Abstract
Extracts obtained from different parts of 10 Centaurea L.. (Asteraceae) species [C. calolepis. Boiss., C. cariensis. Boiss subsp. maculiceps. (O. Schwarz) Wagenitz, C. cariensis. Boiss. subsp. microlepis. (Boiss.) Wagenitz, C. hierapolitana. Boiss., C. cadmea. Boiss., C. reuterana. Boiss. var. reuterana, C. cyanus. L., C. depressa. Bieb., C. urvillei. DC. subsp. urvillei., and C. ensiformis. P.H. Davis], most of them endemic in Turkey, were evaluated for in vitro. antiprotozoal activities (against Plasmodium falciparum. and Leishmania donovani.) and antimicrobial activities (against Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Mycobacterium intracellulare, Aspergillus fumigatus., and methicillin-resistant Staphylococcus aureus.). The chloroform extract of C. hierapolitana. demonstrated activity against chloroquine-sensitive and chloroquine-resistant P. falciparum. clones with IC50 values of 7 and 7.3 µg/ml, respectively. The highest antileishmanial activities among the extracts were observed with the chloroform extract of C. hierapolitana. (IC50 = 8.7 µg/ml, IC90 = 17 µg/ml). Hexane extracts of C. depressa. and C. urvillei. subsp. urvillei. showed antifungal activity against Candida krusei. (IC50 = 15 and 45 µg/ml, respectively). Finally, the chloroform extract of C. urvillei. subsp. urvillei. had activity against Cryptococcus neoformans. with an IC50 value of 40 µg/ml.
Introduction
Plants used in traditional medicine have the potential to provide pharmacologically active natural products that can be used to treat various ailments. The biological properties of several species of Asteraceae have been evaluated for antiprotozoal (Bailey et al., Citation2004; Taleb-Contini et al., Citation2004) and antimicrobial (Salie et al., Citation1996; Stojanović et al., Citation2005) activities. As a result of these evaluations, artemisinin has been isolated from Artemisia annua. L. (Asteraceae) and has well-documented antiplasmodial activity (Dhingra et al., Citation1999). Moreover, drugs derived from artemisinin are now in widespread use in single and combination therapies. Evaluation of native plants in validated activity screens will help determine which plants have a value for new pharmaceutical products.
The genus Centaurea. L. (Asteraceae) comprises about 181 species in the flora of Turkey distributed throughout the Anatolian peninsula, with 61% being endemic (Wagenitz, Citation1975; Güner, Citation2000; Duran & Duman, Citation2002). The aerial parts of several Centaurea. species are used in traditional medicine (Baytop, Citation1999). C. pulchella., C. drabifolia., and C. solstitialis. are reported in Turkish folk medicinal use to treat abcesses, hemorrhoids, peptic ulcers, and the common cold (Honda et al., Citation1996; Sezik et al., Citation2001). In some cases, biological evidence for these activities has been observed: antifungal (Skaltsa et al., Citation2000; Panagouleas et al., Citation2003), antibacterial (Yesilada et al., Citation1999; Karioti et al., Citation2002), cytotoxic (Koukoulitsa et al., Citation2002), anti-inflammatory (Negrete et al., Citation1993), and antiplasmodial (Medjroubi et al., Citation2005). The constituents of Centaurea. are mainly flavonoids (Akkal et al., Citation2003) and lignan glucosides (Gousiado et al., Citation2003), the latter including sesquiterpene lactones (Janaćković et al., Citation2004; Marco et al., Citation2005).
The flower heads of C. cyanus. are commonly used in European traditional medicine for the treatment of minor ocular inflammation (Bruneton, Citation1995). In vivo. anti-inflammatory and immunological activities (Garbacki et al., Citation1999) of C. cyanus. have been reported. Phytochemical studies on C. cyanus. revealed the presence of anthocyanins, flavonoids, flavonoid glycosides, and sesquiterpenes (Sarker et al., Citation2001). Indole alkoloids were also isolated from the seeds of C. cyanus. (Sarker et al., Citation2001).
Chloroform and ethyl acetate concentrates of the leaves of C. urvillei., which is known to contain methoxyflavones, demonstrated cytotoxic activity against L-strain fibroblasts in tissue culture (Ulubelen & Oksuz, Citation1982). Several flavonoids have been isolated from C. cariensis. (Halfon et al., Citation1989). C. urvillei. and C. cariensis. represent different subspecies of flora in Turkey (Wagenitz, Citation1975), and because of nonspecified subspecies names, exact plant materials are not known in these studies.
In this study, n.-hexane, chloroform, and methanol extracts prepared from different parts of 10 Centaurea. species were evaluated for the first time for antifungal, antibacterial, antileishmanial, and antimalarial activities.
Materials and Methods
Plant material
Plants were collected from western and southwestern parts of Turkey in June 2004 and identified by Prof. Dr. Ozcan Secmen, Section of Botany, Department of Biology, Faculty of Science, Ege University. Voucher specimens were deposited in the herbarium of Ege University, Faculty of Pharmacy, Izmir, Turkey ().
Table 1.. Collection sites of Centaurea. species.
Preparation of plant extracts
Dried and powdered plant parts were extracted sequentially with n.-hexane, chloroform, and methanol (3 × 10 ml/g, for each), sonicated at room temperature for 24 h, and then filtered. The combined extracts were evaporated under reduced pressure to dryness at 40°C.
Assay for antimalarial activity
The in vitro. antimalarial activity was determined against two strains of Plasmodium falciparum. (D6, chloroquine-sensitive, and W2, chloroquine-resistant) in 96-well microplate format. The assay is based on the determination of plasmodial lactate dehydrogenase (LDH) activity. For the assay, a suspension of red blood cells infected with D6 or W2 strains of P. falciparum. (200 µl, with 2% parasitemia and 2% hematocrit in RPMI 1640 medium supplemented with 10% human serum and 60 µg/ml amikacin) is added to the wells of a 96-well plate containing 10 µl of test samples diluted in medium at various concentrations. The plate is placed in a modular incubation chamber (Billups-Rothenberg, Del-Mar, CA, USA) and flushed with a gas mixture of 90% N2, 5% O2, and 5% CO2 and incubated at 37°C, for 72 h. Parasitic LDH activity is determined by using Malstat reagent (Flow Inc., Portland, OR, USA) according to the procedure of Makler et al. (Citation1993). Briefly, 20 µl of the incubation mixture is mixed with 100 µl of the Malstat reagent and incubated at room temperature for 30 min. A 1:1 mixture of NBT/PES (20 µl) (Sigma, St. Louis, MO, USA) is then added, and the plate is further incubated in the dark for 1 h. The reaction is then stopped by the addition of 100 µl of a 5% acetic acid solution. The plate is read at 650 nm using the EL-340 Biokinetics Reader (Bio-Tek Instruments, Winooski, VT, USA). IC50 values are computed from the dose-response curves. Artemisinin and chloroquine are included in each assay as the drug controls. DMSO (0.25%) is used as vehicle control.
Assay for antileishmanial activity
Antileishmanial activity of the compounds was tested in vitro. against a culture of Leishmania donovani. promastigotes. They were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (Gibco Chem. Co. Carlsbad, CA, USA) at 26°C. A 3-day-old culture was diluted to 5 × 105 promastigotes/ml. Drug dilutions (50–3.1 µg/ml) were prepared directly in cell suspension in 96-well plates. Plates were incubated at 26°C for 48 h, and growth of leishmania promastigotes was determined by Alamar blue assay as described earlier (Mikes et al., Citation2000). Standard fluorescence was measured on a Fluostar Galaxy plate reader (BMG Lab Technologies Offenburg, Germany) at excitation wavelength of 544 nm and emission wavelength of 590 nm. Pentamidine (Sigma) and amphotericin B (ICN Biomedicals, Cleveland, OH, USA) were used as the standard antileishmanial agents. IC50 and IC90 values were computed from dose curves generated by plotting percent growth versus sample concentration.
Assay for antimicrobial activity
All organisms were obtained from the American Type Culture Collection (Manassas, VA, USA) and included the fungi Candida albicans. ATCC 90028, Candida glabrata. ATCC 90030, Candida krusei. ATCC 6258, Cryptococcus neoformans. ATCC 90113, and Aspergillus fumigatus. ATCC 90906, and the bacteria methicillin-resistant Staphylococcus aureus. ATCC 43300 (MRS) and Mycobacterium intracellulare. ATCC 23068. Susceptibility testing was performed using a modified version of the NCCLS methods (NCCLS, Citation2000aCitationbCitation2002aCitationb). M. intracellulare. was tested using a modified method of Franzblau et al. (Citation1998). Briefly, samples (dissolved in DMSO) were serially diluted using 20% DMSO/normal saline and transferred in duplicate to 96-well flat-bottom microplates. Microbial inocula were prepared by diluting suspensions of cells/spores in assay media (RPMI 1640/2% dextrose/MOPS, pH 4.5) (Cellgro, Herndon, VA, USA) for Candida. spp., Sabouraud dextrose (Difco) for C. neoformans., cation-adjusted Mueller-Hinton (Difco BD, Franklin Lakes, NJ, USA), pH 7.3 for MRS, and 5% Alamar blue (BioSource International, Camarillo, CA, USA) in Middlebrook, 7H9 broth (Difco) with OADC enrichment (BBL), pH = 7.3 for M. intracellulare., using OD630 as a reference to afford final target inocula. The microbial inocula were added to the samples to achieve a final volume of 200 µl and final sample concentrations starting with 200 µg/ml. Drug controls [Ciprofloxacin (ICN Biomedicals) for bacteria and amphotericin B (ICN Biomedicals) for fungi] were included as positive controls. All organisms were read at either 630 nm using the EL-340 Biokinetics Reader (Bio-Tek Instruments) or 544ex/590em (M. intracellulare., A. fumigatus.) using the Polarstar Galaxy Plate Reader (BMG LabTechnologies, Offenburg, Germany) prior to and after incubation: Candida. spp. and MRS at 37°C for 18–24 h, C. neoformans. and A. fumigatus. at 30°C for 72 h, and M. intracellulare. at 37°C and 10% CO2 for 72 h. Percent growth was calculated and plotted versus test concentration to afford the IC50.
Results and Discussion
The current study was carried out on the extracts obtained from 10 Centaurea. species growing in Turkey. The three extraction procedures were used in a sequential manner in order to extract nonpolar (n.-hexane), intermediate (chloroform), and polar (methanol) compounds. We have evaluated these extracts for activities against a panel of pathogenic fungi, bacteria, and the protozoa to explore the beneficial effects of these species.
Primary antimalarial evaluation of all the extracts revealed that only the chloroform extract of all parts of C. hierapolitana., which is an endemic species for Turkey, showed 95% growth inhibiton in P. falciparum. D6 clone at a concentration of 15.9 µg/ml. Further evaluation of the chloroform extract of C. hierapolitana. (as shown in ) revealed moderate dose-dependent activity with IC50 values of 7 µg/ml and 7.3 µg/ml against D6 and W2 clones with a selectivity index ranging from >3.4 and >3.3, respectively.
Table 2.. Antiplasmodial activity of C. hierapolitana..
Activity of the extracts against a culture of Leishmania donovani. promastigotes was also tested in vitro.. The highest antileishmanial activiy among the extracts shown in was observed with the chloroform extract of C. hierapolitana. (IC50 = 8.7 µg/ml, IC90 = 17 µg/ml) followed by the chloroform extract of whole parts of C. calolepis. (IC50 = 9.6 µg/ml, IC90 = 17 µg/ml), another endemic plant in Turkey. C. depressa, C. urvillei. ssp. urvillei, C. cyanus., and C. ensiformis. demonstrated no activity at the highest test concentration of 100 µg/ml.
Table 3.. Antileishmanial activity of Centaurea. species.
Extracts obtained from Centaurea. species were also evaluated for their in vitro. antimicrobial activities, and the results are displayed in . It can be seen that six plants had antifungal activity against Candida krusei.. Results indicated that hexane extracts of C. depressa. and C. urvillei. subsp. urvillei. showed moderate antifungal activity against Candida krusei. (IC50 = 15 µg/ml and 45 µg/ml, respectively). As well, the chloroform extract of C. urvillei. subsp. urvillei. showed mild activity against Cryptococcus neoformans. with an IC50 value of 40 µg/ml.Only the hexane extract of C. hierapolitana. showed low activity against methicillin-resistant Staphylococcus aureus. (IC50 = 100 µg/ml). All of the extracts were inactive against Candida albicans., Mycobacterium intracellulare., and Aspergillus fumigatus.. We did not observe any antimicrobial activity for all extracts of C. cyanus., the most common Centaurea. species because of it is pharmaceutical properties, C. cadmea., or the chloroform extract of C. hierapolitana..
Table 4.. Determination of antimicrobial activity of Centaurea. extracts.
It is well-known that flavonoids comprise a large group of naturally occurring, low-molecular-weight substances found practically in all parts of the plants, with a broad spectrum of biological activity within the group (Pathak et al., Citation1991). It is also known that sesquiterpene lactones possess antibacterial, antifungal, antiplasmodial, cytotoxic and anti-tumourgenic properties (Picman, Citation1986). There is evidence in support of sesquiterpene lactones as potential antibacterial and antifungal agents (Neerman, Citation2003). A large number of sesquiterpene lactones are found in members of Asteraceae where they are characteristic constituents of the family (Neerman, Citation2003). Genus Centaurea. has been extensively studied for its sesquiterpene lactones (Janaćković et al., Citation2004; Marco et al., Citation2005) and flavonoid (Akkal et al., Citation2003) content. The possibility that observed activity of plants studied in this work is due to the presence of sesquiterpene lactones and flavonoids cannot be ruled out.
In conclusion, to our knowledge this is the first report of the antiprotozoal and antimicrobial activity of select Centaurea. species. Thin-layer chromatography (TLC) plates have been run verifying that every solvent extract produced in the laboratory has a number of constituents, and phytochemical studies of some of the active plants are currently in progress in order to isolate pure compounds.
Acknowledgments
We thank Prof. Dr. Özcan Seçmen and Serdar Şenol for their valuable assistance for collection and identification of the plant material and Ms. M. Wright, Mr. J. Trott, and Ms. Mahita Orungati for technical assistance in antimicrobial, antimalarial, and antileishmanial testing, respectively. This research was partly supported by TUBITAK (The Scientific and Technical Council of Turkey) with NATO-B1 postdoctoral scholarship and by the United States Department of Agriculture, Agricultural Research Service Specific Cooperative Agreement No. 58-6408-2-0009. The antimicrobial assays were supported by NIH/NIAID grant Al 27094.
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