research article achyrocline satureioides (lam.) d.c. … · 2019. 7. 31. · puhlmann and...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 787916 12 pageshttpdxdoiorg1011552013787916

Research ArticleAchyrocline satureioides (Lam) DC Hydroalcoholic ExtractInhibits Neutrophil Functions Related to Innate Host Defense

Eric Diego Barioni1 Joseacute Roberto Santin1 Isabel Daufenback Machado1

Stephen Fernandes de Paula Rodrigues1 Viviane Ferraz-de-Paula1

Theodoro Marcel Wagner2 Bruno Cogliati3 Matheus Correcirca dos Santos2

Marina da Silva Machado2 Seacutergio Faloni de Andrade2 Rivaldo Niero2

and Sandra Helena Poliselli Farsky1

1 Department of Clinical and Toxicological Analysis School of Pharmaceutical Sciences University of Sao Paulo Av Prof Lineu Prestes580 Bl 13B 05508-900 Sao Paulo SP Brazil

2 Nucleo de Investigacoes Quımico-Farmaceuticas (NIQFAR) University of Vale do Itajaı (UNIVALI) Rua Uruguai 45888302-202 Itajaı SC Brazil

3 Department of Pathology School of Veterinary Medicine and Animal Science University of Sao PauloAv Prof Dr Orlando Marques de Paiva 87 Cidade Universitaria 05508-270 Sao Paulo SP Brazil

Correspondence should be addressed to Sandra Helena Poliselli Farsky sfarskyuspbr

Received 28 November 2012 Accepted 31 December 2012

Academic Editor David Baxter

Copyright copy 2013 Eric Diego Barioni et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Achyrocline satureioides (Lam) DC is a herb native to South America and its inflorescences are popularly employed to treatinflammatory diseases Here the effects of the in vivo actions of the hydroalcoholic extract obtained from inflorescences ofA satureioides on neutrophil trafficking into inflamed tissue were investigated Male Wistar rats were orally treated with Asatureioides extract and inflammation was induced one hour later by lipopolysaccharide injection into the subcutaneous tissueThe number of leukocytes and the amount of chemotactic mediators were quantified in the inflammatory exudate and adhesionmolecule and toll-like receptor 4 (TLR-4) expressions and phorbol-myristate-acetate- (PMA-) stimulated oxidative burst werequantified in circulating neutrophils Leukocyte-endothelial interactions were quantified in the mesentery tissue Enzymes andtissue morphology of the liver and kidney were evaluated Treatment with A satureioides extract reduced neutrophil influx andsecretion of leukotrieneB4 andCINC-1 in the exudates the number of rolling and adhered leukocytes in themesentery postcapillaryvenules neutrophil L-selectin 1205732-integrin and TLR-4 expression and oxidative burst but did not cause an alteration in themorphology and activities of liver and kidney Together the data show that A satureioides extract inhibits neutrophil functionsrelated to the innate response and does not cause systemic toxicity

1 Introduction

Achyrocline satureioides (Lam) DC is a herb native to SouthAmerica In Brazil it predominantly occurs in southernregions where it is popularly known as Marcela or Macelaand is largely employed in folk medicine [1] The ethnophar-macological use of infusions prepared from inflorescences ofA satureoides leads to the relief of symptoms of inflammatorydisorders asthma anxiety gastric ulcers and other digestivediseases [2ndash7] Investigations on its chemical composition

showed that the extract obtained from inflorescences is richin flavonoids mainly quercetin and luteolin [1 8 9]

Experimental assays in vivo and in vitro have con-firmed the ethnopharmacological employment of the extractsobtained from inflorescences of A satureoides In this con-text the anti-inflammatory activity of aqueous and ethanolicextracts from A satureoides in carrageenan-induced rat pawedema was shown [10] which may be correlated with themodulation of the innate immune response Puhlmann andcoauthors [11] showed enhanced in vivo phagocytic activity

2 Evidence-Based Complementary and Alternative Medicine

of carbon particles by macrophages obtained from ratstreated with A satureoides extract In addition A satureoidesinfusion increased peripheral blood human mononuclearphytohemagglutinin- (PHA-) induced proliferation inter-feron gamma (IFN120574) and interleukin-4 (IL-4) secretion [611] Although the anti-inflammatory effects of A satureoideshave been demonstrated its direct actions on neutrophilfunctions have not been shown

Neutrophils express a wide range of membrane receptorssuch as adhesion molecules and chemoattractant receptorsprompting them to react to exogenous stimuli and endoge-nous chemical mediators In this context neutrophils expressthe toll-like receptor 4 (TLR-4) which is responsible forthe recognition of lipopolysaccharides (LPSs) from Gram-negative bacteria LPS binds to TLR-4 on the cell mem-brane and activates signal-transduction pathways mainly viaMyD88 which is a central adapter protein that leads to acti-vation of the nuclear transcription factor factor-120581B (NF-120581B)NF-120581B is the most important regulator of proinflammatorygene expression and induces a release of critical inflammatorymolecules that are necessary to induce leukocyte migrationinto injured tissue and suitable immune responses [12 13]

Leukocyte influx to the site of the inflammatory lesionis initially dependent on interactions between circulatingcells and the endothelial cells of postcapillary venules medi-ated by expressionactivity of adhesion molecules on thesurface of both cell types L-selectin mediates the rollingof the neutrophils along endothelial cells as it is rapidlyexpressed by activated neutrophils and interacts with consti-tutive carbohydrates or even with P- or E-selectin expressedon endothelial cells [14] L-selectin is cleaved by actionof metalloproteases pointing to the leukocytes becomearrested to the endothelium [15] Therefore integrin familymolecules especially1205732-integrin subfamilymolecules whichare mostly expressed by leukocytes mediate firm adhesionby interacting with a diversity of endothelial membranecomponents and immunoglobulin superfamily moleculesUltimately neutrophils transmigrate between interendothe-lial junctions via heterophilic and homophilic interactionsof immunoglobulin superfamily molecules such as plateletendothelial cell adhesion molecule-1 (PECAM-1) to sub-sequently migrate to the inflammatory focus [16] In theextravascular matrix neutrophils directly move to the siteof the lesion in response to chemotactic chemical mediators[17] At the lesion site they phagocyte the lesion-causingagent and release preformed chemical substances that con-tribute to the destruction of the damaging agent However inthe case of noncontrolled inflammation blockage of phasesof neutrophil mobilization into focus of the inflammatoryreaction is an important therapeutic strategy

This study investigated the in vivo actions of hydroalco-holic extract obtained from inflorescences of A satureoideson neutrophil trafficking from the blood into inflamedtissue Data confirmed quercetin and luteolin to be themain constituents in the hydroalcoholic extract and showedthe absence of systemic toxicity However results clearlyshowed that A satureoides hydroalcoholic extract inhibitsLPS-induced pathways of neutrophil migration via a mecha-nism that might be partially dependent on TLR-4 expression

Additional anti-inflammatorymechanismsmight exist as theextract also inhibited neutrophil activation caused by a directintracellular stimulation of protein kinases

2 Materials and Methods

21 Chemicals Lipopolysaccharide from Escherichia coli(LPS serotype 026B6) indomethacin EDTA and pro-pidium iodide (PI) were purchased from Sigma-Aldrich(St Louis MO USA) Ketamine and xylazine were pur-chased from Vetbrands (Paulinia SP Brazil) and hep-arin (Liquemine) was purchased from Roche Pharma-ceuticals (Brazil) Anti-L-selectin-phycoerythrin (PE) anti-1205732-integrin-fluorescein-isothiocyanate (FITC) Annexin-V-FITC and anti-CD284MD-2-PE (toll-like receptor 4) anti-bodies were obtained from BD Pharmingen (San Diego CAUSA) Leukotriene B4 (LTB4) enzyme-linked immunosor-bent assay (EIA) was obtained fromGEHealthcare (Salt LakeCity UT USA) The biochemical assays aspartate amino-transferase (AST) alanine aminotransferase (ALT) gamma-glutamyl transferase (gamma-GT) urea and creatinine werepurchased from Biotecnica (Varginha MG Brazil) Phorbolmyristate acetate (PMA) was obtained from Calbiochem(San Diego CA USA) and 2101584071015840 dichlorodihydrofluorescein-diacetate (DCFH-DA) was obtained from Molecular Probes(Eugene OR USA) CINC-1 was also determined using EIAkits obtained from RampD Systems (Minneapolis MN USA)Air filters were purchased from TPP Switzerland and allreagents used in preparing the phosphate buffered solution(PBS) and ringer solution were purchased fromMerck USA

22 Plant Material Inflorescences of A satureoides werecollected in Fraiburgo in the State of Santa Catarina BrazilThe material was identified and a voucher specimen wasdeposited at the herbarium of the State University ofMaringa(UEM) with the code HUEM-23568 The material collectionand all experiments were authorized by the Council ofManagement of Genetic Patrimony Brazil (CGEN processnumber 010062-2012-2) Air-dried plant material was cutinto small pieces and macerated with 70 (vv) aqueousethanol at room temperature for 7 days The macerate wasfiltered and the solvent removed by rotary evaporation underreduced pressure

23 Apparatus and Chromatographic Conditions Analysiswas conducted using a high performance liquid chromatog-raphy (HPLC) system (Waters) equipped with a 600-F pump717 plus autosampler followed by a line degasser (AF) andequipped with a UV-Vis detector (PDA 2996) A reverse-phase C18 column (25 cm 46mm id 05 120583m film thicknessand 100A)was employed (Luna Phenomenex) at 25∘C Chro-matographic separation was performed at room temperaturewith a flow rate 08mLmin of gradient elution using twosolvents A (aqueous methanol 50 (vv)) and B (wateracidified with acetic acid at pH 23) The gradient systemused was 50 A (15min) 50ndash60 A (15min) 60ndash70A (10min) 70ndash80 A (10min) 80ndash90 A (10min) and95 A (5min) UV-Vis spectra were recorded at wavelengths

Evidence-Based Complementary and Alternative Medicine 3

200ndash400 nm (detection at 350 nm) Solvents used were ofHPLC grade filtered (02120583m Schleicher amp Schuell Maid-stone Kent UK) and degassed by sonication before useThe samples of hydroalcoholic extract (054mgmL) andstandard quercetin and luteolin (05mgmL) were dissolvedin methanol and filtered through a 045120583mmembrane filterand 20120583L was analyzed in triplicate

24 Gas Chromatography-Mass Spectrometry Analysis (GC-MS) TheGC-MS analysis was carried out using a ShimadzuGas Chromatograph (Model QP-2010S series) equipped withanAOC-20i injectorTheGCwas equippedwith a fused silicacapillary column-TRX-1 (30m times 025mm) film thickness01 120583m The oven temperature was maintained at 220∘C for5min holding time and was then raised from 220 to 300∘Cat a rate of 20∘Cmin then held for 2min and raised furtherto 310∘C at a rate of 10∘Cmin and once more held at thistemperature for 15min employing helium gas (99999)as a carrier gas at a constant flow rate of 080mLminHydroalcoholic extract of A satureoides (1 120583L) at a splitratio of 1 20 was injected An MS transfer line tempera-ture of 250∘C was performed on a Shimadzu (Model QP-2010S series) coupled Gas Chromatograph equipped with anNIST08 Library software databaseMass spectrawere taken ata 70 eV scanning rate of 1 scans Identification of compoundswas conducted using the database of the NIST08 LibraryThe mass spectrum of the individual unknown compoundswas compared with that of known compounds stored in thesoftware database Library

25 Animals Male Wistar rats (180ndash220 g) were obtainedfrom the Central Animal House of the School of Pharma-ceutical Sciences and Chemistry Institute of the Universityof Sao Paulo The animals were housed in standard cagesat room temperature (25 plusmn 3∘C) with 12 h darklight cyclesand supplemented with food and water ad libitum All pro-cedures were performed according to the Brazilian Societyof Science of Laboratory Animal guidelines for the propercare and use of experimental animals and the experimentswere approved by the local ethics committee (protocolnumber CEUAFCF334) The animals were anesthetizedbefore each experimental procedure with ketaminexylazine(80 8mgkg ip) thus preventing stress 5-6 animals wereused in each assay

26 Treatments The doses used in this study were based ondata previously published by Santin et al [2] which in turnapplied doses based on the traditional folk use of this plantIn this study the extract was administered orally at 50 100and 250mgkg Assays were carried out 1 or 2 hours aftertreatments

27 In Vivo Leukocyte Migration Air Pouch Model Animalswere anesthetized and 10mL of sterile air was injectedsubcutaneously into the dorsal region After six days thepouch was refilled with 10mL air On the tenth day followingthe first air injection the animals were divided into sixgroups and received one of the following treatments byoral gavage (1) sham (2) vehicle (PBSethanol 10) (3)

indomethacin (30mgkg positive control) or (4)ndash(6) Asatureoides (50 100 or 250mgkg) After 1 h LPS from E coli(serotype 026B6 1mg2mL PBS) was injected directly intothe pouches At 1 h or 4 h after the LPS injection the animalswere reanesthetized and sacrificedThe pouches were washedwith 2mL ice-cold PBS and the total leukocyte numberwas determined using a Neubauer chamber Differentialcell counts were performed on smears stained with May-Grunwald-Giemsa

28 Cell Viability and TLR-4 Expression by Flow CytometryPeripheral blood samples frommaleWistar rats submitted tothe air pouch model (after 1 h LPS injection) were obtainedvia abdominal aorta punctures Heparin (5000UImL) wasused as an anticoagulant Subsequently the whole blood washemolyzed (hypotonic lysis with NaCl solution at 02 and16) and centrifuged (5min at 600 g) to obtain the leuko-cytes The total number of leukocytes was quantified usinga Neubauer chamber Peripheral leukocytes (1 times 106 cells)were incubated with Annexin V conjugated to FITC (1 100)for 20min and PI (100 120583gmL) was added immediately toevaluate cell viability To quantify TLR-4 expression leuko-cytes (1 times 106 cells) were incubated with anti-CD284MD-2(TLR-4) monoclonal antibodies conjugated to PE (1 100) for60min at room temperature Immediately after incubationsall the samples except for Annexin V were centrifuged(5min 600 g) and resuspended in PBS for analysis in the flowcytometer FACSCalibur (Immunocytometry System SanJose CA USA) Data were obtained from 10000 cells andonly the morphologically viable leukocytes were consideredfor analysis The neutrophil population was characterizedby different size and complexity parameters of different celltypes detected by flow cytometry The optical signals emittedwere converted into electronic signals and were analyzed byFlowJo software (Tree Star Inc Ashland TN USA) Resultsof TLR-4 expression are presented as fluorescence unitsand apoptosis and necrosis are shown as the percentage ofAnnexin V- or PI-positive cells

29 Adhesion Molecule Expression and Oxidative Burst byFlow Cytometry Male Wistar rats (not submitted to the airpouch model) were divided into three groups and receivedone of the following treatments by oral gavage (1) sham (2)vehicle (PBSethanol 10) or (3) A satureoides (100mgkg)Circulating leukocytes (1 times 106) from the whole bloodwere collected as described in Section 28 To measure theadhesion molecules expression cells were incubated withor without LPS (1120583gmL) for 60min at 37∘C Subsequentlythe leukocyte suspension was washed with 1mL ice-coldPBS The supernatant was discarded and leukocytes wereincubated with monoclonal anti-CD62L antibodies conju-gated to PE (1 100) and anti-CD18 conjugated to FITC(1 100) for 20 minutes at room temperature To assess theleukocyte oxidative metabolism leukocytes (1 times 106 cells)were incubated with 100 120583L of PMA (100 ng) 200120583L ofDCFH-DA (03mM) and 700 120583L of PBS for 30min at 37∘C

Immediately after incubations the samples were cen-trifuged (5min 600 g) and were resuspended in PBS forquantification by flow cytometry The neutrophil population


was characterized as described in Section 28 Results ofoxidative metabolism are expressed as units of fluorescenceand data of adhesion molecules are expressed as the percent-age of positive cells

210 Intravital Microscopy Rats were divided into threegroups and received one of the following treatments by oralgavage (1) sham (PBS) (2) vehicle (PBSethanol 10) or(3) A satureoides (100mgkg) The rats were anesthetized1 h after the treatment and 30min later the mesentery wassurgically exteriorized Animals were maintained on a boardthermostatically controlled at 37∘C which included a trans-parent platform on which the tissue to be transilluminatedwas placed The preparation was kept moist and warm byirrigating the tissuewith awarmed (37∘C)Ringer-Locke solu-tion (154mM NaCl 56mM KCl 2mM CaCl

2sdot2H2O 6mM

NaHCO3 5mM glucose and 1 (wv) gelatin and pH 72ndash

74) The rate of solution outflow onto the exposed tissue wascontrolled to keep the preparation in continuous contact witha film of the solution Transilluminated images were obtainedby optical microscopy (Axioplan II Carl-Zeiss equippedwith 50030 times Plan-Neofluar or 100025 times Achroplanlongitudinal distance objectivesnumeric aperture and 10 times125times or 160timesOptovar)The imageswere captured by a videocamera (ZVS 3C75DE Carl-Zeiss) and were transmittedsimultaneously to a TV monitor and to a computer Imagesobtained on the TV monitor were recorded on softwareDigitized images on the computermonitorwere subsequentlyanalyzed by image analyzing software (AxioVision)

The interaction between leukocytes and the vessel wallswas analyzed by determining the number of rolling andadherent leukocytes on the postcapillary venule wall (20ndash30 120583mdiameter 200120583mlength) of themesentery Leukocytesmoving in the periphery of the axial stream in contact withthe endothelium were considered to be rolling and theirnumber was determined in 10min periods The number ofleukocytes that adhered to the endothelium (stopped at thevessel wall) was determined in the same vascular segmentafter 10min The number of rolling and adherent cells wasquantified after topical application of LPS (30 120583g40 120583L inPBS) to the venules of the mesentery microcirculationThreefields were evaluated per animal after applicationThe resultswere then averaged for each animal

211 Inflammatory Mediators The air pouch lavage fluid wascollected 1 h after LPS injection to evaluate the concentra-tion of inflammatory mediators LTB4 and CINC-1 werequantified using EIA Kits according to the manufacturerrsquosinstructions The results were expressed as pgmL or ngmLrespectively

212 Biochemical Parameters Thewhole blood was collected2 hour after treatments without anticoagulant to serum sepa-ration after centrifugation (10min 600 g)The concentrationof kidney and liver markers was analyzed using commercialbiochemical kits for urea creatinine aspartate aminotrans-ferase (AST) alanine aminotransferase (ALT) and gamma-GT

213Histopathology Liver and kidney sampleswere collected2 hour after treatments washed with phosphate-bufferedsaline (PBS) and fixed in 10 buffered formalin for 24hours Tissue samples were dehydrated in graded ethanolsolutions cleared in xylene and embedded in paraffin waxAfter that serial sections (5120583m) were prepared and stainedwith hematoxylin and eosin (HampE) Images were taken atoriginal magnification of 100x (Eclipse E800 MicroscopeNikon Japan)

214 Statistical Analysis Means and the standard error of themean (SEM) for all data are presented and were comparedusing Studentrsquos 119905-tests or ANOVA Tukeyrsquos Multiple Com-parisons test was performed to determine the significance ofthe differences between experimental conditions GraphPadPrism 40 software (San Diego CA USA) was employedValues of 119875 lt 005 were considered significant

3 Results

31 Chromatographic Analysis The phytochemical profileof the A satureoides hydroalcoholic extract showed sixcompounds and two main compounds were identified(Figure 1(a)) The major components of the extract werecompounds 1 and 2 which were identified by direct compar-ison with authentic samples and area peaks as luteolin andquercetin respectively Although these compounds occurredin other tissues of A satureoides the relative content of thesetwo flavonoids from inflorescences was 1231 and 1365 120583gmLfor 1 and 2 respectively The GC-MS analysis showed sevendistinct peaks identified via the NIST08 library softwaredatabase as ethyl ester derivatives of oleic palmitic andstearic acids In addition the steroids stigmasterol gamma-sitosterol and sitostenone were also identified in the Asatureoides extract (Figure 1(b))

32 A satureoides Hydroalcoholic Extract Inhibits In VivoNeutrophil Migration into LPS-Inflamed Tissue The in vivoanti-inflammatory effect of the hydroalcoholic A satureoidesextract on LPS-induced inflammation in the subcutaneoustissue of rats (air pouch model) was investigated Differentdoses were tested and the number of cells migrating intothe pouches was determined 4 h after injection of LPS orPBS Results presented in Figure 2(a) show thatA satureoidestreatment reduced leukocyte migration to the inflamma-tory site which reflected an impaired influx of neutrophils(Figure 2(b)) The effect caused by 100mgKg of A sat-ureoides extract was similar to that evoked by indomethacintreatment (Figures 2(a) and 2(b))

Assays were conducted to evaluate the inhibitory mech-anism of A satureoides extract on cellular mechanismsinvolved in neutrophil migration to the site of the lesionFor this purpose neutrophils were isolated from rats andincubated in vitro with the extract thus the direct action ofthe extract in each phase of neutrophil migration could beinvestigated However in vitro incubation with the extractcaused cell death especially by necrosis (data not shown)For this reason all the following assays were conducted using


AU

0

012

024

036

048

Minutes12 15 18 21 24 27 30 33 36 39

L

Q

1

23

4 5 6

(a)

4

3

2

1

3 4 5 6 7 8 9 10 11

(times1000000)TIC

2pa

lmiti

c aci

d et

hyl e

ster

4ol

eic a

cid

ethy

l este

r

3et

hyl h

epta

deca

noat

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5ste

aric

acid

eth

yl es

ter

6et

hyl i

cosa

noat

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7et

hyl n

onad

ecan

oate

8sti

gmas

tero

l

9ga

mm

a-sit

oste

rol

10s

itoste

none

1n-

hexa

deca

noic

acid

(b)

Figure 1 (a) HPLC overlap chromatogram profile of hydroalcoholic extract of A satureoides L luteolin Q quercetin (b) GC-MSchromatogram showing peaks of the main phytochemicals present in extract of A satureoides

Vehicle 30 50 100 2500

50

100

150

LPS

mgkg poIndomethacin

mgkg poA satureoides

lowast

lowast

lowast

Tota

lleu

kocy

tes(106

cells

mL)

(a)

Vehicle 30 50 100 250 0

50

100

150

LPS

mgkg poIndomethacin


Neu

troph

ils(106

cells

mL)

lowastlowast

lowast

(b)

Figure 2 Effects of A satureoides hydroalcoholic extract on in vivo leukocyte migration induced by LPS Air pouch animals received orally(1) vehicle (PBSethanol 10) (2) indomethacin (30mgkg) or (3)A satureoides (50 100 or 250mgkg) LPSs from E coli 026B6 (1mg2mL)or PBSwere injected after 1 h directly into the air pouch and 4 h later the number of cells in the pouchwas quantifiedThe dotted line indicatesthe number of neutrophils in the noninflamed tissue (a) Number of total leukocytes in the air pouch (b) Number of neutrophils in the airpouch Data are expressed as mean plusmn SEM of 5-6 animals in each group Statistical analysis was performed using ANOVA followed byTukeyrsquos test lowast119875 lt 005 versus vehicle


Sham Vehicle0

100

200

300

100 mgkg

Rolli

ng le

ukoc

ytes

(cel

ls10

min

)

A satureoides

LPS topic 30120583g40120583L

lowast

lowast

(a)

Sham Vehicle0

2

4

6

8

100 mgkgA satureoides

LPS topic 30120583g40120583L

lowastlowastlowast

lowast

Adhe

rent

leuk

ocyt

e(ce

lls2

00120583

m)

(b)Figure 3 Effect of A satureoides hydroalcoholic extract on leukocyte-endothelial interaction in vivo Animals received orally (1) sham(surgical manipulation without treatment) (2) vehicle (PBSethanol 10) or (3) A satureoides (100mgkg) After 1 h LPS from E coli 026B6(30120583g40 120583L) was applied topically into the mesenteric network and the number of rolling and adherent leukocytes was quantified (a)Number of rolling leukocytes (b)Number of adherent leukocytes Data are expressed asmeanplusmn SEM of 5-6 animals in each group Statisticalanalysis was performed using ANOVA followed by Tukeyrsquos test lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 versus sham 119875 lt 005 and 119875 lt 001 versusvehicle

blood and circulating leukocytes collected after in vivo vehicleor A satureoides extract treatments

33 A satureoides Hydroalcoholic Extract In Vivo TreatmentDoes Not Affect Neutrophil Viability Based on cell viabilitydata obtained from in vitro studies it was also relevantto investigate leukocyte viability from circulating bloodfollowing in vivo treatments with A satureoides extractDatapresented in Table 1 show that extract treatment did not causecell death as determined by the Annexin VPI labeled flowcytometry assay

34 A satureoides Hydroalcoholic Extract Impairs In VivoLeukocyte-Endothelial Interactions The behavior of leuko-cytes in the peripheral blood was evaluated by direct obser-vation of the microcirculation network Administration ofA satureoides extract slightly reduced the number of LPS-induced rolling leukocytes in comparison to the num-ber observed in vehicle-treated rats (Figure 3(a)) Howeveradministration of the extract markedly reduced the LPS-induced adherence of leukocytes to the vessel wall of themesentery network (Figure 3(b)) It was further shown thatAsatureoides extract did not induce toxicological effects on themicrocirculation such as hemorrhage thrombus formationor vascular stasis (data not shown)

35 A satureoides Hydroalcoholic Extract Alters NeutrophilAdhesion Molecule Expression Leukocyte-endothelial inter-action is mediated by adhesion molecule expression As Asatureoides extract reduced the in vivo leukocyte-endothelialinteractions assays were performed to investigate the actionsof the extract on the expression of L-selectin and 1205732-integrinon the neutrophil surface Results show that a dose of

100mgkg A satureoides extract reduced the number of 1205732-integrin- (Figure 4(a)) and L-selectin-positive (Figure 4(b))neutrophils after LPS stimulation showing that treatmentwith the extract affected the ability of neutrophils to expressboth molecules

36 A satureoides Hydroalcoholic Extract Reduces CINC-1 and LTB-4 Secretion in LPS-Induced Inflamed ExudatesChemical mediators including neutrophils are secreted ininflammatory condition by different cells To investigate theability of A satureoides extract to inhibit the secretion ofthe chemotactic mediators CINC-1 and LTB-4 exudate wascollected from animals treated with A satureoides hydroal-coholic extract or vehicles 1 h following LPS injection intothe air pouch This experimental strategy was employed toavoid large differences in the number of migrated leukocytesinto air pouches as observed 4 hours after LPS injection(Figure 2(b) vehicle 92 times 106 A satureoides 25 times 106)which could be responsible for altered secretion of thesechemokines As shown in Figure 5(a) treatment with Asatureoides extract reduced neutrophil migration into theair pouch 1 hour after LPS injection (vehicle 115 times 106 Asatureoides 05 times 106) and decreased both LTB-4 and CINC-1levels in the inflammatory exudate (Figures 5(b) and 5(c))

37 A satureoides Hydroalcoholic Extract Reduces TLR-4 Expression and PMA-Induced Oxidative Burst on Neu-trophils To elucidate the molecular mechanism of the anti-inflammatory effect shown by A satureoides extract mem-brane TLR-4 expression was evaluated by flow cytometryThe results showed that neutrophils obtained from animals


Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

A satureoides

lowast

Posit

ivec

ells120573

2-in

tegr

in(

)

(a)

Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

Posit

ive c

ells

L-se

lect

in (

)

A satureoides

lowast

(b)

Figure 4 1205732-integrin and L-selectin expression on neutrophils from animals treated with A satureoides hydroalcoholic extract Animalsreceived orally (1) control (untreated animals) (2) vehicle (PBSethanol 10) or (3) A satureoides (100mgkg) Neutrophils were incubatedwithPBSor LPS fromE coli 026B6 (5 120583gmL) for 60min at 37∘C (a)Thepercentage of1205732-integrin-positive neutrophils and (b) the percentageof L-selectin-positive neutrophils Data are expressed as mean plusmn SEM of 5-6 animals in each group Statistical analysis was performed usingANOVA followed by Tukeyrsquos test lowast119875 lt 005 versus respective LPS-stimulated control

Table 1 Effects of A satureoides hydroalcoholic extract on in vivo cell viability

Treatment Dose ALT AST Gama-GT Urea Creatinine(mgkg) (mgdL) (mgdL) (mgdL) (mgdL) (mgdL)

Sham mdash 3765 plusmn 196 12460 plusmn 946 904 plusmn 220 3702 plusmn 283 053 plusmn 008Vehicle mdash 4160 plusmn 257 11360 plusmn 1745 325 plusmn 234 3359 plusmn 083 053 plusmn 041A satureoides 100 3507 plusmn 192 10660 plusmn 1063 544 plusmn 218 3720 plusmn 257 035 plusmn 006Data are expressed asmeanplusmn SEM of 6 animals in each group Sham (air pouch induced without treatment) Statistical analysis was performed using ANOVAfollowed by Tukeyrsquos test

orally treated with A satureoides extract showed lower TLR-4 expression than animals treated with vehicle (Figure 6(a))The reduction of TLR-4 expression might be totally orpartially responsible for the anti-inflammatory activitiesobserved in this study and might also suggest that thismechanism is a unique pathway of A satureoides extractaction To investigate this hypothesis neutrophils collectedfrom treated rats were stimulated with PMA in vitro PMA islipophilic and directly stimulates the phosphorylation of PKCkinases which are responsible for activation of the respiratoryburst [18 19] Neutrophils from animals treated with Asatureoides extract and incubated with PMA for 30mindemonstrated a significant reduction in the production ofPMA-stimulated reactive oxygen species (ROS) as measuredby DCFH formation (Figure 6(b))

38 A satureoides Hydroalcoholic Extract Treatment Does NotCause Systemic Toxicity Liver and kidney biochemical andhistological parameters were investigated in rats treated withvehicle or A satureoides Data showed that activities of themain hepatic enzymes AST ALT and gamma-GT as wellas levels of creatinine and urea were equivalent in samplescollected from rats treated withA satureoides hydroalcoholicextract or vehicle (Table 2) Furthermore no alteration in

liver and kidney structures was observed in animals fromboth treatments (Figure 7)

4 Discussion

Neutrophils exert an important role in the induction of innateinflammatory reactions and in the transition between innateand immune responses Therefore in the case of exacerbatedreactions blockage of their functions represents a therapeuticstrategy Here we show that in vivo treatment with Asatureoides inflorescence hydroalcoholic extract significantlyimpaired neutrophil migration into LPS-induced inflamedexudates and effects on neutrophil migratory properties andsecretion of chemotactic mediators might be involved in itsanti-inflammatory action Although expression of TLR-4 themain receptor for LPS bindingwas reduced on the neutrophilmembrane and might be responsible for the activity of Asatureoides extract in these cells other forms of action mightbe involved as a lower oxidative burst was observed followingdirect intracellular activation of PKC kinases in neutrophils

Much evidence has shown that plant extracts containingflavonoids exert anti-inflammatory effects [20ndash24] Previous


Table 2 Effects of A satureoides hydroalcoholic extract on biochemical parameters

Treatment Dose Apoptotic Later apoptotic Necrotic Viable(mgkg) cells () cells () cells () cells ()

Sham mdash 223 plusmn 039 690 plusmn 069 1313 plusmn 179 7012 plusmn 555Vehicle mdash 131 plusmn 014 377 plusmn 019 1245 plusmn 115 8271 plusmn 138A satureoides 100 082 plusmn 005 732 plusmn 056 1913 plusmn 125 7401 plusmn 238Data are expressed asmeanplusmn SEM of 6 animals in each group Sham (air pouch induced without treatment) Statistical analysis was performed using ANOVAfollowed by Tukeyrsquos test

Sham Vehicle0

05

1

15

Indomethacin

LPS

100 mgkg30 mgkg

A satureoides

lowastlowastlowast

Neu

troph

ils(106

cells

mL)

(a)

Sham Vehicle0

2

4

6

8

LPS

LTB-

4 (p

gm

L)

100 mgkg30 mgkg

Indomethacin A satureoides

lowast

(b)

Sham Vehicle 30 mgkg 100 mgkg0

20

40

60

80

100

CIN

C-1

(ng

mL)

LPS


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 5 Effects of A satureoides hydroalcoholic extract on in vivo leukocyte migration induced by LPS LTB-4 and CINC-1 secretion Airpouch animals received orally (1) sham (air pouch induced without treatment) (2) vehicle (PBSethanol 10) (3) indomethacin (30mgkg)or (4)A satureoides (100mgkg) After 1 h LPSs from E coli 026B6 (1mg2mL) or PBS were injected directly into the air pouch and 1 h laterthe number of cells in the pouch was quantified (a) Number of neutrophils in the air pouch (b) levels of LTB-4 on air pouch exudate and (c)levels of CINC-1 secretion on air pouch exudate Data are expressed as mean plusmn SEM of 5-6 animals in each group Statistical analysis wasperformed using ANOVA followed by Tukeyrsquos test lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 versus sham 119875 lt 005 119875 lt 001 and 119875 lt 001 versusvehicle

studies have shown that A satureoides extracts contain lute-olin and quercetin [1 2 8 9] and here HPLC analysis cor-roborated that the two flavonoids are the main compoundswithin the inflorescence extract however GC-MS analysisshowed that the A satureoides extract also contains steroidsand fatty acids Based on the anti-inflammatory effects of

flavonoids it is expected that A satureoides inflorescenceextract can reduce neutrophil influx Here we show for thefirst time that A satureoides extract reduces in vivo LPSinduced neutrophil migration which might relate to reducedTLR-4 membrane expression and therefore corroborate theaction of quercetin and luteolin onTLR-4 expression [25ndash28]


Sham Vehicle0

10

20

30

40

50

LPS

100 mgkg

Toll-

like r

ecep

tor 4

(TLR

-4)

fluor

esce

nce i

nten

sity

(au

)

A satureoides

lowast

(a)

Sham Vehicle0

100

200

300

400

PMA

100 mgkg

DCF

H-D

Aflu

ores

cenc

e int

ensit

y (a

u)

A satureoides

lowastlowastlowast

(b)

Figure 6 Effects of A satureoides hydroalcoholic extract on TLR-4 expression and DCFH PMA-induced formation Animals received orally(1) sham (air pouch induced without treatment) (2) vehicle (PBSethanol 10) or (3) A satureoides (100mgkg) (a) After 1 h LPSs from Ecoli 026B6 (1mg2mL) or PBS were injected directly into the air pouch and 1 h later TLR-4 expression was measured in blood leukocytes byflow cytometry (b) Circulating leukocytes were collected and in vitro stimulated by PMA DCFH formation wasmeasured by flow cytometryData are expressed as mean plusmn SEM of 5-6 animals in each group Statistical analysis was performed using ANOVA followed by Tukeyrsquos testlowast119875 lt 005 and lowastlowastlowast119875 lt 0001 versus vehicle 119875 lt 005 and 119875 lt 0001 versus sham

(a) (b) (c)

(d) (e) (f)

Figure 7 Effects of A satureoides hydroalcoholic extract on liver and kidney histology in treated rats Control (sham air pouch inducedwithout treatment) vehicle (PBSethanol 10) and A satureoides extract (100mgkg) There were no histological alterations on liver andkidney tissues Hematoxylin-eosin Scale bar = 20120583m

LPS treatment induced TLR-4 expression as visualized byenhanced membrane density in cells collected from vehicle-treated animals in comparison to those obtained from shamanimals TLR-4 expressions on cells collected from extract-treated rats were reduced However it is notable that reducedTLR-4 is not the only pathway of action of A satureoidesextract on neutrophils as neutrophils collected from ratstreatedwithA satureoides extract showed a reduced oxidative

burst elicited by in vitro PMA stimulation which directlyactivates PKC phosphorylation and phagocyte NADPH oxi-dase leading to the release of reactive metabolites [29]

Data in our study do not show the direct effect ofA satureoides extract on neutrophil function as in vitroneutrophil incubation with hydroalcoholic extract causednecrosis even at low concentrations (data not shown) Celldeath was not detected in vehicle-incubated cells excluding


the action of alcohol in the toxic effect (data not shown) It isrelevant here that cytotoxicity caused by in vitro incubationwith flavonoids is not cited in the literature Many in vitroexperimental studies have shown the direct action of extractscontaining flavonoids on neutrophil function but this hasnot been associated with cell death [30ndash33] It is possiblethat the experimental conditions and technical approach toquantify cell death are responsible for divergent results In thepresent study flow cytometry quantification of Annexin-V-and PI-labeled neutrophils was used to detect cell membranealterations as modifications of the cell surface structure alsoalter the adhesion and locomotory response of neutrophilsNotably in vivo extract treatment did not alter neutrophilviability and subsequent assays were performed using thisexperimental strategy Kinetic studies will be carried out toinvestigate the mechanisms in the absence of extract toxicityin vivo In addition the lack of functional or morphologicalalterations in kidney and liver followingA satureoides extracttreatment strongly supports the absence of in vivo toxicity

As previously mentioned neutrophil migration is depen-dent on initial contact of neutrophils with the endothe-lial cells from postcapillary venules [14] and intravitalmicroscopy assays allow the visualization of leukocyte behav-ior in the microcirculatory network [34ndash36] Data hereclearly show that in vivo administration of the A satureoidesextract reduced the number of rolling and adhered leuko-cytes in LPS-stimulated mesentery These data were furthercorroborated by the altered number of L-selectin- and 1205732-integrin-positive neutrophils suggesting that in vivo extracttreatment modifies the adhesive properties of neutrophils tothe endothelium which then impairs their migration intoinflamed tissue It has been shown that genetic deficiency ofL-selectin or 1205732-integrin on leukocyte membranes reducescell influx into inflamed areas leading to accumulation ofneutrophils in the blood [37ndash39]

Leukocyte adhesion to endogenous substrates such asendothelium and extravascular matrix constituents anddirect migration to the damage site are dependent onthe interaction between chemotactic mediators and specificreceptors mainly those expressed on cell membranes whichactivate pathways involved in adhesion and migration [40ndash43] In this context leukotriene B4 and CINC-1 are chemoat-tractants secreted by different cells in the inflammatory pro-cess including migrated neutrophils resident macrophagesmast cells and fibroblasts [44ndash48] Data in this study showthat in vivo treatment with A satureoides extract reducedthe amount of both mediators in the inflammation exudatesshowing the ability of the extract to inhibit the secretionof inflammatory cells The cells that are responsible for thisreduced secretion have been not established nevertheless Asatureoides treatment might inhibit the secretory activity ofresident cells in the subcutaneous tissue as the reduced levelsof chemoattractants were similar in exudates collected 1 or 4 hafter LPS injections irrespective of the number of neutrophilsin the pouches

Taken together data presented here show the mecha-nisms of A satureoides inflorescence extract on neutrophil

influx using in vivo approaches which might be responsiblefor the ethnopharmacological application of the extract Thismight also be the mechanism involved in antiulcerogenicactivity of the extract [2] as neutrophil influx into damagedstomachs is a hallmark of acute gastric disease

5 Conclusions

Taken together data presented here from different in vivostudies show the mechanisms of the anti-inflammatory effectof A satureoides hydroalcoholic extract Based on thesefindings we have highlighted the inhibitory actions of Asatureoides hydroalcoholic extract on adhesive andmigrationproperties TLR-4 expression and oxidative metabolism ofneutrophils which might contribute to its anti-inflammatoryeffects and help to explain the use of A satureoides extract asa therapeutic agent

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors thank FAPESP for financial support (Grant no201115115-2) S H P Farsky is a fellow of the ConselhoNacional de Pesquisa e Tecnologia (CNPq) E D BarioniJ R Santin and I D Machado are postgraduate fellowsof FAPESP V Ferraz-de-Paula and S Fernandes de PaulaRodrigues are postdoctoral fellows of FAPESP The authorsthank the Neuroimmunomodulation Research Group forflow cytometry measurements (FAPESP no 200951886-3)

References

[1] G Ferraro C Anesini A Ouvina et al ldquoTotal phenolic contentand antioxidant activity of extracts of Achyrocline satureioidesflowers from different zones in Argentinardquo Latin AmericanJournal of Pharmacy vol 27 no 4 pp 626ndash628 2008

[2] J R Santin M Lemos L C K Junior R Niero and S F deAndrade ldquoAntiulcer effects of Achyrocline satureoides (Lam)DC (Asteraceae) (Marcela) a folk medicine plant in differentexperimental modelsrdquo Journal of Ethnopharmacology vol 130no 2 pp 334ndash339 2010

[3] A Gugliucci and T Menini ldquoThree different pathways forhuman LDL oxidation are inhibited in vitro by water extractsof the medicinal herbAchyrocline satureoidesrdquo Life Sciences vol71 no 6 pp 693ndash705 2002

[4] C Kadarian A M Broussalis J Mino et al ldquoHepatoprotectiveactivity of Achyrocline satureioides (Lam) D Crdquo Pharmacologi-cal Research vol 45 no 1 pp 57ndash61 2002

[5] M F Arredondo F Blasina C Echeverry et al ldquoCytoprotectionby Achyrocline satureioides (Lam) DC and some of its mainflavonoids against oxidative stressrdquo Journal of Ethnopharmacol-ogy vol 91 no 1 pp 13ndash20 2004

[6] M Cosentino R Bombelli E Carcano et al ldquoImmunomodula-tory properties ofAchyrocline satureioides (Lam) DC infusiona study on human leukocytesrdquo Journal of Ethnopharmacologyvol 116 no 3 pp 501ndash507 2008


[7] L A Del Vitto E M Petenatti M E Petenatti S M Mazzaand E J Marchevsky ldquoMajor and trace elements contents incrude drug and infusions of two South American species ofAchyrocline (Asteraceae) named rdquomarcelasrdquordquo Latin AmericanJournal of Pharmacy vol 28 no 4 pp 552ndash559 2009

[8] K B C De Souza V L Bassani and E E S SchapovalldquoInfluence of excipients and technological process on anti-inflammatory activity of quercetin and Achyrocline satureoides(Lam) DC extracts by oral routerdquo Phytomedicine vol 14 no2-3 pp 102ndash108 2007

[9] J M Fachinetto M D Bagatini J Durigon A C F Silva and SB Tedesco ldquoEfeito anti-proliferativo das infusoes deAchyroclinesatureoides DC (Asteraceae) sobre o ciclo celular de Alliumcepardquo Revista Brasileira De Farmacognosia vol 17 no 1 pp 49ndash54 2007

[10] C M Simoes E P Schenkel L Bauer and A LangelohldquoPharmacological investigations on Achyrocline satureioides(Lam) DC compositaerdquo Journal of Ethnopharmacology vol22 no 3 pp 281ndash293 1988

[11] J Puhlmann U Knaus L Tubaro W Schaefer and H WagnerldquoImmunologically active metallic ion-containing polysaccha-rides of Achyrocline satureioidesrdquo Phytochemistry vol 31 no 8pp 2617ndash2621 1992

[12] J C Kagan and R Medzhitov ldquoPhosphoinositide-mediatedadaptor recruitment controls toll-like receptor signalingrdquo Cellvol 125 no 5 pp 943ndash955 2006

[13] J H Peng T Cui Z L Sun et al ldquoEffects of puerariae radixextract on endotoxin receptors and TNF-120572 expression inducedby gut-derived endotoxin in chronic alcoholic liver injuryrdquoEvidence Based inComplementary andAlternativeMedicine vol2012 Article ID 234987 12 pages 2012

[14] C E Green D N Pearson R T Camphausen D E Stauntonand S I Simon ldquoShear-dependent capping of L-selectin and P-selectin glycoprotein ligand 1 by E-selectin signals activation ofhigh-avidity 1205732-integrin on neutrophilsrdquo Journal of Immunol-ogy vol 172 no 12 pp 7780ndash7790 2004

[15] D M Smalley and K Ley ldquoL-selectin mechanisms and physio-logical significance of ectodomain cleavagerdquo Journal of Cellularand Molecular Medicine vol 9 no 2 pp 255ndash266 2005

[16] K Ley C Laudanna M I Cybulsky and S NoursharghldquoGetting to the site of inflammation the leukocyte adhesioncascade updatedrdquo Nature Reviews Immunology vol 7 no 9 pp678ndash689 2007

[17] C N Serhan S D Brain C D Buckley et al ldquoResolution ofinflammation state of the art definitions and termsrdquoTheFASEBJournal vol 21 no 2 pp 325ndash332 2007

[18] M AMyers L C McPhail and R Snyderman ldquoRedistributionof protein kinase C activity in human monocytes correlationwith activation of the respiratory burstrdquo Journal of Immunologyvol 135 no 5 pp 3411ndash3416 1985

[19] Y Yasui K Yamada S Takahashi et al ldquoPMA induces GCMaphosphorylation and alters its stability via the PKC- and ERK-dependent pathwayrdquo Biochemical and Biophysical ResearchCommunications vol 417 no 4 pp 1127ndash1132 2012

[20] A R Tapas D M Sakarkar and R B Kadke ldquoFlavonoidsas nutraceuticals a reviewrdquo Tropical Journal of PharmaceuticalResearch vol 7 pp 1089ndash1099 2008

[21] A Garcıa-Lafuente E Guillamon A Villares M A Rostagnoand J A Martınez ldquoFlavonoids as anti-inflammatory agentsimplications in cancer and cardiovascular diseaserdquo Inflamma-tion Research vol 58 no 9 pp 537ndash553 2009

[22] C R Liao Y S Chang W H Peng S C Lai and Y L HoldquoAnalgesic and anti-inflammatory activities of the methanolextract of Elaeagnus oldhamii Maxim in micerdquo AmericanJournal of Chinese Medice vol 40 no 3 pp 581ndash597 2012

[23] T H Quang N T Ngan C V Minh et al ldquoAnti-inflammatoryand PPAR transactivational properties of flavonoids from theroots of Sophora flavescensrdquo Phytotherapy Research 2012

[24] B T ChenWX Li R RHe et al ldquoAnti-inflammatory effects ofa polyphenols-rich extract from tea (Camellia sinensis) flowersin acute and chronic mice modelsrdquo Oxidative Medicine andCellular Longevity vol 2012 Article ID 537923 7 pages 2012

[25] M Kaneko H Takimoto T Sugiyama Y Seki K Kawaguchiand Y Kumazawa ldquoSuppressive effects of the flavonoidsquercetin and luteolin on the accumulation of lipid rafts aftersignal transduction via receptorsrdquo Immunopharmacology andImmunotoxicology vol 30 no 4 pp 867ndash882 2008

[26] J K Lee S Y Kim Y S Kim W H Lee D H Hwang and J YLee ldquoSuppression of the TRIF-dependent signaling pathway oftoll-like receptors by luteolinrdquo Biochemical Pharmacology vol77 no 8 pp 1391ndash1400 2009

[27] S Bhaskar V Shalini and A Helen ldquoQuercetin regulatesoxidized LDL induced inflammatory changes in human PBMCsby modulating the TLR-NF-120581B signaling pathwayrdquo Immunobi-ology vol 216 no 3 pp 367ndash373 2011

[28] HQiao X Zhang C Zhu et al ldquoLuteolin downregulates TLR4TLR5 NF-120581B and p-p38MAPK expression upregulates the p-ERK expression and protects rat brains against focal ischemiardquoBrain Research vol 448 pp 71ndash81 2012

[29] H Lundqvist P Follin L Khalfan and C Dahlgren ldquoPhorbolmyristate acetate-induced NADPH oxidase activity in humanneutrophils only half the story has been toldrdquo Journal ofLeukocyte Biology vol 59 no 2 pp 270ndash279 1996

[30] E S Suyenaga E L Konrath R R Dresch et al ldquoAppraisal ofthe antichemotactic activity of flavonoids on polymorphonu-clear neutrophilsrdquo Planta Medica vol 77 no 7 pp 698ndash7042011

[31] M Ciz P Denev M Kratchanova O Vasicek G Ambrozovaand A Lojek ldquoFlavonoids inhibit the respiratory burst ofneutrophils in mammalsrdquo Oxidative Medicine and CellularLongevity vol 2012 Article ID 181295 6 pages 2012

[32] A K Kiss A KapThlon-Cieslicka K J Filipiak G Opolskiand M Naruszewicz ldquoEx vivo effects of anOenothera paradoxaextract on the reactive oxygen species generation and neutralendopeptidase activity in neutrophils from patients after acutemyocardial infarctionrdquo Phytotherapy Research vol 26 no 4 pp482ndash487 2012

[33] H R Liao J J Chen Y H Chien S Z Lin S Linand C P Tseng ldquo5-Hydroxy-7-methoxyflavone inhibits N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced super-oxide anion production by specific modulate membrane local-ization of Tec with a PI3K independent mechanism in humanneutrophilsrdquo Biochemical Pharmacology vol 84 no 2 pp 182ndash191 2012

[34] S H P Farsky P Borelli R A Fock S Z Proto J M CFerreira Jr and S B V Melo ldquoChronic blockade of nitric oxidebiosynthesis in rats effect on leukocyte endothelial interactionand on leukocyte recruitmentrdquo Inflammation Research vol 53no 9 pp 442ndash452 2004

[35] C B de Lima E K Tamura T Montero-Melendez et alldquoActions of translocator protein ligands on neutrophil adhesionand motility induced by G-protein coupled receptor signalingrdquo


Biochemical and Biophysical Research Communications vol 417no 2 pp 918ndash923 2012

[36] F N Gavins ldquoIntravital microscopy new insights into cellularinteractionsrdquo Current Opinion of Pharmacology vol 12 no 5pp 601ndash607 2012

[37] E Van de Vijver A Maddalena O Sanal et al ldquoHematologi-cally important mutations leukocyte adhesion deficiency (firstupdate)rdquo Blood Cells and Molecular Disease vol 15 no 1 pp53ndash61 2012

[38] Y Shimada M Hasegawa Y Kaburagi et al ldquoL-selectin orICAM-1 deficiency reduces an immediate-type hypersensitivityresponse by preventing mast cell recruitment in repeatedelicitation of contact hypersensitivityrdquo Journal of Immunologyvol 170 no 8 pp 4325ndash4334 2003

[39] Y Li J Brazzell A Herrera and B Walcheck ldquoADAM17deficiency by mature neutrophils has differential effects on L-selectin sheddingrdquo Blood vol 108 no 7 pp 2275ndash2279 2006

[40] S I Simon and C E Green ldquoMolecular mechanics and dynam-ics of leukocyte recruitment during inflammationrdquo AnnalsReview of Biomedical Engineering vol 7 pp 151ndash185 2005

[41] S Y Yuan Q Shen R R Rigor and M H Wu ldquoNeutrophiltransmigration focal adhesion kinase and endothelial barrierfunctionrdquoMicrovascular Research vol 83 no 1 pp 82ndash88 2012

[42] M J Sanz and P Kubes ldquoNeutrophil-active chemokines in invivo imaging of neutrophil traffickingrdquo European Journal ofImmunology vol 42 no 2 pp 278ndash283 2012

[43] S D Chase J L Magnani and S I Simon ldquoE-selectin ligandsas mechanosensitive receptors on neutrophils in health anddiseaserdquo Annals of Biomedical Engineering vol 40 no 4 pp849ndash859 2012

[44] J Palmblad ldquoThe role of granulocytes in inflammationrdquo Scandi-navian Journal of Rheumatology vol 13 no 2 pp 163ndash172 1984

[45] E J Leonard and T Yoshimura ldquoNeutrophilattractantactivation protein-1 (NAP-1 [interleukin-8])rdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 2 no 6 pp 479ndash486 1990

[46] S Koyama E Sato H Numanami K Kubo S Nagai and TIzumi ldquoBradykinin stimulates lung fibroblasts to release neu-trophil and monocyte chemotactic activityrdquo American Journalof Respiratory Cell and Molecular Biology vol 22 no 1 pp 75ndash84 2000

[47] J Witowski H Tayama K Ksiek M Wanic-Kossowska TO Bender and A Jorres ldquoHuman peritoneal fibroblasts are apotent source of neutrophil-targeting cytokines a key role ofIL-1beta stimulationrdquo Laboratory Investigation vol 89 no 4 pp414ndash424 2009

[48] Z Weng B Zhang S Asadi et al ldquoQuercetin is more effectivethan cromolyn in blocking human mast cell cytokine releaseand inhibits contact dermatitis andphotosensitivity in humansrdquoPLoS One vol 7 no 3 Article ID e33805 2012

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


of carbon particles by macrophages obtained from ratstreated with A satureoides extract In addition A satureoidesinfusion increased peripheral blood human mononuclearphytohemagglutinin- (PHA-) induced proliferation inter-feron gamma (IFN120574) and interleukin-4 (IL-4) secretion [611] Although the anti-inflammatory effects of A satureoideshave been demonstrated its direct actions on neutrophilfunctions have not been shown

Neutrophils express a wide range of membrane receptorssuch as adhesion molecules and chemoattractant receptorsprompting them to react to exogenous stimuli and endoge-nous chemical mediators In this context neutrophils expressthe toll-like receptor 4 (TLR-4) which is responsible forthe recognition of lipopolysaccharides (LPSs) from Gram-negative bacteria LPS binds to TLR-4 on the cell mem-brane and activates signal-transduction pathways mainly viaMyD88 which is a central adapter protein that leads to acti-vation of the nuclear transcription factor factor-120581B (NF-120581B)NF-120581B is the most important regulator of proinflammatorygene expression and induces a release of critical inflammatorymolecules that are necessary to induce leukocyte migrationinto injured tissue and suitable immune responses [12 13]

Leukocyte influx to the site of the inflammatory lesionis initially dependent on interactions between circulatingcells and the endothelial cells of postcapillary venules medi-ated by expressionactivity of adhesion molecules on thesurface of both cell types L-selectin mediates the rollingof the neutrophils along endothelial cells as it is rapidlyexpressed by activated neutrophils and interacts with consti-tutive carbohydrates or even with P- or E-selectin expressedon endothelial cells [14] L-selectin is cleaved by actionof metalloproteases pointing to the leukocytes becomearrested to the endothelium [15] Therefore integrin familymolecules especially1205732-integrin subfamilymolecules whichare mostly expressed by leukocytes mediate firm adhesionby interacting with a diversity of endothelial membranecomponents and immunoglobulin superfamily moleculesUltimately neutrophils transmigrate between interendothe-lial junctions via heterophilic and homophilic interactionsof immunoglobulin superfamily molecules such as plateletendothelial cell adhesion molecule-1 (PECAM-1) to sub-sequently migrate to the inflammatory focus [16] In theextravascular matrix neutrophils directly move to the siteof the lesion in response to chemotactic chemical mediators[17] At the lesion site they phagocyte the lesion-causingagent and release preformed chemical substances that con-tribute to the destruction of the damaging agent However inthe case of noncontrolled inflammation blockage of phasesof neutrophil mobilization into focus of the inflammatoryreaction is an important therapeutic strategy

This study investigated the in vivo actions of hydroalco-holic extract obtained from inflorescences of A satureoideson neutrophil trafficking from the blood into inflamedtissue Data confirmed quercetin and luteolin to be themain constituents in the hydroalcoholic extract and showedthe absence of systemic toxicity However results clearlyshowed that A satureoides hydroalcoholic extract inhibitsLPS-induced pathways of neutrophil migration via a mecha-nism that might be partially dependent on TLR-4 expression

Additional anti-inflammatorymechanismsmight exist as theextract also inhibited neutrophil activation caused by a directintracellular stimulation of protein kinases

2 Materials and Methods

21 Chemicals Lipopolysaccharide from Escherichia coli(LPS serotype 026B6) indomethacin EDTA and pro-pidium iodide (PI) were purchased from Sigma-Aldrich(St Louis MO USA) Ketamine and xylazine were pur-chased from Vetbrands (Paulinia SP Brazil) and hep-arin (Liquemine) was purchased from Roche Pharma-ceuticals (Brazil) Anti-L-selectin-phycoerythrin (PE) anti-1205732-integrin-fluorescein-isothiocyanate (FITC) Annexin-V-FITC and anti-CD284MD-2-PE (toll-like receptor 4) anti-bodies were obtained from BD Pharmingen (San Diego CAUSA) Leukotriene B4 (LTB4) enzyme-linked immunosor-bent assay (EIA) was obtained fromGEHealthcare (Salt LakeCity UT USA) The biochemical assays aspartate amino-transferase (AST) alanine aminotransferase (ALT) gamma-glutamyl transferase (gamma-GT) urea and creatinine werepurchased from Biotecnica (Varginha MG Brazil) Phorbolmyristate acetate (PMA) was obtained from Calbiochem(San Diego CA USA) and 2101584071015840 dichlorodihydrofluorescein-diacetate (DCFH-DA) was obtained from Molecular Probes(Eugene OR USA) CINC-1 was also determined using EIAkits obtained from RampD Systems (Minneapolis MN USA)Air filters were purchased from TPP Switzerland and allreagents used in preparing the phosphate buffered solution(PBS) and ringer solution were purchased fromMerck USA

22 Plant Material Inflorescences of A satureoides werecollected in Fraiburgo in the State of Santa Catarina BrazilThe material was identified and a voucher specimen wasdeposited at the herbarium of the State University ofMaringa(UEM) with the code HUEM-23568 The material collectionand all experiments were authorized by the Council ofManagement of Genetic Patrimony Brazil (CGEN processnumber 010062-2012-2) Air-dried plant material was cutinto small pieces and macerated with 70 (vv) aqueousethanol at room temperature for 7 days The macerate wasfiltered and the solvent removed by rotary evaporation underreduced pressure

23 Apparatus and Chromatographic Conditions Analysiswas conducted using a high performance liquid chromatog-raphy (HPLC) system (Waters) equipped with a 600-F pump717 plus autosampler followed by a line degasser (AF) andequipped with a UV-Vis detector (PDA 2996) A reverse-phase C18 column (25 cm 46mm id 05 120583m film thicknessand 100A)was employed (Luna Phenomenex) at 25∘C Chro-matographic separation was performed at room temperaturewith a flow rate 08mLmin of gradient elution using twosolvents A (aqueous methanol 50 (vv)) and B (wateracidified with acetic acid at pH 23) The gradient systemused was 50 A (15min) 50ndash60 A (15min) 60ndash70A (10min) 70ndash80 A (10min) 80ndash90 A (10min) and95 A (5min) UV-Vis spectra were recorded at wavelengths














2sdot2H2O 6mM








3 Results





AU

0

012

024

036

048

Minutes12 15 18 21 24 27 30 33 36 39

L

Q

1

23

4 5 6

(a)

4

3

2

1

3 4 5 6 7 8 9 10 11

(times1000000)TIC

2pa

lmiti

c aci

d et

hyl e

ster

4ol

eic a

cid

ethy

l este

r

3et

hyl h

epta

deca

noat

e

5ste

aric

acid

eth

yl es

ter

6et

hyl i

cosa

noat

e

7et

hyl n

onad

ecan

oate

8sti

gmas

tero

l

9ga

mm

a-sit

oste

rol

10s

itoste

none

1n-

hexa

deca

noic

acid

(b)


Vehicle 30 50 100 2500

50

100

150

LPS

mgkg poIndomethacin


lowast

lowast

lowast

Tota

lleu

kocy

tes(106

cells

mL)

(a)

Vehicle 30 50 100 250 0

50

100

150

LPS

mgkg poIndomethacin


Neu

troph

ils(106

cells

mL)

lowastlowast

lowast

(b)



Sham Vehicle0

100

200

300

100 mgkg

Rolli

ng le

ukoc

ytes

(cel

ls10

min

)

A satureoides

LPS topic 30120583g40120583L

lowast

lowast

(a)

Sham Vehicle0

2

4

6

8


LPS topic 30120583g40120583L

lowastlowastlowast

lowast

Adhe

rent

leuk

ocyt

e(ce

lls2

00120583

m)










Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

A satureoides

lowast

Posit

ivec

ells120573

2-in

tegr

in(

)

(a)

Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

Posit

ive c

ells

L-se

lect

in (

)

A satureoides

lowast

(b)








4 Discussion







Sham Vehicle0

05

1

15

Indomethacin

LPS

100 mgkg30 mgkg

A satureoides

lowastlowastlowast

Neu

troph

ils(106

cells

mL)

(a)

Sham Vehicle0

2

4

6

8

LPS

LTB-

4 (p

gm

L)

100 mgkg30 mgkg


lowast

(b)


20

40

60

80

100

CIN

C-1

(ng

mL)

LPS


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)





Sham Vehicle0

10

20

30

40

50

LPS

100 mgkg

Toll-

like r

ecep

tor 4

(TLR

-4)

fluor

esce

nce i

nten

sity

(au

)

A satureoides

lowast

(a)

Sham Vehicle0

100

200

300

400

PMA

100 mgkg

DCF

H-D

Aflu

ores

cenc

e int

ensit

y (a

u)

A satureoides

lowastlowastlowast

(b)


(a) (b) (c)

(d) (e) (f)











5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























2sdot2H2O 6mM








3 Results





AU

0

012

024

036

048

Minutes12 15 18 21 24 27 30 33 36 39

L

Q

1

23

4 5 6

(a)

4

3

2

1

3 4 5 6 7 8 9 10 11

(times1000000)TIC

2pa

lmiti

c aci

d et

hyl e

ster

4ol

eic a

cid

ethy

l este

r

3et

hyl h

epta

deca

noat

e

5ste

aric

acid

eth

yl es

ter

6et

hyl i

cosa

noat

e

7et

hyl n

onad

ecan

oate

8sti

gmas

tero

l

9ga

mm

a-sit

oste

rol

10s

itoste

none

1n-

hexa

deca

noic

acid

(b)


Vehicle 30 50 100 2500

50

100

150

LPS

mgkg poIndomethacin


lowast

lowast

lowast

Tota

lleu

kocy

tes(106

cells

mL)

(a)

Vehicle 30 50 100 250 0

50

100

150

LPS

mgkg poIndomethacin


Neu

troph

ils(106

cells

mL)

lowastlowast

lowast

(b)



Sham Vehicle0

100

200

300

100 mgkg

Rolli

ng le

ukoc

ytes

(cel

ls10

min

)

A satureoides

LPS topic 30120583g40120583L

lowast

lowast

(a)

Sham Vehicle0

2

4

6

8


LPS topic 30120583g40120583L

lowastlowastlowast

lowast

Adhe

rent

leuk

ocyt

e(ce

lls2

00120583

m)










Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

A satureoides

lowast

Posit

ivec

ells120573

2-in

tegr

in(

)

(a)

Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

Posit

ive c

ells

L-se

lect

in (

)

A satureoides

lowast

(b)








4 Discussion







Sham Vehicle0

05

1

15

Indomethacin

LPS

100 mgkg30 mgkg

A satureoides

lowastlowastlowast

Neu

troph

ils(106

cells

mL)

(a)

Sham Vehicle0

2

4

6

8

LPS

LTB-

4 (p

gm

L)

100 mgkg30 mgkg


lowast

(b)


20

40

60

80

100

CIN

C-1

(ng

mL)

LPS


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)





Sham Vehicle0

10

20

30

40

50

LPS

100 mgkg

Toll-

like r

ecep

tor 4

(TLR

-4)

fluor

esce

nce i

nten

sity

(au

)

A satureoides

lowast

(a)

Sham Vehicle0

100

200

300

400

PMA

100 mgkg

DCF

H-D

Aflu

ores

cenc

e int

ensit

y (a

u)

A satureoides

lowastlowastlowast

(b)


(a) (b) (c)

(d) (e) (f)











5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















AU

0

012

024

036

048

Minutes12 15 18 21 24 27 30 33 36 39

L

Q

1

23

4 5 6

(a)

4

3

2

1

3 4 5 6 7 8 9 10 11

(times1000000)TIC

2pa

lmiti

c aci

d et

hyl e

ster

4ol

eic a

cid

ethy

l este

r

3et

hyl h

epta

deca

noat

e

5ste

aric

acid

eth

yl es

ter

6et

hyl i

cosa

noat

e

7et

hyl n

onad

ecan

oate

8sti

gmas

tero

l

9ga

mm

a-sit

oste

rol

10s

itoste

none

1n-

hexa

deca

noic

acid

(b)


Vehicle 30 50 100 2500

50

100

150

LPS

mgkg poIndomethacin


lowast

lowast

lowast

Tota

lleu

kocy

tes(106

cells

mL)

(a)

Vehicle 30 50 100 250 0

50

100

150

LPS

mgkg poIndomethacin


Neu

troph

ils(106

cells

mL)

lowastlowast

lowast

(b)



Sham Vehicle0

100

200

300

100 mgkg

Rolli

ng le

ukoc

ytes

(cel

ls10

min

)

A satureoides

LPS topic 30120583g40120583L

lowast

lowast

(a)

Sham Vehicle0

2

4

6

8


LPS topic 30120583g40120583L

lowastlowastlowast

lowast

Adhe

rent

leuk

ocyt

e(ce

lls2

00120583

m)










Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

A satureoides

lowast

Posit

ivec

ells120573

2-in

tegr

in(

)

(a)

Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

Posit

ive c

ells

L-se

lect

in (

)

A satureoides

lowast

(b)








4 Discussion







Sham Vehicle0

05

1

15

Indomethacin

LPS

100 mgkg30 mgkg

A satureoides

lowastlowastlowast

Neu

troph

ils(106

cells

mL)

(a)

Sham Vehicle0

2

4

6

8

LPS

LTB-

4 (p

gm

L)

100 mgkg30 mgkg


lowast

(b)


20

40

60

80

100

CIN

C-1

(ng

mL)

LPS


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)





Sham Vehicle0

10

20

30

40

50

LPS

100 mgkg

Toll-

like r

ecep

tor 4

(TLR

-4)

fluor

esce

nce i

nten

sity

(au

)

A satureoides

lowast

(a)

Sham Vehicle0

100

200

300

400

PMA

100 mgkg

DCF

H-D

Aflu

ores

cenc

e int

ensit

y (a

u)

A satureoides

lowastlowastlowast

(b)


(a) (b) (c)

(d) (e) (f)











5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sham Vehicle0

100

200

300

100 mgkg

Rolli

ng le

ukoc

ytes

(cel

ls10

min

)

A satureoides

LPS topic 30120583g40120583L

lowast

lowast

(a)

Sham Vehicle0

2

4

6

8


LPS topic 30120583g40120583L

lowastlowastlowast

lowast

Adhe

rent

leuk

ocyt

e(ce

lls2

00120583

m)










Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

A satureoides

lowast

Posit

ivec

ells120573

2-in

tegr

in(

)

(a)

Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

Posit

ive c

ells

L-se

lect

in (

)

A satureoides

lowast

(b)








4 Discussion







Sham Vehicle0

05

1

15

Indomethacin

LPS

100 mgkg30 mgkg

A satureoides

lowastlowastlowast

Neu

troph

ils(106

cells

mL)

(a)

Sham Vehicle0

2

4

6

8

LPS

LTB-

4 (p

gm

L)

100 mgkg30 mgkg


lowast

(b)


20

40

60

80

100

CIN

C-1

(ng

mL)

LPS


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)





Sham Vehicle0

10

20

30

40

50

LPS

100 mgkg

Toll-

like r

ecep

tor 4

(TLR

-4)

fluor

esce

nce i

nten

sity

(au

)

A satureoides

lowast

(a)

Sham Vehicle0

100

200

300

400

PMA

100 mgkg

DCF

H-D

Aflu

ores

cenc

e int

ensit

y (a

u)

A satureoides

lowastlowastlowast

(b)


(a) (b) (c)

(d) (e) (f)











5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

A satureoides

lowast

Posit

ivec

ells120573

2-in

tegr

in(

)

(a)

Control Vehicle0

20

40

60

80

100

BasalLPS

100 mgkg

Posit

ive c

ells

L-se

lect

in (

)

A satureoides

lowast

(b)








4 Discussion







Sham Vehicle0

05

1

15

Indomethacin

LPS

100 mgkg30 mgkg

A satureoides

lowastlowastlowast

Neu

troph

ils(106

cells

mL)

(a)

Sham Vehicle0

2

4

6

8

LPS

LTB-

4 (p

gm

L)

100 mgkg30 mgkg


lowast

(b)


20

40

60

80

100

CIN

C-1

(ng

mL)

LPS


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)





Sham Vehicle0

10

20

30

40

50

LPS

100 mgkg

Toll-

like r

ecep

tor 4

(TLR

-4)

fluor

esce

nce i

nten

sity

(au

)

A satureoides

lowast

(a)

Sham Vehicle0

100

200

300

400

PMA

100 mgkg

DCF

H-D

Aflu

ores

cenc

e int

ensit

y (a

u)

A satureoides

lowastlowastlowast

(b)


(a) (b) (c)

(d) (e) (f)











5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Sham Vehicle0

05

1

15

Indomethacin

LPS

100 mgkg30 mgkg

A satureoides

lowastlowastlowast

Neu

troph

ils(106

cells

mL)

(a)

Sham Vehicle0

2

4

6

8

LPS

LTB-

4 (p

gm

L)

100 mgkg30 mgkg


lowast

(b)


20

40

60

80

100

CIN

C-1

(ng

mL)

LPS


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)





Sham Vehicle0

10

20

30

40

50

LPS

100 mgkg

Toll-

like r

ecep

tor 4

(TLR

-4)

fluor

esce

nce i

nten

sity

(au

)

A satureoides

lowast

(a)

Sham Vehicle0

100

200

300

400

PMA

100 mgkg

DCF

H-D

Aflu

ores

cenc

e int

ensit

y (a

u)

A satureoides

lowastlowastlowast

(b)


(a) (b) (c)

(d) (e) (f)











5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sham Vehicle0

10

20

30

40

50

LPS

100 mgkg

Toll-

like r

ecep

tor 4

(TLR

-4)

fluor

esce

nce i

nten

sity

(au

)

A satureoides

lowast

(a)

Sham Vehicle0

100

200

300

400

PMA

100 mgkg

DCF

H-D

Aflu

ores

cenc

e int

ensit

y (a

u)

A satureoides

lowastlowastlowast

(b)


(a) (b) (c)

(d) (e) (f)











5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article achyrocline satureioides (lam.) d.c. … · 2019. 7. 31. · puhlmann and...

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