Biologia, Bratislava, 61/3: 275—278, 2006
Section Cellular and Molecular Biology
DOI: 10.2478/s11756-006-0050-8
Antibacterial and antifungal activity of ethanolic extracts
from eleven spice plants
Ömer Ertürk
Department of Biology, Ordu Faculty of Arts and Sciences, Ondokuz Mayıs University, TR-52750 Perşembe, Ordu, Turkey;
e-mail: [email protected]
Abstract: Eleven ethanolic extracts from spices of Melissa officinalis, Mentha piperita, Laurus nobilis, Rhus coriaria,
Dianthus coryophyllum, Piper nigrum, Capsicum annum, Juniperus oxycedrus, Erica arborea, Colutea arborescens, and
Cuminum cyminum collected from various regions of Turkey and local markets were assayed for the in vitro antibacterial
activity against 3 Gram-positive (Bacillus subtilis, Staphylococcus aureus and S. epidermidis) and 2 Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), using agar dilution methods. In addition, their possible toxicity to
Candida albicans and Aspergillus niger was determined, using both agar dilution and disc-diffusion methods. The minimum
inhibition concentration (MIC) of the M. piperita, L. nobilis and J. oxycedrus ethanolic extracts was 5 mg/mL for all the
microorganisms tested. P. aeruginosa was the most sensitive bacterial strain to P. nigrum and E. arborea extracts among
both Gram-positive and Gram-negative bacteria tested with MIC of 5 mg/mL. The extracts of L. nobilis, D. coryophyllum,
J. oxycedrus and C. arborescens showed higher inhibitory activity against the yeast C. albicans and the fungus A. niger
than the standard antifungal nystatin.
Key words: antimicrobial activity, spice plants, plant extracts.
Introduction
Spices and herbs have been used for thousands of centuries by many cultures to enhance the flavor and aroma
of foods. Early cultures also recognized the value of
using spices and herbs in preserving foods and for
their medicinal value. Scientific experiments since the
late 19th century have documented the antimicrobial
properties of some spices, herbs, and their components
(Shelef, 1983; Zaika, 1988). Many herbs and spices
are known to exert antioxidant activity and are useful
for preventing lipid oxidation in living organisms as well
as in foods.
Medicinal plants have been used for a wide variety of purposes for many thousand of years in Turkey
and all over the world. In particular, extracts and oils
of these plants have formed the basis of many applications, including raw and processed food preservation,
pharmaceutical, alternative medicine, and natural therapies. Recently, the antimicrobial activity of various
plant extracts has been studied against many microorganisms in Turkey. Spices have been used to combat
snakebites, poor eyesight, stomach disorders, sleeping
problems, poor circulation, sores, colds, muscular aches,
gout, lumbago, poor digestion, motion sickness, and
hangovers (Baytop, 1984; Hamburger et al., 1991).
Spices exhibit antibacterial and antifungal activity. Microbiologists and food-product developers have
conducted laboratory experiments that involve numerous challenging food-borne bacteria, fungi, and yeasts
with phytochemicals extracted from spice plants. Multiple techniques have been used to investigate antimicrobial activity, and the primary data vary considerably in quality and quantity among different spices.
Nevertheless, it is now clear that many spices have
potent antimicrobial properties (Hargreaves et al.,
1975; Shelef, 1983; Deans et al., 1987; Hirasa et al.,
1993; Nakatani, 1994).
This study explores the antimicrobial activity of
11 ethanolic extracts from spices of aromatic plants
collected in Turkey against Gram-positive and Gramnegative bacteria and fungi. Some spice plants previously screened for biological activity by other investigators were included in this study because different
methods and microorganisms or strains were used in
the study, which provided a comparison base.
Material and methods
Plant material
Spices of Melissa officinalis (Labiatae), Mentha piperita
(Labiatae), Laurus nobilis (Lauraceae), and Rhus coriaria
L. (Anacardiaceeae) were collected during April-May 2000
and March-January and May of 2003-2004 from different parts of Turkey. The identification of these specimens
was carried out using the Flora of Turkey (DAVIS, 19661988). The samples of Dianthus coryophyllum (Caryophyl-
c
2006
Institute of Molecular Biology, Slovak Academy of Sciences
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Ö. Ertürk
276
laceae), Piper nigrum (Piperaceae), Capsicum annum L.
(Solanaceae), Juniperus oxycedrus L. (Cupressaceae), Erica arborea L. (Ericaceae), Colutea arborescens L. (Leguminosae), and Cuminum cyminum L. (Umbelliferae) spices
were obtained from local markets.
Preparation of extracts
Fresh leaves and twigs of the plants were dried at 45 ◦C for
5–6 hours. The extracts of the plants were prepared according to the methods described by ERTÜRK et al. (2003) and
HOLOPAINEN et al. (1988), with slight modification. Dried
leaves and twigs of the plants were extracted with 95%
ethanol (50 g, 1/5 ethanol) at room temperature. The extracts were kept at 4 ◦C for one day, and filtered through 45
µm membrane filter, and then the solutions were dried with
an evaporator. The crude extracts were stored at −20 ◦C
until tested.
Test strains and culture media
Strains of bacteria and fungi were obtained from ATCC
(American Type Culture Collection, Rockville, Maryland).
Antimicrobial activity of 11 spice ethanolic extracts against
Bacillus subtilis (ATCC 6633), Staphylococcus aureus
(ATCC 25923), Staphylococcus epidermidis (ATCC 12228),
Escherichia coli (ATCC 25922), Pseudomonas aeruginosa
(ATCC 10145), Candida albicans (ATTC 60192), and Aspergillus niger was studied. The species of bacteria were
grown in Mueller Hinton Agar (Merck) and Mueller Hinton Broth (Merck). C. albicans and A. niger were grown in
Sabouraud Dextrose Broth (Difco) and Sabouraud Dextrose
Agar (Oxoid). The concentration of bacterial suspensions
were adjusted to 108 cells/mL, and that of fungal suspensions to 107 cells/mL.
Antifungal assay
Antifungal activity was measured using methods of disc diffusion plates on agar (RONALD, 1990). In order to test the
antifungal activity, the fractions of spice extracts were dissolved in 70% ethanol. Twenty mL of Sabouraud Dextrose
Agar (Oxoid) was poured into each 15 cm Petri dish. C.
albicans and A. niger were grown in Sabouraud Dextrose
Broth (Difco) at 27 ◦C for 48 h. Growth was adjusted to
OD (600 nm) of 0.1 by dilution with Sabouraud Dextrose
Broth (Difco). One hundred µL of suspension containing approximately 108 bacteria/mL was placed over agar in Petri
dishes and dispersed. Then, sterile paper discs (6 mm diameter) were placed on agar to load 10 and 15 µL of each spice
sample (1 mg/mL). One hundred units of nystatin, obtained
from a local pharmacy, were used as a positive control and
ethanol as a negative control. Inhibition zones were determined after incubation at 27 ◦C for 48 h. All tests were done
in triplicate.
Antibacterial assay
The agar dilution method, described by VANDEN BERGHE
& VIETINCK (1991) was used for the antibacterial screening with slight modifications. Instead of 96 well microtiter
plates, 24 well tissue culture (Corning) plates were used.
The crude extracts were dissolved in 70% ethanol and physiological Tris buffer (Amresco 0826-500G) (1:4) and mixed
with an equal amount of 3% agar solution at 45 ◦C to obtain a final concentration of 50, 25, 12.5, and 6.25 mg of
extract/mL. From these solutions 400 µL were transferred
into each well of the tissue culture (Corning) plate. After solidification, each well was inoculated with 10 µL of freshly
prepared bacterial suspension of 108 bacteria/mL and incubated at 37 ◦C for 24 h. For bacteria, amoxicillin clavulanate
and cefazolin sodium, obtained from a local pharmacy, were
used at 50, 25, 12.5, and 6.25 mg/mL concentrations (from
1 mg/mL stock) as positive control, and 70% ethanol was
used as negative control. The bacterial growth was assessed
by a stereomicroscope after the incubation period. All tests
were done in triplicate.
Results
The antibacterial and antifungal activities of the extracts from the test samples in terms of minimum inhibitory concentrations (MIC) and diameters of inhibition zones are reported in Table 1. The crude extracts
from M. piperita, L. nobilis, and J. oxycedrus L. were
found to be prominently active against the tested microorganisms at the concentrations less than 5 mg/mL
(MIC). The crude extracts obtained from R. coriaria
L., D. coryophyllum, and P. nigrum showed moderate antibacterial activity against the test organisms in
the same concentration range. The MIC values of the
crude extracts of C. annum, C. arborescens L., and C.
cyminum L. were found to be 10–17.5 mg/mL against
E. coli, S. aureus, S. epidermidis, B. subtilis, P. aeruginosa, A. niger, and C. albicans.
The extracts of M. officinalis, P. nigrum, C.
annum, and C. cyminum showed antifungal activity
against A. niger and C. albicans with inhibition zone
diameter range of 10–16 mm/15µL (Table 1). The extracts of M. piperita and E. arborea L. showed higher
antifungal activity against A. niger and C. albicans
with inhibition zone diameters of 18–23 mm/15µL. The
extracts of L. nobilis, D. coryophyllum, J. oxycedrus,
and C. arborescens L. showed the highest antifungal
activity against A. niger and C. albicans with inhibition zone diameters of 20–32 mm/15µL.
The extracts showed antimicrobial activity against
both Gram-positive and Gram-negative bacteria. It is
interesting to note that extracts were more effective
against bacteria than fungi. The extract of E. arborea
L. was the most effective against the fungi A. niger and
C. albicans.
Discussion
In recent years, although technology and medicine have
developed extensively, some countries have made it
obligatory to use natural products for many different
purposes due to decrease in natural richness and drawbacks. Like in many other countries, the plants known
by people with health benefits are picked up and used
for the treatment of various diseases in Turkey.
In this study, the antimicrobial capacity of the extracts from 11 spices and herbs against bacteria and
fungi were determined. The two test methods have been
widely utilized for the determination of antimicrobial
activity of various samples, including plant extracts,
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Antimicrobial activity of ethanolic spice extracts
277
Table 1. Results of antimicrobial screening of spice plant extracts determined by the agar-well diffusion method (minimum inhibitory
concentration, MIC) and agar diffusion method (inhibition zone).a
Inhibition zone
(mm)
MIC (mg/mL)
Plant species
and family
Melissa officinalis
Labiatae
Mentha piperita
Labiatae
Laurus nobilis
Lauraceae
Rhus coriaria L.
Anacardiaceeae
Dianthus coryophyllum
Caryophyllaceae
Piper nigrum
Piperaceae
Capsicum annum L.
Solanaceae
Juniperus oxycedrus L.
Cupressaceae
Erica arborea L.
Ericaceae
Colutea arborescens L.
Leguminosae
Cuminum cyminum L.
Umbelliferae
Amoxicillin
Cefazolin
Nystatin
70% ethanol
Part used
Local name
Collection site
E.c. B.s. S.a. S.e. P.a.
A.n.
C.a. A.n. C.a.
Fr, Lf
Oğul otu
Hatay
10
15
10
10
15
12.5
25
12
10
Sd,
Nane
Hatay
5
5
5
5
5
5
5
18
20
Lf
Defne
Trabzon
5
5
5
5
5
5
5
22
25
Lf
Sumak, Sanak, Tekri
Hatay
15
15
12.5 15
15
15
15
15
16
Sd, Ft
Karanfil
Hatay
15
20
25
15
15
25
20
25
30
Sd
Karabiber
Market
15
12.5 12.5 15
5
12.5
12.5 15
12
Ft
Kırmızı biber
Market
10
15
10
10
10
15
17.5 10
10
Ft
Katran
Market
5
5
5
5
5
5
5
20
32
Lf
Funda
Ordu
7.5 15
5
2.5
2.5 18
23
Lf
Yalancı sinemaki
Trabzon
10
15
10
10
15
12.5
12.5 20
25
Ft
Kimyon
market
10
10
12.5 15
10
15
15
15
14
5≤
5≤
NT
–
5≤
5≤
NT
–
5≤ 5≤
5≤ 5≤
NT NT
–
–
5≤
NT
NT
5≤
NT
NT
NT 12.5≤ 12.5≤
–
–
–
NT
NT
15
–
NT
NT
16
–
7.5 15
a Parts used: Fr, flower; Ft, fruit; Lf, leaf; Sd, seed. NT: not tested. Microorganisms: E.c., Escherichia coli; B.s., Bacillus subtilis;
S.a., Staphylococcus aureus; S.e, Staphylococcus epidermidis; P.a., Pseudomonas aeruginosa; A.n, Aspergillus niger; C.a., Candida
albicans.
biological fluids and natural products. The antimicrobial activity of the extracts of these spices and herbs
was more effective against bacteria than fungi, similar
to the results of Avato et al. (1997) and Zavala et
al. (1997). The use of some antibiotics is no longer recommended because of the potency of the widespread
resistance to them (Zavala et al, 1997). Thus, these
spices and herbs, like many other plants, can be used
instead of antibiotics.
Some plants previously screened by other investigators were included in this study because different
methods and different microorganisms or strains were
used in the assay. The extracts from M. piperita, L. nobilis, and J. oxycedrus L. showed antibacterial activity
with MIC values less than 5mg/mL (Table 1) (Cowan
et al., 1999; Mrlianova et al. 2002). These plants were
observed to show various inhibitory effects on the microorganisms. Diğrak et al. (2001) have reported the
acetone extract from R. coriaria to possess antibacterial effect against some bacteria.
The activity of some crude extracts used in the
study against P. aeruginosa, S. aureus, B. subtilis, S.
epidermidis, and E. coli was similar to that of amoxicillin clavulanate and cefazolin sodium. Furthermore,
the antifungal activity of some of the crude extracts
tested was more potent than the standard antifungal
nystatin (100 units) against C. albicans and A. niger.
In this study, the antimicrobial influence of the
crude extracts from 11 spices and herbs against bacteria and fungi were determined. The plants investigated are known with healing powers, and used for the
treatment of various diseases among people. The continuance of this study should include the isolation of
the compounds responsible for the antimicrobial activity present in M. piperita, L. nobilis, and J. oxycedrus
L., the plants showing the largest inhibitory activity
over the growth of the microorganisms tested.
The essential oil from L. nobilis leaves has narcotic, antibacterial, and fungicidal properties (Duke &
Ayensu, 1985). The plant is highly resistant to pests
and diseases, and it is said to protect neighboring plants
from insect and health problems (Holtom & Hylton,
1979). The leaves are highly aromatic and can be used
as an insect repellent, the dried leaves protect stored
grains, beans etc. from weevils (Holtom & Hylton,
1979).
Oil of peppermint, M. piperita, is a powerful diffusible stimulant, with carminative, antispasmodic, and
antiemetic properties. It is widely used to relieve flatulence, gastrodynia, nausea, and spasms of the stomach,
and to cover the taste of other drugs. J. oxycedrus L.
plant yields the essential oil “Oil of Cade” by destrucUnauthenticated
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278
tive distillation of the wood. It is used externally in the
treatment of skin diseases such as psoriasis and chronic
eczema. It is a good parasiticide in cases of psora and
favus (Grieve, 1984).
The isolation of the compounds with antimicrobial
and antifungal activity will lower the required doses
compared to the crude extracts. In addition, it is noteworthy that these spices are used best in lukewarm
meals, since the extraction yields will be lower in cold
and the active compounds will be transformed into less
active or inactive products when heated.
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Received May 24, 2005
Accepted January 30, 2006
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