Chemical Composition, Antibacterial and Antimutagenic Activities of Essential Oil from (Tunisian) Cyperus rotundus
Abstract
Essential oil from the tubers of Cyperus ratundus, obtained by steam distillation, was analyzed by GC and GC/MS. In total, 33 compounds were identified. The oil was characterized by its high content of sesquiterpenes with cyperene (30.9%) being major. The antibacterial activity of oil from tubers of Cyperus rotundus, showed more important activity against Gram-positive bacteria specially Staphylococcus aureus than Gram-negative bacteria. The antimutagenic activity was tested by the “SOS Chromotest” and the “Ames” test. C. rotundas oil acted as an antimutagen against Aflatoxin B1 in both Salmonella strains (TA100 and TA98) and Escherichia coli strain (PQ37) and against nifuroxazide in Escherichia coli strain (PQ37), where its mutagenicity is not expressed. The highest rates of AFB1 mutagenesis inhibition tested by Ames assay, ranged from about 82.56% for TA100 strain to 85.47% for TA98 strain at the same dose of 50 µg AFB1 per plate. Whereas, the mutagenic effect of respectively nifuroxazide and AFBl (50 µg/assay) were reduced by aproximately 58.19% and 81.67% when tested by the SOS chromotest assay.
Key Word Index
Cyperus wtundus, Cyperaceae, essential oil composition, antibacterial activity, mutagenicity, antimutagenicity, SOS chromotest, Ames test.
Introduction
Cyperus rotuncliis L., a sedge of the family Cyperaceae, was collected in the region of Monastir situated in the center of Tunisia in June 2003. It is widely distributed in the Mediterranean basin areas. This plant, which grows naturally in tropical, subtropical and temperate regions, is widespread in northeast, center and south (Gabes) of Tunisia (1).
The tuberal part of C, rotuncliis is one of the oldest known medicinal plants used for the treatment of dysmenorrhoea and menstrual irregularities. It was also used as analgesic, sedative, antispasmodic and to relieve diarrhea (2). Cyperus rotundus has been widely investigated by several authors (3,4). It is a medicinal plant appearing among Indian, Chinese and Japanese traditional drugs that are used against spasms, stomach disorders and anti-inflammatory diseases (5,6).
Other pharmacological investigations indicated that C. rotundus had remarkable hypotensive, anti-inflammatory and antipyretic effects. Previous phytochemical studies showed that the major chemical components of this herb were essential oil, flavonoids, sesquiterpenes and cardiac glycosides (7).
In this study, we report on the composition analysis of essential oil from C. rotundus tubers, its antibacterial and antimutagenic activities using two tests of genotoxicity: the “SOS chromotest” and the “Ames test.”
The Aines test was further more chosen as it is a wellknown reference test that proved suitable in many similar investigations (8-11). Whereas the SOS chromotest is a widely used assay for genotoxic/antigenotoxic activity (12) that has been used for testing extracts from medicinal plants (13,14). The SOS chromotest and the Ames test detect genotoxicity by different mechanisms and thus complement each other.
Experimental
Chemicals: O-Nitrophenyl-β-D-galactopyranoside (ONPG) and P-nitrophenyl phosphate (PNPP) were purchased from Sigma, Germany. The positive mutagens Aflatoxin B1 (AFB1) and nifuroxazide were purchased from Sigma, Aldrich USA.
Plant material: Tubers parts of C. rotundas were harvested in the region of Monastir in the center of Tunisia, in June 2003. Botanical identification was carried out by Pr Chaieb (Department of Botany, faculty of Sciences, University of Sfax, Tunisia), according to the flora of Tunisia (1). A voucher specimen (CP-06.03) has been deposited in the Herbarium of the Laboratory of Pharmacognosy, Faculty of Pharmacy of Monastir, Tunisia.
Oil isolation: The oil was isolated from 100 g of the tubers parts by hydrodistillation for 2 h in 500 mL of distilled water, using the apparatus described in the 9th edition of the French Pharmacopeia cited by Bruneton (15). They were kept in vials covered with aluminium foil at 4°C (to prevent the negative effect of light, especially direct sunlight).
Oil analysis: The composition of the oil was investigated by GC and GC/MS. GC analysis was performed with chromatograph HP5890 equipped with a flame ionization detector (HP5 30 m x 0.25 mm fused silica capillary column, film thickness 0.25 µm). Injector and detector temperature were set at 240°C and 280°C, respectively. The oven temperature program was 50°C for 3 min, then 50°-280°C at 9°C /min, and 280°C for 3 min. Nitrogen was employed as carrier gas at a flow rate of 1 mL/min. The volume injected was 0.1 µL, of oil diluted in hexane (10%). The percentage composition of the oils was recorded from the GC peak areas without using any correction factors.
The sample was analyzed by GC/MS using a HP5972/A mass spectrometer recording at 70 eV at the same conditions as described above in GC analysis. The identification of compounds was performed by comparison of retention indexes (determined relatively to the retention times of a series of n-alkanes) with those of authentic standards of a mass spectra library (Wiley 275.1) (16).
Bacterial strains: The following bacteria were used: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076, and Salmonella typhimurium NRRLB 4420.
Esherichia coli PQ37 strain was kindly provided by Pr.Quillardet, (Institut Pasteur of Paris, France). The complete genotype, as well as strain construction details are described by Quillardet et Hofnung (17). Frozen permanent copies of the tester strain were prepared and stored at -80°C.
Histidine-dependent strains, Salmonella typhimurium TA100 and TA98, were provided by Pr Quillardet (Institut Pasteur of Paris, France) and maintained as described by Maron et Ames (18). Strain TA98 allows detection of frameshift mutations. This strain contains the plasmide pKM 101 and the products of the muc AB genes on this plasmid enhance SOS mutagenesis, thereby making strain TA98 more responsive to certain mutagens as AFB1. Whereas, TA100 strain is the most sensitive of all of the Salmonella typhimurium tester strains (19) particulary against AFB1. The genotypes of the test strains were checked routinely for their histidine requirement, deep rough (rfa) character, UV sensitivity (uvr B mutation) and presence of the R factor. They were stored at -80°C.
S9 fraction and S9 mix: The S9 microsome fraction is prepared from rats treated with Aroclor 1254 (18). The composition of the activation mixture for SOS chromotest was the following per 10 mL of S9 mix: salt solution (1.65 M KCl, 0.4 M MgCl^sub 2^ 6H^sub 2^O) 0.2 mL; Glucose-6-Phosphate (1M) 0.05 mL; NADP (0.1 M) 0.15 mL; Tris buffer TRIS (0.4 M pH 7.4) 2.5 mL; L medium (Bacto tryptone 10 g, Bacto yeast extract 5 g, NaCl 10 g/L of distilled water) 6.1 mL; S9 1 mL (17).
The components of S9 mix for Ames test were 0.4M MgCl^sub 2^, 1.65 M KCl, 1 M Glucose-6-Phosphate, 0.1 M NADP, 0.2 M sodium phosphate buffer, pH 7.4, and S9 at a concentration of 0.04 mL/mL of mix. The S9 mix was prepared freshly for each assay (18).
Antibacterial assay: Antibacterial activity of the oil was determined using the microdilution agar technique (20), with some modifications; the emulsion of the oil in agar 1% was obtained by sonication of the mixture at 7 volts power. Overnight grown bacterial suspensions were standardized to approximately 105 cells/mL (21). Ninety-six well-microtiter plates were used. In each well, 100 µL of microbial suspension was added to 100 μL of the test oil dilution. The last row containing only agar and bacterial suspension served as a positive growth control. After incubation at 37°C for 24 h with shaking, the first well without turbidity was determined as the minimal inhibition concentration (CMI) (22, 23).
SOS chromotest assay: The SOS chromotest assay is a bacterial test for detecting DNA-damaging agents. It involves a set of functions known as the SOS responses. We have taken advantage of an operon fusion placing lacZ, the structural gene for β-galactosidase, under control of the SfiA gene, an SOS function involved in cell division inhibition, to devise a simple and direct colorimetric assay of the SOS response to DNA damage (12). It is also constitutive for alkaline phosphatase (AP) synthesis.
An overnight culture of E.coli PQ37 (100 µL) was added to 5 mL of fresh medium and incubated for 2 h at 37°C. One mL of this culture approximate cell density 2×10^sup 8^ cell/mL was diluted with 9 mL of nutrient broth or 9 mL of S9 mix. A fraction of 0.6 mL was transferred into a series of glass test tubes, each containing 20 µL gradual dilutions of the compound to be tested. The mixtures were incubated with shaking for 2 h at 37°C. After this, two equal sets of tubes were filled with 0.3 mL of each incubated mixture.
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