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

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.


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][4][5][6][7][8][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][11][12][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][2][3][4].
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.

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).

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% heatinactivated fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin at 37 ∘ C and 10% CO 2 in a humidified Evidence-Based Complementary and Alternative Medicine 3 incubator [27]. Reagents for cell culture were purchased from ATCC. J774A.1 cells were seeded at a density of 1 × 10 6 /mL cells (2 × 10 5 /well) in 96-well plates at the presence of LPS (200 ng/mL) and IFN-(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 × 10 6 /mL) were seeded 4 h before the experiments in a 96well plate. EEP-B at the concentrations of 5-50 g/mL with or without LPS + IFN-was added to the cells. The final volume was 200 L. After 24 h the medium was removed and 20 L 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%.

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 × 10 6 /mL) cells were treated with 5-50 g/mL EEP-B with or without LPS + IFN-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%.

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 ED 50 value, the concentration required to scavenge 50% of DPPH • . Ascorbic acid was used as a standard.

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 L of 10 mM) and preincubated at 37 ∘ C in the dark for 5 min. Next, ABTS (30 L of 1.25 mM in 0.05 M phosphate-citrate buffer) and peroxidase (30 L 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 ED 50 value, the concentration required to scavenge 50% of ABTS •+ . Ascorbic acid was used as a standard. 2.11. Quantification of NO Production. J774A.1 macrophages (1 × 10 6 /mL) stimulated with LPS + IFN-were incubated with 5-50 g/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 L) were mixed with 100 L 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. . 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-stimulated and native J774A.1 cells (1 × 10 6 /mL) were incubated with or without 25-50 g/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 L 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 Bioplex Array Reader (Bio-Plex 200 System). The fluorescence intensity was evaluated using Bio-Plex Manager software (Bio-Rad) [33,35].

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.

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 fortythree 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).  (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 g/mL did not influence the cell viability and did not exert cytotoxic effect. Therefore for further studies of antiinflammatory properties EEP-B was used at the concentrations of 5-50 g/mL.

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 • (ED 50 of 24.1 g/mL) and ABTS •+ (ED 50 of 40.6 g/mL) compared with ascorbic acid (ED 50 of 15.8 g/mL and 10.1 g/mL, resp.).

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 g/mL suppressed the chemiluminescence in PMA stimulated J774A.1 cells in dose-dependent manner with an ED 50 of 0.02 g/mL ( Figure 6).

Effect of Brazilian Green Propolis Extract on NO Production in LPS + IFN-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-stimulation nitrite concentration markedly increased, but LPS + IFN--induced NO synthesis in J774A.1 cells was significantly decreased by EEP-B in dose-dependent manner (ED 50 = 30.1 g/mL). The inhibitory effect of 5-50 g/mL EEP-B on NO production in LPS + IFN-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 ED 50 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).

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-, or TNF-has detrimental effects on many organ systems of the body, leading to acute or chronic inflammatory diseases [44]. Song [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 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-1 , IL-1 , IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-13, IL-17, TNF-, IFN-, G-CSF, GM-CSF, MCP-1, MIP-1 , MIP-1 , and RANTES in LPS + IFNstimulated 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-by the macrophages [37,48,49]. The upregulation of these inflammatory cytokines in LPS or LPS + IFN-activated macrophages was blocked by propolis extracts. We showed that EEP-B significantly downregulated the production of IL-1 , IL-1 , IL-6, IL-12p40, and TNF-in LPS + IFNtreated J774A. The findings confirm significant anti-inflammatory effect of ethanolic extract of Brazilian green propolis on macrophage in vitro model.

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.