Chemical Composition and Anti-Inflammatory Effect of Ethanolic Extract of Brazilian Green Propolis on Activated J774A.1 Macrophages

Szliszka, Ewelina; Kucharska, Alicja Z.; Sokół-Łętowska, Anna; Mertas, Anna; Czuba, Zenon P.; Król, Wojciech

doi:https://doi.org/10.1155/2013/976415

Evidence-Based Complementary and Alternative Medicine

On this page

Abstract Introduction Materials and Methods Results Discussion Conclusion Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2013 | Article ID 976415 | https://doi.org/10.1155/2013/976415

Chemical Composition and Anti-Inflammatory Effect of Ethanolic Extract of Brazilian Green Propolis on Activated J774A.1 Macrophages

Ewelina Szliszka,¹Alicja Z. Kucharska,²Anna Sokół-Łętowska,²Anna Mertas,¹Zenon P. Czuba,¹and Wojciech Król¹

Academic Editor: Andrzej K. Kuropatnicki

Received04 Apr 2013

Accepted08 May 2013

Published06 Jun 2013

Abstract

The aim of this study was to investigate the chemical composition and anti-inflammatory effect of ethanolic extract of Brazilian green propolis (EEP-B) on LPS + IFN-γ or PMA stimulated J774A.1 macrophages. The identification and quantification of phenolic compounds in green propolis extract were performed using HPLC-DAD and UPLC-Q-TOF-MS methods. The cell viability was evaluated by MTT and LDH assays. The radical scavenging ability was determined using DPPH^• and ABTS^•+. ROS and RNS generation was analyzed by chemiluminescence. NO concentration was detected by the Griess reaction. The release of various cytokines by activated J774A.1 cells was measured in the culture supernatants using a multiplex bead array system based on xMAP technology. Artepillin C, kaempferide, and their derivatives were the main phenolics found in green propolis. At the tested concentrations, the EEP-B did not decrease the cell viability and did not cause the cytotoxicity. EEP-B exerted strong antioxidant activity and significantly inhibited the production of ROS, RNS, NO, cytokine IL-1α, IL-1β, IL-4, IL-6, IL-12p40, IL-13, TNF-α, G-CSF, GM-CSF, MCP-1, MIP-1α, MIP-1β, and RANTES in stimulated J774A.1 macrophages. Our findings provide new insights for understanding the anti-inflammatory mechanism of action of Brazilian green propolis extract and support its application in complementary and alternative medicine.

1. Introduction

Propolis is a resinous substance collected by honeybees from the leaf bud and bark of certain plants. Baccharis dracunculifolia, Eucalyptus citriodora, Araucaria angustifolia, and Myrocarpus frondosus are major botanical sources of propolis from southeast Brazil (States of Minas Gerais and Sao Paulo) called due to its colour green propolis [1, 2]. Extracts of Brazilian green propolis possess antioxidant, antimicrobial, anti-inflammatory, chemopreventive, and anticancer properties [3–9]. Therefore, propolis has been extensively used in food, beverages, and dietary supplements to improve health and prevent diabetes, cancer, inflammatory, and heart diseases [10–13]. The medical application of propolis has led to increased interest in its chemical composition depending on the geographical origin and specific flora of the region, seasonality or methods of harvesting the raw material [1–4].

Our study was designed to investigate the chemical composition and anti-inflammatory effect of ethanolic extract of Brazilian green propolis (EEP-B) on LPS (lipopolysaccharide) + IFN-γ (interferon γ) or PMA (phorbol 12-myristate 13-acetate) stimulated J774A.1 macrophages.

Macrophages are the first effector cells of the immune system that protect against microbial infection and tissues injury. Macrophages have three important functions: antigen presentation, phagocytosis, and synthesis of various inflammatory mediators [14, 15]. Propolis affects the nonspecific immunity via modulation of macrophages activity [3, 13]. During inflammation, upon stimulation by LPS from Gram-negative bacteria and IFN-γ from host immune cells, macrophages release large amounts of reactive oxygen (ROS) and nitrogen species (RNS), nitric oxide (NO), and numerous cytokines [16]. Overexpression of these mediators causes pathological, acute, or chronic inflammatory responses [14].

This is the first comprehensive report explaining an anti-inflammatory potential of Brazilian green propolis extract on macrophage in vitro model. We determined the effect of EEP-B on production of NO, ROS, RNS, and cytokines: interleukin (IL)-IL-1α, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-13, IL-17, TNF-α (tumor necrosis factor α), IFN-γ (interferon γ), G-CSF (granulocyte colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), MCP-1 (monocyte chemotactic protein 1), MIP-1α (macrophage inflammatory protein 1α), MIP-1β (macrophage inflammatory protein 1β), and RANTES (regulated upon activation normal T cell expressed and secreted) in activated J774A.1 cells.

It has been suggested that the immunomodulatory properties of propolis is contributed by its flavonoids and phenolic acids [3, 5, 9, 17]. The typical ingredients of Brazilian green propolis are kaempferide (3,5,7-trihydroxy-4′-methoxyflavone) and cinnamic acid derivatives: p-coumaric acid (4-hydroxycinnamic acid), artepillin C (3,5-diprenyl-4-hydroxycinnamic acid), baccharin (3-prenyl-4-(dihydrocinnamoyloxy)-cinnamic acid) and drupanin (3-prenyl-4-hydroxycinnamic acid) [1, 4, 18]. We performed the detailed identification and quantification of phenolic compounds in propolis extract.

The present study analyzed chemical composition of green propolis and confirmed its significant role in suppressing chronic inflammation and reducing risk of related human health problems. Our findings provide new insights for understanding the anti-inflammatory mechanism of action of Brazilian green propolis extract and support its application in complementary and alternative therapies.

2. Materials and Methods

2.1. General

The Brazilian green propolis sample was kindly supplied by Nihon Natural Foods Co., Ltd. (Tokyo, Japan). LPS (LPS E. coli O111:B4) was obtained from Fluka Chemie GmbH (Buchs, Switzerland) and recombinant mouse IFN-γ was purchased from R&D Systems (Minneapolis, MN, USA). PMA, DMSO (dimethyl sulphoxide), acetonitrile, and formic acid were obtained from Sigma-Aldrich (Steinheim, Germany). Acetonitrile for LC-MS was purchased from POCh (Gliwice, Poland). The following compounds were used as standards: sakuranetin, hesperitin, caffeic acid (Roth, Karlsruhe, Germany), kaempferol, pinocembrin, naringenin, p-coumaric acid (Sigma-Aldrich, Steinheim, Germany), kaempferide, ferulic acid (Serva, Heidelberg, Germany), isosakuranetin (TransMIT GmbH, Giessen, Germany), and artepillin C (Wako Pure Chemicals, Osaka, Japan).

2.2. Preparation of Brazilian Green Propolis Extract

The Brazilian green propolis sample was collected manually from beehive located in southeast Brazil (the state of Minas Gerais) and was kept desiccated prior to processing. The sample was extracted in 95% v/v ethyl alcohol, in a hermetically sealed glass vessel for 4 days at 37°C, under occasional shaking. The ethanolic extract of Brazilian green propolis (EEP-B) was then filtered through Whatman filter paper no. 4 and evaporated in a rotary evaporator, under reduced pressure at 60°C. The same collection and extraction procedures were used throughout all our laboratory studies [6, 12]. EEP-B was dissolved in DMSO (50 mg/mL), and the final concentration of DMSO in the culture medium was controlled at 0.1% (v/v).

2.3. Identification and Quantification of Phenolic Compounds by HPLC-DAD Method

Phenolic compounds were determined using Dionex (USA) HPLC system equipped with diode array detector model Ultimate 3000, a quaternary pump LPG-3400A, autosampler EWPS-3000SI, and thermostated column compartment TCC-3000SD and controlled by Chromeleon v.6.8 software. The reversed phase Cadenza 5CD-C18 (75 mm × 4.6 i.d.) column (Imtakt, Kyoto, Japan) with guard column Cadenza (5 × 4.6 i.d.) guard column (Imtakt, Kyoto, Japan) was used. The mobile phase was composed of (A) 0.1% (v/v) formic acid in water and (B) acetonitrile. The applied elution conditions were 0 min 20% B; 0–10 min linear gradient from 20% to 30% B; 10–40 min linear gradient from 30% to 40% B; 40–60 min, linear gradient from 40% to 60% B; 60–80 min, linear gradient from 60% to 80% B; and then again the initial conditions [19–23]. The flow rate was 1 mL/min, and the injection volume was 20 μL. The column was operated at 30°C. The compounds were monitored at 290 nm, 325 nm, and 370 nm.

2.4. Identification and Quantification of Phenolic Compounds by LC-MS Method

Compounds identification was performed on an Acquity ultra-performance liquid chromatography (UPLC) system coupled with a quadruple time of flight (Q-TOF) MS instrument (UPLC/Synapt Q-TOF MS, Waters Corp., Milford, MA, USA) with an electrospray ionization (ESI) source. Separation was achieved on the Acquity BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm; Waters). Detection wavelengths were set at 290, 325, and 370 nm. A mobile phase was a mixture of 1.5% formic acid (A) and acetonitrile (B). The gradient program was as follows: initial conditions-95% (A), 12 min-5% (A), 13 min-5% (A), 14.5 min-95% (A), and 16 min-95% (A). The flow rate was 0.45 mL/min and the injection volume was 5 μL. The column was operated at 30°C. The major operating parameters for the Q-TOF MS were set as follows: capillary voltage 2.0 kV, cone voltage 45 V, cone gas flow of 11 L/h, collision energy 50 eV, source temperature 100°C, desolvation temperature 250°C, collision gas, argon; desolvation gas (nitrogen) flow rate, 600 L/h; data acquisition range, 100–1.000 Da; ionization mode, negative [24–26]. The data were collected by Mass-Lynx V 4.1 software.

2.5. Cell Culture

Murine macrophage J774A.1 cell line was obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and 10% CO₂ in a humidified incubator [27]. Reagents for cell culture were purchased from ATCC. J774A.1 cells were seeded at a density of 1 × 10⁶/mL cells (2 × 10⁵/well) in 96-well plates at the presence of LPS (200 ng/mL) and IFN-γ (25 U/mL) with or without EEP-B for 24 h.

2.6. Cell Viability Assay

The cell viability was determined by the 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay as described [28, 29]. This test is based on the cleavage of the tetrazolium salt MTT to a blue formazan dye by viable cells. The J774A.1 cells (1 × 10⁶/mL) were seeded 4 h before the experiments in a 96-well plate. EEP-B at the concentrations of 5–50 μg/mL with or without LPS + IFN-γ was added to the cells. The final volume was 200 μL. After 24 h the medium was removed and 20 μL MTT solutions (5 mg/mL) (Sigma Chemical Company, St. Louis, MO, USA) were added to each well for 4 h. The resulting formazan crystals were dissolved in DMSO. The controls included native cells and medium alone. The spectrophotometric absorbance was measured at 550 nm wavelength using a microplate reader (ELx 800, Bio-Tek Instruments Inc., Winooski, VT, USA). The cytotoxicity as percentage of cell death was calculated by the formula: (1 − [absorbance of experimental wells/absorbance of control wells]) × 100%.

2.7. Cytotoxicity Assay

The cytotoxicity of EEP-B was determined by using LDH activity assay kit (Roche Diagnostics GmbH, Mannheim, Germany) [30, 31]. Lactate dehydrogenase (LDH) is a stable cytosolic enzyme released upon membrane damage in necrotic cells. The J774A.1 (1 × 10⁶/mL) cells were treated with 5–50 μg/mL EEP-B with or without LPS + IFN-γ for the indicated period of time. LDH released in culture supernatants is detected with coupled enzymatic assay, resulting in the conversion of a tetrazolium salt into a red formazan product. The maximal release of LDH was obtained after treating control cells with 1% Triton X-100 (Sigma Chemical Company, St. Louis, MO) for 10 min at room temperature. The spectrophotometric absorbance was measured at 490 nm wavelength using a microplate reader (ELx 800, Bio-Tek Instruments Inc., Winooski, VT, USA). The percentage of necrotic cells was expressed using the following formula: (sample value/maximal release) × 100%.

2.8. DPPH Radical Scavenging Activity

Hydrogen-donating activity was measured using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH^•) (Sigma Chemical Company, St. Louis, MO) following a previously reported protocol [32]. EEP-B (0.1 mL) was mixed with 0.9 mL of 0.041 mM DPPH^• in ethanol and stored at room temperature in the dark for 30 min. The absorbance of the resulting solutions was measured at 517 nm wavelength using V-630 Spectrophotometer (Jasko International Co., Tokyo, Japan). The percentage of scavenging activity was calculated by the formula: DPPH^• scavenging activity = 1 − (absorbance of experimental wells/absorbance of control wells) × 100%. The scavenging activity of the sample was expressed as the ED₅₀ value, the concentration required to scavenge 50% of DPPH^•. Ascorbic acid was used as a standard.

2.9. ABTS Cation Radical Scavenging Activity

2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS^•+) (Sigma Chemical Company, St. Louis, MO) scavenging activity was determined according to the previously described procedure [32]. EEP-B (0.1 mL) was mixed with potassium phosphate buffer (0.1 mL of 0.1 M) and hydrogen peroxide (10 μL of 10 mM) and preincubated at 37°C in the dark for 5 min. Next, ABTS (30 μL of 1.25 mM in 0.05 M phosphate-citrate buffer) and peroxidase (30 μL of 1 unit/mL) were added to the mixture and then incubated at 37°C in the dark for 10 min. The absorbance of the resulting solutions was measured at 417 nm wavelength using V-630 Spectrophotometer (Jasko International Co., Tokyo, Japan). The percentage of scavenging activity was calculated by the formula: ABTS^•+ scavenging activity = 1 − (absorbance of experimental well/absorbance of control wells) × 100%. The scavenging activity of the sample was expressed as the ED₅₀ value, the concentration required to scavenge 50% of ABTS^•+. Ascorbic acid was used as a standard.

2.10. Detection of ROS and RNS Production by Chemiluminescence

The chemiluminescence of J774A.1 macrophages was evaluated by microplate method in Hank’s balanced salt solution, pH 7.4, at room temperature. The cells were incubated with 0.01–10 μg/mL EEP-B for 30 min. Next, luminol (Sigma Chemical Company, St. Louis, MO, USA) solution was added to wells containing 2 × 10⁵ cells, giving a final concentration of 110 μM. After 5 min, for macrophages stimulation PMA solution was injected to obtain the concentration of 0.8 μM. The final volume of each sample was 200 μL. The chemiluminescence was determined for 5 min with luminol alone and after stimulation with PMA for 30 min. The measuring system was equipped with LB 960 CentroXS³ microplate luminometer (Berthold Technologies GmbH, Wildbad, Germany) [33].

2.11. Quantification of NO Production

J774A.1 macrophages (1 × 10⁶/mL) stimulated with LPS + IFN-γ were incubated with 5–50 μg/mL EEP-B for 24 h. After this time NO production was determined by measuring the accumulation of nitrite, a stable end product, in the culture supernatant according to the Griess reaction [27, 34]. Equal volumes of culture supernatant from each well or medium (100 μL) were mixed with 100 μL of Griess reagent in a 96-well plate and incubated for 15 min at room temperature. The spectrophotometric absorbance was read at 550 nm wavelength in Eon Microplate Spectrophotometer (BioTek, Winooski, VT, USA) and the nitrite concentration in the medium was calculated using sodium nitrite as a standard. Nitrite was not detectable in cell-free medium.

2.12. Multiplex Bead-Based Cytokine Assay

Cytokines released from J774A.1 macrophages treated with EEP-B were determined in the cell culture supernatants with a Pro Mouse Cytokines 19-plex assay kit for IL-1α, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-13, IL-17, TNF-α, IFN-γ, G-CSF, GM-CSF, MCP-1, MIP-1α, MIP-1β, and RANTES (Bio-Rad Laboratories Inc., Hercules, CA, USA). This test was performed using Bio-Plex 200 System based on xMAP suspension array technology (Bio-Rad Laboratories Inc., Hercules, CA, USA). The LPS + IFN-γ stimulated and native J774A.1 cells (1 × 10⁶/mL) were incubated with or without 25–50 μg/mL EEP-B for 24 h. Standard curves for each cytokine were generated using kit-supplied reference cytokine sample. The assay is designed for the multiplexed quantitative measurement of multiple cytokines in a single well using 50 μL of sample. Briefly, the following procedure was performed: after prewetting the 96-well filter plate with washing buffer, the solution in each well was aspirated using a vacuum manifold. Next, the cell culture supernatants were incubated with antibody-conjugated beads for 30 min. Following the incubational period, detection antibodies and streptavidin-PE were added to each well for 30 min. Then, after washing with buffer to remove the unbound streptavidin-PE, the beads bound to each cytokine were analyzed in the Bio-plex Array Reader (Bio-Plex 200 System). The fluorescence intensity was evaluated using Bio-Plex Manager software (Bio-Rad) [33, 35].

2.13. The Statistical Analysis

The values represent mean ± SD of two, three, or four independent experiments performed in duplicate or quadruplicate. Significant differences were analyzed using Student’s -test and -values <0.05 were considered significant. The concentration-response curves were analyzed using Pharma/PCS version 4 (Pharmacological Calculations System) software.

3. Results

3.1. The Content and Characterization of Phenolic Compounds Identified in Brazilian Green Propolis Extract

The identification and quantification of phenolic compounds in Brazilian green propolis extract were performed using HPLC-DAD and UPLC-Q-TOF-MS methods. Qualitative analysis results obtained by LC-ESI/MS methods and quantitative analysis data obtained by HPLC (quantified using DAD detection) are presented in Figures 1, 2, 3, and 4 and Table 1. A total of forty-three phenolic ingredients were found in tested propolis sample. Thirty-four compounds were identified by comparison of their UV and MS/MS spectra to standards and/or to the literature data, whereas another nine compounds remained unknown. Kaempferide, with its derivatives, and hesperitin, which were characterized by MS from their molecular ions at 299.0572 and 301.0709, respectively, are the major flavonoids identified in Brazilian green propolis. Among the phenolic acids, prevailed p-coumaric acid ( 163.0406 and fragment at 119 resulting from the loss of a COO group) and prenylated cinnamic acid derivatives: artepillin C ( 299.1634), baccharin ( 363.1619), and drupanin ( 231.1025) (Table 1).

3.2. Effect of Brazilian Green Propolis Extract on Viability of J774A.1 Macrophages

The cell viability in the presence of 5–50 μg/mL EEP-B and/or LPS + IFN-γ for 24 h was measured by MTT test (Figure 5). The cytotoxicity of the propolis extract at the same concentrations and incubation time was evaluated by LDH assay. EEP-B at the concentrations of ≤50 μg/mL did not influence the cell viability and did not exert cytotoxic effect. Therefore for further studies of anti-inflammatory properties EEP-B was used at the concentrations of 5–50 μg/mL.

3.3. Antioxidant Activity of Brazilian Green Propolis Extract

Antioxidant activity of EEP-B was investigated by using two different methods for stable DPPH^• and ABTS^•+. The propolis extract exhibited strong scavenging potential against DPPH^• (ED₅₀ of 24.1 μg/mL) and ABTS^•+ (ED₅₀ of 40.6 μg/mL) compared with ascorbic acid (ED₅₀ of 15.8 μg/mL and 10.1 μg/mL, resp.).

3.4. Effect of Brazilian Green Propolis Extract on ROS and RNS Production in PMA Stimulated J774A.1 Cells

Changes in production of ROS and RNS in macrophages were determined by chemiluminescence assay. EEP-B at the concentrations of 0.01–10 μg/mL suppressed the chemiluminescence in PMA stimulated J774A.1 cells in dose-dependent manner with an ED₅₀ of 0.02 μg/mL (Figure 6).

(a)

(b)

3.5. Effect of Brazilian Green Propolis Extract on NO Production in LPS + IFN-γ Stimulated J774A.1 Cells

NO production was determined by measuring the accumulation of nitrite in the culture supernatants using Griess reagent. After 24 h LPS + IFN-γ stimulation nitrite concentration markedly increased, but LPS + IFN-γ-induced NO synthesis in J774A.1 cells was significantly decreased by EEP-B in dose-dependent manner (ED₅₀ = 30.1 μg/mL). The inhibitory effect of 5–50 μg/mL EEP-B on NO production in LPS + IFN-γ stimulated macrophages is presented in Figure 7. The propolis extract did not interfere with the viability of J774A.1 cells, as shown in MTT and LDH test. The ED₅₀ value of the EEP-B within the nontoxic concentration range suggests that the inhibition of nitrite accumulation was specific to responses by macrophages (due to inhibitory activity on NO production and not cytotoxic property of EEP-B).

3.6. Effect of Brazilian Green Propolis Extract on Cytokine Production in LPS + IFN-γ Stimulated J774A.1 Cells

The effect of EEP-B on production of cytokine IL-1α, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-13, IL-17, TNF-α, IFN-γ, G-CSF, GM-CSF, MCP-1, MIP-1α, MIP-1β, and RANTES in LPS + IFN-γ stimulated J774A.1 cells is shown in Figure 8. Specifically, native and activated J774A.1 macrophages were treated with 25–50 μg/mL EEP-B for 24 h. The cytokines released in culture supernatants were analyzed simultaneously by Bio-plex Suspension Array System. This assay is designed for the multiplexed quantitative measurement of cytokines (19-plex) in a single well using 50 μL of sample. EEP-B significantly decreased synthesis of IL-1α, IL-1β, IL-4, IL-6, IL-12p40, IL-13, TNF-α, G-CSF, GM-CSF, MCP-1, MIP-1α, MIP-1β, and RANTES in LPS + IFN-γ stimulated J774A.1 cells in dose-dependent manner. The EEP-B did no influence the concentrations of IL-3, IL-5, IL-9, IL-17, and IFN-γ and only slightly downregulates IL-10 in culture supernatants derived from activated macrophages.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

(m)

(n)

(o)

(p)

(q)

(r)

(s)

Figure 8

Effect of EEP-B on cytokines production in LPS + IFN-γ stimulated J774A.1 macrophages: (a) IL-1α, (b) IL-1β, (c) IL-3, (d) IL-4, (e) IL-5, (f) IL-6, (g) IL-9, (h) IL-10, (i) IL-12p40, (j) IL-13, (k) IL-17, (l) TNF-α, (m) IFN-γ, (n) G-CSF, (o) GM-CSF, (p) MCP-1, (q) MIP-1α, (r) MIP-1β, and (s) RANTES. J774A.1 cells were incubated with 25–50 μg/mL EEP-B and/or LPS + IFN-γ for 24 h. Cytokine concentrations in the culture medium were determined by Multiplex (19-plex) bead-based cytokine assay. The values represent mean ± SD of two independent experiments () * = , ** = , *** = compared to LPS + IFN-γ-stimulated cells.

4. Discussion

In Brazil, twelve distinct groups of propolis have been classified according to their botanical origin and biological properties. Green propolis collected in southern region of Brazil (States of Minas Gerais and Sao Paulo) belongs to Group 12 (propolis G12) [1]. Chemical evidence suggested that Baccharis dracunculifolia is the main plant source for green propolis [36]. Park et al. demonstrated similar profiles of phenolic components identified in green propolis extract and Baccharis dracunculifolia resins [37]. The tested sample of green propolis was rich in hesperitin, kaempferide and its derivatives, and cinnamic acid derivatives: p-coumaric acid, artepillin C, baccharin, and drupanin. Our studies confirmed the results obtained by Park et al. [1, 37] and Banskota et al. [38].

Macrophages are the main cells responsible for innate (nonspecific) immunity. The J774A.1 cells, a murine macrophage cell line, are widely used to establish inflammatory model in vitro [27]. Numerous findings showed antioxidant and anti-inflammatory effects of propolis extract [3, 4, 13, 18, 21, 27, 39, 40]. The biologically active molecules in green propolis are phenolic acids and flavonoids, which act as scavengers of free radicals and inhibitors of nitric oxide and inflammatory cytokines production by macrophages and/or neutrophils [4, 13, 16–18, 27, 34, 39, 40]. Kaempferide, artepillin C, and their derivatives are the major constituents identified in our tested sample of Brazilian green propolis extract.

Brazilian green propolis exhibits significant antioxidant properties by scavenging ROS and inhibiting chemiluminescence reactions [4, 18, 40]. The topical or oral treatment of animals with Brazilian propolis extracts demonstrated potential effect against oxidative stress [41]. DPPH^• and ABTS^•+ tests have been widely used for evaluating antioxidant properties of natural phenolic compounds. Antioxidants intercept the free radical chain oxidation by donating hydrogen from the phenolic hydroxyl groups, thereby forming stable end products, which do not initiate or propagate further oxidation. EEP-B exerted strong free radical scavenging activity based on reduction of DPPH^• and ABTS^•+ [4, 17]. Izuta et al. showed the ability of scavenging DPPH^• by green propolis and artepillin C, in contrast to other prenylated derivatives of cinnamic acid, drupanin and baccharin, which did not cause similar effect. The findings indicate that 3-prenyl chain of cinnamic acid is important for antioxidant activity of artepillin C [42]. Hayashi et al. noticed strong antioxidant effect of two compounds isolated from Brazilian propolis, kaempferide, and artepillin C [43]. In the present study we verified potent scavenging capability of EEP-B against DPPH^• and ABTS^•+.

Regarding the anti-inflammatory property, the effect of EEP-B in ROS and RNS scavenging was also determined by chemiluminescence assay. We evaluated for the first time the role of green propolis in the oxidative metabolism of PMA stimulated macrophages. The ROS and RNS release by activated J774A.1 cells was significantly inhibited by EEP-B in dose-dependent manner. Król et al. and Simões et al. proved that kaempferide and extracts of Polish and Brazilian green propolis decrease the chemiluminescence produced by stimulated neutrophils [18, 39, 40].

NO, a short half-life free radical, is an effector molecule in host defense against pathogens. NO production in macrophages is mediated by the inducible nitric oxide synthase (iNOS). However, excessive release of NO induced by LPS, IFN-γ, or TNF-α has detrimental effects on many organ systems of the body, leading to acute or chronic inflammatory diseases [44]. Song et al. reported that treatment of RAW264.7 cells with extract of Korean propolis markedly suppresses NO production and iNOS mRNA and protein expression induced by LPS + IFN-γ [45]. Blonska et al. observed the inhibition of NO synthesis and iNOS mRNA expression in LPS stimulated J774A.1 macrophages by extract of Polish propolis and its phenolic components: chrysin, galangin, kaempferol, and quercetin [27]. Paulino et al. demonstrated that artepillin C decreases NO concentration in RAW264.7 cells incubated with LPS [46]. Król et al. described downregulation of NO by kaempferide in activated murine peritoneal macrophages [16]. Similar to previous studies, our data confirmed the effect of EEB-P on NO generation in J774A.1 cells. The additional data from in vitro study supplied by Tan-No et al. proved that Brazilian propolis extract suppresses corrageenin-induced paw edema in mouse through inhibition of NO production [47].

Macrophages are a major source of many cytokines involved in immune response, hematopoiesis, inflammation, and other homeostatic processes. Upon stimulation by microorganisms, microbial products (e.g., LPS) or endogenous factors (including cytokines), macrophages synthetize de novo, and release a large variety of cytokines: IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, IL-17, TNF-α, IFN-α, IFN-γ, TGF-β, M-CSF, G-CSF, GM-CSF, MCP-1, MCP-3, MCP-5, MIP-1, MIP-2, RANTES, MIF, and KC. In addition, these cytokines can modulate most of the functions of macrophages, cell surface markers expression, and others cytokines secretion. The cytokine network plays a key role in regulation of macrophages activation [48]. To gain further insight into anti-inflammatory activity of EEP-B, nineteen cytokines secreted by macrophages were analyzed. We investigated the influence of Brazilian green propolis extract on production of cytokine IL-1α, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-13, IL-17, TNF-α, IFN-γ, G-CSF, GM-CSF, MCP-1, MIP-1α, MIP-1β, and RANTES in LPS + IFN-γ stimulated macrophages. Inflammatory cytokines are generated by innate immune cells during infection. For example, LPS induces strong release of IL-1, IL-6, IL-12, and TNF-α by the macrophages [37, 48, 49]. The upregulation of these inflammatory cytokines in LPS or LPS + IFN-γ activated macrophages was blocked by propolis extracts. We showed that EEP-B significantly downregulated the production of IL-1α, IL-1β, IL-6, IL-12p40, and TNF-α in LPS + IFN-γ treated J774A.1 cells. Bachiega et al. described the increase of IL-1β and decrease of IL-6 production by Brazilian green propolis extract and its phenolic acids in peritoneal murine macrophages challenged with LPS [50]. Shi et al. and Wang et al. noticed that extracts from Chinese propolis suppress mRNA and protein expression of IL-1β and IL-6 induced by LPS in RAW264.7 cells [51, 52]. Blonska et al. reported the inhibition of IL-1β mRNA and protein expression in LPS-stimulated J774A.1 macrophages by extract of Polish propolis and its flavones [27]. We observed that the secretion of interleukin IL-4 and IL-13 in LPS + IFN-γ activated J774A.1 cells was also reduced by EEP-B. Whereas the concentrations of IL-3, IL-5, IL-9, IL-10, IL-17, and IFN-γ were only slightly or not at all affected after incubation with EEP-B. The in vivo experiment performed by Franchin et al. showed down-regulation of IL-1β and TNF-α production by extract of geopropolis from Melipona scutellaria [53]. By contrast, Orsatti et al. demonstrated upregulation of IL-1β and IL-6 in in vivo model [54]. In other in vivo test Hu et al. found out that Chinese propolis extracts suppressed carrageenan induced peritonitis and pleurisy or acute lung damage in rats through decrease of the number of neutrophils. Chinese propolis also down-regulated IL-6 in Freund’s complete adjuvant (FCA) induced arthritis in rats [55]. Paulino et al. reported a significant inhibition of paw edema in carrageenan induced model of murine peritonitis by artepillin C also via decrease of neutrophils’ percentage in the peritoneal cavity [46].

The colony-stimulating factors, G-CSF, and GM-CSF release in activated J774A.1 macrophages were decreased by EEP-B. Chemokines (MCP-1, MCP-3, MIP-1, MIP-2, and RANTES) are chemotactic cytokines contributing to the recruitment of circulating monocytes within tissues. Our study extended to macrophage chemokines demonstrated that EEP-B downregulates the expression of MCP-1, MIP-1α, MIP-1β, and RANTES in LPS + IFN-γ stimulated J774A.1 cells. These results suggest a crucial contribution of Brazilian green propolis in the modulation of chemokine-mediated inflammation.

The findings confirm significant anti-inflammatory effect of ethanolic extract of Brazilian green propolis on macrophage in vitro model.

5. Conclusion

Propolis has become a subject of special interest as a source of valuable phenolic compounds to developed food components, dietary supplements, or even pharmaceuticals for the prevention or treatment of inflammatory diseases. Artepillin C, drupanin, baccarin, p-coumaric acid, and kaempferide are the main ingredients of Brazilian green propolis. Our recent study provide new evidence-based proofs of anti-inflammatory properties of Brazilian green propolis extract.

Conflict of Interests

No conflict of interests was declared in relation to this paper.

Acknowledgments

This work was supported by a Research Grant KNW-1-063/P/2/0 from the Medical University of Silesia in Katowice (Poland). The authors thank Mr. Rindai Yamamoto, the President of Nihon Natural Foods Co., Ltd. (Tokyo, Japan), and Mrs. Etsuko Yamamoto, the President of Komyodo Kampo Shoyaku Co., Ltd. (Tokyo, Japan), for the sample of Brazilian green propolis.

References

Y. K. Park, S. M. Alencar, and C. L. Aguiar, “Botanical origin and chemical composition of Brazilian propolis,” Journal of Agricultural and Food Chemistry, vol. 50, pp. 2502–2506, 2002.
View at: Google Scholar
F. C. Lopes, V. Bankova, and J. M. Sforcin, “Effect of three vegetal sources of propolis on macrophages activation,” Phytomedicine, vol. 10, no. 4, p. 343, 2003.
View at: Publisher Site | Google Scholar
J. M. Sforcin, “Propolis and the immune system: a review,” Journal of Ethnopharmacology, vol. 113, pp. 1–14, 2007.
View at: Google Scholar
E. W. Teixeira, D. Message, G. Negri, A. Salatino, and P. C. Stringheta, “Seasonal variation, chemical composition and antioxidant activity of brazilian propolis samples,” Evidence-Based Complementary and Alternative Medicine, vol. 7, no. 3, pp. 307–315, 2010.
View at: Publisher Site | Google Scholar
J. M. Sforcin and V. Bankova, “Propolis: is there a potential for the development of new drugs?” Journal of Ethnopharmacology, vol. 133, no. 2, pp. 253–260, 2011.
View at: Publisher Site | Google Scholar
E. Szliszka, G. Zydowicz, B. Janoszka, C. Dobosz, G. Kowalczyk-Ziomek, and W. Król, “Ethanolic extract of Brazilian green propolis sensitizes prostate cancer cells to TRAIL-induced apoptosis,” International Journal of Oncology, vol. 38, no. 4, pp. 941–953, 2011.
View at: Publisher Site | Google Scholar
D. Sawicka, H. Car, M. H. Borawska, and J. Niklinski, “The anticancer activity of propolis,” Folia Histochemica et Cytobiologica, vol. 50, pp. 25–37, 2012.
View at: Google Scholar
G. C. Franchi, C. S. Moraes, V. C. Toreti, A. Daugsch, A. E. Nowill, and Y. K. Park, “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, vol. 2012, Article ID 918956, 6 pages, 2012.
View at: Publisher Site | Google Scholar
E. Szliszka and W. Król, “Polyphenols isolated from propolis augment TRAIL-induced apoptosis in cancer cells,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 731940, 10 pages, 2013.
View at: Publisher Site | Google Scholar
A. H. Banskota, Y. Tezuka, and S. Kadota, “Recent progress in pharmacological research of propolis,” Phytotherapy Research, vol. 15, no. 7, pp. 561–571, 2001.
View at: Publisher Site | Google Scholar
E. Szliszka, Z. P. Czuba, M. Domino, B. Mazur, G. Zydowicz, and W. Król, “Ethanolic extract of propolis (EEP) enhances the apoptosis-inducing potential of TRAIL in cancer cells,” Molecules, vol. 14, no. 2, pp. 738–754, 2009.
View at: Publisher Site | Google Scholar
E. Szliszka, Z. P. Czuba, J. Bronikowska, A. Mertas, A. Paradysz, and W. Król, “Ethanolic extract of propolis (EEP) augments TRAIL-induced apoptotic death in prostate cancer cells,” Evidence-Based Complementary and Alternative Medicine, vol. 2011, Article ID 535172, 11 pages, 2011.
View at: Publisher Site | Google Scholar
G. C. Chan, K. W. Cheung, and D. M. Sze, “The immunomodulatory and anticancer properties of propolis,” Clinical Reviews in Allergy & Immunology, vol. 44, no. 3, pp. 262–273, 2013.
View at: Publisher Site | Google Scholar
D. L. Laskin and K. J. Pendino, “Macrophages and inflammatory mediators in tissue injury,” Annual Review of Pharmacology and Toxicology, vol. 35, pp. 655–677, 1995.
View at: Google Scholar
A. Aderem and D. M. Underhill, “Mechanisms of phagocytosis in macrophages,” Annual Review of Immunology, vol. 17, pp. 593–623, 1999.
View at: Publisher Site | Google Scholar
W. Król, Z. P. Czuba, G. Pietsz, M. D. Threadgill, and B. D. Cunningham, “Modulation of the cytotoxic activity of murine macrophages by flavones,” Current Topics in Biophysics, vol. 20, pp. 88–93, 1996.
View at: Google Scholar
M. R. Ahn, K. Kunimasa, S. Kumazawa et al., “Correlation between antiangiogenic activity and antioxidant activity of various components from propolis,” Molecular Nutrition and Food Research, vol. 53, no. 5, pp. 643–651, 2009.
View at: Publisher Site | Google Scholar
L. M. C. Simões, L. E. Gregório, A. A. Da Silva Filho et al., “Effect of Brazilian green propolis on the production of reactive oxygen species by stimulated neutrophils,” Journal of Ethnopharmacology, vol. 94, no. 1, pp. 59–65, 2004.
View at: Publisher Site | Google Scholar
A. A. Carvalho, D. Finger, C. S. Machado et al., “In vivo antitumoural activity and composition of an oil extract of Brazilian propolis,” Food Chemistry, vol. 126, pp. 1239–1245, 2011.
View at: Google Scholar
S. A. Moura, G. Negri, A. Salatino et al., “Aqueous extract of Brazilian green propolis: primary components, evaluation of inflammation and wound healing by using subcutaneous implanted sponges,” Evidence-Based Complementary and Alternative Medicine, vol. 2011, Article ID 748283, 8 pages, 2011.
View at: Publisher Site | Google Scholar
F. Pellati, G. Orlandini, D. Pinetti, and S. Benvenuti, “HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts,” Journal of Pharmaceutical and Biomedical Analysis, vol. 55, no. 5, pp. 934–948, 2011.
View at: Publisher Site | Google Scholar
S. I. Falcao, N. Vale, and P. Gomes, “Phenolic profiling of Portuguese propolis by LC-MS spectrometry: uncommon propolis rich in flavonoid glycosides,” Phytochemical Analysis, 2012.
View at: Publisher Site | Google Scholar
C. C. Fernandes-Silva, A. Salatino, M. L. Salatino, E. D. Breyer, and G. Negri, “Chemical profiling of six samples of Brazilian propolis,” Quimica Nova, vol. 36, pp. 237–240, 2013.
View at: Google Scholar
A. C. H. F. Sawaya, D. M. Tomazela, I. B. S. Cunha et al., “Electrospray ionization mass spectrometry fingerprinting of propolis,” Analyst, vol. 129, no. 8, pp. 739–744, 2004.
View at: Publisher Site | Google Scholar
D. Wojnicz, A. Z. Kucharska, A. Sokół-Łętowska, M. Kicia, and D. Tichaczek-Goska, “Medicinal plants extracts affect virulence factors expression and biofilm formation by the uropathogenic Escherichia coli,” Urological Research, vol. 40, pp. 683–697, 2012.
View at: Google Scholar
S. Keckes, U. Gasic, T. Cirkovic-Velickovic, D. Milojkovic-Opsenica, M. Natic, and Z. Tesic, “The determination of phenolic profiles of Serbian unifloral honeys using ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry,” Food Chemistry, vol. 138, pp. 32–40, 2013.
View at: Google Scholar
M. Blonska, J. Bronikowska, G. Pietsz, Z. P. Czuba, S. Scheller, and W. Król, “Effects of ethanol extract of propolis (EEP) and its flavones on inducible gene expression in J774A.1 macrophages,” Journal of Ethnopharmacology, vol. 91, no. 1, pp. 25–30, 2004.
View at: Publisher Site | Google Scholar
E. Szliszka, Z. P. Czuba, Ł. Sȩdek, A. Paradysz, and W. Król, “Enhanced TRAIL-mediated apoptosis in prostate cancer cells by the bioactive compounds neobavaisoflavone and psoralidin isolated from Psoralea corylifolia,” Pharmacological Reports, vol. 63, no. 1, pp. 139–148, 2011.
View at: Google Scholar
E. Szliszka, D. Jaworska, M. Kłósek, Z. P. Czuba, and W. Król, “Targeting death receptor TRAIL-R2 by chalcones for TRAIL-induced apoptosis in cancer cells,” International Journal of Molecular Sciences, vol. 13, pp. 15343–15359, 2012.
View at: Google Scholar
E. Szliszka, K. J. Helewski, E. Mizgala, and W. Król, “The dietary flavonol fisetin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells,” International Journal of Oncology, vol. 39, no. 4, pp. 771–779, 2011.
View at: Publisher Site | Google Scholar
E. Szliszka, E. Kostrzewa-Susłow, J. Bronikowska et al., “Synthetic flavanones augment the anticancer effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),” Molecules, vol. 17, pp. 11693–11711, 2012.
View at: Google Scholar
I. C. Jang, E. K. Jo, M. S. Bae et al., “Antioxidant and antigenotoxic activities of different parts of persimmon (Diospyros kaki cv. Fuyu) fruit,” Journal of Medicinal Plant Research, vol. 4, no. 2, pp. 155–160, 2010.
View at: Google Scholar
E. Szliszka, D. Skaba, Z. P. Czuba, and W. Król, “Inhibition of inflammatory mediators by neobavaisoflavone in activated RAW264.7 macrophages,” Molecules, vol. 16, pp. 3701–3712, 2011.
View at: Google Scholar
W. Król, Z. P. Czuba, M. D. Threadgill, B. D. M. Cunningham, and G. Pietsz, “Inhibition of nitric oxide (NO.) production in murine macrophages by flavones,” Biochemical Pharmacology, vol. 50, no. 7, pp. 1031–1035, 1995.
View at: Publisher Site | Google Scholar
S. B. Yoon, Y. J. Lee, S. K. Park et al., “Anti-inflammatory effects of Scutellaria baicalensis water extract on LPS-activated RAW 264.7 macrophages,” Journal of Ethnopharmacology, vol. 125, no. 2, pp. 286–290, 2009.
View at: Publisher Site | Google Scholar
E. Szliszka, G. Zydowicz, E. Mizgala, and W. Król, “Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) sensitizes prostate cancer LNCaP cells to TRAIL-induced apoptosis,” International Journal of Oncology, vol. 41, pp. 818–828, 2012.
View at: Google Scholar
Y. K. Park, J. F. Paredes-Guzman, C. L. Aguiar, S. M. Alencar, and F. Y. Fujiwara, “Chemical constituents in Baccharis dracunculifolia as the main botanical origin of Southeastern Brazilian propolis,” Journal of Agricultural and Food Chemistry, vol. 52, no. 5, pp. 1100–1103, 2004.
View at: Google Scholar
A. H. Banskota, Y. Tezuka, J. K. Prasain, K. Matsushige, I. Saiki, and S. Kadota, “Chemical constituents of Brazilian propolis and their cytotoxic activities,” Journal of Natural Products, vol. 61, no. 7, pp. 896–900, 1998.
View at: Publisher Site | Google Scholar
W. Król, S. Scheller, Z. Czuba et al., “Inhibition of neutrophils' chemiluminescence by ethanol extract of propolis (EEP) and its phenolic components,” Journal of Ethnopharmacology, vol. 55, no. 1, pp. 19–25, 1996.
View at: Publisher Site | Google Scholar
L. M. C. Simões-Ambrosio, L. E. Gregório, J. P. B. Sousa et al., “The role of seasonality on the inhibitory effect of Brazilian green propolis on the oxidative metabolism of neutrophils,” Fitoterapia, vol. 81, no. 8, pp. 1102–1108, 2010.
View at: Publisher Site | Google Scholar
M. J. V. Fonseca, Y. M. Fonseca, F. Marquele-Oliveira et al., “Evaluation of the potential of Brazilian propolis against UV-induced oxidative stress,” Evidence-Based Complementary and Alternative Medicine, vol. 2011, Article ID 863917, 2011.
View at: Publisher Site | Google Scholar
H. Izuta, Y. Narahara, M. Shimazawa, S. Mishima, S. I. Kondo, and H. Hara, “1,1-diphenyl-2-picrylhydrazyl radical scavenging activity of bee products and their constituents determined by ESR,” Biological and Pharmaceutical Bulletin, vol. 32, no. 12, pp. 1947–1951, 2009.
View at: Publisher Site | Google Scholar
K. Hayashi, S. Komura, N. Isaji, N. Ohishi, and K. Yagi, “Isolation of antioxidative compounds from Brazilian propolis: 3,4-dihydroxy-5-prenylcinnamic acid, a novel potent antioxidant,” Chemical and Pharmaceutical Bulletin, vol. 47, no. 11, pp. 1521–1524, 1999.
View at: Google Scholar
J. MacMacking, Q. Xie, and C. Nathan, “Nitric oxide and macrophages function,” Annual Review of Immunology, vol. 15, pp. 323–350, 1997.
View at: Google Scholar
Y. S. Song, E. H. Park, G. M. Hur, Y. S. Ryu, Y. M. Kim, and C. Jin, “Ethanol extract of propolis inhibits nitric oxide synthase gene expression and enzyme activity,” Journal of Ethnopharmacology, vol. 80, no. 2-3, pp. 155–161, 2002.
View at: Publisher Site | Google Scholar
N. Paulino, S. R. L. Abreu, Y. Uto et al., “Anti-inflammatory effects of a bioavailable compound, Artepillin C, in Brazilian propolis,” European Journal of Pharmacology, vol. 587, no. 1–3, pp. 296–301, 2008.
View at: Publisher Site | Google Scholar
K. Tan-No, T. Nakajima, T. Shoji et al., “Anti-inflammatory effect of propolis through inhibition of nitric oxide production on carrageenin-induced mouse paw edema,” Biological and Pharmaceutical Bulletin, vol. 29, no. 1, pp. 96–99, 2006.
View at: Publisher Site | Google Scholar
J. M. Cavaillon, “Cytokines and macrophages,” Biomedicine and Pharmacotherapy, vol. 48, pp. 445–453, 1994.
View at: Google Scholar
T. Hanada and A. Yoshimura, “Regulation of cytokine signaling and inflammation,” Cytokine and Growth Factor Reviews, vol. 13, no. 4-5, pp. 413–421, 2002.
View at: Publisher Site | Google Scholar
T. F. Bachiega, C. L. Orsatti, A. C. Pagliarone, and J. M. Sforcin, “The effects of propolis and its isolated compounds on cytokine production by murine macrophages,” Phytotherapy Research, vol. 26, pp. 1308–1313, 2012.
View at: Google Scholar
H. Shi, H. Yang, X. Zhang, and L. Yu, “Identification and quantification of phytochemical composition and anti-inflammatory and radical scavenging properties of methanolic extract of Chinese propolis,” Journal of Agricultural and Food Chemistry, vol. 60, pp. 12403–12410, 2012.
View at: Google Scholar
K. Wang, S. Ping, S. Huang et al., “Molecular mechanisms underlying the in vitro anti-inflammatory effects of flavonoid-rich ethanol extract from Chinese propolis,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 127672, 11 pages, 2013.
View at: Publisher Site | Google Scholar
M. Franchin, M. G. Cunha, C. Denny et al., “Geopropolis from Melipona scutellaris decreases the mechanical inflammatory hypernociception by inhibiting the production of IL-1β and TNF-α,” Journal of Ethnopharmacology, vol. 143, pp. 709–715, 2012.
View at: Google Scholar
C. L. Orsatti, F. Missima, A. C. Pagliarone et al., “Propolis immunomodulatory action in vivo on toll-like receptors 2 and 4 expression and on pro-inflammatory cytokines production in mice,” Phytotherapy Research, vol. 24, no. 8, pp. 1141–1146, 2010.
View at: Publisher Site | Google Scholar
F. Hu, H. R. Hepburn, Y. Li, M. Chen, S. E. Radloff, and S. Daya, “Effects of ethanol and water extracts of propolis (bee glue) on acute inflammatory animal models,” Journal of Ethnopharmacology, vol. 100, no. 3, pp. 276–283, 2005.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2013 Ewelina Szliszka et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

4220

Downloads

2479

Citations