Evaluation of antitumoral and antimicrobial activity of Morinda citrifolia L. grown in Southeast Brazil1

PURPOSE: To evaluate the antitumor and antimicrobial activity of ethanolic extract of Morinda citrifolia L. fruit cultivated in southeastern Brazil. METHODS: Preparation ethanolic extract of the fruit of Morinda citrifolia L. Culture of melanoma cells B16-F10 for treatment with ethanolic extract of Morinda citrifolia L. fruit to determine cell viability by MTT and determination temporal effect of ethanolic extract fruit on the cell growth B16-F10 for 8 days. Evaluation of antimicrobial activity of ethanolic extract fruit against Staphylococcus aureus and Escherichia coli by determination of Minimum Inhibitory Concentration (MIC). RESULTS: The ethanolic extract of Morinda citrifolia L. fruit (10mg/mL) decreased cellular activity and inhibited 45% the rate of cell proliferation of B16-F10 melanoma treated during period studied. The ethanolic extract of Morinda citrifolia L. fruit demonstrated antimicrobial activity inhibiting the growth of both microorganisms studied. Staphylococcus aureus was less resistant to ethanolic extract of Morinda citrifolia L. fruit than Escherichia coli, 1 mg/mL and 10 mg/mL, respectively. CONCLUSION: What these results indicate that the ethanolic extract of the fruit of Morinda citrifolia L. showed antitumor activity with inhibition of viability and growth of B16-F10 cells and also showed antibacterial activity as induced inhibition of growth of Staphylococcus aureus and Escherichia coli.


Introduction
Cutaneous melanoma is considered the most serious type of skin cancer. It is a highly lethal and very invasive neoplasm, accounting for less than 5 % of all skin cancer cases. Despite its low incidence, it is considered a problem for public health due to the significant raise in the number of cases, exceeding other malignancies growth rate 1-3 . In the last few years there has been growing interest in natural products with biological activity, with relevance to anticancer activity. A large diversity of plants has been extensively investigated, these plants being secondary metabolite producers.
These studies on the biotechnological potential of plants sources promising therapeutic agents are mentioned, with antibacterial, antiviral, antitumor and immunossupressive potential 3,4 .
Morinda citrifolia L, known as Noni, belongs to the Rubiaceae family native to Southeast Asia and secularly used in Polynesian traditional medicine. Noni juice is widely used in complementary medicine due to its probable antioxidant, antiinflammatory and antitumor effects against diseases such as cancer, atherosclerosis, diabetes and ulcer 5 To what e ffxtent the findings from experimental pharmacological studies are of potential clinical relevance is not clear at present 7 .
Many pharmacological studies of Morinda citrifolia L juice and isolated compounds from the fruit has been published.
These compounds including iridoids, flavonoids, lignans, coumarins and anthraquinones 7,8 . The purposes of this study were to evaluate antioxidant, antitumoral and antimicrobial activity of ethanolic extract from Morinda citrifolia L. fruit grown in Southeast Brazil. Plant materials used in this study were fresh fruits (seedless without core) of Morinda citrifolia L.. The preparation of 70% ethanolic extract of dried fruit (50 g) was obtained by grinding and exposure to organic solvent. A suspension of dried fruit (50 g) in water (150 mL) was extracted with ethanol (350 mL) for 8 days. The aqueous layer was evaporated and then followed by lyophilization to give a water-soluble fraction.

Cell Culture
The mouse melanoma B16-F10 cell line was purchased from Rio de Janeiro Cell Bank (BCRJ/UFRJ). The cells were maintained at 37 °C in an incubator with a humidified atmosphere of 5 % CO 2 and cultured in DMEM/F12 supplemented with 10 % heat-inactivated FBS, streptomycin (100 μg/mL) and penicillin (100 units/mL).

B16-F10 cell growth inhibition
B16-F10 cells at 80% confluence, the cells were harvested with trypsin, and serum-free medium was used to obtained a singlecell suspension. The cells were then seeded in 06-well plates at a density of 200,000 cells/well. After 24 h, the wells were replaced with fresh medium, including FBS. Next, the wells were treated with 10mg/mL ethanolic extract of Morinda citrifolia L. and the cell numbers were counted following 1-8 days. A control group was prepared simultaneously and a growth curve was generated.

Cell Viability Assay
The effects of Noni extract treatment on cell viability were determined by MTT assay, which is based on the reduction of a tetrazolium salt by mitochondrial dehydrogenase in viable cells.
For all experimental groups, cells were seeded in 96-well plates at a density of 1 × 104 cells/well and treated with Noni extract at a final concentration of 1 to 80mg/mL . After 48 h, 50 μL of MTT stock solution (2 mg/mL) was added to each well to reach a total reaction volume of 250 μL, and the plates were incubated for an additional 4 h.
Supernatants were aspirated, and the resulting formazan crystals were dissolved in 150 μL isopropyl alcohol. Absorbance was measured at 540 nm using a colorimetric MTT ELISA assay (VERSAmax Tunable microplate reader, Molecular Devices, CA, USA).

Statistical Analysis
Data are presented as mean ± SD of four independent experiments. Statistical analysis among groups was performed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls Multiple Range Test. GraphPad Prism v.3.0 software was used, p < 0.05 was considered to be statistically significant.

Discussion
Malignant melanoma is a cancer with a high incidence, malignancy and poor prognosis. This cancer is highly metastatic and high mortality rate. Currently, there are no methods or effective drugs for treatment and thus new methods are necessarily    can have a major role in the anticancer therapy 10 .

Conclusions
The ethanolic extract Morinda citrifolia L. fruit induce cell growth inhibition on Staphylococcus aureus and Escherichia coli and cell growth inhibition on B16-F10 cells. Considering the acquire resistance to antibiotics used and chemoresistance exhibited by melanoma towards conventional chemotherapy drugs, this novel compound may provide promising improvements in the therapeutic approach to infectious diseases and melanoma treatment.