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Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 976415 13 pageshttpdxdoiorg1011552013976415

Research ArticleChemical Composition and Anti-InflammatoryEffect of Ethanolic Extract of Brazilian Green Propolis onActivated J774A1 Macrophages

Ewelina Szliszka1 Alicja Z Kucharska2 Anna SokoacuteB-Awtowska2 Anna Mertas1

Zenon P Czuba1 and Wojciech Kroacutel1

1 Department of Microbiology and Immunology Medical University of Silesia in Katowice Jordana 19 41 808 Zabrze Poland2Department of Fruit and Vegetables and Cereals Technology Wrocław University of Environmental and Life SciencesChełmonskiego 3741 51 630 Wrocław Poland

Correspondence should be addressed to Wojciech Krol wkrolsumedupl

Received 4 April 2013 Accepted 8 May 2013

Academic Editor Andrzej K Kuropatnicki

Copyright copy 2013 Ewelina Szliszka et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of this study was to investigate the chemical composition and anti-inflammatory effect of ethanolic extract of Braziliangreen propolis (EEP-B) on LPS + IFN-120574 or PMA stimulated J774A1 macrophagesThe identification and quantification of phenoliccompounds in green propolis extract were performed using HPLC-DAD and UPLC-Q-TOF-MS methods The cell viability wasevaluated by MTT and LDH assays The radical scavenging ability was determined using DPPH∙ and ABTS∙+ ROS and RNSgeneration was analyzed by chemiluminescence NO concentration was detected by the Griess reaction The release of variouscytokines by activated J774A1 cells was measured in the culture supernatants using a multiplex bead array system based onxMAP technology Artepillin C kaempferide and their derivatives were the main phenolics found in green propolis At the testedconcentrations the EEP-B did not decrease the cell viability and did not cause the cytotoxicity EEP-B exerted strong antioxidantactivity and significantly inhibited the production of ROS RNS NO cytokine IL-1120572 IL-1120573 IL-4 IL-6 IL-12p40 IL-13 TNF-120572G-CSF GM-CSF MCP-1 MIP-1120572 MIP-1120573 and RANTES in stimulated J774A1 macrophages Our findings provide new insightsfor understanding the anti-inflammatory mechanism of action of Brazilian green propolis extract and support its application incomplementary and alternative medicine

1 Introduction

Propolis is a resinous substance collected by honeybees fromthe leaf bud and bark of certain plants Baccharis dracun-culifolia Eucalyptus citriodora Araucaria angustifolia andMyrocarpus frondosus are major botanical sources of propolisfrom southeast Brazil (States of Minas Gerais and Sao Paulo)called due to its colour green propolis [1 2] Extracts ofBrazilian green propolis possess antioxidant antimicrobialanti-inflammatory chemopreventive and anticancer prop-erties [3ndash9] Therefore propolis has been extensively usedin food beverages and dietary supplements to improvehealth and prevent diabetes cancer inflammatory and heartdiseases [10ndash13] The medical application of propolis has ledto increased interest in its chemical composition depending

on the geographical origin and specific flora of the regionseasonality or methods of harvesting the raw material [1ndash4]

Our study was designed to investigate the chemical com-position and anti-inflammatory effect of ethanolic extract ofBrazilian green propolis (EEP-B) on LPS (lipopolysaccha-ride) + IFN-120574 (interferon 120574) or PMA (phorbol 12-myristate13-acetate) stimulated J774A1 macrophages

Macrophages are the first effector cells of the immunesystem that protect against microbial infection and tissuesinjury Macrophages have three important functions antigenpresentation phagocytosis and synthesis of various inflam-matory mediators [14 15] Propolis affects the nonspe-cific immunity via modulation of macrophages activity [313] During inflammation upon stimulation by LPS fromGram-negative bacteria and IFN-120574 from host immune cells

2 Evidence-Based Complementary and Alternative Medicine

macrophages release large amounts of reactive oxygen (ROS)and nitrogen species (RNS) nitric oxide (NO) and numerouscytokines [16] Overexpression of these mediators causespathological acute or chronic inflammatory responses [14]

This is the first comprehensive report explaining an anti-inflammatory potential of Brazilian green propolis extracton macrophage in vitro model We determined the effectof EEP-B on production of NO ROS RNS and cytokinesinterleukin (IL)-IL-1120572 IL-1120573 IL-3 IL-4 IL-5 IL-6 IL-9IL-10 IL-12p40 IL-13 IL-17 TNF-120572 (tumor necrosis factor120572) IFN-120574 (interferon 120574) G-CSF (granulocyte colony-stimu-lating factor) GM-CSF (granulocyte-macrophage colony-stimulating factor) MCP-1 (monocyte chemotactic protein1) MIP-1120572 (macrophage inflammatory protein 1120572) MIP-1120573(macrophage inflammatory protein 1120573) and RANTES (regu-lated upon activation normal T cell expressed and secreted)in activated J774A1 cells

It has been suggested that the immunomodulatory prop-erties of propolis is contributed by its flavonoids and phenolicacids [3 5 9 17] The typical ingredients of Brazilian greenpropolis are kaempferide (357-trihydroxy-41015840-methoxyfla-vone) and cinnamic acid derivatives p-coumaric acid (4-hydroxycinnamic acid) artepillin C (35-diprenyl-4-hydrox-ycinnamic acid) baccharin (3-prenyl-4-(dihydrocinnamoyl-oxy)-cinnamic acid) and drupanin (3-prenyl-4-hydroxycin-namic acid) [1 4 18] We performed the detailed identifica-tion and quantification of phenolic compounds in propolisextract

The present study analyzed chemical composition ofgreen propolis and confirmed its significant role in suppress-ing chronic inflammation and reducing risk of related humanhealth problems Our findings provide new insights forunderstanding the anti-inflammatory mechanism of actionof Brazilian green propolis extract and support its applicationin complementary and alternative therapies

2 Materials and Methods

21 General The Brazilian green propolis sample was kindlysupplied by Nihon Natural Foods Co Ltd (Tokyo Japan)LPS (LPS E coli O111B4) was obtained from Fluka ChemieGmbH (Buchs Switzerland) and recombinant mouse IFN-120574 was purchased from RampD Systems (Minneapolis MNUSA) PMA DMSO (dimethyl sulphoxide) acetonitrile andformic acid were obtained from Sigma-Aldrich (SteinheimGermany) Acetonitrile for LC-MS was purchased fromPOCh (Gliwice Poland) The following compounds wereused as standards sakuranetin hesperitin caffeic acid (RothKarlsruhe Germany) kaempferol pinocembrin naringeninp-coumaric acid (Sigma-Aldrich Steinheim Germany)kaempferide ferulic acid (Serva Heidelberg Germany)isosakuranetin (TransMIT GmbH Giessen Germany) andartepillin C (Wako Pure Chemicals Osaka Japan)

22 Preparation of Brazilian Green Propolis Extract TheBrazilian green propolis sample was collected manually frombeehive located in southeast Brazil (the state ofMinas Gerais)and was kept desiccated prior to processing The sample was

extracted in 95 vv ethyl alcohol in a hermetically sealedglass vessel for 4 days at 37∘C under occasional shakingThe ethanolic extract of Brazilian green propolis (EEP-B)was then filtered through Whatman filter paper no 4 andevaporated in a rotary evaporator under reduced pressureat 60∘CThe same collection and extraction procedures wereused throughout all our laboratory studies [6 12] EEP-B wasdissolved in DMSO (50mgmL) and the final concentrationof DMSO in the culture mediumwas controlled at 01 (vv)

23 Identification and Quantification of Phenolic Compoundsby HPLC-DAD Method Phenolic compounds were deter-mined using Dionex (USA) HPLC system equipped withdiode array detector model Ultimate 3000 a quaternarypump LPG-3400A autosampler EWPS-3000SI and ther-mostated column compartment TCC-3000SD and controlledby Chromeleon v68 software The reversed phase Cadenza5CD-C18 (75mm times 46 id) column (Imtakt Kyoto Japan)with guard column Cadenza (5 times 46 id) guard column(Imtakt Kyoto Japan) was used The mobile phase wascomposed of (A) 01 (vv) formic acid in water and (B)acetonitrile The applied elution conditions were 0min 20B 0ndash10min linear gradient from 20 to 30 B 10ndash40minlinear gradient from 30 to 40 B 40ndash60min lineargradient from40 to 60B 60ndash80min linear gradient from60 to 80 B and then again the initial conditions [19ndash23]The flow rate was 1mLmin and the injection volume was20120583L The column was operated at 30∘C The compoundswere monitored at 290 nm 325 nm and 370 nm

24 Identification and Quantification of Phenolic Compoundsby LC-MS Method Compounds identification was per-formed on an Acquity ultra-performance liquid chromatog-raphy (UPLC) system coupled with a quadruple time of flight(Q-TOF) MS instrument (UPLCSynapt Q-TOF MS WatersCorp Milford MA USA) with an electrospray ionization(ESI) source Separation was achieved on the Acquity BEHC18 column (100mm times 21mm id 17 120583m Waters) Detec-tion wavelengths were set at 290 325 and 370 nm A mobilephase was a mixture of 15 formic acid (A) and acetonitrile(B) The gradient program was as follows initial conditions-95 (A) 12min-5 (A) 13min-5 (A) 145min-95 (A)and 16min-95 (A) The flow rate was 045mLmin and theinjection volume was 5 120583LThe columnwas operated at 30∘CThe major operating parameters for the Q-TOF MS were setas follows capillary voltage 20 kV cone voltage 45V conegas flow of 11 Lh collision energy 50 eV source temperature100∘C desolvation temperature 250∘C collision gas argondesolvation gas (nitrogen) flow rate 600 Lh data acquisitionrange119898119911 100ndash1000Da ionization mode negative [24ndash26]The data were collected by Mass-Lynx V 41 software

25 Cell Culture Murine macrophage J774A1 cell line wasobtained from ATCC (American Type Culture CollectionManassas VA USA) Cells were cultured in Dulbeccorsquosmodified Eaglersquos medium supplemented with 10 heat-inactivated fetal bovine serum 100UmL penicillin and100 120583gmL streptomycin at 37∘Cand 10CO

2

in a humidified

Evidence-Based Complementary and Alternative Medicine 3

incubator [27] Reagents for cell culture were purchased fromATCC J774A1 cells were seeded at a density of 1 times 106mLcells (2 times 105well) in 96-well plates at the presence of LPS(200 ngmL) and IFN-120574 (25UmL)with or without EEP-B for24 h

26 Cell Viability Assay The cell viability was determined bythe 3-(45-dimethyl-2-thiazyl)-25-diphenyl-2H-tetrazoliumbromide (MTT) reduction assay as described [28 29] Thistest is based on the cleavage of the tetrazolium salt MTT toa blue formazan dye by viable cells The J774A1 cells (1 times106mL) were seeded 4 h before the experiments in a 96-well plate EEP-B at the concentrations of 5ndash50120583gmL withor without LPS + IFN-120574 was added to the cells The finalvolume was 200120583L After 24 h the medium was removed and20120583LMTT solutions (5mgmL) (Sigma Chemical CompanySt Louis MO USA) were added to each well for 4 hThe resulting formazan crystals were dissolved in DMSOThe controls included native cells and medium alone Thespectrophotometric absorbance was measured at 550 nmwavelength using a microplate reader (ELx 800 Bio-TekInstruments Inc Winooski VT USA) The cytotoxicity aspercentage of cell death was calculated by the formula(1 minus [absorbance of experimental wellsabsorbance of controlwells]) times 100

27 Cytotoxicity Assay The cytotoxicity of EEP-B was deter-mined by using LDH activity assay kit (Roche DiagnosticsGmbH Mannheim Germany) [30 31] Lactate dehydro-genase (LDH) is a stable cytosolic enzyme released uponmembrane damage in necrotic cellsThe J774A1 (1 times 106mL)cells were treated with 5ndash50120583gmL EEP-B with or withoutLPS + IFN-120574 for the indicated period of time LDH releasedin culture supernatants is detected with coupled enzymaticassay resulting in the conversion of a tetrazolium salt intoa red formazan product The maximal release of LDH wasobtained after treating control cells with 1 Triton X-100(Sigma Chemical Company St Louis MO) for 10min atroom temperature The spectrophotometric absorbance wasmeasured 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 thefollowing formula (sample valuemaximal release) times 100

28 DPPH Radical Scavenging Activity Hydrogen-donatingactivity was measured using 11-diphenyl-2-picrylhydrazylradical (DPPH∙) (Sigma Chemical Company St Louis MO)following a previously reported protocol [32] EEP-B (01mL)was mixed with 09mL of 0041mM DPPH∙ in ethanol andstored at room temperature in the dark for 30min Theabsorbance of the resulting solutions was measured at 517 nmwavelength using V-630 Spectrophotometer (Jasko Inter-national Co Tokyo Japan) The percentage of scavengingactivity was calculated by the formula DPPH∙ scavengingactivity = 1 minus (absorbance of experimental wellsabsorbanceof control wells) times 100 The scavenging activity of thesample was expressed as the ED

50

value the concentration

required to scavenge 50 of DPPH∙ Ascorbic acid was usedas a standard

29 ABTS Cation Radical Scavenging Activity 221015840-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation(ABTS∙+) (Sigma Chemical Company St Louis MO) scav-enging activity was determined according to the previouslydescribed procedure [32] EEP-B (01mL) was mixed withpotassium phosphate buffer (01mL of 01M) and hydrogenperoxide (10 120583L of 10mM) and preincubated at 37∘C in thedark for 5min Next ABTS (30 120583L of 125mM in 005Mphosphate-citrate buffer) and peroxidase (30 120583L of 1 unitmL)were added to the mixture and then incubated at 37∘Cin the dark for 10min The absorbance of the resultingsolutions was measured at 417 nm wavelength using V-630Spectrophotometer (Jasko International Co Tokyo Japan)The percentage of scavenging activity was calculated by theformula ABTS∙+ scavenging activity = 1 minus (absorbance ofexperimental wellabsorbance of control wells) times 100 Thescavenging activity of the sample was expressed as the ED

50

value the concentration required to scavenge 50of ABTS∙+Ascorbic acid was used as a standard

210 Detection of ROS and RNS Production by Chemilumines-cence The chemiluminescence of J774A1 macrophages wasevaluated bymicroplate method in Hankrsquos balanced salt solu-tion pH 74 at room temperature The cells were incubatedwith 001ndash10120583gmL EEP-B for 30min Next luminol (SigmaChemical Company St Louis MO USA) solution was addedtowells containing 2times 105 cells giving a final concentration of110 120583M After 5min for macrophages stimulation PMA solu-tion was injected to obtain the concentration of 08120583M Thefinal volume of each sample was 200120583L The chemilumines-cence was determined for 5min with luminol alone and afterstimulation with PMA for 30minThemeasuring systemwasequipped with LB 960 CentroXS3 microplate luminometer(Berthold Technologies GmbH Wildbad Germany) [33]

211 Quantification of NO Production J774A1 macrophages(1 times 106mL) stimulated with LPS + IFN-120574 were incubatedwith 5ndash50120583gmL EEP-B for 24 h After this time NO pro-duction was determined by measuring the accumulationof nitrite a stable end product in the culture supernatantaccording to the Griess reaction [27 34] Equal volumes ofculture supernatant from each well or medium (100120583L) weremixed with 100 120583L of Griess reagent in a 96-well plate andincubated for 15min at room temperature The spectropho-tometric absorbance was read at 550 nm wavelength in EonMicroplate Spectrophotometer (BioTekWinooski VT USA)and the nitrite concentration in the medium was calculatedusing sodium nitrite as a standard Nitrite was not detectablein cell-free medium

212 Multiplex Bead-Based Cytokine Assay Cytokines re-leased from J774A1 macrophages treated with EEP-B weredetermined in the cell culture supernatants with a ProMouseCytokines 19-plex assay kit for IL-1120572 IL-1120573 IL-3 IL-4 IL-5 IL-6 IL-9 IL-10 IL-12p40 IL-13 IL-17 TNF-120572 IFN-120574


G-CSF GM-CSF MCP-1 MIP-1120572 MIP-1120573 and RANTES(Bio-Rad Laboratories Inc Hercules CA USA) This testwas performed using Bio-Plex 200 System based on xMAPsuspension array technology (Bio-Rad Laboratories IncHercules CA USA) The LPS + IFN-120574 stimulated and nativeJ774A1 cells (1 times 106mL) were incubated with or without 25ndash50120583gmL EEP-B for 24 h Standard curves for each cytokinewere generated using kit-supplied reference cytokine sampleThe assay is designed for the multiplexed quantitative mea-surement of multiple cytokines in a single well using 50 120583Lof sample Briefly the following procedure was performedafter prewetting the 96-well filter plate with washing bufferthe solution in each well was aspirated using a vacuummanifold Next the cell culture supernatants were incubatedwith antibody-conjugated beads for 30min Following theincubational period detection antibodies and streptavidin-PE were added to each well for 30min Then after washingwith buffer to remove the unbound streptavidin-PE thebeads bound to each cytokine were analyzed in the Bio-plex Array Reader (Bio-Plex 200 System) The fluorescenceintensity was evaluated using Bio-Plex Manager software(Bio-Rad) [33 35]

213The Statistical Analysis The values represent mean plusmn SDof two three or four independent experiments performedin duplicate or quadruplicate Significant differences wereanalyzed using Studentrsquos 119905-test and 119875-values lt005 wereconsidered significant The concentration-response curveswere analyzed using PharmaPCS version 4 (Pharmacolog-ical Calculations System) software

3 Results

31 The Content and Characterization of Phenolic CompoundsIdentified in Brazilian Green Propolis Extract The identifica-tion and quantification of phenolic compounds in Braziliangreen propolis extract were performed using HPLC-DADand UPLC-Q-TOF-MS methods Qualitative analysis resultsobtained by LC-ESIMS methods and quantitative analysisdata obtained byHPLC (quantified usingDADdetection) arepresented in Figures 1 2 3 and 4 and Table 1 A total of forty-three phenolic ingredients were found in tested propolis sam-ple Thirty-four compounds were identified by comparisonof their UV and MSMS spectra to standards andor to theliterature data whereas another nine compounds remainedunknown Kaempferide with its derivatives and hesperitinwhich were characterized by MS from their molecular ionsat 119898119911 2990572 and 3010709 respectively are the majorflavonoids identified in Brazilian green propolis Among thephenolic acids prevailed p-coumaric acid (119898119911 1630406and fragment at 119898119911 119 resulting from the loss of a COOgroup) and prenylated cinnamic acid derivatives artepillinC (119898119911 2991634) baccharin (119898119911 3631619) and drupanin(119898119911 2311025) (Table 1)

32 Effect of Brazilian Green Propolis Extract on Viability ofJ774A1 Macrophages The cell viability in the presence of 5ndash50120583gmL EEP-B andor LPS + IFN-120574 for 24 h was measured

by MTT test (Figure 5) The cytotoxicity of the propolisextract at the same concentrations and incubation time wasevaluated by LDH assay EEP-B at the concentrations ofle50120583gmL did not influence the cell viability and did notexert cytotoxic effect Therefore for further studies of anti-inflammatory properties EEP-B was used at the concentra-tions of 5ndash50120583gmL

33 Antioxidant Activity of Brazilian Green Propolis ExtractAntioxidant activity of EEP-B was investigated by using twodifferentmethods for stableDPPH∙ andABTS∙+Thepropolisextract exhibited strong scavenging potential against DPPH∙(ED50

of 241120583gmL) andABTS∙+ (ED50

of 406 120583gmL) com-pared with ascorbic acid (ED

50

of 158120583gmL and 101 120583gmLresp)

34 Effect of Brazilian Green Propolis Extract on ROS and RNSProduction in PMAStimulated J774A1 Cells Changes in pro-duction of ROS and RNS in macrophages were determinedby chemiluminescence assay EEP-B at the concentrations of001ndash10 120583gmL suppressed the chemiluminescence in PMAstimulated J774A1 cells in dose-dependent manner with anED50

of 002 120583gmL (Figure 6)

35 Effect of Brazilian Green Propolis Extract on NO Produc-tion in LPS + IFN-120574 Stimulated J774A1 Cells NOproductionwas determined by measuring the accumulation of nitritein the culture supernatants using Griess reagent After 24 hLPS + IFN-120574 stimulation nitrite concentration markedlyincreased but LPS + IFN-120574-induced NO synthesis in J774A1cells was significantly decreased by EEP-B in dose-dependentmanner (ED

50

= 301 120583gmL) The inhibitory effect of 5ndash50120583gmL EEP-B on NO production in LPS + IFN-120574 stim-ulated macrophages is presented in Figure 7 The propolisextract did not interfere with the viability of J774A1 cells asshown in MTT and LDH test The ED

50

value of the EEP-B within the nontoxic concentration range suggests that theinhibition of nitrite accumulation was specific to responsesbymacrophages (due to inhibitory activity onNOproductionand not cytotoxic property of EEP-B)

36 Effect of Brazilian Green Propolis Extract on CytokineProduction in LPS + IFN-120574 Stimulated J774A1 Cells Theeffect of EEP-B on production of cytokine IL-1120572 IL-1120573IL-3 IL-4 IL-5 IL-6 IL-9 IL-10 IL-12p40 IL-13 IL-17TNF-120572 IFN-120574 G-CSF GM-CSF MCP-1 MIP-1120572 MIP-1120573and RANTES in LPS + IFN-120574 stimulated J774A1 cells isshown in Figure 8 Specifically native and activated J774A1macrophages were treated with 25ndash50120583gmL EEP-B for 24 hThe cytokines released in culture supernatants were analyzedsimultaneously by Bio-plex Suspension Array System Thisassay is designed for the multiplexed quantitative measure-ment of cytokines (19-plex) in a single well using 50 120583Lof sample EEP-B significantly decreased synthesis of IL-1120572 IL-1120573 IL-4 IL-6 IL-12p40 IL-13 TNF-120572 G-CSF GM-CSF MCP-1 MIP-1120572 MIP-1120573 and RANTES in LPS + IFN-120574


2

15

2025

21

1819

24

22

28

26

11

10

1

8

3

9

7

6

5

4

Time2 3 4 5 6 7 8 9 10 11

(au

)

0

112141618

222242628

332

17

16121314

232927

31

30323334

35

36

37

39

38 40

41

43

428eminus1

6eminus1

4eminus1

2eminus1

Figure 1 UPLC-DAD chromatogram (290 nm) of compounds of ethanol extract from Brazilian green propolis

363

Time2 3 4 5 6 7 8 9 10 11

0

112141618

222242628

3

2

25

15

21

18

19

24

26

22

2811

10

1

8

9

7

6

5

4

17

161213

1423

29

27

31

30 32

33

3435

3739

38

40

41

43

42

20

(au

)

8eminus1

6eminus1

4eminus1

2eminus1


stimulated J774A1 cells in dose-dependentmannerTheEEP-B did no influence the concentrations of IL-3 IL-5 IL-9 IL-17 and IFN-120574 and only slightly downregulates IL-10 in culturesupernatants derived from activated macrophages

4 Discussion

In Brazil twelve distinct groups of propolis have been clas-sified according to their botanical origin and biologicalproperties Green propolis collected in southern region ofBrazil (States of Minas Gerais and Sao Paulo) belongs toGroup 12 (propolis G12) [1] Chemical evidence suggestedthat Baccharis dracunculifolia is the main plant source forgreen propolis [36] Park et al demonstrated similar profiles

of phenolic components identified in green propolis extractand Baccharis dracunculifolia resins [37] The tested sampleof green propolis was rich in hesperitin kaempferide and itsderivatives and cinnamic acid derivatives p-coumaric acidartepillin C baccharin and drupanin Our studies confirmedthe results obtained by Park et al [1 37] and Banskota et al[38]

Macrophages are the main cells responsible for innate(nonspecific) immunity The J774A1 cells a murine macro-phage cell line are widely used to establish inflammatorymodel in vitro [27] Numerous findings showed antioxidantand anti-inflammatory effects of propolis extract [3 4 1318 21 27 39 40] The biologically active molecules in greenpropolis are phenolic acids and flavonoids which act as


Time2 3 4 5 6 7 8 9 10 11

(au

)

0

112141618

222242628

13

2526

14

8eminus1

6eminus1

4eminus1

2eminus1


2

Time2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 105

0

100 2311

3011

54311630

1790

5151

48922631

677248722471

5431

2471

7052

3772

2851

32123292

3152

2992

4772

3212

61334332

8

6

3 3122

26

18

15

41

363310

17

42

20

11

()

Figure 4 UPLC-ESI-MS (negative ion) chromatogram of main compounds of ethanol extract from Brazilian green propolis

scavengers of free radicals and inhibitors of nitric oxideand inflammatory cytokines production by macrophagesandor neutrophils [4 13 16ndash18 27 34 39 40] Kaempferideartepillin C and their derivatives are the major constituentsidentified in our tested sample of Brazilian green propolisextract

Brazilian green propolis exhibits significant antioxidantproperties by scavenging ROS and inhibiting chemilumines-cence reactions [4 18 40] The topical or oral treatment ofanimals with Brazilian propolis extracts demonstrated poten-tial effect against oxidative stress [41] DPPH∙ and ABTS∙+tests have been widely used for evaluating antioxidant prop-erties of natural phenolic compounds Antioxidants interceptthe free radical chain oxidation by donating hydrogen from

the phenolic hydroxyl groups thereby forming stable endproducts which do not initiate or propagate further oxida-tion EEP-B exerted strong free radical scavenging activitybased on reduction of DPPH∙ and ABTS∙+ [4 17] Izuta etal showed the ability of scavenging DPPH∙ by green propolisand artepillin C in contrast to other prenylated derivativesof cinnamic acid drupanin and baccharin which did notcause similar effect The findings indicate that 3-prenyl chainof cinnamic acid is important for antioxidant activity ofartepillin C [42] Hayashi et al noticed strong antioxidanteffect of two compounds isolated from Brazilian propoliskaempferide and artepillin C [43] In the present study weverified potent scavenging capability of EEP-B againstDPPH∙and ABTS∙+


Table 1The content [mgg] and characterization of phenolic compounds of the ethanol extract of Brazilian green propolis using their spectralcharacteristic in negative ions in LC-ESIMS

PeakRetention time119905119903

(min)[MminusH]minus MSMS fragments Compound namelowast Quantity

[mgg of propolis]lowastlowast

(1) 196 3530879 1910553173044616102411350440 Caffeoylquinic acidisomerbc 649e

(2) 226 1790349 16102411350440 Caffeic acida 129(3) 279 1630406 1190510 p-Coumaric acida 1990(4) 302 1930492 1930492 Ferulic acida 148

(5) 330 5151196 35308791910553179034916102411350440 Dicaffeoylquinic acidizomerbc 222e


(7) 365 2310652 187076016303801450653 Coumaric acid prenylesterb 014d

(8) 384 4871597 263091117903491610241 Caffeic acid derivativec 055e

(9) 427 6771512 515119635308791910553179034916102411350441 Tricaffeoylquinic acidbc 046e

(10) 443 5431486 381117317903491610241Dimethyl-

dicaffeoylquinicacidc

300e

(11) 465 5431486 381117317903491610241Dimethyl-


361e

(12) 489 2710616 151001713306541070134 Naringenina 054(13) 496 2850394 2850394 Kempferola 335(14) 501 3150512 30107091510017 Isorhamnetinbc 318h

(15) 508 3010709 1520119 Hesperitina 842(16) 525 3011455 239142620209921320568 Ni mdash(17) 543 7051837 5431486381117317903491610241 Caffeic acid derivativec 083e

(18) 563 2470982 20310721480535 34-Dihydroxy5-prenylcinnamic acidb 241d

(19) 571 3311565 3311565 Ni 275d

(20) 589 2311025 187112413205684-Hydroxy

3-prenylcinnamic acid(drupanin)b

2563d

(21) 605 3151602 253160920112861460728 Coumaric acidderivativec 361d

(22) 644 2850775 16401711190510 Sakuranetina 243(23) 646 2850775 1640116151004213501621080215 Izosakuranetina 339(24) 651 2550663 2550663 Pinocembrina 312(25) 659 2990572 284031320112861510042 Kaempferidea 1881(26) 672 3771958 29905722011286 Kaempferide derivativec 1018g

(27) 678 3931335 163038014502841190510 Dicoumaric prenyl esterc 371d

(28) 687 3151602 299053625316091981039 Ni 416d

(29) 703 3131433 211112615605831490618 Ni 061d

(30) 710 5291495 29905722840313 Kaempferide derivativec 007g

(31) 722 3151602 20112861460728 Ni 006d


(33) 749 3291780 29916342551742 Artepillin C derivativec mdash


Table 1 Continued




(34) 761 5452541 33119002691174 Ni mdash


(36) 806 2991634 25517422001197 Artepillin Ca 5144(37) 840 5851772 30114551250974 Ni mdash(38) 891 4472171 29714891490613 Ni mdash

(39) 913 3631619 1871124149061313104973-Prenyl-4-

(dihydrocinnamoyloxy)-cinnamic acid(baccharin)b

966d

(40) 932 2971489 253157719810391529951 Ni 062d

(41) 951 3212426 29916342551742 Artepillin derivativec 089e



Ni not identifiedlowastaConfirmed by standardbConfirmed by reference [19ndash21 23 24 26]cConfirmed by MS fragmentationlowastlowastdExpressed as p-coumaric acideExpressed as caffeic acidfExpressed as artepillin CgExpressed as kaempferidehExpressed as rhamnetin

Cyto

toxi

city

(MTT

) (

)

109876543210

minus

minus minus

minus minus minus minus + + + + +

5 10 25 50 5 10 25 50LPS + IFN-120574

EEP-B (120583gmL)

Figure 5 Effect of EEP-B on viability of J774A1 macrophages Thecytotoxicity was evaluated by MTT assay after 24 h incubation ofJ774A1 cells with 5ndash50120583gmL EEP-B andor LPS + IFN-120574 Thevalues represent mean plusmn SD of three independent experiments (119899 =12)

Regarding the anti-inflammatory property the effect ofEEP-B in ROS and RNS scavenging was also determinedby chemiluminescence assay We evaluated for the first timethe role of green propolis in the oxidative metabolism ofPMA stimulated macrophages The ROS and RNS releaseby activated J774A1 cells was significantly inhibited by EEP-B in dose-dependent manner Krol et al and Simoes et alproved that kaempferide and extracts of Polish and Braziliangreen propolis decrease the chemiluminescence produced bystimulated neutrophils [18 39 40]

NO a short half-life free radical is an effector mole-cule in host defense against pathogens NO production in

macrophages is mediated by the inducible nitric oxide syn-thase (iNOS) However excessive release of NO inducedby LPS IFN-120574 or TNF-120572 has detrimental effects on manyorgan systems of the body leading to acute or chronicinflammatory diseases [44] Song et al reported that treat-ment of RAW2647 cells with extract of Korean propolismarkedly suppresses NO production and iNOS mRNA andprotein expression induced by LPS + IFN-120574 [45] Blonskaet al observed the inhibition of NO synthesis and iNOSmRNA expression in LPS stimulated J774A1 macrophagesby extract of Polish propolis and its phenolic componentschrysin galangin kaempferol and quercetin [27] Paulinoet al demonstrated that artepillin C decreases NO concen-tration in RAW2647 cells incubated with LPS [46] Krolet al described downregulation of NO by kaempferide inactivated murine peritoneal macrophages [16] Similar toprevious studies our data confirmed the effect of EEB-P onNO generation in J774A1 cells The additional data from invitro study supplied by Tan-No et al proved that Brazilianpropolis extract suppresses corrageenin-induced paw edemain mouse through inhibition of NO production [47]

Macrophages are a major source of many cytokinesinvolved in immune response hematopoiesis inflamma-tion and other homeostatic processes Upon stimulation bymicroorganisms microbial products (eg LPS) or endoge-nous factors (including cytokines) macrophages synthetizede novo and release a large variety of cytokines IL-1 IL-3IL-4 IL-5 IL-6 IL-8 IL-9 IL-10 IL-12 IL-13 IL-17 TNF-120572 IFN-120572 IFN-120574 TGF-120573 M-CSF G-CSF GM-CSF MCP-1MCP-3 MCP-5 MIP-1 MIP-2 RANTES MIF and KC In


200018001600140012001000

800600400200

0

Chem

ilum

ines

cenc

e (co

unts

s)

PMA

0 200 400 600 800 1000 1200 1400 1600 1800Time (s)

ControlEEP-B-01 120583gmL

(a)

100908070605040302010

0

Chem

ilum

ines

cenc

e (

of c

ontro

l)

EEP-B (120583gmL)001 01 1 10

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

(b)

Figure 6 Effect of EEP-B on chemiluminescence of PMA activated J774A1 macrophages (a) time course of chemiluminescence (b)chemiluminescence of PMA activated J774A1 macrophages treated with 001ndash10120583gmL EEP-B for 30min Chemiluminescence wasdetermined using microplate luminometer and expressed as a percentage of the PMA-stimulated cells The values represent mean plusmn SDof four independent experiments (119899 = 8) lowastlowastlowast = 119875 lt 0001 compared to PMA-stimulated cells

Nitr

ite (

of c

ontro

l)

120

100

80

60

40

20

0


lowastlowastlowast

lowastlowastlowast

LPS + IFN-120574EEP-B (120583gmL)

+ + + + +

minus 5 10 25 50

Figure 7 Effect of EEP-B on nitrite (NO) production in LPS + IFN-120574 stimulated J774A1macrophages J774A1 cells were incubatedwith5ndash50 120583gmLEEP-B andor LPS+ IFN-120574 for 24 hNOproductionwasmeasured by the Griess reaction assay and expressed as a percentageof LPS + IFN-120574-stimulated cells The values represent mean plusmn SD ofthree independent experiments (119899 = 12) lowastlowastlowast = 119875 lt 0001 comparedto LPS + IFN-120574-stimulated cells

addition these cytokines can modulate most of the functionsof macrophages cell surface markers expression and otherscytokines secretion The cytokine network plays a key role inregulation of macrophages activation [48] To gain furtherinsight into anti-inflammatory activity of EEP-B nineteencytokines secreted by macrophages were analyzed We inves-tigated the influence of Brazilian green propolis extract onproduction of cytokine IL-1120572 IL-1120573 IL-3 IL-4 IL-5 IL-6 IL-9 IL-10 IL-12p40 IL-13 IL-17 TNF-120572 IFN-120574 G-CSF GM-CSF MCP-1 MIP-1120572 MIP-1120573 and RANTES in LPS + IFN-120574stimulated macrophages Inflammatory cytokines are gener-ated by innate immune cells during infection For exampleLPS induces strong release of IL-1 IL-6 IL-12 and TNF-120572

by the macrophages [37 48 49] The upregulation of theseinflammatory cytokines in LPS or LPS + IFN-120574 activatedmacrophages was blocked by propolis extracts We showedthat EEP-B significantly downregulated the production ofIL-1120572 IL-1120573 IL-6 IL-12p40 and TNF-120572 in LPS + IFN-120574treated J774A1 cells Bachiega et al described the increaseof IL-1120573 and decrease of IL-6 production by Brazilian greenpropolis extract and its phenolic acids in peritoneal murinemacrophages challenged with LPS [50] Shi et al and Wanget al noticed that extracts from Chinese propolis suppressmRNA and protein expression of IL-1120573 and IL-6 induced byLPS in RAW2647 cells [51 52] Blonska et al reported theinhibition of IL-1120573 mRNA and protein expression in LPS-stimulated J774A1 macrophages by extract of Polish propolisand its flavones [27] We observed that the secretion ofinterleukin IL-4 and IL-13 in LPS + IFN-120574 activated J774A1cells was also reduced by EEP-B Whereas the concentrationsof IL-3 IL-5 IL-9 IL-10 IL-17 and IFN-120574 were only slightlyor not at all affected after incubation with EEP-B Thein vivo experiment performed by Franchin et al showeddown-regulation of IL-1120573 and TNF-120572 production by extractof geopropolis from Melipona scutellaria [53] By contrastOrsatti et al demonstrated upregulation of IL-1120573 and IL-6 inin vivomodel [54] In other in vivo testHu et al found out thatChinese propolis extracts suppressed carrageenan inducedperitonitis and pleurisy or acute lung damage in rats throughdecrease of the number of neutrophils Chinese propolisalso down-regulated IL-6 in Freundrsquos complete adjuvant(FCA) induced arthritis in rats [55] Paulino et al reporteda significant inhibition of paw edema in carrageenan inducedmodel of murine peritonitis by artepillin C also via decreaseof neutrophilsrsquo percentage in the peritoneal cavity [46]

The colony-stimulating factors G-CSF and GM-CSFrelease in activated J774A1 macrophages were decreasedby EEP-B Chemokines (MCP-1 MCP-3 MIP-1 MIP-2


120100

80604020

0

lowastlowastlowast

lowastlowastlowastIL

-1120572

( o

f con

trol)

(a)

120100

80604020

0

lowastlowastlowastlowastlowastlowast

IL-1120573

( o

f con

trol)

(b)

120100

80604020

0

IL-3

( o

f con

trol)

(c)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-4

( o

f con

trol)

(d)

140120100

80604020

0

IL-5

( o

f con

trol)

(e)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-6

( o

f con

trol)

(f)

120100

80604020

0

IL-9

( o

f con

trol)

(g)

120100

80604020

0

lowast

IL-1

0 (

of c

ontro

l)

(h)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-1

2p40

( o

f con

trol)

(i)

120100

80604020

0

lowastlowastlowast

IL-1

3 (

of c

ontro

l)

(j)

120100

80604020

0

IL-1

7 (

of c

ontro

l)

LPS + IFN-120574 minus

minus minus

+ ++

25 50EEP-B (120583gmL)

(k)

120100

80604020

0


TNF-120572

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(l)

Figure 8 Continued


120100

80604020

0

IFN

-120574(

of c

ontro

l)

(m)

120100

80604020

0lowastlowastlowast

lowastlowastlowast

G-C

SF (

of c

ontro

l)

(n)

120100

80604020

0 lowastlowastlowastlowastlowastlowastGM

-CSF

( o

f con

trol)

(o)

120100

80604020

0

lowastlowastlowastlowastlowast

MCP

-1 (

of c

ontro

l)

(p)

120100

80604020

0


MIP

-1120572

( o

f con

trol)

(q)

120100

80604020

0

lowastlowastM

IP-1120573

( o

f con

trol)

(r)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

RAN

TES

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(s)

Figure 8 Effect of EEP-B on cytokines production in LPS + IFN-120574 stimulated J774A1 macrophages (a) IL-1120572 (b) IL-1120573 (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-120572 (m) IFN-120574 (n) G-CSF (o) GM-CSF (p) MCP-1 (q) MIP-1120572 (r)MIP-1120573 and (s) RANTES J774A1 cells were incubated with 25ndash50 120583gmL EEP-B andor LPS + IFN-120574 for 24 h Cytokine concentrations inthe culture medium were determined by Multiplex (19-plex) bead-based cytokine assay The values represent mean plusmn SD of two independentexperiments (119899 = 8) lowast = 119875 lt 005 lowastlowast = 119875 lt 001 lowastlowastlowast = 119875 lt 0001 compared to LPS + IFN-120574-stimulated cells

and RANTES) are chemotactic cytokines contributing tothe recruitment of circulating monocytes within tissues Ourstudy extended to macrophage chemokines demonstratedthat EEP-B downregulates the expression of MCP-1 MIP-1120572MIP-1120573 and RANTES in LPS + IFN-120574 stimulated J774A1cells These results suggest a crucial contribution of Braziliangreen propolis in the modulation of chemokine-mediatedinflammation

The findings confirm significant anti-inflammatory effectof ethanolic extract of Brazilian green propolis on macro-phage in vitromodel

5 Conclusion

Propolis has become a subject of special interest as a sourceof valuable phenolic compounds to developed food com-ponents dietary supplements or even pharmaceuticalsfor the prevention or treatment of inflammatory diseasesArtepillin C drupanin baccarin p-coumaric acid andkaempferide are the main ingredients of Brazilian greenpropolis Our recent study provide new evidence-basedproofs of anti-inflammatory properties of Brazilian greenpropolis 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-1-063P20 from the Medical University of Silesia in Katowice(Poland) The authors thank Mr Rindai Yamamoto thePresident of Nihon Natural Foods Co Ltd (Tokyo Japan)and Mrs Etsuko Yamamoto the President of KomyodoKampo Shoyaku Co Ltd (Tokyo Japan) for the sample ofBrazilian green propolis

References

[1] Y K Park S M Alencar and C L Aguiar ldquoBotanical originand chemical composition of Brazilian propolisrdquo Journal ofAgricultural and Food Chemistry vol 50 pp 2502ndash2506 2002

[2] F C Lopes V Bankova and JM Sforcin ldquoEffect of three vegetalsources of propolis on macrophages activationrdquo Phytomedicinevol 10 no 4 p 343 2003

[3] J M Sforcin ldquoPropolis and the immune system a reviewrdquoJournal of Ethnopharmacology vol 113 pp 1ndash14 2007

[4] E W Teixeira D Message G Negri A Salatino and PC Stringheta ldquoSeasonal variation chemical composition andantioxidant activity of brazilian propolis samplesrdquo Evidence-Based Complementary and AlternativeMedicine vol 7 no 3 pp307ndash315 2010

[5] J M Sforcin and V Bankova ldquoPropolis is there a potential forthe development of new drugsrdquo Journal of Ethnopharmacologyvol 133 no 2 pp 253ndash260 2011

[6] E Szliszka G Zydowicz B Janoszka C Dobosz G Kowalczyk-Ziomek and W Krol ldquoEthanolic extract of Brazilian greenpropolis sensitizes prostate cancer cells to TRAIL-inducedapoptosisrdquo International Journal of Oncology vol 38 no 4 pp941ndash953 2011

[7] D Sawicka H Car M H Borawska and J Niklinski ldquoThe anti-cancer activity of propolisrdquo Folia Histochemica et Cytobiologicavol 50 pp 25ndash37 2012

[8] G C Franchi C S Moraes V C Toreti A Daugsch A ENowill and Y K Park ldquoComparison of effects of the ethanolicextracts of Brazilian propolis on human leukemic cells asassessed with the MTT assayrdquo Evidence-Based Complementaryand Alternative Medicine vol 2012 Article ID 918956 6 pages2012

[9] E Szliszka and W Krol ldquoPolyphenols isolated from propolisaugment TRAIL-induced apoptosis in cancer cellsrdquo Evidence-Based Complementary and Alternative Medicine vol 2013Article ID 731940 10 pages 2013

[10] A H Banskota Y Tezuka and S Kadota ldquoRecent progress inpharmacological research of propolisrdquo Phytotherapy Researchvol 15 no 7 pp 561ndash571 2001

[11] E Szliszka Z P Czuba M Domino B Mazur G Zydowiczand W Krol ldquoEthanolic extract of propolis (EEP) enhancesthe apoptosis-inducing potential of TRAIL in cancer cellsrdquoMolecules vol 14 no 2 pp 738ndash754 2009

[12] E Szliszka Z P Czuba J Bronikowska A Mertas A Paradyszand W Krol ldquoEthanolic extract of propolis (EEP) augmentsTRAIL-induced apoptotic death in prostate cancer cellsrdquo

Evidence-Based Complementary and Alternative Medicine vol2011 Article ID 535172 11 pages 2011

[13] G C Chan K W Cheung and D M Sze ldquoThe immunomod-ulatory and anticancer properties of propolisrdquo Clinical Reviewsin Allergy amp Immunology vol 44 no 3 pp 262ndash273 2013

[14] D L Laskin andK J Pendino ldquoMacrophages and inflammatorymediators in tissue injuryrdquoAnnual Review of Pharmacology andToxicology vol 35 pp 655ndash677 1995

[15] A Aderem and D M Underhill ldquoMechanisms of phagocytosisin macrophagesrdquo Annual Review of Immunology vol 17 pp593ndash623 1999

[16] W Krol Z P Czuba G Pietsz M D Threadgill and B DCunningham ldquoModulation of the cytotoxic activity of murinemacrophages by flavonesrdquo Current Topics in Biophysics vol 20pp 88ndash93 1996

[17] M R Ahn K Kunimasa S Kumazawa et al ldquoCorrelationbetween antiangiogenic activity and antioxidant activity ofvarious components from propolisrdquo Molecular Nutrition andFood Research vol 53 no 5 pp 643ndash651 2009

[18] LMC Simoes L EGregorio AADa Silva Filho et al ldquoEffectof Brazilian green propolis on the production of reactive oxygenspecies by stimulated neutrophilsrdquo Journal of Ethnopharmacol-ogy vol 94 no 1 pp 59ndash65 2004

[19] A A Carvalho D Finger C S Machado et al ldquoIn vivoantitumoural activity and composition of an oil extract ofBrazilian propolisrdquo Food Chemistry vol 126 pp 1239ndash12452011

[20] S A Moura G Negri A Salatino et al ldquoAqueous extractof Brazilian green propolis primary components evaluationof inflammation and wound healing by using subcutaneousimplanted spongesrdquo Evidence-Based Complementary and Alter-native Medicine vol 2011 Article ID 748283 8 pages 2011

[21] F Pellati G Orlandini D Pinetti and S Benvenuti ldquoHPLC-DAD and HPLC-ESI-MSMS methods for metabolite profilingof propolis extractsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 55 no 5 pp 934ndash948 2011

[22] S I Falcao N Vale and P Gomes ldquoPhenolic profiling of Por-tuguese propolis by LC-MS spectrometry uncommon propolisrich in flavonoid glycosidesrdquo Phytochemical Analysis 2012

[23] C C Fernandes-Silva A Salatino M L Salatino E D Breyerand G Negri ldquoChemical profiling of six samples of Brazilianpropolisrdquo Quimica Nova vol 36 pp 237ndash240 2013

[24] A C H F Sawaya D M Tomazela I B S Cunha et alldquoElectrospray ionization mass spectrometry fingerprinting ofpropolisrdquo Analyst vol 129 no 8 pp 739ndash744 2004

[25] D Wojnicz A Z Kucharska A Sokoł-Łętowska M Kicia andD Tichaczek-Goska ldquoMedicinal plants extracts affect virulencefactors expression and biofilm formation by the uropathogenicEscherichia colirdquoUrological Research vol 40 pp 683ndash697 2012

[26] S Keckes U Gasic T Cirkovic-Velickovic D Milojkovic-Opsenica M Natic and Z Tesic ldquoThe determination of phe-nolic profiles of Serbian unifloral honeys using ultra-high-performance liquid chromatographyhigh resolution accuratemass spectrometryrdquo Food Chemistry vol 138 pp 32ndash40 2013

[27] M Blonska J Bronikowska G Pietsz Z P Czuba S SchellerandW Krol ldquoEffects of ethanol extract of propolis (EEP) and itsflavones on inducible gene expression in J774A1 macrophagesrdquoJournal of Ethnopharmacology vol 91 no 1 pp 25ndash30 2004

[28] E Szliszka Z P Czuba Ł Sedek A Paradysz and W KrolldquoEnhanced TRAIL-mediated apoptosis in prostate cancer cellsby the bioactive compounds neobavaisoflavone and psoralidin


isolated from Psoralea corylifoliardquo Pharmacological Reports vol63 no 1 pp 139ndash148 2011

[29] E Szliszka D Jaworska M Kłosek Z P Czuba and W KrolldquoTargeting death receptor TRAIL-R2 by chalcones for TRAIL-induced apoptosis in cancer cellsrdquo International Journal ofMolecular Sciences vol 13 pp 15343ndash15359 2012

[30] E Szliszka K J Helewski E Mizgala andW Krol ldquoThe dietaryflavonol fisetin enhances the apoptosis-inducing potential ofTRAIL in prostate cancer cellsrdquo International Journal of Oncol-ogy vol 39 no 4 pp 771ndash779 2011

[31] E Szliszka E Kostrzewa-Susłow J Bronikowska et al ldquoSyn-thetic flavanones augment the anticancer effect of tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)rdquoMolecules vol 17 pp 11693ndash11711 2012

[32] I C Jang E K Jo M S Bae et al ldquoAntioxidant and antigeno-toxic activities of different parts of persimmon (Diospyros kakicv Fuyu) fruitrdquo Journal of Medicinal Plant Research vol 4 no2 pp 155ndash160 2010

[33] E Szliszka D Skaba Z P Czuba and W Krol ldquoInhibitionof inflammatory mediators by neobavaisoflavone in activatedRAW2647macrophagesrdquoMolecules vol 16 pp 3701ndash3712 2011

[34] W Krol Z P Czuba M D Threadgill B D M Cunninghamand G Pietsz ldquoInhibition of nitric oxide (NO) production inmurine macrophages by flavonesrdquo Biochemical Pharmacologyvol 50 no 7 pp 1031ndash1035 1995

[35] S B Yoon Y J Lee S K Park et al ldquoAnti-inflammatory effectsof Scutellaria baicalensis water extract on LPS-activated RAW2647macrophagesrdquo Journal of Ethnopharmacology vol 125 no2 pp 286ndash290 2009

[36] E Szliszka G Zydowicz E Mizgala and W Krol ldquoArtepillinC (35-diprenyl-4-hydroxycinnamic acid) sensitizes prostatecancer LNCaP cells to TRAIL-induced apoptosisrdquo InternationalJournal of Oncology vol 41 pp 818ndash828 2012

[37] Y K Park J F Paredes-Guzman C L Aguiar S M Alencarand F Y Fujiwara ldquoChemical constituents in Baccharis dracun-culifolia as the main botanical origin of Southeastern Brazilianpropolisrdquo Journal of Agricultural and Food Chemistry vol 52no 5 pp 1100ndash1103 2004

[38] A H Banskota Y Tezuka J K Prasain K Matsushige I Saikiand S Kadota ldquoChemical constituents of Brazilian propolis andtheir cytotoxic activitiesrdquo Journal ofNatural Products vol 61 no7 pp 896ndash900 1998

[39] W Krol S Scheller Z Czuba et al ldquoInhibition of neutrophilsrsquochemiluminescence by ethanol extract of propolis (EEP) and itsphenolic componentsrdquo Journal of Ethnopharmacology vol 55no 1 pp 19ndash25 1996

[40] L M C Simoes-Ambrosio L E Gregorio J P B Sousa et alldquoThe role of seasonality on the inhibitory effect of Braziliangreen propolis on the oxidative metabolism of neutrophilsrdquoFitoterapia vol 81 no 8 pp 1102ndash1108 2010

[41] M J V Fonseca Y M Fonseca F Marquele-Oliveira et alldquoEvaluation of the potential of Brazilian propolis against UV-induced oxidative stressrdquo Evidence-Based Complementary andAlternative Medicine vol 2011 Article ID 863917 2011

[42] H Izuta Y Narahara M Shimazawa S Mishima S I Kondoand H Hara ldquo11-diphenyl-2-picrylhydrazyl radical scavengingactivity of bee products and their constituents determined byESRrdquo Biological and Pharmaceutical Bulletin vol 32 no 12 pp1947ndash1951 2009

[43] K Hayashi S Komura N Isaji N Ohishi and K Yagi ldquoIso-lation of antioxidative compounds from Brazilian propolis

34-dihydroxy-5-prenylcinnamic acid a novel potent antioxi-dantrdquo Chemical and Pharmaceutical Bulletin vol 47 no 11 pp1521ndash1524 1999

[44] J MacMacking Q Xie and C Nathan ldquoNitric oxide andmacrophages functionrdquo Annual Review of Immunology vol 15pp 323ndash350 1997

[45] Y S Song EH Park GMHur Y S Ryu YMKim andC JinldquoEthanol extract of propolis inhibits nitric oxide synthase geneexpression and enzyme activityrdquo Journal of Ethnopharmacologyvol 80 no 2-3 pp 155ndash161 2002

[46] N Paulino S R L Abreu Y Uto et al ldquoAnti-inflammatoryeffects of a bioavailable compound Artepillin C in Brazilianpropolisrdquo European Journal of Pharmacology vol 587 no 1ndash3pp 296ndash301 2008

[47] K Tan-No T Nakajima T Shoji et al ldquoAnti-inflammatoryeffect of propolis through inhibition of nitric oxide productionon carrageenin-induced mouse paw edemardquo Biological andPharmaceutical Bulletin vol 29 no 1 pp 96ndash99 2006

[48] J M Cavaillon ldquoCytokines andmacrophagesrdquo Biomedicine andPharmacotherapy vol 48 pp 445ndash453 1994

[49] T Hanada and A Yoshimura ldquoRegulation of cytokine signalingand inflammationrdquoCytokine andGrowth Factor Reviews vol 13no 4-5 pp 413ndash421 2002

[50] T F Bachiega C L Orsatti A C Pagliarone and J M SforcinldquoThe effects of propolis and its isolated compounds on cytokineproduction by murine macrophagesrdquo Phytotherapy Researchvol 26 pp 1308ndash1313 2012

[51] H Shi H Yang X Zhang and L Yu ldquoIdentification and quan-tification of phytochemical composition and anti-inflammatoryand radical scavenging properties of methanolic extract ofChinese propolisrdquo Journal of Agricultural and Food Chemistryvol 60 pp 12403ndash12410 2012

[52] K Wang S Ping S Huang et al ldquoMolecular mechanismsunderlying the in vitro anti-inflammatory effects of flavonoid-rich ethanol extract from Chinese propolisrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID127672 11 pages 2013

[53] M Franchin M G Cunha C Denny et al ldquoGeopropolis fromMelipona scutellaris decreases the mechanical inflammatoryhypernociception by inhibiting the production of IL-1120573 andTNF-120572rdquo Journal of Ethnopharmacology vol 143 pp 709ndash7152012

[54] C L Orsatti F Missima A C Pagliarone et al ldquoPropolisimmunomodulatory action in vivo on toll-like receptors 2 and4 expression and on pro-inflammatory cytokines production inmicerdquo Phytotherapy Research vol 24 no 8 pp 1141ndash1146 2010

[55] F Hu H R Hepburn Y Li M Chen S E Radloff andS Daya ldquoEffects of ethanol and water extracts of propolis(bee glue) on acute inflammatory animal modelsrdquo Journal ofEthnopharmacology vol 100 no 3 pp 276ndash283 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


macrophages release large amounts of reactive oxygen (ROS)and nitrogen species (RNS) nitric oxide (NO) and numerouscytokines [16] Overexpression of these mediators causespathological acute or chronic inflammatory responses [14]

This is the first comprehensive report explaining an anti-inflammatory potential of Brazilian green propolis extracton macrophage in vitro model We determined the effectof EEP-B on production of NO ROS RNS and cytokinesinterleukin (IL)-IL-1120572 IL-1120573 IL-3 IL-4 IL-5 IL-6 IL-9IL-10 IL-12p40 IL-13 IL-17 TNF-120572 (tumor necrosis factor120572) IFN-120574 (interferon 120574) G-CSF (granulocyte colony-stimu-lating factor) GM-CSF (granulocyte-macrophage colony-stimulating factor) MCP-1 (monocyte chemotactic protein1) MIP-1120572 (macrophage inflammatory protein 1120572) MIP-1120573(macrophage inflammatory protein 1120573) and RANTES (regu-lated upon activation normal T cell expressed and secreted)in activated J774A1 cells

It has been suggested that the immunomodulatory prop-erties of propolis is contributed by its flavonoids and phenolicacids [3 5 9 17] The typical ingredients of Brazilian greenpropolis are kaempferide (357-trihydroxy-41015840-methoxyfla-vone) and cinnamic acid derivatives p-coumaric acid (4-hydroxycinnamic acid) artepillin C (35-diprenyl-4-hydrox-ycinnamic acid) baccharin (3-prenyl-4-(dihydrocinnamoyl-oxy)-cinnamic acid) and drupanin (3-prenyl-4-hydroxycin-namic acid) [1 4 18] We performed the detailed identifica-tion and quantification of phenolic compounds in propolisextract

The present study analyzed chemical composition ofgreen propolis and confirmed its significant role in suppress-ing chronic inflammation and reducing risk of related humanhealth problems Our findings provide new insights forunderstanding the anti-inflammatory mechanism of actionof Brazilian green propolis extract and support its applicationin complementary and alternative therapies

2 Materials and Methods

21 General The Brazilian green propolis sample was kindlysupplied by Nihon Natural Foods Co Ltd (Tokyo Japan)LPS (LPS E coli O111B4) was obtained from Fluka ChemieGmbH (Buchs Switzerland) and recombinant mouse IFN-120574 was purchased from RampD Systems (Minneapolis MNUSA) PMA DMSO (dimethyl sulphoxide) acetonitrile andformic acid were obtained from Sigma-Aldrich (SteinheimGermany) Acetonitrile for LC-MS was purchased fromPOCh (Gliwice Poland) The following compounds wereused as standards sakuranetin hesperitin caffeic acid (RothKarlsruhe Germany) kaempferol pinocembrin naringeninp-coumaric acid (Sigma-Aldrich Steinheim Germany)kaempferide ferulic acid (Serva Heidelberg Germany)isosakuranetin (TransMIT GmbH Giessen Germany) andartepillin C (Wako Pure Chemicals Osaka Japan)

22 Preparation of Brazilian Green Propolis Extract TheBrazilian green propolis sample was collected manually frombeehive located in southeast Brazil (the state ofMinas Gerais)and was kept desiccated prior to processing The sample was

extracted in 95 vv ethyl alcohol in a hermetically sealedglass vessel for 4 days at 37∘C under occasional shakingThe ethanolic extract of Brazilian green propolis (EEP-B)was then filtered through Whatman filter paper no 4 andevaporated in a rotary evaporator under reduced pressureat 60∘CThe same collection and extraction procedures wereused throughout all our laboratory studies [6 12] EEP-B wasdissolved in DMSO (50mgmL) and the final concentrationof DMSO in the culture mediumwas controlled at 01 (vv)

23 Identification and Quantification of Phenolic Compoundsby HPLC-DAD Method Phenolic compounds were deter-mined using Dionex (USA) HPLC system equipped withdiode array detector model Ultimate 3000 a quaternarypump LPG-3400A autosampler EWPS-3000SI and ther-mostated column compartment TCC-3000SD and controlledby Chromeleon v68 software The reversed phase Cadenza5CD-C18 (75mm times 46 id) column (Imtakt Kyoto Japan)with guard column Cadenza (5 times 46 id) guard column(Imtakt Kyoto Japan) was used The mobile phase wascomposed of (A) 01 (vv) formic acid in water and (B)acetonitrile The applied elution conditions were 0min 20B 0ndash10min linear gradient from 20 to 30 B 10ndash40minlinear gradient from 30 to 40 B 40ndash60min lineargradient from40 to 60B 60ndash80min linear gradient from60 to 80 B and then again the initial conditions [19ndash23]The flow rate was 1mLmin and the injection volume was20120583L The column was operated at 30∘C The compoundswere monitored at 290 nm 325 nm and 370 nm

24 Identification and Quantification of Phenolic Compoundsby LC-MS Method Compounds identification was per-formed on an Acquity ultra-performance liquid chromatog-raphy (UPLC) system coupled with a quadruple time of flight(Q-TOF) MS instrument (UPLCSynapt Q-TOF MS WatersCorp Milford MA USA) with an electrospray ionization(ESI) source Separation was achieved on the Acquity BEHC18 column (100mm times 21mm id 17 120583m Waters) Detec-tion wavelengths were set at 290 325 and 370 nm A mobilephase was a mixture of 15 formic acid (A) and acetonitrile(B) The gradient program was as follows initial conditions-95 (A) 12min-5 (A) 13min-5 (A) 145min-95 (A)and 16min-95 (A) The flow rate was 045mLmin and theinjection volume was 5 120583LThe columnwas operated at 30∘CThe major operating parameters for the Q-TOF MS were setas follows capillary voltage 20 kV cone voltage 45V conegas flow of 11 Lh collision energy 50 eV source temperature100∘C desolvation temperature 250∘C collision gas argondesolvation gas (nitrogen) flow rate 600 Lh data acquisitionrange119898119911 100ndash1000Da ionization mode negative [24ndash26]The data were collected by Mass-Lynx V 41 software

25 Cell Culture Murine macrophage J774A1 cell line wasobtained from ATCC (American Type Culture CollectionManassas VA USA) Cells were cultured in Dulbeccorsquosmodified Eaglersquos medium supplemented with 10 heat-inactivated fetal bovine serum 100UmL penicillin and100 120583gmL streptomycin at 37∘Cand 10CO

2

in a humidified






50




50








3 Results







50





50


50




2

15

2025

21

1819

24

22

28

26

11

10

1

8

3

9

7

6

5

4

Time2 3 4 5 6 7 8 9 10 11

(au

)

0

112141618

222242628

332

17

16121314

232927

31

30323334

35

36

37

39

38 40

41

43

428eminus1

6eminus1

4eminus1

2eminus1


363

Time2 3 4 5 6 7 8 9 10 11

0

112141618

222242628

3

2

25

15

21

18

19

24

26

22

2811

10

1

8

9

7

6

5

4

17

161213

1423

29

27

31

30 32

33

3435

3739

38

40

41

43

42

20

(au

)

8eminus1

6eminus1

4eminus1

2eminus1



4 Discussion





Time2 3 4 5 6 7 8 9 10 11

(au

)

0

112141618

222242628

13

2526

14

8eminus1

6eminus1

4eminus1

2eminus1


2

Time2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 105

0

100 2311

3011

54311630

1790

5151

48922631

677248722471

5431

2471

7052

3772

2851

32123292

3152

2992

4772

3212

61334332

8

6

3 3122

26

18

15

41

363310

17

42

20

11

()

















(10) 443 5431486 381117317903491610241Dimethyl-


300e

(11) 465 5431486 381117317903491610241Dimethyl-


361e




(19) 571 3311565 3311565 Ni 275d

(20) 589 2311025 187112413205684-Hydroxy


2563d




(28) 687 3151602 299053625316091981039 Ni 416d

(29) 703 3131433 211112615605831490618 Ni 061d


(31) 722 3151602 20112861460728 Ni 006d




Table 1 Continued




(34) 761 5452541 33119002691174 Ni mdash



(39) 913 3631619 1871124149061313104973-Prenyl-4-


966d

(40) 932 2971489 253157719810391529951 Ni 062d





Cyto

toxi

city

(MTT

) (

)

109876543210

minus

minus minus


5 10 25 50 5 10 25 50LPS + IFN-120574

EEP-B (120583gmL)







200018001600140012001000

800600400200

0

Chem

ilum

ines

cenc

e (co

unts

s)

PMA

0 200 400 600 800 1000 1200 1400 1600 1800Time (s)


(a)

100908070605040302010

0

Chem

ilum

ines

cenc

e (

of c

ontro

l)

EEP-B (120583gmL)001 01 1 10

lowastlowastlowast


(b)


Nitr

ite (

of c

ontro

l)

120

100

80

60

40

20

0


lowastlowastlowast

lowastlowastlowast

LPS + IFN-120574EEP-B (120583gmL)

+ + + + +

minus 5 10 25 50






120100

80604020

0

lowastlowastlowast


-1120572

( o

f con

trol)

(a)

120100

80604020

0


IL-1120573

( o

f con

trol)

(b)

120100

80604020

0

IL-3

( o

f con

trol)

(c)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-4

( o

f con

trol)

(d)

140120100

80604020

0

IL-5

( o

f con

trol)

(e)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-6

( o

f con

trol)

(f)

120100

80604020

0

IL-9

( o

f con

trol)

(g)

120100

80604020

0

lowast

IL-1

0 (

of c

ontro

l)

(h)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-1

2p40

( o

f con

trol)

(i)

120100

80604020

0

lowastlowastlowast

IL-1

3 (

of c

ontro

l)

(j)

120100

80604020

0

IL-1

7 (

of c

ontro

l)


minus minus

+ ++

25 50EEP-B (120583gmL)

(k)

120100

80604020

0


TNF-120572

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(l)

Figure 8 Continued


120100

80604020

0

IFN

-120574(

of c

ontro

l)

(m)

120100

80604020

0lowastlowastlowast

lowastlowastlowast

G-C

SF (

of c

ontro

l)

(n)

120100

80604020


-CSF

( o

f con

trol)

(o)

120100

80604020

0


MCP

-1 (

of c

ontro

l)

(p)

120100

80604020

0


MIP

-1120572

( o

f con

trol)

(q)

120100

80604020

0

lowastlowastM

IP-1120573

( o

f con

trol)

(r)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

RAN

TES

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(s)




5 Conclusion





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















50




50








3 Results







50





50


50




2

15

2025

21

1819

24

22

28

26

11

10

1

8

3

9

7

6

5

4

Time2 3 4 5 6 7 8 9 10 11

(au

)

0

112141618

222242628

332

17

16121314

232927

31

30323334

35

36

37

39

38 40

41

43

428eminus1

6eminus1

4eminus1

2eminus1


363

Time2 3 4 5 6 7 8 9 10 11

0

112141618

222242628

3

2

25

15

21

18

19

24

26

22

2811

10

1

8

9

7

6

5

4

17

161213

1423

29

27

31

30 32

33

3435

3739

38

40

41

43

42

20

(au

)

8eminus1

6eminus1

4eminus1

2eminus1



4 Discussion





Time2 3 4 5 6 7 8 9 10 11

(au

)

0

112141618

222242628

13

2526

14

8eminus1

6eminus1

4eminus1

2eminus1


2

Time2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 105

0

100 2311

3011

54311630

1790

5151

48922631

677248722471

5431

2471

7052

3772

2851

32123292

3152

2992

4772

3212

61334332

8

6

3 3122

26

18

15

41

363310

17

42

20

11

()

















(10) 443 5431486 381117317903491610241Dimethyl-


300e

(11) 465 5431486 381117317903491610241Dimethyl-


361e




(19) 571 3311565 3311565 Ni 275d

(20) 589 2311025 187112413205684-Hydroxy


2563d




(28) 687 3151602 299053625316091981039 Ni 416d

(29) 703 3131433 211112615605831490618 Ni 061d


(31) 722 3151602 20112861460728 Ni 006d




Table 1 Continued




(34) 761 5452541 33119002691174 Ni mdash



(39) 913 3631619 1871124149061313104973-Prenyl-4-


966d

(40) 932 2971489 253157719810391529951 Ni 062d





Cyto

toxi

city

(MTT

) (

)

109876543210

minus

minus minus


5 10 25 50 5 10 25 50LPS + IFN-120574

EEP-B (120583gmL)







200018001600140012001000

800600400200

0

Chem

ilum

ines

cenc

e (co

unts

s)

PMA

0 200 400 600 800 1000 1200 1400 1600 1800Time (s)


(a)

100908070605040302010

0

Chem

ilum

ines

cenc

e (

of c

ontro

l)

EEP-B (120583gmL)001 01 1 10

lowastlowastlowast


(b)


Nitr

ite (

of c

ontro

l)

120

100

80

60

40

20

0


lowastlowastlowast

lowastlowastlowast

LPS + IFN-120574EEP-B (120583gmL)

+ + + + +

minus 5 10 25 50






120100

80604020

0

lowastlowastlowast


-1120572

( o

f con

trol)

(a)

120100

80604020

0


IL-1120573

( o

f con

trol)

(b)

120100

80604020

0

IL-3

( o

f con

trol)

(c)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-4

( o

f con

trol)

(d)

140120100

80604020

0

IL-5

( o

f con

trol)

(e)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-6

( o

f con

trol)

(f)

120100

80604020

0

IL-9

( o

f con

trol)

(g)

120100

80604020

0

lowast

IL-1

0 (

of c

ontro

l)

(h)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-1

2p40

( o

f con

trol)

(i)

120100

80604020

0

lowastlowastlowast

IL-1

3 (

of c

ontro

l)

(j)

120100

80604020

0

IL-1

7 (

of c

ontro

l)


minus minus

+ ++

25 50EEP-B (120583gmL)

(k)

120100

80604020

0


TNF-120572

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(l)

Figure 8 Continued


120100

80604020

0

IFN

-120574(

of c

ontro

l)

(m)

120100

80604020

0lowastlowastlowast

lowastlowastlowast

G-C

SF (

of c

ontro

l)

(n)

120100

80604020


-CSF

( o

f con

trol)

(o)

120100

80604020

0


MCP

-1 (

of c

ontro

l)

(p)

120100

80604020

0


MIP

-1120572

( o

f con

trol)

(q)

120100

80604020

0

lowastlowastM

IP-1120573

( o

f con

trol)

(r)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

RAN

TES

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(s)




5 Conclusion





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















3 Results







50





50


50




2

15

2025

21

1819

24

22

28

26

11

10

1

8

3

9

7

6

5

4

Time2 3 4 5 6 7 8 9 10 11

(au

)

0

112141618

222242628

332

17

16121314

232927

31

30323334

35

36

37

39

38 40

41

43

428eminus1

6eminus1

4eminus1

2eminus1


363

Time2 3 4 5 6 7 8 9 10 11

0

112141618

222242628

3

2

25

15

21

18

19

24

26

22

2811

10

1

8

9

7

6

5

4

17

161213

1423

29

27

31

30 32

33

3435

3739

38

40

41

43

42

20

(au

)

8eminus1

6eminus1

4eminus1

2eminus1



4 Discussion





Time2 3 4 5 6 7 8 9 10 11

(au

)

0

112141618

222242628

13

2526

14

8eminus1

6eminus1

4eminus1

2eminus1


2

Time2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 105

0

100 2311

3011

54311630

1790

5151

48922631

677248722471

5431

2471

7052

3772

2851

32123292

3152

2992

4772

3212

61334332

8

6

3 3122

26

18

15

41

363310

17

42

20

11

()

















(10) 443 5431486 381117317903491610241Dimethyl-


300e

(11) 465 5431486 381117317903491610241Dimethyl-


361e




(19) 571 3311565 3311565 Ni 275d

(20) 589 2311025 187112413205684-Hydroxy


2563d




(28) 687 3151602 299053625316091981039 Ni 416d

(29) 703 3131433 211112615605831490618 Ni 061d


(31) 722 3151602 20112861460728 Ni 006d




Table 1 Continued




(34) 761 5452541 33119002691174 Ni mdash



(39) 913 3631619 1871124149061313104973-Prenyl-4-


966d

(40) 932 2971489 253157719810391529951 Ni 062d





Cyto

toxi

city

(MTT

) (

)

109876543210

minus

minus minus


5 10 25 50 5 10 25 50LPS + IFN-120574

EEP-B (120583gmL)







200018001600140012001000

800600400200

0

Chem

ilum

ines

cenc

e (co

unts

s)

PMA

0 200 400 600 800 1000 1200 1400 1600 1800Time (s)


(a)

100908070605040302010

0

Chem

ilum

ines

cenc

e (

of c

ontro

l)

EEP-B (120583gmL)001 01 1 10

lowastlowastlowast


(b)


Nitr

ite (

of c

ontro

l)

120

100

80

60

40

20

0


lowastlowastlowast

lowastlowastlowast

LPS + IFN-120574EEP-B (120583gmL)

+ + + + +

minus 5 10 25 50






120100

80604020

0

lowastlowastlowast


-1120572

( o

f con

trol)

(a)

120100

80604020

0


IL-1120573

( o

f con

trol)

(b)

120100

80604020

0

IL-3

( o

f con

trol)

(c)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-4

( o

f con

trol)

(d)

140120100

80604020

0

IL-5

( o

f con

trol)

(e)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-6

( o

f con

trol)

(f)

120100

80604020

0

IL-9

( o

f con

trol)

(g)

120100

80604020

0

lowast

IL-1

0 (

of c

ontro

l)

(h)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-1

2p40

( o

f con

trol)

(i)

120100

80604020

0

lowastlowastlowast

IL-1

3 (

of c

ontro

l)

(j)

120100

80604020

0

IL-1

7 (

of c

ontro

l)


minus minus

+ ++

25 50EEP-B (120583gmL)

(k)

120100

80604020

0


TNF-120572

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(l)

Figure 8 Continued


120100

80604020

0

IFN

-120574(

of c

ontro

l)

(m)

120100

80604020

0lowastlowastlowast

lowastlowastlowast

G-C

SF (

of c

ontro

l)

(n)

120100

80604020


-CSF

( o

f con

trol)

(o)

120100

80604020

0


MCP

-1 (

of c

ontro

l)

(p)

120100

80604020

0


MIP

-1120572

( o

f con

trol)

(q)

120100

80604020

0

lowastlowastM

IP-1120573

( o

f con

trol)

(r)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

RAN

TES

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(s)




5 Conclusion





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















2

15

2025

21

1819

24

22

28

26

11

10

1

8

3

9

7

6

5

4

Time2 3 4 5 6 7 8 9 10 11

(au

)

0

112141618

222242628

332

17

16121314

232927

31

30323334

35

36

37

39

38 40

41

43

428eminus1

6eminus1

4eminus1

2eminus1


363

Time2 3 4 5 6 7 8 9 10 11

0

112141618

222242628

3

2

25

15

21

18

19

24

26

22

2811

10

1

8

9

7

6

5

4

17

161213

1423

29

27

31

30 32

33

3435

3739

38

40

41

43

42

20

(au

)

8eminus1

6eminus1

4eminus1

2eminus1



4 Discussion





Time2 3 4 5 6 7 8 9 10 11

(au

)

0

112141618

222242628

13

2526

14

8eminus1

6eminus1

4eminus1

2eminus1


2

Time2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 105

0

100 2311

3011

54311630

1790

5151

48922631

677248722471

5431

2471

7052

3772

2851

32123292

3152

2992

4772

3212

61334332

8

6

3 3122

26

18

15

41

363310

17

42

20

11

()

















(10) 443 5431486 381117317903491610241Dimethyl-


300e

(11) 465 5431486 381117317903491610241Dimethyl-


361e




(19) 571 3311565 3311565 Ni 275d

(20) 589 2311025 187112413205684-Hydroxy


2563d




(28) 687 3151602 299053625316091981039 Ni 416d

(29) 703 3131433 211112615605831490618 Ni 061d


(31) 722 3151602 20112861460728 Ni 006d




Table 1 Continued




(34) 761 5452541 33119002691174 Ni mdash



(39) 913 3631619 1871124149061313104973-Prenyl-4-


966d

(40) 932 2971489 253157719810391529951 Ni 062d





Cyto

toxi

city

(MTT

) (

)

109876543210

minus

minus minus


5 10 25 50 5 10 25 50LPS + IFN-120574

EEP-B (120583gmL)







200018001600140012001000

800600400200

0

Chem

ilum

ines

cenc

e (co

unts

s)

PMA

0 200 400 600 800 1000 1200 1400 1600 1800Time (s)


(a)

100908070605040302010

0

Chem

ilum

ines

cenc

e (

of c

ontro

l)

EEP-B (120583gmL)001 01 1 10

lowastlowastlowast


(b)


Nitr

ite (

of c

ontro

l)

120

100

80

60

40

20

0


lowastlowastlowast

lowastlowastlowast

LPS + IFN-120574EEP-B (120583gmL)

+ + + + +

minus 5 10 25 50






120100

80604020

0

lowastlowastlowast


-1120572

( o

f con

trol)

(a)

120100

80604020

0


IL-1120573

( o

f con

trol)

(b)

120100

80604020

0

IL-3

( o

f con

trol)

(c)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-4

( o

f con

trol)

(d)

140120100

80604020

0

IL-5

( o

f con

trol)

(e)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-6

( o

f con

trol)

(f)

120100

80604020

0

IL-9

( o

f con

trol)

(g)

120100

80604020

0

lowast

IL-1

0 (

of c

ontro

l)

(h)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-1

2p40

( o

f con

trol)

(i)

120100

80604020

0

lowastlowastlowast

IL-1

3 (

of c

ontro

l)

(j)

120100

80604020

0

IL-1

7 (

of c

ontro

l)


minus minus

+ ++

25 50EEP-B (120583gmL)

(k)

120100

80604020

0


TNF-120572

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(l)

Figure 8 Continued


120100

80604020

0

IFN

-120574(

of c

ontro

l)

(m)

120100

80604020

0lowastlowastlowast

lowastlowastlowast

G-C

SF (

of c

ontro

l)

(n)

120100

80604020


-CSF

( o

f con

trol)

(o)

120100

80604020

0


MCP

-1 (

of c

ontro

l)

(p)

120100

80604020

0


MIP

-1120572

( o

f con

trol)

(q)

120100

80604020

0

lowastlowastM

IP-1120573

( o

f con

trol)

(r)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

RAN

TES

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(s)




5 Conclusion





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Time2 3 4 5 6 7 8 9 10 11

(au

)

0

112141618

222242628

13

2526

14

8eminus1

6eminus1

4eminus1

2eminus1


2

Time2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 105

0

100 2311

3011

54311630

1790

5151

48922631

677248722471

5431

2471

7052

3772

2851

32123292

3152

2992

4772

3212

61334332

8

6

3 3122

26

18

15

41

363310

17

42

20

11

()

















(10) 443 5431486 381117317903491610241Dimethyl-


300e

(11) 465 5431486 381117317903491610241Dimethyl-


361e




(19) 571 3311565 3311565 Ni 275d

(20) 589 2311025 187112413205684-Hydroxy


2563d




(28) 687 3151602 299053625316091981039 Ni 416d

(29) 703 3131433 211112615605831490618 Ni 061d


(31) 722 3151602 20112861460728 Ni 006d




Table 1 Continued




(34) 761 5452541 33119002691174 Ni mdash



(39) 913 3631619 1871124149061313104973-Prenyl-4-


966d

(40) 932 2971489 253157719810391529951 Ni 062d





Cyto

toxi

city

(MTT

) (

)

109876543210

minus

minus minus


5 10 25 50 5 10 25 50LPS + IFN-120574

EEP-B (120583gmL)







200018001600140012001000

800600400200

0

Chem

ilum

ines

cenc

e (co

unts

s)

PMA

0 200 400 600 800 1000 1200 1400 1600 1800Time (s)


(a)

100908070605040302010

0

Chem

ilum

ines

cenc

e (

of c

ontro

l)

EEP-B (120583gmL)001 01 1 10

lowastlowastlowast


(b)


Nitr

ite (

of c

ontro

l)

120

100

80

60

40

20

0


lowastlowastlowast

lowastlowastlowast

LPS + IFN-120574EEP-B (120583gmL)

+ + + + +

minus 5 10 25 50






120100

80604020

0

lowastlowastlowast


-1120572

( o

f con

trol)

(a)

120100

80604020

0


IL-1120573

( o

f con

trol)

(b)

120100

80604020

0

IL-3

( o

f con

trol)

(c)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-4

( o

f con

trol)

(d)

140120100

80604020

0

IL-5

( o

f con

trol)

(e)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-6

( o

f con

trol)

(f)

120100

80604020

0

IL-9

( o

f con

trol)

(g)

120100

80604020

0

lowast

IL-1

0 (

of c

ontro

l)

(h)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-1

2p40

( o

f con

trol)

(i)

120100

80604020

0

lowastlowastlowast

IL-1

3 (

of c

ontro

l)

(j)

120100

80604020

0

IL-1

7 (

of c

ontro

l)


minus minus

+ ++

25 50EEP-B (120583gmL)

(k)

120100

80604020

0


TNF-120572

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(l)

Figure 8 Continued


120100

80604020

0

IFN

-120574(

of c

ontro

l)

(m)

120100

80604020

0lowastlowastlowast

lowastlowastlowast

G-C

SF (

of c

ontro

l)

(n)

120100

80604020


-CSF

( o

f con

trol)

(o)

120100

80604020

0


MCP

-1 (

of c

ontro

l)

(p)

120100

80604020

0


MIP

-1120572

( o

f con

trol)

(q)

120100

80604020

0

lowastlowastM

IP-1120573

( o

f con

trol)

(r)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

RAN

TES

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(s)




5 Conclusion





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























(10) 443 5431486 381117317903491610241Dimethyl-


300e

(11) 465 5431486 381117317903491610241Dimethyl-


361e




(19) 571 3311565 3311565 Ni 275d

(20) 589 2311025 187112413205684-Hydroxy


2563d




(28) 687 3151602 299053625316091981039 Ni 416d

(29) 703 3131433 211112615605831490618 Ni 061d


(31) 722 3151602 20112861460728 Ni 006d




Table 1 Continued




(34) 761 5452541 33119002691174 Ni mdash



(39) 913 3631619 1871124149061313104973-Prenyl-4-


966d

(40) 932 2971489 253157719810391529951 Ni 062d





Cyto

toxi

city

(MTT

) (

)

109876543210

minus

minus minus


5 10 25 50 5 10 25 50LPS + IFN-120574

EEP-B (120583gmL)







200018001600140012001000

800600400200

0

Chem

ilum

ines

cenc

e (co

unts

s)

PMA

0 200 400 600 800 1000 1200 1400 1600 1800Time (s)


(a)

100908070605040302010

0

Chem

ilum

ines

cenc

e (

of c

ontro

l)

EEP-B (120583gmL)001 01 1 10

lowastlowastlowast


(b)


Nitr

ite (

of c

ontro

l)

120

100

80

60

40

20

0


lowastlowastlowast

lowastlowastlowast

LPS + IFN-120574EEP-B (120583gmL)

+ + + + +

minus 5 10 25 50






120100

80604020

0

lowastlowastlowast


-1120572

( o

f con

trol)

(a)

120100

80604020

0


IL-1120573

( o

f con

trol)

(b)

120100

80604020

0

IL-3

( o

f con

trol)

(c)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-4

( o

f con

trol)

(d)

140120100

80604020

0

IL-5

( o

f con

trol)

(e)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-6

( o

f con

trol)

(f)

120100

80604020

0

IL-9

( o

f con

trol)

(g)

120100

80604020

0

lowast

IL-1

0 (

of c

ontro

l)

(h)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-1

2p40

( o

f con

trol)

(i)

120100

80604020

0

lowastlowastlowast

IL-1

3 (

of c

ontro

l)

(j)

120100

80604020

0

IL-1

7 (

of c

ontro

l)


minus minus

+ ++

25 50EEP-B (120583gmL)

(k)

120100

80604020

0


TNF-120572

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(l)

Figure 8 Continued


120100

80604020

0

IFN

-120574(

of c

ontro

l)

(m)

120100

80604020

0lowastlowastlowast

lowastlowastlowast

G-C

SF (

of c

ontro

l)

(n)

120100

80604020


-CSF

( o

f con

trol)

(o)

120100

80604020

0


MCP

-1 (

of c

ontro

l)

(p)

120100

80604020

0


MIP

-1120572

( o

f con

trol)

(q)

120100

80604020

0

lowastlowastM

IP-1120573

( o

f con

trol)

(r)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

RAN

TES

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(s)




5 Conclusion





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1 Continued




(34) 761 5452541 33119002691174 Ni mdash



(39) 913 3631619 1871124149061313104973-Prenyl-4-


966d

(40) 932 2971489 253157719810391529951 Ni 062d





Cyto

toxi

city

(MTT

) (

)

109876543210

minus

minus minus


5 10 25 50 5 10 25 50LPS + IFN-120574

EEP-B (120583gmL)







200018001600140012001000

800600400200

0

Chem

ilum

ines

cenc

e (co

unts

s)

PMA

0 200 400 600 800 1000 1200 1400 1600 1800Time (s)


(a)

100908070605040302010

0

Chem

ilum

ines

cenc

e (

of c

ontro

l)

EEP-B (120583gmL)001 01 1 10

lowastlowastlowast


(b)


Nitr

ite (

of c

ontro

l)

120

100

80

60

40

20

0


lowastlowastlowast

lowastlowastlowast

LPS + IFN-120574EEP-B (120583gmL)

+ + + + +

minus 5 10 25 50






120100

80604020

0

lowastlowastlowast


-1120572

( o

f con

trol)

(a)

120100

80604020

0


IL-1120573

( o

f con

trol)

(b)

120100

80604020

0

IL-3

( o

f con

trol)

(c)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-4

( o

f con

trol)

(d)

140120100

80604020

0

IL-5

( o

f con

trol)

(e)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-6

( o

f con

trol)

(f)

120100

80604020

0

IL-9

( o

f con

trol)

(g)

120100

80604020

0

lowast

IL-1

0 (

of c

ontro

l)

(h)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-1

2p40

( o

f con

trol)

(i)

120100

80604020

0

lowastlowastlowast

IL-1

3 (

of c

ontro

l)

(j)

120100

80604020

0

IL-1

7 (

of c

ontro

l)


minus minus

+ ++

25 50EEP-B (120583gmL)

(k)

120100

80604020

0


TNF-120572

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(l)

Figure 8 Continued


120100

80604020

0

IFN

-120574(

of c

ontro

l)

(m)

120100

80604020

0lowastlowastlowast

lowastlowastlowast

G-C

SF (

of c

ontro

l)

(n)

120100

80604020


-CSF

( o

f con

trol)

(o)

120100

80604020

0


MCP

-1 (

of c

ontro

l)

(p)

120100

80604020

0


MIP

-1120572

( o

f con

trol)

(q)

120100

80604020

0

lowastlowastM

IP-1120573

( o

f con

trol)

(r)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

RAN

TES

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(s)




5 Conclusion





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















200018001600140012001000

800600400200

0

Chem

ilum

ines

cenc

e (co

unts

s)

PMA

0 200 400 600 800 1000 1200 1400 1600 1800Time (s)


(a)

100908070605040302010

0

Chem

ilum

ines

cenc

e (

of c

ontro

l)

EEP-B (120583gmL)001 01 1 10

lowastlowastlowast


(b)


Nitr

ite (

of c

ontro

l)

120

100

80

60

40

20

0


lowastlowastlowast

lowastlowastlowast

LPS + IFN-120574EEP-B (120583gmL)

+ + + + +

minus 5 10 25 50






120100

80604020

0

lowastlowastlowast


-1120572

( o

f con

trol)

(a)

120100

80604020

0


IL-1120573

( o

f con

trol)

(b)

120100

80604020

0

IL-3

( o

f con

trol)

(c)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-4

( o

f con

trol)

(d)

140120100

80604020

0

IL-5

( o

f con

trol)

(e)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-6

( o

f con

trol)

(f)

120100

80604020

0

IL-9

( o

f con

trol)

(g)

120100

80604020

0

lowast

IL-1

0 (

of c

ontro

l)

(h)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-1

2p40

( o

f con

trol)

(i)

120100

80604020

0

lowastlowastlowast

IL-1

3 (

of c

ontro

l)

(j)

120100

80604020

0

IL-1

7 (

of c

ontro

l)


minus minus

+ ++

25 50EEP-B (120583gmL)

(k)

120100

80604020

0


TNF-120572

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(l)

Figure 8 Continued


120100

80604020

0

IFN

-120574(

of c

ontro

l)

(m)

120100

80604020

0lowastlowastlowast

lowastlowastlowast

G-C

SF (

of c

ontro

l)

(n)

120100

80604020


-CSF

( o

f con

trol)

(o)

120100

80604020

0


MCP

-1 (

of c

ontro

l)

(p)

120100

80604020

0


MIP

-1120572

( o

f con

trol)

(q)

120100

80604020

0

lowastlowastM

IP-1120573

( o

f con

trol)

(r)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

RAN

TES

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(s)




5 Conclusion





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















120100

80604020

0

lowastlowastlowast


-1120572

( o

f con

trol)

(a)

120100

80604020

0


IL-1120573

( o

f con

trol)

(b)

120100

80604020

0

IL-3

( o

f con

trol)

(c)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-4

( o

f con

trol)

(d)

140120100

80604020

0

IL-5

( o

f con

trol)

(e)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-6

( o

f con

trol)

(f)

120100

80604020

0

IL-9

( o

f con

trol)

(g)

120100

80604020

0

lowast

IL-1

0 (

of c

ontro

l)

(h)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

IL-1

2p40

( o

f con

trol)

(i)

120100

80604020

0

lowastlowastlowast

IL-1

3 (

of c

ontro

l)

(j)

120100

80604020

0

IL-1

7 (

of c

ontro

l)


minus minus

+ ++

25 50EEP-B (120583gmL)

(k)

120100

80604020

0


TNF-120572

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(l)

Figure 8 Continued


120100

80604020

0

IFN

-120574(

of c

ontro

l)

(m)

120100

80604020

0lowastlowastlowast

lowastlowastlowast

G-C

SF (

of c

ontro

l)

(n)

120100

80604020


-CSF

( o

f con

trol)

(o)

120100

80604020

0


MCP

-1 (

of c

ontro

l)

(p)

120100

80604020

0


MIP

-1120572

( o

f con

trol)

(q)

120100

80604020

0

lowastlowastM

IP-1120573

( o

f con

trol)

(r)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

RAN

TES

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(s)




5 Conclusion





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















120100

80604020

0

IFN

-120574(

of c

ontro

l)

(m)

120100

80604020

0lowastlowastlowast

lowastlowastlowast

G-C

SF (

of c

ontro

l)

(n)

120100

80604020


-CSF

( o

f con

trol)

(o)

120100

80604020

0


MCP

-1 (

of c

ontro

l)

(p)

120100

80604020

0


MIP

-1120572

( o

f con

trol)

(q)

120100

80604020

0

lowastlowastM

IP-1120573

( o

f con

trol)

(r)

120100

80604020

0

lowastlowastlowast

lowastlowastlowast

RAN

TES

( o

f con

trol)


minus minus

+ ++

25 50EEP-B (120583gmL)

(s)




5 Conclusion





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Research Article Chemical Composition and Anti ...downloads.hindawi.com/journals/ecam/2013/976415.pdf · Research Article Chemical Composition and Anti-Inflammatory Effect of Ethanolic