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ANTIMITOTIC AND GENOTOXICITY EFFECTS OF WILD LIBYAN ARTICHOKE Cynara cornigera LEAVES AQUEOUS EXTRACT

Allium cepa root tip cells were used to find out the effect of Cynara cornigera extract on chromosomal structure and behavior during cell division. To measure the mitotic index and chromosome aberration in treated cells. The root meristem cells were treated with different concentrations of Cynara cornigera leaves aqueous extract (0.1, 0.01 and 0.001 mg/ml) for 4, 6 and 12 hours, respectively. The results indicate that aqueous extracts of C. cornigera plant have the ability to decrease the (MI) values with increasing the concentration at (P<0.005). The treatments have caused different kinds of mitotic abnormalities and chromosomal aberrations, such as: Change percentage of mitotic phases, C-metaphase, Stickiness, Disturbed nuclear membrane, Highly condensation of prophase and Accumulation of cell in interphase.


INTRODUCTION
According to the World Health Organization (WHO), about 80% of developing countries use traditional folk medicines, 85% of which are plant extracts. Furthermore, the international center of commerce reported that medicinal plants are still vitally important in the preparation of pharmaceutical products (Fransworth, 1994;Nune and Caravalho, 2003).
Artichoke has been known since the 4 th century B.C. as a food and remedy. This plant was appreciated by the ancient Egyptians, Greeks, and Romans, who used it both as a food and as a medicine (for their beneficial effects against hepatobiliary diseases and as a digestive aid) (Marzi et al., 1975;Sonnante et al., 2002).
In Libya, 4 species of Artichoke are reported and they include Cynara cornigera, Cynara cadunculus, Cynara scolymus and the endemic Cynara cyrenaica (Jafri and El-gadi, 1983). Cynara cornigera is dwarf plant, 10-20 (30) cm and it is flowering in Spring (Plate 1). This plant is distributed in the eastern part of Libya and using widely in folk medicine. Shortage and no enough information about its effect on human use was led to shade some light on this plant. There for, this study was aimed and designed to evaluate the antimitotic and antimutagenic activities of aqueous extract of the leaves of C. cornigera on A. cepa root tip cells to find out the effect of C. cornigera on chromosomal structure and behavior during cell division by studying the mitotic index (MI) in the treated cells.

MATERIALS AND METHODS
Plant material: C. cornigera plants were collected from the borders of Benghazi city and identified according to the mentioned references, beside consultation of the Faculty of Science, Benghazi University, Libya. leaves were washed with distilled water, dried with filter paper in dark place. they were grind by electric blender and were stored as powder in dark flask until used. Commercial variety bulbs of the common onion (Allium cepa) were selected in shape and purchased from local market for plant bioassay.
Aqueous extract: 5g of C. cornigera powder was added to 100 ml boiled distilled water after cooling to 60 C°. the content of the flask was shaken and left at room temperature (Tawab et al,2004). A buchner funnel lined with whatman No.1 filter paper was employed to filtrate the content of the flask. The filtrate was used as stock solution and three different concentration (0.1, 0.01 and 0.001 mg/ml) were prepared by serial dilution method (Jain and Sethi, 1991;Eltorki, 2006;Iranbakhsh et al., 2010).

Procedure of plant chromosomes technique:
The preparation of plant chromosome achieved using standard methods described by (Grant, 1982), with minor modification. Onion bulb germination and treatment: Onion bulbs were grown in distilled water at room temperature (25±2 C°) in the darkness. They were placed in small (50 ml ) glass cups containing distilled water for approximately 3 to 4 days, when the primary root tips were 0.5-1.5 cm long, the bulbs were transferred to clean and dried containers included the treatments with a series of concentration. In this experiment two groups were used. group 1 included negative control (distilled water) and positive control treated only with colchicines solution 0.05%. In treated samples 5 ml of colchicines solution 0.05mg/ml were added to each treatment for 3 hours prior to harvesting except the negative control. The group 2 treatment with series of concentration stock solution, 0.1, 0.01and 0.001 for 4, 6 and 12 h. The controls were treated with distilled water only. Ten root tips were cut and examined for each treatment, the experiment repeated for five times. Fixation and slide preparation: For cytological preparation, the root tips were fixed in 95% ethanol /glacial acetic acid 3:1 (v/v) from 1 to 24 hrs. at 5 C°. The technique which use is described by Fiskesjö (1994). Slides were prepared using the aceto-orcein squash technique, by hydrolyzing the root tips in 1N HCL at 60 C° for 12 min. and stained with acetoorcein stain for 45min. The meristematic region of the root was removed and washed with 45% glacial acetic acid on glass slide and squashed with flattened glass rod in Glycerol and mounted with cover slip and temporary sealed with clear finger nail polish. Slides from each treatment examined by light microscope under 40x and 100x to find out the mitotic aberrations. The mitotic index MI was calculated as the percentage of dividing cells to the total number of cells examined. Abnormality cells was calculated as the percentage of abnormal cells to the total number of cells examined. Data analysis: The data of mitotic index MI and Cmetaphase of plant chromosomes technique were analyzed by one way analysis of variance (ANOVA), Multiple comparisons were performed by least significance (LSD) using statistical package for social science (SPSS) with significance level less than 0.05 (P<0.05).

Antioxidant activity of C. cornigera leaves aqueous extracts
The effects of C. cornigera aqueous extract on mitotic index (MI) and c-metaphase of A. cepa root tip cells at different exposure time intervals are illustrated in table (1). The obtained data showed significant differences between groups and within group (p<0.005). The results of short hours treatments (4 hours) showed significant differences, decrease in MI by decreasing the plant extract concentration compared to (negative and positive) control. Lowest mitotic index values were in samples treated with 0. 001 plant extract, its value was 7.49% compared to the negative control which was 45.60%, the low concentration of 0.001 had more inhibition effect on MI than 0.1 plant extract concentration but close to 0.01 concentration action. The lowest number of cmetaphase cells (2.00) obtained in samples treated with 0.01 plant extract for four hours (4 hours) compared to the positive control which was (26.33) cells in c-metaphase.  The statistical analysis revealed no significant differences between groups and within groups total cells (TC) at time intervals. But there were significant differences between groups and within groups of dividing cells (DC) at 4 hours treatment with different plant extract concentrations (P= 0.019). There were highly significant inhibitory effects on dividing cells in samples treated with different plant extract concentrations compared to positive and negative control between groups and within groups (P= 0.003) by ANOVA analysis at time of 6 hours.
Table (1) showed a decline in C-metaphase cells at 0.1mg/ml concentration which was 1.33cells in cells treated to 6 hours compared to positive control which was 35.33 cells. By increasing the time to 12 hours, there were significant differences (P= 0.012) between groups within groups on c-metaphase at different concentration (p<0.05), but there were not significant differences between groups within groups on dividing cells (P=0.193). ANOVA test indicated significant effect (p<0.05) of plant extract on number of c-metaphase in treated sample with all concentrations compared to (positive and negative) control at all-time treatments, also it indicated a significant effect (p<0.05) of plant extract on M.I.% in treated samples compared to (positive and negative) control at (4 and 6) hours of time treatment.

Genotoxicity of Cynara cornigera leaves aqueous extract
The treatment of A. cepa root tip cells with different concentration of C. cornigera crude water extract (0.1, 0.01 and 0.001 mg/ml), for different exposure times (4, 6 and 12), the resulted data were presented in table (2).
No abnormal cells were observed at negative control treatment samples at all applied interval times. While at high concentration (positive control)   (2) and (3) and chromosomal stickiness at metaphase is more observed in all treatments.  Disturbed nuclear shape (stock solution at 12hr).

DISCUSSION
A. cepa root tip meristems have been widely used for evaluation of cytotoxic and antimitotic activity of various compounds (Fiskesjo, 1988;Soliman andGhoneam, 2004 andSehgel et al. , 2006). Such mitotic inhibition could result of the inhibition of DNA synthesis which is considered as one of major perquisites for a cell to divide (El Garabulli and Mohamed, 2007). However the reduction of mitotic activity after treatment could be associated with a reduction in the amount of both DNA and RNA (Badr, 1986;Soliman and Ghonium, 2004). Many other investigators attributed the depression in mitotic index values to the inhibition of protein synthesis, or due to inhibition of certain types of nuclear proteins essential in mitotic cycle (Haliem, respectively. The percentage of chromosomal abnormalities decreased at lower extract concentration but it was found to be increased with high concentration. Such results agree with those obtained by Kaushik et al., (1993), who tested water extract of Datura stramonium on A. cepa root tip cells. They found that the mitodepressive effect of plant extract were increased with increase the concentration and exposure time.
The present experiment also showed that The mitotic index inhibition of root tips of A. cepa L. at different concentrations and exposure period times, indicated that C. cornigera plant has positive antimitotic effects. The data obtained from this study suggested that using extracts of C. cornigera plant is a good and safe method and can be used as antioxidant and anticancer agent. Therefore, more investigation using molecular methods most take in account with more attention.