Moringa oleifera Lam. leaves prevent Cyclophosphamide-induced micronucleus and DNA damage in mice Sathya T.N.1*, Aadarsh P.1, Deepa V.1, Balakrishna Murthy P.1
Abstract Chemoprotective effect of ethanolic extract of Moringa oleifera Lam leaves was evaluated on cyclophosphamide (CP)-induced genotoxicity in the mouse. Animals were pre-treated with the extract for seven consecutive days at doses of 250, 500, 1000 and 2000 mg/kg b.w. Micronucleus in bone marrow and comet (DNA damage) in the liver were performed. Cyclophosphamide was administered intra-peritoneally on day 7 and Mice were sacrificed after 24 hours. In CP treated animals, statistically significant induction of micronuclei in polychromatic erythrocytes (PCE) was recorded. However, in the animals pre-treated with the extract, the percentage of CP-induced MN decreased with increasing concentration of the extract. Results of comet assay showed similar decrease in DNA damage in mice pre-dosed with the extract. These results point out to the presence of chemopreventive phytoconstituents in the crude extract offering protection against CP-induced genotoxicity in the mouse.
Introduction Much focus has been laid on the role of dietary constituents as anti-mutagens and anti-carcinogens since they are non-toxic in nature . Moringa species have long been recognized by folk medicine practitioners as having value in tumor therapy . Moringa oleifera Lam. is the most extensively cultivated species of the Moringaceae family, found in various parts of the world. Many phytochemicals have been isolated from various parts of the plant, viz., phenolic compounds such as quercetin and kaempferol, flavonoids, anthocyanins, carotenoids, vitamins, minerals, amino acids, sterols, glycosides and alkaloids. It contains unique group of compoundscalled glucosinolates and isothiocyanates [3,4]. Recently isothiocyanates and niacimicin from this plant were shown to be potent inhibitors of cancer [5-7].Niazimicin also inhibited tumor promotion in a mouse two-stage DMBA-TPA tumor model. Seed pod extracts of Moringa have been demonstrated to prevent skin tumors in mice . The leaves are highly nutritious, being a significant source of β-carotene, Vitamin C, protein, iron and potassium and have diverse curative properties [9 -17]. Its leaves are also used as nutritional supplement and growth promoters [18-22]. An immuno enhancing polysaccharide  has been reported from the leaves. Rich source of ascorbic acid and flavonoid pigments such as kaempferol, rhamnetin, isoquercitrin, and kaempferitrin in leaves of M. oleifera are known for their antioxidant properties. [24,25]. Ethanolic extract of leaves of M.oleifera have shown antimicrobial activity [26,27]. Radio protective effect of M.oleifera leaves has been established wherein radiation-induced chromosomal aberrations and micronuclei were suppressed by pre-treatment with methanolic extract . Given its rich nutritional value and abundant therapeutic efficiency, we attempted to study the chemo protective effect of Moringa oleifera leaves on CP-induced DNA damage using micronucleus and comet assays. Ethanol was used for extraction of the phytochemicals since it is a regarded as a powerful solvent capable of isolating majority of the plant constituents.........
Discussion The results of the present study clearly show that ethanolic extract of M.oleifera leaves have a dose-dependent modulatory effect on CP-induced micronuclei. Cyclophosphamide metabolizes into its mutagenic intermediate phosphoramide mustard through enzymatic and nonenzymatic metabolic activation. It is initially acted upon by the mixed function oxygenases to form 4-hydroxy-CP which exists as aldophosphamide. Aldophosphamide further metabolizes to form cytotoxic (acrolein and phosphoramide mustard) and non-cytotoxic (4-ketocyclophosphamide, carboxyphosphamide and aldophosphamide) intermediates. The protective effect may be attributed to the potential involvement of the phytomolecules of the extract to interfere with the enzymes participating in the biotransformation of CP to cytotoxic metabolites. Free radical scavenging represents one of the important strategies in antimutagenesis and anticarcinogenesis. Leaves of M. oleifera contain rich amount of antioxidants [36, 37]. It is reported that M.oleifera has 46 antioxidants and 36 anti-inflammatory compounds naturally occurring in it [38-41]. A possible explanation for the protective effect recorded in the present investigation could be the involvement of its antioxidant and scavenging properties. Antioxidants provide protection by scavenging reactive oxidative species (ROS) that damage DNA and initiate diseases such as cancer. Ethanolic extract of M.olerifera leaves have been reported to contain tannins, saponins, flavonoids, glycosides and terpenoids . Antioxidant vitamins, flavonoids, glucosinolates and organo-sulfur compounds have been proven to have antimutagenic or anticarcinogenic potential [42, 43]
Therefore protection against the clastogenic effects of CP could arise from the scavenging ability of M.oleifera leaves to trap hydroxyl radicals originating from metabolites of CP with an OH functional group. A similar decrease in MnPCEs induced by CP has been described for other antioxidants like stobadine, eugenol which possess the potential to protect DNA from reactive oxygen species and metabolism-dependent mutagens . Aqueous and methanolic extract of M. oleifera leaves have been reported only to limited extent for their antioxidant properties . In that regard, the anti-genotoxic effect delivered by the ethanolic extract of M.oleifera leaves could probably be attributed to the appreciable amount of antioxidant constituents. However, such comparative studies need to be conducted. Our study shows that in the mouse micronucleus test M. oleifera ethanolic extract prevents the genotoxic effects of CP when administered for a period of one week. Furthermore, the extract was non-clastogenic because it did not induce chromosome breakage in the bone marrow cells. Similar results were recorded in the comet assay. It reduces the percentage of DNA damage induced by CP in the liver cells. The results show that the extract has anti-genotoxic effects on CP-induced lesions in mice. The present results eventually lead us to conclude that ethanolic extract of M.oleifera leaves possess anti-genotoxic phytoconstituents.