ABSTRACT
The genus Cistus. L., mainly growing in the Mediterranean region, is represented by five species in Turkey: C. creticus. L., C. laurifolius. L., C. monspeliensis. L., C. parviflorus. Lam., and C. salviifolius. L. In this study, in vitro. antimicrobial activity of water, methanol, chloroform, ethyl acetate, and buthanol extracts, prepared from dried and powdered leaves and the fruits of the five Cistus. species, were investigated by the disk diffusion method against the following microorganisms: Staphylococcus aureus. (ATCC 29213 and ATCC 25923), Streptococcus faecalis. (ATCC 29212), Bacillus subtilis. (ATCC 6633), Bacillus cereus. (RSKK 1122), Pseudomonas aeruginosa. (ATCC 27853), Escherichia coli. (ATCC 25922), and Candida albicans. (ATCC 10231). Chloroform, ethyl acetate, butanol, and the remaining aqueous extracts, obtained from the fractionation of methanol extract, were also tested. All of the extracts showed some activity against B. subtilis. and B. cereus.. Lyophylized extracts showed the highest activity against Staphylococcus aureus. 25923 and 29213. Buthanol extracts of the leaves and fruits of C. creticus. showed the highest activity. Extracts of all Cistus. species did not show any activity against Pseudomonas aeruginosa. and Candida albicans..
Keywords:
Introduction
The genus Cistus. (Cistaceae), growing naturally in the Mediterranean region, is represented with 20 species in the world (Zohary, Citation1987) and with five species (C. creticus. L., C.. laurifolius. L., C. monspeliensis. L., C. parviflorus. Lam., and C. salviifolius. L.) in Turkey (Davis Citation1965Citation1988). These plants are shrubs and are identified mainly according to the color of their flowers. Inflorescense is a simple or compound cyme, rarely solitary; calix consists of 3–5 sepals, petals pink or white, stamens many and all fertile; carpels 5–10, capsule splitting down to the middle into 5–10 valves, seeds minute (Davis, Citation1965).
Chemical studies, conducted on different species of the genus, revealed that their components consist of mainly terpenoids (usually labdane type) (Tabacik & Bard, Citation1971; Calabuig et al., Citation1981; Demetzos et al., Citation1990Citation1994a), polyphenols (flavonoids and proanthocynanins) (Demetzos et al., Citation1989; Petereit, Citation1991; Danne et al., Citation1993aCitationbCitation1994; Vogt & Gülz, Citation1994; Qa'dan et al., Citation2003), essential oils (0.02–0.08%) (Guenther, Citation1952; Hegnauer, Citation1964; Guy & Vernin, Citation1996; Demetzos et al., Citation1999Citation1997; Angelopoulou et al., Citation2002), resin (Demetzos et al., Citation1994bCitation1999), gum (Mrabet et al., Citation1997), and lipids (from the seeds) (Krollmann & Guelz, Citation1983).
Extracts obtained from some Cistus. species have been reported to possess antiulcer (Yeşilada et al., Citation1997, Citation1999), wound healing, antimicrobial (Chinou et al., Citation1994; Demetzos et al., Citation1997Citation1999; Kalpoutzakis et al., Citation2001), vasodilatator (Somoza et al., Citation1996), antispasmodic, and cytotoxic activity (Chinou et al., Citation1994; Demetzos et al., 1994; Dimas et al., Citation1998; Angelopoulou et al., 2001) and have also been described as regulators of blood circulation and reported to have depressant and analgesic effects on the central nervous system (de Andres et al., Citation1999). The alcohol-soluble absolutes are widely used in the perfume and cosmetics industry (Guenther, Citation1952; Baytop, Citation1999).
Cistus. species are used traditionally for various purposes in Turkey. For example, decoction of roots are used to cure sterility in women in Artvin (Tabata et al., Citation1988). Decoction of flowers and buds of C. laurifolius. are used as antiulcer in Afyon province (Yeşilada et al., Citation1999); C. laurifolius. is used for rheumatism in Eğirdir (Isparta) (Erol & Tuzlacı, Citation1996); fruits of the same species are also used as antidiabetic (Baytop, Citation1999); tea prepared from the leaves of C.. salviifolius. is used for the treatment of cancer.
A literature survey reveals some studies about the antimicrobial activity of Cistus. species. An oleoresin called “ladanum” is obtained from C. creticus. in Crete and Cyprus, but not in Turkey, although C. creticus. has high volatile oil. In another study, C. incanus. subsp. creticus. (L.) Heywood herb was investigated for its antibacterial activity (Chinou et al., Citation1994). To make a comparison with prior studies, we have studied the antimicrobial activity of leaves and fruits of the species growing in Turkey.
Materials and Methods
Plant material
The leaves of Cistus. species used in this study were collected during the flowering periods from Aegean, Mediterranean, and Inner Anatolian regions of Turkey. Voucher specimens are deposited at the Ankara Üniversitesi Eczacılık Fakültesi Herbaryumu (AEF). The collection locations of the research material are listed in .
Table 1.. Locations of the studied Cistus. species.
Extraction
Plant materials (leaves and fruits) were dried at room temperature and then powdered. Powdered materials (20 g) were extracted with 250 ml of distilled water in a glass bottle shaking for approximately 8 h (approximately 700 rpm/min and 50°C). The extracts were filtrated and then freezed and lyophylized (extract 1). Powdered materials (20 g) were extracted with 250 ml MeOH (Merck 1.06008-extra pure) in a Soxhlet apparatus for 8 h. Then, the filtered extracts were evaporated to dryness at 40°C using a rotary evaporator (extract 2). The same procedure was applied before fractionation. The residues were dissolved in a 200 ml mixture of H2O: MeOH (90:10), then extracted with three portions of chloroform (Merck 1.02431-extra pure) (3 × 20 ml) in a separatory funnel. The chloroform layer was collected and evaporated at 40°C (extract 3). Then the same extraction procedure was applied using ethyl acetate (Merck 1.00864-extra pure) (extracts were evaporated at 40°C) and n.-butanol (Merck Art. 988) (extract evaporated at 60°C) to remaining layer in H2O: MeOH mixture, respectively (extracts 4 and 5). The remaining aqueous solution was lyophylized (extract 6). All extracts (extracts 1–6) were used for microbiological tests.
Activity studies
All microorganisms were obtained from the Refik Saydam National Type Culture Collection of Turkey. Antimicrobial activities of the samples were determined using the disk diffusion technique (Shadomy & Pfaller, Citation1991; National Committee for Clinical Laboratory Standards, Citation1998). Mueller-Hinton agar (Difco, Detroit, MI, USA) plates were used for bacteria, and yeast nitrogen base (YNB) (Difco, Becton, Dickinson and Co., Spaks, MD, USA) supplemented with glucose (1%) and asparagines (0.15%) agar plates were used for Candida albicans..
Preparation of disks
Solution of 1/9 w/v concentrations of extracts were prepared with sterile distilled water and the solvents used for the extraction, and 0.02 ml of these portions were impregnated to sterile paper disks. Solvents are also applied to test their activities. Then, all disks were left to dry for a night. Cefazolin, ciprofloxacin, and fluconazole were used as standards (see ). Colonies (4–5), choosen from fresh cultures of the microorganisms used for microbiological studies, were taken into tryptic soy broth (Oxoid) and incubated for 2–4 h at 35°C. The broth cultures having 0.5 McFarland turbidity were inoculated to the surface of agar plates with sterile cotton swabs. After 10 min, all disks were dispensed on the surface of the inoculated agar plates. Petri dishes were incubated for 16–18 h at 35°C for bacteria and 24 h at 35°C for C. albicans. and inhibition zones were measured.
Results
The results of antimicrobial activity of different extracts obtained from five Cistus. species and standard antibiotics are listed in Tables .
Table 2.. Results of the antimicrobial activity of aqueous extracts (extract 1) of Cistus. species (inhibition zones in mm).
Table 3.. Results of the antimicrobial activity of methanol extracts (extract 2) of Cistus. species (inhibition zones in mm).
Table 4.. Results of the antimicrobial activity of chloroform extracts (extract 3) of Cistus. species (inhibition zones in mm).
Table 5.. Results of the antimicrobial activity of ethyl acetate extracts (extract 4) of Cistus. species (inhibition zones in mm).
Table 6.. Results of the antimicrobial activity of n.-butanol extracts (extract 5) of Cistus. species (inhibition zones in mm).
Table 7.. Results of the antimicrobial activity of remaining aqueous extracts (extract 6) of Cistus. species (inhibition zones in mm).
Table 8.. Inhibition zones of standards antibiotics (inhibition zones in mm).
Discussion
In this study, six different extracts were prepared from leaves and fruits of five Cistus. species. All extracts showed activity against S. aureus. 29213. Only butanol extracts showed activity against E. coli.. Although methanol, chloroform, ethyl acetate, and the remaining aqueous extracts did not show any activity against S. faecalis., butanol extracts of the leaves and fruits of C. creticus., butanol extracts of C. laurifolius. and C.. salviifolius., and aqueous extracts of the leaves of C. creticus. showed low activity against this microorganism. All of the extracts showed some activity against B. subtilis. and B.. cereus.. Fruit extracts of C. parviflorus. were not effective against these two microorganisms except for the butanol extract against B. subtilis.. Furthermore, the fruit extracts of C. salviifolius. were not effective against these two microorganisms except for the methanol extracts. Especially chloroform and ethyl acetate extracts of the leaves of all species were found to be active against B. subtilis. and B. cereus.. Extracts of all Cistus. species were not found effective against C. albicans.. Compounds that are soluble in polar solvents (water and butanol) are more effective than nonpolar solvents. Infusions or decoctions of most of Cistus. species are used in folk medicine in Turkey. Furthermore, results of this study showed that leaves and fruits of C. creticus. has the highest activity among other species.
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