Evaluation of Antimicrobial Activity of Some Endemic Verbascum., Sideritis., and Stachys. Species from Turkey

Abstract

Methanol extracts obtained from endemic Verbascum pseudoholotrichum. Hub.-Mor., Verbascum cymigerum. Hub.-Mor., Verbascum cholorostegium. Bornm. & 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., and Stachys pinardii. Boiss. have been investigated for their antimicrobial activity. Antimicrobial activity was determined with Escherichia coli. ATCC 11230, Staphylococcus aureus. ATCC 6538P, Klebsiella pneumoniae. UC57, Pseudomonas aeruginosa. ATCC 27853, Proteus vulgaris. ATCC 8427, Bacillus cereus. ATCC 7064, Mycobacterium smegmatis. CCM 2067, Listeria monocytogenes. ATCC 15313, Micrococcus luteus. CCM 169, Candida albicans. ATCC 10231, Rhodotorula rubra. DSM 70403, and Kluyveromyces fragilis. ATCC 8608 by the disk diffusion method. Verbascum. L. extracts had strong antimicrobial activity against Gram-positive bacteria and yeast cultures. The extracts of Stachys. L. were effective only against bacteria. The extracts of Sideritis. L. demonstrated antimicrobial effects against the bacteria and the yeast cultures used in this study.

Keywords:

• Antimicrobial activity
• Verbascum pseudoholotrichum. Hub.-Mor
• Verbascum cymigerum. Hub.-Mor
• Verbascum cholorostegium. Bornm. & 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
• Stachys pinardii. Boiss

Introduction

Higher plants have been the source of medicinal agents since early times. They continue to play a dominant role in the primary health care of about 80% of the world's population (Baker et al., 1995). Some species of Verbascum. L. (Scrophulariaceae) have widely been used throughout centuries to treat internal and external infections. Many internal and external uses of the leaves and flowers of several Verbascum. L. species have been documented in many societies in Europe, Asia, Africa, and North America (Maurer-Grimes et al., 1996). Verbascum pseudoholotrichum. Hub.-Mor., Verbascum cymigerum. Hub.-Mor., Verbascum cholorostegium. Bornm. & Murb., Verbascum linguifolium. Hub.-Mor., and Verbascum pellitum. Hub.-Mor., have been used for their ethnopharmacological effects among the common folk in Turkey. Especially their flower has been used as a diuretic, expectorant, and sedative. Seeds of Verbascum. species are poisonous because of the saponins contained. People use these poisonous seeds for hunting fish. Verbascum. species are called “fishplant” in northern Anatolia because of that property (Zeybek, 1985).

Some species of Stachys. L. and Sideritis. L. (Labiatae) have widely been used for their diuretic and expectorant effects. In addition, leaves of plants have also been used against abdominal pains in Turkey (Baytop, 1999). The genus Sideritis. (Labiatae) is widely distributed in subtropical and moderate regions (Davis, 1982). Sideritis. species 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 number of Sideritis. species are used as tonics, carminatives, as 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., Verbascum cymigerum. Hub.-Mor., Verbascum cholorostegium. Bornm. & 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., and Stachys pinardii. Boiss. are endemic to Turkey. (Davis, 1978; Guner et al., 2000). Although there are many investigations on Sideritis., Stachys., and Verbascum. species, these plants have not been previously investigated. Therefore, our aim was to determine the antimicrobial effects of plant extracts obtained from these endemic species against microorganisms.

Materials and Methods

Plant materials

Aerial parts of Verbascum pseudoholotrichum. Hub.-Mor., Verbascum cymigerum. Hub.-Mor., Verbascum cholorostegium. Bornm. & 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., and Stachys pinardii. Boiss. were collected from different localities in Turkey during the months of September–October 2001. Voucher specimens of the plants were deposited in the Biology Department at Canakkale Onsekiz Mart University, Canakkale, Turkey, and identified by Emin Ugurlu.

Preparation of extracts

The plant parts were air-dried. Each dry powdered plant material (20 g) was extracted with 150 ml of 80% methanol (Merck, Darmstadt, Germany) for 24 h by using Soxhlet equipment (Khan et al., 1988). The extract was filtered using Whatman filter paper no. 1, and the filtrates were then evaporated under reduced pressure and dried using a rotary evaporator at 55°C. Dried extracts were stored in labeled sterile screw-capped bottles at –20°C.

Microorganisms

Escherichia coli. ATCC 11230, Staphylococcus aureus. ATCC 6538P, Klebsiella pneumoniae. UC57, Pseudomonas aeruginosa. ATCC 27853, Proteus vulgaris. ATCC 8427, Bacillus cereus. ATCC 7064, Mycobacterium smegmatis. CCM 2067, Listeria monocytogenes. ATCC 15313, Micrococcus luteus. CCM 169, Candida albicans. ATCC 10231, Rhodotorula rubra. DSM 70403, and Kluyveromyces fragilis. ATCC 8608 were used as test microorganisms.

Screening for antimicrobial activities

The dried plant extracts were dissolved in 10% aqueous dimethylsulfoxide (DMSO) to a final concentration of 200 mg/ml and sterilized by filtration through an 0.45 µm membrane filter. Empty sterilized antibiotic disks having a diameter of 6 mm (Schleicher & Schull No. 2668, Dassel, Germany) were each impregnated with 50 µl of extract (10 mg/disk) at a concentration of 200 mg/ml. All the bacteria mentioned above were incubated at 35 ± 0.1°C for 24 h by inoculation into nutrient broth (Difco), and the yeast cultures studied were incubated in malt extract broth (Difco Laboratories, MI, USA) at 25 ± 0.1°C for 48 h. An inoculum containing 10⁶ bacterial cells or 10⁸ yeast cells/mL was spread on Mueller-Hinton agar (Oxoid Ltd., Hampshire, UK) plates (1 ml inoculum/plate). The disks injected with extracts were placed on the inoculated agar by pressing slightly. Petri dishes were placed at 4°C for 2 h, plaques injected with the yeast cultures were incubated at 25 ± 0.1°C, and bacteria were incubated at 35 ± 0.1°C for 24 h (Collins et al., 1989; Ali-Stayeh et al., 1998). At the end of the period, inhibition zones formed on the medium were evaluated in millimeters. Studies were performed in triplicate. On each plate, an appropriate reference antibiotic disk was applied, depending on the test microorganisms for comparison.

Results and Discussion

Table 1 shows antimicrobial activities of the plant extracts, and the inhibition zones formed by standard antibiotic disks are indicated in Table 2. As can clearly be seen from Table 1, the extracts of Stachys. have an antibacterial effect against tested bacteria, but no significant activity was found against the yeast cultures. Activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris., and Bacillus cereus. were found from extracts of all Stachys. L. species, showing inhibition zones of 8.0–18.8 mm. As can be seen from Tables 1 and 2, the extract obtained from Stachys aleurites. species has much higher inhibition zones than the standard antibacterial antibiotics, except for OFX5 and TE30 against Bacillus cereus.. In addition, the extract of Stachys pinardii. has a greater antibacterial effect than the standard antibacterial antibiotics P10, SAM20, and CTX30 against Proteus vulgaris.. Although there is no information on Stachys. species used in this study, there are many studies on the chemical contents of other Stachys. species. Sesquiterpenes-α-copaene (16.5%), spathulenol (16.1%), β-caryophyllene (14.3%), and β-cubebene were identified by gas chromatography (GC) and GUMS analyses from aerial parts of Stachys byzanthina. (Khanavi et al., 2003). Similarly, α -pinene (20.1%), β-pinene (12.1%), and spathulenol (7.2%) were determined from Stachys lavandulifolia. (Feizbaksh et al., 2003). According to Mirza and Baher (2003), Stachys lanata. has α-thyjone, α-humulene, β-caryophyllene, and viridiflorol. Digrak et al. (2001) reported that the chloroform extract of Stachys annua. subsp. annua. and Stachys pumila. had little antibacterial effect against used bacteria, and both of them were also found not to be effective against the fungi studied. Our findings concur with this study, despite the different extraction method used.

Table 1.. Summary of antimicrobial activity of studied plants

	Microorganisms/inhibition zone (mm)^a.
Plant species	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	15.2
V. cymigerum.	—	17.4	—	—	—	14.4	10.8	11.8	15.2	18.2	19.4	17.2
V. cholorostegium.	—	19.2	—	—	—	17.8	12.2	15.2	16.4	14.2	17.2	20.6
V. linguifolium.	—	16.8	—	—	—	16.6	10.6	10.8	12.8	11.6	14.6	14.2
V. pellitum.	—	17.8	—	—	—	14.2	11.8	16.8	14.8	10.8	12.6	11.4
S. brevidens.	12.6	—	14.2	10.6	—	14.6	15.6	16.2	—	14.4	10.6	12.2
S. cilicica.	14.4	10.8	15.8	—	—	18.6	—	18.8	—	10.2	11.8	15.8
S. vuralii.	16.4	11.6	16.4	11.2	—	17.4	15.2	14.8	—	11.8	12.8	14.6
S. 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 cereus.; 7, Mycobacterium smegmatis.; 8, Listeria monocytogenes.; 9, Micrococcus luteus.; 10, Candida albicans.; 11, Kluyveromyces fragilis.; 12, Rhodotorula rubra..

^a.Includes diameter of disk (6 mm).

Table 2.. Antimicrobial activities of some standard antibiotics

	Inhibition zone (mm)
Microorganisms	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.	—	—	—	—	—	—	20
Kluyveromyces fragilis.	—	—	—	—	—	—	18
Rhodotorula rubra.	—	—	—	—	—	—	18

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

As also listed in Table 1, it was found that extracts of Verbascum. L. species have no significant activity against Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris., and Pseudomonas aeruginosa.. Activity against the Gram-positive bacteria such as Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Micrococcus luteus., and acid-fast bacterium Mycobacterium smegmatis. were found. Besides, extracts of Verbascum. L. species have an antiyeast activity against all tested yeast cultures. Extracts of V. cholorostegium. have more antibacterial and antifungal effects than other Verbascum. L. species. Staphylococcus aureus. is more susceptible to the extracts of all Verbascum. L. species, as compared to standard antibiotics, except for OFX5 and TE 30. Similarly, in comparison to P10, SAM 20, and CTX30 standard, it was seen that Bacillus cereus. is more susceptible. In addition, the extract of V. cholorostegium. has a strong antiyeast effect againts all the yeast cultures.

Verbascum. L. species contain a wide range of compounds, such as glycosides (Klimek, 1996; Skaltsounis et al., 1996; Kalpoutzakis et al., 1999; Elgindi & Mabry, 2000), alkaloids (Youhnovski et al., 1999), and saponins (Hartleb & Seifert, 1994). Members of the family Scrophulariaceae have been reported to contain a group of unusual macrocyclic spermine alkaloids (Seifert et al., 1982; Koblikova et al., 1983). The antimicrobial activities of nine Verbascum. L. species have previously been reported by Meurer-Grimes et al. (1996). They used extracts from flowers, seeds, leaves, and roots and detected a strong growth inhibition. As a result of that study, antimicrobial activity was more consistently detected and activity against the Gram-positive bacterium Staphylococcus aureus. and the yeast cultures had been found. In our previously study, the extracts obtained from three endemic Verbascum. species (Verbascum olympicum. Boiss, Verbascum prusianum. Boiss., and Verbascum bombyciferum. Boiss.) showed similar results against specific bacteria (Dulger et al., 2002). It is determined that Verbascum. L. species showed antimicrobial activity against Gram-positive bacteria and yeasts. Our results are similar to those reported in the mentioned study. In general, Gram-negative bacteria have been found to be more resistant to extracts than Gram-positive bacteria, possibly because of their cell wall lipopolysaccharide (Farbood et al., 1976; Cetin & Gurler, 1989; Outtara et al., 1997).

The extracts obtained from Sideritis. L. were found to be effective against all tested microorganisms used in this study, at different levels, showing inhibition zones of 10.2–18.8 mm. However, all Sideritis. L. species were not effective against Micrococcus luteus. and Proteus vulgaris.. When the results obtained with S. cilicica. were compared with those of the standard antibiotics, it was determined that Listeria monocytogenes, Bacillus cereus., and Klebsiella pneumoniae. are especially susceptible to the extract of S. cilicica.. Futhermore, the antifungal effect of Sideritis. L. species was found to be much less in comparison with the standard antifungal antibiotics. It was reported that Sideritis brevidens. and Sideritis vuralii. have monoterpene hydrocarbons and Sideritis pisidica. has sesquiterpene hydrocarbons (Baser, 2002). In addition, water-distilled essential oils from the flowering spike of S. vuralii. were analyzed by GC-MS. β-Pinene (35%), 1,8-cineole (15%) and α-pinene (15%), were the main components in the oil of S. vuralii. (Kirimer et al., 1999).

It is not surprising that there are differences in the antimicrobial effects of plant groups, due to phytochemical properties and differences among species. For the evaluation of plants that are naturally grown in Turkey and are potential useful resources, additional studies will be beneficial from medicinal and economic standpoints.