Introduction

In the last few years, several vegetable products capable of modifying the activity of mutagens and carcinogens in various test systems have been identified; these include extracts of plants as well as plant derived products: pigments, vitamins, carotenes, phenolic, lactose, flavonoids and tannins (Dhir et al., 1993; Cozzi et al., 1997 and Lanar et al., 1999).

To date, a rather limited number of plants particularly of medicinal relevance have been screened for possible cytogenetic and genotoxic properties. Additional studies are needed to evaluate the possible antimutagenic, anticarcenogenic, and/or anticarcenogenic properties of plants and their derivatives.

Ginger (Zingiber officinale Roscoe, Zingiberaceae) is among the most frequently and heavily consumed dietary condiments throughout the world. Plants of ginger family have been widely used as spices and also traditional medicine. Ginger has been used extensively for more than 2500 years in China for conditions including headaches, nausea and colds (Grand and Lutz, 2000 and Dedov et al., 2002). Ginger extract failed to induce any mutagenicity (Ungsurungsie and Suthienkul, 1982), and even suppressed the genotoxicity of several carcinogens in bacterial and mammalian cells (Tarjan and Csukas, 1989).

The present study carried out in the framework of wider toxicological investigations, were designed to test the genotoxic and anti-mutagenic potential of tenoxicam and ginger plant extract. Cytogenetic test was performed on root tips of Vicia faba in order to study tenoxicam and ginger effects on chromosome aberrations as well as its genotoxic and/or anti-mutagenic action.

Materials and Methods

Chemicals

– Tenoxicam (C13H11N3O4S2) non-steroidal anti-inflammatory drug from F. Ttoffmann-LaRoche Ltd. Basel, Switzerland.

– Ginger plant extract from Kahira Pharmaceuticals and Chemical Industries Company, Egypt.

Cytogenetic assays

The cytogenetic Vicia test is recommended for detection of genotoxicity effects of environmental chemicals in plants by (Kanaya et al., 1994). It enables the simultaneous evaluation of numeric and structural chromosome changes. Vicia faba seeds were obtained from local nurseries. The roots were exposed for 6, 12, and 24 hours to 10, 20 and 40 mg/L of tenoxicam and ginger plant extract singly and combined.

Root meristems were examined at various intervals by thoroughly washing root tips and fixing them in carryon fixative (1:3 – ethanol:glacial acetic acid) for 24 hours followed by washing in 70 % ethanol. Then, staining in acetocarmine and preserving them in 70 % ethanol. The root tips were squashed in 45 % acetic acid after macerating them in 1 N HCl for 5 – 10 minutes at maintained temperature of about 60 ^oC (Dyer, 1979).

Cells were screened under a light microscope for mitotic phase and chromosomal aberrations. The mitotic index was expressed as the percentage of the number of divided cells to the total number of cells examined. The total number of chromosomal aberration was estimated in dividing cells. The abnormalities included cells with stickiness, fragments, breakage, brigdes and micronuclei.

The collected data of chromosomal aberrations were statistical analysis using (SPSS program).

Results and Discussion

In the present work we have characterized two properties of ginger extract, genotoxicity and antimutagenicity. Ginger (Zingiber officinale roscoe, Zingiber aceae) is among the most frequently and heavily consumed dietary condiments throughout the world. Besides its extensive use as a spice, the rhizome of ginger has also been used in traditional oriental herbal medicine for the management of such symptoms as common cold, digestive disorders, rheumatism, neurologia, colic and motion – sickness (Mascolo et al., 1989; Mustafa et al., 1993 and Surh, 1999).

Table (1) shows the types and number of chromosomal aberrations obtained by exposing root tips cells of Vicia faba. We found that ginger extract did not influence mitotic activity of Vicia faba and did not significantly increase the frequency of chromosomal aberrations compared to control (in the three concentrations used for three different times). Similar results in other organisms have been reported by (Ungsurungsie and Suthienkul, 1982; Tarjan and Csukas, 1989; Surh et al., 1998; Surh, 2000; Someya et al., 2003; Bhandari et al., 2005 and White, 2007).

Table (1): Percentage of chromosomal aberrations induced by ginger plant extract in root meristams of Vicia faba.

In other studies, however, the ginger extracts were found to induced chromosome breakage and other aberrations in root tip cells of onion (Abraham et al., 1976).

Nakamura and Yamamoto (1982) reported the mutagenicity of gingerol in Escherichia Coli B/r. The aliphatic chain moiety containing a hydroxyl group was proposed as an active part of gingerol responsible for its pungent constituent’s gingerol and shogaol were tested in Salmonella typhimurium TA100 and TA1535. The gingerol and shogoal induced His⁺ reversion in these bacteria in the presence of rat S-9 mix. (Nagabhushan et al., 1987). The ethanol extract of ginger was mutagenic in S. typhimurium TA98 and TA102 without metabolic activation (Mahmoud et al., 1992).

Tenoxicam is one of the oxicams a special group of non-steroidal anti-inflammatory drugs. Tenoxicam is a potent inhibitor of prostaglandin biosynthesis (Dammann, 1999). It is widely used in the treatment of arthritis and pain (Vilegas et al., 2002). Table (2) shows the significant increase of chromosomal aberrations rates was scored after treatment with the same three different concentrations and three different times of tenoxicam.

Table 2: Percentage of chromosomal aberrations induced by tenoxicam drug in root meristams of Vicia faba.

Also, genotoxic effects (mitotic index reduction and cell growth inhibition) were observed after treatment with tenoxicam concentrations above 40 mg/L.

This is agreement with the genotoxicity found of piroxicam (the same chemical class as Tenoxicam) in Saccharomyces cerevisia (Badawy and Ali, 2000). Also, Giri and Mu Khopadhyay (1998) tested in vivo sister chromatid exchange (SCE) in bone marrow cells of mice for four pyrazolone derivatives (anti-inflammatory drugs) and they found that all four drugs showed a statistically significant increase in SCE in bone marrow cells when compared with control.

Moreover, an investigation was undertaken to determine anti-inflammatory drugs (4 compounds) had mutagenicity. Kuboyama and Fujii (1992) found that the (4 compounds) of anti-inflammatory drugs showed a DNA – damaging tendency.

On the other hand, many tests have been carried out on anti-inflammatory drugs about their safety. Kadotani et al., 1984 they were examined the tenoxicam drug by using in vitro bacterial systems (repair test and reversion test) and it was not mutagenic. And, Kullich et al., 1990 reported that tenoxicam and lornoxicam showed no influence on the SCE frequencies in therapeutic dosages in bone marrow cells of mice. However, Philipose et al., 1997 tested three anti-inflammatory drugs ibuprofen, ketoprofen and naproxen in the Ames mutagenicity assay and in vivo genotoxicity was tested by SCE in bone marrow cells of mice. Results showed no mutagenic effects in both testes.

Recently, there is interest in the development of chemoprevention agents against environmental mutagens. Natural products and naturally derived compounds from plants may have applications in controlling mutagenicity of some drugs. The protective action of fruits and vegetables has been attributed to the presence of anti-oxidants (Katt and Kushad, 2000; Prior and Cao, 2000 and Kaur and Kapoor, 2002).

Also, some flavonoids (a group of natural products) are able to cause DNA damage (Said et al., 1992 and Yamashita et al., 1999). The aim of this investigation was to determine if extract of ginger reduce the genotoxic damage induced by tenoxicam drug using the cytogenetic test (chromosomal aberrations and mitotic index) in root tips of Vicia faba plant.

From our results ginger plant extract failed to induce any anti-mutagenic effect of tenoxicam drug in Vicia faba.

Combined treatment with ginger plant extract and tenoxicam drug significantly increase,

Table (3) showed that the combined treatment with ginger plant extract and tenoxicam drug significantly increases. The combination effect did not bring about antagonistic effect in comparison with the single treatment by tenoxicam alone but it showed synergistic effect. Ginger extract may have pro-oxidant actions. These results demonstrated that the pro-oxidant properties of ginger extract which are generally considered to be anti-oxidant may be involved with their mutagenic activities in combined treatment. These, results are in agreement with those reported by Yamashita et al. (1999) who fount that quartering induced extensive DNA damage via reacting with Cu (II).

Table 3: Percentage of chromosomal aberrations induced by the combined treatments of ginger and tenoxicam in root meristams of Vicia faba.

Addition, Soudamini et al., 1995 found that spices like pepper, pippali, ginger and mustard increased the number of revertants in Salmonella Typhimurium strains TA100 and TA1535. However, treating root meristems of Vicia faba with aqueous extract of black pepper was fount to give clastogenic effects (Abraham and John, 1989). Furthermore, ginger extract were found to induce chromosome breakage and other aberrations in root tip cells of onion (Abraham et al., 1976).

From mitotic index data (Table 4) it is evident that ginger plant extract did not influence mitotic activity of Vicia faba. But tenoxicam singly or combined exhibited mitotic activity and the percentage of mitotic index decreased at highest concentration.

Table 4: Mitotic index in root meristams of Vicia faba treated by tenoxicam, ginger plant extract and Combination of them.

The cytogenetic Vicia faba test documented that tenoxicam single or combined with ginger plant extract exhibited the clastogenic effect on Vicia faba root tip meristams.

Finally, we conclude that ginger extract even though it showed genotoxic activity at combination with tenoxicam drugs is not a genotoxicant alone in root tips of Vicia faba and suggest that it is advisable to extend studies on this matter using other biological models.

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