Evaluation of Antimicrobial Activity of Some Endemic Scrophulariaceae. Members from Turkey

Abstract

The methanol extracts obtained from endemic Scrophulariaceae. members Verbascum protractum. Fenel ex Tchihat., Verbascum bellum. Hub.-Mor., Verbascum dalamanicum. Hub.-Mor., Scrophularia mersinensis. Lall, Scrophularia cryptophila. Boiss. & Heldr., Pedicularis olympica. Boiss., and Veronica lycica. E. Lehm. 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. The extracts of all Scrophulariaceae members used in this study had strong antimicrobial activity against the Gram-positive bacteria and yeast cultures.

Keywords:

• Antimicrobial activity
• Scrophulariaceae
• Verbascum protractum. Fenel ex Tchihat
• Verbascum bellum. Hub.-Mor
• Verbascum dalamanicum. Hub.-Mor
• Scrophularia mersinensis. Lall
• Scrophularia cryptophila. Boiss. & Heldr
• Pedicularis olympica. Boiss
• Veronica lycica. E. Lehm

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 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. L. species have been used for their ethnopharmacological effects among the common folk in Turkey. Especially their flower has been used as 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).

Many species belonging to Scrophularia. L. have been used since ancient times as folk remedies for some medical treatments including scrophula, scabies, tumors and inflammatory affections (Paris & Moyse, 1976; Heather & Hendersoni, 1994).

The Scrophulariaceae members Verbascum protractum. Fenel ex Tchihat., Verbascum bellum. Hub.-Mor., Verbascum dalamanicum. Hub.-Mor., Scrophularia mersinensis. Lall, Scrophularia cryptophila. Boiss. & Heldr., Pedicularis olympica. Boiss., and Veronica lycica. E. Lehm. are endemic to Turkey (Davis, 1978). Although there are many investigations on Verbascum, Scrophularia., Pedicularis., and Veronica. 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 protractum. Fenel ex Tchihat., Verbascum bellum. Hub.-Mor., Verbascum dalamanicum. Hub.-Mor., Scrophularia mersinensis. Lall, Scrophularia cryptophila. Boiss. & Heldr., Pedicularis olympica. Boiss., and Veronica lycica. E. Lehm. were collected from different localities in Turkey during the months of September–October 2001. Voucher specimen of the plants was 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 extract 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 Laboratories, MI, USA), and the yeast cultures studied were incubated in malt extract broth (Difco) 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.

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.. protractum.	—	16.2	—	—	—	14.2	13.8	13.6	12.6	11.4	15.4	14.8
V.. bellum.	—	16.8	—	—	—	15.2	14.2	15.0	13.4	16.4	18.2	19.2
V.. dalamanicum.	—	17.0	—	—	—	13.8	12.8	14.2	11.6	12.8	13.2	14.4
S. mersinensis.	—	12.8	—	—	—	12.6	13.4	14.2	15.2	—	10.2	—
S. cryptophila.	—	11.2	—	—	—	10.2	11.8	12.8	10.8	—	10.8	—
P. olympica.	—	13.6	—	—	—	14.2	15.0	16.8	12.8	14.6	15.2	15.6
V. lycica.	—	10.8	—	—	—	11.6	10.6	12.2	13.8	—	11.0	13.4
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 can clearly be seen from Table 1, the extracts obtained from all Scrophulariaceae members used in this study have no significant activity against Gram-negative bacteria such as Escherichia coli., Klebsiella pneumoniae., Proteus vulgaris., and Pseudomonas aeruginosa.. Notably, 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, the plant extracts have an antiyeast activity against all tested yeast cultures in different levels.

It was found that extracts of V. bellum have. more antibacterial and antifungal effects than the 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 VA30 standard, it was seen that Bacillus cereus. is more susceptible. In addition, the extract of V. bellum. has a strong antiyeast effect against all the yeast cultures. Notably, the acid-fast bacterium Mycobacterium smegmatis. is more resistant to the extracts in comparison to the standard antibacterial antibiotics, except for CTX30.

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 previous 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 Scrophularia. L. were found to be weakly effective against Gram-positive bacteria and the yeast cultures in different level, showing inhibition zones of 10.2–15.2 mm. Both Scrophularia. L. species were not effective against Candida albicans. and Kluyveromyces fragilis..

Many Scrophularia. species have been investigated and found to contain many classes of secondary metabolites including iridoids, phenylpropanoids, phenolic acids, flavanoids, and saponins. Some of these compounds were shown to have anti-inflammatory, antibacterial, fungicidal, protozoocidal, molluscicidal, cytotoxic, hepatoprotective, immunomodulator, cardiovascular, diuretic, and antitumor activities (J de Santos Galindez et al., 2002). According to the literature, antibacterial activity of Scrophularia. L. species can be attributed to the presence of phenolic acids (ferutic, isovanillic, p.-hydroxybenzoic, syringic, caffeic, gentisic, protocetechuric, p.-coumaric, and vanillic acids) (Fernandez et al., 1996). The plant species could be considered as potentially antiseptic agents on bacteriological infections, especially in processes where Gram-positive bacteria are involved (J de Santos Galindez et al., 2002).

When the results obtained with Pedicularis olympica. were compared to those of the standard antibiotics, it was determined that Listeria monocytogenes., Bacillus cereus., and the acid-fast bacterium Mycobacterium smegmatis. are more susceptible. Furthermore, the antifungal effect of P. olympica. extracts was found to be much less in comparison to the standard antifungal antibiotics. The extracts obtained from V. lycica. have weak antimicrobial effect against the tested microorganisms. It has no effect against Candida albicans.. Although there is no information on Pedicularis. and Veronica. species used in this study, there are many studies on the chemical contents of other Pedicularis. and Veronica. species. New iridoid glycosides from Pedicularis kansuensis. f. albiflora. were identified and elucidated by spectroscopic methods (Yuan et al., 2003). Artselaenins were isolated from Pedicularis artselaeri. (Su et al., 1998). A new iridoid glucoside, proceroside, was isolated from the leaves of Pedicularis procera. (Su et al., 1997). Acylated flavone glycosides from Veronica liwanensis. and Veronica longifolia. were isolated and identified by NMR spectroscopy (Albach et al., 2003). A nuatigenin-type steroidal saponin, multifidoside, was isolated from the aerial parts of Veronica fuhsii. and V. multifida,. and its structure was identified (Ozipek et al., 2002).

Undoubtedly, the plant kingdom still holds many species of plants containing substances of medicinal value that have yet to be discovered; large numbers of plants are constantly being screened for their antimicrobial effects. Some of these plants belong to the Scrophulariaceae family. This family may prove to be a richer source of compounds with possible antimicrobial activities, but more pharmacological investigations are necessary.