Evaluation of the Genotoxicity of the Aerial Parts of Iraqi Euphorbia cyathophora on Bone Marrow and Spleen Cells in Mice

Euphorbia cyathophora Family: Euphorbiaceae, is an ornamental annual herb that has a poisonous latex. Traditionally it was used for fever, and mouth sores. Various phytochemicals were identified in this plant; flavonoids, tannins, glycosides, and others that resulted in its proven pharmacological effects. Evaluation the Genotoxic effect of methanolic fraction of aerial part of Euphorbia cyathophora on bone marrow cells and spleen cells in mice. 200 gm of E. cyathophora fine powder extracted by cold maceration 80% ethanol for seven days. The extract was filtered and dried in a rotary evaporator then the dried extract was suspended with water and consecutively extracted using chloroform, ethyl acetate for each. The aqueous layer was then mixed with 100ml methanol. Methanol fraction was dried under reduced pressure to obtain the dry extract. Twenty-four Albino mice were divided into four groups: Group 1: Mice were treated with distilled water daily for seven successive days. Group 2: Mice were treated with a single dose (20mg/kg) of methotrexate (positive control). Group 3: Mice were treated with (100mg/kg) of menthol fraction for seven successive days. Group 4: Mice were treated with (200mg/kg) of methanol fraction for seven successive days. Mice were sacrificed by (spinal dislocation). Samples of bone marrow cells and spleen cells were taken and genotoxic analyses. Methanol fraction of Euphorbia cyathophora at a dose of 100mg/kg demonstrated a significant decrease in mitotic index and a significant increase in total chromosomal aberrations as compared to distilled water in both bone marrow cells and spleen cells ( p<0.05 ). 100 mg/kg and 200 mg/kg of methanol fraction of Euphorbia cyathophora that showed to be significantly higher in mitotic index and significantly lower in total chromosomal aberration as compared to methotrexate ( p<0.05 ). The present study revealed that the methanol fraction of the aerial parts of Euphorbia cyathophora is genotoxic but its genotoxicity is less than that of methotrexate


Introduction
For thousands of years, the majority of the world's population used traditional medicine, and about 80% of developing countries people rely on traditionally used medicinal plants for disease management (1) .The discovery of the medicinal or toxic potential of the plant was assessed during their use for therapeutic purposes (2) .Plants hold certain therapeutic components with remarkable physiological effects which can be extracted and used in drug preparation (3) .Lots of the medicinal plants possessed cytotoxic, genotoxic, and mutagenic potential, therefor their uses might be associated with a higher incidence of tumor formation (4) .Further investigations are required before the consumption of these plants, particularly in the terms of their mutagenic potential (2) .Euphorbia cyathophora is also called Poinsettia cyathophora Murray (5) , it is family Euphorbiaceae (Spurge family).In the wild world, about 300 genera and 8000 species are included in this family, but most members of this family have poisonous milky latex (6) .Euphorbia cyathophora is naturally grown or indigenous in tropical America and cultivated in tropical and subtropical areas as ornamental.This annual herb rises to one-meter height (7) .Traditionally the airy parts of the plant were used for fever, jaundice, mouth sores, and improved milk production (8) .The previous study for the aerial part shows that there are different types of active constituents which include flavonoids, tannins, glycosides, saponins, and cardiac glycosides are present in aerial parts of the plant, other studies show the anti-inflammatory, antioxidant, antidiabetic, antibacterial, hepatoprotective and significant anti-Parkinson activity (9,10) .Silver nanoparticles of aqueous extract of Euphorbia cyathophora leaves exhibit an anticancer effect against HT-29 cell line (11) .Genotoxicity is the destruction of cellular genetic materials which may result in undesirable consequences as mutagenesis or tumor induction (12) . ).Genotoxicity was evaluated by different tests such as the bacterial Ames test, chromosomal aberration (cytogenetic parameter), and other tests.Chromosomal aberration can be performed through vivo and in vitro studies (13) .Methotrexate (MTX) is an anti-metabolite drug widely used in the treatment of neoplastic disorders, rheumatoid arthritis, and psoriasis.Developed as an analog of folic acid, it inhibits purine and pyrimidine synthesis that accounts for its therapeutic efficacy as well as for its toxicities.MTX has a narrow therapeutic index and its toxicity has been reported in various organ systems including gastrointestinal, haematologic, and central nervous systems (14) .The basis for its therapeutic efficacy is the inhibition of dihydrofolate reductase (DHFR), a key enzyme in the folic acid (FA) metabolism.FA is reported to have protective effects on MTX-induced genotoxicity in the somatic cells (15) .This study was intended to evaluate the genotoxic potential of Euphorbia cyathophora methanolic fraction by measuring mitotic index and the total chromosomal aberration in both bone marrow cells and spleen cells in mice.

Collection and authentication
Euphorbia cyathophora aerial parts were obtained from the medicinal plants garden in the college of pharmacy/ University of Baghdad during May/2021, authenticated by Assist.Prof. Dr. Sokaena Abass in the College of the Science / University of Baghdad.The desired aerial parts were rinsed with tap water and then dried at room temperature for fourteen days, grounded in a grinder to a fine powder.200 gm of E. cyathophora fine powder was defatted twice in 800 ml hexane for seven days, the defatted plant material was then extracted twice by cold maceration using 800 ml 80% ethanol for seven days.The extract was filtered and dried in a rotary evaporator yielding dry extract.The dried extract was suspended in 100ml water and consecutively extracted using chloroform and ethyl acetate (100ml x 3) for each.The aqueous layer was then mixed with 100ml methanol.These fractions (chloroform, ethyl acetate, and methanol) are dried under reduced pressure to obtain dry extract (16) .

Experimental model
Twenty-four Albino Swiss mice (Mus musculus) were used for each experiment.They were supplied by the animal house/Department of Pharmacology and Toxicology/ University of Baghdad.Their weights were 20-25 grams.They were divided into four groups; each was kept in a separate plastic cage.The animals were maintained at a temperature of 23 -25°C, and they had free excess to food (standard pellets) and water (ad libitum).The animals were divided into four groups (six mice of each) as follow: Group 1: Mice were treated with distilled water.This group was served as normal control the dose was given (I.P.) daily for seven successive days.Group 2: Mice were treated with a single dose (20mg/kg) of methotrexate.This group was served as a positive control (20) .Group 3: Mice were treated (oral) with (100mg/kg) of menthol fraction of Euphorbia cyathophora for seven successive days.Group 4: Mice were treated (oral) with (200mg/kg) of methanol fraction of Euphorbia cyathophora for seven successive days.After seven days of treatment, all animals were injected intraperitoneally with 1mg/kg colchicine, and then two hours later they are sacrificed by cervical dislocation.Bone marrow samples were aspirated from the femur bone and processed using an aseptic technique for evaluation of mitotic index and total chromosomal aberration as previously reported elsewhere (21) .

Statistical analysis
Data are expressed as Mean ± SD; unless otherwise indicated, statistical analyses were performed using ANOVA test.If the overall P value was found statistically significant (P<0.05),further comparisons among groups were made according to post hoc Tukey's test.

Results and Discussion
The phytochemical analysis of the methanol fraction of Euphorbia cyathophora (Table 1) revealed the existence of flavonoids, tannins, sugar, proteins, and saponins, while the alkaloids are absent (10) .In the Table (2), the methanol fraction of Euphorbia cyathophora at a dose of 100mg/kg caused a significant decrease in the mitotic index as compared to distilled water in both bone marrow cells and spleen cells (p<0.05).100 mg/kg and 200 mg/kg of methanol fraction of Euphorbia cyathophora showed to be significantly higher in the mitotic index as compared to methotrexate (p<0.05), also there were significant differences between methanol fraction doses (p<0.05).In Table (3), the methanol fraction of Euphorbia cyathophoraat both doses caused a significant increase in total chromosomal aberration as compared to distilled water in both bone marrow cells and spleen cells (p<0.05).100 mg/kg and 200 mg/kg of methanol fraction of Euphorbia cyathophora showed to be significantly lower in total chromosomal aberration as compared to methotrexate (p<0.05) also there were significant differences between methanol fraction doses (p<0.05).In the Table (2) both doses of methanol fraction, mitotic index was significantly higher as compared to MTX in both bone marrow and spleen cells in a dose-dependent manner(p<0.05), in the Table (3), there was a significant decrease in total chromosomal abrasion as compared to MTX (p<0.05).
The mitotic index is an indicator for cell genome health, a previous study showed that any damage or disruption in the genetic material (chromosomes) will activate a repair mechanism, this activation leads to prevent cell division and a decrease in the mitotic index as a result (22) .According to phytochemical investigation for Euphorbia cyathophora, tannins and flavonoids are major active constituents present in the plant.A previous study has been shown that tannin has a genotoxic activity which manifested by an increase in chromosomal aberration and a decrease in mitotic index (23) , this explain the increase in chromosomal aberration in both bone marrow cells and spleen cells when compared with negative control at the same time it explains the decrease in the mitotic index in both bone marrow cells and spleen cells as compared to the negative control.
Other important active constituents that are present according to the phytochemical investigation are flavonoids.Flavonoids constitute a group of polyphenolic compounds characterized by a common gamma-benzo-pyrone structure considered in numerous biological systems to possess an antioxidant capacity.A previous study has been investigating the effect of flavonoids on chromosome integrity, and found that flavonoids had a protective effect on chromosomes and decrease in the chromosomal aberration (24) .
In the present study, the presence of genoprotective flavonoids with genotoxic tannin explain the significant decrease in total chromosomal aberration in both methanol fraction as compared with methotrexate, besides the increase in the mitotic index in both doses as compared to methotrexate also related to the mixture of both compounds (flavonoids and tannin).Besides the increase in total chromosomal aberration for the methanolic fraction in both bone marrow cells and spleen cells as compared with the healthy group give a hint that either the quantity of tannic acid as higher than the quantity of flavonoids or the clastogenic effect of tannic was higher as compared to the anticlastogenic effect of flavonoids.

Conclusion
The current study demonstrated that the methanol fraction of aerial parts of Iraqi Euphorbia cyathophora is genotoxic but its genotoxicity is less than that of methotrexate.

Table 3 . Total chromosomal aberration in the bone marrow and spleen cells of albino mice treated with different doses of the methanol extract of Euphorbia cyathophora compared to methotrexate and distilled water. Treatment Groups Total Chromosome Aberration Bone Marrow Cells Spleen Cells
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