Antiproliferative effect of hydroalcoholic brown propolis extract on tumor and non-tumor cellsAbstract
Studies show that propolis has antimicrobial, antifungal, antiviral, anti-inflammatory, antioxidant, antitumor, and immunomodulatory properties, and may protect against diseases such as diabetes, cardiovascular disease, and cancer. We aimed to extract compounds of brown propolis with hydroalcoholic solvents and evaluate their cytotoxic activity on tumor and non-tumor cells by MTT test. We tested the solute:solvent ratio (ethanol:water) and extraction time in a Shaker incubator (710 rpm) before conducting a central composite rotational design (CCRD) to optimize time and solvent mixture. We found that a temperature of 80 °C and a solvent concentration of 90:10 were the best extraction conditions for phenolic compounds, especially pinocembrin and cinnamic acid. The extract showed antioxidant capacity, acid characteristics, low humidity, and the presence of ash, lipids, and soluble solids. The cytotoxicity test with normal kidney cells of Macaca mullata (LLC-MK2) and human lung tumor cells (A549) showed no statistical difference from the negative control. For liver tumor cells (HuH7.5), different concentrations showed cytotoxic/antiproliferative activity. Thus, the data indicate that this product deserves prominence in the search for new applications.
Keywords:
brown propolis; cancer; cytotoxicity test; antioxidant; phenolic compounds; functional food
Resumo
Estudos mostram que a própolis possui propriedades antimicrobianas, antifúngicas, antivirais, anti-inflamatórias, antioxidantes, antitumorais e imunomoduladoras, podendo proteger contra doenças como diabetes, doenças cardiovasculares e câncer. O objetivo deste estudo foi extrair compostos de própolis marrom com solventes hidroalcoólicos e avaliar sua atividade citotóxica para células tumorais e não-tumorais, pelo teste MTT. A relação soluto:solvente (etanol:água) e o tempo de extração foram testados em uma incubadora Shaker (710 rpm) antes de realizar um projeto composto rotacional central (DCCR) para otimização do tempo e mistura do solvente. Verificou-se que a temperatura de 80 oC e a concentração de solvente de 90:10 foram as melhores condições de extração em termos de compostos fenólicos, especialmente pinocembrina e ácido cinâmico. O extrato apresentou capacidade antioxidante, característica ácida, baixa umidade, presença de cinzas, lipídios e sólidos solúveis. O teste de citotoxicidade com células normais de rim de Macaca mullata (LLC-MK2) e de tumor pulmonar humano (A549) não apresentou diferença estatística do controle negativo. Para os tumores hepáticos (HuH7.5), diferentes concentrações apresentaram citotoxicidade/atividade antiproliferativa. Assim, os dados do presente estudo demonstraram que este produto merece destaque na busca por novas aplicações.
Palavras-chave:
própolis marrom; câncer; teste de citotoxicidade; antioxidantes; compostos fenólicos; alimento funcional
1. Introduction
Functional foods are so called because they include compounds that are physiologically active in the body, producing metabolic effects capable of regulating body functions to promote health and, consequently, assist in the protection and treatment of diseases (Maciel et al., 2018).
Among these foods, propolis stands out for having biological and therapeutic properties, such as antimicrobial, antioxidant (Mouhoubi-Tafinine et al., 2016; Zabaiou et al., 2017), anti-inflammatory (Moura et al., 2011), anti-ulcer (Hata et al., 2012), antiseptic, healing (Freires et al., 2019), antiviral (Refaat et al., 2021) and antitumoral activities and may help prevent chronic diseases such as diabetes, cancer and cardiovascular diseases (Pasupuleti et al., 2017). Propolis has been proven to contain compounds such as phenolic acids and flavonoids (Mirbagheri et al., 2023).
The chemical structure and characteristics of propolis are influenced by the regions covered by the bees, climatic conditions, genetic variety of the hive, botanical source, and harvest time in the year (Novak et al., 2014). Brown propolis, for example, comes from southern Brazil and is found in Paraná and Santa Catarina (Machado et al., 2016a).
The search for compounds with anticancer effects is of great importance since cancer is one of the main causes of death in the world (WHO, 2021). As a result, many people seek new antitumor agents or combinations that are more effective for the treatment/cure, including medicinal plants or functional foods rich in compounds with cytotoxic/antitumor and pharmacological activities (El-Lakkany et al., 2021).
One of the purposes of this work was to help search for new molecules that are effective in treating cancer and add value to propolis, a food with pharmaceutical potential. Thus, we evaluated the cytotoxic/antiproliferative activity of hydroalcoholic extracts of brown propolis produced in benchtop, through an optimized process, in culture of hepatocellular carcinoma cells (HuH7.5), non-small cell pulmonary adenocarcinoma (A549) and normal kidney cells of Macaca mullata (LLC-MK2). Additionally, we characterized the pollen grains of brown propolis taxonomically to assess the botanical origin of the extracted compounds.
2. Material and Methods
2.1. Extract production
About 1 kg of crude brown propolis was obtained in its solid state at a fair of rural producers in the municipality of Francisco Beltrão, Paraná, Brazil (26°08’27” S, 53°09’21” W), in May 2021. Propolis samples were kept in the Plant Micromorphology Laboratory of the State University of Feira de Santana (#PUEFS 2898).
The propolis sample underwent chemical treatment following the method outlined by Matos et al. (2014). A minimum of 500 pollen grains was set as the criterion for the pollen census. To determine the botanical affinity of the pollen types, we followed the guidelines by Santos (2011). Taxonomic identification of the pollen types was carried out by comparing them with the pollen library of the Laboratory of Plant Micromorphology (UEFS) and consulting published pollen catalogs (Roubik and Moreno, 1991; Mouga and Dec, 2012).
2.2. Definition of solute: solvent ratio and extraction time
Initially, we carried out an extraction experiment following Machado et al. (2016b) with adaptations, under fixed conditions of time, temperature, and solvent type to define the most appropriate amount of brown propolis solute to the ethanol solvent by determining the content of phenolic compounds.
We mixed 0.01 L of P.A. ethanol, carefully heated at 60 °C, with different concentrations of propolis (0.05, 0.10, 0.25, 0.5, 1.0, 1.5, 2.0 and 3.0 g). The extraction occurred at this same temperature for 30 minutes under constant agitation in a Shaker incubator (710 rpm). The extract was centrifuged at 3,700 rpm for 10 min at room temperature. After centrifugation, the supernatant was transferred to a 0.05 L beaker, and 0.01 L of P.A. ethanol was added to the residue, with vigorous vortex agitation for homogenization, and the same centrifugation process was repeated.
Due to the varied extraction times of propolis in the literature, this parameter was evaluated using a uni-variable analysis with different values (5, 15, 30, 45, 60, and 300 min), with the response variable being the phenolic content.
The statistical difference between the tests for the solute:solvent ratio and for the extraction time was evaluated by an analysis of variance (ANOVA) followed by Duncan’s test (α = 0.05) in the free software SASM-Agri.
2.3. Optimization of the extraction conditions
After defining the solute:solvent ratio and the most appropriate extraction time, a central rotational composite design was designed for time optimization and solvent mixing. To find the yield range of higher phenolic extraction, we conducted several trials using five levels and three factors, with three replications of the central point, called star planning according to Barros Neto et al. (2001). The independent variables were temperature (°C) and solvent mixture (proportion (%) ethanol:water).
We processed and interpreted the experimental data in the program Statistica for Windows 5.0 to analyze the factorial variance and generate the response surface graphs. After defining the optimal extraction parameters, we used the optimum point to obtain enough extract to analyze physicochemical properties, phenolic profile, bioactive compounds, and biological activity in cell culture.
2.4. Analysis of phenolic compounds
To determine the total phenolic compounds of propolis extracts, the Folin-Ciocalteu spectrophotometric method was used. The results were expressed in milligrams of gallic acid per 100 g of sample (mg AG 100 g-1).
The phenolic profile in the propolis samples was produced using high-performance liquid chromatography (Varian – 900 LC) coupled with a photodiode array detector (DAD) and a C18 reversed-phase column (ACE column 250 x 4.6 mm x 5 μm). Standards were used for the following compounds: gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, cinnamic acid, rutin, myricetin, catechin, epicatechin, quercetin, kaempferol, pinocembrin, crisin, and glucosylxanthone mangiferin. The mobile phase consisted of H2O:H3PO4 (99.8:0.2 v v-1) (A) and CH3OH 100% (B) eluted in a gradient mode starting at 30% of B; 64% of B in 15 min; 75% of B in 26 min; 95% of B in 28 min; 30% of B in 32 min and maintaining it up to 42 min for conditioning. UV spectra were recorded from 200 to 390 nm and, for quantification purposes, peak areas were determined in concentrations ranging from 1.0 to 50 mg L−1.
2.5. Analysis of the antioxidant capacity
We used the sequestration method of the free radical DPPH (1.1-diphenyl-2-picrilidrazil) to determine the propolis extract’s antioxidant activity. To determine the total antioxidant activity (AAT), we replaced the absorbance equivalent to 50% of the concentration of DPPH and found the result corresponding to the sample needed to reduce by 50% the initial concentration of the DPPH radical (EC50).
2.6. Physicochemical analysis
For the physicochemical analysis (n = 3) of the hydroalcoholic extract of brown propolis, we followed the techniques recommended in the manual of Physicochemical Methods for Food Analysis of the Adolfo Lutz Institute (IAL, 2008), in which pH, total acidity, humidity, lipid content, soluble solids, minerals, and total sugars are assessed.
2.7. Test for cytotoxicity/antiproliferative activity
The extract produced was rotated and evaporated until becoming a paste and, afterward, diluted with 100 μL of PA ethanol and 900 μL of Dulbecco's Modified Eagle's Medium - high glucose (DMEM). We then diluted it with a culture medium supplemented with fetal bovine serum at the following concentrations: 0.25; 0.5; 1.0; 2.5; 5.0; 10; 25; 50; 100 μg mL-1.
Cell lines (HuH7.5, A549, and LLC-MK2) were grown in 25 cm2 culture flasks, containing 0.010 L of DMEM culture medium, supplemented with 10% fetal bovine serum and incubated in an incubator with 5% CO2 and temperature control at 37 °C until reaching the required volume for each strain to be sown.
The cytotoxicity/antitumor activity assay of MTT [3-(4.5-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide] was performed as described by Mosmann (1983), with modifications. Culture plates of 96 wells were used, in which 1.0 x 104 HuH7.5 cells, 3.0 x 104 cells A549, or 1.0 x 105 cells LLC-MK2 were sown. After 24 hours, to allow stabilization, the culture medium was discarded and 100 µL of the treatments were added: culture medium (negative control), methyl-methane-sulfonate cytotoxic agent (MMS - 500 µM) (positive control), solvent control (10 µL ethanol per mL of culture medium supplemented with serum) and treatments with different concentrations of propolis extract. After 24, 48, and 72 hours, we measured the absorbances in microplate beds (Thermo Plate) at 560 nm in a UV-Vis spectrophotometer.
The mean and standard deviation of absorbances were compared using the Dunnet mean comparison test (n = 4, α = 0.05) in the Action Stat software. We estimated the percentage of cell viability (VC) by the ratio between the absorbances of the treatment and the negative control.
3. Results and Discussions
3.1. Pollen spectrum
The pollen spectrum of the analyzed propolis sample comprises 29 pollen types, with abundance ranging from 1% to 25%. The most prevalent pollen types in the propolis sample, each representing at least 5% of the pollen census, include Eucalyptus sp. - Myrtaceae (25%); Cataranthus sp. - Apocynaceae (9.2%); Monocotyledon type (8.5%); Sphagneticola sp. - Asteraceae (7.8%); Scoparia sp. - Plantaginaceae (7.2%), and Psidium sp. – Myrtaceae (5.9%).
The highly represented pollen types belong to plants known to be nectariferous or polleniferous, suggesting a trophic relationship with bees. Resiniferous and oleiferous plants were also documented, primarily represented by the pollen of Anacardiaceae (4.6%), Malpighiaceae (3.9%), Cecropia sp.- Urticaceae (2.0%), and Ilex sp. – Aquifoliaceae (1.3%). Most of the identified pollen types indicate the broad trophic foraging behavior of bees, encompassing nectar and pollen sources, including Allophylus sp.- Sapindaceae (4.6%), Arecaceae (1.3%), Borreria sp.- Rubiaceae, Citrus sp. - Rutaceae, and Foeniculum sp. - Apiaceae (0.7% each).
We did not detect any pollen from Copaifera langsdorffii in the analyzed sample. This absence raises questions about the role of this particular species in the production of brown propolis in southern Brazil. While Copaifera langsdorffii is often associated with certain types of propolis, our findings indicate that other plant species may contribute more significantly to the composition of the propolis in this region. The presence of resiniferous and oleiferous plants further supports the idea of diverse botanical sources contributing to the properties of propolis. This diverse pollen composition reflects the broad foraging behavior of bees, which encompasses various nectar and pollen sources, and indicates that the composition of brown propolis can vary significantly depending on the local flora and the foraging habits of the bees.
3.2. Solute concentration
The results of the phenolic compounds concentrations for the determination of the solute:solvent ratio (Table 1) indicate a statistically significant difference between the weights evaluated, and the test with 0.25 g of crude propolis showed a better result, with 40.65 μg mg-1 of phenolic compounds.
Mean content and standard deviation of phenolic compounds obtained for each extraction assay with the different concentrations of brown propolis at 60 °C for 30 minutes.
3.3. Extraction time
The differences between the results of the concentrations of phenolic compounds for the determination of the extraction time (Table 2) were statistically significant, and the assay with the extraction of 5 hours (300 minutes) showed a higher value, of 45.66 μg mg-1 of phenolic compound. However, the concentration of phenolic compounds in the extraction of 60 minutes (42.38 μg mg-1) was very close numerically to the extraction of 5 hours. Thus, considering the cost-benefit of the process, the ideal extraction time for this work was 60 minutes.
Mean content and standard deviation of phenolic compounds obtained from each extraction assay with different extraction times at a solute:solvent ratio of 0.25 g:mL-1 at 60 °C.
3.4. Ethanol concentration and extraction temperature by experimental design
The results of the concentrations of phenolic compounds, obtained from the central experimental design rotational compound, to determine the optimum condition of ethanol concentration and extraction temperature (Table 3) indicate that the different assays showed very different values from each other, and the highest content was obtained in assay 4 (44.36 μg mg-1 of phenolic compounds).
Mean content and standard deviation of phenolic compounds obtained from each trial of the extractions of the experimental design.
Statistically, the factors temperature, % ethanol, and interaction between % ethanol and temperature showed a p-value less than or equal to 0.05. The correlation coefficient of the model was R2 = 0.99666, showing the adequacy of the model. The Pareto chart demonstrates that the variables temperature, ethanol concentration, and interaction between ethanol concentration and temperature were significant for the content of phenolic compounds.
The contour curve plot (Figure 1) shows that the greatest response regarding phenolic compounds occurred at 80 °C with solvent concentration corresponding to 90% ethanol and 10% water.
Response surface with two factors (ethanol concentration and temperature) for the content of phenolic compounds.
Thus, the parameters chosen for the hydroalcoholic extraction of brown propolis were: 0.25 g of crude brown propolis for 0.01 L of solvent (90% ethanol and 10% water) at 80 °C for 60 minutes.
3.5. Determination of the antioxidant capacity and phenolic profile of the extract
The produced hydroalcoholic extract of brown propolis presented EC50 of 3.34 ± 0.29 mg mL-1. This value was close to the EC50 of 3.97 mg mL-1 for propolis extract from Alagoas (Silva et al., 2019), but lower than that of Araújo et al. (2020a), which had a high value of EC50 (93.92 ± 21.76 mL-1) for propolis extract using methanol as solvent.
It is widely known that endogenous stimuli, such as cellular metabolism, and exogenous agents, such as UV radiation, toxins, and drugs, generate reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide anion (O2-) and hydroxyl ion (HO-), in addition to reactive nitrogen species (RNS), such as nitric oxide (NO). These reactive species can induce oxidative modifications in biomolecules such as carbohydrates, proteins, lipids, and nucleic acids, resulting in cell damage and eventually cell death. The excess of ROS in cells can trigger harmful effects such as cancer, neurodegenerative diseases, anemia, ischemia, and low-density lipoprotein oxidation, the latter leading to cardiovascular problems (Viuda-Martos et al., 2008; Sosa et al., 2013; Arfin et al., 2021). The antioxidant activity of propolis is particularly relevant, because the antioxidants present may contribute to its chemotherapeutic and antitumor effects. In addition, the choice of solvent can influence the results since different solvents can extract bioactive compounds with variable antioxidant capacities.
The phenolic compounds identified in the propolis sample by using HPLC-DAD (Table 4) were, in decreasing order of concentration: Pinocembrin, Cinnamic acid, Ferulic acid, Chlorogenic acid, Caffeic acid, p-Coumaric acid, Catechin, Crisin, Gallic acid. Waller et al. (2017) and Zhang et al. (2023) also identified and quantified these compounds in propolis.
The phenolic composition of brown propolis is highly variable and depends on its botanical and geographical origin, as demonstrated in some studies (Bankova et al., 2002; Tiveron et al., 2016; Ribeiro et al., 2023). Identifying propolis botanical sources is one of the major challenges in this field of study. Although some species such as Baccharis dracunculifolia (green propolis) and Dalbergia ecastaphyllum (red propolis) are well established as primary sources, brown propolis can include a variety of plant sources (Santos et al., 2021).
Determining total phenolic and flavonoid content in propolis samples has been widely used to assess biological properties (Sawaya et al., 2011). Herein, we identified compounds such as caffeic acid, ferulic acid, chlorogenic acid, catechin, and gallic. These compounds are known for their antioxidant properties and potential chemotherapeutic effects (Waller et al., 2017). Although pinocembrin is commonly associated with propolis from Populus spp. (Bankova et al., 2002; Park et al., 2002), our sample, which is not derived from Populus, also presented this compound, reflecting the chemical diversity of brown propolis from other botanical sources, such as Pinus spp., Eucalyptus spp., and Baccharis dracunculifolia and Araucaria angustifolia (Fonseca et al., 2011; Santos et al., 2021).
Additionally, compounds such as caffeic acid, ferulic acid, and catechin reinforce the relevance of our sample since they are associated with important antioxidant and antitumor properties (Tiveron et al., 2016; Ribeiro et al., 2023). The presence of prenylated phenolic acids and flavonols, such as artepillin C and quercetin, also highlights the contribution of regional plant sources, as observed in propolis from southern Brazil (Machado et al., 2021; Araújo et al., 2020b). These phenolic characteristics confirm the potential of brown propolis as a promising therapeutic agent due to its complex bioactive composition.
3.6. Physical and chemical characteristics of the extract
The physical-chemical analysis for the hydroalcoholic extract of brown propolis shows that the extract is acidic (pH = 4.77 ± 0.11, total acidity = 15.85 ± 0.39 mEq L-1). The pH of the alcohol extracts of propolis from Africanized Apis mellifera bees produced by three different production techniques also presented acidic characteristics (Souza et al., 2010).
Regarding total soluble solids, a high index (21.75 ± 0.20 °Brix) can be observed in the propolis extract produced. The percentage of humidity found in the hydroalcoholic extract of brown propolis was low, 0.72 ± 0.09%. However, compared with the results of crude propolis, the value is similar to the results in the literature. Nunes et al. (2018) obtained a result of 8.03 ± 0.12% humidity in the samples of crude brown propolis and Silva et al. (2006) found results ranging from 1.94 to 2.78% in propolis samples from Paraíba.
For the analysis of lipids, a percentage of 4.44 ± 0.19% was found in the hydroalcoholic propolis extract. Nunes et al. (2018) found higher values for crude samples of brown (11.04 ± 0.12%), green (8.19 ± 0.64%), and red propolis (15.61 ± 1.01%).
Regarding the analysis of total ash, a small percentage was found in the hydroalcoholic extract of brown propolis: 0.017 ± 0.005%. Compared with the result obtained by Nunes et al. (2018) in crude brown propolis, the result is low, given that their study found a value of 1.35 ± 0.19% of total ash.
3.7. Cytotoxic/antiproliferative activity of the extract
Absorbance data obtained with normal kidney cells of Macaca mullata (LLC-MK2) (Figure 2A) indicate that no concentration of the extract (μg mL-1) in none of the evaluated times (24, 48, 72 hours) presented a cytotoxic effect for these cells. Moreover, it is worth mentioning that the cell viability of the groups treated with the extract was greater than 80% (24 hours), 84% (48 hours), and 68% (72 hours).
Mean absorbances and standard deviations of normal kidney cells of Macaca mullata (LLC-MK2) (A), human liver tumor cells (HuH7.5) (B) and human lung tumor cells (A549) (C) treated for 24, 48 and 72 hours with the concentrations (μg mL-1) of the hydroalcoholic extract of brown propolis. NC: Negative Control; PC: Positive Control; SC: Solvent Control. *Result statistically different from the negative control (Dunnet test, p < 0.05).
Campos (2019), testing concentrations higher than those of the present study (250; 500; 750; 1000; 2000 µg mL-1) with methanolic extract (70%) of Scaptotrigona affinis postica propolis showed its toxicity to the cell line LLC-MK2. Araújo et al. (2011), performing an acute toxicity test with hydroalcoholic propolis extract of Scaptotrigona affinis postica, found that it did not induce the death of mice, even when receiving high doses (1000, 2000, and 4000 mg kg-1) indicating, as in the present study, the absence of toxicity of propolis hydroalcoholic extracts, and this effect may be related to the solvent used.
For hepatocellular carcinoma cells (HuH7.5) (Figure 2B), concentrations above 10 µg mL-1, at times of 24 and 48 hours, and all concentrations tested of the extract (0.25 to 100 µg mL-1) at the time of 72 hours showed cytotoxic/antiproliferative effect. Even at the lowest concentration (0.25 µg mL-1), one can observe this effect, and viability reaches 0% at the concentration of 100 µg mL-1 at all evaluated times. In general, cell viability decreased with increasing concentration at all times.
According to Kim et al. (2013), a phenethylic ester of caffeic acid (CAPE), a phenolic compound present in the hydroalcoholic extract of brown propolis in the present study (Table 4), has anticancer activity in various types of malignant diseases. The authors describe that treatment with CAPE induces apoptosis of hepatocellular carcinoma (SK-Hep1) cells. As the present study also used the same type of tissue, it is possible that the cytotoxic effect occurred by activating apoptosis induced by caffeic acid.
Furthermore, cinnamic acid also present in the present extract (Table 4) has antitumor potential in melanoma (Lima et al., 2018) and hepatocellular, colon, and breast carcinoma cells (Ruwizhi and Aderibigbe, 2020). Several action mechanisms have been proposed for cinnamic acid, such as induction of apoptosis in cells with irreversible DNA damage, cytoskeletal damage (Liang et al., 2017), prevention of cell multiplication (Sova et al., 2013), inhibition of NF-κB, AP-1, and STAT3, as well as decrease of MAPK and PI3K signaling/Akt (Tsai et al., 2013), interruption of metastasis stages (Lima et al., 2018) and reduction of the invasive capacity of cancer cells (Yen et al., 2011). These mechanisms may have acted in the HuH7.5 cells of our study, proving the antitumor activity of the hydroalcoholic extract of brown propolis.
Regarding antioxidant activities, Machado et al. (2016a, b) conclude that these may assist in the antitumor activity of brown propolis, and `Popolo et al. (2009) demonstrate the antiproliferative action of Cuban brown propolis extract against human breast cancer cells.
For human lung tumor cells (A2549) (Figure 2C), one can observe that no concentration of the extract, in none of the evaluated times (24, 48, 72 hours), presented a cytotoxic effect, as cell viability was greater than 80% (24 hours), 89% (48 hours) and 94% (72 hours).
4. Conclusion
The results of this study demonstrate the successful extraction of bioactive compounds from brown propolis using a hydroalcoholic extraction method with a solute ratio of 0.25 g of crude propolis to 10 mL of solvent at 80 °C with 90% ethanol and 10% water. Our study presents unprecedented findings regarding the physicochemical properties of brown propolis, including its solids content and acidity, while confirming its antioxidant activity through the DPPH method. Additionally, we observed selective cytotoxic and antiproliferative effects against human liver carcinoma cells, with no cytotoxicity toward normal cells.
Thus, the data of this study indicate that brown propolis is a promising source of bioactive compounds with potential anticancer properties. The presence of specific phenolic compounds such as pinocembrin, caffeic acid, and ferulic acid, known for their cytotoxic effects on cancer cells, highlights its relevance in the search for new therapeutic molecules. Importantly, the observed selective cytotoxicity towards cancer cells without significant effects on healthy cells underscores the potential of brown propolis as a safer alternative in cancer treatment strategies.
Acknowledgements
The authors thank beekeeper Ademir Cuba, the Federal Technological University of Paraná (UTFPR) Francisco Beltrão, Paraná, Brazil, and the National Council for Scientific and Technological Development for the Productivity Grant to Profa. Dra.. Elisângela Düsman (CNPq #305029/2022-3).
References
-
ARAÚJO, A.B.C., DIAS, A.J.S., FREITAS, R.L.S., NUNES, A.R., SILVA, G.A. and SANTOS, D.C., 2020a. Chemical prospecting and evaluation of biological activity of propolis from Salinópolis, Pará. Revista Virtual de Química, vol. 12, no. 2, pp. 492-499. http://doi.org/10.21577/1984-6835.20200039
» http://doi.org/10.21577/1984-6835.20200039 -
ARAÚJO, C., MAYWORM, M.A.S., YATSUDA, R., NEGRI, G., SALATINO, M.L.F., SALATINO, A., TIMENETSKY, J. and CAMPOS, G.B., 2020b. Chemical composition and antimycoplasma activity of a brown propolis from Southern Brazil. Journal of Food Science and Technology, vol. 57, no. 11, pp. 4228-4235. http://doi.org/10.1007/s13197-020-04461-y PMid:33071344.
» http://doi.org/10.1007/s13197-020-04461-y -
ARAÚJO, M.J.A.M., DUTRA, R.P., COSTA, G.C., REIS, A.S., ASSUNÇÃO, A.K.M., LIBÉRIO, S.A., SILVA, L., GUERRA, R.N.M., RIBEIRO, M.N.S. and NASCIMENTO, F.R.F., 2011. Pharmacognostic and acute toxicological evaluation of Scaptotrigona aff. postica propolis extract in pre-clinical assays. Natural Product Research, vol. 25, no. 11, pp. 1037-1046. http://doi.org/10.1080/14786419.2010.482059 PMid:21500093.
» http://doi.org/10.1080/14786419.2010.482059 -
ARFIN, S., JHA, N.K., JHA, S.K., KESARI, K.K., RUOKOLAINEN, J., ROYCHOUDHURY, S. and KUMAR, D., 2021. Oxidative stress in cancer cell metabolism. Antioxidants, vol. 10, no. 5, pp. 642. http://doi.org/10.3390/antiox10050642 PMid:33922139.
» http://doi.org/10.3390/antiox10050642 -
BANKOVA, V., POPOVA, M., BOGDANOV, S. and SABATINI, A., 2002. Chemical composition of european propolis: expected and unexpected results. Zeitschrift für Naturforschung. C, A Journal of Biosciences, vol. 57, no. 5-6, pp. 530-533. http://doi.org/10.1515/znc-2002-5-622 PMid:12132697.
» http://doi.org/10.1515/znc-2002-5-622 - BARROS NETO, B., SCARMINIO, I.S. and BRUNS, R.E., 2001. How to do experiments Campinas: UNICAMP, 401 p.
- CAMPOS, T.M.C., 2019. Evaluation of antimicrobial and antiparasitic activities of alcoholic and aqueous extracts of propolis Scaptotrigona affinis postica. São Paulo: Instituto Butantan. Master's Dissertation in Science.
-
EL-LAKKANY, N.M., ELKATTAN, H.H. and ELSISI, A.E., 2021. The ponatinib/gossypol novel combination provides enhanced anticancer activity against murine solid Ehrlich carcinoma via triggering apoptosis and inhibiting proliferation/angiogenesis. Toxicology and Applied Pharmacology, vol. 432, pp. 115767. http://doi.org/10.1016/j.taap.2021.115767 PMid:34699866.
» http://doi.org/10.1016/j.taap.2021.115767 -
FONSECA, M.J.V., FONSECA, Y.M., MARQUELE-OLIVEIRA, F., VICENTINI, F.T.M.C., FURTADO, N.A.J.C., SOUSA, J.P.B. and LUCISANO-VALIM, Y.M., 2011. Evaluation of the potential of Brazilian propolis against UV-induced oxidative stress. Evidence-Based Complementary and Alternative Medicine, vol. 863917, no. 1, pp. 863917. http://doi.org/10.1155/2011/863917 PMid:20953396.
» http://doi.org/10.1155/2011/863917 - FREIRES, M.A.L., MELO, W.F., LACERDA, L.E., ROLIM, F.D., MARACAJÁ, P.B., NÓBREGA, K.G., FERREIRA, A.E.E. and MELO, R., 2019. Os tipos de própolis e sua utilização em lesões cutâneas. Revista Brasileira de Gestão Ambiental, vol. 13, no. 4, pp. 25-29.
-
HATA, T., TAZAWA, S., OHTA, S., RHYU, M.R., MISAKA, T. and ICHIHARA, K., 2012. Artepillin C, a major ingredient of Brazilian propolis, induces a pungent taste by activating TRPA1 channels. PLoS One, vol. 7, no. 11, e48072. http://doi.org/10.1371/journal.pone.0048072 PMid:23133611.
» http://doi.org/10.1371/journal.pone.0048072 -
INSTITUTO ADOLFO LUTZ – IAL, 2008 [viewed 3 April 2024]. Métodos físico químicos para análises de alimentos [online]. Available from: https://wp.ufpel.edu.br/nutricaobromatologia/files/2013/07/NormasADOLFOLUTZ.pdf
» https://wp.ufpel.edu.br/nutricaobromatologia/files/2013/07/NormasADOLFOLUTZ.pdf -
KIM, E.Y., RYU, J.H. and KIM, A.K., 2013. CAPE promotes TRAIL-induced apoptosis through the upregulation of TRAIL receptors via activation of p38 and suppression of JNK in SK-Hep1 hepatocellular carcinoma cells. International Journal of Oncology, vol. 43, no. 4, pp. 1291-1300. http://doi.org/10.3892/ijo.2013.2018 PMid:23857473.
» http://doi.org/10.3892/ijo.2013.2018 -
LIANG, C., PEI, S., JU, W., JIA, M., TIAN, D., TANG, Y. and MAO, G., 2017. Synthesis and in vitro and in vivo antitumour activity study of of 11-hydroxyl esterified bergenin/cinnamic acid hybrids. European Journal of Medicinal Chemistry, vol. 133, pp. 319-328. http://doi.org/10.1016/j.ejmech.2017.03.053 PMid:28395218.
» http://doi.org/10.1016/j.ejmech.2017.03.053 -
LIMA, G.D.A., RODRIGUES, M.P., MENDES, T.A.O., MOREIRA, G.A., SIQUEIRA, R.P., SILVA, M.A., VAZ, B.G., FIETTO, J.L.R., BRESSAN, G.C., MACHADO-NEVES, M. and TEIXEIRA, R.R., 2018. Synthesis and antimetastatic activity evaluation of cinnamic acid derivatives containing 1,2,3-triazolic portions. Toxicology In Vitro, vol. 53, pp. 1-9. http://doi.org/10.1016/j.tiv.2018.07.015 PMid:30048736.
» http://doi.org/10.1016/j.tiv.2018.07.015 -
MACHADO, B.A.S., SILVA, R.P.D., BARRETO, G.D.A., COSTA, S.S., DA SILVA, D.F., BRANDO, H.N., ROCHA, J.L.C., DELLAGOSTIN, O.A., HENRIQUES, J.A.P., UMSZA-GUEZ, M.A. and PADILHA, F.F., 2016b. Chemical composition and biological activity of extracts obtained by supercritical extraction and ethanolic extraction of brown, green and red propolis derived from different geographic regions in Brazil. PLoS One, vol. 11, no. 1, e0145954. http://doi.org/10.1371/journal.pone.0145954 PMid:26745799.
» http://doi.org/10.1371/journal.pone.0145954 -
MACHADO, C.S., FINGER, D., LIMA, T.C., MODESTO, T., MONTEIRO, M.C., OLIVEIRA, F.C.E., PESSOA, C. and TORRES, Y.R., 2021. Seeking the regional identity of brown propolis produced in southern brazil and linked to total levels of bioactive compounds and biological activity. ACS Food Science & Technology, vol. 1, no. 2, pp. 176-185. http://doi.org/10.1021/acsfoodscitech.0c00052
» http://doi.org/10.1021/acsfoodscitech.0c00052 -
MACHADO, C.S., MOKOCHINSKI, J.B., LIRA, T.O., DE OLIVEIRA, F.D.C.E., CARDOSO, M.V., FERREIRA, R.G., SAWAYA, A.C.H.F., FERREIRA, A.G., PESSOA, C., CUESTA-RUBIO, O., MONTEIRO, M.C., CAMPOS, M.S.D. and TORRES, Y.R., 2016a. Comparative study of chemical composition and biological activity of yellow, green, brown, and red Brazilian propolis. Evidence-Based Complementary and Alternative Medicine, vol. 2016, no. 12, pp. 6057650. http://doi.org/10.1155/2016/6057650 PMid:27525023.
» http://doi.org/10.1155/2016/6057650 -
MACIEL, F.F.C., SILVA, E.B., LIMA, R., OLIVEIRA, N.D., DANTAS, E.N.A., CORDEIRO, S.A., SOUZA, G.S.F., MACEDO, N.L.S., OLIVEIRA, D.A. and DONATO, N.R., 2018. Alimentos funcionais e câncer de mama: breve revisão. International Journal of Nutrition, vol. 11, no. 1, pp. S24-S237. http://doi.org/10.1055/s-0038-1674376
» http://doi.org/10.1055/s-0038-1674376 -
MATOS, V.R., ALENCAR, S.M. and SANTOS, F.A.R., 2014. Pollen types and levels of total phenolic compounds in propolis produced by Apis mellifera L. (Apidae) in an area of the Semiarid Region of Bahia, Brazil. Anais da Academia Brasileira de Ciências, vol. 86, no. 1, pp. 407-418. http://doi.org/10.1590/0001-376520142013-0109 PMid:24676176.
» http://doi.org/10.1590/0001-376520142013-0109 -
MIRBAGHERI, M.S., AKHAVAN-MAHDAVI, S., HASAN, A., KHARAZMI, M.S. and JAFARI, S.M., 2023. Propolis-loaded nanofiber scaffolds based on polyvinyl alcohol and polycaprolactone. International Journal of Pharmaceutics, vol. 642, pp. 123186. http://doi.org/10.1016/j.ijpharm.2023.123186 PMid:37385356.
» http://doi.org/10.1016/j.ijpharm.2023.123186 -
MOSMANN, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, vol. 65, no. 1-2, pp. 55-63. http://doi.org/10.1016/0022-1759(83)90303-4 PMid:6606682.
» http://doi.org/10.1016/0022-1759(83)90303-4 - MOUGA, D.M.D.S. and DEC, E., 2012. Catálogo polínico de plantas medicinais apícolas Florianópolis: DIOESC, 156 p.
-
MOUHOUBI-TAFININE, Z., OUCHEMOUKH, S. and TAMENDJARI, A., 2016. Antioxydant activity of some algerian honey and propolis. Industrial Crops and Products, vol. 88, pp. 85-90. http://doi.org/10.1016/j.indcrop.2016.02.033
» http://doi.org/10.1016/j.indcrop.2016.02.033 -
MOURA, S.A.L., FERREIRA, M.A.N.D., ANDRADE, S.P., REIS, M.L.C., NOVIELLO, M.L. and CARA, D.C., 2011. Brazilian green propolis inhibits inflammatory angiogenesis in a murine sponge model. Evidence-Based Complementary and Alternative Medicine, vol. 2011, no. 1, pp. 182703. http://doi.org/10.1093/ecam/nep197 PMid:20007259.
» http://doi.org/10.1093/ecam/nep197 - NOVAK, E.M., SILVA, M.S.C., MARCUCCI, M.C., SAWAYA, A.C.H.F., GIMÉNEZ-CASSINA LÓPEZ, B., FORTES, M.A.H.Z., GIORGI, R.R., MARUMO, K.T., RODRIGUES, R.F. and MARIA, D.A., 2014. Antitumoural activity of Brazilian red propolis fraction enriched with xanthochymol and formononetin: an in vitro and in vivo study. Journal of Functional Foods, vol. 2014, no. 11, pp. 91-102.
-
NUNES, D., MACHADO, B.A.S., BARRETO, G.A., SILVA, J.R., SILVA, D.F., ROCHA, J.L.C., BRANDÃO, H.N., BORGES, V.M. and UMSZA-GUEZ, M.A., 2018. Chemical characterization and biological activity of six different extracts of propolis through conventional methods and supercritical extraction. PLoS One, vol. 13, no. 12, e0207676. http://doi.org/10.1371/journal.pone.0207676 PMid:30513100.
» http://doi.org/10.1371/journal.pone.0207676 -
PARK, Y.K., ALENCAR, S.M. and AGUIAR, C.L., 2002. Botanical origin and chemical composition of Brazilian propolis. Journal of Agricultural and Food Chemistry, vol. 50, no. 9, pp. 2502-2506. http://doi.org/10.1021/jf011432b PMid:11958612.
» http://doi.org/10.1021/jf011432b -
PASUPULETI, V.R., SAMMUGAM, L., RAMESH, N. and GAN, S.H., 2017. Honey, propolis, and royal jelly: a comprehensive review of their biological actions and health benefits. Oxidative Medicine and Cellular Longevity, vol. 2017, no. 1, pp. 1259510. http://doi.org/10.1155/2017/1259510 PMid:28814983.
» http://doi.org/10.1155/2017/1259510 -
POPOLO, A., PICCINELLI, L.A., MORELLO, S., CUESTA-RUBIO, O., SORRENTINO, R., RASTRELLI, L. and PINTO, A., 2009. Antiproliferative activity of brown Cuban propolis extract on human breast cancer cells. Natural Product Communications, vol. 4, no. 12, pp. 1711-1716. http://doi.org/10.1177/1934578X0900401221 PMid:20120113.
» http://doi.org/10.1177/1934578X0900401221 -
REFAAT, H., MADY, F.M., SARHAN, H.A., RATEB, H.S. and ALAAELDIN, E., 2021. Optimization and evaluation of propolis liposomes as a promising therapeutic approach for COVID-19. International Journal of Pharmaceutics, vol. 592, pp. 120028. http://doi.org/10.1016/j.ijpharm.2020.120028 PMid:33166584.
» http://doi.org/10.1016/j.ijpharm.2020.120028 -
RIBEIRO, V.P., MEJIA, J.A.A., RODRIGUES, D.M., ALVES, G.R., DE FREITAS PINHEIRO, A.M., TANIMOTO, M.H., BASTOS, J.K. and AMBRÓSIO, S.R., 2023. Brazilian brown propolis: an overview about its chemical composition, botanical sources, quality control, and pharmacological properties. Revista Brasileira de Farmacognosia, vol. 33, no. 2, pp. 288-299. http://doi.org/10.1007/s43450-023-00374-x PMid:36908300.
» http://doi.org/10.1007/s43450-023-00374-x -
ROUBIK, D.W. and MORENO, P.J.E., 1991. Pollen and spores of Barro Colorado Island. Monographs in Systemat. Kew Bulletin, vol. 36, pp. 1-270. http://doi.org/10.2307/4110734
» http://doi.org/10.2307/4110734 -
RUWIZHI, N. and ADERIBIGBE, B.S., 2020. Cinnamic acid derivatives and their biological efficacy. International Journal of Molecular Sciences, vol. 21, no. 16, pp. 5712. http://doi.org/10.3390/ijms21165712 PMid:32784935.
» http://doi.org/10.3390/ijms21165712 - SANTOS, F.A.R., 2011. Botanical identification of bee pollen. Magistra, vol. 23, pp. 5-9.
-
SANTOS, M.F.C., OLIVEIRA, L.C., RIBEIRO, V.P., SOARES, M.G., MORAES, G.O.I., SARTORI, A.G.O., ROSALEN, P.L., BASTOS, J.K., ALENCAR, S.M., VENEZIANI, R.C.S. and AMBRÓSIO, S.R., 2021. Isolation of diterpenes from Araucaria sp. Brazilian brown propolis and development of a validated HPLC method for its analysis. Journal of Separation Science, vol. 44, pp. 3089-3097. http://doi.org/10.1002/jssc.202100374 PMid:34169651.
» http://doi.org/10.1002/jssc.202100374 -
SAWAYA, A.C.H.F., CUNHA, B.S. and MARCUCCI, M.C., 2011. Analytical methods applied to diverse types of Brazilian propolis. Chemistry Central Journal, vol. 5, no. 1, pp. 27. http://doi.org/10.1186/1752-153X-5-27 PMid:21631940.
» http://doi.org/10.1186/1752-153X-5-27 -
SILVA, F.R.G., MATIAS, T.M.S., SOUZA, L.I.O., MATO-ROCHA, L.I.O., FONSECA, S.A., MOUSINHO, K.C. and SANTOS, A.F., 2019. Phytochemical screening and in vitro antibacterial, antifungal, antioxidant and antitumor activities of the red própolis Alagoas. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 79, no. 3, pp. 3. http://doi.org/10.1590/1519-6984.182959 PMid:30379200.
» http://doi.org/10.1590/1519-6984.182959 -
SILVA, R.A., RODRIGUES, A.E., RIBEIRO, M.C.M., CUSTÓDIO, A.R., ANDRADE, N.E.D. and PEREIRA, W.E., 2006. Características físico-químicas e atividade antimicrobiana de extratos de própolis da Paraíba, Brasil. Ciência Rural, vol. 36, no. 6, pp. 1842-1848. http://doi.org/10.1590/S0103-84782006000600027
» http://doi.org/10.1590/S0103-84782006000600027 -
SOSA, V., MOLINÉ, T., SOMOZA, R., PACIUCCI, R., KONDOH, H. and LLEONART, M.E., 2013. Oxidative stress and cancer: an overview. Ageing Research Reviews, vol. 12, no. 1, pp. 376-390. http://doi.org/10.1016/j.arr.2012.10.004 PMid:23123177.
» http://doi.org/10.1016/j.arr.2012.10.004 -
SOUZA, E.A., INOUE, H.T., GOMES, S.M.A., FUNARI, S.R.C. and ORSI, R.O., 2010. Propriedade físico-química da própolis em função da sazonalidade e método de produção. Archivos de Zootecnia, vol. 56, no. 228, pp. 571-576. http://doi.org/10.21071/az.v59i228.4712
» http://doi.org/10.21071/az.v59i228.4712 -
SOVA, M., ZIZAC, Z., STANKOVIC, J.A.A., PRIJATELJ, M., TURK, S., JURANI, Z.D., MLINARI–RASCAN, I. and GOBEC, S., 2013. Cinnamic acid derivatives induce cell cycle arrest in carcinoma cell lines. Medicinal Chemistry (Shariqah, United Arab Emirates), vol. 9, no. 5, pp. 633-641. http://doi.org/10.2174/1573406411309050002 PMid:23140579.
» http://doi.org/10.2174/1573406411309050002 -
TIVERON, A.P., ROSALEN, P.L., FRANCHIN, M., LACERDA, R.C.C., BUENO-SILVA, B., BENSO, B., DENNY, C., IKEGAKI, M. and DE ALENCAR, S.M., 2016. Chemical characterization and antioxidant, antimicrobial, and anti-inflammatory activities of South Brazilian organic propolis. PLoS One, vol. 11, no. 11, e0165588. http://doi.org/10.1371/journal.pone.0165588 PMid:27802316.
» http://doi.org/10.1371/journal.pone.0165588 -
TSAI, C.M., YEN, G.C., SUN, F.M., YANG, S.F. and WENG, C.J., 2013. Assessment of the antiinvasion potential and mechanism of select cinnamic acid derivatives on human lung adenocarcinoma cells. Molecular Pharmaceutics, vol. 10, no. 5, pp. 1890-1900. http://doi.org/10.1021/mp3006648 PMid:23560439.
» http://doi.org/10.1021/mp3006648 -
VIUDA-MARTOS, M., RUIZ-NAVAJAS, Y., FERNÁNDEZ-LÓPEZ, J. and PÉREZ-ALVAREZ, J.A., 2008. Functional properties of honey, propolis, and royal jelly. Journal of Food Science, vol. 73, no. 9, pp. 117-124. http://doi.org/10.1111/j.1750-3841.2008.00966.x
» http://doi.org/10.1111/j.1750-3841.2008.00966.x -
WALLER, S.B., PETER, C.M., HOFFMANN, J.F., PICOLI, T., OSORIO, L.G., CHAVES, F., ZANI, J.L., FARIA, R.O., MELLO, J.R.B. and MEIRELES, M.C.A., 2017. Chemical and cytotoxic analyses of brown Brazilian própolis (Apis mellifera) and its in vitro activity against itraconazole resistant Sporothrix brasiliensis. Microbial Pathogenesis, vol. 105, pp. 117-121. http://doi.org/10.1016/j.micpath.2017.02.022 PMid:28219829.
» http://doi.org/10.1016/j.micpath.2017.02.022 -
WORLD HEALTH ORGANIZATION – WHO, 2021 [viewed 3 April 2024]. Cancer [online]. Available from: https://www.who.int/news-room/fact-sheets/detail/cancer
» https://www.who.int/news-room/fact-sheets/detail/cancer -
YEN, G.C., CHEN, Y.L., SUN, F.M., CHIANG, Y.L., LU, S.H. and WENG, C.J., 2011. A comparative study on the effectiveness of cis- and trans-form of cinnamic acid treatments for inhibiting invasive activity of human lung adenocarcinoma cells. European Journal of Pharmaceutical Sciences, vol. 44, no. 3, pp. 281-287. http://doi.org/10.1016/j.ejps.2011.08.006 PMid:21871959.
» http://doi.org/10.1016/j.ejps.2011.08.006 -
ZABAIOU, N., FOUACHE, A., TROUSSON, A., BARON, S., ZELLAGUI, A., LAHOUEL, M. and LOBACCARO, J.M.A., 2017. Biological properties of propolis extracts: something new from an ancient product. Chemistry and Physics of Lipids, vol. 207, no. Pt B, pp. 214-222. http://doi.org/10.1016/j.chemphyslip.2017.04.005 PMid:28411017.
» http://doi.org/10.1016/j.chemphyslip.2017.04.005 -
ZHANG, Y., CAO, C., YANG, Z., JIA, G., LIU, X., LI, X., CUI, Z. and LI, A., 2023. Simultaneous determination of 20 phenolic compounds in propolis by HPLC-UV and HPLC-MS/MS. Journal of Food Composition and Analysis, vol. 115, pp. 104877. http://doi.org/10.1016/j.jfca.2022.104877
» http://doi.org/10.1016/j.jfca.2022.104877
