Brazilian Red Propolis: Extracts Production, Physicochemical Characterization, and Cytotoxicity Profile for Antitumor Activity

Abstract

Brazilian red propolis has been proposed as a new source of compounds with cytotoxic activity. Red propolis is a resinous material of vegetal origin, synthesized from the bees of the Appis mellifera family, with recognized biological properties. To obtain actives of low polarity and high cytotoxic profile from red propolis, in this work, we proposed a new solvent accelerated extraction method. A complete 2³ factorial design was carried out to evaluate the influence of the independent variables or factors (e.g., temperature, number of cycles, and extraction time) on the dependent variable or response (i.e., yield of production). The extracts were analyzed by gas chromatography coupled with mass spectrometry for the identification of chemical compounds. Gas chromatography analysis revealed the presence of hydrocarbons, alcohols, ketones, ethers, and terpenes, such as lupeol, lupenone, and lupeol acetate, in most of the obtained extracts. To evaluate the cytotoxicity profile of the obtained bioactives, the 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2-H-tetrazolium bromide colorimetric assay was performed in different tumor cell lines (HCT116 and PC3). The results show that the extract obtained from 70 °C and one cycle of extraction of 10 min exhibited the highest cytotoxic activity against the tested cell lines. The highest yield, however, did not indicate the highest cytotoxic activity, but the optimal extraction conditions were indeed dependent on the temperature (i.e., 70 °C).

Keywords: antitumor activity; extraction; red propolis; supercritical liquids.

Figure 1

(a) Surface response chart of the 2³ factorial design, representing the interactions of two distinct factors (time and cycle) favoring the determination of the best extraction conditions. (b) Surface response chart representing the interactions between the temperature and number of cycles. (c) Surface response chart representing the interactions between the time and temperature variables. (d) Pareto graph of 2 interactions between the variables applied to determine the yield as a response of the developed factorial planning. The vertical interrupted red line (in d) represents the significance of the compared variables; the right-hand side of the cut has significant representativity and the left-hand side does not present significance.

Figure 2

Evaluation of the yield of red propolis extract samples obtained by the solvent accelerated extraction method, where the vertical interrupted red line represents the cutoff of 11%.

Figure 3

Cytotoxic analysis against tumor cell lines derived from human malignant neoplasms HCT116 (colon adenocarcinoma) and PC3 (prostate adenocarcinoma). - - - - Minimum percentage limit of cytotoxic activity considered to be at least moderate. ^______ Minimal percentage limit of cytotoxic activity considered strong.

Figure 4

Average inhibitory concentration graph capable of causing 50% of the maximal effect in HCT116 cells.