At the molecular level, oxygen is one of the most important causes of cancer, oxidants, and free radicals. Through mechanisms such as telomerase inhibition, changes in cell permeability, and DNA damage, these compounds lead to cell damage and the development of many diseases, including cancer (Lin,2018). For this reason, cancer treatment with the approach of strengthening the body's antioxidant system is one of the favorite paths of researchers. Plants have also been widely used as a natural and rich source of antioxidant compounds in the treatment of diseases. Research has shown the presence of 46 different antioxidant compounds and 36 natural anti-inflammatory agents in Moringa (Bose,2007), including zeitin (a powerful antioxidant), quercetin (a flavonoid known to neutralize free radicals), beta-sitosterol (anti-inflammatory cholesterol in the body), caffeic acid (another strong anti-inflammatory compound) and kaempferol (Lin,2018).
In addition, various parts of the Moringa oleifera plant (leaves, seeds, and sprout) are high in calcium, iron, potassium; amino acids and proteins, vitamins B1, B2, B3, B6, C, E, riboflavin, folic acid, and nutrients that strengthen the body, which is essential for the health of living things (Karim,2016). Moringa oleifera has also been shown to have anticancer, hypoglycemic, anti-inflammatory, anti-bacterial, anti-fungal, anti-viral, anti-allergic, pain-relieving, anti-infective, and anticoagulant effects. The role of this plant against Alzheimer's disease, gastric ulcer, cholesterol control, and reduction, and wound healing has been studied and proven (Tragulpakseerojn,2017). Researchers have reported that Moringa strengthens cells against chemotherapy and increases the cell tolerance threshold for chemicals (Tragulpakseerojn,2017). Therefore, in this study, the main purpose of this study was to evaluate the effects of Moringa oleifera on melanoma cancer in vitro and in vivo.
In this study, first, the total flavonoid and phenolic content of the prepared Moringa leaf extract was obtained, and then the dominant flavonoid compounds in the extract were identified, and the concentration was determined. In this study, three phenolic and flavonoid compounds with high antioxidant properties (quercetin, gallic acid, and caffeic acid) were identified. The results showed that in extraction by soxhlet with methanol solvent, the flavonoid content of the extract was 60.65±1.75 mg quercetin acid per gram of dry extract. In Ishaqzadeh's study, the total flavonoid content of Moringa hydroalcoholic extract amplified in glass was 78.189±0.01 mg/g (Asieh Ishaqzadeh Torbati,2015). The results of their study showed a higher concentration of total flavonoid content in Moringa, which is due to the expression of flavonoid concentration. In this study, expression was based on the amount of quercetin, which was the highest type of flavonoids, but in Ishaqzadeh's study, the total flavonoids were reported (Asieh Ishaqzadeh Torbati,2015). The results show that Moringa extract has a high total flavonoid content, and due to the antioxidant properties of flavonoid compounds, it can be used in the treatment of various diseases, including cancer.
In this study, two free radical scavenging tests, DPPH and FRAP tests, were used to evaluate the antioxidant power of the extract. The potency of Moringa leaf extract in inhibiting DPPH free radicals was 42±0.73. On the other hand, in this study, it was found that with increasing the concentration of Moringa leaf extract, both the free radical scavenging power of DPPH and the regenerative power of the extract increased significantly (P<0.05). Another study performed a linear and direct relationship between the antioxidant activity of the extract and the presence of phenolic and flavonoid compounds. These results were consistent with the study of Jeyakumar et al. (Jeyakumar,2020). They showed that DPPH inhibition for ethanolic extracts of Moringa leaves was 29.49, 43.72, and 54.59 at 500, 1000, and 1500 ppm, respectively, which was close to the average obtained in our study. Abdolshahi et al.( Abdolshahi,2015), in their study, evaluated the antioxidant activity of methanolic extract of medicinal plants by the DPPH method and concluded that methanolic extract of medicinal plants has the potential to be used as a natural antioxidant.
In the present study, melanoma cancer cells (B16F10) were treated with different concentrations of Moringa extract between 1024-32 μM, and a dose-dependent growth reduction was identified in them. The IC50 of Moringa extract in this study was 73 μM/mol. This result showed that the extract obtained in this study has very good activity against melanoma cancer cells.
In a study, Abdulrahman Khazim Al-Asmari et al. evaluated the effects of Moringa olifera extracts on breast cancer cell lines (MDA-MB-231) and colorectal (HCT-8). They found that treating cancer cells with Moringa extract reduced cell viability. Then, using the anxin V staining method, they proved that this extract leads to apoptosis in cancer cells. The effects of the extract on cell cycle arrest were evaluated, and the cells were treated with 500 μg/ml extract. After 24 hours, it was found that the cell cycle in cancer cells had stopped at the G2/M stage (Al-Asmari,2015). Our results were in line with the results of Al-Asmari studies and previous studies that reported the anticancer effects of Moringa. In previous studies, the anticancer effects of Moringa have been attributed to compounds other than quercetin in Moringa. Lacroix et al. investigated the role of eugenol composition in inducing apoptosis in MDA-MB-231 cells and found that Bax apoptotic protein expression was increased in these cells (Lacroix,2006). On the other hand, Al-Sharif et al. reported that the combination of eugenol reduces the expression of E2F1 protein, which can have promising consequences for the treatment of breast cancer (Al-Sharif,2013).
In the study of Bich Hang Do et al. (Hoang,2020), the effects of the phenolic extract of Moringa leaves on human melanoma cells (cell lines A375 and A2058) were investigated. During their study, it was found that Moringa extract could induce apoptosis from a pathway dependent on increased oxygen species. This result has been replicated in a number of other studies (Tiloke,2013). In tae Eun Guon's study, it was found that Moringa extract leads to the production of hydroxyl radicals and hydrogen peroxide in A2058 melanoma cells and, in this way, can induce apoptosis (Guon,2017). Given that all studies have pointed to the antioxidant effects of Moringa extract and its compounds, and in addition to the fact that active oxygen species cause cancer and many diseases, the results of tae Eun Guon seem to be a contradiction, but to clarify it, two points must be noted: first, in studies that have shown the induction of apoptosis by the pathway dependent on reactive oxygen species, this pathway has been observed in cancer cells, not healthy cells, and second, reactive oxygen species, along with their destructive effects, can act as an intercellular signaling mediator (Stadtman,2001). On the other hand, overproduction of reactive oxygen species leads to increased oxidative stress as a result of cell damage and inhibition of cell function and cycle, and ultimately apoptosis (Kuo,2007). Activated oxygen species are produced by active processes in normal cells and are associated with various biological processes, including cell differentiation and gene expression (Rhee,2006). For this reason, the homeostasis of reactive oxygen species is of particular importance for cell survival. However, the relationship between the dual antioxidant effects and the production of reactive oxygen species in Moringa oleifera extract has not yet been determined.
In this study, after tumor induction in mice, the tumors were treated with Moringa extract at doses of 0.02, 0.04, and 0.08 g/kg body weight. These results showed that Moringa extract could significantly reduce tumor growth over a period of one to two weeks. On the other hand, the tumor volume at a dose of 0.02 g/kg body weight decreased more than the other two doses. In a similar study, L. Purwal et al. evaluated the anticancer effects of Moringa fruit and left on mouse model melanoma. They injected methanolic and hydroalcoholic extracts of Moringa fruits and leaves at a dose of 500 mg/kg for 15 days orally into C57 mice infected with B16F10 cells. By examining the kinetics of tumor growth, they showed that none of the treatments prepared with the mentioned concentration gave a complete response to the extract. Delay in tumor growth was observed in all groups with increasing volume doubling time, but the inhibitory effect on tumor growth was not sufficient to treat tumors (Purwal,2010). These results were contrary to the results of the present study. One of the main reasons for the difference in the results is how the extract was administered, which in the present study was done by intratumoral injection, while in the L. Purwal study, it was given orally. In the oral method, the drug enters the stomach and intestines and undergoes changes in pH and decomposition by digestive enzymes, and the amount of active ingredients decreases. On the other hand, with the changes that occur on it, the effective compounds that have anticancer effects may be reduced or disappear. In the present study, by direct injection into the tumor, the extract was directly available to the tumor without any changes and was effective. L. Purwal et al. eventually concluded that consuming Moringa as a plant food could delay tumor growth and increase the life expectancy of cancer patients (Purwal,2010).
This study found that Moringa extract with phenolic and flavonoid compounds can both cause melanoma cell death in vitro, and injecting into tumors after one week can reduce the volume of tumors.