International Journal of Microbiology and Mycology | IJMM |

pISSN: 2309-4796
http://www.innspub.net
Vol. 13, No. 1, p. 9-17, 2021

Open Access RESEARCH PAPER

Antibacterial activity of selected medicinal plants extracts

against Escherichia coli isolates causing urinary tract infections

Muhammad Junaid Khan1, Asmat Ullah2, Uroosa Naseem3, Inam Ullah4,

Nadeem Ullah*5, Muhammad Sameer ishaq6, Mohammad Nasar7, Basit Ali Khan8,
Shakeel Ahmad9, Muhammad Zahid1, Nadia Mubarik11

1
Department of Microbiology, Abdul Wali Khan University, Mardan, Pakistan
2
Department of Microbiology, Government College University, Faislabad, Pakistan
3
Department of Microbiology, Federal Urdu University, Gulshan Campus, Karachi, Pakistan
4
Department of Microbiology and Immunology, Hebei Medical University, Shijiajhuang, China
5
Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, People’s Republic of China
6
Key Laboratory of Chemical Biology, School of Pharmaceutical Sciences,
Shangdong University, Jinan, China
7
Department of Biosciences, COMSATS University, Islamabad, Pakistan
8
Government College University, Lahore, Pakistan
9
Department of Biological Science, Gomal University, Dera Ismail Khan, Pakistan
10
Institute of Biotechnology and Genetic Engineering, University of Agriculture, Peshawar, Pakistan

Keywords: Antimicrobials, Paganum harmala, Fogonia cretica, Ajuga bracteosa, Olea feroogenia,
Urinary Tract Infections

Publication date: July 30, 2021

9 Khan et al.
Abstract
Antibiotic resistance was firstly observed soon after Alexander Fleming discovered penicillin in 1928.
Resistance to commonly used antibiotics is one of the most threatening issues to public health across
the globe. There has been an increasing demand to search for novel antimicrobial agents to combat the
growing resistance. Medicinal plants are a rich source of many drugs especially antimicrobial drugs and
these plants have been used from years to treat different types of diseases. The main goal of our study
was evaluation of antimicrobial activity of the selected plant extracts against clinical isolates and then to
compare resistance level of bacteria to commonly used antibiotics and medicinal plants. Methanolic
extract was taken and dried in water bath; dried extracts were dissolved in Dimethyl Sulfoxide (DMSO)
in different concentration (25mg/mL, 50mg/mL, and 100mg/mL). The zones of inhibition were
measured by using agar well diffusion method. Methanolic Extracts of Paganum harmala, Fogonia
cretica, Ajuga bracteosa and Olea feroogenia showed varying level of activities against E. coli. The
results of the study shows that the antibacterial effect of selected plant extracts increased with increase
in concentration. The extracts also showed activity against those isolates, which shows resistance to
commonly used antibiotics. Therefore, it is suggested that antibacterial agents present in these plants
should be isolated through advanced biochemical techniques such as HPLC for the evaluation of
potency. These compounds may be evaluated as potential antimicrobial therapeutic agents against a
wide range of human pathogens.
* Corresponding Author: Nadeem Ullah  [email protected]

10 Khan et al.
Introduction Botanists are working on medicinal plants and the
Scientists defined the antimicrobial resistance as horizons of knowledge about phyto-extracts are
ineffectiveness of drug against microbes (Coenen, increasing with the passage of time. Such studies
Ferech et al., 2007). For the first time the are very much important for making the
resistant cases were noted when new pharmaceutical research and development strong
antimicrobial agents were introduced to medium (Shakeri, Hazeri et al., 2012). Plants having
(Alekshun and Levy 2007). Antimicrobial compounds that helps to modulate immune
resistance is the result of evolution and is responses and eliminate pathogens. Immune
considered as the part of natural selection in the modulation is limited to one because modulations
survival promotion. Certain antibiotics of specific and non-specific responses are done
(Sulfamethoxazole-Trimethoprim, Ampicillin by phyto-extracts (Pandey and Chawdhry 2006).
Tetracycline) were widely used before which are Phyto-extracts are very significant for treatment
now no more used in many diseases (Hoge, of wide range of infections. Recently many
Gambel et al., 1998). Microbes can resist more scientists proved the medici nal importance of
antimicrobials drugs at a time, which is termed as plants. Plants synthesize secondary metabolites,
multidrug resistance. Staphylococcus spp has
which are important antimicrobials and extracted
been reported to have resistant against multiple
from plants to use in clinical setups (Nascimento,
antibiotics. Resistance mechanisms include
Locatelli et al., 2000). Reports from Palestine
alteration in membrane permeability, mutation in
shows that use of herbal medicines are on the top
genes, which cause alteration in target site,
there. Mostly herbal medicines have very low or
enzymatic modification of drugs, efflux pumps
no side effects therefore they are used mostly.
that transfer the antimicrobials outside the cell.
Herbal medicines are very much common as it
The bacteria not only resist the action of only one
merged in their culture. In Palestine, medicinal
antibiotic while it also resists the action of
plants commonly found on mountains, many of
structurally same antibiotics such as tetracycline
them exploited in agricultural use (pesticides)
resistant strains of bacteria can resist the action
(Jaradat 2005). Seeds of Peganum harmala
of oxytetracycline, chlortetracycline, doxycycline,
reported to cause intoxication and nausea (MM
and minocycline (Chopra and Roberts 2001).
2007). They are useful for epilepsy, psychosis,
From the ancient times, medicinal plants play an
loss of memory, chronic headache, kidney stone,
important role in human health. About 80% of
dropsy, jaundice, colic and sciatica. These plant’s
the word population use medicinal plants for
seeds are used to detoxify toxins in human body.
traditional health therapies according to the
Flax seed and honey in combination are useful for
World Health Organization report. Medicinal
the patients of dyspnea. The decoction has been
plants have been used for the derivation of more
used for numbness, lung and liver diseases
than half of the available chemotherapeutic
(Mikaili, Maadirad et al., 2013).
agents (Kirbağ, Zengin et al., 2009).

P. harmala is mainly used for disinfection

Antimicrobial and antioxidant activity of the
phytochemicals like vitamins A, C, E and K, purposes (Almasirad, Hosseini et al., 2006). A.

flavonoids, terpenoids, polyphenols, carotenoids, bracteosa is used in medicine since from very

pigments, saponins, enzymes and minerals have old time and has various utilizations. In ethno

been reported (Madhuri and Pandey 2009). The medicine, its use is reported as anthelmintic,

exact mechanisms of action of many plant astringent, antibacterial, anti-fungal, anti-

extracts are not clear. The antibacterial inflammatory, and hypoglycemic and it

compounds of phyto-extracts are rays of hope for remediates intestinal ailments (Israili and

the better future of medicine. Lyoussi 2009).

11 Khan et al.
Chinese use the above-mentioned plant in Identification
treatment of phlegm and pyrexia, as it is part of These plants were transferred to Microbiology
their tradition (Shen, Isogai et al., 1993). It is Laboratory where a Botanist from the
recommended in Ayurveda to treat rheumatism, Department of Botany Abdul Wali Khan University
amenorrhea, and gout and palsy (Kaithwas and Mardan Pakistan identified it.
Majumdar 2012). It is known that most of the
plant-derived compounds have substantial Preparation of the extracts

analgesic properties. Oil of Olive ferrugenia royle Eight Grams of air-dried powder of different parts

is the main fatty component of natural olive and of the selected plants were placed in 70 mL of

mediates positive effects on cardiovascular, pure methanol (100%) in a conical flask, plugged

metabolic, inflammatory and autoimmune with cotton. The material was then kept at room

diseases (La Lastra, Barranco et al., 2001). temperature for one week. After one week, it was

Extracts from olive have antioxidants properties, filtered with the Whatman No.1 filter paper and

for this purpose, we can use it in pharmaceuticals the filtrate was evaporated to dryness in water

as well as food industry (Savarese, De Marco et bath at 65℃ as reported (Setif 2011). The dried

al., 2007). Recently rate of olive usage increased extract was then dissolved in Dimethyl sulfoxide

many folds. Mostly people use their leaves as (DMSO) as solvent at different concentration

antidote in the recurring malarial fever. (25mg/mL, 50mg/mL, and 100mg/mL) and

Hypertension could be prevented with use of stored at -4 ℃ in the refrigerator for further use.

leaves extract of olive (Ranalli, Contento et al.,

Test Microorganisms
2006). The fresh fruits of O. ferrugenia royale are
The bacterial strains were obtained from
collected in summer and then dehydrated which is
Hayatabad Medical Complex Peshawar. Different
useful in lowering of glucose levels in diabetes
strains of E. coli were tested in this study.
(Ahmed et al., 2015). Fagonia indica is useful in
blood purification. Fruits of given plant are rich in
Maintenance of Bacterial Isolates
ascorbic acid (Shinwari et al., 2010). Extracts from
The bacterial strains were isolated by providing
given plants were tested for anticancer properties
standard temperature and pressure using CLED
and were reported positive for anticancer
media for Urine sample, MacConkey Agar and
properties (Graham, Quinn et al., 2000). For that
Blood Agar for all other samples. Colony
reason, the present study was conducted to
morphology and different chemical biochemical
determine the antibacterial activity of selected
testing were used to confirm the species
medicinal plants extracts against Escherichia coli
identification. The isolated strains were preserved
isolates causing urinary tract infections
in LB broth with Glycerol in Eppendorf tubes and
stored at -20℃ in the refrigerator for further use.
Material and methods
The present study was conducted in the
Agar Well Diffusion Method
Laboratory of Department of Microbiology, Abdul
Suspension of inoculum was swabbed uniformly on
Wali Khan University Mardan, Pakistan. The
solidified plates having 25 mL Mueller-Hinton Agar
plants used in this study were Paganum harmala
(MHA). Six mm in diameter holes were made in the
(seeds), Fogonia cretica (whole plant), Ajuga
agar by using glass Pasteur pipettes. Forty microliter
bracteosa (whole plant) and Olea feroogenia
from each plant crude extract of different
royle (Leaf).
concentration (25 mg/mL, 50mg/mL and
Collection of plants 100mg/mL) were added into each well made on the
The powder form of these plants were collected medium. For proper diffusion, it was allowed to stand
from local market in Mardan city. on the bench.

12 Khan et al.
Finally, for 24 hour it was incubated at 37℃. Zone of Olea feroogenia royle also shows inhibitory
inhibition were then measured. This experiment was activity against all strain of E. coli. (Table 4) Zone
carried out in duplicate. of inhibition were increased as increased occur in
concentration. (Fig. 4) Synthetic antibiotics (CAZ-
Results 30, SXT-25, CN-10, TE-30 and E-15) were also
Well diffusion method was used to determine the checked comparatively with the methanolic
antibiogram assay of selected medicinal plants. extract of Olea feroogenia royle. Maximum strain
According to our results, P.harmala extracts of E. coli shows resistance to synthetic antibiotics
showed high inhibitory activity against all tested accept SXT25 and CN-10, which effectively inhibit
strains of E. coli. (Table 1) some strains of E. coli. (Table 4)

Zone of inhibition were increased as increased Table 1. Antibacterial activity of Paganum

occur in concentration. (Fig. 1) Synthetic harmala methanolic extracts against different

antibiotics such as CAZ-30, SXT-25, CN-10, TE- isolates of E. coli.

30 and E-15 were also checked comparatively Synthetic

Inhibitory zone of Antibiotics
with the plants extract of P. harmala and most of plant extract (mm) Inhibitory
Isolates
the tested strain E. coli shows resistance to zone (mm)
25 50 100
antibiotic except some strain, which were CAZ E SXT TE CN
mg/mLmg/mLmg/mL
sensitive to SXT-25, CN-10. (Table 1) E1 15 16 18 0 0 20 15 15
E2 18 20 25 0 0 0 20 0
E3 19 22 25 20 0 0 0 10
Methanolic extract of Fogonia cretica also shows
E4 22 25 29 17 0 0 0 25
significant inhibition against all strain of E. coli.
E5 16 13 11 0 0 0 0 0
Some strains were less sensitive while some are E6 15 12 10 0 0 0 0 0
more sensitive to plants extract. (Table 2) Zone E7 14 11 10 16 0 17 17 16
of inhibition were increased as increased occur in E8 14 12 11 15 0 18 15 17

concentration. (Fig. 2) Synthetic antibiotics (CAZ- CAZ- trimethoprim, SXT- Ceftazidime, TE-

30, SXT-25, CN-10, TE-30 and E-15) were also Tetracycline, CN-Gentamycin, E- Erythromycin

checked comparatively with the methanolic

extract of Fogonia cretica. Table 2. Antibacterial activity of Fagonia cretica
methanolic extracts against different isolates of
E. coli by.
Most of the strains were resistant to antibiotic
Synthetic
except some strain, which were sensitive to SXT- Antibiotics
Inhibitory zone of
Inhibitory
25, CN-10. (Table 2) Methanolic extract of Ajuga plant extract (mm)
Isolates zone
bracteosa also showed inhibitory activity against (mm)
25 50 100
all strains of E. coli. (Table 3) Zone of inhibition CAZ E SXT TE CN
mg/mL mg/mL mg/mL
were increased as increased occur in E1 8 10 14 0 0 19 17 13
concentration. (Fig. 3) E2 15 18 20 0 0 0 19 0
E3 13 15 18 21 0 0 0 11
E4 12 13 15 16 0 0 0 24
Synthetic antibiotics (CAZ-30, SXT-25, CN-10,
E5 15 17 20 0 0 0 0 0
TE-30 and E-15) were also checked
E6 12 14 15 0 0 0 0 0
comparatively with the plants extract, in which all E7 10 11 13 17 0 15 16 15
strains show resistant to all antibiotics other than E8 13 14 16 14 0 19 17 15
SXT-25 and CN-10, which inhibit the growth CAZ- trimethoprim, SXT- Ceftazidime, TE-
some strains. (Table 3) Tetracycline, CN-Gentamycin, E- Erythromycin

13 Khan et al.
Table 3. Antibacterial activity of Ajuga Bracteosa
50
methanolic extracts against different isolates of
E. coli. 0
Synthetic E1 E2 E3 E4 E5 E6 E7 E8
Antibiotics
Inhibitory zone of Zone of inhibition of plant extract (mm) 25
Inhibitory
Isolates plant extract (mm)
zone
(mm)
Zone of inhibition of plant extract (mm) 50
25 50 100
CAZ E SXT TE CN
mg/mL mg/mL mg/mL
E1 14 17 18 0 0 21 17 13 Zone of inhibition of plant extract (mm) 100
E2 12 14 16 0 0 0 19 0 mg/mL
E3 10 12 15 19 0 0 0 11
E4 10 12 14 18 0 0 0 26 Fig. 2. Relative Zone of inhibition of
E5 10 13 15 0 0 0 0 0
E6 12 15 17 0 0 0 0 0 different concentration of Fagonia cretica
E7 9 10 12 17 0 16 15 15 methanolic extracts.
E8 11 12 14 13 0 20 15 18
CAZ- trimethoprim, SXT- Ceftazidime, TE-
Tetracycline, CN-Gentamycin, E- Erythromycin 20
0
E1 E2 E3 E4 E5 E6 E7 E8
Table 4. Antibacterial activity of Olea ferrugenia
Zone of inhibition of plant extract (mm) 25
Royale methanolic extracts against different
isolates of E. coli. Zone of inhibition of plant extract (mm) 50
Synthetic
Inhibitory zone of Antibiotics Zone of inhibition of plant extract (mm) 100
Isolates plant extract (mm) Inhibitory mg/mL
zone (mm)
25 50 100
CAZ E SXT TE CN Fig. 3. Relative Zone of inhibition of
mg/mLmg/mLmg/mL
E1 15 16 18 0 0 18 14 16 different concentration of Ajuga B racteosa
E2 15 18 20 0 0 0 22 0
methanolic extracts.
E3 15 18 20 23 0 0 0 12
E4 14 17 21 19 0 0 0 22
E5 13 11 9 0 0 0 0 0
E6 17 15 13 0 0 0 0 0 40
E7 13 10 8 15 0 18 18 17
E8 18 15 13 15 0 20 14 19 20
CAZ- trimethoprim, SXT- Ceftazidime, TE-
0
Tetracycline, CN-Gentamycin, E- Erythromycin E1 E2 E3 E4 E5 E6 E7 E8

Zone of inhibition of plant extract (mm) 25

40
Zone of inhibition of plant extract (mm) 50
20
0
Zone of inhibition of plant extract (mm) 100
E1 E2 E3 E4 E5 E6 E7 E8
mg/mL
Zone of inhibition of plant extract (mm) 25 Fig. 4. Relative Zone of inhibition of different
concentration of Olea ferrugenia royale
Zone of inhibition of plant extract (mm) 50 methanolic extracts.

Zone of inhibition of plant extract (mm) 100

Discussion
mg/mL
Both for conventional and modern medicines,
Fig. 1. Relative Zone of inhibition of medicinal plants are used as potential active
different concentration of Paganum source (Shariff 2001). The use of medicinal plants
harmala methanolic extracts. As an alternative of chemical antimicrobial agents

14 Khan et al.
to treat bacterial infections are advantageous Fogonia cretica. Most of the strains were resistant
because many of the medicinal plants have the to antibiotic except some strain, which were
ability of agreeing the biological toxicity and have sensitive to SXT-25, CN-10. Methanolic extract of
rarer side effects. Easy access, low price and no Ajuga bracteosa also showed inhibitory activity
reported resistance of herbs to bacteria make it against all strains of E. coli. Zone of inhibition
advantageous (Hayacibara, Koo et al., 2005). In were increased as increased occur in
the present study the antibacterial activity of concentration. Similar result are reported in
methanolic extract of Paganum harmala, Fagonia another study (Khan, Prakash et al., 2011) using
cretica, Ajuga bracteosa and Olea ferrugenia aqueous extracts of flowers part of this plant and
royle against different isolates of E. coli were give significant results. Another study (Shad,
studied by using well diffusion method. According Zeeshan et al., 2016) concluded the highest
to our results, P.harmala extracts showed high percent zone of inhibition of hexane fraction of A.
inhibitory activity against all tested strains of E. bracteosa against E. coli. Synthetic antibiotics
coli. Zone of inhibition were increased as (CAZ-30, SXT-25, CN-10, TE-30 and E-15) were
increased occur in concentration. also checked comparatively with the plants
extract, in which all strains show resistant to all
Mohsenipour et al obtained similar results antibiotics other than SXT-25 and CN-10, which
(Mohsenipour and Hassanshahian 2016) and the inhibit the growth some strains.
study reported low efficacy to prevent biofilm
formation of E. coli. This result is also in Olea feroogenia royle also shows inhibitory

agreement with the previous study done by activity against all strain of E. coli. Zone of
inhibition were increased as increased occur in
Arshad et al., who reported the antibacterial
concentration. Another study also reported
activity of seed extract of P. hamala against
appreciable bactericidal activity against E. coli. It
O1:K1 serotype of E. coli (Arshad, Neubauer et
is evident that the Olea feroogenia royle show
al., 2008). Synthetic antibiotics such as CAZ-30,
activity against Escherichia coli and other clinical
SXT-25, CN-10, TE-30 and E-15 were also
isolates (Mehmood and Murtaza 2018). Synthetic
checked comparatively with the plants extract of
antibiotics (CAZ-30, SXT-25, CN-10, TE-30 and
P. harmala and most of the tested strain E. coli
E-15) were also checked comparatively with the
shows resistance to antibiotic except some strain,
methanolic extract of Olea feroogenia royle.
which were sensitive to SXT-25, CN-10.
Maximum strain of E. coli shows resistance to
Methanolic extract of Fogonia cretica also shows
synthetic antibiotics accept SXT25 and CN-10,
significant inhibition against all strain of E. coli.
which effectively inhibit some strains of E. coli.
Some strains were less sensitive while some are
more sensitive to plants extract. Zone of Conclusion
inhibition were increased as increased occur in Plants have been used as medicinal sources in
concentration. Our study is in accordance with the developing countries from long time. In the
the previous study who use whole plant current study antibacterial activity of Paganum
methanolic extract of Fagonia cretica in various harmala, Fogonia cretica, Ajuga bracteosa and
concentrations against Escherichia coli. Olea feroogenia royle were checked against
clinical isolates E. coli. The plant extracts showed
The extract indicated more activity against all the activity against some pathogens while some
tested bacterial strains of E. coli (Sajid, Alia et pathogens are resistant to the plants extract. The
al., 2011). Synthetic antibiotics (CAZ-30, SXT-25, results of the study shows that the antibacterial
CN-10, TE-30 and E-15) were also checked effect of plants extracts increased after increase
comparatively with the methanolic extract in concentration.

15 Khan et al.
Synthetic antibiotics were also used to check Graham J, Quinn M, Fabricant D, Farnsworth
their antibiotic sensitivity testing in which most of N. 2000. Plants used against cancer–an
the clinical isolates were resistant strains. extension of the work of Jonathan Hartwell.
Possibly these plants have bioactive compounds Journal of ethnopharmacology 73, 347-377.
which inhibit the growth of bacteria. Therefore, it
is recommended to isolate and separate these Hayacibara MF, Koo H, Rosalen PL, Duarte S,
bioactive compounds responsible for this Franco EM, Bowen WH, Ikegaki M, Cury JA.
antibacterial activity using advanced scientific 2005. In vitro and in vivo effects of isolated
techniques. They might have a potential for new fractions of Brazilian propolis on caries
drug development against wide range of human development. Journal of ethnopharmacology
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