2. PARTE II: ARTIGO CIENTÍFICO
2.2. Material and methods
2.2.2. Evaluation of antiproliferative activity
In this study, three different tumor cell lines were used: human breast adenocarcinoma (MCF-7), human cervical adenocarcinoma (HeLa) and human glioblastoma (U343). A normal human cell line (lung fibroblasts, GM07492A) was included to evaluate possible
22 selective activity of the natural medicinal product under investigation. Different cell lines were maintained as monolayers in plastic culture medium (HAM-F10 + DMEM, 1:1, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Nutricell), antibiotics (0.01 mg/mL streptomycin and 0.005 mg/mL penicillin; Sigma-Aldrich) and 2.38 mg/mL Hepes (SigmaAldrich). Cells were incubated at 36.5°C in humidified 5% CO2 atmosphere.
Evaluation of the antiproliferative activity of the samples was performed using the in vitro toxicology colorimetric assay-XTT Kit (Roche Diagnostics) according to the manufacturer's guidelines. For the experiments, 1 x 104 cells were seeded in 96-well microplates, each well received a maximum of 100 μL culture medium (HAM F10 + DMEM, 1:1) supplemented with 10% fetal bovine serum containing different sample concentrations, which ranged from 9.77 to 1250 µg/mL. Negative (untreated), solvent (1% DMSO) and positive (25% DMSO) control wells were also included. After incubation with the substances at 37 °C for 24 h, the culture medium was removed, the cells were washed with 100 μL PBS to remove treatments and exposed to 100 μL HAM-F10 culture medium without phenol red. Then, 25 μL of XTT were added to each well and the microplates were incubated at 37 °C for 17 h. The absorbance of the samples was determined using a multiplate reader (Asys-UVM 340/Microwin 2000 ELISA) at a wavelength of 450 nm and a reference length of 620 nm. The number of soluble products formed (formazan) was proportional to the number of viable cells. The negative control group was designated as 100% and the results were expressed as a percentage of the negative control. The experiments were performed in triplicate.
23 2.2.3 Statistical analysis
Cytotoxicity was assessed using the IC50 value (50% cell growth inhibition) as a response parameter, which was calculated with the GraphPad Prism 5.0 program (GraphPad Sofware, San Diego, CA, USA) by plotting cell survival against the respective concentrations of the natural products tested. One-way ANOVA was used for comparison of means (p < 0.05).
Figure 2. HPLC-DAD profile of red propolis hydroalcoholic extract (RPHE; A) and its fractions obtained in n-hexanes (HF; B), dichloromethane (DF; C), ethyl acetate (AF;
D), and n-butanol (BF; E). Identity of main constituents: liquiritigenin (1), formononetin (2), liquiritigenin (3), vestitol (4), neovestitol (5), medicarpin (6), 7-O-methylvestitol (7), mixture of guttiferone E and xanthochymol (8), oblongifolin B (9).
2.3 RESULTS
Table 1 presents the results expressed by inhibitory concentration of 50% cell growth (IC50).The results showed that, for each cell line-GM07492A (human lung fibroblasts, non-tumor cells), MCF-7 (breast adenocarcinoma), U343 (glioblastoma) and HeLa (cervix adenocarcinoma)-RPHE revealed an IC50 of 137.0, 262.7, 138.2 and 137.4 µg/mL, HF an IC50 of 33.8, 133.3, 33.8 and 34.5 µg/mL, DF an IC50 of 78.9, 237.6, 71.6 and 99.6 µg/mL, and AF and BF an IC50 of higher than 1250 µg/mL in all cell lines.
RPHE, HF and DF therefore showed cytotoxic activity to all tumor cell lines, but also to
24 the normal cell line, indicating selectivity absence. HF presented the highest cytotoxic potential with the lowest IC50, being significantly different from those observed for RPHE. The cytotoxicity of HF might be attributed to its chemical composition, consisting predominantly of polyprenylated benzophenones 8 and 9 (guttiferone E, xanthochymol and oblongifolin B), described in the literature as pro apoptotic compounds.
Table 1. Inhibitory concentrations (IC50, µg/mL) obtained from RPHE-treated non-tumoral (GM07492A) and non-tumoral (MCF-7, U343 and HeLa) cell cultures and its
Note: RPHE: red propolis hydroalcoholic extract; HF: hexanes fraction; DF: dichloromethane fraction; AF:
ethyl acetate fraction; BF: n-butanol fraction. GM07492A: non-tumor human lung fibroblast; MCF-7:
human breast adenocarcinoma; U343: human glioblastoma; HeLa: human cervical adenocarcinoma. Values are mean ± SD, n: 3. a Significantly different from the RPHE treatment (p < 0.05).
2.4 Discussion
In this sense, Lin et al. (2019) reported cytotoxicity of a mixture of guttiferone E and xanthochymol in five types of human cancer cell lines: leukemic (HEL, IC50 = 11.27 μg/mL; K562, IC50 = 10.92 μg/mL), cervical (HeLa, IC50 = 5.46 μg/mL), breast (MCF-7, IC50 = 4.68 μg/mL) and lung (A549, IC50 = 6.10 μg/mL). These results demonstrate that these two substances present in the hexanes fraction of red propolis corroborate their cytotoxic potential. Novak et al. (2014) observed that a fraction of the ethanolic extract of Brazilian red propolis, containing xanthochymol and formononetin, showed antiproliferative effect in acute promyelocytic leukemia cell lines (HL-60, IC50 = 20.5 μg/mL), human chronic myeloid leukemia (K562). IC50 = 30.3 μg/mL), multiple myeloma (RPMI8226, IC50 = 32.6 μg/mL) and murine melanoma (B16F10, IC50 = 25.7 μg/mL). The fraction showed more promising antiproliferative effect than that observed by the ethanolic extract. Through the MTT test, the ethyl acetate fractions of the red
25 propolis extract revealed cytotoxic activity in HT-29 (human colorectal adenocarcinoma) and HCT-116 (human colorectal carcinoma) and non-tumor cell line Vero (monkey kidney epithelium). IC50 values ranged from 40.32 to 105.23 µg/mL, with the lowest IC50
values corresponding to the tumoral lines tested, indicating selectivity. Chemical analysis of the fractions indicated the presence of formononetin, vestitol, biochanin A, liquiritigenin and the guttiferone E xanthochymol mixture (Santos et al. 2019). The antiproliferative activity of the hydroethanolic extract of red propolis and two of its fractions (J and L) was evaluated in human laryngeal epidermoid carcinoma cells (Hep-2) by Da Silva Frozza et al. (2017). The chemical profile of fraction J revealed the presence of formononetin, liquiritigenin, medicarpin, vestitol, isovestitol and (3S)-ferreirin, and of fraction L, only liquiritigenin. The results showed an IC50 of 145.40 μg/mL for the extract, 60.96 μg/mL for the J fraction and 74.60 μg/mL for the L fraction.
Thus, the fractions showed a greater cytotoxic effect than the extract. Fractionation of the extract leads to a decrease in the number of chemicals present in the fraction and may increase the concentration of active compounds when compared to the exposure of cells to the crude extract (da Silva Frozza et al. 2017). The results showed that red propolis presents substances with antiproliferative activity, indicating that the hexanes fraction may have substances with antitumor potential. Further studies should be conducted with guttiferone E, xanthochymol and oblongifolin B, substances predominantly identified in the hexanes fraction.
ACKNOWLEDGEMENTS
This work was supported by the São Paulo Research Foundation (FAPESP, Brazil; grants #2016/24269-7 and #2017/04138-8) and by Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil). K.S. Freitas was recipient
26 of a master fellowship from FAPESP (grants #2018/02370-3). The authors are grateful to the National Council for Scientific and Technological Development (CNPq, Brazil) for the fellowships granted. The authors declare that there is no conflict of interest.
27
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4. ANEXO
BIOFARMASI J NAT PROD BIOCHEM
Volume 18, Number 2, August 2020 E-ISSN: 2580-2550
Pages: 66-69 DOI: 10.13057/biofar/f180203
Evaluation of the antiproliferative activity of red propolis hydroalcoholic extract and its fractions obtained by partition
IARA SILVA SQUARISI1,*, KAROLINE SOARES DE FREITAS1,*, DANIELI CRISTINA LEMES1,
IARA SILVA SQUARISI1,*, KAROLINE SOARES DE FREITAS1,*, DANIELI CRISTINA LEMES1,