Because colorectal cancer is an important cause of death in many countries, we evaluated the effect of the hydroalcoholic extract of C. langsdorffii leaves on this neoplasia. The sequence of events that culminates in colon cancer helped us select the bioassays and protocols for this study. We employed the two endpoints − chemical-induced DNA damage and ACF − because they help determine in which step of colon carcinogenesis the C. langsdorffii extract acts.
ACF are putative preneoplastic lesions of colonic neoplasia in rodents and humans ; they are useful intermediate biomarkers that help one to assess the modifying effects of certain natural and synthetic compounds on chemically induced carcinogenesis . The advantage of studying the pathogenesis of cancer of the colon in animal models induced by chemical carcinogens, is that the tumor induction is rapid and allows to reproduce the adenoma-carcinoma sequence, as in humans. The high frequency of tumors that develop in the distal colon of rats, and the histogenesis of multiple adenomas, with consequent formation of adenocarcinomas, justify the importance of this species in the study of the pathogenesis of colon cancer .
Treatment with the C. langsdorffii extract reduced DNA damage and the number of ACF in the colon tissue of DMH-treated rats. ACF appear to arise from gene mutations, so an increased number of ACF reflects the initiation step of colorectal carcinogenesis. Our results indicate that the C. langsdorffii extract prevents the DNA damage and formation of preneoplastic lesions involved in the initial phase of colon cancer. In a previous study, treatments with C. langsdorffii extract diminished the genotoxicity induced by the chemotherapeutic agent doxorubicin as revealed by the Swiss mice peripheral blood micronucleus test .
In vivo studies have shown that DMH is metabolized to azomethane, azoxymethane, methylazoxymethanol, ethane, and carbon dioxide . Furthermore, DMH has been reported to induce carcinogenesis in rats and mice due to the high production of reactive free radicals [33, 34], which react with DNA, thus demonstrating its genotoxic effect. Although the mechanisms underlying the protective effect against DNA damage and ACF induction are not clearly understood, the putative antioxidant activity of the C. langsdorffii extract might explain its inhibitory action at least in part.
Phenolic compounds display antioxidant activity as a result of their capacity to scavenge free radicals . The most important natural phenolics are flavonoids, which present a broad spectrum of chemical and biological activities, including antioxidant and free radical scavenging properties. Flavonoids act as antioxidants, scavengers of a wide range of reactive oxygen species, and inhibitors of lipid peroxidation .
The hydroalcoholic extract of C. langsdorffii leaves contains the flavonoids quercetin-3-O-α-L-rhamnopyranoside and kaempferol-3-O-α-L-rhamnopyranoside as major compounds; these flavonoids constitute about 10% of the crude extract (Figure 3). The high water solubility of this extract facilitates its dissolution in the gastrointestinal tract, enhancing its antioxidant potential. Studies using a model system for food products have demonstrated that flavonoids such as quercetin, rhamnetin, kaempferol, rutin, and quercetrin are one of the most effective antioxidants .
Serpeloni et al.  stated that studying the chemical constituents of plants and their mechanisms of action is a major challenge: plant species contain various compounds, and their extracts can produce biological effects that may arise from the combined effects of the individual compounds. Phytochemical screening considers that every compound, regardless of their proportion in the plant, known or not, may be an active ingredient . Studies suggest that combinations of phenolics naturally present in fruits and vegetables as a whole inhibit cancer cell growth more effectively than the individual compounds .
Flavonoids and other phenolic compounds might exert direct protective effects on the gastrointestinal tract, by scavenging reactive species and/or preventing their formation. Polyphenols can inhibit hemeprotein-induced peroxidation in the stomach and decrease DNA base deamination or nitrosamine formation by HNO2-derived reactive nitrogen species .