Jundishapur J Nat Pharm Prod. 2015 August; 10(3): e23004. DOI: 10.17795/jjnpp-23004
Published online 2015 August 29. Research Article
Antimicrobial Activity of Aqueous-Alcoholic Extracts and the Essential Oil of
Verbascum thapsus L.
Fatemeh Ghasemi 1; Fakhreddin Rezaei 1; Atefeh Araghi 2; Mohaddeseh Abouhosseini
1Young Researchers and Elite Club, Babol Branch, Islamic Azad University, Babol, IR Iran
2Department of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, IR Iran
*Corresponding author: Mohaddeseh Abouhosseini Tabari, Department of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, IR Iran. Tel: +98-1144265008,
Fax: +98-1144271054, E-mail: email@example.com
Received: September 9, 2014; Revised: February 25, 2015; Accepted: March 11, 2015
Background: The increasing resistance of human pathogens to the available antimicrobials is a serious threat, resulting in the need for
novel antibiotic resources such as plants. Some species of the genus Verbascum have been used by mankind since ancient times as an
eﬀective remedy for infectious diseases.
Objectives: This study was designed to determine the antimicrobial eﬃcacy of the aqueous-alcoholic extracts and the essential oil of
Verbascum thapsus L. against diﬀerent kinds of bacterial and fungal strains, viz. Streptococcus pyogenes, Escherichia coli, Staphylococcus
aureus, Candida albicans, and Aspergillus fumigatus.
Materials and Methods: The antimicrobial activities of the V. thapsus extracts were examined in the present study on the basis of disc
diﬀusion and microdilution assays, and their potency was quantitatively assessed in terms of inhibition zone diameters and minimum
inhibitory concentration (MIC) values.
Results: The disk diﬀusion test showed that the methanol extract of V. thapsus had more growth inhibitory eﬀects on E. coli and S. pyogenes
than the aqueous and ethanol extracts. The methanol and aqueous extracts had no eﬀects on S. aureus. The maximal inhibition zone for
the microorganisms sensitive to the methanol extract was in the range of 7 - 16.8 mm, and the MIC value was 31.25 μg/mL. For the ethanol
extract, the maximal inhibition zone was 5.3 - 11 mm and the MIC value was 62.5 - 125 μg/mL. The essential oil of V. thapsus did not exhibit any
antibacterial and antifungal activities.
Conclusions: The ﬁndings of the present study revealed the V. thapsus extract possesses compounds with antibacterial properties that can
be used as novel antimicrobial agents in the development of new drugs for the treatment of infectious diseases.
Keywords: Antimicrobial Activity; Aqueous Extract; Alcoholic Extract; Essential Oil; Verbascum Thapsus L.
Copyright © 2015, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences. This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the mate-
rial just in noncommercial usages, provided the original work is properly cited.
Plants play an essential role in the everyday needs of hu-
man life. They are used as food, cosmetics, ﬂavors, medi-
cines, and ornaments (1). Medicinal plants because of
their potential health eﬀects have become part of comple-
mentary medicine worldwide (2). Various plant extracts
have been widely used for therapeutic purposes, includ-
ing battling infectious diseases (3). Verbascum L. is a mem-
ber of the family Scrophulariaceae represented by 5,100
species, all of which are grown in temperate and tropical
areas (4). The genus Verbascum comprises 360 species (5).
Scrophulariaceae are a source of saponins, monoterpene
glycosides, iridoids, phenylethanoid glycosides, neolig-
nan glycosides, ﬂavonoids, steroids, spermine alkaloids,
phenolic acids, and fatty acids (6). Plants within the ge-
nus of Verbascum have beneﬁcial therapeutic eﬀects and
are employed in traditional medicine. It is reported that
the leaves and ﬂowers of Verbascum show mucolytic and
expectorant eﬀects and are utilized to treat respiratory
diseases such as dry cough, bronchitis, tuberculosis, and
asthma in traditional medicine. Also, these plants are
drawn upon to treat superﬁcial fungal infections, rheu-
matic pain, hemorrhoids, diarrhea, and wounds. It is also
reported that these plants have inhibitory eﬀects against
inﬂuenza viruses A2 and B and murine lymphocytic leu-
kemia (7). In Asia, Europe, and Northern America, several
Verbascum species have been indicated as having antioxi-
dant, narcotic, antiseptic, emollient, astringent, expec-
torant, sedative, and diuretic properties; moreover, they
are used as a treatment for inﬂammations, tumors, and
migraine (8). The ever-increasing resistance of human
pathogens to the available antimicrobial agents is a seri-
ous threat, resulting in an urgent need for novel antibi-
otic resources such as plants (9). Some species of the ge-
nus Verbascum have been used by mankind since ancient
times to treat internal and external infections. The genus
Verbascum has been tested for antimicrobial and antifun-
Ghasemi F et al.
Jundishapur J Nat Pharm Prod. 2015;10(3):e230042
gal activity; however, the results have revealed that the
extracts of the diﬀerent species of the genus Verbascum
do not exhibit similar antimicrobial eﬀects against diﬀer-
ent kinds of bacteria and fungi (10).
The aim of the present study was to compare the eﬃcacy
of the methanol, ethanol, and aqueous extracts of Verbas-
cum thapsus against Streptococcus pyogenes (S. pyogenes),
Escherichia coli (E. coli), Staphylococcus aureus (S. aureus),
Candida albicans (C. albicans), and Aspergillus fumigatus (A.
fumigatus) via the disk diﬀusion method and determina-
tion of minimal inhibitory concentration (MIC) values.
3. Materials and Methods
3.1. Collection, Identiﬁcation, and Preparation of
the Plant Material
Aerial parts of V. thapsus were collected from the sub-
urbs of Arak, Markazi Province, center of Iran, and iden-
tiﬁed by the Research Center of Agriculture and Natural
Resources of Sari, Mazandaran, Iran. A voucher specimen
(Herbarium No. 448) was deposited at the herbarium of
the Research Center of Agriculture and Natural Resourc-
es of Sari, Mazandaran. The collected plant material was
dried in the shade, and the leaves were separated from
the stem and ground in a grinder with a 2-mm diameter
mesh. The aqueous extract of V. thapsus was obtained by
blending 10 g of V. thapsus powder in 100 mL of boiling
sterile distilled water, centrifuged at 5,000 rpm, and ster-
ilized by ltration (0.45 μm). The aqueous extract was
concentrated in vacuum at 40°C using a rotary evapora-
tor. The residues obtained were stored at 4°C. The metha-
nol extract of V. thapsus was prepared by taking 10 g of V.
thapsus powder in Soxhlet extraction with methanol (3X)
for 6 hours. Then, the methanol was evaporated under
vacuum, and the residue was dissolved in dimethyl sulf-
oxide (DMSO) to give a concentration of 100 mg mL-1. The
ethanol extract of V. thapsus was obtained by blending 10
g of V. thapsus powder with 50% ethanol (3X) at 50°C. The
ethanol was evaporated under vacuum, and the residue
was dissolved in DMSO to give a concentration of 100 mg
mL-1. For the isolation of essential oil, 100 g of V. thapsus
powder was hydrodistillated in a Clevenger-type appara-
tus for 3 hours. The oil was dried over anhydrous sodium
sulfate and kept at 4°C in a sealed brown vial (10).
3.2. Antimicrobial Assay
E. coli ACCT 25922, S. aureus ACCT 1112, S. pyogenes PTTC
1447, C. albicans ATCC 10231, and A. fumigatus ATCC 26606
were obtained from the Industrial Research and Training
Center of Iran to examine the antimicrobial activity of V.
The disk diﬀusion method with a 6-mm ﬁlter paper
disk (Roshd Research Laboratory, Iran) was used for the
screening of antibacterial and antifungal activities (11).
The bacterial and fungal strains were respectively tested
on Müller-Hinton and Sabouraud dextrose agars. Steril-
ized paper disks were loaded with diﬀerent amounts of
V. thapsus extracts (ethanol, methanol, and water) and oil
(100, 200, 300, and 400 μg, respectively) and then placed
on the inoculated agars. Antimicrobial tests by the disc
diusion method were carried out using 100μL of sus-
pension containing 108 CFU/mL of the microorganisms.
All the plates were incubated at 37°C for 24 hours for bac-
teria; at 25°C for 24 hours for C. albicans; and at 29°C for
2 days for A. fumigatus. Inhibition zone diameters were
measured after a conventional incubation period. Genta-
micin (10 μL) and nystatin (10 μL) (obtained from Sigma)
were used as positive reference standards respectively
for bacterial and fungal strains. Antimicrobial tests were
thereafter carried out via the disc diﬀusion method using
100 μL of suspension containing 108 CFU/mL of bacteria.
Antimicrobial activity was evaluated by measuring the
zone of inhibition against the test organisms. Each assay
in this experiment was repeated 3 times.
The MIC values of the V. thapsus extracts and oil against
the bacterial strains and the A. fumigatus and C. albicans
isolates were also determined based on a micro-well dilu-
tion method (12, 13). The inocula of the microorganisms
were prepared from broth cultures, and suspensions
were adjusted to 0.5 McFarland standard turbidity. The
96-well plates were prepared by dispensing into each
well 1 mL of nutrient broth. Also, 1 mL of V. thapsus ex-
tracts initially prepared at a concentration of 1000 μg/mL
was added into the ﬁrst and second wells. Subsequently,
1 mL from 1 mL of the content of test tube number 2 was
added to test tube number 3 and mixed completely. This
process was performed serially to the last test tube. At the
end, 1 mL content of the last test tube (number 12) was dis-
carded. Finally, 100 μL of the inoculums was added to test
tubes number 2 to 12; and in order to have equal amounts
of material in all the test tubes, 0.9 mL of test tube num-
ber 1 was discarded. The plate was covered with a sterile
plate sealer. The contents of each well were incubated in
a shaking incubator at 200 rpm at 37°C for an appropri-
ate time period (24 hours for bacteria and 72 hours for
fungi). Microbial growth was determined by absorbance
at 600 nm using the microplate reader. The extracts and
essential oil tested in this study were screened 3 times
against each organism. The MIC was deﬁned as the lowest
concentration of the compounds to inhibit the growth of
the microorganisms (12).
DMSO 10%, which was used for diluting the alcoholic ex-
tracts and oil of V. thapsus to a concentration of 100 mg
mL-1, was tested against the microbial strains of the pres-
ent study and showed no antimicrobial activity.
The inhibition zone against S. aureus, S. pyogenes, and E.
coli for all the 3 extracts is shown in Table 1. The disk diﬀu-
sion test showed that the methanol extract of V. thapsus
Ghasemi F et al.
Jundishapur J Nat Pharm Prod. 2015;10(3):e23004
had more growth inhibitory eﬀects on E. coli and S. pyo-
genes than the aqueous and ethanol extracts. On the other
hand, the methanol and aqueous extracts had no eﬀects
on S. aureus, while the ethanol extract had growth inhibi-
tory eﬀects on this bacterium. A. fumigatus and C. albicans
were not susceptible to the antimicrobial compounds of
the extracts. Surprisingly, no antibacterial or antifungal ac-
tivity was observed for the essential oil of V. thapsus.
The MIC value for the aqueous extract was 62.5 μg/mL
for E. coli and 250 μg/mL for S. aureus and S. pyogenes. The
MIC value of the ethanol extract was 62.5 μg/mL for E. coli
and S. pyogenes and 125 μg/mL for S. aureus. The MIC value
for the methanol extract for E. coli and S. pyogenes was
31.25 μg/mL (Table 2).
Table 1. Antimicrobial Activity of Verbascum thapsus Extracts and Oil Against the Tested Microbial Strains Based on the Disk Diﬀusion Method a
Strains Aqueous Extract, μL Ethanol Extract, μL Methanol Extract, μL Essential
10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40
E. coli 5.83 ± 0.2 6.83 ± 0.2 8.93 ± 0.08 - 6.03 ± 0.04 8 ± 0.35 11 ± 0.0 7 ± 0.0 10 ± 0.0 13.87 ± 0.11 16.83 ± 0.2 - - - - 33.67 ± 0.22 -
S. aureus - - - - - - 5.3 ± 0.19 7 ± 0.14 - - - - - - - - 25.17 ± 0.2 -
S. pyogenes - 5.93 ± 0.08 9.07 ± 0.15 10 ± 0.0 - 5.83 ± 0.2 8 ± 0.35 10.93 ± 0.36 - 8 ± 0.14 10.23 ± 0.18 13 ± 0.0 - - - - 25 ± 0.0 -
- - - - - - - - - - - - - - - - - 37 ± 2.12
C. albicans - - - - - - - - - - - - - - - - - 23.33 ± 1.08
a Inhibition zone is expressed in diameter around the test disk (mm ± SEM).
Table 2. Minimum Inhibitory Concentration Values of Verbascum thapsus Against the Microorganisms Tested in the Microdilution
Strains Aqueous Extract, μg/mL Ethanolic Extract, μg/mL Methanolic Extract, μg/mL Essential Oil, μg/mL
E. coli 62.5 62.5 31.25 -
S. aureus - 125 - -
S. pyogenes 250 62.5 31.25 -
A.fumigatus - - - -
C. albicans - - - -
Today more than ever, the increasing occurrence of
antibiotic-resistant strains has led to an urgent demand
for new antibiotics (14). The majority of the available
drugs in developing countries are driven from medicinal
plants; and in industrialized countries, plants make up
the raw material for the synthesis of pure chemical de-
In the current study, 2 Gram-positive strains (S. pyogenes
and S. aureus), 1 Gram-negative strain (E. coli), and 2 fun-
gal strains (A. fumigatus and C. albicans) were used to com-
pare the antimicrobial activity of the aqueous-alcoholic
extracts and the essential oil of V. thapsus. Some previous
investigations have indicated that there is no antimicro-
bial activity for the oil obtained from the dried ﬂowering
aerial parts of V. thapsus (16). In line with these studies,
the results of the present study showed that the essential
oil of V. thapsus had no antimicrobial activity against any
of the bacterial or fungal isolates tested in the study.
The maximal inhibition zone for the microorganisms
sensitive to the methanol extract was in the range of
7 - 16.8 mm, and the MIC value was 31.25 mg/mL. For the
ethanol extract, the maximal inhibition zone was 5.3 - 11
mm and the MIC value was 62.5 - 125 mg/mL. These re-
sults clearly indicated that the alcoholic extracts of V.
thapsus were able to inhibit the growth of some patho-
genic bacteria; however, the eﬀectiveness varied against
the diﬀerent microorganisms tested. The ﬁndings of the
present study conﬁrmed the reported results of some
other investigations insofar as some Verbascum species
contain substances with antimicrobial properties (17,
18). Some studies have been conducted on the antimicro-
bial properties of several Verbascum species extracts (16,
19-22). The results of the present study are in agreement
with a study performed by Guarino (23), who examined
the antimicrobial activity of V. macrurum leaves extracts
and demonstrated that the ethanol-aqueous extract ex-
hibited the most activity against Gram-positive bacteria
such as S. aureus.
In addition, the methanol extracts of the seeds, roots,
leaves, and ﬂowers of V. blattaria, V. bombyciferum, V. ni-
grum, V. chaixii, V. dumulosum, V. phlomoides, V. olympicum,
V. roripifolium, and V. phoeniceum have been previously
studied for their antimicrobial activities. One study re-
ported that the extracts had a strong antimicrobial ac-
tivity against E. coli ATCC 11230 (24). Turker and Camper
(10) studied the antibacterial activity of V. thapsus extracts
(aqueous, ethanolic, and methanolic) and commercial
products of Common Mullein (an alcoholic extract, a
ﬂower oil, tea bags, and swallow capsules) against some
bacterial strains (E. coli, P. aeruginosa, K. pneumoniae, S.
Ghasemi F et al.
Jundishapur J Nat Pharm Prod. 2015;10(3):e230044
aureus, and S. epidermidis) and reported antibacterial
activity with S. epidermidis, S. aureus, K. pneumonia, and
E coli. The commercially Mullein ﬂower oil sample (ﬂow-
ers extracted in pure olive oil) had growth inhibitory ef-
fects on all the test organisms except S. epidermidis and
S. pyogenes (10). Another study showed that the V. thapsus
oil exhibited concentration-dependent antimicrobial ac-
tivity against B. subtilis, S. typhi, S. aureus, A. niger, and P.
aeruginosa. The results also demonstrated that the essen-
tial oil had no antimicrobial activity against E. coli and C.
albicans, which chimes in with the results of the present
These kinds of diﬀerences in susceptibility against anti-
microbial agents in plant extracts may be related to the
inheritance of antimicrobial-resistance genes in bacteri-
al strains and/or the diﬀerences in cell-wall composition
of bacterial strains (19). Chemical composition may dif-
fer between essential oils and extracts from the same or
taxonomically similar species (25) due to external factors.
These diﬀerences will in turn aﬀect the biocidal activity
of oils (26).
Finally, the results of the present study indicated that
V. thapsus possesses compounds with antibacterial prop-
erties that can be used as novel antimicrobial agents in
the development of new drugs for the treatment of infec-
tious diseases. The presence of these medicinal eﬀects
suggests that this plant, V. thapsus, may be a source of bio-
active substances with multifaceted activities. Further
phytochemical analyses such as fractionation should be
performed on these extracts to isolate active constituents
and conduct subsequent pharmacological evaluations.
We express our special thanks to Dr. Mohammad Reza
Yousseﬁ, Head of Young Researchers’ and Elite Club,
Babol Branch, Islamic Azad University, for his kind techni-
cal advice and support.
Fatemeh Ghasemi and Fakhreddin Rezaei developed
the original idea and the protocol and analyzed data.
Mohaddeseh Abouhosseini Tabari and Atefeh Araghi con-
tributed to the development of the protocol, abstracted
the data, and prepared the manuscript.
This study was supported in part by Grant#bpj91035,
Young Researchers and Elite Club, Babol Branch, Islamic
Azad University, Babol, IR Iran.
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