Evaluation of Antimicrobial Activity of Some Endemic Verbascum,

Sideritis, and Stachys Species from Turkey

Basaran Dulger1, Emin Ugurlu2, Cuneyt Aki1, Tulay Bican Suerdem1, Aytac Camdeviren1, and Gorkem Tazeler1

1Department of Biology, Faculty of Science & Arts, Canakkale Onsekiz Mart University, Canakkale, Turkey;2Department of Biology, Faculty of Science & Arts, Celal Bayar University, Manisa, Turkey

Abstract

Methanol extracts obtained from endemic Verbascumpseudoholotrichum Hub.-Mor., Verbascum cymigerumHub.-Mor., Verbascum cholorostegium Bornm. & Murb.,Verbascum linguifolium Hub.-Mor., Verbascum pellitumHub.-Mor., Sideritis brevidens P.H. Davis, Sideritis cili-cica Boiss. & Bal., Sideritis vuralii H. Duman & Baser,Stachys aleurites Boiss. & Heldr., and Stachys pinardiiBoiss. have been investigated for their antimicrobialactivity. Antimicrobial activity was determined withEscherichia coli ATCC 11230, Staphylococcus aureusATCC 6538P, Klebsiella pneumoniae UC57, Pseudomo-nas aeruginosa ATCC 27853, Proteus vulgaris ATCC8427, Bacillus cereus ATCC 7064, Mycobacterium smeg-matis CCM 2067, Listeria monocytogenes ATCC 15313,Micrococcus luteus CCM 169, Candida albicans ATCC10231, Rhodotorula rubra DSM 70403, and Kluyvero-myces fragilis ATCC 8608 by the disk diffusion method.Verbascum L. extracts had strong antimicrobial activityagainst Gram-positive bacteria and yeast cultures. Theextracts of Stachys L. were effective only against bac-teria. The extracts of Sideritis L. demonstrated anti-microbial effects against the bacteria and the yeastcultures used in this study.

Keywords: Antimicrobial activity, Verbascum pseudo-holotrichum Hub.-Mor., Verbascum cymigerum Hub.-Mor., Verbascum cholorostegium Bornm. & Murb.,Verbascum linguifolium Hub.-Mor., Verbascum pellitumHub.-Mor., Sideritis brevidens P.H. Davis, Sideritiscilicica Boiss. & Bal., Sideritis vuralii H. Duman &Baser, Stachys aleurites Boiss. & Heldr., Stachys pinardiiBoiss.

Introduction

Higher plants have been the source of medicinal agentssince early times. They continue to play a dominant rolein the primary health care of about 80% of the world’spopulation (Baker et al., 1995). Some species of Verbas-cum L. (Scrophulariaceae) have widely been usedthroughout centuries to treat internal and external infec-tions. Many internal and external uses of the leaves andflowers of several Verbascum L. species have been docu-mented in many societies in Europe, Asia, Africa, andNorth America (Maurer-Grimes et al., 1996). Verbascumpseudoholotrichum Hub.-Mor., Verbascum cymigerumHub.-Mor., Verbascum cholorostegium Bornm. & Murb.,Verbascum linguifolium Hub.-Mor., and Verbascumpellitum Hub.-Mor., have been used for their ethnophar-macological effects among the common folk in Turkey.Especially their flower has been used as a diuretic, expec-torant, and sedative. Seeds of Verbascum species arepoisonous because of the saponins contained. Peopleuse these poisonous seeds for hunting fish. Verbascumspecies are called ‘‘fishplant’’ in northern Anatoliabecause of that property (Zeybek, 1985).

Some species of Stachys L. and Sideritis L. (Labiatae)have widely been used for their diuretic and expectoranteffects. In addition, leaves of plants have also been usedagainst abdominal pains in Turkey (Baytop, 1999). Thegenus Sideritis (Labiatae) is widely distributed in sub-tropical and moderate regions (Davis, 1982). Sideritisspecies are a group of plants known as ‘‘mountain tea’’in Turkey. Some species are used as tea, flavoring agents,and for medicinal purposes in several regions of Turkey(Ozcan et al., 2001). Infusion of aerial parts of a numberof Sideritis species are used as tonics, carminatives, as

Accepted: December 29, 2004

Address correspondence to: Basaran Dulger, Department of Biology, Faculty of Science & Arts, Canakkale Onsekiz Mart University,17100 Canakkale, Turkey. Fax: þ 90-286-2180533; E-mail: dbasaran@comu.edu.tr

DOI: 10.1080/13880200590928861 # 2005 Taylor & Francis Ltd.

Pharmaceutical Biology2005, Vol. 43, No. 3, pp. 270–274

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anti-inflammatory agents, antispasmodics, diuretics,digestives, and in the treatment of colds (Koedam,1986; Yesilada & Ezer, 1989; Ezer et al., 1991).

Verbascum pseudoholotrichum Hub.-Mor., Verbascumcymigerum Hub.-Mor., Verbascum cholorostegiumBornm. & Murb., Verbascum linguifolium Hub.-Mor.,Verbascum pellitum Hub.-Mor., Sideritis brevidens P.H.Davis, Sideritis cilicica Boiss. & Bal., Sideritis vuralii H.Duman & Baser, Stachys aleurites Boiss. & Heldr., andStachys pinardii Boiss. are endemic to Turkey. (Davis,1978; Guner et al., 2000). Although there are manyinvestigations on Sideritis, Stachys, and Verbascum spe-cies, these plants have not been previously investigated.Therefore, our aim was to determine the antimicrobialeffects of plant extracts obtained from these endemicspecies against microorganisms.

Materials and Methods

Plant materials

Aerial parts of Verbascum pseudoholotrichum Hub.-Mor., Verbascum cymigerumHub.-Mor., Verbascum cho-lorostegium Bornm. & Murb., Verbascum linguifoliumHub.-Mor., Verbascum pellitum Hub.-Mor., Sideritisbrevidens P.H. Davis, Sideritis cilicica Boiss. & Bal.,Sideritis vuralii H. Duman & Baser, Stachys aleuritesBoiss. & Heldr., and Stachys pinardii Boiss. were col-lected from different localities in Turkey during themonths of September–October 2001. Voucher specimensof the plants were deposited in the Biology Departmentat Canakkale Onsekiz Mart University, Canakkale,Turkey, and identified by Emin Ugurlu.

Preparation of extracts

The plant parts were air-dried. Each dry powdered plantmaterial (20 g) was extracted with 150ml of 80%methanol(Merck, Darmstadt, Germany) for 24h by using Soxhletequipment (Khan et al., 1988). The extract was filteredusing Whatman filter paper no. 1, and the filtrates werethen evaporated under reduced pressure and dried usinga rotary evaporator at 55�C. Dried extracts were storedin labeled sterile screw-capped bottles at –20�C.

Microorganisms

Escherichia coli ATCC 11230, Staphylococcus aureusATCC 6538P, Klebsiella pneumoniaeUC57, Pseudomonasaeruginosa ATCC 27853, Proteus vulgaris ATCC 8427,Bacillus cereus ATCC 7064, Mycobacterium smegmatisCCM 2067, Listeria monocytogenes ATCC 15313,Micro-coccus luteus CCM 169, Candida albicans ATCC 10231,Rhodotorula rubraDSM 70403, and Kluyveromyces fragi-lis ATCC 8608 were used as test microorganisms.

Screening for antimicrobial activities

The dried plant extracts were dissolved in 10% aqueousdimethylsulfoxide (DMSO) to a final concentration of200mg=ml and sterilized by filtration through an0.45 mm membrane filter. Empty sterilized antibioticdisks having a diameter of 6mm (Schleicher & SchullNo. 2668, Dassel, Germany) were each impregnated with50 ml of extract (10mg=disk) at a concentration of200mg=ml. All the bacteria mentioned above were incu-bated at 35� 0.1�C for 24 h by inoculation into nutrientbroth (Difco), and the yeast cultures studied were incu-bated in malt extract broth (Difco Laboratories, MI,USA) at 25� 0.1�C for 48 h. An inoculum containing106 bacterial cells or 108 yeast cells=mL was spread onMueller-Hinton agar (Oxoid Ltd., Hampshire, UK)plates (1ml inoculum=plate). The disks injected withextracts were placed on the inoculated agar by pressingslightly. Petri dishes were placed at 4�C for 2 h, plaquesinjected with the yeast cultures were incubated at25� 0.1�C, and bacteria were incubated at 35� 0.1�Cfor 24 h (Collins et al., 1989; Ali-Stayeh et al., 1998).At the end of the period, inhibition zones formed onthe medium were evaluated in millimeters. Studies wereperformed in triplicate. On each plate, an appropriatereference antibiotic disk was applied, depending on thetest microorganisms for comparison.

Results and Discussion

Table 1 shows antimicrobial activities of the plantextracts, and the inhibition zones formed by standardantibiotic disks are indicated in Table 2. As can clearlybe seen from Table 1, the extracts of Stachys have anantibacterial effect against tested bacteria, but no signifi-cant activity was found against the yeast cultures.Activity against Escherichia coli, Staphylococcus aureus,Pseudomonas aeruginosa, Proteus vulgaris, and Bacilluscereus were found from extracts of all Stachys L. species,showing inhibition zones of 8.0–18.8mm. As can be seenfrom Tables 1 and 2, the extract obtained from Stachysaleurites species has much higher inhibition zones thanthe standard antibacterial antibiotics, except for OFX5and TE30 against Bacillus cereus. In addition, the extractof Stachys pinardii has a greater antibacterial effect thanthe standard antibacterial antibiotics P10, SAM20, andCTX30 against Proteus vulgaris. Although there is noinformation on Stachys species used in this study, thereare many studies on the chemical contents of otherStachys species. Sesquiterpenes-a-copaene (16.5%),spathulenol (16.1%), b-caryophyllene (14.3%), andb-cubebene were identified by gas chromatography(GC) and GUMS analyses from aerial parts of Stachysbyzanthina (Khanavi et al., 2003). Similarly, a -pinene(20.1%), b-pinene (12.1%), and spathulenol (7.2%) weredetermined from Stachys lavandulifolia (Feizbaksh et al.,

Antimicrobial activity of Turkish plants 271

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2003). According to Mirza and Baher (2003), Stachyslanata has a-thyjone, a-humulene, b-caryophyllene, andviridiflorol. Digrak et al. (2001) reported that the chloro-form extract of Stachys annua subsp. annua and Stachyspumila had little antibacterial effect against used bac-teria, and both of them were also found not to be effec-tive against the fungi studied. Our findings concur withthis study, despite the different extraction method used.

As also listed in Table 1, it was found that extracts ofVerbascum L. species have no significant activity againstGram-negative bacteria such as Escherichia coli,Klebsiella pneumoniae, Proteus vulgaris, and Pseudomo-nas aeruginosa. Activity against the Gram-positivebacteria such as Staphylococcus aureus, Bacillus cereus,Listeria monocytogenes, Micrococcus luteus, and acid-fastbacterium Mycobacterium smegmatis were found.

Besides, extracts of Verbascum L. species have anantiyeast activity against all tested yeast cultures.Extracts of V. cholorostegium have more antibacterialand antifungal effects than other Verbascum L. species.Staphylococcus aureus is more susceptible to the extractsof all Verbascum L. species, as compared to standardantibiotics, except for OFX5 and TE 30. Similarly, incomparison to P10, SAM 20, and CTX30 standard, itwas seen that Bacillus cereus is more susceptible. Inaddition, the extract of V. cholorostegium has a strongantiyeast effect againts all the yeast cultures.

Verbascum L. species contain a wide range of com-pounds, such as glycosides (Klimek, 1996; Skaltsouniset al., 1996; Kalpoutzakis et al., 1999; Elgindi & Mabry,2000), alkaloids (Youhnovski et al., 1999), and saponins(Hartleb & Seifert, 1994). Members of the family

Table 2. Antimicrobial activities of some standard antibiotics.

Microorganisms

Inhibition zone (mm)

P10 SAM20 CTX30 VA30 OFX5 TE30 NY100

Escherichia coli 18 12 10 22 30 28 —Staphylococcus aureus 13 16 12 13 24 26 —Klebsiella pneumoniae 18 14 13 22 28 30 —Pseudomonas aeruginosa 8 10 54 10 44 34 —Proteus vulgaris 10 16 18 20 28 26 —Bacillus cereus 14 12 14 18 30 25 —Mycobacterium smegmatis 15 21 11 20 32 24 —Listeria monocytogenes 10 12 16 26 30 28 —Micrococcus luteus 36 32 32 34 28 22 —Candida albicans — — — — — — 20Kluyveromyces fragilis — — — — — — 18Rhodotorula rubra — — — — — — 18

P10, penicillin G (10 units); SAM20, ampicillin 10 mg; CTX30, cefotaxime 30 mg; V30, vancomycin 30mg; OFX 5, oflaxacin 5mg; TE30,tetracycline 30 mg; NY100, nystatin 100mg.

Table 1. Summary of antimicrobial activity of studied plants.

Plant species

Microorganisms=inhibition zone (mm)a

1 2 3 4 5 6 7 8 9 10 11 12

V. pseudoholotrichum — 18.2 — — — 15.6 11.2 12.6 14.6 12.8 14.6 15.2V. cymigerum — 17.4 — — — 14.4 10.8 11.8 15.2 18.2 19.4 17.2V. cholorostegium — 19.2 — — — 17.8 12.2 15.2 16.4 14.2 17.2 20.6V. linguifolium — 16.8 — — — 16.6 10.6 10.8 12.8 11.6 14.6 14.2V. pellitum — 17.8 — — — 14.2 11.8 16.8 14.8 10.8 12.6 11.4S. brevidens 12.6 — 14.2 10.6 — 14.6 15.6 16.2 — 14.4 10.6 12.2S. cilicica 14.4 10.8 15.8 — — 18.6 — 18.8 — 10.2 11.8 15.8S. vuralii 16.4 11.6 16.4 11.2 — 17.4 15.2 14.8 — 11.8 12.8 14.6S. aleurites 10.8 16.8 — 10.4 15.6 18.8 — — 14.6 — — —S. pinardii 8.0 14.6 — 16.2 18.4 15.2 — — 12.8 — — —Methanol (control) — — — — — — — — — — — —

1, Escherichia coli; 2, Staphylococcus aureus; 3, Klebsiella pneumoniae; 4, Pseudomonas aeruginosa; 5, Proteus vulgaris; 6, Bacillus cer-eus; 7, Mycobacterium smegmatis; 8, Listeria monocytogenes; 9, Micrococcus luteus; 10, Candida albicans; 11, Kluyveromyces fragilis;12, Rhodotorula rubra.aIncludes diameter of disk (6mm).

272 B. Dulger et al.

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Scrophulariaceae have been reported to contain a groupof unusual macrocyclic spermine alkaloids (Seifert et al.,1982; Koblikova et al., 1983). The antimicrobial activi-ties of nine Verbascum L. species have previously beenreported by Meurer-Grimes et al. (1996). They usedextracts from flowers, seeds, leaves, and roots anddetected a strong growth inhibition. As a result of thatstudy, antimicrobial activity was more consistentlydetected and activity against the Gram-positivebacterium Staphylococcus aureus and the yeast cultureshad been found. In our previously study, the extractsobtained from three endemic Verbascum species (Verbas-cum olympicum Boiss, Verbascum prusianum Boiss., andVerbascum bombyciferum Boiss.) showed similar resultsagainst specific bacteria (Dulger et al., 2002). It isdetermined that Verbascum L. species showed antimicro-bial activity against Gram-positive bacteria and yeasts.Our results are similar to those reported in the mentionedstudy. In general, Gram-negative bacteria have beenfound to be more resistant to extracts than Gram-positive bacteria, possibly because of their cell walllipopolysaccharide (Farbood et al., 1976; Cetin &Gurler, 1989; Outtara et al., 1997).

The extracts obtained from Sideritis L. were found tobe effective against all tested microorganisms used in thisstudy, at different levels, showing inhibition zones of10.2–18.8mm. However, all Sideritis L. species were noteffective against Micrococcus luteus and Proteus vulgaris.When the results obtained with S. cilicica were comparedwith those of the standard antibiotics, it was determinedthat Listeria monocytogenes, Bacillus cereus, andKlebsiella pneumoniae are especially susceptible to theextract of S. cilicica. Futhermore, the antifungal effectof Sideritis L. species was found to be much less in com-parison with the standard antifungal antibiotics. It wasreported that Sideritis brevidens and Sideritis vuralii havemonoterpene hydrocarbons and Sideritis pisidica hassesquiterpene hydrocarbons (Baser, 2002). In addition,water-distilled essential oils from the flowering spike ofS. vuralii were analyzed by GC-MS. b-Pinene (35%),1,8-cineole (15%) and a-pinene (15%), were the maincomponents in the oil of S. vuralii (Kirimer et al., 1999).

It is not surprising that there are differences in theantimicrobial effects of plant groups, due to phytochem-ical properties and differences among species. For theevaluation of plants that are naturally grown in Turkeyand are potential useful resources, additional studies willbe beneficial from medicinal and economic standpoints.

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