Biologia, Bratislava, 61/3: 275278, 2006Section Cellular and Molecular BiologyDOI: 10.2478/s11756-006-0050-8

Antibacterial and antifungal activity of ethanolic extractsfrom eleven spice plants

Omer Erturk

Department of Biology, Ordu Faculty of Arts and Sciences, Ondokuz Mays University, TR-52750 Persembe, Ordu, Turkey;e-mail: oseerturk@hotmail.com

Abstract: Eleven ethanolic extracts from spices of Melissa ocinalis, Mentha piperita, Laurus nobilis, Rhus coriaria,Dianthus coryophyllum, Piper nigrum, Capsicum annum, Juniperus oxycedrus, Erica arborea, Colutea arborescens, andCuminum cyminum collected from various regions of Turkey and local markets were assayed for the in vitro antibacterialactivity against 3 Gram-positive (Bacillus subtilis, Staphylococcus aureus and S. epidermidis) and 2 Gram-negative bac-teria (Escherichia coli and Pseudomonas aeruginosa), using agar dilution methods. In addition, their possible toxicity toCandida albicans and Aspergillus niger was determined, using both agar dilution and disc-diusion methods. The minimuminhibition concentration (MIC) of the M. piperita, L. nobilis and J. oxycedrus ethanolic extracts was 5 mg/mL for all themicroorganisms tested. P. aeruginosa was the most sensitive bacterial strain to P. nigrum and E. arborea extracts amongboth 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. nigerthan the standard antifungal nystatin.

Key words: antimicrobial activity, spice plants, plant extracts.

Introduction

Spices and herbs have been used for thousands of cen-turies by many cultures to enhance the avor and aromaof foods. Early cultures also recognized the value ofusing spices and herbs in preserving foods and fortheir medicinal value. Scientic experiments since thelate 19th century have documented the antimicrobialproperties of some spices, herbs, and their components(Shelef, 1983; Zaika, 1988). Many herbs and spicesare known to exert antioxidant activity and are usefulfor preventing lipid oxidation in living organisms as wellas in foods.

Medicinal plants have been used for a wide vari-ety of purposes for many thousand of years in Turkeyand all over the world. In particular, extracts and oilsof these plants have formed the basis of many appli-cations, including raw and processed food preservation,pharmaceutical, alternative medicine, and natural ther-apies. Recently, the antimicrobial activity of variousplant extracts has been studied against many microor-ganisms in Turkey. Spices have been used to combatsnakebites, poor eyesight, stomach disorders, sleepingproblems, poor circulation, sores, colds, muscular aches,gout, lumbago, poor digestion, motion sickness, andhangovers (Baytop, 1984; Hamburger et al., 1991).

Spices exhibit antibacterial and antifungal activ-ity. Microbiologists and food-product developers have

conducted laboratory experiments that involve numer-ous challenging food-borne bacteria, fungi, and yeastswith phytochemicals extracted from spice plants. Mul-tiple techniques have been used to investigate antimi-crobial activity, and the primary data vary consider-ably in quality and quantity among dierent spices.Nevertheless, it is now clear that many spices havepotent antimicrobial properties (Hargreaves et al.,1975; Shelef, 1983; Deans et al., 1987; Hirasa et al.,1993; Nakatani, 1994).

This study explores the antimicrobial activity of11 ethanolic extracts from spices of aromatic plantscollected in Turkey against Gram-positive and Gram-negative bacteria and fungi. Some spice plants previ-ously screened for biological activity by other inves-tigators were included in this study because dierentmethods and microorganisms or strains were used inthe study, which provided a comparison base.

Material and methods

Plant materialSpices of Melissa ocinalis (Labiatae), Mentha piperita(Labiatae), Laurus nobilis (Lauraceae), and Rhus coriariaL. (Anacardiaceeae) were collected during April-May 2000and March-January and May of 2003-2004 from dier-ent parts of Turkey. The identication of these specimenswas carried out using the Flora of Turkey (DAVIS, 1966-1988). The samples of Dianthus coryophyllum (Caryophyl-

c2006 Institute of Molecular Biology, Slovak Academy of Sciences

276 O. Erturk

laceae), Piper nigrum (Piperaceae), Capsicum annum L.(Solanaceae), Juniperus oxycedrus L. (Cupressaceae), Er-ica arborea L. (Ericaceae), Colutea arborescens L. (Legu-minosae), and Cuminum cyminum L. (Umbelliferae) spiceswere obtained from local markets.

Preparation of extractsFresh leaves and twigs of the plants were dried at 45C for56 hours. The extracts of the plants were prepared accord-ing to the methods described by ERTURK et al. (2003) andHOLOPAINEN et al. (1988), with slight modication. Driedleaves and twigs of the plants were extracted with 95%ethanol (50 g, 1/5 ethanol) at room temperature. The ex-tracts were kept at 4C for one day, and ltered through 45m membrane lter, and then the solutions were dried withan evaporator. The crude extracts were stored at 20Cuntil tested.

Test strains and culture mediaStrains of bacteria and fungi were obtained from ATCC(American Type Culture Collection, Rockville, Maryland).Antimicrobial activity of 11 spice ethanolic extracts againstBacillus subtilis (ATCC 6633), Staphylococcus aureus(ATCC 25923), Staphylococcus epidermidis (ATCC 12228),Escherichia coli (ATCC 25922), Pseudomonas aeruginosa(ATCC 10145), Candida albicans (ATTC 60192), and As-pergillus niger was studied. The species of bacteria weregrown in Mueller Hinton Agar (Merck) and Mueller Hin-ton Broth (Merck). C. albicans and A. niger were grown inSabouraud Dextrose Broth (Difco) and Sabouraud DextroseAgar (Oxoid). The concentration of bacterial suspensionswere adjusted to 108 cells/mL, and that of fungal suspen-sions to 107 cells/mL.

Antifungal assayAntifungal activity was measured using methods of disc dif-fusion plates on agar (RONALD, 1990). In order to test theantifungal activity, the fractions of spice extracts were dis-solved in 70% ethanol. Twenty mL of Sabouraud DextroseAgar (Oxoid) was poured into each 15 cm Petri dish. C.albicans and A. niger were grown in Sabouraud DextroseBroth (Difco) at 27C for 48 h. Growth was adjusted toOD (600 nm) of 0.1 by dilution with Sabouraud DextroseBroth (Difco). One hundred L of suspension containing ap-proximately 108 bacteria/mL was placed over agar in Petridishes and dispersed. Then, sterile paper discs (6 mm diam-eter) were placed on agar to load 10 and 15 L of each spicesample (1 mg/mL). One hundred units of nystatin, obtainedfrom a local pharmacy, were used as a positive control andethanol as a negative control. Inhibition zones were deter-mined after incubation at 27C for 48 h. All tests were donein triplicate.

Antibacterial assayThe agar dilution method, described by VANDEN BERGHE& VIETINCK (1991) was used for the antibacterial screen-ing with slight modications. Instead of 96 well microtiterplates, 24 well tissue culture (Corning) plates were used.The crude extracts were dissolved in 70% ethanol and phys-iological Tris buer (Amresco 0826-500G) (1:4) and mixedwith an equal amount of 3% agar solution at 45C to ob-tain a nal concentration of 50, 25, 12.5, and 6.25 mg ofextract/mL. From these solutions 400 L were transferredinto each well of the tissue culture (Corning) plate. After so-lidication, each well was inoculated with 10 L of freshly

prepared bacterial suspension of 108 bacteria/mL and incu-bated at 37C for 24 h. For bacteria, amoxicillin clavulanateand cefazolin sodium, obtained from a local pharmacy, wereused at 50, 25, 12.5, and 6.25 mg/mL concentrations (from1 mg/mL stock) as positive control, and 70% ethanol wasused as negative control. The bacterial growth was assessedby a stereomicroscope after the incubation period. All testswere done in triplicate.

Results

The antibacterial and antifungal activities of the ex-tracts from the test samples in terms of minimum in-hibitory concentrations (MIC) and diameters of inhibi-tion zones are reported in Table 1. The crude extractsfrom M. piperita, L. nobilis, and J. oxycedrus L. werefound to be prominently active against the tested mi-croorganisms at the concentrations less than 5 mg/mL(MIC). The crude extracts obtained from R. coriariaL., D. coryophyllum, and P. nigrum showed moder-ate antibacterial activity against the test organisms inthe same concentration range. The MIC values of thecrude extracts of C. annum, C. arborescens L., and C.cyminum L. were found to be 1017.5 mg/mL againstE. coli, S. aureus, S. epidermidis, B. subtilis, P. aerug-inosa, A. niger, and C. albicans.

The extracts of M. ocinalis, P. nigrum, C.annum, and C. cyminum showed antifungal activityagainst A. niger and C. albicans with inhibition zonediameter range of 1016 mm/15L (Table 1). The ex-tracts of M. piperita and E. arborea L. showed higherantifungal activity against A. niger and C. albicanswith inhibition zone diameters of 1823 mm/15L. Theextracts of L. nobilis, D. coryophyllum, J. oxycedrus,and C. arborescens L. showed the highest antifungalactivity against A. niger and C. albicans with inhibi-tion zone diameters of 2032 mm/15L.

The extracts showed antimicrobial activity againstboth Gram-positive and Gram-negative bacteria. It isinteresting to note that extracts were more eectiveagainst bacteria than fungi. The extract of E. arboreaL. was the most eective against the fungi A. niger andC. albicans.

Discussion

In recent years, although technology and medicine havedeveloped extensively, some countries have made itobligatory to use natural products for many dierentpurposes due to decrease in natural richness and draw-backs. Like in many other countries, the plants knownby people with health benets are picked up and usedfor the treatment of various diseases in Turkey.

In this study, the antimicrobial capacity of the ex-tracts from 11 spices and herbs against bacteria andfungi were determined. The two test methods have beenwidely utilized for the determination of antimicrobialactivity of various samples, including plant extracts,

Antimicrobial activity of ethanolic spice extracts 277

Table 1. Results of antimicrobial screening of spice plant extracts determined by the agar-well diusion method (minimum inhibitoryconcentration, MIC) and agar diusion method (inhibition zone).a

Inhibition zoneMIC (mg/mL) (mm)

Plant speciesand family

Part used Local name Collection siteE.c. B.s. S.a. S.e. P.a. A.n. C.a. A.n. C.a.

Melissa ocinalisLabiatae

Fr, Lf Ogul otu Hatay 10 15 10 10 15 12.5 25 12 10

Mentha piperitaLabiatae

Sd, Nane Hatay 5 5 5 5 5 5 5 18 20

Laurus nobilisLauraceae

Lf Defne Trabzon 5 5 5 5 5 5 5 22 25

Rhus coriaria L.Anacardiaceeae

Lf Sumak, Sanak, Tekri Hatay 15 15 12.5 15 15 15 15 15 16

Dianthus coryophyllumCaryophyllaceae

Sd, Ft Karanl Hatay 15 20 25 15 15 25 20 25 30

Piper nigrumPiperaceae

Sd Karabiber Market 15 12.5 12.5 15 5 12.5 12.5 15 12

Capsicum annum L.Solanaceae

Ft Krmz biber Market 10 15 10 10 10 15 17.5 10 10

Juniperus oxycedrus L.Cupressaceae

Ft Katran Market 5 5 5 5 5 5 5 20 32

Erica arborea L.Ericaceae

Lf Funda Ordu 7.5 15 7.5 15 5 2.5 2.5 18 23

Colutea arborescens L.Leguminosae

Lf Yalanc sinemaki Trabzon 10 15 10 10 15 12.5 12.5 20 25

Cuminum cyminum L.Umbelliferae

Ft Kimyon market 10 10 12.5 15 10 15 15 15 14

Amoxicillin 5 5 5 5 5 NT NT NT NTCefazolin 5 5 5 5 5 NT NT NT NTNystatin NT NT NT NT NT 12.5 12.5 15 1670% ethanol

a Parts used: Fr, ower; 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., Candidaalbicans.

biological uids and natural products. The antimicro-bial activity of the extracts of these spices and herbswas more eective against bacteria than fungi, similarto the results of Avato et al. (1997) and Zavala etal. (1997). The use of some antibiotics is no longer rec-ommended because of the potency of the widespreadresistance to them (Zavala et al, 1997). Thus, thesespices and herbs, like many other plants, can be usedinstead of antibiotics.

Some plants previously screened by other inves-tigators were included in this study because dierentmethods and dierent microorganisms or strains wereused in the assay. The extracts from M. piperita, L. no-bilis, and J. oxycedrus L. showed antibacterial activitywith MIC values less than 5mg/mL (Table 1) (Cowanet al., 1999;Mrlianova et al. 2002). These plants wereobserved to show various inhibitory eects on the mi-croorganisms. Digrak et al. (2001) have reported theacetone extract from R. coriaria to possess antibacte-rial eect against some bacteria.

The activity of some crude extracts used in thestudy against P. aeruginosa, S. aureus, B. subtilis, S.epidermidis, and E. coli was similar to that of amox-icillin clavulanate and cefazolin sodium. Furthermore,the antifungal activity of some of the crude extractstested was more potent than the standard antifungal

nystatin (100 units) against C. albicans and A. niger.In this study, the antimicrobial inuence of the

crude extracts from 11 spices and herbs against bac-teria and fungi were determined. The plants investi-gated are known with healing powers, and used for thetreatment of various diseases among people. The con-tinuance of this study should include the isolation ofthe compounds responsible for the antimicrobial activ-ity present in M. piperita, L. nobilis, and J. oxycedrusL., the plants showing the largest inhibitory activityover the growth of the microorganisms tested.

The essential oil from L. nobilis leaves has nar-cotic, antibacterial, and fungicidal properties (Duke &Ayensu, 1985). The plant is highly resistant to pestsand diseases, and it is said to protect neighboring plantsfrom insect and health problems (Holtom & Hylton,1979). The leaves are highly aromatic and can be usedas an insect repellent, the dried leaves protect storedgrains, beans etc. from weevils (Holtom & Hylton,1979).

Oil of peppermint, M. piperita, is a powerful dif-fusible stimulant, with carminative, antispasmodic, andantiemetic properties. It is widely used to relieve atu-lence, 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 destruc-

278 O. Erturk

tive distillation of the wood. It is used externally in thetreatment of skin diseases such as psoriasis and chroniceczema. It is a good parasiticide in cases of psora andfavus (Grieve, 1984).

The isolation of the compounds with antimicrobialand antifungal activity will lower the required dosescompared to the crude extracts. In addition, it is note-worthy that these spices are used best in lukewarmmeals, since the extraction yields will be lower in coldand the active compounds will be transformed into lessactive or inactive products when heated.

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Received May 24, 2005Accepted January 30, 2006

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