction methods (Table 2). Alkaloids
were not detected in all plant extracts. In contrast, previous studies
demonstrated the presence of alkaloids in S. madagascariensis leaf
extract (ethanolic and aqueous) (Sani et al., 2016; Hassan et al.,
2008), X. caffra leaves (Maroyi, 2016) and F. indica stem bark extract
(Swati et al., 2009). Differences in extraction methods, the season of
plant collection as well as the differences in location could have
accounted for the absence of alkaloids. According to Palombo (2006),
alkaloids harbour anti-diarrhoeal properties, however, from the cur-
rent findings it can be suggested that anti-bacterial effects were due
to the presence of other bioactive constituents such as tannins, sapo-
nins and flavonoids.
Of importance was the detection of tannins in S. madagascariensis
leaf extracts (THWE, CWEC and EEC), X. caffra leaf extracts (EEC and
THWE) and F. indica bark extracts (CWEC). This study revealed that the
traditional THWE extraction method was not inferior to the CWEC and
the EEC methods in terms of its extraction of tannins. Previous studies
also reported the presence of tannins in S. madagascariensis ethanolic
and aqueous leaf extracts as well as X. caffra stem bark extracts (Sani
et al., 2016;Maroyi, 2016). Tannins could have contributed to the
observed antimicrobial activities of the extracts against E. coli,Salmo-
nella and Shigella sp as noted in the study of Bamisaye et al. (2013).
Considerable amounts of saponins and reducing sugars were accu-
mulated with all extraction methods with the exception of S. madagas-
cariensis leaf ethanolic extracts. Additionally, flavonoids were shown
to be present in the F. indica bark extract (THWE), S. madagascariensis
ethanolic leaf extract (EEC) and X. caffra cold water leaf extract
(CWEC). Saponins and reducing sugars have been reported to harbour
important pharmacological properties (Bamisaye et al., 2013).
Overall, Ahmed et al. (2014), in a study on the effect of hot versus
cold water extraction of Hibiscus sabdariffa calyxes revealed greater
accumulation of total phenolics, total flavonoids and tannins with
short time high temperature extraction process. The antimicrobial
activity of plant extracts may be attributed to the presence of a num-
ber of phytochemicals such as flavonoids, tannins and saponins that
are reported to harbour antimicrobial properties (Bamisaye et al.,
2013).Yung et al. (2010) demonstrated an increase in the phenolic
acid content and antioxidant activities of Pegaga (Centella asiatica)
extracts with boiling temperature (90 °C). Eramma and Devaraja
(2013) demonstrated high content of flavonoids, saponins and tan-
nins in F. indica back, as well as alkaloids, tannins, terpenoids,
Table 4
Minimum inhibition concentration (MIC) of extracts obtained using hot water
extraction (THWE), cold water extraction with concentration (CWEC) and etha-
nolic extraction with concentration (EEC).
Plant species Part Extraction Microorganism MIC (mg/ml)
F. indica Bark CWEC Shigella spp. spp. 3.125
S. typhi 6.25
E. coli 25
EEC Shigella spp. spp. 3.125
S. typhi 1.562
E. coli 3.125
THWE Shigella spp. spp. 1.562
S. typhi 1.562
E. coli 0
Leaves CWEC Shigella spp. spp. 3.125
S. typhi 0.781
E. coli 0.781
EEC E. coli 3.125
Shigella spp. spp. 1.562
S. typhi 12.5
THWE Shigella spp. spp. 1.562
S. typhi 1.562
E. coli 0
X. caffra Leaves CWEC Shigella spp. spp. 3.125
S. typhi 0.781
E. coli 1.562
EEC Shigella spp. spp. 0.781
S. typhi 0.781
E. coli 0.781
THWE Shigella spp. spp. 1.562
S. typhi 1.562
E. coli 0
Table 5
Extracts with ZOI (at 100 mg/ml) greater than that of Streptomycin against E. coli
O157:H7, Salmonella typhi and Shigella spp.
Plant/Part Extract ZOI (100 mg/ml)
E. coli O157:H7 S. typhi Shigella spp.
F. indica bark THWE 12 24
CWEC 34 24 14
EEC 13 20 10
S. madagascariensis leaves THWE 23 23
CWEC 23 26 28
EEC 24 27 27
X. caffra leaves THWE 11 27
CWEC 26 22 27
EEC 26 20 31
Table 6
Effect of extraction method (hot water extraction - THWE, cold water extraction
with concentration - CWEC and ethanolic extraction with concentration - EEC)
on antimicrobial activities (zone of inhibition) against E. coli, S. typhi and Shigella
spp. spp.
Plant species and extract Fvalue Pvalue Comment
F. indica bark 0.31 0.75 No significant association
S. madagascariensis bark 1.19 0.38 No significant association
X. caffra leaves 1.48 0.31 No significant association
C. Chingwaru et al. / South African Journal of Botany 128 (2020) 119127 125
glycosides and phenolic compounds. Flavonoids have been shown to
harbour high antimicrobial activities against Salmonella species
(Dzoyem et al., 2017). Examples of flavonoids with high antimicrobial
activities include quercetin (Wang et al., 2017), rutin (Arima et al.,
2002) and others. These phytochemicals may have underscored the
observed high antimicrobial activities of the THWE against S. typhi.
Interestingly, all THWE extracts did not yield antimicrobial effects
against E. coli. The observed high accumulation of phenolic substan-
ces (flavonoids and tannins) as well as saponins may be due to the
increased dissolution of these substances with the hot water extrac-
tion method in the present study.
Hot water extracts (THWE) of S. madagascariensis leaves were
shown to harbour significantly greater inhibition against Shigella spp.
than CWEC extracts (F= 150, p= 0.0003) and EEC extracts (F= 294,
p= 0.0001) and compared to CWEC against S. typhi (F= 544,
p<0.0001) and EEC (F= 32, p= 0.005) (Table 3). On the contrary,
CWEC extracts of S. madagascariensis leaves were shown to have sig-
nificantly greater inhibition than EEC against Shigella spp.(F= 24,
p= 0.008), and significantly greater inhibition compared to EEC
extracts against E. coli O157:H7 (F= 661, p<0.0001) (Table 3). The
ethanolic (EEC) of S. madagascariensis leaves yielded significantly
greater antimicrobial activities compared to CWEC against S. typhi
(F= 312, p<0.0001), while EEC extract of X. caffra leaves exhibited
significantly greater inhibition than CWEC (F= 42.25, p= 0.003) and
THWE (F= 42.25, p= 0.003) against Shigella spp (Table 3). These find-
ings show that the aqueous extraction methods (THWE and CWEC)
yielded extracts with significantly greater antimicrobial activities
than the ethanolic exctracts. At the same time, the traditional extrac-
tion method (THWE) yielded significantly greater antimicrobial activ-
ities than the cold water extraction method (CWEC).
When antimicrobial activities of the extracts wee compared to that
of Streptomycin, the following were observed: (i) CWEC and EEC
extracts of the 3 plants yielded greater inhibition (ZOI) against the test
microorganisms, while (ii) THWE extracts of F. indica bark and X. caffra
leaves yielded greater inhibition against S. typhi than the antibiotic.
Streptomycin is a pure aminoglycoside antibiotic with broad activities
particularly against isolates of microbial species that include Yersinia
spp. (Fois et al., 2018), E. coli (Aasmae et al., 2019), S. typhi (Almeida
et al., 2018)andShigella (Baker and H.C., 2018).The THWE method,
which did not include a desiccation step to concentrate the extracts
as done with the other two methods (CWEC and EEC), attained reason-
ably high inhibitory activities. This could have been due to the fact that
the THWE extracts were used fresh (within 24 h from extraction)
which minimised oxidative degradation (Azwanida, 2015). The con-
centration step (dessication) as done with the CWEC and EEC methods
could have increased the oxidative degradation of the active ingre-
dients in the extracts, hence negatively affect their activities. The fact
that CWEC and THWE extracts attained inhibitory activities that were
close to or greater than that of the pure antimicrobial Streptomycin,
demonstrates the validity of the aqueous extraction methods in tradi-
tional medicinal practices. Crude plant extracts frequently attain lower
antimicrobial activities compared to pure antibiotics.
5. Conclusion
In conclusion the traditional extraction method (THWE) and the
cold water extraction method (with desiccation) (CWEC) were shown
to have greater inhibitory activities than the EEC obtained extracts
against the microorganisms used in this study. The chemical compo-
sition of the extracts obtained with the THWE method was shown to
be comparable to that of extracts obtained using the other two meth-
ods (CWEC and EEC) albeit the former having excluded desiccation as
performed with the latter two. This study therefore validates the
widespread use of steeping (THWE) as a method of choice in tradi-
tional medicine as it yields extracts with high antimicrobial activities
and chemical composition that are comparable to extraction methods
(CWEC and EEC). We recommend the use of the THWE method for
the preparation of medicinal products from F. indica bark, S. madagas-
cariensis leaves, and X. caffra leaves for the management of diarrhoea
that is caused by S. typhi or Shigella spp, but not by E. coli. We also rec-
ommend the domestication of F. indica,P. angolensis and S. madagas-
cariensis as well as other species that are used in traditional medicine
in view of the continued or increased reliance on medicinal plants for
the management of ailments by in LIC.
Supplementary materials
Supplementary material associated with this article can be found
in the online version at doi:10.1016/j.sajb.2019.10.022.
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