Cyperus rotundus aerial parts were collected from the region of Monastir, located in the Center of Tunisia, in October 2008. The botanical identification was carried out by Prof. Med Chaieb (Department of Botany, Faculty of Sciences, University of Sfax, Tunisia), according to the flora of Tunisia . A voucher specimen (Cp.10-08) is kept in the laboratory of Pharmacognosy, Faculty of Pharmacy of Monastir, for future references.
Preparation of plant extracts
The fresh aerial parts of Cyperus rotundus were dried at room temperature and reduced to coarse powder. 100 g of the powdered leaves were boiled in water for 15 to 20 min. The crude extract obtained was filtered and lyophilized (aqueous extract). The residue was dissolved in water.
In order to obtain an extract enriched with Total Oligomer Flavonoids (TOF), 100 g of powder were macerated in water/acetone mixture (1v/2v) for 24 hours with continuous stirring. The extract was filtered and the acetone was evaporated under low pressure in order to obtain an aqueous phase. The tannins were partially removed by precipitation with an excess of NaCl for 24 hours at 5°C, then we recovered the supernatant. The latter was extracted with ethyl acetate, concentrated and precipitated with an excess of chloroform. The precipitate was separated and yielded TOF enriched extract which was dissolved in water.
Ethyl acetate and methanol extracts were obtained by soxhlet extraction (6 h). The two types of extracts, with different polarities, were concentrated to dryness and the residue was kept at 4°C. These two extracts were resuspended in DMSO.
Quantitative analysis of extracts
Determination of total polyphenol and flavonoid content
The polyphenol content of Cyperus rotundus extracts was quantified by the Folin-Ciocalteau reagent [11, 12]. Aliquots of test samples (100 μl) were mixed with 2.0 ml of 2% Na2CO3 and incubated at room temperature for 2 min. After the addition of 100 μl 50% Folin-Ciocalteau phenol reagent, the reaction tube was further incubated for 30 min at room temperature, and finally absorbance was read at 720 nm. Gallic acid (0.2 mg/ml) was used as a standard. A known volume of each extract was placed in a 10 ml volumetric flask to estimate flavonoid content according to the modified method of Zhishen et al. . Distilled water was added to make the volume 5 ml, and then 0.3 ml NaNO2 aliquot (1:20 w/v) was added to this dilution. Three milliliters of AlCl3 (1:10 w/v) were added 5 min later. After a 6- min incubation, 2 ml of 1 N NaOH was added to the mixture and the total absorbance was measured at 510 nm . Quercetin was used as standard for constructing a calibration curve.
Determination of tannin content
The method described by Pearson  was used for the determination of tannin content of the samples. The extraction of tannin in the sample was achieved by dissolving 5 g of the sample in 50 ml of distilled water in a conical flask, allowing the mixture to stand for 30 min with shaking of the flask at 10-min intervals, and then centrifuging at 5000 rpm for 15 min to obtain a supernatant (tannin extract). The extract was diluted to 100 ml in a standard flask using distilled water. Five milliliters of the diluted extract and 5 ml of standard tannic acid (0.1 g/L) were measured into different 50-ml volumetric flasks. One milliliter of Folin- Denis reagent was added to each flask, followed by 2.5 ml of saturated sodium carbonate solution. The solutions were made up to the 50-ml mark with distilled water and incubated at room temperature for 90 min . The absorption of these solutions was measured against that of the reagent blank (containing only 5 ml of distilled water) in a Beckmann spectrophotometer at 760 nm wavelength. Tannin content (tannic acid equivalents) was calculated in triplicate and the concentration of tannin in the extract was determined using pure tannic acid as standard.
Balb/c mice (6–8 week-old males, 20–30 g) were obtained from the Pasteur Institute (Tunis, Tunisia). The animals were housed in polypropylene cages with stainless steel grill tops and provided with bedding of clean paddy husk. The animals were acclimatized to laboratory conditions for one week prior to treatment. The temperature in the animal room was maintained between 25 ± 2°C with a relative humidity of 30–70% and illumination cycle set to 12 h light and 12 h dark. The mice were fed with standard laboratory pelleted feed. All animal experiments were performed in accordance with the guidelines for the care and use of laboratory animals published by the US National Institutes of Health. The study protocol was approved by the Ethics Committee of the University Hospital Fattouma-Bourguiba of Monastir, Tunisia.
Mice were randomly divided into groups of 5 animals and treated by i.p. injection: a first control group was given solvent Tween-80 (1%) and a second control group was treated with Tween-80 (5%). In fact, aqueous, methanol and TOF-enriched extracts were dissolved in Tween-80 (1%), whereas ethyl acetate extract was dissolved in Tween-80 (5%). The other groups of animals were treated by different doses of extracts, ranging from 125 mg/kg to 1 g/kg body weights. The number of dead animals was followed every day for one week after treatment.
Analgesic study: acetic acid-induced writhing assay
The analgesic effect was tested according to the method described by Shibata et al. . The abdomen writhing is a model of visceral pain and it was produced by i.p. injection of 1% (v/v) acetic acid solution (volume of injection 0.1 ml/10 g b.w.) to each mouse, 30 min after the administration of the the aqueous, the TOF-enriched extract, ethyl acetate and methanol extracts (50, 150, 300 mg/kg, b.w, respectively). Tween-80 (1%, v/v) was used as negative control and diclofenac sodium was used as positive control (100 mg/kg, b.w.). After the immediate injection of acetic acid, each mouse was isolated in an individual observation box and the number of writhes produced in these animals was counted for 15 min.
The percentage analgesic activity was calculated as follows:
Where N is the average number of stretching of control animals per group
And Nt is the average number of stretching of treated animals per group.
Anti-inflammatory study: xylene-induced ear oedema
The mice were divided into groups of five. Thirty minutes after i.p. injection of the extract (50 to 300 mg/kg, b.w.) or dexamethasone (positive control) (300 mg/kg, b.w.), 0.03 ml of xylene was applied to the anterior and posterior surfaces of the right ear. The left ear was considered as control. Two hours after xylene application, the mice were sacrificed and both ears were removed. Circular sections were taken using a cork borer with a diameter of 7 mm, and were later weighed. The increase in weight caused by the irritant was measured by subtracting the weight of the untreated left ear section from that of the treated right ear sections. The oedema was quantified as the weight difference between the two earplugs. The anti-inflammatory activity was evaluated as percent oedema reduction/inhibition in the treated animals relative to the control animals [17, 18] using the relation:
Where R t = mean weight of the right ear plug of the treated animals; Lt = mean weight of the left ear plug of the treated animals; Rc = mean weight of the right ear plug of the control animals; Lc = mean weight of the left earplug of the control animals.
Evaluation of lipid peroxidation by the thiobarbituric acid (TBA) assay
TBA reacts with malondialdehyde (MDA) to form a diadduct, pink chromogen, which can be detected spectrophotometrically at 532 nm . Normal male mice were used for the preparation of liver homogenate. The perfused liver was isolated, and 10% (w/v) homogenate was prepared with homogenizer at 0-4°C with 0.15 M KCl. The homogenate was centrifuged at 800 rpm for 15 min and clear cell-free supernatant was used for the study of in vitro lipid peroxidation. One ml of 0.15 M KCl, 0.1 ml of rat liver homogenate, 100 μl of ascorbic acid (0.5 mM) and 0.2 ml of each extract concentration (1 to 4 mg/ml) were mixed and the peroxidation reaction was initiated by adding 100 μl of 15 mM FeSO4. After the incubation at 37°C for 1 h, the reaction was stopped by adding 500 μl of trichloroacetic acid (TCA) (28%, w/v) and 380 μl TBA (2%, w/v). This final mixture was heated in a water bath for 20 min at 80°C, then it was cooled, centrifuged and the absorbance of the supernatant was measured at 532 nm. The inhibition percentage of lipid peroxidation was calculated by comparing the results of lipid peroxidation obtained with liver homogenates treated with FeSO4 and extracts to those of controls treated only with FeSO4, by using the following formula:
Cell preparation from mice
Spleen Balb/c lymphocytes were obtained as previously reported . Briefly, 8-week-old male Balb/c mice were killed by cervical dislocation. The spleen was isolated, aseptically removed and homogenized by mincing with a sterile forceps. The splenocytes were harvested by centrifugation (1500 rpm, for 10 min), and red blood cells were lysed with a lysis buffer (144 mM NH4Cl, 1.7 mM Tris Base). The cells were washed twice with phosphate-buffered saline (PBS), resuspended in complete RPMI medium and adjusted to 5×106cell/ml. The cell incubation was conducted at 37°C, in 5% CO2 enriched atmosphere.
Cell proliferation assay
The evaluation of lymphocyte proliferation was carried out by using the MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] coloration method . One hundred microliter of splenocyte suspension (3×106 cells/ml) in a 96-well plate were preincubated in RPMI 1640 medium (Control) for 24 h before the addition of mitogens (LPS and lectin at 5 μg/ml), alone or with increasing concentrations of each extract (1, 0.1, 0.01, 0.001 mg/ml) solubilized in RPMI. The cells were incubated at 37°C in humidified 5% CO2 atmosphere for additionally 24 h. After centrifugation at 1500 rpm for 10 min, the cell pellet was re-suspended in 50 μl of MTT (5 mg/ml) in RPMI media and incubated for 4 h at 37°C in humidified 5% CO2 atmosphere. The culture medium was removed after centrifugation and dimethylsulfoxide was added. The absorbance was measured at 570 nm.
The percentage of proliferation was calculated by the following equation:
Chromosome aberrations assay
The aim of this experiment was to investigate if C. rotundus extracts exhibit a genotoxic effect, using animal models. Blab/c mice were used (average body weight 20 ± 0.3 g; age: 6 weeks old). These mice were given a standard granulated food and drinking water, and were divided into four groups with 5 mice each, as follows:
Group 1: Mice given Tween-80 (1%).
Group 2: Mice given Tween-80 (5%).
Group 3: Mice given 40 mg/kg b.w. of methyl methane sulfonate (MMS), as positive control.
Group 4: Mice given 300 mg/kg b.w. of Cyperus rotundus extracts.
The animals within the different treatment groups were given a subcutaneous injection of yeast suspension, dissolved in water (20 mg/ml), to accelerate the mitosis of bone marrow cells . Ten minutes later, they received a single intraperitoneal (I/P) injection of the tested substance solution (200 μl of MMS, Cyperus rotundus extracts or Tween-80). Twenty-four hours later, the different animals were sacrificed by cervical dislocation. 200 μl of vinblastin (250 μg/ml) were injected (I/P injection) to animals 45 minutes before they were sacrificed in order to block the dividing cells in metaphase. The cells of the bone marrow were collected from femurs and tibias, shielded with a hypotonic shock solution (KCl 0.075 M), then harvested in the presence of a methanol–acetic acid mixture 3/1 (v/v) (3 repetitions) by centrifugation at 1200 rpm for 10 minutes, according to the technique described by Evans et al. . The cells were spread on glass slides that were blazed on a flame for 5 s, then air-dried for conservation at room temperature and finally stained with a 4% (v/v) Giemsa solution in water, for 15 min . After coding slides, the chromosomes of 100 cells blocked in metaphase were examined for chromosome abnormalities at a magnification of 100 × using an optical microscope (Olympus, France). Three replicates (300 metaphases per dose level) for each controls and treated groups were conducted. The chromosome aberrations were identified according to the criteria described by Savage . The metaphases with chromosome breaks, gaps, rings and centric fusions were recorded and expressed as percentage of total metaphases per group.
Data are expressed as mean ± SD (Standard Deviation). The biological activity was examined in three individual experiments, performed in triplicate for each dose. The statistical significance was determined using the Student’s t-test. P<0.05 was considered as indicative of significance as compared to the control group.