ArticlePDF Available

Evaluation of Antimicrobial and Genotoxic Activity of Ephedra foeminea Ethanolic and Aqueous Extracts on Escherichia coli

Authors:

Abstract and Figures

This study was conducted to evaluate the antimicrobial activity and the genotoxic effects of ethanolic and aqueous extracts of aerial parts of Ephedra foeminea (E. foeminea) plant on Escherichia coli (E. coli ATCC 25922). Antimicrobial activity was investigated using microbroth dilution method, while the genotoxic effect was determined using enterobacterial repetitive intergenic consensus (ERIC)-PCR. MIC value of both ethanolic and aqueous extracts of E. foeminea plant was found to be 50 mg/ml. Genotoxic effects of both extracts, showed an alteration in (ERIC)-PCR profiles of E. coli strain treated with extracts compared to untreated control. These alterations included a decreased intensity or absence of some amplified fragments. Such findings strongly indicate the genotoxic effects of both ethanolic and aqueous extracts from E. foeminea plant on E. coli. The findings draw attention to the unsafe, use of E. foeminea plant in folkloric medicine and point out the capability of using E. foeminea to treat bacterial infections. Future studies are required to know the exact molecules as well as the mechanisms responsible for the genotoxicity of this plant. In vivo genotoxicity studies are recommend for assessment of the safety of using E. foeminea plant for therapeutic purposes.
Content may be subject to copyright.
JJBS
Volume 13, Number 1,March 2020
ISSN 1995-6673
Pages 123 - 126
Jordan Journal of Biological Sciences Short Communication
Evaluation of Antimicrobial and Genotoxic Activity of Ephedra
foeminea Ethanolic and Aqueous Extracts on Escherichia coli
Shurooq M. Ismail*, Ghaleb M. Adwan, and Naser R. Jarrar
Department of Biology and Biotechnology, An-Najah National University,P.O. Box (7)-Nablus, Palestine
Received February 21, 2019; Revised April 23, 2019; Accepted May 7, 2019
Abstract
This study was conducted to evaluate the antimicrobial activity and the genotoxic effects of ethanolic and aqueous extracts
of aerial parts of Ephedra foeminea (E. foeminea) plant on Escherichia coli (E. coli ATCC 25922). Antimicrobial activity
was investigated using microbroth dilution method, while the genotoxic effect was determined using enterobacterial
repetitive intergenic consensus (ERIC)-PCR. MIC value of both ethanolic and aqueous extracts of E. foeminea plant was
found to be 50 mg/ml. Genotoxic effects of both extracts, showed an alteration in (ERIC)-PCR profiles of E. coli strain
treated with extracts compared to untreated control. These alterations included a decreased intensity or absence of some
amplified fragments. Such findings strongly indicate the genotoxic effects of both ethanolic and aqueous extracts from E.
foeminea plant on E. coli. The findings draw attention to the unsafe, use of E. foeminea plant in folkloric medicine and point
out the capability of using E. foeminea to treat bacterial infections. Future studies are required to know the exact molecules
as well as the mechanisms responsible for the genotoxicity of this plant. In vivo genotoxicity studies are recommend for
assessment of the safety of using E. foeminea plant for therapeutic purposes.
Keywords: Ephedra foeminea, Genotoxicity potential, Plant extracts, Antimicrobial activity.
* Corresponding author e-mail: shurooq.ismail@najah.edu.
1. Introduction
The family Ephedraceae consists of only one genus
called Ephedra L. It has a group of approximately fifty
species of perennials, evergreen, and dioecious sub-shrubs
species growing up to four feet tall, with slender and
joined stems. In general, species of this genus adapted to
grow wild in arid and semiarid conditions and
disseminated mainly in the moderate zones of Asia,
Europe and North America (O'Dowd et al., 1998;
Pirbalouti et al., 2013). Approximately 25 species of
Ephedra are found in the drier regions of the Old World
covering the area westwards from Central Asia across
southwest Asia and into North Africa and Mediterranean
Europe (Caveney et al., 2001). In the New World, about
24 species of Ephedra are found ranging from the
southwestern United States to the central plateau of
Mexico, and in South America occur in an area from
Ecuador to Patagonia (Caveney et al., 2001). Ephedra
grows widely in Palestine. In the flora Palestina, 5 species
of Ephedra has been reported, included E. foeminea, E.
alata, E. aphyla, E. ciliata and E. fragilis (Danin, 2018).
Approximately, all commercial applications of Ephedra
extracts derived from the ephedrine alkaloids found in the
stems in many Eurasian Ephedra species. These extracts
are used in traditional medicine to treat several diseases
such as bronchial asthma, coughs, chills, allergies, colds,
edema, headaches, fever, flu and gastric disorders. In
addition, Ephedra shows anticancer and antimicrobial
activities (Parsaeimehr et al., 2010; Pirbalouti et al., 2013;
Dehkordi at al., 2015; Dosari et al., 2016; Al-Rimawi et
al., 2017; Mendelovich et al., 2017). Besides, it was
shown that hydro-alcoholic extract of E. pachyclada was
effective in experimentally healing rat ulcers (Pirbalouti et
al., 2013).
Ephedra possesses a high antioxidant potential since it
has been considered as a source of different phenolic
compounds, as well as, a natural source of alkaloids such
as ephedrine, pseudoephedrine, and other related
compounds. (Eberhardt et al., 2000; Parsaeimehr et al.
2010; Amakura et al., 2013; Dehkordi et al., Ibragic and
Sofić 2015; Al-Rimawi et al., 2017). The studies
conducted on the cytotoxicity of Ephedra showed that
ephedrine derivatives and ground ma-huang extracts were
more cytotoxic than those of the whole herb extracts. A
study on Neuro-2a cell line showed the cell line was more
sensitive to the cytotoxicity (Lee et al., 2000). Ethanolic
leaf extract and fruit juice of E. foeminea reduced viability
of cancer cells in vitro, whereas the aqueous extract
reduced the cytotoxicity in all cell lines (Mendelovich et
al., 2017). Since there is no scientific report to date about
the genetoxicity of E. foeminea on prokaryotes, the current
study was performed to determine the antimicrobial effect
of ethanolic and aqueous extracts from E. foeminea plant
growing wild in Palestine as well as evaluate the genotoxic
effect of these extracts on E. coli strain using
enterobacterial repetitive intergenic consensus (ERIC)-
PCR.
© 2020 Jordan Journal of Biological Sciences. All rights reserved - Volume 13, Number 1
124
2. Materials and Methods
2.1. Plant collection and identification
The aerial parts of E. foeminea were collected from a
natural habitat in Tulkarm province, West Bank-Palestine,
during September, 2018. Identification of the plant was
carried out by the plant taxonomist Dr. Ghadeer Omar,
Department of Biology and Biotechnology, An-Najah
National University, Palestine.
The collected aerial parts of E. foeminea were washed
with water to remove soil and dust particles then dried.
Exposure to light was avoided to prevent possible loss of
effective ingredients. The dried aerial parts were powdered
finely using a blender to make them ready for ethanolic
and aqueous extract preparation.
2.2. Plant extract preparation
2.2.1. Ethanolic extract
Approximately 50 g of dried aerial parts powder were
mixed thoroughly using magnetic stirrer in 200 mL of 80%
ethanol. The ethanol-aerial parts 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 4°C to remove fine particles. Then, the
supernatant extract was dried and concentrated by using
rotary evaporator at 50°C. The obtained dried plant extract
powder was stored in refrigerator at 4°C. Before starting
the experiments, this material was dissolved in 10%
Dimethyl Sulfoxide (DMSO) to obtain a concentration of
200 mg/mLand stored at 4ºC for further assays.
2.2.2. Aqueous extract
Aqueous aerial parts extract was prepared by mixing
approximately 50 g of dried aerial parts powder thoroughly
using magnetic stirrer in 200 ml of cold (room
temperature) sterile distilled water. The water-aerial parts
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 4°C to remove
fine particles. Then the supernatant extract was dried and
concentrated by freeze dryer (lyophilizer). The obtained
dried plant extract powder was stored in refrigerator at
4°C. Before starting the experiments, this dried plant
extract powder was dissolved in a sterile distilled water to
obtain a concentration of 200 mg/mL and stored at 4ºC for
further assays.
2.3. Determination of MIC for plant extracts by broth
microdilution method
MIC of plant extracts was determined by the broth
microdilution method in sterile 96- wells microtiter plates
according to the CLSI instructions (CLSI, 2017). The plant
extract (200 mg/mL of 10% DMSO) and 10% DMSO
(negative control) were two fold-serially diluted in nutrient
broth directly in the wells of the plates in a final volume of
100μL. After that, a bacterial inoculum size of 104
CFU/mL was added to each well. Negative control wells
containing either 100μL nutrient broth only, or 100μL
DMSO with bacterial inoculum, or plant extracts and
nutrient broth without bacteria were included in this
experiment. Each plant extract was run in duplicate. The
microtiter plate was then covered and incubated at 37°C
for 24h. The MIC was taken as the minimum concentration
of the dilutions that inhibited the growth of the test
microorganism. MIC was determined by visual inspection.
2.4. Evaluation of the genotoxic potential of Ephedra
foeminea aerial extracts on E. coli
Few colonies from a 24 hour old E. coli strain growth
culture plated on EMB agar medium were sub-cultured
under sterile conditions into a bottle containing 20-mL of
nutrient broth, then incubated at 37°C for 1 hour with
continuous shaking. After that, aseptically, 1 mL of one
hour old E. coli culture was added to each of eight sterile
bottles each containing 25 mL broth medium. These
bottles were incubated at 37°C for 1 hour with continuous
shaking. Then three different concentrations of ethanolic
extract (3.5 mg/mL, 1.75 mg/mL and 0.875 mg/mL of
10% DMSO), and other three different concentrations of
aqueous extract (3.5 mg/mL, 1.75 mg/mL and 0.875
mg/mL of distilled water) were added into six bottles of
the E. coli broth culture. The other two bottles were
considered as a negative or untreated control by adding a
specific volume of 10% DMSO and distilled water into
each bottle.
Genome of E. coli was prepared for enterobacterial
repetitive intergenic consensus (ERIC) PCR according to
the method described previously (Adwan et al., 2013).
Three mL samples were taken from the E. coli growth
culture after 2 hours, 6 hours, and 24 hours, centrifuged for
five minutes at 14,000 rpm 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 five minutes at
14,000 rpm; after that, the supernatant was discarded. The
pellet of each bacterial sample was re-suspended in a 300
µL of sterile distilled water and boiled for 15 minutes.
Then the mixture was incubated in ice for 10 minutes. The
samples were pelleted by centrifugation at 14,000 rpm for
five minutes, and each sample supernatant was transferred
into a new Eppendorf tube. The DNA concentration for
each sample was determined by using nanodrop
spectrophotometer (GenovaNano, Jenway) and the DNA
samples were stored at -20ºC for ERIC-PCR analysis. The
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`. Each PCR
reaction mix (25 µL) was composed of 10 mM PCR buffer
pH 8.3; 3 mM MgCl2; 0.4 mM of each dNTP; 0.8 µM
primer; 1.5U of Taq DNA polymerase and fixed amount of
DNA template (60 ng). Then, DNA amplification was
carried out using the thermal cycler (Mastercycler
personal, Eppendorf, Germany) according to the following
thermal conditions: initial denaturation for 3 min at 94 ºC;
followed by 40 cycles of denaturation at 94 ºC for 50 s,
annealing at 50 ºC for 1 min and extension at 72 ºC for 1
min, followed by a final extension step at 72ºC for 5 min.
The PCR products were analyzed by electrophoresis
through 1.8% agarose gel. The ERIC-PCR profile was
visualized using UV trans-illuminator and photographed.
Changes in ERIC-PCR banding pattern profiles following
plant extracts treatments, including variations in band
intensity as well as gain or loss of bands, were taken into
consideration (Lalrotluanga et al., 2011; Atienzar et al.,
2002).
© 2020 Jordan Journal of Biological Sciences. All rights reserved - Volume 13, Number 1 125
3. Results
Results of this study showed that both aqueous and
ethanolic aerial parts extracts of E. foeminea had an
antibacterial activity. The MIC value of both aqueous and
ethanolic aerial parts extracts of E. foeminea on E. coli
strain were found to be 50 mg/ml.
DNA genome which was extracted from each E. coli
strain which was treated with different concentrations of
both aqueous and ethanolic aerial parts extracts of E.
foeminea at various time intervals. Changes in extracted
DNA genome from E. coli strain were evaluated and
compared with untreated controls at the same time
intervals.
The effect of aqueous aerial parts extract on genome of
E. coli strain was evaluated by using ERIC-PCR. ERIC-
PCR profile showed that a band with an amplicon length
of about 800-bp was less intense in E. coli strain treated
with 3.5 mg/mL and 1.75 mg/mL (Figure 1A, lanes 1 and
2) of aqueous aerial parts extract for 2h. Besides, this
band disappeared in E. coli strain treated with 0.875 mg/ml
of the same extract (Figure 1A, lane 3), in comparison with
the same band appeared in un-treated control. Moreover,
all bands disappeared after 6h in the E. coli strain treated
with 3.5 mg/mL aqueous aerial parts extract (Figure 1A,
lane 4). The band with an amplicon length of about 800-bp
was less intense in E. coli strain treated with 1.75 mg/mL
(Figure 1A, lane 5) disappeared in E. coli strain treated
with 0.875 mg/mL of the same extract for 6h (Figure 1A,
lane 6), in comparison with the same band appeared in the
un-treated control. The band with an amplicon length of
about 800-bp disappeared in E. coli strain treated with 3.5
mg/ml, 1.75 mg/mL and 0.875 mg/mL aqueous aerial parts
extract for 24h (Figure 1A, lanes 7,8 and 9). Moreover, the
band with an amplicon length of about 300-bp was less
intense (Figure 1A, lanes 7, 8 and 9) in comparison with
the same band appeared in un-treated control. It was
observed that in lane number four most bands disappeared
when treated with aqueous aerial parts extract of E.
foeminea of 3.5 mg/mL concentration. ERIC-PCR profiles
for E. coli strain untreated and treated with different
concentrations of aqueous aerial parts extract of E.
foeminea at the different time intervals are shown in
Figure 1A.
Comparing the ERIC-PCR profile of untreated control
samples with the profile of the E. coli treated with
ethanolic aerial parts extract showed decreasing of
intensity or loss of some bands from the profile. ERIC-
PCR profile showed that a band with an amplicon length
of about 800-bp was less intense in E. coli strain treated
with 3.5 mg/mL (Figure 1B, lane 2 and 5) and disappeared
in E. coli strain treated with 1.75 mg/mL (Figure 1B, lane
3 and 6) of ethanolic aerial parts extract for 2h and 6h. The
band with an amplicon length of about 800-bp was less
intense in E. coli strain treated with 3.5 mg/mL (Figure
1B, lane 7), and disappeared in E. coli strain treated with
1.75 mg/mL and 0.875 mg/mL (Figure 1B, lane 8 and 9)
aqueous aerial parts extract for 24h. In addition, the band
with an amplicon length of about 300-bp was less intense
(Figure 1B, lanes 8 and 9) in comparison with the same
band appeared in untreated control. ERIC-PCR profiles for
E. coli strain untreated and treated with different
concentrations of ethanolic aerial parts extract of E.
foeminea at the different time intervals are shown in
Figure 1A.
Figure 1. ERIC-PCR profile of E. coli strain untreated and treated
with different aerial parts extract concentrations. A: aqueous aerial
parts extract, B: ethanolic aerial parts extract of E. foeminea at
different time intervals. Lanes C1, C2 and C3 are untreated
(negative controls); lanes 1, 4 and 7 treated with 3.5 mg/ml; Lanes
2, 5 and 8 treated with 1.75 mg/ml; Lanes 3, 6 and 9 treated with
0.875 mg/ml of plant extract.
4. Discussion
In the present study broth microdilution method was
used to examine the potential antimicrobial activity of both
aqueous and ethanolic aerial parts extracts of E. foeminea
against E. coli. The results confirmed that both aqueous
and ethanolic aerial parts extracts of E. foeminea exhibited
antibacterial activity against E. coli strain. Antimicrobial
activity of some Ephedra species has been reported using
different types of extracts (Al-Khalil et al., 1998; Feresin
et al., 2001; Cottiglia et al., 2005; Parsaeimehr et al.,
2010; Rustaiyan et al., 2011; Dehkordi et al., 2015; Dosari
et al., 2016). According to previously conducted studies,
phenolic compounds are the active ingredients of Ephedra
plant (Dehkordi et al., 2015; Dosari et al., 2016).
In this study, the potential genotoxic effect of the
aqueous and ethanolic aerial parts extracts of E. foeminea
against E. coli was examined using ERIC-PCR technique.
Reviewing the scientific literature showed that this study is
the first of its kind that studied the genetoxicity of E.
foeminea extracts on prokaryotes using ERICPCR
technique. Besides, many plants were previously tested to
detect their genotoxicity potential by different techniques
(Basaran et al., 1996; Lalrotluanga et al., 2011; El-Tarras
et al., 2013; Hajar and Gumgumjee, 2014; Ciğerci et al.,
2016; Abu-Hijleh et al., 2018). ERIC-PCR profiles
showed significant differences between the treated and
untreated E. coli strain used in this study. The changes in
the treated E. coli strain with both aqueous and ethanolic
aerial parts extracts included the disappearance of certain
bands as well as the change in the band intensity in
comparison with untreated control. The changes in the
profile of the treated E. coli strain in comparison with the
untreated control samples could be explained due to the
effect of the genotoxic molecules that were present in the
plant extracts. These molecules can induce different
© 2020 Jordan Journal of Biological Sciences. All rights reserved - Volume 13, Number 1
126
changes such as breakdown in DNA strands, point
mutations and/or rearrangements in chromosomes. These
changes in the DNA might have a potential effect on the
primer binding sites and/or inter-priming distances (Abu-
Hijleh et al., 2018). DNA sequencing or probing and other
techniques can help in understanding the proposed
mechanisms that lead to such differences in ERIC-PCR
profiles (Lalrotluanga et al., 2011). Ma-huang is a
traditional Chinese medicinal preparation derived from
Ephedra sinica Stapf and other Ephedra species that are
used to treat different diseases. Studies on cytotoxity of the
ma-huang extracts showed that, cytotoxicity of all ma-
huang extracts could not be totally accounted for by their
ephedrine contents, suggesting the presence of other toxins
in the extracts which may modify its pharmacological and
toxicological activities (Lee et al., 2000).
5. Conclusion
The results of this study showed that aqueous and
ethanolic aerial parts extracts of E. foeminea possess
genotoxic and mutagenic potential against E. coli. In
addition, the results also point out the capability of using
E. foeminea to treat infections caused by E. coli. More
studies are recommended to reveal the exact molecules
that are responsible for E. foeminea genotoxicity as well as
the mechanisms responsible for that genotoxicity.
Competing Interests
Authors have declared that no competing interests
exist.
References
Abu-Hijleh A, Adwan G and Abdat W. 2018. Biochemical and
molecular evaluation of the plant Ecballium elaterium extract
effects on Escherichia coli. J Adv Biol Biotechnol., 9 (2): 1-11.
Adwan G, Adwan K, Jarrar N, Salama Y and Barakat A. 2013.
Prevalence of seg, seh and sei genes among clinical and nasal
Staphylococcus aureus isolates. Br Microbiol Res J., 3(2):139-
149.
Al-Khalil S, Alkofahi A, El-Eisawi D and Al-Shibib A. 1998.
Transtorine, a new quinoline alkaloid from Ephedra transitoria. J
Nat Prod., 61(2):262-263.
Al-Rimawi F, Abu-Lafi S, Abbadi J, Alamarneh AAA, Sawahreh
RA and Odeh I. 2017. Analysis of phenolic and flavonoids of wild
Ephedra alata plant extracts by LC/PDA and LC/MS and their
antioxidant activity. Afr J Tradit Complement Altern Med., 14(2):
130-141.
Amakura Y, Yoshimura M, Yamakami S, Yoshida T, Wakana D,
Hyuga M, Hyuga S, Hanawa T and Goda Y.2013.
Characterization of phenolic constituents from ephedra herb
extract. Molecules, 18(5):5326-5334.
Andrews, J. M. 2006. BSAC standardized disc susceptibility
testing method (version 5). J Antimicrob Chemother., 58(3): 511-
529.
Atienzar FA, Venier P, Jha AN and Depledge MH. 2002.
Evaluation of the random amplified polymorphic DNA (RAPD)
assay for the detection of DNA damage and mutations. Mutation
Research/Genetic Toxicol and Environ Mutagen., 521(1-2): 151-
163.
Basaran AA, Yu TW, Plewa MJ and Anderson D. 1996. An
investigation of some Turkish herbal medicines in Salmonella
typhimurium and in the COMET assay in human lymphocytes.
Teratogen, Carcinogen, and Mutagen., 16(2):125-138.
Caveney S, Charlet DA, Freitag H, Maier-Stolte M and Starratt
AN. 2001. New observations on the secondary chemistry of world
Ephedra (Ephedraceae). Am J Bot., 88(7):1199-1208.
Ciğerci IH., Cenkci S, Kargıoğlu M and Konuk M. 2016.
Genotoxicity of Thermopsis turcica on Allium cepa L. roots
revealed by alkaline comet and random amplified polymorphic
DNA assays. Cytotechnol., 68(4):829-838.
Clinical and Laboratory Standards Institute (CLSI). 2017.
Performance Standards for Antimicrobial Susceptibility
Testing. 27th ed. CLSI supplement. M100S. Wayne, PA, USA.
Cottiglia F, Bonsignore L, Casu L, Deidda D, Pompei R, Casu M
and Floris C. 2005. Phenolic constituents from Ephedra
nebrodensis. Nat Prod Res., 19(2):117-23.
Danin A. 2018. Flora of Israel on line. http://flora.huji.ac.il/
browse asp. Accessed (Dec. 10, 2018).
Dehkordi NV, Kachouie MA, Pirbalouti AG, Malekpoor F and
Rabei M. 2015.Total phenolic content, antioxidant and
antibacterial activities of the extract of Ephedra procera fisch. et
mey. Acta Pol Pharm., 72(2):341-345.
Dosari AS, Amin Norouzi A, Moghadam MT and Satarzadeh N.
2016. Antimicrobial activity of Ephedra pachyclada methanol
extract on some enteric gram negative bacteria which causes
nosocomial infections by agar dilution method. Zahedan J Res
Med Sci., 18(11):1-4.
El-Tarras AA, Hassan MM and El-Awady MA. 2013. Evaluation
of the genetic effects of the in vitro antimicrobial activities of
Rhazya stricta leaf extract using molecular techniques and
scanning electron microscope. Afr J Biotechnol., 12(21):3171-
3180.
Eberhardt MV, Lee CY and Liu RH 2000. Antioxidant activity of
fresh apples. Nature 405:903904.
Feresin GE, Tapia A, López SN and Zacchino SA. 2001.
Antimicrobial activity of plants used in traditional medicine of
San Juan province, Argentine. J Ethnopharmacol., 78(1):103-107.
Hajar AS and Gumgumjee NM. 2014. Antimicrobial activities and
evaluation of genetic effects of Moringa peregrina (forsk) fiori
using molecular techniques. Inter J Plant and Animal Environ
Sci., 4(1):65-72.
Ibragic S and Sofić E. 2015. Chemical composition of various
Ephedra species. Bosn J Basic Med Sci., 15(3): 21-27.
Lalrotluanga, Kumar NS and Gurusubramanian G. 2011.
Evaluation of the random amplified polymorphic DNA (RAPD)
assay for the detection of DNA damage in mosquito larvae treated
with plant extracts. Science Vision, 11(3): 155-158.
Lee MK, Cheng BW, Che CT and Hsieh DP. 2000. Cyto-
toxicity assessment of Ma-huang (Ephedra) under different
conditions of preparation. 28TToxicol Sci28T., 56(2):424-430.
Mendelovich M, Shoshan M, Fridlender M, Mazuz M, Namder D,
Nallathambi R, Selvaraj G, Kumari P, Ion A, Wininger S, Nasser
A, Samara M, Sharvit Y, Kapulnik Y, Dudai N and Koltai H.
2017. Effect of Ephedra foeminea active compounds on cell
viability and actin structures in cancer cell lines. J Med Plants
Res., 11(43): 690-702.
O'Dowd NA, McCauley G, Wilson JAN, Parnell TAK and
Kavanaugh D. 1998. In vitro culture, micropropagation and the
production of ephedrine and other alkaloids. In: Biotechnology in
Agriculture and Forestry; Bajaj YPS. (Ed.), p. 41, Springer,
Berlin.
Parsaeimehr A, Sargsyan E and Javidnia K. 2010. A 24Tcomparative24T
24Tstudy24T of the 24Tantibacterial24T, 24Tantifungal24T and 24Tantioxidant24T 24Tactivity24T and
24Ttotal24T 24Tcontent24T of 24Tphenolic24T 24Tcompounds24T of 24Tcell24T 24Tcultures24T and 24Twild24T
24Tplants24T of 24Tthree24T 24Tendemic24T 24Tspecies24T of 24TEphedra24T. Molecules, 15(3):
1668-1678.
Pirbalouti AG, Azizi S, Amirmohammadi M and Craker L. 2013.
Healing effect of hydro-alcoholic extract of Ephedra pachyclada
Boiss. in experimentally gastric ulcer in rat. Acta Pol Pharm., 70
(6):1003-1009.
© 2020 Jordan Journal of Biological Sciences. All rights reserved - Volume 13, Number 1 127
Rustaiyan A, Javidnia K, Farjam MH, Aboee-Mehrizi F and
Ezzatzadeh E. 2011. Antimicrobial and antioxidant activity of the
Ephedra sarcocarpa growing in Iran. J Med Plants Res., 5
(17):4251-4255.
... 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 150 ml of 80% ethanol. ...
... 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. ...
... 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][35][36][37][38][39]. In this study, RAPD-PCR and ERIC-PCR profiles showed many significant differences between the treated and untreated E. coli strain. ...
Article
Full-text available
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.
... 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 150 ml of 80% ethanol. ...
... 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. ...
... 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][35][36][37][38][39]. In this study, RAPD-PCR and ERIC-PCR profiles showed many significant differences between the treated and untreated E. coli strain. ...
Article
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. 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.
Article
Full-text available
Aims: This study was conducted to evaluate the genotoxic effects of fruit and leaf ethanolic extracts of Ecaballium elateruim on clinical and reference strains of E. coli (E. coli ATCC 25922). Methodology: The genotoxic effects of fruit and leaf ethanolic extracts were determined by using enterobacterial repetitive intergenic consensus (ERIC-PCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: The results showed an alteration in DNA and protein profiles of both tested Escherichia coli strains treated with fruit and leaf extracts compared with untreated control. The alterations ranged between decreased or increased intensity of some bands, absence or appearance of new amplified fragments. Moreover, increased concentrations of E. elaterium extracts and increased time intervals seems to yield a more profound increase in total protein concentrations in both tested E. coli strains. Conclusions: Such findings strongly indicate the genotoxic effects of E. elaterium extracts on both E. coli strains. The results draw attention to the unsafe, improper use of E. elaterium extracts in folkloric medicine and point out the capability of using E. elaterium to treat E. coli infections. Future studies should be needed to find out the exact mechanisms responsible for the observed genotoxicity.
Article
Full-text available
Rhazya stricta plants have always played a major role in the treatment of human and animal diseases and it has main role in the folk medicine. The aim of this study was to explore the potential antimicrobial activities of the aqueous leaves extract of R. stricta on Gram-negative and Gram-positive food-borne bacteria and evaluate the antimicrobial effect at the molecular level. The results indicate that the aqueous leaves extract of R. stricta exhibited the antimicrobial activity against tested microorganisms. A clear, but significantly smaller, inhibition zones were formed after the treatment of two Gram-negative bacteria (Escherichia coli and Aeromonas hydrophila) and one Gram-positive bacteria (Staphylococcus aureus) with the aqueous leaves extract of R. stricta (50 mg) comparing with those formed after the treatment with streptomycin (15 mg). Moreover, the results obtained after the treatments of bacterial strains with elevated concentrations of aqueous extracts of the wild plant of R. stricta leaves reveled that the extract has potent lethal activities as the growth turbidity decreased as the concentration or time of exposure increased. In addition, the observation by the scanning electron microscope showed that cells of the bacterial strains were damaged after the treatment with plant extracts. The noticed antimicrobial effect was explored at the molecular level, using restriction fragment length polymorphism (RFLP) analysis of the plasmid DNA and random amplification of polymorphic DNA (RAPD) analysis of the genomic DNA extracted from the control (untreated) and R. stricta leaf extract-treated bacterial strains. The results demonstrate polymorphic band pattern for most treated microbes compared with the wild type (untreated) strain. Concerning gene expression under the same conditions, total protein contents of the three treated bacteria showed significantly gradual increase in all of the treatment doses compared to control. In addition, the SDS-PAGE of the bacterial cellular proteins resulted in the induction of some protein bands under the treatment conditions. All these results strongly point out the mutagenicity, lethal and antimicrobial effect of the leaves extract of R. stricta. The results indicate the possibility of using the leaves extract of R. stricta as a source of antibacterial compounds for treatment of infections caused by multi-drug resistant (MDR) bacterial pathogens.
Article
Full-text available
Background Ephedra is among Palestinian medicinal plants that are traditionally used in folkloric medicine for treating many diseases. Ephedra is known to have antibacterial and antioxidant effects. The goal of this study is to evaluate the antioxidant activity of different extracts from the Ephedra alata plant growing wild in Palestine, and to analyze their phenolic and flavonoid constituents by HPLC/PDA and HPLC/MS. Materials and Methods Samples of the Ephedra alata plant grown wild in Palestine were extracted with three different solvents namely, 100% water, 80% ethanol, and 100% ethanol. The extracts were analyzed for their total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (AA), as well as phenolic and flavonoids content by HPLC/PDA/MS. Results The results revealed that the polarity of the extraction solvent affects the TPC, TFC, and AA of extracts. It was found that both TPC and AA are highest for plant extracted with 80% ethanol, followed by 100% ethanol, and finally with 100% water. TFC however was highest in the following order: 100% ethanol > 80% ethanol > water. Pearson correlation indicated that there is a significant correlation between AA and TPC, but there is no correlation between AA and TFC. Simultaneous HPLC-PDA and UHPLC-MS analysis of the ethanolic plant extracts revealed the presence of Luteolin-7-O-glucuronide flavone, Myricetin 3-rhamnoside and some other major polyphenolic compounds that share myricetin skeleton. Conclusion Ephedra alata extract is rich in potent falvonoid glycosidic compounds as revealed by their similar overlaid UV-Vis spectra and UHPLC-MS results. On the basis of these findings, it is concluded that Ephedra alata constitutes a natural source of potent antioxidants that may prevent many diseases and could be potentially used in food, cosmetics, and pharmaceutical products.
Article
Full-text available
The medicinal significance of Ephedra is based on the sympathomimetic properties of ephedrine (E) alkaloids. Pharmacological effects depend on the phytocomposition of individual Ephedra species. The aim of this study was to measure the total alkaloids content (TAC), total phenolics content (TPC), and total flavonoids content (TFC) and determine their relationship in dry herb of Ephedra major, Ephedra distachya subsp. helvetica, Ephedra monosperma, Ephedra fragilis, Ephedra foeminea, Ephedra alata, Ephedra altissima and Ephedra foliata. Nowadays, medicinal use of Ephedrae herba is limited, but the abuse of its psychostimulants is rising. In this study, TAC, TPC and TFC were determined using spectrophotometric methods. For the first time, ultra-performance liquid chromatography with ultraviolet detection (UPLC-UV) was used for separation and quantification of E-type alkaloids of various Ephedra species. The highest TPC and TFC were found in E. alata (53.3 ± 0.1 mg Gallic acid equivalents/g dry weight, 2.8 mg quercetin equivalents/g dry weight, respectively). The total content of E and pseudoephedrine determined by UPLC-UV varied between 20.8 mg/g dry weight (E. distachya subsp. helvetica) and 34.7 mg/g dry weight (E. monosperma). The variable content and ratio between secondary metabolites determined in different Ephedra species reflects their metabolic activities. Utilization of UPLC-UV unveiled that this technique is sensitive, selective, and useful for separation and quantification of different alkaloids in complex biological matrixes. The limit of detection was 5 ng. Application of UPLC-UV can be recommended in quick analyses of E-type alkaloids in forensic medicine and quality control of pharmaceutical preparations.
Article
Full-text available
Ephedra procera belonging to the family Ephedraceae is a poison and medicinal plant. The main aim of present study was to determine total phenolic content and antioxidant and antibacterial activities of ethano-lic extract from the aerial parts of E. procera collected from a natural habitat in Chaharmahal va Bakhtiari province, Southwestern Iran. The total phenolic content of the extract by Folin-Ciocalteu method and the ntioxidant activity using DPPH assay were determined. The antibacterial activity, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of the extract were evaluated against five bacteria, including Proteus vulgaris, Pseudomonas aeruginosa, Enterobacter aerogenes, Bacillus cereus and Staphylococcus aureus . Total phenolic content in the extract of E. procera was 0.718 mg tannic acid/g dry weight extract. The results indicated that the ethanolic extract of E. procera exhibited radical scavenging activ-ity. In addition, the results of this study confirmed that the ethanolic extract of E. procera exhibited antibacter-ial activity. In conclusion, the extract of E. procera could be an important source of phenolic components with antioxidant capacity and antibacterial activity.
Article
Full-text available
This study was undertaken to evaluate genotoxic potential of Thermopsis turcica aqueous extracts on the roots of onion bulb (Allium cepa L.) by comet assay and random amplified polymorphic DNA technique. The Allium root growth inhibition test indicated that the EC50 and 2×EC50 values were 8 and 16 mg/ml concentrations of T. turcica aqueous extracts, respectively. The negative control (distilled water), positive control (methyl methane sulfonate, 10 mg/l) and 8 and 16 mg/ml concentrations of T. turcica extracts were introduced to the roots of onion bulbs for 24 and 96 h. The root growth, DNA damage in root cells and randomly amplified polymorphic DNA (RAPD) profiles of root tissue were used as endpoints of the genotoxicity. The comet assay clearly indicated that dose-dependent single strand DNA breaks in the root nuclei of onions were determined for the treatment concentrations of T. turcica extracts. In comparison to RAPD profile of negative control group, RAPD polymorphisms became evident as disappearance and/or appearance of RAPD bands in treated roots. The diagnostic and phenetic numerical analyses of RAPD profiles obviously indicated dose-dependent genotoxicity induced by Thermopsis extracts. In conclusion, the results clearly indicated that water extract of T. turcica has genotoxic potential on the roots of onion bulbs as shown by comet assay and RAPD technique.
Article
Full-text available
The random amplified polymorphic DNA (RAPD) assay was used to assess the level of DNA damage in various exposed and unexposed Culex quinquefasciatus larvae to acetone and chloroform extracts of Curcuma longa and Melia azedarach at different concentrations (6.25, 12.5 and 25 ppm). This is the first report of an analysis of genomic alterations in plant extracts-treated mosquito larvae using RAPD-PCR fingerprinting. In comparison to the control larvae, larvae treated with the plant extracts caused greater changes in the RAPD patterns. DNA strand breakage was more in the larvae of C. quinquefasciatus.
Article
Full-text available
Ephedra pachyclada Boiss. (family Ephedraceae) is a medicinal plant very frequently cited as acting against gastrointestinal disorders in ethno-pharmacological inventories of the Kerman region of Iran. This study was done to evaluate the effect of hydro-alcoholic extract from the stems of E. pachyclada for treatment of gastric ulcers induced by ethanol in Wistar rats. Experimental treatments were the hydro-alcoholic extract of E. pachyclada (250, 500, and 1000 mg/kg, orally), omperazole as standard drug (20 mg/kg, orally), and control group. Ulcer index in mm2 and histological examination were evaluated. On 3, 6, 9 and 12 day after treatments, the hydro-alcoholic extract of E. pachyclata (1000 mg/kg) produced 51, 72, 98.8 and 100% and omperazole also produced 53, 79, 93 and 100% curative effect for gastric mucosal damage in ethanol model, respectively. The results of the histopalogical analysis indicated the hydro-alcoholic extract of E. pachyclada at 1000 mg/kg was effective in experimentally healing rat ulcers. E. pachyclada accelerated ulcer healing in rats and, thus supports its folk medicine use by Kerman people.
Article
Ma-huang is a traditional Chinese medicinal herb derived from Ephedra sinica Stapf and other Ephedra species, used to treat asthma, nose and lung congestion, and fever with anhidrosis. It contains 0.5‐2.5% by weight of total alkaloids, of which ephedrine accounts for 30 to 90%. Recently, large amounts of ma-huang were used as a source of ephedrine in many dietary supplements formulated for weight reduction, because ephedrine has been found effective in inducing weight loss in diet-restricted obese patients. However, indiscriminate consumption of ma-huang‐ containing products has resulted in many cases of poisoning, some of which were fatal. The objective of this study is to investigate the relative toxicity of ma-huang extracted under different conditions. The toxicities of various extracts were assayed using MTT colorimetry on a battery of cell lines, while ephedrine alkaloids were analyzed with HPLC. The results are summarized as follows. (1) The cytotoxicity of all ma-huang extracts could not be totally accounted for by their ephedrine contents, suggesting the presence of other toxins in the extracts. (2) Grinding was a significant condition enhancing the toxicity of the extracts. (3) The relatively high sensitivity of the Neuro-2a cell line to the toxicity of ma-huang extracts suggests that the toxic principles were acting on neuronal cells. (4) One condition to produce a ma-huang extract with high ephedrine-to-toxins ratio would be to boil the whole herb for two h.