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Evaluation of Antimicrobial activity and Genotoxic Potential of Capparis spinosa (L.) Plant Extracts by Adwan and Omar

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Objective: The aims of this study were to evaluate the antimicrobial activity and the genotoxic effect of both ethanolic and aqueous extracts of stem and leaf of Capparis spinosa (C. spinosa) plant on Escherichia coli (E. coli) ATCC 25922, Staphylococcus aureus (S. aureus) ATCC 6538P, clinical isolate of Methicillin-resistant S. aureus (MRSA) and Klebsiella pneumoniae (K. pneumoniae) and Candida albicans (C. albicans) ATCC 90028. Materials and Methods: The antimicrobial activity was determined using microbroth dilution method, while the genotoxic effect was investigated using randomly amplified polymorphic DNA (RAPD)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR. Results: The MIC values of both ethanolic and aqueous leaf and stem extracts of C. spinosa plant had a range 6.25 mg/ml to 100 mg/ml. In addition, it was found that ethanolic extract more effective than aqueous extract. The genotoxic activity of aqueous leaf extract, showed changes in both Random Amplified Polymorphic DNA (RAPD)-PCR and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR profiles of E. coli strain treated with extract compared to untreated (negative) control. These changes included an alteration in the intensity, absence or appearance of new amplified fragments. Conclusions: Results of this study strongly show the genotoxic effect of aqueous leaf extract from C. spinosa plant on E. coli. The findings draw awareness to the possible toxic effect use of C. spinosa plant in traditional medicine and point out the capability of using C. spinosa to treat bacterial or fungal infections. More studies are needed to detect the exact ingredients of this plant as well as the mechanisms responsible for genotoxicity. Further in vivo genotoxicity studies are recommended to ensure and to evaluate the safety of using plants for therapeutic purposes. In addition, results of this study showed that molecular fingerprinting based on ERIC-PCR can be used to evaluate the genotoxic effect in the model bacterial species E. coli.
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*Corresponding author: E-mail: adwang@najah.edu;
Microbiology Research Journal International
31(1): 48-57, 2021; Article no.MRJI.67085
ISSN: 2456-7043
(Past name: British Microbiology Research Journal, Past ISSN: 2231-0886, NLM ID: 101608140)
Evaluation of Antimicrobial activity and Genotoxic
Potential of Capparis spinosa (L.) Plant Extracts
Ghaleb M. Adwan
1*
and Ghadeer Ibrahim Omar
1
1
Department of Biology and Biotechnology, An-Najah National University, P.O.Box 7, Nablus,
Palestine.
Authors’ contributions
This work was carried out in collaboration between both authors. Authors GMA and GIO designed the
study, wrote the protocol, conducted experimental work, managed the analysis and wrote the first
draft of the manuscript. Both authors read and approved the final manuscript.
Article Information
DOI: 10.9734/MRJI/2021/v31i130297
Editor(s):
(1)
Dr. Ana Cláudia Coelho, University of Trás-os-Montes and Alto Douro, Portugal.
Reviewers:
(1)
Ajay Vikram Singh, Federal Institute for Risk Assessment, Germany.
(2)
Maria Atanassova, University of Chemical Technology and Metallurgy, Bulgaria.
Complete Peer review History:
http://www.sdiarticle4.com/review-history/67085
Received 15 January 2021
Accepted 21 March 2021
Published 22 March 2021
ABSTRACT
Objective:
The aims of this study were to evaluate the antimicrobial activity and the genotoxic
effect of both ethanolic and aqueous extracts of stem and leaf of Capparis spinosa (C. spinosa)
plant on Escherichia coli (E. coli) ATCC 25922, Staphylococcus aureus (S. aureus) ATCC 6538P,
clinical isolate of Methicillin-resistant S. aureus (MRSA) and Klebsiella pneumoniae (K.
pneumoniae) and Candida albicans (C. albicans) ATCC 90028.
Materials and Methods: The antimicrobial activity was determined using microbroth dilution
method, while the genotoxic effect was investigated using randomly amplified polymorphic DNA
(RAPD)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR.
Results: The MIC values of both ethanolic and aqueous leaf and stem extracts of C. spinosa plant
had a range 6.25 mg/ml to 100 mg/ml. In addition, it was found that ethanolic extract more effective
than aqueous extract. The genotoxic activity of aqueous leaf extract, showed changes in both
Random Amplified Polymorphic DNA (RAPD)-PCR and Enterobacterial Repetitive Intergenic
Consensus (ERIC)-PCR profiles of E. coli strain treated with extract compared to untreated
(negative) control. These changes included an alteration in the intensity, absence or appearance of
new amplified fragments.
Original Research Article
Adwan and Omar; MRJI, 31(1): 48-57, 2021; Article no.MRJI.67085
49
Conclusions:
Results of this study strongly show the genotoxic effect of aqueous leaf extract from
C. spinosa plant on E. coli. The findings draw awareness to the possible toxic effect use of C.
spinosa plant in traditional medicine and point out the capability of using C. spinosa to treat
bacterial or fungal infections. More studies are needed to detect the exact ingredients of this plant
as well as the mechanisms responsible for genotoxicity. Further in vivo genotoxicity studies are
recommended to ensure and to evaluate the safety of using plants for therapeutic purposes. In
addition, results of this study showed that molecular fingerprinting based on ERIC-PCR can be
used to evaluate the genotoxic effect in the model bacterial species E. coli.
Keywords: Capparis spinosa; antimicrobial; genotoxic effect; ethanolic extract; aqueous extract.
1. INTRODUCTION
Plants are considered a rich source of medicinal
and nutraceutical agents for centuries [1-2]. In
the modern age, approximately 25% of the new
drugs originated from plant sources. Among
valuable flora, wild plants have gained much
awareness in recent decades because of their
functional food and potential health benefits [3-4].
Capparis L. is considered the largest genus of
the family Capparaceae (or Capparidaceae).
This genus includes 350 species and is
distributed in many parts of the world, in arid and
semi-arid regions of the tropical and subtropical
world, many of them distributed in the
Mediterranean regions [5]. The Caper (Capparis
spinosa (C. spinosa)) is naturally widely
distributed from the Atlantic coast of the Canary
Island and Morocco to the Black Sea, in Crimea
and Armenia, and to the east side of the Caspian
Sea and Iran. It is also spread in Europe, North
Africa, Australia, West Asia and Afghanistan.
This plant might have emerged in the tropic
areas, and then extended to other
parts of the world such as the Mediterranean
basin and Central Asia [5]. Capparis spinosa
plant is a perennial shrub, thorny, 0.3–1 m tall
and has deep roots, which can extend up to 6-10
m [6-7].
Capparis spinosa is considered a future source
of invaluable nutrient materials for human food
and has been used in traditional medicine to treat
several human infections [6]. Phytochemical
analysis showed that this plant has high
quantities of numerous bioactive ingredients and
molecules, which are responsible for different
pharmacological activities. These activities
include antioxidant effect [6,8-10], antifungal
effect [11], phytotoxic effect [11-12],
anticancer [9,13-14], nephrotoxicity and
hepatotoxicity effects [15], antibacterial effect
[12,16-19], antimutagenic effect [20]. Other
pharmacological effects have also been reported.
Since C. spinosa has several beneficial health
effects on human diseases, the adverse effects
of using or consumption certain parts of this plant
is not studied [3]. This study was conducted to
evaluate the genotoxic potential of the aqueous
extract from C. spinosa growing wild in Palestine
on Escherichia coli (E. coli) ATCC 25922 strain
using Random Amplified Polymorphic DNA
(RAPD)-PCR and Enterobacterial Repetitive
Intergenic Consensus (ERIC)-PCR as well as to
determine the antimicrobial activity of both
ethanolic and aqueous extracts of stem and leaf
of C. spinosa plant.
2. MATERIALS AND METHODS
2.1 Plant Collection and Identification
The stem and leaf parts of C. spinosa plant were
collected from a natural habitat in Tulkarm
province, West Bank-Palestine, during summer,
2019. Identification of the plant was conducted
by the plant taxonomist Dr. Ghadeer Omar,
Department of Biology and Biotechnology, An-
Najah National University, Palestine.
The collected stem and leaf parts of C. spinosa
were washed with water to eliminate soil and
dust particles, then they were dried. Light
exposure was avoided to minimize or prevent
possible loss of active molecules. To obtain a
fine powder that was ready for ethanolic and
aqueous extract preparation, the air dried stem
and leaf parts were powdered using an electric
blender.
2.2 Plant Extract Preparation
2.2.1 Ethanolic extract
Ethanolic extract was prepared as described
previously [21-22] with some modifications.
Briefly, approximately 30 g of dried plant powder
was mixed thoroughly using a magnetic stirrer in
Adwan and Omar; MRJI, 31(1): 48-57, 2021; Article no.MRJI.67085
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150 ml of 80% ethanol. The ethanol-plant powder
part mixture was incubated on a shaker at room
temperature for 48h. The mixture was filtered
using muslin cloth to remove large insoluble
particles. After that, the plant mixture was
centrifuged at 5,000 rpm for 15 min at 4°C, to
remove fine particles. Then, the supernatant
extract was dried in an incubator at 40°C. The
dried plant extract powder was kept in a
refrigerator at 4°C. Before starting the assays,
the dried plant extract powder was dissolved in
10% dimethyl sulfoxide (DMSO) to obtain a final
concentration of 200 mg/ml and stored at 4°C for
further experiments.
2.2.2 Aqueous extract
Aqueous extract was prepared as described
previously [21,22] with some modifications.
Briefly, approximately 30 g of dried plant powder
was mixed thoroughly using a magnetic stirrer in
150-ml cold (room temperature) sterile distilled
water. The water-plant powder part mixture was
incubated on a shaker at room temperature for
48h. The mixture was filtered using muslin cloth
to remove large insoluble particles. After that, the
mixture was centrifuged at 5,000 rpm for 15 min
at C, to remove fine particles. Then, the
supernatant extract was dried and concentrated
by freeze dryer (lyophilizer). The dried plant
extract powder was kept in a refrigerator at 4°C.
Before starting the assays, the dried plant extract
powder was dissolved in sterile distilled water to
obtain a final concentration of 200 mg/ml and
stored at 4°C for further experiments.
2.3 Determination of Antimicrobial
Activity of C. spinosa Extracts
2.3.1 Determination of MIC for plant extracts
by the broth microdilution method
MIC of plant extracts was determined by the
broth microdilution method in sterile 96-well
microtiter plates according to the CLSI
instructions [23]. The plant extract (200 mg/ml of
10% DMSO, 200 mg/ml of sterile distilled water)
and 10% DMSO (negative control) were two-fold-
serially diluted in Mueller Hinton broth directly in
the wells of the plates in a final volume of 100 μl.
After that, a bacterial inoculum size of 10
5
CFU/ml (Candida albicans (C. albicans) inoculum
size of 0.5 to 2.5 × 10
5
CFU/ml) was added to
each well. Negative control wells containing
either 100μl Mueller Hinton broth only, or 100 μl
DMSO with microorganism inoculum, or plant
extracts and Mueller Hinton broth without
microorganism were also included in these
experiments. Each plant extract was performed
in duplicate. The microtiter plates were then
covered and incubated at 37°C for 24h. The MIC
was taken as the lowest concentration of plant
extract, which inhibits the visible growth of the
test microorganism.
2.3.2 Evaluation of the genotoxic potential of
C. spinosa aqueous leaf extract on E.
coli ATCC 25922 strain
2.3.2.1 Inoculation of Escherichia coli ATCC
25922 strain
Few colonies from a 24h old E. coli strain growth
culture plated on Eosin Methylene Blue (EMB)
agar medium were subcultured under sterile
conditions into a bottle containing 20 mL of
nutrient broth, then incubated at 37°C for 1h with
continuous shaking. After that, aseptically, 1 ml
of E. coli culture was added to each of the four
sterile bottles each containing 25 ml nutrient
broth medium. These bottles were incubated at
37°C for 1h with continuous shaking. Then, three
concentrations of aqueous leaf extract (250
μg/ml, 125 μg/ml and 62.5 μg/ml of distilled
water) were added to three bottles of the E. coli
broth culture. The fourth bottle was considered a
negative or untreated control by adding 1 ml of
sterile distilled water.
2.3.2.2 DNA extraction
The DNA genome of E. coli was prepared for
randomly amplified polymorphic DNA (RAPD)-
PCR and enterobacterial repetitive intergenic
consensus (ERIC) PCR according to the method
described previously [24]. Three ml samples
were taken from the E. coli growth culture after 2
h, 5 h, and 24 h, centrifuged for five minutes at
14,000 x g where the supernatant of each
sample was discarded. Then, each bacterial
sample pellet was re-suspended in 0.8 ml of Tris-
EDTA (10 mM Tris-HCl, 1 mM EDTA [pH 8]),
centrifuged for 5 min at 14,000 x g; after that, the
supernatant was discarded. The pellet of each
bacterial sample was re-suspended in 300 μl of
sterile distilled water and boiled for 15 min. Then,
the mixture was incubated in ice for 10 min. The
samples were pelleted by centrifugation at
14,000 x g for 5 min, and each sample
supernatant was transferred into a new
Eppendorf tube. The DNA concentration for each
sample was determined using nanodrop
spectrophotometer (GenovaNano, Jenway) and
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the DNA samples were kept at -20°C for RAPD-
PCR and ERIC-PCR- based DNA fingerprinting
techniques.
2.3.2.3 RAPD-PCR assay and ERIC-PCR assay
The RAPD-PCR was conducted using RAPD
primer 208 5ʹ-ACG GCC GAC C-3ʹ [25], while
ERIC-PCR was performed using Primer ERIC1:
5`-ATG TAA GCT CCT GGG GAT TCA C-3` and
Primer ERIC2: 5-AAG TAA GTG ACT GGG GTG
AGC G-3` [26]. Each PCR reaction mix (25 μL)
was composed of 10 mM PCR buffer pH 8.3; 3
mM MgCl
2
; 0.4 mM of each dNTP; 0.8 μM
primer; 1.5 U of Taq DNA polymerase and fixed
amount of DNA template (30 ng). Then, DNA
amplification was carried out using the thermal
cycler (Mastercycler personal, Eppendorf,
Germany) according to the following thermal
conditions for RAPD-PCR: initial denaturation for
3 min at 94°C; followed by 35 cycles of
denaturation at 94°C for 1 min, annealing at
32°C for 1 min and extension at 72°C for 2 min,
followed by a final extension at 72°C for 5 min.
The thermal conditions for ERIC-PCR were initial
denaturation for 3 min at 94°C; followed by 40
cycles of denaturation at 94°C for 50 s,
annealing at 50°C for 1 min and extension at 72
°C for 1 min, followed by a final extension step at
72°C for 5 min. The PCR products were
analyzed by electrophoresis through 1.8%
agarose gel. The PARD-PCR and ERIC-PCR
profiles were visualized using UV trans-
illuminator and photographed. Changes in
PARD-PCR or ERIC-PCR banding pattern
profiles following plant extract treatments,
including variations in band intensity as well as
gain or loss of bands, were taken into
consideration [21,22,27-28].
3. RESULTS
3.1 Antimicrobial Activity of C. spinosa
Extracts
Results of this study showed that both ethanolic
and aqueous extracts of stem and leaf of C.
spinosa plant had antimicrobial activity. The MIC
value of both aqueous and ethanolic extracts of
C. spinosa on different bacterial strains had a
range 6.25 mg/ml to 100 mg/ml. However, the
MIC value of both aqueous and ethanolic
extracts of C. spinosa on C. albicans had a range
25 mg/ml to 50 mg/mL. The MIC profile of both
ethanolic and aqueous extracts of stem and leaf
of C. spinosa plant against different
microorganisms is shown in Table 1.
3.2 Evaluation of the Genotoxic Potential
of C. spinosa Aqueous Leaf Extract
DNA genome was extracted from each E. coli
strain, which was treated with different
concentrations of aqueous leaf extract of C.
spinosa at various time intervals. Changes in the
extracted DNA genome from treated E. coli strain
were evaluated and compared with negative
(untreated) controls at the same time intervals.
The effect of aqueous leaf extract on E. coli
genome was evaluated using molecular
fingerprinting based on PARD-PCR and ERIC-
PCR techniques. RAPD-PCR profile showed that
a band with an amplicon length of about 700-bp
was less intense in E. coli strain treated with 3
doses (250 μg/ml, 125 μg/ml and 62.5 μg/ml) of
aqueous leaf extract for 2h (Fig. 1, lanes 1, 2 and
3), compared with the same band that appeared
in the negative control. However, the profile
showed that a band with an amplicon length of
about 1500-bp was more intense in E. coli strain
treated with 3 doses for 2h of the same extract
(Fig. 1, lanes 1, 2 and 3), compared with the
same band that appeared in the negative control.
The bands with an amplicon length of about 300-
bp and 400-bp were less intense in E. coli strain
treated with 250 μg/mL and 125 μg/ml of the
aqueous extract for 2h (Fig. 1, lanes 1 and 2),
compared with the same bands that appeared in
the negative control. In addition, a band with an
amplicon length of about 900-bp was less
intense in E. coli strain treated with 250 μg/ml for
2h (Fig. 1, lane 1), compared with the same band
that appeared in the negative control. Besides, a
band with an amplicon length of about 200-bp
appeared in E. coli strain treated with 250 μg/ml
of aqueous leaf extract for 2h (Fig. 1, lane 1),
compared with negative control. In addition,
RAPD-PCR profile showed that a band with an
amplicon size of more than 1500-bp appeared in
E. coli strain treated with 3 doses of the same
extract for 2h (Fig. 1, lanes 1, 2 and 3),
compared with negative control. Results of
RAPD-PCR showed that bands with an amplicon
length of about 1500-bp, 700-bp, 400-bp and
300-bp were less intense in E. coli strain treated
with 250 μg/ml of aqueous leaf extract for 5h
(Fig. 1, lane 4), compared with the same bands
that appeared in the negative control.
Additionally, a band with an amplicon length of
about 1200-bp was less intense in E. coli strain
treated with 3 doses of aqueous leaf extract for
5h (Fig. 1, lane 4, 5 and 6), compared with the
same band that appeared in the negative control.
Besides, a new band with an amplicon size of
about 200-bp appeared in E. coli strain treated
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with 250 μg/ml and 125 μg/ml of the same
extract for 5h (Fig. 1, lanes 4 and 5), compared
with negative control. Results of RAPD-PCR also
showed that bands with an amplicon length of
about 700-bp and 300-bp were less intense in E.
coli strain treated with 3 doses of the aqueous
leaf extract for 24h (Fig. 1, lanes 7, 8 and 9),
compared with the same bands that appeared in
the negative control. RAPD-PCR profiles of E.
coli strain treated with different concentrations of
aqueous leaf extract of C. spinosa and negative
control at the different time intervals are shown in
Fig. 1.
ERIC-PCR profile showed that two bands with an
amplicon fragment size of than 1500-bp
appeared in E. coli strain treated with 250 μg/ml
and 125 μg/ml of aqueous leaf extract of C.
spinosa for 2h (Fig. 2, lanes 1 and 2), compared
with negative control. Additionally, the band with
an amplicon fragment size of about 700-bp was
more intense, while the band with an amplicon
fragment size of about 300-bp was less intense
in E. coli strain treated with both doses 125 μg/ml
and 62.5 μg/ml of the same extract for 2h (Fig. 2,
lanes 2 and 3), compared with the same bands
that appeared in the negative control. Besides,
the bands with an amplicon fragment length of
about 300-bp and 700-bp disappeared in E. coli
strain treated with 125 μg/ml of aqueous leaf
extract of C. spinosa for 2 h (Fig. 2, lane 1),
compared with the same bands that appeared in
the negative control. Results of ERIC-PCR
showedthat band with an amplicon fragment size
of more than 1500-bp was less intense in E. coli
strain treated with 3 doses of aqueous leaf
extract for 5 h (Fig. 1, lane 4, 5 and 6), compared
with the same band that appeared in the
negative control. Besides, a new band with an
amplicon fragment size of about 1500-bp
appeared in E. coli strain treated with 250 μg/ml
of the same extract for 5 h (Fig. 1, lane 4),
compared with negative control. Additionally, the
band with an amplicon fragment size of about
700-bp was more intense in E. coli strain treated
with both doses 125 μg/ml and 62.5 μg/ml of
aqueous leaf extract, while the same band that
disappeared in E. coli strain treated with both
doses 250 μg/ml of the same extract for 5 h (Fig.
2, lanes 4, 5 and 6), compared with the same
bands that appeared in the negative control. The
band with an amplicon fragment length of about
450-bp was less intense in E. coli strain treated
with 250 μg/ml and 125 μg/ml for 5 h (Fig. 2,
lanes 5 and 6), compared with the same band
that appeared in the negative control. In addition,
a new band with an amplicon fragment size of
about 100-bp appeared in E. coli strain treated
with 3 doses for 5h of the same extract (Fig. 2,
lanes 4, 5 and 6), compared with negative
control. ERIC-PCR profile also showed that the
band with an amplicon fragment length more
than 1500-bp was more intense in E. coli strain
treated with 3 doses of aqueous leaf extract for
24 h (Fig. 2, lane 7, 8 and 9), compared with the
same band that appeared in the negative control.
Besides, the band with an amplicon fragment
length of about 1300-bp appeared in E. coli strain
treated with 250 μg/ml and 125 μg/ml for 24h
(Fig. 2, lanes 7 and 8), compared with negative
control. Additionally, the band with an amplicon
fragment length of about 450-bp was less
intense, while the same band was more intense
in E. coli strain treated with 250 μg/ml and 62.5
μg/ml of the same extract for 24 h, respectively
(Fig. 2, lanes 7 and 9), compared with the same
bands that appeared in the negative control.
Also, the band with an amplicon fragment size of
about 100-bp was less intense in E. coli strain
treated with 250 μg/ml and 125 μg/ml for 24 h
(Fig. 2, lanes 7 and 8), compared with the same
band that appeared in the negative control.
ERIC-PCR profiles of E. coli strain treated with
different concentrations of aqueous leaf extract
of C. spinosa and negative control at the different
time intervals are shown in Fig. 2.
Table 1. MIC profile of both ethanolic and aqueous extracts of leaf and stem of C. spinosa
plant against different microorganisms
Microorganism
MIC (mg/ml)
Aqueous extract
Ethanolic extract
Leaf
Stem
E. coli ATCC 25922 6.25 25 6.25 12.5
S. aureus ATCC 6538P 12.5 25 12.5 25
MRSA 50 100 12.5 6.25-12.5
K. pneumoniae 50 100 25 50
C. albicans ATCC 90028 25 50 25 50
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Fig. 1. RAPD-PCR profile of E. coli strain untreated (negative control) and treated with different
concentrations of C. spinosa leaf aqueous extract at different time intervals. Lanes C1, C2 and
C3 are negative controls; lanes 1, 4 and 7 treated with 250 μg/ml; Lanes 2, 5 and 8 treated with
125 μg/ml; Lanes 3, 6 and 9 treated with 62.5 μg/ml of plant extract; lanes L (ladder)
Fig. 2. ERIC-PCR profile of E. coli strain untreated (negative control) and treated with different
concentrations of C. spinosa leaf aqueous extract at different time intervals. Lanes C1, C2 and
C3 are negative controls; lanes 1, 4 and 7 treated with 250 μg/ml; Lanes 2, 5 and 8 treated with
125 μg/ml; Lanes 3, 6 and 9 treated with 62.5 μg/ml of plant extract; lanes L (ladder).
Results of the current study showed that
molecular fingerprinting based on ERIC-PCR can
be used to evaluate the genotoxic effects to
estimate the chemical compounds or molecules
risk connected with their potential mutagenic
effects in the model bacterial species E. coli.
Molecular fingerprinting based on ERIC-PCR has
sensitivity to evaluate genotoxicity as well as
molecular fingerprinting based on RAPD-PCR
Figs. 1 and 2.
4. DISCUSSION
In this study broth microdilution method was
used to detect the potential antimicrobial effect of
both ethanolic and aqueous leaf and stem
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extracts of C. spinosa against different species of
microorganisms. These species included E. coli,
S. aureus, MRSA, K. pneumoniae and C.
albicans. The results of the current confirmed
that both ethanolic and aqueous leaf and stem
extracts of C. spinosa showed antimicrobial
effect on these microorganisms. Antimicrobial
activity of C. spinosa has been reported
previously using different types of extracts and
plant parts against different types of
microorganisms [11,12,16-19]. According to the
previously conducted studies, diverse
phytochemical compounds are the active
ingredients of C. spinosa plant [6-10,13].
Most nutraceutical plants are used without any
standard safety and toxicological trials. The
common hypothesis that the products of these
plants are nontoxic. However, this hypothesis is
incorrect, so toxicological tests should be
conducted for herbal drugs [29]. Recently
advances in machine learning and artificial
intelligence immensely decoded and
empowered, the herbal drug discovery and
modeling, which gave medicine modern tool to
predict the biosafety and efficacy [30-31] and in-
silico methods [32-33] to potentially decipher the
quantitative nanostructure activity-relationship
(Nano-QSAR).
In this study, the potential genotoxic effect of the
aqueous extract of C. spinosa plant against E.
coli was tested using molecular fingerprinting
based on ERIC-PCR and RAPD-PCR
techniques. Reviewing the scientific literature
showed that this study is the first of its kind that
studied the genotoxicity of C. spinosa extract on
prokaryotes using molecular fingerprinting based
on ERIC-PCR and RAPD-PCR techniques.
Besides, many plants were previously examined
to investigate their genotoxic potential using
different techniques [21,22,28,34-39]. In this
study, RAPD-PCR and ERIC-PCR profiles
showed many significant differences between the
treated and untreated E. coli strain. The
alterations in the treated E. coli strain with
aqueous leaf extract at different time intervals
included the appearance and disappearance of
certain bands and the alteration in the band
intensity compared with negative control. These
alterations in both the RAPD-PCR and ERIC-
PCR profiles of the treated E. coli strain
compared with the negative control could be
explained due to the effect of the genotoxic
ingredients that were present in the aqueous leaf
extract. These ingredients can induce different
alterations and changes such as point mutations
and/or rearrangements in chromosomes,
damage and chromosomal aberrations. These
alterations in the DNA might have a potential
change on the primer binding sites and/or inter-
priming distances [21,22]. Using other
techniques such as DNA sequencing or probing
can help understand the correct mechanisms
that lead to such differences in RAPD-PCR and
ERIC-PCR profiles [7,18,24]. Findings of the
current study were in contrast to study published
previously [20], which showed that C. spinosa
buds aqueous extract is non-genotoxic and their
study reveals that C. spinosa aqueous extract
had antimutagenic potential against Ethyl
Methane sulfonate induced chromosomal
aberrations in A. cepa root meristem cells. This
may be due to differences in plant parts and the
techniques used to evaluate genotoxicity. In a
literature survey, it is also showed that plant
extracts can be mutagenic and antimutagenic
depending on the test system used. This
indicates that a group of assays is needed before
any conclusion can be reached about the
genotoxic effect [34].
Results of the current study showed that
molecular fingerprinting based on ERIC-PCR is
an effective and sensitive technique that can be
used to evaluate the genotoxic effects to
estimate the chemical compounds or molecules
risk connected with their potential mutagenic
effects in the model bacterial species E. coli.
5. CONCLUSION
The results of the current study showed that
aqueous leaf extract of C. spinosa possesses
genotoxic and mutagenic potential effects on E.
coli. In addition, the results also point out the
capability of using C. spinosa to treat and
prevent infections caused by several
microorganisms. Further studies are
recommended to determine the specific
ingredients in this plant as well as the correct
mechanisms responsible for that genotoxicity. In
addition, findings of this study showed that
molecular fingerprinting based on ERIC-PCR is
an effective and sensitive technique that can be
used to evaluate the genotoxic effect in the
model bacterial species E. coli.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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55
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... In any case, the DNA was damaged by the treatment concentrations in the larvae. Similar study made on microbes from the extract of Capparis spinosa showed a genotoxic effect which was confirmed through RAPD-PCR [33]. ...
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