Evaluation of antimicrobial and antitumoral activity of Garcinia mangostana L. (mangosteen) grown in Southeast Brazil1

PURPOSE: To characterize the anatomy of the fruit and leaf and the presence of phytocompounds. To evaluate the antitumor and antimicrobial activity of ethanolic extract of Garcinia mangostana L. (mangosteen) cultivated in southeastern Brazil. METHODS: Anatomical characterization and histochemical reactions were performed for structural identification and the presence of phytocompounds. Preparation of ethanolic extract of the fruit, leaf and resin of mangosteen. Culture B16-F10 melanoma cells for treatment with mangosteen ethanolic extract to determine cell viability by MTT and genotoxic effect by comet assay. Evaluation by antimicrobial activity against Staphylococcus aureus and Escherichia coli by agar diffusion test and by determination of Minimum Inhibitory Concentration (MIC). RESULTS: Our results showed many secretory canals in resin fruit and leaf; identifying lipids, starch, lignin and phenolic compounds. The leaf extract induced genotoxicity and apoptosis in B16-F10 cells, since the fragmentation of DNA in the comet assay. The ethanolic extract of mangosteen obtained in the resin, leaf and fruit showed antimicrobial activity against Staphylococcus aureus and Escherichia coli with a MIC at 0.1 mg/mL. CONCLUSION: In conclusion, we have demonstrated both antimicrobial and antitumor activity of ethanol extract of mangosteen emphasizing its therapeutic potential in infectious diseases and in cancer, such as melanoma.


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 .

Garcinia mangostana L. (mangosteen) is a plant of
Asian origin, belonging to the family Clusiaceae. It was introduced into Brazil in 1935 in Bahia, where it is still cultivated 5 . In some populations, especially in their countries of origin, the dried and ground mangosteen rind is used for medicinal purposes against dysentery and chronic diarrhea, besides being used as a homemade dye due to its color 6,7 . The biological activity of the mangosteen is subject of major scientific research, and its pericarp (rind) has been the main structure under study, demonstrating antioxidant 8 , antimicrobial 9 and antidepressant activities 10 . Considering the therapeutic potential of the plant, this study aimed to characterize the morphological aspects of Garcinia mangostana L. cultivated in Southern Bahia and to evaluate its antimicrobial and antitumoral activity.

Plant materials and preparation of ethanolic extract
The leaf, fruit and resin of Garcinia mangostana L (mangosteen) collection was carried out in the city of Una, Southern region of Bahia, Brazil. Voucher specimens were deposited in the Herbarium of Department of Biological Sciences, University Estadual de Santa Cruz, Bahia, Brazil. The samples were washed with running tap water and separated before the fruit was chopped into pieces. They were oven-dried at 42°C for 5 days and ground to powder.
Plant materials used in this study were fresh fruits (seedless without core), leaf and resin of Garcinia mangostana L. The preparation of 70% ethanolic extract of dried plant was obtained by grinding and exposure to organic solvent for 8 days.
A suspension of dried fruit (50 g) in water (150 mL) was extracted with ethanol (350 mL). Then, a rotary evaporator was used to remove the remaining alcohol and the aqueous layer and then followed by lyophilization to give water-soluble fractions.

Extraction of gamboge resin
The yellow exudate from the pericarp of the mangosteen fruit known as gamboge 11 resin was obtained after cooling and centrifuging the extract and removing alcohol, which resulted in the precipitation of resin and an aqueous supernatant purple color similar to the external appearance of mangosteen. To ensure the quality of separation of the sample, successive washes and centrifugations was taken until the characteristic yellow color prevailed and there were no other traces of dye.

Anatomical study of the mangosteen pericarp
For the anatomical description of the mangosteen pericarp, 10 μm thick sections were obtained with a rotatory microtome, and the mounted slides were stained with Astra blue and Safranin. The sections were analyzed in Photonic Microscopy (Axiostar model plus, ZEISS), x 200 magnification.

Histochemical of mangosteen pericarp and leaves
The mangosteen pericarp of the fruit and leaves were analyzed for compounds by histochemical reactions with Sudam III, Ferric chloride, Lugol and phloroglucinol acidified for identification of lipids phenolics, starch and lignin, respectively. The freehand cuts were performed with the help of razor (Gillette ®) and placed in contact with the reagent. The micrographs were obtained in Photonic Microscopy (Axiostar model plus, ZEISS), x200 magnification.

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).

Cell viability assay
The effects of mangosteen 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 × 10 4 cells/well and treated with mangosteen extract concentration 1 to 80 mg/mL for 48 h. After treatment, B16-F10 cells were briefly washed with PBS. A serumfree medium containing 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 at 37° C for 4 h. Supernatants were aspirated, and the resulting formazan crystals were dissolved in 150 μL isopropyl alcohol. Absorbance of the product was measured at 540 nm using a colorimetric MTT ELISA assay (VERSAmax Tunable microplate reader, Molecular Devices, CA, USA). The absorbance of the negative control was considered as corresponding to a viability of 100%, and the values of treated cells were calculated as percentage of the control.

Comet assay
The alkaline comet assay was performed as described by Singh et al. (1988) 13 was adapted as follows. The melanoma cell line (B16-F10) was subcultured and seeded in culture bottles.
After the process trypsinization, they were transferred to 15 mL Falcon tubes, centrifuged at 1500 rpm for 5 min. Then, the supernatant was discarded and a new aliquot of fresh DMEM/ The slides were analyzed in photonic microscope (model Axiostar plus, ZEISS) at 10X magnification. Images of 100 randomly selected cells (50 cells from each of two replicate slides). Cells were scored visually according to tail size (from undamaged -0, to maximally damaged -4). Visual scoring of comets is a valid evaluation method determined by international guidelines and recommendations for the comet assay 14,15 .

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.

Fruit and leaf anatomy and histochemistry
The analysis of paraffin-embedded sections and stained with Astra Blue and Safranin demonstrated the presence of large amounts of secretory ducts in the pericarp of fruit, primarily in the mesocarp (Figure 1 A, B).
In the evaluation of histochemical reactions were identified in secretory parenchyma of the central rib, whose contents exhibited the same appearance oxidation occurring in the pericarp (Figure 1 C) the region ducts.
The reaction with Lugol was positive for starch that can be observed in the parenchyma cells of the vascular tissue in the midrib of the leaf. The reaction to Sudam III showed not well defined, with characteristic reaction in the cuticle and uncertainty as to the content of the ducts as seen in fruit.
At midrib was also observed the presence of calcium oxalate crystals.

Antimicrobial activity
The agar diffusion test was used for antimicrobial activity against Staphylococcus aureus and Escherichia coli strains of the ethanolic extract of resin (Figure 2A), leaves ( Figure 2B) and pericarp ( Figure 2C). For these extracts, the minimum inhibitory concentration (MIC) was also determined, Chart 1.

Genotoxicity analysis by comet assay
The comet assay with ethanolic extract mangosteen leaf showed apoptosis induction in B16-F10, which was evidenced by DNA fragmentation and formation of apoptotic bodies mainly at the concentration of 1 mg/mL (Figure 4).

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 expected 1