Pharmaceutical Biology, 2009; 47(8): 697–700

RESEARCH ARTICLE

Antibacterial, antifungal, and antimycobacterial activity

of Ilex aquifolium leaves
Nurgün Erdemoglu1, Gökalp Iscan2, Bilge Sener1, and Prasit Palittapongarnpim3
1
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara, Turkey, 2Department of
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Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskişehir, Turkey, and 3Department of Microbiology,
Faculty of Science, Mahidol University, Bangkok, Thailand

Abstract
The objective of this study was to examine antibacterial, antifungal, and antimycobacterial properties of
Ilex aquifolium L. (Aquifoliaceae) growing in Turkey. The ethanol, ethyl acetate, chloroform, and n-hexane
extracts prepared from the leaves of I. aquifolium were tested against Escherichia coli, Staphylococcus
aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, Proteus vulgaris, Salmonella typhimurium, and
Candida albicans for antibacterial and antifungal evaluation using the microdilution broth susceptibility
assay. In addition, antimycobacterial activity of the crude extracts of I. aquifolium was evaluated by micro-
plate Alamar blue assay. The results showed that the extracts tested, except n-hexane, possessed moder-
ate antibacterial and antifungal activity varying from 62.5 to 250 μg/mL. On the other hand, the ethanol
For personal use only.

extract of the leaves exhibited a minimum inhibitory concentration value (MIC) against Mycobacterium
tuberculosis H37Ra strain of 200 μg/mL.
Keywords:  Antibacterial activity; Aquifoliaceae; Ilex aquifolium

Introduction in Turkey, namely I. aquifolium and I. colchica Poj.

Tuberculosis (TB) still remains among serious public
health problems. In the world, TB ranks second among
the leading infectious diseases, with ca. 2–3 million
deaths in 7–8 million new cases of active TB each year
(World Health Organization, 2004). The presence of a
number of highly effective medications for treating TB
has increased in the last three decades, yet the morbid-
ity and mortality of TB continue to increase, especially
when coupled with immunodeficiency virus (HIV).
Furthermore, multidrug resistant TB strains have led to
the search for new classes of potent antimycobacterial
drugs, including natural products, against this infectious
disease (Duncan, 2003).
Ilex aquifolium L. (Aquifoliaceae) (European or
English holly), one of the most well known species
of genus Ilex, is a dioecious broad-leaved evergreen
perennial shrub or tree with red drupes (Harder et al.,
1965; Yaltırık, 1966). The genus has two species growing
(Yaltırık, 1966). Today, hollies are grown comm­­ercially
for decorative purposes during Christmas because of
their colored berries and shiny green leaves (Grieve,
1982).
I. aquifolium leaves have been used in folk medicine
for several purposes, such as in intermittent fevers and
rheumatisms, for their antipyretic properties, and as
astringent, diuretic, and expectorant agents (Grieve,
1982). Muller et al. (1998) reported the inhibitory effect
of I. aquifolium against lipid peroxidation. There have
been several studies on the chemical composition of
I. aquifolium which reported that anthocyanins, flavo-
noids, terpenoids, sterols, amino acids, alkaloids, fatty
acids, alcohols, carbohydrates, carotenoids, cyanogenic
glucoside, phenols, and phenolic acids were present in
the plant (Valadon et  al., 1975; Knights & Smith 1977;
Catalano et  al., 1978; Budzikiewicz & Thomas, 1980;
Alikaridis, 1987; Nahrstedt & Wray 1990; Van Genderen &
Jaarsma, 1990; Erdemoglu et al., 2000; Sun et al., 2000).

Address for Correspondence:  N. Erdemoglu, Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler 06330, Ankara, Turkey. Fax:
+90-312-2235018. E-mail: nurgun@gazi.edu.tr
(Received 12 February 2008; revised 27 May 2008; accepted 28 May 2008)

ISSN 1388-0209 print/ISSN 1744-5116 online © 2009 Informa UK Ltd

DOI: 10.1080/13880200902930431 http://www.informapharmascience.com/phb
 698   Nurgün Erdemoglu et al.
As a part of our ongoing research on the screening of
biologically active Turkish plants, we have investigated
the antibacterial and antifungal activity of crude extracts
of the leaves of I. aquifolium, as well as tested for antimy-
cobacterial activity against Mycobacterium tuberculosis
H37Ra strain.
Materials and methods
Plant material
Ilex aquifolium was collected from the vicinity of
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Amasya, Turkey, in February 1998. Plant material was
collected by one of us (N.E.) and identified by Prof.
Dr. Mecit Vural (Department of Biology, Faculty of
Science, Gazi University, Ankara). A voucher speci-
men (2302) is kept in the Herbarium of the Faculty of
Pharmacy, Gazi University, Ankara, Turkey.
Preparation of crude extracts
Air-dried and powdered leaves of Ilex aquifolium were
accurately weighed (10 g) and separately extracted with
For personal use only.
96% ethanol, ethyl acetate (Merck), chloroform (Merck),
and n-hexane (Merck) using maceration at room tem-
perature, in duplicate (× 50 mL). After filtration, the com-
bined extracts were evaporated to dryness in vacuo and
the crude EtOH, EtOAc, CHCl3, and n-hexane extracts
were obtained. The percentage yields of the extracts are
given in Table 2.
Antimicrobial assay
Microorganisms were stored at +4°C on agar slants.
Standard strains of the following bacteria, namely
Escherichia coli (ATCC 25922), Staphylococcus aureus
(ATCC 6538), Pseudomonas aeruginosa (ATCC 27853),
Enterobacter aerogenes (NRRL 3567), Proteus vulgaris
(NRRL B-123), and Salmonella typhimurium (NRRL
B-4420) for the determination of antibacterial activ-
ity, and standard strains of Candida albicans (clinical
isolate; Osmangazi University, Faculty of Medicine,
Eskisehir, Turkey) for the determination of antifungal
activity were used.
A microdilution broth susceptibility assay (Koneman
et al., 1997; Zgoda & Porter, 2001) was used for antimi-
crobial evaluation of the extracts. Stock solution was
prepared in dimethylsulfoxide (DMSO; Carlo-Erba,
France). Dilution series using sterile distilled water were
prepared from 4 to 0. 007 mg/mL in micro-test tubes
(Eppendorf ), and were transferred to 96-well microtiter
plates. Overnight grown bacterial and C. albicans sus-
pensions in Mueller-Hinton broth were standardized
(for bacteria and C. albicans app. 108 and 106 cfu/mL,
respectively) using McFarland No. 0.5 standard solu-
tion. Each microorganism suspension was then added
into the wells. The last well column with medium and
microorganism served as a positive growth control.
After incubation at 37°C for 18–24 h, the first well with-
out turbidity was determined as the minimal inhibitory
concentration (MIC). Chloramphenicol was used as the
standard antibacterial agent whereas ketoconazole was
used as the antifungal.
Antimycobacterial assay
Antimycobacterial bioassay was performed using
the microplate Alamar blue assay (MABA) (Collins &
Franzblau, 1997). Suspensions of Mycobacterium tuber-
culosis H37Ra strain were prepared at approximately
105 cells/mL concentration. Samples were dissolved in
dimethylsulfoxide (DMSO) at  20 mg/mL in sterile con-
ditions and subsequently diluted in sterile Middlebrook
liquid medium 7H9 broth to 200 g/mL. Two-fold
dilutions were prepared. The assay was performed in
clear 96-well sterile microplates. One hundred microlit-
ers of bacterial suspension were added to each well of
the microtiter plate together with the plant extracts in
Middlebrook 7H9 medium to a final volume of 200 L,
and the final concentrations of the plant extracts were
50, 100, and 200 g/mL. Following incubation for 7 days
at 37°C, one control without sample was developed with
20 L of ×10 Alamar blue solution and 12 L of sterile
10% Tween 80. The plates were reincubated at 37°C for
24 h. After this incubation period, if the dye turned pink,
indicating bacterial growth, Alamar blue and Tween
solutions were then added to all remaining wells in the
plate. The results were read the following day and the
MIC values of the extracts were calculated. Rifampin,
isoniazid, and kanamycin were used as references. The
MIC ranges of the references were 0.0047–0.0095, 0.05–
0.1, and 2.5–5.0 g/mL, respectively.
Results and discussion
Four extracts prepared from the leaves of Ilex aquifolium
were screened against the standard strains of Escherichia
coli, Staphylococcus aureus, Pseudomonas aeruginosa,
Enterobacter aerogenes, Proteus vulgaris, Salmonella
typhimurium, and Candida albicans for their antibacte-
rial and antifungal evaluation using the microdilution
broth susceptibility assay, and the results compared
to those obtained with reference agents (Table 1). The
microorganisms were inhibited with moderate activ-
ity, having MIC values of 62.5–250 g/mL, lower than
those of the antimicrobial standard agents. However, the
EtOH extract showed stronger inhibition against Proteus
vulgaris at a concentration of 62.5 g/mL. None of the
 Antimicrobial activity of Ilex aquifolium   699

Table 1.  Antibacterial and antifungal activities of Ilex aquifolium leaf Table 2.  Antimycobacterial activity results for extracts of Ilex
extracts. aquifolium against Mycobacterium tuberculosis H37Ra.
MIC (g/mL) Results against TB at MIC value
EtOH EtOAc CHCl3 n-Hexane Extract Yield (w/w, %) 200 g/mL (g/mL)
Microorganism Source extract extract extract extract Std EtOH extract 18.3 Active 200
Bacteria EtOAc extract 7.4 Inactive —
Escherichia coli NRRL — 250 — — 31.25* CHCl3 extract 8.4 Inactive —
B-3008 n-Hexane 6.4 Inactive —
Staphylococcus ATCC 250 250 — — 3.9* extract
aureus 6538
Pseudomonas ATCC — — — — 62.5*
the chemical composition of the plant. Spraying of TLC
aeruginosa 27853
Enterobacter NRLL — — 250 — 62.5*
plates with 30% H2SO4 solution, followed by heating at
aerogenes B-3567 105°C for 1–2 min, showed some regions corresponding
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Proteus vulgaris NRLL 62.5 — 250 250 15.6* to triterpene type compounds that were visualized as
B-123 red spots with UV light (254 nm). Therefore, it can be
Salmonella NRRL — — 250 — 31.25* stated that triterpenes in the ethanol extract could be
typhimurium B-4420 responsible for antimicrobial activity. To the best of our
Fungi knowledge, this is the first study describing antibacte-
Candida Clinically 250 250 125 — 62.5** rial, antifungal, and antimycobacterial activities of
albicans isolated
I. aquifolium.
Tests were done in triplicate. Test concentration of extracts  2 mg/mL
in DMSO.
MIC, minimum inhibitory concentration; —, no inhibition; Std: Declaration of interest: The authors report no conflicts
*chloramphenicol; of interest.
**ketoconazole.
For personal use only.

extracts showed inhibitory effects on Pseudomonas
aeruginosa.
On the other hand, the crude extracts obtained
from the leaves of Ilex aquifolium were also tested
for evaluation of the minimum inhibitory concentra-
tion to Mycobacterium tuberculosis H37Ra strain with a
microplate technique using Alamar blue. Active extracts
exhibiting varying degrees of inhibition in the in vitro
primary screen test at 200 g/mL were retested at a
lower concentration to determine the actual MIC values.
The results of antimycobacterial activity assay of these
extracts are summarized in Table 2. The antimycobacte-
rial activity of the extracts was compared with rifampin,
isoniazid, and kanamycin. The ethanol extract of the
leaves showed activity at a concentration of 200 g/mL.
The rest of the extracts were found to be inactive.
In recent years, the crude extracts of many plants,
especially those with ethnomedicinal uses, have been
assessed for antimicrobial potency using in vitro assays.
These studies showed that a number of antifungal, anti-
bacterial, and antimycobacterial natural compounds
have been isolated (Cowan, 1999; Cantrell et  al., 2001;
Rios & Recio, 2005). In fact, the triterpenes, widely dis-
tributed in I. aquifolium, especially ursolic and oleanolic
acid derivatives (Knights & Smith 1977; Catalano et al.,
1978; Van Genderen & Jaarsma, 1990), have been also
reported to illustrate antimycobacterial (Cantrell et al.,
2001; Vik et  al., 2007) and antifungal (Cowan, 1999)
activities. In this study, the ethanol extract was subjected
to thin layer chromatography (TLC) for investigation of
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