Original Article

Genotoxicity of Ecballium elaterium (L) A Rich Cucurbitaceae Fruit Juice Using Micronucleus Assay and DNA Single Strand Break Techniques.

I Muhammad Said Shabbar, A Maslat

Citation

I Muhammad Said Shabbar, A Maslat. Genotoxicity of Ecballium elaterium (L) A Rich Cucurbitaceae Fruit Juice Using Micronucleus Assay and DNA Single Strand Break Techniques.. The Internet Journal of Health. 2006 Volume 6 Number 2.

Abstract

This study was designed to investigate the genotoxicity of the Ecballium elaterium crude fruit juice, which is applied for treatment of jaundice as a traditional folk medicine in Jordan. The LD50 was estimated to be 61µl of the juice administered via the gastric route. The potential genotoxicity of E. elaterium was examined by micronucleus (MN) assay and DNA single strand break (SSB) techniques. The results showed that the fruit juice significantly induced the MN formation and SSB in the DNA by the oral administration of E. elaterium extract in the test groups as compared to the control. Thus, the data suggest that E. elaterium extract may have the potential to induce genotoxicity.

 

Introduction

Medicinal plants play an important role in the life of people in many countries and its use increases more and more all over the world. Green plants are generally containing mutagenic and carcinogenic substances, but there is little information about the biological activities of herbal medicine.[1]

. Ecballium elaterium (L.) (‘‘squirting cucumber'‘ or ‘‘donkey's green'‘) is a perennial plant from the family Cucurbitaceae, a mediterranean medicinal plant that has been investigated for its several pharmacological properties.[2,3,4,5,6,7] Ecballium elaterium has a large fleshy root, from which several round, thick, rough stems rise, branching and trailing like the common cucumber, but without tendrils. The leaves are petiolate, large, rough, irregularly cordate, and of a grayish-green color. The flowers are yellow and axillary. The fruit has the shape of a small oval cucumber, about an inch and a half long and one inch thick. It has a greenish or grayish color, and is covered with stiff hairs or prickles. When fully ripe, it separates from the peduncle, and throws out its juice and seeds with considerable force through an opening at the base, where it was attached to the footstalk. The name of squirting cucumber was derived from this circumstance, and the scientific and official title (elaterium) is supposed to have had a similar origin, though some authors maintain that the term elaterium was applied to the drug rather from the mode of its operation upon the bowels than from the projectile property of the fruit. The word elaterium was used by Hippocrates to signify any active purge. Dioscorides applied it to the medicine of which we are treating. [8, 9]

The ripe fruit is about 4 cm long of yellow-green color. The name ‘‘squirting cucumber'‘ comes from the tendency of the ripe fruit to explode upon the slightest touch, with its dark seeds and juice ejected at a distance of several meters. [10, 11] Herbal medicine recommends it for the treatment of chronic sinusitis or rhinosinusitis [7, 12] It is also recommended for liver cirrhosis as well as other conditions thought to be inflammatory in nature including rheumatism and infections [13] The juice of Ecballium elaterium fruit is a well-known powerful hydragogue cathartic in folk medicine and is also used for its diuretic activity, especially in edema caused by kidney troubles.

Ecballium elaterium roots were used, in Anatolian folk medicine, as analgesic and in treatment of hemorrhoids; fruits in sinusitis, jaundice, nocturia, lumbago and otalgia [7, 11, 14, 15]The active antiinflammatory principal is cucurbitacin B, a triterpene derivative isolated from fruits and seeds of the plant [11, 13] The building block of terpenes is isoprene or 2-methyl 1,3-butadiene, precursor of essential oils found in many plants. [15] The significance of this study depends on the facts that Ecballium elaterium is a mediterranean medicinal plant used as an analgesic and in treatment of hemorrhoids; fruits in sinusitis, jaundice, nocturia, lumbago and otalgia as well as other conditions thought to be inflammatory in nature including rheumatism and infections. Accordingly, we found it of special interest to investigate the effect, if there is any, of its fruit juice on the genetic material.

The objectives of the study aimed at determining the LD50 and investigating the genotoxicity of Ecballium elaterium fruit juice administered orally to mice.

The elicit effect of the E. elaterium fruit juice on the DNA.

Materials And Methods

The protocol of the study was approved by research and animal testing ethics committee of Yarmouk University Deanship of Researches and Postgraduate studies,

Animal subjects

Balb/c male mice 2-3 months old weighing 25-30 g were used. The mice were placed in cages (6-8/each) and maintained under controlled conditions (temperature 20-22°C, relative humidity 60-80% and on 12 hrs light-dark cycle) and on diet ad libitum and water. The mice were supplied by the animal house unit at Yarmouk University.

Plant materials

The ripe fruits of Ecballium elaterium were collected from Aban district and Yarmouk University campus in Irbid during late November and December of 2005 and stored at -20°C until needed. The classification of which was assessed by Prof. Dr. Jamil N. Lahham (Professor of Flowering Plants Taxonomy, Yarmouk University, Irbid, Jordan). The frozen fruits were quickly thawed at 37 °C in an air blowing incubator and then the juice were extracted and chilled on ice in a dark closed glass container.

Oral administration of the juice

This process needed two persons; one handled the mouse, taking it from the cage and semi- anesthize it with diethyl ether and holding it tight and vertical and the other one holding a forceps in one hand and the micropipette (containing the specific dose that should be given) in the other hand. The forceps was used to open the mouse mouth and smoothly pulling its tongue, then in the other hand the micropipette was inserted into the mouse oral cavity slightly into the beginning of the esophagus after the throat, then waiting for the mouse to begin waking up. At this point the dose was given, waiting for the mouse to swallow the dose while holding the tongue.

Determination of LD

Sixty four Balb/c male mice were divided into eight groups. Each group received orally 160, 113, 100, 87, 74, 61, 48, and 24 µl of the Ecballium elaterium crude fruit juice (0.69 mg triterpenes/ml juice). The animals were monitored for 24 hrs for mortality. The number of animals survived a specific dose S and the number of those died at that dose D was determined. The percent mortality was calculated for each dose group as in the following:

Figure 1

Micronucleus assay

Cell preparation

Frozen fruits were quickly thawed at 37 °C in an air blowing incubator and then the juice were extracted and chilled on ice. A total number of 36 mice were used in the first and the second experiment, given a single dose via the gastric route using 74, 61 and 48 µl doses of the Ecballium elaterium crude fruit juice. Peripheral blood was then obtained in heparinized capillaries from the retro-orbital vein after 36 hrs of the juice administration.

A total of 12 mice were used as negative and positive controls in the first and the second experiment that were applied according to González Borroto et al. (2003), Mengs et al. (1999) and Heddle et al. (1983). Mitomycin C, at a concentration of 14 mg/kg, was used as a positive control which was dissolved in sterile distilled water.

Preparation of the blood smears

Blood smears were prepared on clean pre-washed glass slides. The prepared blood films were air-dried and fixed in methanol for about 3 minutes (Schmid, 1975; Heddle et al., 1983). At least four slides were prepared from each mouse.

Staining and micronucleus evaluation

The smeared preparations were stained with Acridine orange (AO) according to the method of Stockert and Lisanti (1972) with some modifications according to Hayashi et al. (1983). The A.O. stock solution was prepared as a 0.1% aqueous solution that was available for several weeks at 4°C. A.O., 0.24 mM in 1/15 M Sörensen's phosphate buffer (pH 6.8) which is 1/15 M Na2HPO4 and 1/15 M KH2PO4 prepared separately and mixed together in a ratio to have pH 6.8 (2 parts of stock solution and 30 parts of the buffer), was used as a working solution. The fixed slides were stained in this solution for 3 min at room temperature (the stored preparations required less time for staining depending on the storage period). The slides were rinsed in the buffer 3 times for 2-3 min each time. If the nuclei emitted a reddish fluorescence, the slides should be rinsed for another several minutes to leave nuclei with green fluorescence. The preparations were mounted with the same buffer, and sealed with Canada balsam. Observations were made within 1 hr using NIKON microscope ECLIPSE E400 with 40X objective and Y-FL EPI-FLUORESCENCE attachment that has 420-490 nm excitation filter and a 520 nm barrier filter for observation and microphotography (Hayashi et al., 1983).

From each animal, 2000 normochromatic erythrocytes were screened for micronuclei.

Single strand breaks

Thirty two Balb/c male mice were divided into eight groups; each group has four mice in it. Frozen fruits were quickly thawed at 37 °C in an air blowing incubator and then the juice was extracted and chilled on ice. The first three groups received a single dose via the gastric route of 61 µl of the Ecballium elaterium crude fruit juice and the other three groups received 48 µl of the juice, the animals were killed by cervical dislocation at 3 different time periods; after 1 hr, 24 hrs and 36 hrs of the juice administration via the gastric route. Peripheral blood was obtained in EDTA tubes after cutting off the head and then the tubes were gently inverted 8-10 times. The livers were taken surgically to isolate the liver genomic DNA from them.

The other two groups, one was used as a negative control (without treatment) on normal diet, and the other group received a single dose of methyl methanesulfonate (MMS) at a concentration of (150 mg/kg, ip) which was used as a positive control (Lee and Garner, 1991). Solveig Walles and Erixon (1984) stated that the relative level of SSB in DNA was determined in various organs (liver, kidney, lung, spleen, testis and brain) 1-24 hrs after administration of the agent. After MMS-treatment, the number of SSB in DNA increased to about the same extent in all organs 1 hr post-treatment but then decreased by time; the SSB persisted for the longest time in brain- and lung-DNA. The DNA alkylating agent MMS is used as a DNA damaging agent to induce mutagenesis and in recombination experiments. MMS modifies both guanine (to 7-methylguanine) and adenine (to 3-methyladenine) to cause base mispairing and replication blocks, respectively (Lundin et al., 2005).

DNA extraction from the blood

Promega Wizard® Genomic DNA Purification Kit (Cat. number A1120, lot number 198647) was used for the DNA purification. The blood samples were freshly collected in EDTA (4%) treated. Then the tubes were gently rocked until thoroughly mixed (about 10 times); 300 µl of the fresh blood samples were transferred to marked 1.5 ml tubes containing 900 µl Cell Lysis Solution (the tubes were inverted 5-6 times to mix). The mixture was incubated for 10 minutes at room temperature (the tubes were inverted 2-3 times once during the incubation to lyse the red blood cells). The tubes were centrifuged at 13,000-16,000 x g for 20 seconds at room temperature. Then the supernatant was removed and discarded as much as possible without disturbing the visible white pellet (approximately 10-20 µl of residual liquid remained in the 1.5 ml tubes that we have). After that, the tubes were vortexed vigorously until the white blood cells were resuspended (10-15 seconds) and 300 µl of Nuclei Lysis Solution were added to the tubes containing the resuspended cells and the solution was pipetted 5-6 times for each tube to lyse the white blood cells (the solution should become very viscous). 1.5 µl of RNase Solution was added to the nuclear lysate. The samples were mixed by inverting the tubes 2-5 times and the mixture was incubated at 37 °C for 15 minutes, and then cooled to room temperature (or chilled on ice for 5 min.). 100 µl Protein Precipitation Solution was added to the nuclear lysate and vortexed vigorously for 10-20 seconds. The samples were centrifuged at 13,000-16,000 x g for 3 minutes at room temperature (a dark brown protein pellet was visible). The supernatant for each sample was transferred separately to a clean marked 1.5 ml microcentrifuge tubes containing 300 µl of room temperature isopropanol (the solutions were gently mixed by inversion until the white thread-like strands of DNA form a visible mass). Then the samples were centrifuged at 13,000-16,000 x g for 1 minute at room temperature (the DNA was visible as a small white pellet). The supernatant was decanted and one sample volume of room temperature 70% ethanol was added to the DNA (to wash the DNA pellet and the sides of the microcentrifuge tube, the tube was gently inverted several times). Then the samples were centrifuged at 13,000-16,000 x g for 1 minute at room temperature. Ethanol was carefully aspirated, the tubes were inverted on clean absorbent paper and the pellet was air-dried for 10-15 minutes. Finally, 100 µl of DNA Rehydration Solution (10 mM Tris, 1 mM EDTA) was added to each tube and the DNA was rehydrated by incubating the samples at 65°C for 1 hour and the DNA samples were stored at 2-8 °C.

Genomic DNA isolation from liver

Promega Wizard® Genomic DNA Purification Kit (Cat. number A1120, lot number 198647) was used for the DNA purification. The liver was surgically removed from each mouse and instantly immersed in liquid nitrogen and grounded using an autoclaved ceramic pestle. About 17 mg of the grounded tissue was added to 600 µl of Nuclei Lysis Solution that was previously added to an ice chilled 1.5 ml marked centrifuge tubes. After all samples were collected, the samples were incubated at 65 °C for 15-30 min.; 3 µl of RNase Solution was add to the nuclear lysate and the samples were mixed by inverting the tubes 2-5 times. The mixture was incubated at 37°C for 15-30 minutes, and then cooled to room temperature (or chilled on ice for 5 min.). Then, 200 µl Protein Precipitation Solution was added to the nuclear lysate and vortexed vigorously for 20 seconds, chilled on ice for 5 min. and were centrifuged at 13,000-16,000 x g for 4 minutes at room temperature (a dark brown protein pellet was visible). The supernatants for each sample were transferred separately to a clean marked 1.5 ml microcentrifuge tubes containing 600 µl of room temperature isopropanol (the solutions were gently mixed by inversion until the white thread-like strands of DNA form a visible mass). The samples were centrifuged at 13,000-16,000 x g for 1 minute at room temperature (the DNA was visible as a small white pellet). The supernatant was decanted and 600 µl room temperature 70% ethanol was added to the DNA (to wash the DNA pellet and the sides of the microcentrifuge tube the tube was gently inverted several times). Then the samples were centrifuged at 13,000-16,000 x g for 1 minute at room temperature. Ethanol was carefully aspirated; then the tubes were inverted on clean absorbent paper and the pellet was air-dried for 10-15 minutes. Finally, 100 µl of DNA Rehydration Solution (Tris- EDTA) was added to each tube and the DNA was rehydrated by incubating the samples at 65°C for 1 hour and the DNA samples were stored at 2-8 °C.

Alkaline agarose gel electrophoresis

DNA single-strand breaks were assayed by using alkaline agarose gel electrophoresis as previously described by Freeman et al. (1986) with modifications. Briefly, DNA samples were denatured by incubation with an alkaline stop mix [25% (v/v) glycerol, 0.125% (w/v) bromocresol green, 0.5N NaOH] for at least 15 min. DNA mixtures were then loaded into wells (1 mm X 2.5 mm) of a 0.6% (w/v) agarose gel (Sigma II agarose prepared in 50 mM NaCl, 4 mM EDTA, 50 ml gel volume, in a BioRad minisub-cell apparatus) presoaked in 2 mM EDTA, 30 mM NaOH. Molecular length marker was added and electrophoresis was carried out for 1.5-3 hrs ? 3 V/cm in 2 mM EDTA, 30 mM NaOH. The gel was neutralized in 250 ml of 0.1 M Tris, pH 8, for 15 min, and then stained in 250 ml ethidium bromide (1 µg/ml) in distilled H2O for 15 min. The gel was destined for 30 min in distilled H2O and visualized under gel documentation system (BioRad).

In the in vitro study, mouse DNA that was rehydrated in TE buffer was incubated with the fruit juice at a ratio of 1:1. The reaction was carried out at 37 °C. After 2 hrs incubation, the reaction was terminated by chilling the mixture in an ice bath according to Lin et al. (2001) with modifications; i.e. instead of using PBS, TE was used and we did not use methoxylamine which if used can protect against depurination/depyrimidination which forms apurinic/apyrimidinic sites. The electrophoresis was carried out at once according to Freeman et al. (1986).

In the in vivo study, the DNA samples from Balb/c mice treated with the Ecballium elaterium crude fruit juice were electrophoresed by Alkaline Gel Electrophoresis. The preparation of the alkaline gel electrophoresis was done according to Freeman et al. (1986), which disperses single strand DNA according to molecular length whereas double stranded DNA was denatured by treatment with alkali, electrophoresed in alkaline agarose, and after neutralization of the gel, the DNA was stained with ethidium bromide.

Results

Determination of LD

The mice were separated into groups; each group consisted of 8 mice. Each mouse received a specific amount of the fruit juice that was freshly thawed and given to the mouse via the gastric route. Then, the animals were monitored for mortality after 24 hrs and percent mortality were calculated.

Figure 2
Table 1: Percent mortality of the mice exposed to different concentrations of the fruit juice.

So, the LD50 was estimated to be 61 µl of the fruit juice according to table (1). Determination of LD50 was used as a guide for the determination of doses, which were used in the next experiments.

Micronucleus assay

The mice were separated into five groups in each experiment; three groups consist of 12 mice and two groups consist of three mice each. The mice in the first three groups received different amount of the fruit juice (74, 61 and 48 µl) that was freshly thawed and given to the mice via the gastric route; the second two groups, one was the negative control which remained without treatment and the second one was the positive control that received a single dose of MMC 14 mg/kg, dissolved in sterile distilled water. After 36 hrs of the dose administration via the gastric route, peripheral blood was harvested from each mouse in heparinized capillary tubes from the retro-orbital vein. After that, blood smears were made for each mouse in quadruplicate and some cases in pentuplicate, air-dried and fixed with methanol. Figures 3-5, present microphotographs of AO-stained peripheral blood films of mouse at different treatments.

Figure 3
Figure 4: Microphotograph of AO-stained peripheral blood film of mouse without treatment, showing two micronucleated cells.

Figure 4
Figure 5: Microphotograph of AO-stained peripheral blood film from the positive control mouse treated with MMC (14 mg/kg). Many cells appear micronucleated.

Figure 5
Figure 6: Microphotograph of AO-stained peripheral blood film from experimented mouse treated with 74 µl of the fruit juice. Most cells appear micronucleated.

To interpret the results, it should be emphasized that there are several criteria for determining a positive response. One of which is a statistically significant dose-related increase in the number of micronucleated normochromatic erythrocytes. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one concentration of the test substance. A test substance which does not produce either a statistically significant dose-related increase in the number of micronucleated normochromatic erythrocytes or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system. Both biological and statistical significance should be considered together in the evaluation (EPA, 1996). Under the test conditions, the fruit juice succeeded in producing a dose-response increase in the number of micronucleated normochromatic erythrocytes and a significant response as seen in tables 3 through 6.

Looking at table (3), since ? ? 0.05, the results indicate that there are statistically significant differences in the means of MN formation due to different doses compared to the group without treatment. From the Post Hoc tests for multiple comparisons (Table 4), it is noticed that there are also statistically significant differences in the means of MN formation in the mice that received a 74, 61 and 48 µl of the juice compared to the negative control group.

Table (5) shows that ? ? 0.05 indicating that the results are statistically significant in respect to MN formation due to different doses compared to the group that was treated with MMC. From the Post Hoc tests for multiple comparisons in table (6), it is clear that there are also statistically significant differences in the means of MN formation in the mice that received a 74, 61 and 48 µl of the juice compared to the group that was treated with MMC. For the group that received a 74 µl of the fruit juice, it's observed that the mean difference is in negative that is because the MN formation in the group that was treated with the juice was more than that of the group treated with MMC and has a statistical significance. On the other hand, the group that was treated with 61 µl of the juice had a positive mean difference in table (6) which means that it's lower than the mean of the group treated with MMC, it has a biological significance but no statistical significance (because it's around the mean of the MMC treated group), it has a statistical significance difference in the mean of MN formation compared to the untreated group (the negative control). Concerning the group that received a 48 µl of the juice, it showed a statistically significant difference compared to the negative control.

Figure 6
Table 2: Results of the pooled data from the two experiments with fruit juice administration via the gastric route at 74, 61 and 48 µl/mouse

Figure 7
Table 3: ANOVA (testing differences of treatments means with micronucleated cells number) between the without treatment group and the different juice amounts administered via the gastric route to the mice.

Figure 8
Table 4: Post Hoc Tests for multiple comparisons between the without treatment group and the different juice amounts administered via the gastric route to the mice.

Figure 9
Table 5: ANOVA (testing differences of treatments means with micronucleated cells number) between the MMC-treated group and the different juice amounts administered via the gastric route to the mice.

Figure 10
Table 6: Post Hoc Tests for multiple comparisons between the MMC treated group and the different juice amounts administered via the gastric route to the mice.

Single strand breaks

The mice were separated into eight groups; two of the first three groups received a single dose, via the gastric route, of 61 and 48 µl of the Ecballium elaterium crude fruit juice. The other two groups, one was used as a positive control and the other one was used as a negative control. The animals from the first six groups were killed by cervical dislocation at 3 different time periods; namely 1 hr, 24 hrs and 36 hrs after the juice administration via the gastric route. Peripheral blood was obtained in EDTA tubes after cutting off the head; the liver was taken surgically to isolate the liver genomic DNA from it.

We conducted a pilot experiment using an alkaline gel electrophoresis described by Lin et al. (2001). Briefly, the extracted DNA was denatured at l00 °C for 3 min in the presence of 5 µl 10 mM NaOH, 95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol. The denatured DNA was electrophoresed on a 0.7% agarose gel with 0.5 µg/ml ethidium bromide in 40 mM Tris-borate buffer containing 1 mM EDTA. The DNA was visualized under UV irradiation of a gel documentation system from BioRad (Figure 7). As can be seen, lane 2-4 show a dose-dependent induction of DNA fragmentation compared to lane 6 & 7 which is the lower concentrations used after 1 hr of juice administration. Over time (after 24 hrs) the higher juice dose gave a lower DNA fragmentation in lane 11-13 while the lower dose gave a higher DNA fragmentation 14-16.

Figure 11
Figure 7: Electrophoretic analysis of DNA single-strand breaks in livers of mice that were given the fruit juice at different amounts and killed at different periods. Lane 1, O'GeneRuler 1kb DNA Ladder; lane 2-4, 61 µl juice and was killed after 1 hr; lane 5, -ve control and was killed after 1 hr; lane 6&7, 48 µl juice and was killed after 1 hr; lane 8-10, +ve control and was killed after 1 hr; lane 11-13, 61 µl juice and was killed after 24 hrs; lane 14-16, 48 µl juice and was killed after 24 hrs; lane 17, +ve control and was killed after 12 hrs.

The electrophoretic analysis of DNA single-strand breaks was conducted using alkaline agarose gel electrophoresis as previously described by Freeman et al. (1986). In figure (8), the mice liver DNA was tested in alkaline gel electrophoresis, whereas in figure (9), the mice blood DNA was tested by the same technique for the presence of SSB.

In figure (8), after 1 hr of juice administration, the group that received a highest concentration of the juice produced a higher DNA fragmentation in the mice liver DNA in lane 1, whereas the lower concentration had a lower effect on the DNA fragmentation at this time in lane 2 & 3. This indicates a dose dependent induction of DNA fragmentation. After 24 hrs of exposure, the higher dose effect on the DNA fragmentation was lower than that of the first time period in lane 6, but the lower concentration of the fruit juice had a higher effect on the DNA fragmentation in lane 7-9. After 36 hrs, however, the effect of the higher dose of the juice on the DNA fragmentation remained low in lane 11 and the lower concentration of the juice produced slightly lower DNA fragmentation than at the second time period in lane 12.

In figure (9), the same results can be seen concerning the blood DNA. About in vitro test, the results showed that the fruit juice induces a drastic DNA fragmentation.

Figure 12
Figure 8: Electrophoretic analysis of DNA single-strand breaks in livers of mice that were given the fruit juice at different amounts and killed at different periods. Lane 1, 61 µl juice and was killed after 1 hr; lane 2 & 3, 48 µl juice and was killed after 1 hr; lane 4, +ve control and was killed after 1 hr; lane 5, -ve control and was killed after 1 hr; lane 6, 61 µl juice and was killed after 24 hrs; lane 7-9, 48 µl juice and was killed after 24 hrs; lane 10, +ve control and was killed after 1 hr; lane 11, 61 µl juice and was killed after 36 hrs; lane 12, 48 µl juice and was killed after 36 hrs.

Figure 13
Figure 9: Electrophoretic analysis of DNA single-strand breaks in blood of mice that were given the fruit juice at different amounts and killed at different periods. lane 1, O'GeneRuler 1kb DNA Ladder; lane 2, 61 µl juice and was killed after 1 hr; lane 3, 48 µl juice and was killed after 1 hr; lane 4, +ve control and was killed after 1 hr; lane 5, -ve control and was killed after 1 hr; lane 6, 61 µl juice and was killed after 36 hrs; lane 7, 48 µl juice and was killed after 36 hrs; lane 8, 61 µl juice and was killed after 24 hrs; lane 9, 48 µl juice and was killed after 24 hrs; lane 10 & 11, test of the juice extract.

Discussion

This study represents the first attempt to investigate the genotoxic effects of Ecballium elaterium fruit juice in mammalian cells. For this reason, it is unfortunate that our results can not be compared and evaluated in relation to other studies. However, the historical usage of Ecballium elaterium fruit juice in folk medicine should be remembered. Further, its presumed efficiency in the treatment of several diseases was emphasized in published literature. From the micronucleus test that was conducted on Balb/c male mice, it was clear that the frequency of the MN presence per 2000 normochromatic erythrocytes, as shown in table (2), ranged between the negative and slightly above the positive control in one of the concentrations used for the fruit juice. This can be explained by looking at the positive control as any compound that has a positive effect on the induction of MN formation in normochromatic erythrocytes in the mice. But in the juice, it is presumed that there were synergistic effects of the juice cocktail on the induction of MN formation. As indicated in tables (3 & 4), there was a statistically significant difference in this study between different concentrations that were used compared to the negative control. All different concentrations used from the fruit juice were faraway from the results of the negative control. The same thing applies (Table 5 & 6), when different concentrations used from the fruit juice are compared to the positive control. One group exceeded the positive control, and another one had about the same results as in the positive control in regard to the induction of MN formation in normochromatic erythrocytes and the last group was lower than the positive control. In our opinion, those were all significant because they were around the positive control results and so those different concentrations had a positive effect on the induction of MN formation taking into consideration that the juice cocktail had synergistic effect as mentioned earlier.

Overall, the collected data indicate that the fruit juice had a significant effect on the induction of MN formation in mice because there were statistically significant dose-related increases in the number of micronucleated normochromatic erythrocytes. Another criterion was based upon detection of a reproducible and statistically significant positive response for the test substance concentrations.

The electrophoretic analysis of DNA single-strand breaks was conducted using alkaline agarose gel electrophoresis as previously described by Freeman et al. (1986). Freeman's method permits the measurement of low levels of DNA lesions in nanogram quantities of non-radioactive DNA. It offers a good precision and accuracy. It is ideal for measuring DNA lesions in organisms, tissues, or cells not easily radioactively labeled, especially those available only in limited quantities.

For testing of the fruit juice ability to produce a DNA single-strand breaks in the mice DNA in the liver or in the blood in vivo or even in vitro, a pilot experiment was conducted using a method for the electrophoretic analysis of mice liver DNA single-strand breaks and the results for this pilot experiment were manifested in figure (7). It's concluded that after one hour of the fruit juice administration via the gastric route, the mice had a dose-dependent induction of DNA fragmentation. Whereas at the lower concentration, there was a slight increase in the DNA fragmentation by the juice. After 24 hrs, it was observed that the mice that received the higher dose were stabilizing (decrease in the DNA fragmentation) but an increase of the DNA fragmentation was manifested and clearly visible in lane 14-16. This increase in the DNA fragmentation over time at the lower concentration is expected to be due to the mice tolerance to juice effect on the DNA SSB but after the first period, it reaches a point that it was not able to resist this breakage as we saw in the second time period where the higher concentration of the juice had a harsh effect on the induction of DNA fragmentation in the first period. The mice that survived this attack were able to heal its DNA, not that the fix was correct or not but at least it decreased the DNA fragmentation over time.

In the electrophoretic analysis of DNA single-strand breaks of mice liver DNA, a dose-dependent induction of DNA fragmentation was noted. The group that received a higher concentration of the juice exhibited a higher DNA fragmentation in the mice liver DNA, whereas the lower concentration had a lower effect on the DNA at this time. After 24 hrs of exposure, the higher dose effect on the DNA fragmentation was lower than that of the first time period but the lower concentration of the fruit juice had an increase in the DNA fragmentation. After 36 hrs, the effect of the higher dose of the juice on the DNA fragmentation remained low and the lower concentration of the juice produced slightly lower DNA fragmentation than at the second time period due to the same criteria that were discussed earlier on (figure 8).

In the electrophoretic analysis of DNA single-strand breaks of mice blood DNA, a dose-dependent induction of DNA fragmentation was also recorded. The group that received a higher concentration of the juice produce a higher DNA fragmentation in the mice blood DNA, whereas the lower concentration had a lower effect on the DNA fragmentation at this time. After 24 hrs of exposure, the higher dose effect on the DNA fragmentation was lower than that of the first time period but the lower concentration of the fruit juice had an increase in the DNA fragmentation. After 36 hrs, the effect of the higher dose of the juice on the DNA fragmentation remained low and the lower concentration of the juice produced slightly lower DNA fragmentation than at the second time period. This is due to the same criteria that were discussed earlier; an induction of DNA fragmentation was observed for the in vitro test.

Conclusions

The conclusion of the present study was made taking into consideration both in vivo and in vitro tests that was implemented on the mice using micronucleus assay and SSB techniques:

LD50 was estimated to be 61 µl of Ecballium elaterium crude fruit juice per 27g mice body weight (0.69 mg triterpenes/ml juice).

Ecballium elaterium crude fruit juice induced statistically significant MN formation in normochromatic erythrocytes in the treated mice at different doses.

Ecballium elaterium crude fruit juice induced dose-dependent induction of DNA fragmentation at different periods, which were depleted over time in some groups due to DNA repair which may not be completely efficient.

At concentrations higher than LD50, it was observed that the mice blood was thicker than normal controls.

Acknowledgments Of The First Author

I Dedicate this work to the memory of my late father Muhammad Said Shabbar who inspired me to do such work and to My dear Mother for her care and support Lady Malak Al Moujtahed Shabbar

With the help of Almighty Allah this work has been finished and the caring and supervision of my supervisor Dr. Ahmed Maslat, thanks to him for his supervision, guidance and encouragement through out this work. Also my deep thanks and great appreciation goes to Prof. Dr. Ahmad Khalil for his help and encouragement, critical suggestions and invaluable comments. My thanks are extended to Prof. Dr. Mahmoud Abussaud and Dr. Saleem Abderrahman for their kindness being members of my committee and for their invaluable comments and suggestions.

I'm grateful to Dr. Ammar Omran from faculty of Pharmacy at Al-Zaytoona University for his help in the quantitative determination of triterpenes in the fruit juice. Thanks to Prof. Dr. Jamil N. Lahham for the classification of the investigated plant.

Correspondence to

Ibrahim Muhammad Said Shabbar B Sc Post graduate Student Yarmouk University Irbid Jordan e-mail: omega@go.com.jo

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Author Information

Ibrahim Muhammad Said Shabbar, B.Sc.
Post graduate Student, Yarmouk University

Ahmed Othman Maslat, Ph.D., Sc.
Assistant Professor, Yarmouk University

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