Antimicrobial Activity of Some Endemic Scrophulariaceaefrom Turkey

Abstract

Methanol extracts obtained from endemic Scrophulariaceae, Verbascum chianophyllum. Hub.-Mor., V. cilicium. Boiss., V. trapifolium. (Stapf) Hub.-Mor., V. meinckeanum. Murb., V. lyratifolium. Köchel, Scrophularia trichopoda. Boiss. & Bal., S. candelabrum. Heywood, and Pedicularis cadmea. Boiss., have been investigated for their antimicrobial activity. Antimicrobial activity was determined with Escherichia coli. ATCC 11230, Staphylococcus aureus. 6538-P, Klebsiella pneumoniae. UC57, Pseudomonas aeruginosa. ATCC 27583, 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 plant species had strong antimicrobial activity against the Gram-positive bacteria and yeasts, but no activity was seen against the Gram-negative bacteria used in this study.

Keywords:

• Antimicrobial activity
• Pedicularis cadmea. Boiss
• Scrophularia candelabrum. Heywood
• Scrophularia trichopoda. Boiss. & Bal
• Verbascum chianophyllum. Hub.-Mor
• Verbascum cilicium. Boiss
• Verbascum lyratifolium. Köchel
• Verbascum meinckeanum. Murb
• Verbascum trapifolium. (Stapf) Hub.-Mor

Introduction

In recent years, multiple resistances in pathogens have developed due to the in discriminate use of antimicrobial drugs commonly employed in the treatment of infectious diseases. The undersirable side effects of some antibiotics and the emergence of previously uncommon infections have forced researchers to look for new antimicrobial substances from various sources like medicinal plants. The screening of plant extracts for antimicrobial activity has shown that higher plants represent a potential source of new anti-infective agents (Poole, 2001; Salvat et al., 2001; Arias et al., 2004).

Some species of Verbascum. L. (Scrophulariaceae) have been used widely 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 may societies in Europe, Asia, Africa, and North America (Meurer–Grimes et al., 1996).

Many Scrophularia. L. species have been used since ancient times as folk remedies for scrophula, scabies, tumors, and inflammatory affections (Paris & Moyse, 1976; Hetaher & Hendersoni, 1994).

Verbascum phlomoides. L., Verbascum densiflorum. Bertol., and Verbascum thapsus. L. have been used for ethnopharmacological effects among the common people in Turkey. Especially their flowers have been used. The drug, prepared from their flowers, has diuretic and expectorant effects. Leaves of plants have also been used for their diuretic, expectorant, and sedative effects. Seeds of Verbascum. species are used as poisonous seeds for hunting fish. Verbascum. species are called “fish plant” in the northern Anatolia because of that property (Zeybek, 1985).

Verbascum chianophyllum. Hub.-Mor., V. cilicium. Boiss., V. trapifolium. (Stapf) Hub.-Mor., V. meinckeanum. Murb., V. lyratifolium. Köchel, Scrophularia trichopoda. Boiss. & Bal., S. candelabrum. Heywood, and Pedicularis cadmea. Boiss. are endemic to Turkey (Guner et al., 2000; Davis, 1978). Although there are many investigations on this species, these particular 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., Scrophularia., and Pedicularis. 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 Çanakkale Onsekiz Mart University (Çanakkale, Turkey) and identified by Emin Uğurlu from Celal Bayar University (Manisa, Turkey).

Preparation of extracts

The plants 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. 6538-P, Klebsiella pneumoniae. UC57, Pseudomonas aeruginosa. ATCC 27583, 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 impregrated 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 Laboratories) 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 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 microorganism for comparison.

Results and Discussion

Table 1 shows antimicrobial activity of the plant extracts, and the inhibition zones formed by standard antibiotic discs are indicated in Table 2. As can clearly be seen from Table 1, no significant activity was found against Gram-negative bacteria such as Escherichia coli., Proteus vulgaris., Klebsiella pneumoniae., 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. was found. Besides, the plant extracts have an antiyeast activity against all tested yeast cultures in different levels (mm).

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. chianophyllum.	—	16.4	—	—	—	15.6	14.2	10.6	18.8	14.8	16.8	15.4
V. cilicium.	—	20.2	—	—	—	24.0	18.8	15.4	25.4	18.0	17.2	20.8
V. trapifolium.	—	19.2	—	—	—	20.0	14.2	9.8	16.8	15.2	15.4	19.2
V. meinckeanum.	—	16.8	—	—	—	16.2	10.8	10.2	17.2	15.0	16.2	14.2
V. lyratifolium.	—	17.2	—	—	—	14.4	15.0	10.8	15.8	14.8	15.2	13.4
S. trichopoda.	—	13.2	—	—	—	13.4	12.8	15.2	15.4	10.2	10.4	—
S. candelabrum.	—	12.8	—	—	—	11.2	14.0	14.2	15.2	—	—	10.8
P. cadmea.	—	13.2	—	—	—	11.6	11.4	11.8	14.0	11.0	12.2	11.8
Methanol (control)	—	—	—	—	—	—	—	—	—	—	—	—

1, Escherichia coli.; 2, Micrococcus luteus.; 3, Klebsiella pneumoniae.; 4, Pseudomonas aeruginosa.; 5, Proteus vulgaris.; 6, Bacillus cereus.; 7, Mycobacterium smegmatis.; 8, Listeria monocytogenes.; 9, Staphylococcus aureus.; 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; OFX5, oflaxacin 5 µg; TE30, tetracycline 30 µg; NY100, nystatin 100 µg.

It was found that extracts of V. cilicium. have more antibacterial and antifungal activity than the other Verbascum. L. species. Staphylococcus aureus. is more susceptible to the extracts of all Verbascum. L. species, as compared with standard antibiotics, except for TE30. Similarly, in comparison with P10, SAM20, CTX30, and VA30 standard, it was seen that Bacillus cereus. is more susceptible. In addition, the extracts of V. cilicium. and V. trapifolium. have a greater antiyeast effect than the standard antifungal antibiotic nystatin against Rhodotorula rubra.. Notably, the extract of V. bellum. has a greater antibacterial effect than those of the standards P10 and CTX30 against acid-fast bacterium Mycobacterium smegmatis..

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 studies, the extracts obtained from some Verbascum. species showed similar results against specific bacteria (Dulger et al., 20022005; Dulger & Gonuz, 2004; Dulger & Ugurlu, 2005). It is determined that Verbascum. L. species showed antimicrobial activity against Gram-positive bacteria and yeasts and no antibacterial activity was found against Gram-negative bacteria. The results in this study are similar to those reported in the mentioned studies. 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; Çetin & Gurler, 1989; Outtara et al., 1997).

The extracts obtained from Scrophularia. L. were found to be effective against Gram-positive bacteria, but they were weakly active against the yeast cultures used in this study. When the results obtained with both Scrophularia. species were compared with those of some standard antibiotics, it was determined that Staphylococcus aureus. and Listeria monocytogenes. are susceptible to the extract. The acid-fast bacterium Mycobacterium smegmatis. is more resistant to the extracts in comparison with the standard antibacterial antibiotics, except for CTX30.

Many Scrophularia. species have been investigated and found to contain several 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, cardivascular, diuretic, and antitumor acitivities (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.-hydroxy benzoic, syringic, caffeic, gentisic, protocetechuric, p.-coumaric, and vanillic acids) (Fernandez et al., 1996). The plant species could be considered as potential antiseptic agents on bacteriological infections, especially in processes where Gram-positive bacteria are involved (J de Santos Galindez et al., 2002).

The extract obtained from P. cadmea. was found to be weakly effective against Gram-positive bacteria and the yeast cultures, showing inhibition zones of 11.0–12.2 mm. Although there is no information on P. cadmea., there are many investigations on the chemical contents of other Pedicularis. 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).

Phytochemical and further pharmacological studies are important tasks for the future in order to better understand the effects of these important pharmaceutical resources. Herbal medicines are a valuable and readily available resource for primary health care and complementary health care systems. 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. These plants belong to the Scrophulariaceae family, which may prove to be a rich source of compounds with possible antimicrobial activities, but more pharmacological investigations are necessary.