Propolis is a complex bioactive mixture produced by bees, known to have different biological activities, especially in countries where there is a rich biodiversity of plant species. The objective of this study was to determine the chemical composition and evaluate the antioxidant and cytotoxic properties of Brazilian propolis from the species Plebeia droryana and Apis mellifera found in Mato Grosso do Sul, Brazil. In the ethanolic extracts of P. droryana propolis (ExEP-P) and A. mellifera (ExEP-A) acids, phenolic compounds, terpenes and tocopherol were identified as major compounds. Both extracts presented antioxidant activity against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, the maximum activities being 500 μg/mL (ExEP-P) and 300 μg/mL (ExEP-A). However, only ExEP-A was able to inhibit lipid peroxidation induced by the oxidizing agent 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which inhibited oxidative hemolysis and reduced the levels of malondialdehyde (MDA) in human erythrocytes for 4 h of incubation. The extracts also reduced the cell viability of the K562 erythroleukemia tumour line, with a predominance of necrotic death. Thus, it is concluded that the propolis produced by P. droryana and A. mellifera contain important compounds capable of minimizing the action of oxidizing substances in the organism and reducing the viability of erythroleukemia cells.
Citation: Bonamigo T, Campos JF, Oliveira AS, Torquato HFV, Balestieri JBP, Cardoso CAL, et al. (2017) Antioxidant and cytotoxic activity of propolis of Plebeia droryana and Apis mellifera (Hymenoptera, Apidae) from the Brazilian Cerrado biome. PLoS ONE 12(9): e0183983. https://doi.org/10.1371/journal.pone.0183983
Editor: Horacio Bach, University of British Columbia, CANADA
Received: April 6, 2017; Accepted: August 15, 2017; Published: September 12, 2017
Copyright: © 2017 Bonamigo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by grants from Foundation to Support to Development of Education, Science and Technology of Mato Grosso do Sul State – FUNDECT and Brazilian National Research Council – CNPq, Instituto Nacional de Pesquisa do Pantanal - INPP, and Fundação de Amparo e Desenvolvimento da Pesquisa – Fadesp. Thaliny Bonamigo was supported by FUNDECT and CAPES. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Propolis is a bee product resulting from the collection of resin from different parts of plants, such as buds of leaves, branches, flowers and pollen, with the addition of mandibular secretions from bees. Many bee species are capable of producing propolis, among them Apis mellifera  and some species of stingless bees known as meliponine bees [2,3]. In the hive, this resin is used to repair cracks or damage, to defend against microorganisms and to mummify the dead bodies of invading insects, preventing their decomposition and the contamination of the hive by fungi and bacteria [4,5].
Propolis is a complex mixture known to exhibit great chemical diversity, especially in tropical climate countries, where the richness of plant species is responsible for the presence of a wide variety of substances in propolis, such as phenolic compounds, flavonoids and terpenes [6,7]. However, the chemical composition of propolis depends on factors such as botanical origin, temperature variation and seasonality, as well as the salivary secretions and enzymes added to propolis by bees [5,8]. These changes can qualitatively and quantitatively alter the compounds, modifying their therapeutic properties [5,8,9].
Thus, propolis produced by different species of bees that cohabit the same region can present different biological substances and activities. Propolis from different parts of the world has been reported to have antioxidant [10,11], antibiofilm [12,13], antimicrobial [14–16], anti-inflammatory [17–19] and antitumour [20–22] activities.
For this reason, this bee product is of great interest to the pharmaceutical and food industries . Studies have been conducted on propolis produced by different species of bees, to evaluate their chemical composition and their potential pharmacological activities [24,25].
The species of stingless bee Plebeia droryana, belonging to the subfamily Meliponinae, native to Brazil, Paraguay, Uruguay and Bolivia, is popularly known as the Mirim bee and produces a viscous propolis . There are few studies on the existing compounds and biological activities of this product. Sawaya et al.  report some species of medicinal plants in which these bees collect resin for the production of propolis, such as Schinus terebinthifolius Raddi. In addition, studies with P. droryana propolis from the southeastern region of Brazil show that this product includes phenolic compounds and terpenes in its composition .
The species Apis mellifera, belonging to the subfamily Apinae, known as the European honey bee, is exotic in Brazil and is a major producer of propolis, which has been reported to present important antioxidant , antimicrobial [14,29] and antitumour [30,31] activities.
Thus, this study aimed to determine the chemical composition and the antioxidant and cytotoxic properties of the propolis of P. droryana and A. mellifera found in the Cerrado biome, in the Midwest region of Brazil.
Materials and methods
Ethics of experimentation
No specific permits were required for the described field studies. All field work to collect the propolis samples was conducted on private land and with owner permission. The field studies did not involve endangered or protected species. The protocol to collect human peripheral blood was approved by the Research Ethics Committee (Comitê de Ética em Pesquisa; CEP) of the University Center of Grande Dourados (Centro Universitário da Grande Dourados; UNIGRAN), Brazil (CEP process number 123/12). All subjects provided written informed consent for participation.
Preparation of the ethanol extract of propolis (ExEP)
Propolis samples were collected from P. droryana and A. mellifera in the state of Mato Grosso do Sul, in the Midwest region of Brazil (22° 13’ 12” S—54° 49’ 2” W). For this, the identity of the bees species were authenticated by entomologist Professor José Benedito Perrella Balestieri, and four sample of propolis were collected in different seasons of the year of 2015, totalling 12.02 g (P. droryana) and 21.27 g (A. mellifera) of samples for each specie.
Ethanol extracts of propolis (ExEP) were prepared in the proportion of 4.5 mL of 80% ethanol per g of propolis. This solution was maintained at 70°C in a closed container in a water bath until complete dissolution and then filtered on 80 g/m2 qualitative filter paper (Prolab, São Paulo, Brazil) to obtain the ethanolic extract of propolis of P. droryana (ExEP- P) and A. mellifera (ExEP-A) . After the extracts were prepared, they were identified, stored in closed containers and kept at -20°C until analysis.
Preparation of the samples.
The samples (1 mg) was fractionated with hexane and water in proportion 1:1 v:v and fraction soluble in hexane was analyzed by GC-MS and fraction soluble in water by HPLC. In addition, the GC-MS technique was employed to analyze highly volatilizable compounds that by the detector employed in this HPLC study would not be detected in the analysis.
Samples were injected and analyzed by gas chromatography-mass spectrometry (GC-MS). The GC-MS analysis was performed on a gas chromatograph (GC-2010 Plus, Shimadzu, Kyoto, Japan) equipped with a mass spectrometer detector (GC-MS Ultra 2010) using LM-5 (5% phenyl dimethylpolysiloxane) capillary column (15 m length × 0.2 mm i.d. and 0.2 μm film thickness) with initial oven temperature set at 150°C and heating from 150°C to 280°C at 15°C min−1 and a hold at 280°C for 15 min. Carrier gas of helium (99.999% and flow rate 1.0 mL min−1), 1 μL injection volume, split ratio (1:20). The injector temperature was 280°C and the quadrupole detector temperature was 280°C. The MS scan parameters included an electron-impact ionization voltage of 70 eV mass range of 45–600 m/z and scan interval of 0.3 s. The identifications were completed by comparing the mass spectra obtained in the NIST21 and WILEY229 libraries. In some cases, the compound was confirmed by comparison of standards. The standards from Sigma-Aldrich with purity ≥ 97%. Standards of the stigmasterol, β-sitosterol, β-amyrin, α-amyrin, β-amyrin acetate, α-amyrin acetate, tocopherol were prepared in the concentration initial of 1 mg/mL. The concentrations of compounds were determined by extern calibration after dilutions appropriated in the range of 0.1–50 μg/mL. The quantification of campesterol and taraxasterol were performed in relation to stigmasterol. All the samples were previously filtrated in 0.45 μm (Millex® Syringe Filter Millipore, Merck). The analysis was performed in triplicate.
The extracts were analyzed in an analytical HPLC (LC-6AD, Shimadzu, Kyoto, Japan) system with a diode array detector (DAD) monitored at λ = 200–600 nm. The HPLC column was a C-18 (25 cm x 4.6 mm; particle size, 5 μm; Luna, Phenomenex, Torrance, CA, USA), with a small pre-column (2.5 cm x 3 mm) containing the same packing, used to protect the analytical column. In each analysis, the flow rate and the injected volume were set as 1.0 mL min-1 and 20 μL, respectively. All chromatographic analyses were performed at 22°C. Elution was carried out using a binary mobile phase of water with 6% acetic acid and 2 mM sodium acetate (eluent A) and acetonitrile (eluent B). The following applied gradient: 5% B (0 min), 15% B (30 min), 50% B (35 min) and 100% B (45 min). Standards of the vanillic acid, p-methylbenzoic acid, caffeic acid, ferrulic acid, p-coumaric acid, benzoic acid, cinnamic acid, rutin, sinapic acid, quercetin, luteolin, apigenin and vanilline (Sigma, ≥ 97%) were prepared in the concentration initial of 1 mg/mL. The concentrations of compounds were determined by extern calibration after dilutions appropriated in the range of 0.01–10 μg/mL. All the samples were previously filtrated in 0.45 μm (Millex® Syringe Filter Millipore, Merck). The analysis was performed in triplicate.
Free radical-scavenger activity.
Free radical-scavenger activity was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, as described previously by Gupta and Gupta , with some modifications. The antiradical activity of the extracts was evaluated using a dilution series, which involved the mixing of 1.8 mL of DPPH solution (0.11 mM DPPH in 80% ethanol) with 0.2 mL of ExEP-P or ExEP-A (1–500 μg/mL). After 30 min, the remaining DPPH radicals were quantified by absorption at 517 nm. The absorbance of each concentration of the ExEP (only sample with 80% ethanol) was subtracted from absorbance of the samples with DPPH solution. Ascorbic acid and butylated hydroxytoluene (BHT) were used as reference antioxidants. The tests were performed in duplicate in three independent experiments. DPPH solution without the tested sample was used as a control. The percentage inhibition was calculated from the control with the following Eq 1: (1)
Protection against lipid peroxidation using a human erythrocyte model
Preparation of erythrocyte suspensions.
Following approval by the Research Ethics Committee, 20 mL samples of peripheral blood were collected from healthy donors into sodium citrate-containing tubes and subsequently centrifuged at 1500 rpm for 10 min. After centrifugation, the blood plasma and leukocyte layers were discarded, and the erythrocytes were washed three times with 0.9% sodium chloride solution (NaCl) and centrifuged at 1500 rpm for 10 min. Finally, 10% erythrocyte suspensions were prepared in 0.9% NaCl.
Oxidative hemolysis inhibition assay.
The antioxidant activity in biological model was evaluated using human erythrocytes subjected to hemolysis via the oxidation of lipids and proteins of the cell membranes by the action of peroxyl free radicals generated by the oxidizing agent AAPH. The protective effect of the propolis extracts was evaluated according to the method described by Campos et al. , with minor modifications. The assays were conducted with erythrocyte suspensions. The erythrocytes were preincubated at 37C for 30 min in the presence of different concentrations of ascorbic acid or ExEP (50–125 μg/mL). Then, 50 mM 2,2’-azobis-(2-amidinopropane) dihydrochloride (AAPH) solution was added. Total hemolysis was induced by incubating erythrocytes with distilled water. Basal hemolysis caused by ExEP was assessed by incubating erythrocytes with the extract without the presence of AAPH, and the negative controls were assessed in erythrocytes incubated with 0.9% NaCl or 1% ethanol. This mixture was incubated at 37°C, with periodical stirring. Hemolysis was determined after 120, 180 and 240 min of sample incubation; specifically, samples were centrifuged at 1500 rpm for 10 min and aliquots of there were transferred to tubes with 0.9% NaCl, after which the absorbance of the supernatant was read spectrophotometrically at 540 nm. The percentage hemolysis was measured with the formula A/B × 100, where (A) is the sample absorbance and (B) is the total hemolysis. Three independent experiments were performed in duplicate.
Dosage of malondialdehyde (MDA).
The inhibition of lipid peroxidation was determined by the quantification of the levels of malondialdehyde, a marker of oxidative damage of the membrane lipids. For this, 10% erythrocyte suspension was used to assess the protective effects of ExEP against lipid peroxidation, evaluated through the dosage of malondialdehyde (MDA), as described by Campos et al. , with some modifications. Erythrocytes were preincubated at 37°C for 30 min with different concentrations of ascorbic acid or ExEP (50–125 μg/mL). The negative controls were assessed in erythrocytes incubated with 0.9% NaCl or 1% ethanol. Next, 50 mM AAPH was added to the erythrocyte solution, which was then incubated at 37°C for 4 hours with periodical stirring. At 120, 180 and 240 min of incubation, the samples were centrifuged at 1500 rpm for 10 min, and 500 μL aliquots of the supernatant were transferred to tubes with 1 mL of 10 nmol thiobarbituric acid (TBA), dissolved in 75 mM monobasic potassium phosphate buffer at pH 2.5. As a standard control, 500 μL of 20 mM MDA solution were added to 1 mL of TBA. The samples were incubated at 96°C for 45 min. The samples were then cooled, 4 mL of n-butyl alcohol were added and the samples were centrifuged at 1500 rpm for 10 min. The absorbance of supernatants sample was read at 532 nm. Three independent experiments were performed in duplicate. MDA levels in the samples were expressed in nmol/mL, obtained with the following formula 2: (2)
Cell line and culture conditions
Human peripheral blood mononuclear cells from healthy donors were collected after informed patient consent. Separation of mononuclear cells was performed by gradient centrifugation methods using Ficoll Histopaque-1077 (1.077 g/cm3) (Sigma–Aldrich, Germany) follow the manufacturer’s instructions at 400 x g for 30 min. The use of human samples was approved by the local Ethical Committee of the University Center of Grande Dourados under protocol number 123/12. The K562 human cell line derived by chronic myelogenous leukemia was grown is suspension in RPMI 1640 media (Cultilab, Campinas, São Paulo, Brazil) supplemented with 10% fetal bovine serum (FBS; Cultilab), 100 U/mL of penicillin (Sigma-Aldrich, Germany) and 100 μg/mL of streptomycin (Sigma-Aldrich, Germany) in a humidified atmosphere at 37°C in 5% CO2.
Cytotoxic activity and cell death profile
The cytotoxicity and possible mechanisms of death promoted by ExEP were determined by cytotoxic activity and cell death profile, evaluated according to the method described by Paredes-Gamero et al. , with minor modifications. Peripheral blood mononuclear cells and K562 cells were seeded into 96-well plates (5 x105 cell/well) and cultured in medium with 10% FBS for 24 h with different concentrations (0.0625–1 mg/mL) or IC50 of ExEP-P (0.38 mg/mL) or ExEP-A (0.36 mg/mL). For dilution of the highest concentration of extract was used 0.2% ethanol, which was tested as a negative control (data not shown). All other concentrations were diluted only in culture medium. The positive control was only culture medium. After this period, the K562 cells were washed with PBS and resuspended in annexin-labeling buffer (0.01 M HEPES, pH 7.4, 0.14 M NaCl and 2.5 mM CaCl2). The suspensions were stained with annexin-FITC and propidium iodide (PI) (Becton Dickinson, Franklin Lakes, NJ, USA), according to the manufacture’s instructions. The cells were incubated at room temperature for 15 min. Three thousand events were collected per sample, and the analyses were performed on a FACSCalibur flow cytometer (Becton Dickinson) with CellQuest software (Becton Dickinson).
K562 cells were seeded at 5 x 105 cells/mL in 12-well microplates and treated with IC50 of the ExEP-P or ExEP-A and incubated for 48 h. The cells were observed after 0, 4, 8, 24 and 48 h of incubation using an inverted microscope under 10X objective (Nikon Eclipse TS 100) connected to digital camera (Nikon DS-1).
Caspase-3 activity was measured by flow cytometer according to the method described by Moraes et al. . K562 erythroleukemia cells were treated with IC50 of the ExEP-P or ExEP-A in 24-well microplates (5 x 105 cells/mL) for 4, 8, 24 and 48 h. Then the cells were fixed with 2% paraformaldehyde in PBS for 30 min and permeabilized with 0.01% saponin for 15 min at room temperature. Next, the cells were incubated for 1 h at 37∘C with anti-cleaved-caspase 3-FITC antibody (Becton Dickinson, USA). After incubation for 40 min, the fluorescence was analyzed by Accuri C6 flow cytometer (Becton Dickinson, USA). A total of 10,000 events were acquired. Alternations in the fluorescence intensity were determined by comparing the levels of the treated cells to those of the controls.
Effect of inhibitors on ExEP-induced cell death
K562 cells were seeded in 96-well microplates (5 x 105 cells/mL) containing RPMI 1640 supplemented with 10% FBS in the presence of 20 μM of necrosis inhibitor necrostatin-1 (NEC-1), and they were incubated in a humidified atmosphere at 37°C and 5% CO2 for 60 min. Afterwards, the IC50 of the ExEP-P or ExEP-A were added to each sample, and the mixture was incubated for 24 h. Then, the cells were washed with PBS, resuspended in Annexin buffer (0.01 M HEPES, pH 7.4, 0.14 M NaCl and 2.5 mM CaCl2) and incubated for 20 min at room temperature after the addition of annexin V-FITC and propidium iodide (Becton Dickinson, Franklin Lakes, NJ) according to the manufacturer's instructions. The analyses were performed using an Accuri C6 flow cytometer (Becton Dickinson) and Accuri C6 software (Becton Dickinson), with 4000 events collected per sample.
All data are represented by the mean ± standard error of the mean (SEM), based on at least two independent experiments. To establish the half-maximal inhibitory concentration (IC50) of DPPH free radical scavenging, the samples were tested in serial dilutions (1, 10, 50, 100, 200, 300 and 500 μg/mL) and analyzed by means of nonlinear regression using the Prism 6 GraphPad Software. The significant differences between the different groups were evaluated by analysis of variance (ANOVA) followed by Dunnett's post-test, using the GraphPad prism 6 program. The results were considered significant when p < 0.05.
The propolis extracts produced by different bee species had a similar chemical profile (Tables 1 and 2 and S1 Fig), although some compounds were identified exclusively in ExEP-A. The major compounds identified in ExEP-P were tocopherol, β-amyrin, ferulic acid and β-amyrin acetate, and the major compounds in ExEP-A were cinnamic acid, tocopherol, β-amyrin and apigenin. Both extracts presented similar amounts of p-methylbenzoic acid and caffeic acid, but ExEP-P contains higher amounts of amyrin, tocopherol, vanillin and ferulic acid analogues. In contrast, ExEP-A presented approximately 2.5 times more cinnamic acid and 2 times more p-coumaric acid and exclusively the compounds apigenin, luteolin, rutin, sinapic acid, α-amyrin acetate, taraxasterol, campesterol and stigmasterol.
Free radical-scavenger activity
Both ExEP presented antioxidant activity against the DPPH free radical. ExEP-P was able to inhibit 50% of free radicals (IC50) at a concentration of 182.4 ± 58.9 μg/mL and had a maximum activity of 94.6 ± 0.9% of DPPH radical capture at 500 μg/mL, being approximately 3.7 times less efficient than ExEP-A, which presented an IC50 of 49.8 ± 4.99 μg/mL and a maximum activity of 94.6 ± 0.3% at 300 μg/mL (Table 3). ExEP-A showed similar antioxidant activity as the synthetic antioxidant BHT, which presented IC50 of 52.8 ± 19.3 μg/mL and a maximum activity of 93.5 ± 0.5% at 500 μg/mL. The antioxidant standard ascorbic acid showed IC50 of 3.16 ± 0.6 μg/mL and maximum activity of 96.8 ± 0.4% at 10 μg/mL (S2 Fig).
Oxidative hemolysis inhibition assay
The antioxidant activity was also evaluated by an inhibition assay against AAPH-induced hemolysis. ExEP-P was not able to inhibit hemolysis induced by the oxidizing agent AAPH. The ascorbic acid control and ExEP-A showed concentration- and time-dependent anti-hemolytic activity. Ascorbic acid and ExEP-A inhibited 52.9 ± 15.6% and 24.6 ± 12.3% of hemolysis compared to the AAPH control, respectively, at a concentration of 125 μg/mL, after 240 min of incubation (Fig 1). The ascorbic acid control and ExEP, in the different concentrations evaluated, did not show hemolytic action when incubated with red blood cells alone, in the absence of the oxidizing agent AAPH (S3 Fig).
Protective effect of ascorbic acid (standard antioxidant) and ethanolic extracts of P. droryana (ExEP-P) and A. mellifera (ExEP-A) propolis against hemolysis induced by AAPH in human erythrocyte suspension at (A) 120 (B) 180 and (C) 240 min evaluation. NaCl (0.9%) and 1% ethanol was employed as negative controls. The results are expressed as mean ± SEM (standard error of the mean), n = 3. *Significantly different (p < 0.05) compared to the AAPH control group. With the exception of the negative control, all treatments were incubated with the oxidizing agent AAPH.
Dosage of malondialdehyde (MDA)
The efficiency of ExEP in inhibiting AAPH-induced lipid peroxidation was assessed based on its ability to reduce levels of MDA, a by-product of lipid peroxidation. ExEP-P was not able to inhibit the MDA content generated by the action of the oxidizing agent AAPH. The ascorbic acid control and ExEP-A reduced the levels of MDA by 65.7 ± 9.0 and 38.4 ± 7.3%, respectively, compared to the AAPH control, after 240 min of incubation at the highest concentration evaluated (Fig 2).
Concentrations of malondialdehyde (nmol/mL) after incubation of human erythrocytes with ascorbic acid (standard antioxidant) and ethanolic extracts of propolis from P. droryana (ExEP-P) and A. mellifera (ExEP-A), induced by oxidizing agent AAPH at (A) 120 (B) 180 and (C) 240 min of evaluation. NaCl (0.9%) and 1% ethanol was employed as negative controls. The results are expressed as mean ± SEM (standard error of the mean), n = 3. *Significantly different (p < 0.05) compared to the AAPH control group. With the exception of the negative control, all treatments were incubated with the oxidizing agent AAPH.
Cytotoxic activity and cell death profile
Peripheral blood mononuclear and K562 cells were treated with ExEP-P and ExEP-A to assess cell cytotoxicity. Both extracts of propolis showed lower cytotoxicity against peripheral blood mononuclear cells than K562 cells. The ExEP-P (IC50 = 0.38 mg/mL) and ExEP-A (IC50 = 0.36 mg/mL) promoted the cell death in K562 cells (Fig 3) after 24 h of treatment, the main mechanisms of death observed in both extracts was necrosis (Fig 4). The results show that propolis produced by different species of bees induce the same cell death mechanism.
Cytotoxic activity of ExEP from (A) P. droryana and (B) A. mellifera against the peripheral blood mononuclear cells (PBMC) and K562 erythroleukemia cell lines.
(A) Dot plots indicating the flow cytometry, and (B) representative diagrams obtained via flow cytometry of cells stained with annexin V-FITC/PI; Anx–/PI–: viable cells; Anx+/PI–: apoptotic cells; Anx–/PI+: necrotic cells, and Anx+/PI+: cells in late apoptosis. ***p < 0.001 treated group versus control viable cells. ###p < 0.001 treated group versus control apoptosis. +++p < 0.001 treated group versus control necrosis. xxxp < 0.001 and xp < 0.05 treated group versus control late apoptosis.
Effect of ExEP on the K562 cell viability
The viability of the K562 cells without and with treatment (IC50) of the ExEP-P or ExEP-A, was observed under an inverted microscope. No significant change was observed in control cells, however, for both ExEP, the cell survival decreased with increasing time (Fig 5). The results showed that the extracts of propolis were antiproliferative agents against K562 cells.
A monoclonal anti-cleaved caspase-3 antibody was used to evaluate caspase-3 activation in cells K562 incubated with IC50 of the ExEP-P and ExEP-A, and the cells were analyzed via flow cytometry. Both extracts resulted in the cleavage of procaspase 3 in 4, 8, 24 and 48 h, as indicated by a shift in fluorescence to the right (Fig 6) compared with the untreated control.
Effect of inhibitors on ExEP-induced cell death
The necrosis inhibitor necrostatin-1 (NEC-1) was effective in inhibiting the ExEP-P-induced death (IC50 = 0.38 mg/mL) of K562 cells treated for 24 h. However, the ExEP-A (IC50 = 0.36 mg/mL) was ineffective in inhibiting cell death (Fig 7).
Propolis is among the oldest natural products described for medicinal purposes and used by ancient societies. This bee product is a promising bioactive blend incorporating phenolic compounds, terpenes and tocopherol. These classes of compounds have already been identified in samples of propolis from different species of bees because they present anti-inflammatory [17–19], antimicrobial [14,34], antibiofilm [12,13], antioxidant [10,36] and antitumour effects [15,16,31,37].
In propolis from Brazilian meliponinae, phenolic compounds and triterpenes have been identified among the major constituents . In addition, compounds such as cinnamic acid, coumaric acids and caffeates are among the main bioactive compounds of propolis [8,36,38–41] and have been described as potential antioxidants [32,36,42,43] due to their chemical structures [36,44].
The structures of phenolic compounds have at least one aromatic ring with one or more attached hydroxyl groups, which are capable of donating hydrogen or electrons, preventing the oxidation of other substances, particularly lipids [39,45]. Another mechanism by which these compounds exert antioxidant activity is via the inactivation of enzymes (xanthine oxidase, protein kinase C, ascorbic acid oxidase) involved in the production of reactive oxygen species (ROS) . Thus, the phenolic compounds may be related to the antioxidant activity exerted by the ExEP of P. droryana and A. mellifera. The extracts of propolis evaluated were able to inhibit the DPPH free radical. In the body, ROS are produced during the cell cycle and functional activities, and they play important roles in various biological processes, such as cell signalling, apoptosis and gene expression [46–48].
However, excessive ROS production may result in oxidative stress, which is characterized by an imbalance between the production of oxidizing substances and endogenous antioxidants, and may cause the oxidation of biomolecules present in the cells, mitochondrial dysfunction and the activation of caspase cascades, resulting in cell death [47,49]. Oxidative stress has been found to be a trigger for chronic diseases such as diabetes, cardiovascular disease and cancer .
The antioxidant defense system of the human organism involves a set of enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as, a set of non-enzymatic substances such as glutathione, composed of an active thiol group and acts in the elimination of reactive species and as cofactor for several antioxidant enzymes. Besides this, ascorbic acid, carotenoids, α-tocopherol, and other dietary antioxidants also act against damage induced by high concentrations of ROS. However, these protection systems are sometimes insufficient to completely prevent oxidative damage . These factors demonstrate the importance of identifying natural compounds and/or new substances that can neutralize these free radicals to prevent oxidative stress.
In this study, the antioxidant activity of ExEP was also evaluated by testing its protection against oxidative hemolysis and the ability to reduce the levels of MDA, a product of lipid peroxidation due to oxidative stress . The cell membrane is one of the structures most susceptible to the action of ROS due to lipid peroxidation, which causes changes in its structure and permeability [36,40].
However, unlike the results obtained by the direct DPPH free radical capture assay, only A. mellifera propolis extract was able to protect red blood cells against damage by the oxidizing agent AAPH. These results may be related to the chemical composition of this propolis, since ExEP-A presented higher amounts of cinnamic acid than ExEP-P, in addition to campesterol, stigmasterol, taraxasterol, rutin, luteolin and apigenin identified exclusively in ExEP-A. Rutin and apigenin, have already been described as antioxidant agents [51, 52], and may be related to the antioxidant activity of this extract.
Although ExEP-A showed higher antioxidant activity than ExEP-P, the cytotoxic activity of the both extracts was similar. This result suggests that compounds that promote antioxidant action may not be responsible for the antitumor action of the extracts. Thus, the compounds caffeic acid, quercetin and tocopherol described by their antitumor activities [53–55] were identified in both extracts and may be related to this biological activity of both extracts.
In addition, other compounds found in the extracts were also reported as cinnamic acid, rutin, apigenin and taraxasterol have also been reported as potential antitumour agents [51,52,56,57]. Studies have shown that apigenin is an important oncogenesis blocker .
In evaluating the cytotoxic activity of propolis, it was observed that the ExEP could reduce the cellular viability of leukemic cells (K562). Thus, the compounds present in ExEP may be relevant in inhibiting tumour cells. Apigenin has been reported to inhibit the growth of laryngeal carcinoma cells . Taraxasterol showed antitumour activity in glioblastoma cells . In addition, caffeic acid, considered the main constituent of propolis, has already been reported to exhibit cytotoxic action in human myeloid leukemia cells .
For both extracts, the main mechanism of death observed was necrosis. Other cytotoxicity studies of stingless bee propolis showed the same mechanism of killing against K562 cells [15,16]. Franchi Jr. et al.  found that extracts of green and red propolis produced by Apis mellifera, in the southeastern and northeastern regions of Brazil, respectively, were cytotoxic against erythroleukemic strains; however, they promoted cell death via apoptosis.
Although apoptosis is among the main mechanisms of action of the drugs currently on the market, some ex vivo studies show that propolis extracts present different responses against tumour cells, regarding the mechanism of cell death [58,59]. In this context, necrosis-induced cell death may be an alternative for the treatment of tumour lines that show resistance to death by apoptosis.
In summary, our results show for the first time that the propolis produced by P. droryana and A. mellifera from the Brazilian Cerrado present potential use in the pharmaceutical and food industries, considering their antioxidant and cytotoxic properties against erythroleukemia cells.
Chromatogram by GC-MS of the ExEP (A) P. droryana, (B) A. mellifera, and HPLC of the ExEP (C) P. droryana and (D) A. mellifera.
S2 Fig. Nonlinear regression to establish the half-maximal inhibitory concentration (IC50) of DPPH free radical scavenging for ascorbic acid, BHT, ethanolic extracts of propolis of P. droryana and A. mellifera.
Effect of ascorbic acid (standard antioxidant) and ethanolic extracts of P. droryana (ExEP-P) and A. mellifera (ExEP-A) propolis in human erythrocyte suspension at (A) 120 (B) 180 and (C) 240 min evaluation. NaCl (0.9%) and 1% ethanol was employed as negative controls. The results are expressed as mean ± SEM (standard error of the mean), n = 3. *Significantly different (p < 0.05) compared to the NaCl (0.9%) control group.
We thank Luis Carlos Rossini from Apiarios Carbonari, for assistance in collect of propolis samples from A. mellifera in the state of Mato Grosso do Sul. This work was supported by Grants from Foundation to Support to Development of Education, Science and Technology of Mato Grosso do Sul State–FUNDECT and Brazilian National Research Council–CNPq, Instituto Nacional de Pesquisa do Pantanal—INPP, and Fundação de Amparo e Desenvolvimento da Pesquisa–Fadesp. Thaliny Bonamigo was supported by FUNDECT and CAPES.
- 1. Castaldo S, Capasso F. Propolis, an old remedy used in modern medicine. Fitoterapia. 2002; 73(1).
- 2. Cortopassi-Laurino M, Imperatriz-Fonseca VL, Roubik DW, Dollin A, Heard T, Aguilar I, et al. Global meliponiculture: challenges and opportunities. Apidologie 2006, 37: 1–18.
- 3. Camargo JMF, Pedro SRM. Meliponini Lepeletier, 1836. In Moure, J. S., Urban, D., Melo, G. A. R. (Orgs), 2014. Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region–versão online. Available in: <http://www.moure.cria.org.br/catalogue> Accessed on 3 nov. 2016.
- 4. Roubik DW. Ecology and Natural History of Tropical Bees. Cambridge University Press, New York, 1989. 514 p.
- 5. Bankova V, Popova M, Trusheva B. Propolis volatile compounds: chemical diversity and biological activity: a review. Chemistry Central Journal. 2014; 8(28): 1–8. pmid:24812573
- 6. Bankova V. Chemical diversity of propolis and the problem of standardization. Journal of Ethnopharmacology. 2005; 100: 114–117. pmid:15993016
- 7. Žižić JB, Vukovic NL, Jadranin MB, Andelkovic BD, Tesevic VV, Kacaniova MM, et al. Chemical composition, cytotoxic and antioxidative activities of ethanolic extracts of propolis on HCT-116 cell line. Journal of the Science of Food and Agriculture. 2013; 93: 3001–3009. pmid:23504630
- 8. Salatino A, Teixeira EW, Negri G, Message D. Origin and Chemical Variation of Brazilian Propolis. Evid-Based Complementary and Alternative Medicine. 2005; 2(1): 33–38. pmid:15841276
- 9. Huang S, Zhang CP, Wang K, Li GQL, Hu FL. Recent Advances in the Chemical Composition of Propolis. Molecules. 2014; 19: 19610–19632. pmid:25432012
- 10. Russo A, Longo R, Vanella A. Antioxidant activity of propolis: role of caffeic acid phenethyl ester and galangin. Fitoterapia. 2002; 73(Suppl. 1): S21–S29.
- 11. Kumazawa S, Hamasaka T, Nakayama T. Antioxidant activity of propolis of various geographic origins. Food Chemistry. 2004; 84: 329–339.
- 12. Ong TH, Chitra E, Ramamurthy S, Siddalingam RP, Yuen KH, Ambu SP, et al. Chitosan-propolis nanoparticle formulation demonstrates anti-bacterial activity against Enterococcus faecalis biofilms. PLoS ONE. 2017; 12(3): 1–22. pmid:28362873
- 13. De Marco S, Piccioni M, Pagiotti R, Pietrella D. Antibiofilm and antioxidant activity of propolis and bud poplar resins versus Pseudomonas aeruginosa. Evid-Based Complementary and Alternative Medicine. 2017; 2017: 1–11.
- 14. Afrouzan H, Amirinia C, Mirhadi SA, Ebadollahi A, Vaseji N, Tahmasbi G. Evaluation of antimicrobial activity of propolis and nanopropolis against Staphylococcus aureus and Candida albicans. African Journal of Microbiology Research. 2012; 6(2): 421–425.
- 15. Campos JF, Santos UP, Macorini LF, de Melo AM, Balestieri JB, Paredes-Gamero EJ, et al. Antimicrobial, antioxidant and cytotoxic activities of propolis from Melipona orbignyi (Hymenoptera, Apidae). Food and Chemical Toxicology. 2014; 65: 374–380. pmid:24412556
- 16. Campos JF, Santos UP, Rocha PS, Damião MJ, Balestieri JBP, Cardoso CAL, et al. Antimicrobial, Antioxidant, Anti-Inflammatoty, and Cytotoxic Activies of Propolis from the Stingless Bee Tetragonisca fiebrigi (Jataí). Evidence-Based Complementary and Alternative Medicine. 2015; 2015: 1–11. pmid:26185516
- 17. Machado JL, Assunção AKM, Silva MCP, Dos Reis AS, Costa GC, Arruda DS, et al. Brazilian Green Propolis: Anti-Inflammatory Property by an Immunomodulatory Activity. Evidence-Based Complementary and Alternative Medicine. 2012; 2012: 1–10 pmid:23320022
- 18. Bueno-Silva B, Alencar SM, Koo H, Ikegaki M, Silva GVJ, Napimoga MH, et al. Anti-Inflammatory and Antimicrobial Evaluation of Neovestitol and Vestitol Isolated from Brazilian Red Propolis. Journal of Agriculture and Food Chemistry. 2013; 61(19): 4546–4550. pmid:23607483
- 19. Funakoshi-Tago M, Okamoto K, Izumi R, Tago K, Yanagisawa K, Narukawa Y. Anti-inflammatory activity of flavonoids in Nepalese propolis is attributed to inhibition of the IL-33 signaling pathway. International Immunopharmacology. 2015; 25: 189–198. pmid:25614224
- 20. Cinegaglia NC, Bersano PRO, Araújo MJAM, Búfalo MC, Sforcin JM. Anticancer Effects of Geopropolis Produced by Stingless Bees on Canine Osteosarcoma Cells In Vitro. Evidence-Based Complementary and Alternative Medicine. 2013; 2013: 1–6. pmid:23690851
- 21. Calhelha RC, Falcão S, Queiroz MJRP, Vilas-Boas M, Ferreira ICFR. Cytotoxicity of Portuguese Propolis: The Proximity of the In Vitro Doses for Tumor and Normal Cell Lines. BioMed Research International. 2014; 2014: 1–7. pmid:24982911
- 22. Lopez ABGC, Lourenço CC, Alvesa DA, Machado D, Lancellottia M, Sawaya CHF. Antimicrobial and cytotoxic activity of red propolis: an alert as to its safe use. Journal of Applied Microbiology. 2015; 119(3): 677–687. pmid:26086953
- 23. Moreira L, Dias LG, Pereira JA, Estevinho L. Antioxidant properties, total phenols and pollen analysis of propolis samples from Portugal. Food and Chemical Toxicology. 2008; 46: 3482–3485. pmid:18804144
- 24. Valente MJ, Baltazar AF, Henrique R, Estevinho L, Carvalho M. Biological activities of Portuguese propolis: Protection against free radical-induced erythrocyte damage and inhibition of human renal cancer cell growth in vitro. Food and Chemical Toxicology. 2011; 49: 86–92. pmid:20934479
- 25. Ewnetu Y, Lemma N, Birhane W. Antibacterial effects of Apis mellifera and stingless bees honeys on susceptible and resistant strains of Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae in Gondar, Northwest Ethiopia. Complementary and Alternative Medicine. 2013; 13(269): 2–7. pmid:24138782
- 26. Sawaya ACHF, Cunha IBS, Marcucci MC, Aidard DS, Silva ECA, Carvalho CAL, et al. Electrospray ionization mass spectrometry fingerprinting of propolis of native Brazilian stingless bees. Apidologie. 2007; 38: 93–103.
- 27. Velikova M, Bankova V, Marcucci MC, Tsvetkova I, Kujumgiev A. Chemical Composition and Biological Activity of Propolis from Brazilian Meliponinae. Zeitschrift für Naturforsch. 2000; 55c: 785–789.
- 28. Bueno-Silva B, Marsola A, Ikegaki M, Alencar SM, Rosalen PL. The effect of seasons on Brazilian red propolis and its botanical source: chemical composition and antibacterial activity. Natural Product Research. 2016; 4:1–7. pmid:27701899
- 29. Cabral ISR, Oldoni TLC, Prado A, Bezerra RMN, Alencar SM. Composição fenólica, atividade antibacteriana e antioxidante da própolis vermelha brasileira. Quimica Nova. 2009; 32(6) 1523–1527.
- 30. Teerasripreecha D, Phuwapraisirisan P, Puthong S, Kimura K, Okuyama M, Mori H, et al. In vitro antiproliferative/cytotoxic activity on cancer cell lines of a cardanol and a cardol enriched from Thai Apis mellifera propolis. BMC Complementary and Alternative Medicine. 2012;12(27): 2–17.
- 31. Milošević-Đorđević O, Grujičić D, Radović M, Vuković N, Žižić J, Marković S. In vitro chemoprotective and anticancer activities of propolis in human lymphocytes and breast cancer cells. Archives of Biological Sciences Belgrade. 2015; 67(2): 571–581.
- 32. Alencar SM, Oldoni TLC, Castro ML, Cabral ISR, Costa-Neto CM, Cury JA, et al. Chemical composition and biological activity of a new type of Brazilian propolis: red propolis. Journal of Ethnopharmacology. 2007; 113: 278–283. pmid:17656055
- 33. Gupta D, Gupta RK. Bioprotective properties of Dragon’s blood resin: In vitro evaluation of antioxidant activity and antimicrobial activity. BMC Complementary Alternative and Medicine. 2011; 11: 1–9. pmid:21329518
- 34. Paredes-Gamero EJ, Martins MNC, Cappabianco FAM, Ide JS, Miranda A. Characterization of dual effects induced by antimicrobial peptides: Regulated cell death or membrane disruption. Biochimica et Biophysica Acta. 2012; 1820: 1062–1072. pmid:22425533
- 35. Moraes VWR, Caires ACF, Paredes-Gamero EJ, Rodrigues T, Organopalladium compound 7b targets mitochondrial thiols and induces caspase-dependent apoptosis in human myeloid leukemia cells. Death & Disease. 2013; 4 (e658): 1–8. pmid:23744358
- 36. KureK-Górecka A, Stojko AR, Górecki M, Stojko J, Sosada M, Zięba GS. Structure and antioxidant activity of polyphenols derived from propolis. Molecules. 2014; 19: 78–101. pmid:24362627
- 37. Jin UH, Song KH, Motomura M, Suzuki I, Gu YH, Kang YJ, et al. Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells. Molecular and Cellular Biochemistry. 2008; 310: 43–48. pmid:18060475
- 38. Awale S, Li F, Onozuka H, Tezuka Y, Kadota S. Constituents of Brazilian red propolis and their preferential cytotoxic activity against human pancreatic PANC-1 cancer cell line in nutrient-deprived condition. Bioorganic & Medicinal Chemistry. 2008; 16: 181–189. pmid:17950610
- 39. Carocho M, Ferreira ICFR. The role of phenolic compounds in the fight against cancer–A Review. Anti-Cancer Agents in Medicinal Chemistry. 2013; 13: 1236–1258. pmid:23796249
- 40. Lü JM, Lin PH, Yao Q, Chen C. Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. Journal of Cellular and Molecular Medicine. 2010; 14(4): 840–860. pmid:19754673
- 41. Kasai H, Kawai K. Oxidative DNA damage: mechanisms and significance in health and disease. Antioxidants & Redox Signaling. 2006; 8: 981–983. pmid:16771687
- 42. Kostova I. Synthetic and natural coumarins as antioxidants. Mini-Reviews in Medicinal Chemistry. 2006; 6(4) 365–374. pmid:16613573
- 43. Scalbert A, Johnson IT, Saltmarsh M. Polyphenols: antioxidants and beyond. The American Journal of Clinical Nutrition. 2005; 81(1): 215S–217S.
- 44. Duthie GG, Gardner PT, Kyle JAM. Plant polyphenols: are they the newmagic bullet? Proceedings of the Nutrition Society. 2003; 62(3): 599–603. pmid:14692595
- 45. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaft Technologie. 1995; 28: 25–30.
- 46. Apel K, Hirt H. Reactive oxygen species: Metabolism, Oxidative Stress, and Signal Transduction. Annual Review of Plant Biology. 2004; 55: 373–99. pmid:15377225
- 47. Circu LM, Aw TY. Reactive oxygen species, cellular redox systems and apoptosis. Free Radical Biology & Medicine. 2010; 48(6): 749–762. pmid:20045723
- 48. Dröge W. Free Radicals in the Physiological Control of Cell Function. Physiological Reviews. 2002;, 82: 47–95. pmid:11773609
- 49. Boonstra J, Post JA. Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene. 2004; 337: 1–13. pmid:15276197
- 50. Dalle-Donne I, Rossi R, Colombo R, Giustarini D, Milzani A. Biomarkers of oxidative damage in human disease. Clinical Chemistry. 2006; 52: 601–623. pmid:16484333
- 51. Boyle SP, Dobson VL, Duthie SJ, Hinselwood DC, Kyle JA, Collins AR. Bioavailability and efficiency of rutin as an antioxidant: a human supplementation study. European Journal of Clinical Nutrition. 2000; 54(10): 774–82. pmid:11083486
- 52. Shaal LAL, Shegokar R, Müller RH. Production and characterization of antioxidant apigenin nanocrystals as a novel UV skin protective formulation. International Journal of Pharmaceutics. 2011; 420(1):133–40. pmid:21871547
- 53. Chang WC, Hsieh CH, Hsiao MW, Lin WC, Hung YC, Ye JC. Caffeic acid induces apoptosis in human cervical cancer cells through the mitochondrial pathway. Taiwan Journal of Obstetrics Gynecology. 2010; 49(4): 419–424.
- 54. Srivastava S, Somasagara RR, Hegde M, Nishana M, Tadi SK, Srivastava M, et al. Quercetin, a natural flavonoid interacts with DNA, arrests cell cycle and causes tumor regression by activating mitochondrial pathway of apoptosis. Scientific Reports. 2016; 6(24049): 1–13 pmid:27068577
- 55. Bak MJ, Das Gupta S, Wahler J, Lee HJ, Li X, Lee MJ, et al. Inhibitory effects of γ- and δ-tocopherols on estrogen-stimulated breast cancer in vitro and in vivo. Cancer Prevention Research. 2017; 10(3): 1–33
- 56. Bao YY, Zhou SH, Fan J, Wang QY. Anticancer mechanism of apigenin and the implications of GLUT-1 expression in head and neck cancers. Future Oncology. 2013; 9 (9):1353–64. pmid:23980682
- 57. Hong JF, Song YF, Liu Z, Zheng ZC, Chen HJ, Wang SS. Anticancer activity of taraxerol acetate in human glioblastoma cells and a mouse xenograft model via induction of autophagy and apoptotic cell death, cell cycle arrest and inhibition of cell migration. Molecular Medicine Reports. 2016;13: 4541–4548. pmid:27081915
- 58. Franchi Jr. GC, Moraes CS, Toreti VC, Daugsch A, Nowill AE, Park YK. Comparison of Effects of the Ethanolic Extracts of Brazilian Propolis on Human Leukemic Cells As Assessed with the MTT Assay. Evidence-Based Complementary and Alternative Medicine. 2012; 2012: 1–6. pmid:21966298
- 59. Sawicka D, Car H, Borawska MH, Nikliński J. The anticancer activity of propolis. Folia Histochemica et Cytobiologica. 2012; 50(1): 25–37. pmid:22532133