Research Article Immunobiologic and …downloads.hindawi.com/journals/bmri/2013/451679.pdfResearch Article Immunobiologic and Antiinflammatory Properties of a Bark Extract from Ampelozizyphus

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 451679 11 pageshttpdxdoiorg1011552013451679

Research ArticleImmunobiologic and Antiinflammatory Properties of a BarkExtract from Ampelozizyphus amazonicus Ducke

Ligia Maria Torres Peccedilanha1 Patricia Dias Fernandes2 Tatiana Jotha-Mattos Simen3

Danilo Ribeiro de Oliveira3 Priscilla Vanessa Finotelli3 Marina Vieira Agostinho Pereira1

Fernanda Ferreira Barboza1 Thays da Silva Almeida2 Stephanie Carvalhal2

Anna Paola Trindade Rocha Pierucci4 Gilda Guimaratildees Leitatildeo5 Luca Rastrelli6

Anna Lisa Piccinelli6 and Suzana Guimaratildees Leitatildeo3

1 Departamento de Imunologia Instituto de Microbiologia Paulo de Goes Universidade Federal do Rio de JaneiroCCS Bloco I 2∘ andar 21941-590 Rio de Janeiro RJ Brazil

2 Instituto de Ciencias Biomedicas Universidade Federal do Rio de Janeiro CCS Bloco K 21941-590 Rio de Janeiro RJ Brazil3 Faculdade de Farmacia Universidade Federal do Rio de Janeiro CCS Bloco A 2∘ andar Ilha do Fundao21941-590 Rio de Janeiro RJ Brazil

4Universidade Federal do Rio de Janeiro Instituto de Nutricao Josue de Castro CCS Bloco JSS08 21941-590 Rio de Janeiro RJ Brazil5 Nucleo de Pesquisas de Produtos Naturais Universidade Federal do Rio de Janeiro CCS Bloco H Rio de Janeiro RJ Brazil6Universita di Salerno Dipartimento di Farmacia Via Ponte Don Melillo 84084 Fisciano Italy

Correspondence should be addressed to Suzana Guimaraes Leitao sgleitaosuperigcombr

Received 7 October 2012 Revised 28 December 2012 Accepted 29 December 2012

Academic Editor Fabio Ferreira Perazzo

Copyright copy 2013 Ligia Maria Torres Pecanha et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Ampelozizyphus amazonicus is used in the treatment and prevention of malaria The effect of an aqueous extract from this plant(SART) on the immune response was investigated by measuring immunoglobulin production induced by immunization with theantigen TNP-Ficoll in Plasmodium chabaudi-infectedmice SART treatment increased antigen-specific IgM and IgG levels in TNP-Ficoll-immunized mice The B cell response during malarial infection was also modified by SART There was an increase in totalserum IgM and IgG and a decrease in the percentage of splenic plasma cells (CD138+ cells) in P chabaudi-infected SART-treatedanimals SART (1 3 or 10mgkg po) and the reference drug dexamethasone (5mgkg) were also tested in carrageenan-inducedleukocyte migration to the subcutaneous air pouch (SAP) All SART doses significantly reduced leukocyte migration into theSAPThe protein concentration resulting from extravasation into the peritoneum was also significantly reduced Our data indicatethat SART possesses immunomodulatory properties inducing an in vivo modification of the B lymphocyte response and anti-inflammatory properties which are partly due to a reduction in cell migration and are most likely due to an inhibition of theproduction of inflammatory mediators Preliminary HPLC-ESI-MSMS analysis of SART shows a complex saponin profile withdeprotonated molecule [M-H]minus ions in the range ofmz 800ndash1000

1 Introduction

Ampelozizyphus amazonicus Ducke (Rhamnaceae) is anAmazonian medicinal plant popularly known as ldquosaracura-mirardquo that is found in the Amazon forest territories of BrazilVenezuela Colombia Peru and Ecuador In Brazil it isrestricted to the states of Amazonas Para and Roraima and

grows mainly in the ldquoterra firmerdquo forests near waterfalls orldquoigarapesrdquo [1] An aqueous drink can be prepared from thebark and roots of A amazonicus This drink has a verybitter taste and forms abundant foam when shaken sim-ilar to beer which also gives rise to its other popularnames ldquocervejinhardquo ldquocervejeirardquo ldquocerveja-do-matordquo ldquocerveja-de-ındiordquo and ldquocerveja-de-pretordquo [2ndash5] These properties can

2 BioMed Research International

be explained by the high saponin content in the speciesIndeed Silva et al [6] showed the presence of approximately48 saponins in an aqueous extract from the roots of theplant So far only three saponins with a dammarane-typeaglycone have been described in the literature [7 8] as wellas the presence of free triterpenes such as melaleucic acid312057327120572-dihydroxylup-20(29)-en-28120573-oic acid betulinic acidbetulin lupeol and phytosteroids [7ndash9]

Several ethnobotanical studies have shown that A ama-zonicus is useful in the treatment and prevention of malaria[2 3 10ndash12] Previous investigations of the antimalarial prop-erties of an extract from this plant have shown that it does nothave a direct action on Plasmodium blood stage forms eitherin vivo or in red blood cell cultures [13] However this naturalproduct could be effective in controlling infection induced bysporozoite forms [13] Based on the findings that A amazon-icus does not have a direct effect upon blood stage forms ofthe protozoan it might be possible to suggest that the controlof the infection induced by this plant could be obtained byan overall augmentation of the immunological response Infact ethnopharmacological studies indicate both stimulatoryand energetic properties for A amazonicus [5 12] In anethnopharmacological study conducted by our group in theldquoquilombolardquo communities ofOriximina State of Para Brazilthe plant is used in the treatment of liver disorders gastritisinflammation of the prostate and ldquoinflammation of awomanrdquoand as a fortifying tonic and aphrodisiac among other uses[12 14] In the Rio Negro valley and Jau National Park inthe state of Amazonas A amazonicus is used against rheu-matism and other types of pain and for the general treatmentof inflammation [5 11]

Based on the reported properties of this plant and its usesin folkmedicine our group suggests thatA amazonicus couldact as an adaptogen by enhancing immune system functionand could alleviate the inflammatory disorders caused bymalaria In the present work we aimed to investigate thetoxicity of A amazonicus and its effects on the immuneresponse as well as its anti-inflammatory properties

2 Material and Methods

21 Plant Material and Preparation of Extracts Ampel-ozizyphus amazonicus Ducke was collected in August2008 in the Brazilian Amazon region of Oriximina (Parastate) at the Pancada community (S 01∘04101584009410158401015840 and W056∘02101584040910158401015840) Plants were collected as a part of a bio-prospecting project in ldquoquilombolardquo communities fromOrix-imina that received authorization by the Brazilian Direct-ing Council of Genetic Heritage (Conselho de Gestaodo Patrimonio Genetico) through Resolution number 213(6122007) published in the Federal Official Gazette of Brazilon December 27 2007 Plants were identified by Mr JoseRamos (parataxonomist) A voucher specimenwas depositedat the Instituto Nacional de Pesquisas da Amazonia INPAherbarium (Manaus AMBrazil) under the registration INPA224161

Dried and ground bark (250 g) of A amazonicus wasused for the preparation of the extracts The bark was

submitted to extraction with boiling water (5 wv) for 15minutes and filtered A second extraction was performedwith boiling water (25 wv 30 minutes) The extracts weremixed and infused into a spray-dryer nozzle unit of BuchiMini Spray Dryer B-290 (Buchi Laboratorius-Technik AGSwitzerland)The conditions of the spray-drying processwereas follows nozzle diameter 03mm aspirator pressure 80flow rate 6mLmin inlet temperature 190 plusmn 3∘C and outlettemperature 88 plusmn 15∘C The atomized powder was collectedby a cyclone and is designated as SART throughout the text

22 Estimation of Daily Dose for Animal Assays The dailyoral dose of A amazonicus (SART) used was determinedbased on its traditional use A drink was prepared accordingto the ldquoquilombolardquo traditional method as described byOliveira et al [14] Briefly one tablespoon of ground bark(83980 g) was added to 200mL of cold water and ldquoshakenrdquoseven times The foam produced after each shaken was dis-cardedThe extractwas filtered and a total solid yield of 021(wv) was obtained [15] Considering that the prophylacticuse of the drink (to prevent from malaria) is 0630 gdayor 300mLday of extract at 021 (wv) of total solid yieldthe daily oral dose for an adult weighing 70 kg would be9mgKgTherefore all biological assays were standardized toa 10mgkg oral dose of SART as obtained by spray dryer

23 HPLC-DAD Profile of SART HPLC analysis of SART(aqueous atomized extract of A amazonicus 2mgmL inacetonitrile water 95 5) was performed on a VWR-HitachiElite LaChrom system consisting of an L-2130 quaternarypumpwith online degasser a Rheodyne injector (20 120583L loop)L-2455 diode array detector (DAD) equipped with a Zic-Hilic PEEK column (SeQuant-Merck 250 times 46mm id par-ticle size 5120583m 200A) coupled with a security guard column(4 times 300mm id C-18 cartridge) The elution solvents usedwere ammonium acetate 10mM pH 58 corrected with aceticacid (solvent A) and acetonitrile (solvent B) and the flow ratewas 05mLmin detectionDAD (210 nm)The elutionwas asfollows 0ndash10min isocratic 4 (solvent A) 96 (solvent B)101ndash65min linear gradient where solvent B decreased from96 to 50 Betulinic acid from SIGMA (0125mgmL inacetonitrile water 95 5) was used as standard

24 HPLC-DAD-ESI-MS Preliminary Profile of SART AnHPLC system including a Surveyor Autosampler a SurveyorLC pump and an LCQ Advantage ion-trap mass spectrome-ter (Thermo Finningan San Jose CA USA) equipped withan electrospray ion source was used for the preliminaryanalysis of SART Chromatographic conditions were thesame reported above MS data were acquired in negativeionization mode and the spectra were recorded in full scan(mz 250ndash1500) and tandem mass (collision energy of 40of the instrument maximum) scanning modes Instrumentalparameters were optimized using a purified saponin mixtureisolated from SART by countercurrent chromatography (datanot shown) Saponin mixture was dissolved (14 120583gsdotmLminus1) inACN H

2O (1 1 vv) and infused in the ESI source at the flow

rate of 5120583gsdotminminus1 by a syringe pump

BioMed Research International 3

25 Animals Experiments were performed with male Swissmice (20ndash25 g) obtained from InstitutoVital Brazil or BALBcmice with ages ranging from 6 to 8 weeks supplied by theCentral Animal Facility from Instituto de Microbiologiafrom Federal University of Rio de Janeiro Animals weremaintained in a room with controlled temperature (22 plusmn2∘C) with a 12 h lightdark cycle and free access to foodand water Animal care and research protocols were inaccordance with the principles and guidelines adopted bythe Brazilian College of Animal Experimentation (COBEA)They were approved by the Ethical Committee for AnimalResearch (Biomedical Science InstituteUFRJ) and receivedthe numbers ICBDFBC-015 and IMIPPG-012

26 Drugs and Extract Administration Carrageenan anddexamethasone were purchased from Sigma (St Louis MOUSA) All drugs were dissolved in phosphate buffer saline(PBS) just before use SARTwas dissolved in sterile water andadministered by oral gavage at doses of 1 3 and 10mgkg in afinal volume of 01mL Dexamethasone (5mgkg) was usedas reference drug and was administered via oral gavage aswellThe negative control group was composed of mice giventhe vehicle (sterile water) significant effects due to the waterper se were not observed throughout the study The dose ofdexamethasone was chosen based on previous experimentsdone by our group

27 Determination of SART Toxicity In Vitro Cells from thecell line A20 (3 times 104) were plated in flat bottom 96-welltissue culture plates Cultures were performed in RPMI 1640medium supplemented with 10 fetal calf serum (Gibco-BRL Grand Island NY USA) L-glutamine (2mM) 2-ME(50120583M) and gentamicin (50 120583gmL) Reagents were ob-tained from SIGMA Chemical Co (St Louis MO USA)Some cultures received different doses of SART An incuba-tion of 48 hours was performed at 37∘C in CO

2Incubator

(Forma Scientific Marietta OH USA) Afterwards cellviability was determined by the XTT-based viability assay[16] Plates were read in a microplate reader (model 550reader Bio-Rad Boston MA USA) to determine A

450

28 Anti-TNP-Ficoll Immunoglobulin Production TNP-Ficoll was obtained from Bioresearch Technologie Inc(Novato CA USA) TNP-Ficoll was diluted in PBS andadministered by the intravenous injection of 50 120583g of thepolysaccharide per animal Oral treatment with SART wasbegun ten days before immunization and each animal wastreated daily with a 10mgkg dose of SARTThe animals werebled through the tail vein weekly and serum samples wereprepared Serum anti-TNP-Ficoll IgM and IgG titers weremeasured by ELISA as previously described [17] Brieflyflexible polyvinyl chloride round bottom microtest plates(BD Falcon BD Labware Franklin Lakes NJ USA) werecovered with a solution of TNP-Ficoll diluted in 1M pH83 Tris buffer Serially diluted samples were added to theplates and were performed in duplicate Secondary alkalinephosphatase-labeled anti-IgM or IgG antibodies (SIGMAChemical Co St Louis MO USA) were used to reveal

antigen-specific antibody binding Plates were read using amicroplate reader to determine A

405(model 550 plate reader

Bio-Rad Boston MA USA) The titer of each sample wasthe serum dilution giving an A

405reading midway on the

linear portion of the titration curve

29 Malarial Infection and Immunoglobulin MeasurementP chabaudi infection was established by the intraperitonealinjection of 106 P chabaudi-infected red blood cells Serumsamples were obtained as described above and were usedfor measuring total IgM and IgG circulating concentra-tions This measurement was performed by a sandwichELISA as previously described [18] Unlabeled goat anti-IgM or anti-IgG antibodies (SIGMA Chemical Co St LouisMO USA) were used as primary antibodies Standardcurves were set up using purified mouse IgM or IgG fromICN Biomedicals (Irvine CA USA) Secondary alkalinephosphatase-labeled antibodies were also obtained fromSIGMA Chemical Co The reaction was quantified by theaddition of p-nitrophenolphosphate (SIGMA Chemical Co)and absorbance was measured at 405 nm with a model 550plate reader (BioRad Boston MA USA)

210 Determination of Antibody-Producing Cells by FlowCytometry Asplenic cell suspensionwas obtained from indi-vidual animals and cells were diluted in RPMI 1640 mediumsupplemented with 5 FCS Samples of 106 cells were labeledwith FITC-conjugated anti-B220 and PE-conjugated anti-CD138 antibodies (BD Biosciences San Jose CA USA) Thesamples were washed fixed with PBS1 formaldehyde andwere analyzed by flow cytometry using a FACSCalibur anddata were analyzed using the CellQuest software (both arefrom BD Biosciences San Jose CA USA)

211 Formalin Test This procedure was similar to themethoddescribed by Gomes et al [19] Mice received an injectionof 20120583L of formalin (25 vv) into the dorsal surface ofthe left hind paw The time that the animal spent licking theinjected paw was immediately recordedThe nociceptive andinflammatory responses consists of the following two phasesthe first phase lasts until 5min after the formalin injection(first-phase neurogenic pain response) and the second phaseoccurs 15ndash30min after the formalin injection (second-phaseinflammatory pain response) The animals were pretreatedwith oral doses of SART or vehicle for 60min before theadministration of formalin

212 Subcutaneous Air Pouch (SAP) The method wasadapted from Raymundo et al [20] Air pouches wereproduced by subcutaneous injection of 10mL of sterile airinto the intrascapular area of the backs of the mice After3 days another 10mL of air were injected to maintain thepouches The animals received an injection of 05mL of asterile carrageenan suspension (1) into the SAP three daysafter the last injection The animals were pretreated with oraldoses SART (1 3 and 10mgkg) 60min before carrageenaninjection The animals were killed 24 h after carrageenaninjection and the cavity was washed with 2mL of sterile PBS


The liquid in the SAP was collected and quantified Totalcell count was done in an automatic cell counter (pocH-100iV Diff Sysmex) The exudates were centrifuged at 170timesgfor 10min at 4∘C and the supernatants were collected andstored at minus20∘C until dosages The protein content of eachsupernatant was determined using the BCA method (BCAProtein Assay Kit Pierce)

213 Reduction of Spontaneous Activity Spontaneous activitywas evaluated as described by Barros et al [21] and Figueiredoet al [22] Mice received 10mgkg of SART by oral admin-istration and were immediately placed individually in anobservation chamber with a floor that was divided into 50squares (5 cm times 5 cm) The total number of squares that themouse walked through during a 5min period was countedThe effect of SART on locomotor performancewas also testedon the rotarod apparatus as described previously [22 23]All animals were trained on the rotarod (37 cm in diameter8 rpm) twenty-four hours before the experiments until theycould remain in the apparatus for 60 s without falling Onthe day of the experiment mice were treated with SART (at10mgkg po) and tested on the rotarod from05 to 35 h afterSART administrationThe number of falls from the apparatuswas recorded with a stopwatch for up to 240 s

214 Acute Toxicity The parameters were determined asdescribed by Lorke [24] A single oral dose of SART(10mgkg) was administered to a group of ten mice (fivemales and five females) We observed various behaviourparameters including convulsion hyperactivity sedationgrooming loss of righting reflex increased or decreasedrespiration and food and water intake over a period of15 days After this period the animals were euthanized bycervical dislocation their stomachs removed an incisionalong the greater curvature was made and the number ofulcers (single or multiple erosion ulcer or perforation) wascounted Hyperaemia was also evaluated

215 Statistical Analysis For in vivo assays all experimentalgroups consisted of 6ndash10 mice For the in vitro assays allstudies were done in triplicate and each protocol was repeatedat least 4 times The results are presented as mean plusmn SDStatistical significance between groups was performed byapplying analyses of variance (ANOVA) followed by Bonfer-ronirsquos test or Studentrsquos t-test for independent samplesP valuesless than 005 (119875 lt 005) were considered significant

3 Results and Discussion

31 HPLC-DAD Profile of SART In a previous study fromour group [14] we demonstrated that an aqueous extractfrom A amazonicus prepared by the traditional quilombolamethod contained free betulinic acid After acid hydrolysisfollowed by gas chromatography-mass spectrometry analysisthe presence of a dammarane-type triterpene skeleton wasalso characterized in that extract [14] In the present studya different methodology was used for extraction which usedhot water instead of tap water to raise the extraction yields

Because we have suggested that some of the use indicationsof this plant for instance as a tonic and for treating malariamight be related to its properties as an adaptogen andto the immunostimulatory properties of the saponins andbetulinic acid in the drink we investigated the presenceof betulinic acid in SART The HPLC profile of SART wasobtained in a Zic-Hilic column in which two different zonescan be found the first refers to triterpenic and nonpolarcompounds while the second refers to saponins and polarcompounds (Figure 1(a)) thus showing the efficiency of thiszwitterionic column in the separation of different compoundsand polarities The presence of betulinic acid in SART (119905

119877

8387min) was confirmed by standard injection

32 HPLC-DAD-ESI-MSMS Profile of SART To moreclosely investigate the chemical composition of SART weperformed a preliminary HPLC-ESI-MSMS analysis of theextract The resulting (minus)-MS chromatogram (Figure 1(b))and its expansion (30ndash41min) show a complex saponinprofile with deprotonated molecule [M-H]minus ions in the rangeofmz 800ndash1000 Analysis of the product ion mass spectra of[M-H]minus ions implied that the compounds belong to the classof triterpenoidal saponins

The peaks at 350 and 320min ([M-H]minus at mz 897and 983) showed a fragmentation pattern that suggested thepresence of triglycosides of jujubogeninThe peak at 350minyielded product ions atmz 765 735 603 and 471 correspond-ing to [M-H-pentose]minus [M-H-hexose]minus [M-H-pentose-hexose]minus and [M-H-2 times pentose-hexose]minus The diagnosticsignals of the peaks at 320min observed in the MSn spectraatmz 941 ([M-H-42]minus) 923 ([M-H-CH

3COOH]minus) 821 ([M-

H-hexose]minus) 633 ([M-H-42-hexose-deoxyhexose]minus) and 471([M-H-42-2 times hexose-deoxyhexose]minus) indicated the struc-ture of an acetylated jujubogenin triglycoside

The peak at 360min ([M-H]minus atmz 959) showed in theMSn spectra a product ion at mz 817 ([M-H-C

8H14O2]minus)

due to a McLafferty rearrangement that is characteristic ofthe keto-dammarane-type triterpene saponins [25] Otherproduct ions at mz 655 ([M-H-C

8H14O2-hexose]minus) 509

([M-H-C8H14O2-hexose-deoxyhexose]minus) and 347 ([M-H-

C8H14O2-2 times hexose-deoxyhexose]minus) confirmed the pres-

ence of a trisaccharide chain The peak at 357min ([M-H]minus at mz 973) displayed MSn spectra very similar tothose of the previous peak product ions at mz 817 ([M-H-C9H16O2]minus) 655 ([M-H-C

9H16O2-hexose]minus) 509 ([M-H-

C9H16O2-hexose-deoxyhexose]minus) and 347 ([M-H-C

9H16O2-

2 times hexose-deoxyhexose]minus) The difference in themz valuesof the [M-H]minus ions of these two compounds was equivalentto 14 (corresponding to a CH

2unit) indicating that the struc-

tural difference is in the alkyl chain of the keto-dammaraneskeletonAn elucidation of the structures of these compoundsis currently in progress

33 Immunobiologic Properties of A amazonicus To evaluatethe hypothesis that A amazonicus could act as an adaptogenby enhancing immune system function its immunobiologicalproperties were examined in different in vitro and in vivomodels The daily oral dose of A amazonicus used in all bio-logical experiments was estimated based on its traditional use


400

200

00 10 20 30 40 50 60

Minutes

mA

U

400

200

0

mA

U

DAD-CH1 210 nm

(a)

0 5 10 15 20 25 30 35 40Time (min)

1210

8642

Inte

nsity

times 106

(b)

1210

8642

Inte

nsity

Time (min)

times 106

2849 3063107

3199 3338

3501

357

3708

37783872

39184012 4059

28 29 30 31 32 33 34 35 36 37 38 39 40 41

(c)

Figure 1 (a) HPLC-DAD chromatogram of SART (detection 210 nm) and betulinic acid at Rt = 8387min (b) HPLC-DAD-ESI-MS (negativemode) chromatogram of SART Triterpene zone between 5 and 10min and saponin zone between 30 and 40min (c) HPLC-DAD-ESI-MSchromatogram of SART expansion of the saponin zone between 30 and 44min

by the preparation of a drink according to the ldquoquilombolardquotraditional method

331 Determination of SART Toxicity and Its Effect on B CellsIn Vitro We initially measured the toxic effects of differentdoses of SART on A20 lymphoma cell cultures SART did notmodify cell viability in cultures incubatedwith this extract foreither 48 h (Figure 2) or 72 h (data not shown)We also testedwhether SART would modify the B lymphocyte response incultureWe employed in this investigation cultures of purifiedB cells stimulated with LPS and measured the stimulationof two parameters associated with B cell activation by LPSimmunoglobulin secretion and the membrane expressionof the protein CD86 We did not observe any significantmodification of either immunoglobulinM (IgM) secretion orCD86 cell surface expression in B cell cultures incubated withdifferent doses of SART (data not shown)

332 Anti-TNP-Ficoll Immunoglobulin Production We nextinvestigated the effect of SART on the in vivo immuneresponse We used immunization with the T-independenttype 2 antigen TNP-Ficoll which induces antigen-specificimmunoglobulin production as amodelThis class of antigenstimulates B cells in the absence of T cell help and induces theproduction of both IgM and IgG [26] Mice were immunizedby intravenous injection of TNP-Ficoll and antigen-specificimmunoglobulin was measured weekly after immunizationAs observed in Figure 3 oral treatment with SART inducedan increase in both IgM (Figure 3(a)) and IgG (Figure 3(b))anti-TNP-Ficoll antibody titers

Ctr deadCtr viable

0

1

2

3

150 50 167 55 19 06 03 01 003 001

48 hs

mdash mdash

A 450

Dose of SM extract added to the culture ( gmL)

Figure 2 Evaluation of the toxicity of SART in vitro in cultured cellsThe cell line A20 (3 times 104 cellswell) was cultured in the presenceof the indicated concentrations of SART Control cultures wereestablished without the addition of SART Cultures were incubatedfor 48 h at 37∘C in a 7CO

2

atmosphere Cell viability wasmeasuredby the XTT reduction measurement Ctr viable indicates untreatedcultures incubated with medium alone Ctr dead indicates controlcultures to which 1120583L of Triton X-100 was added prior to theaddition of XTT Mean values plusmn SD of absorbance (A

450

) are shown

The finding that SART treatment could amplify theresponse of murine B cells to a T-independent type 2 antigensuggests that components of the plant extract might haveimmunopotentiating effects Saponins are the major com-ponents of the extract obtained from SART Several studieshave shown that these compounds contain adjuvant proper-ties [27] including the ability to enhance immunoglobulin


0

2000

4000

6000

8000

CTR SM TF TF + SM

0

1000

2000

3000

4000

CTR SM TF TF + SM

0

1000

2000

3000

4000

CTR SM TF TF + SM CTR SM TF TF + SM0

2000

4000

6000

8000

CTR SM TF TF + SM0

2000

4000

6000

8000

CTR SM TF TF + SM0

4000

8000

12000

16000

lowast

Day 7 Day 7

Day 14 Day 14

Day 21 Day 21

Seru

m an

ti-TN

P-Fi

coll

titer

sSe

rum

anti-

TNP-

Fico

ll tit

ers

Seru

m an

ti-TN

P-Fi

coll

titer

s

(a) IgM (b) IgG

Figure 3 Antigen-specific immunoglobulin production in animals treatedwith SARTThe four indicated groupswere established CTR (PBS-treatedmice) SART (mice treated daily with an oral dose of SART) TF (mice immunizedwith TNP-Ficoll) andTF+ SART (mice immunizedwith TNP-Ficoll and treated daily with SART) Oral treatment with SART was onset 10 days before immunization Serum was obtained fromindividual mice at days 7 14 and 21 after immunization as indicated in the figure Anti-TNP-Ficoll serum titers were determined by ELISA(a) show data on IgM antibodies and (b) the one on IgG levels Data indicate mean values plusmn SD of 6 animals per group Data are representativeof two independent experiments lowast119875 le 0 05 when compared to the TF group

production [28] and also to stimulate the release of immunemediators and the proliferation of immune cells in vitro [29]

333 Malaria InfectionMeasurement of Serum Immunoglob-ulin Levels and Numbers of Splenic Antibody-Producing CellsAs described above SART treatment is popularly used tocontrol malaria infection The role of B cells in this infectionwas previously investigated and it was shown that micedeficient in mature B cells had reduced primary acuteinfections but were unable to eliminate parasites indicatingthat B cells are required for final parasite clearance [30] Itwas also described thatPlasmodium-infected animals showedan increase in circulating antibody levels when compared

to normal animals [31] Therefore we next investigated theeffects of SART treatment on the course of the B cell responsein P chabaudi-infected mice We examined whether SARTtreatment could induce a modification in immunoglobulinproduction during infection Treatment with SART inducedan increase in the levels of circulating total IgM and IgG inP chabaudi-infected mice (Figure 4) We next investigatedthe levels of antibody-secreting cells in the spleen of Pchabaudi-infected SART-treated mice To perform this studywe measured the percentage of cells expressing the surfaceprotein CD138 a marker for antibody-secreting cells [32]Despite the increase in circulating immunoglobulin levelsthere was a decrease in the number of antibody-producing


0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

G (m

gm

L)

0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

M (

gm

L) Day 5

Day 5

Day 8

Day 8 lowast

lowast

Figure 4 Serum total IgM and IgG levels in mice infected with P chabaudi and treated with SART The four indicated groups were set upCTR (vehicle-treated animals) SART (mice treated with a daily oral dose of SART) Pc (animals injected with 106P chabaudi-infected redblood cells) and Pc + SART (P chabaudi-infected SART-treatedmice) Groups of 8 animals were used and two independent experiments wereperformed Serum was obtained at the indicated periods after infection and total IgM (top panels) and IgG (bottom panels) were determinedby ELISA Data indicate mean immunoglobulin levels plusmn SD lowast119875 le 0 05 when compared to the Pc group

plasma cells in the spleens of P chabaudi-infected SART-treatedmice 8 days after infectionwhen compared to infecteduntreated animals (Figure 5)

Taken together our data indicate that SART modulatesmalaria infection because SART increases immunoglobulinproduction during infection and regulates the appearance ofantibody-secreting cells The relevance of this phenomenonon resistance and the progression of infection need to befurther analyzed

Due to the effect of SART on immunoglobulin produc-tion the saponins may have antimalarial properties thatare potentiated through other mechanisms Dammarane-type saponins have shown potent hepatoprotective effectsin different models [33ndash35] and these effects could justifythe use of this plant for the treatment of liver disorders andmalaria infection

34 Evaluation of the Anti-Inflammatory Activity of SARTBased on the ethnopharmacological information thatA ama-zonicus is indicated to treat ldquoinflammation of the prostaterdquoldquoinflammation of a womanrdquo and liver disorders that could berelated to inflammatory processes we decided to evaluate thepossible anti-inflammatory activity of this plant in differentmodels

341 Formalin Test The injection of formalin (25) inducesa biphasic licking response in the injected paw of mice Thefirst phase occurs until 5min after injection and the secondphase occurs between 15 and 30min after formalin injection

Pretreatment of mice with 1 3 or 10mgkg SART did notreduce the time that the animal spent licking the formalin-injected paw in the first or second phase (data not shown)

342 SubcutaneousAir Pouch (SAP) To evaluate the possibleanti-inflammatory activity of SART the carrageenan-inducedinflammation model in the SAP was used This modelinvolves synovial inflammation caused by carrageenan injec-tion into the air pouch that forms in the back of mice Thisprocedure induces the proliferation of cells that stratify onthe surfaceThe injection of carrageenan drastically increasedthe exudate volume into the pouch up to twice the level ofmice that received PBS in the SAP In the absence of SART thenumbers of leukocytes in the carrageenan-injected air pouchexudates were markedly increased up to 57-fold higher thancontrol levels Figure 6(a) (125plusmn 11times 106 cellsmL versus 72plusmn124 times 106 cellsmL) These numbers were markedly reducedby approximately 56 24 776 and 47 by pretreatmentwith increasing doses of SARTThe pretreatment ofmicewithSART significantly reduced the volume of exudate recoveredfrom the air pouches Carrageenan treatment also caused a14-fold increase in the exudate protein concentration andthe pretreatment of mice with SART significantly inhibitedcarrageenan-induced protein leakage with all doses testedFigure 6(b) Using an in vivo air pouch inflammation modelwe showed that carrageenan increased both the exudatevolume and the exudate protein concentration which indi-cates vascular leakage of the serum contents Carrageenanis also an important chemotactic agent because it induces


Coun

ts30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PE

M1

M1M1

M1

074 064

966 409

Coun

ts

30

25

20

15

10

5

0

100 104

CD138-PE

CTR SM

Pc Pc + SM

(a)

0

3

6

9

12

15

18

Ctr SM Pc Pc + SM

lowast

CD138 expression

CD13

8+ce

lls (

)

(b)

Figure 5 Levels of antibody-producing (CD138+) cells in the spleen of P chabaudi-infected animals treated with SART Four different groupsCTR SART Pc and Pc + SART were established as detailed in Figure 4 Splenic cells were obtained from individual animals and 106 cellswere labeled with anti-B220-FITC and anti-CD138-PE antibodies Samples were run in a FACScalibur Flow Cytometer Data shown in (a)indicate a typical profile of CD138+ cells in the B220+ lymphoid population in each experimental group The percentage of CD138+ cells isincluded in each graphic Data in (b) indicate the mean value plusmn SD of the percentage of CD138+ cells from each group Six animals were usedper group and data are representative of two independent experiments lowast119875 le 0 05 when compared to the Pc group

0

100

200

300

400

500

PBS Carrageenan

Prot

ein

(mg

mL)

Veh

icle

Veh

icle

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowast

lowast

(a)

Veh

icle

Veh

icle

0

50

100

150

PBS Carrageenan

Leuk

ocyt

es (times

106

cell

mL)

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowastlowast

(b)

Figure 6 Effect of SART on leukocyte migration (a) and protein extravasation (b) into the subcutaneous air pouch Animals were pretreatedwith oral administration of different doses of SART 1 h prior to carrageenan (1) injection into the SAP The results are presented as mean plusmnSD (119899 = 6minus10) of number of leukocyte (times106 cellmL) (a) or protein (mgmL) (b) Statistical significance was calculated by ANOVA followedby Bonferronirsquos test lowast119875 lt 005 when comparing latex-treated mice to the vehicle-treated group 119875 lt 005 when comparing vehicle-treatedmice to the PBS-treated group


the migration of inflammatory cells such as neutrophils andmacrophages [36] In this study SART reduced leukocytemigration exudate volume and protein extravasation evenat a dose of 1mgkg suggesting a suppression of vascularleakage These results show an anti-inflammatory effect forSART which was demonstrated by reduced cell migrationand liquid and protein extravasation Acute inflammationsuch as carrageenan-induced cell migration involves thesynthesis or release of mediators at the injured site Thesemediators include prostaglandins particularly those of the Eseries histamine bradykinins leukotrienes and serotoninall of which also cause pain and fever [37] Inhibiting thesemediators to prevent them from reaching the injured siteand exerting their pharmacological effects will normallyameliorate inflammation and other symptoms This studyhas shown that SART possesses the ability to significantlyaffect cell migration and protein leakage induced by car-rageenan suggesting a possible modulation of some eventsrelated to inflammatory process Because the carrageenan-induced inflammation model is a significant predictive testfor anti-inflammatory agents that act on mediators of acuteinflammation [38 39] these results are an indication thatSART may be effective in treating acute inflammatory dis-orders Moreover this effect could explain the reduction inthe inflammatory processes that accompany malaria whichhave already been discussed for other plant species usedin the treatment of this disease [40] Triterpene saponinshave demonstrated anti-inflammatory properties through theinhibition of iNOS and proinflammatory cytokine expression[41]These compounds exhibited significant xanthine oxidaseinhibitory activity preventing the overaccumulation anddeposition of uric acid in the joints which leads to painfulinflammation [42]

More assays such as assays that determine the dosing ofsome inflammatory mediators are underway to reveal theexact mechanisms of the anti-inflammatory action of SART

35 Acute Toxicity and Reduction of Spontaneous ActivityThe oral administration of a 10mgkg dose of SART did notinduce any toxic effects No behavioral alterations lesionsor gastric bleeding was observed Additionally no signs ofintoxication including convulsion death or gastric ulcerwere observed even after 5 days of a single dose (data notshown) It is noteworthy that the oral administration of SARTdid not show any gastric disorders because the irritation ofthe gastric mucosa is an important side effect that is expectedwith the oral use of saponin-containing herbs such as HorseChestnut Ruscus [43]

4 Conclusions

HPLC-ESI-MSMS analysis of SART showed a complexdammarane-type saponin profile with molecular weights inthe range ofmz 800ndash1000The biological results shown hereindicate that SART possesses immunomodulatory propertiesthat induce an in vivo modification of the B lymphocyteresponse as well as anti-inflammatory properties partly dueto a reduction in cell migration and most likely due to an

inhibition of inflammatory mediator production Thereforethe results presented in this work support our hypothesisthat A amazonicus could act as an adaptogen by enhancingimmune system function and could mitigate the inflam-matory disorders caused by malaria Future studies on thechemical structures of the saponins isolated from SARTand their roles in the B cell response resistance to malariainfection and the anti-inflammatory activity of the extractwill be important

Acknowledgments

The authors are deeply indebted to (Associacao de Comu-nidades Remanescentes de Quilombos do Municıpio de Or-iximina) ARQMO Oriximina PA Brazil for supervisingplant collection and for providing housing during the fieldtrips This work was supported by FAPERJ (E-261103762010) and CNPq (scholarships and grants)

References

[1] R B Lima ldquoFlora da reserva Ducke Amazonas Brasil Rham-naceaerdquo Rodriguesia no 57 pp 247ndash249 2006

[2] J A Ducke and R V MartinezAmazonian Ethnobotanical Dic-tionary CRC Press Boca Raton Fla USA 1994

[3] A U Krettli and V F Andrade-Neto ldquoBusca de antimalaricosna medicina popularrdquo Ciencia Hoje no 35 pp 70ndash73 2004

[4] J Revilla Plantas Uteis Da Bacia Amazonica vol 1 INPASE-BRAE-AM Manaus Brazil 2002

[5] A M S Santos C C Kahwage M R C Ferreira and N ASampaio ldquoMedicinas Tradicionais no Vale do Rio Negro (Ama-zonas Brasil) Observacoes sobre Etnofarmacologia e o Uso daPlanta Saracura-Mira (Ampelozizyphus amazonicus) AtividadeFarmacologica eou Eficacia Simbolicardquo Boletim do MuseuParaense Emılio Goeldi Serie Ciencias Humanas vol 1 pp 137ndash147 2005

[6] J R D A Silva G M Correa R Carvalho et al ldquoAnalyses ofAmpelozizyphus amazonicus a plant used in folk medicine ofthe Amazon Regionrdquo Pharmacognosy Magazine vol 4 no 17pp 75ndash80 2009

[7] M G Brandao M A Lacaille-Dubois M A Teixera and HWagner ldquoTriterpene saponins from the roots ofAmpelozizyphusamazonicusrdquo Phytochemistry vol 31 no 1 pp 352ndash354 1992

[8] M G L BrandaoM A Lacaille-Dubois M A Teixeira andHWagner ldquoA dammarane-type saponin from the roots of Ampel-ozizyphus amazonicusrdquo Phytochemistry vol 34 no 4 pp 1123ndash1127 1993

[9] L V Rosas M S C Cordeiro F R Campos et al ldquoInvitro evaluation of the cytotoxic and trypanocidal activities ofAmpelozizyphus amazonicus (Rhamnaceae)rdquo Brazilian Journalof Medical and Biological Research vol 40 no 5 pp 663ndash6702007

[10] A U Krettli V F Andrade-Neto M D G L Brandao andW M S Ferrari ldquoThe search for new antimalarial drugs fromplants used to treat fever and malaria or plants ramdomlyselected a reviewrdquoMemorias do Instituto Oswaldo Cruz vol 96no 8 pp 1033ndash1042 2001

[11] E Rodrigues ldquoPlants and animals utilized as medicines inthe Jau National Park (JNP) Brazilian Amazonrdquo PhytotherapyResearch vol 20 no 5 pp 378ndash391 2006


[12] D R Oliveira G G Leitao N G Castro M N VieiraARQMO and S G Leitao ldquoEthnomedical Knowledge amongthe ldquoQuilombolasrdquo from the Amazon Region of Brazil witha special focus on plants used as nervous system tonicsrdquo inMedicinal Plants Biodiversity and Drugs vol 1 pp 142ndash178Science Publishers Enfield NH USA 1st edition 2012

[13] V F Andrade-Neto M G L Brandao F Nogueira V ERosario and A U Krettli ldquoAmpelozyziphus amazonicus Ducke(Rhamnaceae) a medicinal plant used to prevent malaria inthe Amazon Region hampers the development of Plasmodiumberghei sporozoitesrdquo International Journal for Parasitology vol38 no 13 pp 1505ndash1511 2008

[14] D R Oliveira A A L M Costa G G Leitao N G CastroJ P Santos and S G Leitao ldquoEstudo etnofarmacognostico dasaracuramira (Ampelozizyphus amazonicusDucke) uma plantamedicinal usada por comunidades quilombolas do Municıpiode Oriximina-PA BrasilrdquoActa Amazonica vol 41 pp 383ndash3922011

[15] Instituto Adolf Lutz (IAL) ldquoNormas analıticas do InstitutoAdolfo LutzmdashMetodos quımicos e fısicos para analise de ali-mentosrdquo Sao Paulo vol 1 p 553 2005

[16] N W Roehm G H Rodgers S M Hatfield and A L Glase-brook ldquoAn improved colorimetric assay for cell proliferationand viability utilizing the tetrazolium salt XTTrdquo Journal ofImmunological Methods vol 142 no 2 pp 257ndash265 1991

[17] J J Mond K Hunter J J Kenny F Finkelman and K With-erspoon ldquo8-Mercaptoguanosine-mediated enhancement of invivo IgG1 IgG2 and IgG3 antibody responses to polysaccharideantigens in normal and xid micerdquo Immunopharmacology vol18 no 3 pp 205ndash212 1989

[18] C M Snapper and W E Paul ldquoB cell stimulatory factor-1(interleukin 4) prepares resting murine B cells to secrete IgG1upon subsequent stimulation with bacterial lipopolysaccha-riderdquo Journal of Immunology vol 139 no 1 pp 10ndash17 1987

[19] NM Gomes CM Rezende S P FontesM EMatheus and PD Fernandes ldquoAntinociceptive activity of Amazonian Copaibaoilsrdquo Journal of Ethnopharmacology vol 109 no 3 pp 486ndash4922007

[20] L J R P Raymundo C C Guilhon D S Alviano et al ldquoChar-acterisation of the anti-inflammatory and antinociceptive activ-ities of the Hyptis pectinata (L) Poit essential oilrdquo Journal ofEthnopharmacology vol 134 no 3 pp 725ndash732 2011

[21] H M T Barros M A L Tannhauser S L Tannhauser andM Tannahuser ldquoEnhanced detection of hyperactivity afterdrug withdrawal with a simple modification of the open-fieldapparatusrdquo Journal of Pharmacological Methods vol 26 no 4pp 269ndash275 1991

[22] G S M Figueiredo R S Zardo B V Silva F A Violante A CPinto and P D Fernandes ldquoConvolutamydine A and syntheticanalogues have antinociceptive properties in micerdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 3 pp 431ndash439

[23] M C M Godoy M R Figuera A E Flores et al ldquo1205722-Adreno-ceptors and 5-HT receptors mediate the antinociceptive effectof new pyrazoles but not of dipyronerdquo European Journal ofPharmacology vol 496 pp 93ndash97 2004

[24] D Lorke ldquoA new approach to practical acute toxicity testingrdquoArchives of Toxicology vol 54 no 4 pp 275ndash287 1983

[25] J J Ma L P Kang W B Zhou H S Yu P Liu and B L MaldquoIdentification and characterization of saponins in extract ofZiziphi spinosae Semen (ZSS) by ultra-performance liquidchromatography-electrospray ionization-quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MSE)rdquo

Journal of Medicinal Plants Research vol 5 no 26 pp 6152ndash6159 2011

[26] J JMond and J F Kokai-Kun ldquoThemultifunctional role of anti-bodies in the protective response to bacterial T cell-independ-ent antigensrdquo Current Topics in Microbiology and Immunologyvol 319 pp 17ndash40 2008

[27] X Song and S Hu ldquoAdjuvant activities of saponins from tra-ditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36 pp4883ndash4890 2009

[28] H Sun L Chen J Wang K Wang and J Zhou ldquoStructure-function relationship of the saponins from the roots of Platy-codon grandiflorum for hemolytic and adjuvant activityrdquo Inter-national Immunopharmacology vol 11 pp 2047ndash2056 2011

[29] A K Hule and A R Juvekar ldquoIn vitro immune response ofsaponin rich fraction of Bacopa monnieri Linnrdquo InternationalJournal of PharmTech Research vol 1 no 4 pp 1032ndash1038 2009

[30] T Von Der Weid N Honarvar and J Langhorne ldquoGene-targeted mice lacking B cells are unable to eliminate a bloodstage malaria infectionrdquo Journal of Immunology vol 156 no 7pp 2510ndash2516 1996

[31] C T Daniel-Ribeiro and G Zanini ldquoAutoimmunity and malar-ia what are they doing togetherrdquo Acta Tropica vol 76 no 3pp 205ndash221 2000

[32] E W Nduati D H L Ng F M Ndungu P Gardner B CUrban and J Langhorne ldquoDistinct kinetics of memory B-cell and plasma-cell responses in peripheral blood followinga blood-stage plasmodium chabaudi infection in micerdquo PLoSONE vol 5 no 11 Article ID e15007 2010

[33] W Ding F Zeng L Xu Y Chen Y Wang and X Wei ldquoBioac-tive dammarane-type saponins from Operculina turpethumrdquoJournal of Natural Products vol 9 no 74 pp 1868ndash1874 2011

[34] M Yoshikawa T Morikawa Y Kashima K Ninomiya and HMatsuda ldquoStructures of new dammarane-type triterpene sapo-nins from the flower buds of Panax notoginseng and hepatopro-tective effects of principal ginseng saponinsrdquo Journal of NaturalProducts vol 66 no 7 pp 922ndash927 2003

[35] Q Le Tran I K Adnyana Y Tezuka T Nagaoka Q K Tranand S Kadota ldquoTriterpene saponins from Vietnamese ginseng(Panax vietnamensis) and their hepatocytoprotective activityrdquoJournal of Natural Products vol 64 no 4 pp 456ndash461 2001

[36] M Guslandi ldquoNitric oxide and inflammatory bowel diseasesrdquoEuropean Journal of Clinical Investigation vol 28 no 11 pp904ndash907 1998

[37] E A Asongalem H S Foyet S Ekobo T Dimo and PKamtchouing ldquoAntiinflammatory lack of central analgesia andantipyretic properties of Acanthus montanus (Ness) T Ander-sonrdquo Journal of Ethnopharmacology vol 95 no 1 pp 63ndash682004

[38] W R Sawadogo R Boly M Lompo et al ldquoAnti-inflammatoryanalgesic and antipyretic activities of dicliptera verticillatardquoInternational Journal of Pharmacology vol 2 no 4 pp 435ndash4382006

[39] G J XuThe Chinese Materia Medica vol 1 Chinese Medicineand Technology Press Beijing China 1996

[40] P M Magalhaes I Dupont A Hendrickx et al ldquoAnti-inflam-matory effect and modulation of cytochrome P450 activities byArtemisia annua tea infusions in human intestinal Caco-2 cellsrdquoFood Chemistry vol 134 pp 864ndash871 2012

[41] Y Lee J Jung Z Ali I A Khan and S Oh ldquoAnti-inflammatoryeffect of triterpene saponins isolated from blue cohosh (Caulo-phyllum thalictroides)rdquo Evidence-Based Complementary andAlternative Medicine vol 2012 Article ID 798192 8 pages 2012


[42] L P LinWQu and J Y Liang ldquoTriterpene saponins with XODinhibitory activity from the roots of Ilex pubescensrdquo ChineseChemical Letters vol 22 no 6 pp 697ndash700 2011

[43] S Mills and H Bone Principles and practice of phytother-apy modern herbal medicine Churchill livingstone TorontoCanada 2000

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


be explained by the high saponin content in the speciesIndeed Silva et al [6] showed the presence of approximately48 saponins in an aqueous extract from the roots of theplant So far only three saponins with a dammarane-typeaglycone have been described in the literature [7 8] as wellas the presence of free triterpenes such as melaleucic acid312057327120572-dihydroxylup-20(29)-en-28120573-oic acid betulinic acidbetulin lupeol and phytosteroids [7ndash9]

Several ethnobotanical studies have shown that A ama-zonicus is useful in the treatment and prevention of malaria[2 3 10ndash12] Previous investigations of the antimalarial prop-erties of an extract from this plant have shown that it does nothave a direct action on Plasmodium blood stage forms eitherin vivo or in red blood cell cultures [13] However this naturalproduct could be effective in controlling infection induced bysporozoite forms [13] Based on the findings that A amazon-icus does not have a direct effect upon blood stage forms ofthe protozoan it might be possible to suggest that the controlof the infection induced by this plant could be obtained byan overall augmentation of the immunological response Infact ethnopharmacological studies indicate both stimulatoryand energetic properties for A amazonicus [5 12] In anethnopharmacological study conducted by our group in theldquoquilombolardquo communities ofOriximina State of Para Brazilthe plant is used in the treatment of liver disorders gastritisinflammation of the prostate and ldquoinflammation of awomanrdquoand as a fortifying tonic and aphrodisiac among other uses[12 14] In the Rio Negro valley and Jau National Park inthe state of Amazonas A amazonicus is used against rheu-matism and other types of pain and for the general treatmentof inflammation [5 11]

Based on the reported properties of this plant and its usesin folkmedicine our group suggests thatA amazonicus couldact as an adaptogen by enhancing immune system functionand could alleviate the inflammatory disorders caused bymalaria In the present work we aimed to investigate thetoxicity of A amazonicus and its effects on the immuneresponse as well as its anti-inflammatory properties

2 Material and Methods

21 Plant Material and Preparation of Extracts Ampel-ozizyphus amazonicus Ducke was collected in August2008 in the Brazilian Amazon region of Oriximina (Parastate) at the Pancada community (S 01∘04101584009410158401015840 and W056∘02101584040910158401015840) Plants were collected as a part of a bio-prospecting project in ldquoquilombolardquo communities fromOrix-imina that received authorization by the Brazilian Direct-ing Council of Genetic Heritage (Conselho de Gestaodo Patrimonio Genetico) through Resolution number 213(6122007) published in the Federal Official Gazette of Brazilon December 27 2007 Plants were identified by Mr JoseRamos (parataxonomist) A voucher specimenwas depositedat the Instituto Nacional de Pesquisas da Amazonia INPAherbarium (Manaus AMBrazil) under the registration INPA224161

Dried and ground bark (250 g) of A amazonicus wasused for the preparation of the extracts The bark was

submitted to extraction with boiling water (5 wv) for 15minutes and filtered A second extraction was performedwith boiling water (25 wv 30 minutes) The extracts weremixed and infused into a spray-dryer nozzle unit of BuchiMini Spray Dryer B-290 (Buchi Laboratorius-Technik AGSwitzerland)The conditions of the spray-drying processwereas follows nozzle diameter 03mm aspirator pressure 80flow rate 6mLmin inlet temperature 190 plusmn 3∘C and outlettemperature 88 plusmn 15∘C The atomized powder was collectedby a cyclone and is designated as SART throughout the text

22 Estimation of Daily Dose for Animal Assays The dailyoral dose of A amazonicus (SART) used was determinedbased on its traditional use A drink was prepared accordingto the ldquoquilombolardquo traditional method as described byOliveira et al [14] Briefly one tablespoon of ground bark(83980 g) was added to 200mL of cold water and ldquoshakenrdquoseven times The foam produced after each shaken was dis-cardedThe extractwas filtered and a total solid yield of 021(wv) was obtained [15] Considering that the prophylacticuse of the drink (to prevent from malaria) is 0630 gdayor 300mLday of extract at 021 (wv) of total solid yieldthe daily oral dose for an adult weighing 70 kg would be9mgKgTherefore all biological assays were standardized toa 10mgkg oral dose of SART as obtained by spray dryer

23 HPLC-DAD Profile of SART HPLC analysis of SART(aqueous atomized extract of A amazonicus 2mgmL inacetonitrile water 95 5) was performed on a VWR-HitachiElite LaChrom system consisting of an L-2130 quaternarypumpwith online degasser a Rheodyne injector (20 120583L loop)L-2455 diode array detector (DAD) equipped with a Zic-Hilic PEEK column (SeQuant-Merck 250 times 46mm id par-ticle size 5120583m 200A) coupled with a security guard column(4 times 300mm id C-18 cartridge) The elution solvents usedwere ammonium acetate 10mM pH 58 corrected with aceticacid (solvent A) and acetonitrile (solvent B) and the flow ratewas 05mLmin detectionDAD (210 nm)The elutionwas asfollows 0ndash10min isocratic 4 (solvent A) 96 (solvent B)101ndash65min linear gradient where solvent B decreased from96 to 50 Betulinic acid from SIGMA (0125mgmL inacetonitrile water 95 5) was used as standard

24 HPLC-DAD-ESI-MS Preliminary Profile of SART AnHPLC system including a Surveyor Autosampler a SurveyorLC pump and an LCQ Advantage ion-trap mass spectrome-ter (Thermo Finningan San Jose CA USA) equipped withan electrospray ion source was used for the preliminaryanalysis of SART Chromatographic conditions were thesame reported above MS data were acquired in negativeionization mode and the spectra were recorded in full scan(mz 250ndash1500) and tandem mass (collision energy of 40of the instrument maximum) scanning modes Instrumentalparameters were optimized using a purified saponin mixtureisolated from SART by countercurrent chromatography (datanot shown) Saponin mixture was dissolved (14 120583gsdotmLminus1) inACN H

2O (1 1 vv) and infused in the ESI source at the flow

rate of 5120583gsdotminminus1 by a syringe pump





2Incubator


450



















119877







8H14O2]minus)








9H16O2-






400

200

00 10 20 30 40 50 60

Minutes

mA

U

400

200

0

mA

U

DAD-CH1 210 nm

(a)

0 5 10 15 20 25 30 35 40Time (min)

1210

8642

Inte

nsity

times 106

(b)

1210

8642

Inte

nsity

Time (min)

times 106

2849 3063107

3199 3338

3501

357

3708

37783872

39184012 4059

28 29 30 31 32 33 34 35 36 37 38 39 40 41

(c)





Ctr deadCtr viable

0

1

2

3

150 50 167 55 19 06 03 01 003 001

48 hs

mdash mdash

A 450



2


450

) are shown



0

2000

4000

6000

8000

CTR SM TF TF + SM

0

1000

2000

3000

4000

CTR SM TF TF + SM

0

1000

2000

3000

4000


2000

4000

6000

8000

CTR SM TF TF + SM0

2000

4000

6000

8000

CTR SM TF TF + SM0

4000

8000

12000

16000

lowast

Day 7 Day 7

Day 14 Day 14

Day 21 Day 21

Seru

m an

ti-TN

P-Fi

coll

titer

sSe

rum

anti-

TNP-

Fico

ll tit

ers

Seru

m an

ti-TN

P-Fi

coll

titer

s

(a) IgM (b) IgG






0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

G (m

gm

L)

0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

M (

gm

L) Day 5

Day 5

Day 8

Day 8 lowast

lowast










Coun

ts30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PE

M1

M1M1

M1

074 064

966 409

Coun

ts

30

25

20

15

10

5

0

100 104

CD138-PE

CTR SM

Pc Pc + SM

(a)

0

3

6

9

12

15

18

Ctr SM Pc Pc + SM

lowast

CD138 expression

CD13

8+ce

lls (

)

(b)


0

100

200

300

400

500

PBS Carrageenan

Prot

ein

(mg

mL)

Veh

icle

Veh

icle

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowast

lowast

(a)

Veh

icle

Veh

icle

0

50

100

150

PBS Carrageenan

Leuk

ocyt

es (times

106

cell

mL)

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowastlowast

(b)






4 Conclusions



Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2Incubator


450



















119877







8H14O2]minus)








9H16O2-






400

200

00 10 20 30 40 50 60

Minutes

mA

U

400

200

0

mA

U

DAD-CH1 210 nm

(a)

0 5 10 15 20 25 30 35 40Time (min)

1210

8642

Inte

nsity

times 106

(b)

1210

8642

Inte

nsity

Time (min)

times 106

2849 3063107

3199 3338

3501

357

3708

37783872

39184012 4059

28 29 30 31 32 33 34 35 36 37 38 39 40 41

(c)





Ctr deadCtr viable

0

1

2

3

150 50 167 55 19 06 03 01 003 001

48 hs

mdash mdash

A 450



2


450

) are shown



0

2000

4000

6000

8000

CTR SM TF TF + SM

0

1000

2000

3000

4000

CTR SM TF TF + SM

0

1000

2000

3000

4000


2000

4000

6000

8000

CTR SM TF TF + SM0

2000

4000

6000

8000

CTR SM TF TF + SM0

4000

8000

12000

16000

lowast

Day 7 Day 7

Day 14 Day 14

Day 21 Day 21

Seru

m an

ti-TN

P-Fi

coll

titer

sSe

rum

anti-

TNP-

Fico

ll tit

ers

Seru

m an

ti-TN

P-Fi

coll

titer

s

(a) IgM (b) IgG






0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

G (m

gm

L)

0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

M (

gm

L) Day 5

Day 5

Day 8

Day 8 lowast

lowast










Coun

ts30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PE

M1

M1M1

M1

074 064

966 409

Coun

ts

30

25

20

15

10

5

0

100 104

CD138-PE

CTR SM

Pc Pc + SM

(a)

0

3

6

9

12

15

18

Ctr SM Pc Pc + SM

lowast

CD138 expression

CD13

8+ce

lls (

)

(b)


0

100

200

300

400

500

PBS Carrageenan

Prot

ein

(mg

mL)

Veh

icle

Veh

icle

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowast

lowast

(a)

Veh

icle

Veh

icle

0

50

100

150

PBS Carrageenan

Leuk

ocyt

es (times

106

cell

mL)

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowastlowast

(b)






4 Conclusions



Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









119877







8H14O2]minus)








9H16O2-






400

200

00 10 20 30 40 50 60

Minutes

mA

U

400

200

0

mA

U

DAD-CH1 210 nm

(a)

0 5 10 15 20 25 30 35 40Time (min)

1210

8642

Inte

nsity

times 106

(b)

1210

8642

Inte

nsity

Time (min)

times 106

2849 3063107

3199 3338

3501

357

3708

37783872

39184012 4059

28 29 30 31 32 33 34 35 36 37 38 39 40 41

(c)





Ctr deadCtr viable

0

1

2

3

150 50 167 55 19 06 03 01 003 001

48 hs

mdash mdash

A 450



2


450

) are shown



0

2000

4000

6000

8000

CTR SM TF TF + SM

0

1000

2000

3000

4000

CTR SM TF TF + SM

0

1000

2000

3000

4000


2000

4000

6000

8000

CTR SM TF TF + SM0

2000

4000

6000

8000

CTR SM TF TF + SM0

4000

8000

12000

16000

lowast

Day 7 Day 7

Day 14 Day 14

Day 21 Day 21

Seru

m an

ti-TN

P-Fi

coll

titer

sSe

rum

anti-

TNP-

Fico

ll tit

ers

Seru

m an

ti-TN

P-Fi

coll

titer

s

(a) IgM (b) IgG






0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

G (m

gm

L)

0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

M (

gm

L) Day 5

Day 5

Day 8

Day 8 lowast

lowast










Coun

ts30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PE

M1

M1M1

M1

074 064

966 409

Coun

ts

30

25

20

15

10

5

0

100 104

CD138-PE

CTR SM

Pc Pc + SM

(a)

0

3

6

9

12

15

18

Ctr SM Pc Pc + SM

lowast

CD138 expression

CD13

8+ce

lls (

)

(b)


0

100

200

300

400

500

PBS Carrageenan

Prot

ein

(mg

mL)

Veh

icle

Veh

icle

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowast

lowast

(a)

Veh

icle

Veh

icle

0

50

100

150

PBS Carrageenan

Leuk

ocyt

es (times

106

cell

mL)

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowastlowast

(b)






4 Conclusions



Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


400

200

00 10 20 30 40 50 60

Minutes

mA

U

400

200

0

mA

U

DAD-CH1 210 nm

(a)

0 5 10 15 20 25 30 35 40Time (min)

1210

8642

Inte

nsity

times 106

(b)

1210

8642

Inte

nsity

Time (min)

times 106

2849 3063107

3199 3338

3501

357

3708

37783872

39184012 4059

28 29 30 31 32 33 34 35 36 37 38 39 40 41

(c)





Ctr deadCtr viable

0

1

2

3

150 50 167 55 19 06 03 01 003 001

48 hs

mdash mdash

A 450



2


450

) are shown



0

2000

4000

6000

8000

CTR SM TF TF + SM

0

1000

2000

3000

4000

CTR SM TF TF + SM

0

1000

2000

3000

4000


2000

4000

6000

8000

CTR SM TF TF + SM0

2000

4000

6000

8000

CTR SM TF TF + SM0

4000

8000

12000

16000

lowast

Day 7 Day 7

Day 14 Day 14

Day 21 Day 21

Seru

m an

ti-TN

P-Fi

coll

titer

sSe

rum

anti-

TNP-

Fico

ll tit

ers

Seru

m an

ti-TN

P-Fi

coll

titer

s

(a) IgM (b) IgG






0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

G (m

gm

L)

0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

M (

gm

L) Day 5

Day 5

Day 8

Day 8 lowast

lowast










Coun

ts30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PE

M1

M1M1

M1

074 064

966 409

Coun

ts

30

25

20

15

10

5

0

100 104

CD138-PE

CTR SM

Pc Pc + SM

(a)

0

3

6

9

12

15

18

Ctr SM Pc Pc + SM

lowast

CD138 expression

CD13

8+ce

lls (

)

(b)


0

100

200

300

400

500

PBS Carrageenan

Prot

ein

(mg

mL)

Veh

icle

Veh

icle

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowast

lowast

(a)

Veh

icle

Veh

icle

0

50

100

150

PBS Carrageenan

Leuk

ocyt

es (times

106

cell

mL)

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowastlowast

(b)






4 Conclusions



Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

2000

4000

6000

8000

CTR SM TF TF + SM

0

1000

2000

3000

4000

CTR SM TF TF + SM

0

1000

2000

3000

4000


2000

4000

6000

8000

CTR SM TF TF + SM0

2000

4000

6000

8000

CTR SM TF TF + SM0

4000

8000

12000

16000

lowast

Day 7 Day 7

Day 14 Day 14

Day 21 Day 21

Seru

m an

ti-TN

P-Fi

coll

titer

sSe

rum

anti-

TNP-

Fico

ll tit

ers

Seru

m an

ti-TN

P-Fi

coll

titer

s

(a) IgM (b) IgG






0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

G (m

gm

L)

0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

M (

gm

L) Day 5

Day 5

Day 8

Day 8 lowast

lowast










Coun

ts30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PE

M1

M1M1

M1

074 064

966 409

Coun

ts

30

25

20

15

10

5

0

100 104

CD138-PE

CTR SM

Pc Pc + SM

(a)

0

3

6

9

12

15

18

Ctr SM Pc Pc + SM

lowast

CD138 expression

CD13

8+ce

lls (

)

(b)


0

100

200

300

400

500

PBS Carrageenan

Prot

ein

(mg

mL)

Veh

icle

Veh

icle

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowast

lowast

(a)

Veh

icle

Veh

icle

0

50

100

150

PBS Carrageenan

Leuk

ocyt

es (times

106

cell

mL)

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowastlowast

(b)






4 Conclusions



Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

G (m

gm

L)

0

400

800

1200

CTR SM Pc Pc + SM

0

7

14

21

28

35

CTR SM Pc Pc + SM

Tota

l ser

um Ig

M (

gm

L) Day 5

Day 5

Day 8

Day 8 lowast

lowast










Coun

ts30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PE

M1

M1M1

M1

074 064

966 409

Coun

ts

30

25

20

15

10

5

0

100 104

CD138-PE

CTR SM

Pc Pc + SM

(a)

0

3

6

9

12

15

18

Ctr SM Pc Pc + SM

lowast

CD138 expression

CD13

8+ce

lls (

)

(b)


0

100

200

300

400

500

PBS Carrageenan

Prot

ein

(mg

mL)

Veh

icle

Veh

icle

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowast

lowast

(a)

Veh

icle

Veh

icle

0

50

100

150

PBS Carrageenan

Leuk

ocyt

es (times

106

cell

mL)

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowastlowast

(b)






4 Conclusions



Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Coun

ts30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PECo

unts

30

25

20

15

10

5

0

100 104

CD138-PE

M1

M1M1

M1

074 064

966 409

Coun

ts

30

25

20

15

10

5

0

100 104

CD138-PE

CTR SM

Pc Pc + SM

(a)

0

3

6

9

12

15

18

Ctr SM Pc Pc + SM

lowast

CD138 expression

CD13

8+ce

lls (

)

(b)


0

100

200

300

400

500

PBS Carrageenan

Prot

ein

(mg

mL)

Veh

icle

Veh

icle

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowast

lowast

(a)

Veh

icle

Veh

icle

0

50

100

150

PBS Carrageenan

Leuk

ocyt

es (times

106

cell

mL)

1 mg

kg

3 mg

kg

10 m

gkg

lowast

lowastlowast

(b)






4 Conclusions



Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





4 Conclusions



Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Immunobiologic and …downloads.hindawi.com/journals/bmri/2013/451679.pdfResearch Article Immunobiologic and Antiinflammatory Properties of a Bark Extract from Ampelozizyphus