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Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 862763 8 pageshttpdxdoiorg1011552013862763

Research ArticlePhytochemical and Antibacterial Investigations of the Extractsand Fractions from the Stem Bark of Hymenaea stigonocarpaMart ex Hayne and Effect on Ultrastructure of Staphylococcusaureus Induced by Hydroalcoholic Extract

Gustavo Santiago Dimech1 Luiz Alberto Lira Soares2

Magda Assunccedilatildeo Ferreira2 Anne Gabrielle Vasconcelos de Oliveira3

Maria da Conceiccedilatildeo Carvalho3 and Eulaacutelia Azevedo Ximenes1

1 Laboratorio de Fisiologia e Bioquımica de Microorganismos Departamento de Antibioticos Centro de Ciencias BiologicasUniversidade Federal de Pernambuco 50670-901 Recife PE Brazil

2 Laboratorio de Farmacognosia Departamento de Farmacia Universidade Federal de PernambucoCentro de Ciencias da Saude 50670-901 Recife PE Brazil

3 Laboratorio de Biologia Celular e Ultraestrutura Centro de Tecnologias Estrategicas do Nordeste (CETENE)50670-901 Recife PE Brazil

Correspondence should be addressed to Eulalia Azevedo Ximenes eulaliaximenesyahoocombr

Received 2 October 2013 Accepted 13 November 2013

Academic Editors J B Gurtler and Z Shi

Copyright copy 2013 Gustavo Santiago Dimech 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

The aim of this study was to investigate the antimicrobial activity of different extracts and fractions obtained from Hymenaeastigonocarpa stem barks The cyclohexanic ethyl acetate ethanol aqueous and hydroalcoholic extracts were obtained bymaceration The hydroalcoholic extract was partitioned which resulted in the ethyl acetate and aqueous fractions All extractsand fractions were subjected to phytochemical screening and evaluation of total phenol and tannin contents An HPLC-DAD andultrastructural alterations analysis were performed Terpenes and coumarins were detected in the cyclohexanic extract Flavonoidsand condensed tannins were present in the other extracts and fractions The extracts with the highest contents of tannins ethanol(EE) hydroalcoholic (HE) and aqueous fraction (AF) showed also the highest antimicrobial activity TheMIC values ranged from64 to 526 120583gmLThe chromatographic fingerprints suggest the presence of astilbin and other flavonoids in EE and HE Presence ofthe thick cell wall undulating outer layer abnormal septa and leakage of the cytoplasmic contents and absence of cell wall and celllyses were the main alterations observed on Staphylococcus aureus ATCC 33591 after treatment with the Hymenaea stigonocarpahydroalcoholic extractThe presence of phenolic compounds like flavonoids and tannins is possibly the reason for the antimicrobialactivity

1 Introduction

Nosocomial infections caused by a multidrug resistant phe-notype posed a major public health problem in the lastdecades [1] These infections cause a prolonged period ofhospitalization and high mortality rates [2]

It is estimated that nearly 16 billion dollars are beingspent annually to treat the nosocomial infections caused

by multidrug resistant microorganisms whose therapy islimited to the use of few antimicrobial agents which are oftenineffective [3] Indeed the search of antimicrobial agents isan important strategy for the establishment of new alter-native therapies in infections which are difficult to handle[4]

Natural products derived from bacteria algae plantsand animals are known to produce a variety of secondary

2 The Scientific World Journal

metabolites and are promising sources of new therapeuticagents including antimicrobials [5]

Approximately 60 to 80 of the worldrsquos populationstill relies on traditional medicines for treatment of severalillnesses In Brazil plants are used as naturalmedicinemainlyby low-income populations and contribute significantly toprimary health care [6]

Hymenaea stigonocarpa Mart ex Hayne (Fabaceae) isa medicinal plant found in the Brazilian savannah andpopularly known as ldquoJatoba-do-cerradordquo [7] Its stem barksare widely used in infusion or decoction to treat stomachpain asthma bronchitis ulcers diarrhea flu and cough [8ndash10]

Phytochemical studies ofHymenaea stigonocarpa showedthe presence of terpenes and sesquiterpenes fatty acidsflavonoids and tannins These metabolites are recognized byits biological activities [11ndash13]

Little research has focused on the pharmacological activ-ity from Hymenaea stigonocarpa stem barks

The antidiarrheal activity and healing properties of theH stigonocarpa stem bark methanolic extract were recentlydemonstrated on experimental gastric and duodenal ulcers[13] However studies on the antimicrobial activity fromHymenaea stigonocarpa stem barks have not yet been pub-lished

Based on this report the aim of the present work was toperform a phytochemical and antibacterial study of extractsand fractions from the Hymenaea stigonocarpa stem barkto establish the fingerprint profile by HPLC-DAD and toidentify the ultrastructural alterations caused by the hydroal-coholic extract on Staphylococcus aureusmethicillin resistantstrain

2 Material and Methods

21 Plant Material Stem barks fromHymenaea stigonocarpaMart ex Hayne were collected in Camocim de Sao Felixcity Pernambuco Brazil (latitude 8∘211015840300010158401015840S and longitude35∘471015840567010158401015840W) A voucher specimen was deposited in theherbariumDardano de Andrade Lima-Instituto Agronomicode Pernambuco (IPA) and registered under number 53563

22 Extracts Preparation The stem barks were air-driedpowdered using a hammer mill (Tigre ASN5) and stored inamber bottles at room temperature Serial exhaustive mac-eration 50 g (1 2 wv) using cyclohexane (CE) ethyl acetate(EAE) ethanol (EE) and water (AE) was performed Ahydroalcoholic extract (HE) was also obtained bymacerationof 60 g (1 2 wv) in ethanolwater (1 1) The hydroalcoholicextract dry residue (5 g) was partitioned (1 1) with water andethyl acetate to obtain the aqueous (AF) and ethyl acetate(EAF) fractions All extracts and fractions were filteredthrough a Whatman No 1 paper filter and evaporated at50∘C under reduced pressure except the aqueous extract andfraction which were lyophilized

23 Phytochemical Screening The phytochemical screen-ing was carried out by thin layer chromatography (TLC)

on silica gel (Merck Germany) following the proceduresdescribed by Wagner and Harborne [14 15] 15120583L aliquotsof each extract solution (05wv) were applied to evaluatethe presence of monoterpenes sesquiterpenes triterpenessteroids alkaloids iridoids coumarins saponins hydrolyzedand condensed tannins phenylpropanoids flavonoids andsugars

231 Total Phenol Contents Total of phenol contents (TPC)was performed in accordance with Folin-Ciocalteu methodas described in the European Pharmacopoeia [16]

The extracts and fractions of the Hymenaea stigonocarpastem bark were solubilized in ethanol (10) to obtain afinal concentration of 05mgmL Five milliliters of eachsolution was diluted to 25mL with distilled water Fromthese solutions 2mL was transferred to a 25mL volumetricflask containing distilled water (10mL) and added to 1mL ofundiluted Folin-Ciocalteu reagent so the volume was madeto 25mL with 10 aqueous Na

2CO3(wv) The blend was

left at room temperature for 30min Then absorbance of thesamples was read at 760 nm in an UVVis spectrophotometer(Evolution 60S Thermo Scientific) Distilled water was usedas a blank

TPC was determined from the extrapolation of thecalibration curve (119910 = 01037119909+00024 1198772 = 09992) whichwas obtained from gallic acid (Sigma Chemical Co St LouisUSA) solution (20ndash80 120583gmL)

The determination of total phenol contents was carriedout in triplicate The TPC was expressed as milligrams ofgallic acid equivalents (GAE) per gram of dried samples

232 Total Tannin Contents In order to determine the totaltannins casein (100mg)was added to 10mLof each extract orfraction solution and homogenized for 60min These blendswere filtered through a Whatman No 1 paper filter Theprocedure for the determination of tanninswas similar to thatdescribed above

The phenol contents in the nontannin fractions werecalculated by standard curve of gallic acid The total tannincontent was calculated as follows total tannins = totalphenols minus nontannin phenols [17]

24 High Performance Liquid Chromatography AnalysisChromatographic fingerprints of Hymenaea stigonocarpastem bark extracts and fractions that present the highesttannin contents and antibacterial activity (EE HE and AF)were obtained by high performance liquid chromatographycoupled with diode array detection (HPLC-DAD)

Extracts and fractions were solubilized in methanol toobtain solutions at the 400120583gmL concentration Astilbinsolution was used as a standard at a concentration of25 120583gmL Then these solutions were filtered through a020120583m polytetrafluoroethylene membrane (Chromafil) andstored in amber vials

Samples analysis was performed on a HPLC-DAD device(Ultimate 3000 Thermo Scientific) equipped with binarypump HPG-3x00RS 3000 (RS) diode array detector andACC-3000 autosampler

The Scientific World Journal 3

Separations were achieved with a column (C18) 250 times46mm id 5120583m (Acclaim 120 Dionex equipped with aguard column (C18 4 times 3mm id Phenomenex)

The mobile phase was composed of ultrapure water(eluent A MilliQ) and acetonitrile (eluent B Tedia) bothacidified with 01 trifluoroacetic acid solution and rangingfrom 10ndash23 in 35min to 23ndash90 in 50min of eluent B at alow rate of 1mLminThe Chromeleon 60 (Dionex) softwarewas used for data acquisition and processing

25 Determination of Antibacterial Activity

251 Bacterial Strains and Inocula Standardization Bacteria(119899 = 28) were obtained from stock cultures and maintainedat our laboratory (Laboratorio de Fisiologia e Bioquımica deMicroorganismos (LFBM))

For this study we used the following bacterial strains iso-lated from clinical specimens Staphylococcus aureus (LFBM05 LFBM 11 LFBM 13 LFBM 15 LFBM 16 LFBM 25 LFBM26 LFBM 28 LFBM 29 and LFBM 30) Enterococcus faecalis(LFBM 02 LFBM 07 LFBM 08 and LFBM 18) Klebsiellapneumoniae LFBM 01 Pseudomonas aeruginosa LFBM 01Proteus mirabilis LFBM 01 and LFBM 02 Salmonella entericaLFBM 01 Shigella sonnei LFBM 01 and Vibrio cholerae LFBM01

Staphylococcus aureus ATCC 25923 Staphylococcusaureus ATCC 33591 Klebsiella pneumoniae ATCC 700603Pseudomonas aeruginosa ATCC 9027 Escherichia coli ATCC25922 and Escherichia coli ATCC 35218 were used asstandard strains

These microorganisms were cultured onto Mueller-Hinton agar (MHA) (Acumedia Manufacturers BaltimoreUSA) and incubated at 37∘C for 18 hours Single colonieswere selected and inoculated into Mueller-Hinton broth(Acumedia Manufacturers Baltimore USA) with turbiditycomparable to that of 05 McFarland standards which isequivalent to a bacterial count of approximately 108 CFUmLIn sequence these bacterial suspensions were diluted toobtain a final inoculum of 107 CFUmL

252 Antibacterial Agents Eurofarma Laboratorio and San-ofi-Aventis provided oxacillin ciprofloxacin teicoplanin andpolymyxin B respectively Resistance was defined for eachcase oxacillin (OXA MIC ge 4120583gmL) ciprofloxacin (CIPMIC ge 1 120583gmL) teicoplanin (TEI ge 32) and polymyxin B(POL MIC ge 8120583gmL) according to the criteria establishedby the Clinical Laboratory Standard Institute [18]

The CE and EAE were solubilized in dimethyl sul-foxideTween80water (118) EE AE HE EAF and AFin ethanolwater (19) while the antimicrobial agents weresolubilized in water The stock solutions were sterilized byfiltration through a Millipore membrane with a 022mMporosity

253 Antibacterial Activity Theminimal inhibitory concen-tration (MIC) determination was performed by brothmicrodilution method following the recommendationsestablished by CLSI 2012 with some modifications Serial

twofold dilutions of extracts fractions and antimicrobialagents were prepared in sterile 96-well microplatescontaining Mueller-Hinton broth (MHB) Five microliters ofthe bacterial suspension was inoculated in each well to givea final concentration of 104 CFU The concentrations rangedfrom 1024 to 0125 120583gmL for all antimicrobial agents Wellscontaining Mueller-Hinton broth with added solvents wereused as a negative control The plates were incubated at 37∘Cfor 24 h Bacterial viability was detected by adding 20 120583Lof 235-triphenyl-2H-tetrazolium chloride (TTC) 05 inaqueous solution The plates were reincubated at 36∘C for2 h and in those wells where bacterial growth occurredthe TTC changed to red The absence of a color changeindicated absence of growth MIC was defined as the lowestconcentration of antibacterial agents that inhibited visiblegrowth as indicated by TTC staining All experiments werecarried out in duplicate

26 Electron TransmissionMicroscopy Staphylococcus aureusATCC 33591 was cultured in Mueller-Hinton broth (control)and inMueller-Hinton broth added to theHymenaea stigono-carpa hydroalcoholic extract at 256 or 128120583gmL (MIC and12 MIC) respectively

The cultures were incubated at 37∘C for 24 hours Thesecultures were centrifuged and the pellets were fixed in 4paraformaldehyde and 25 glutaraldehyde in 01M sodiumcacodylate buffer (pH 72) for 4 hours at room temperature

Then pellets were washed and centrifuged for 10 minutesin the same buffer This procedure was repeated three timesThe pellets were postfixed in 2 osmium tetroxide 08potassium ferricyanide and 5mM calcium chloride in 01Msodium cacodylate buffer for 1 hour and washed in sodiumcacodylate buffer The samples were dehydrated through agradient of acetone solutions and embedded in Epon 812 resin(SigmaUSA) Subsequently ultrathin sectionswere obtainedusing an ultramicrotome (Leica UC6) stained with uranylacetate and lead citrate and then examined under aMorgani-FEI transmission electron microscope (TEM)

27 Statistical Analysis The statistical analysis was madethrough a one-way ANOVA and Newman-Keuls multiplecomparison test using GraphPad Prism version 50 forWindows GraphPad software (San Diego CA USA) and119875 lt 005 was used as the level of significance

3 Results and Discussion

31 Phytochemical Screening The phytochemical profile ofthe Hymenaea stigonocarpa stem bark showed the presenceof terpenes and coumarins in the cyclohexanic extract (CE)flavonoids and condensed tannins in ethyl acetate (EAE)and ethanolic (EE) aqueous (AE) and hydroalcoholic (HE)extracts in the ethyl acetate fraction (EAF)The aqueous frac-tion (AF) showed only the presence of condensed tanninsThese results are in accordance with studies that demon-strated the presence of flavonoids terpenes steroids andtannins in the heartwood stem bark and seeds ofHymenaeastigonocarpa [11ndash13 19]


Table 1 Yields total phenols and tannins from Hymenaea stigonocarpa extracts and fractions

Extractsfractions Yield () Total phenol content Total tanninCyclohexane (CE) 035 plusmn 002 000 000Ethyl acetate (EAE) 265 plusmn 022 663 plusmn 013 182 plusmn 017

Ethanolic (EE) 1199 plusmn 075 726 plusmn 004 276 plusmn 010lowast

Aqueous (AE) 209 plusmn 006 495 plusmn 002 146 plusmn 006

Hydroalcoholic (HE) 1549 plusmn 056 696 plusmn 009 296 plusmn 008lowast

Aqueous (AF) 5700 plusmn 500 616 plusmn 003 271 plusmn 005lowast

Ethyl acetate (EAF) 2210 plusmn 220 712 plusmn 003 200 plusmn 004

Yield expressed in percentage ()Total phenols and total tannin contents expressed in mg GAEg of dried sampleslowastMeans are not significantly different (119875 lt 005)

50 at 2967 min 99984minus50 at 2993 min 99986

minus100

200

400

600

190 250 300 350 400

()

(nm)

1983

2902

(A)


400

1984

1912 2905

minus100

200

400

600

190 250 300 350

()

(nm)

(B)


1975

19312939

minus100

200

400

600

190 250 300 350 400

()

(nm)

(C)


1910 2943

minus100

200

400

600

190 250 300 350 400

()

(nm)

(D)

HE + astilbin

HE

EE

AF

Mill

iabs

orba

nce u

nits

(mAU

)

00 50 100 150 200 250Time (min)

A

A

AE

E

E

E

B

BC

C

CD

D

D

B

300 350 400 450 500minus100

minus50

0

50

120WVL280 nm

Figure 1 Chromatographic fingerprints from Hymenaea stigonocarpa stem barks Hydroalcoholic extract (HE) + astilbin (98)hydroalcoholic extract (HE) ethanolic extract (EE) and aqueous fraction (AF) obtained by HPLC DAD at 280 nm Each chromatographicpeak appears with its corresponding retention time UV spectrum pattern derivatives flavonoids (A C and D) identified as astilbin (B)

The presence of terpenes steroids and mainly polyphe-nols was described previously in Hymenaea courbarilHymenaea parvifolia Hymenaea palustris and Hymenaeamartiana [20ndash24]

The yields expressed regarding dry weight plant materialas well as the total phenols and tannin contents are shownin Table 1 The yield of the extracts and fractions varied from570 plusmn 500 to 035 plusmn 002 with a descending order of AF gtEAF gtHE gt EE gt EAE gt AE gt CE

The partition of hydroalcoholic extract with waterresulted in the highest amount of total extractive compoundsTotal phenols content ranged from 495plusmn002 to 712plusmn003mg

GAEg of dried samplesThese results indicate a high contentof these bioactive compounds in the polar extractsThe TPCsare about 20 to 29 times higher than the value found inethanolic extract from Hymenaea stigonocarpa wood [19]

32 HPLC-DAD Analysis The EE HE and AF that showedthe highest tannin contents were selected to be analyzedby HPLC-DAD The chromatography fingerprints of theseextracts are showed in Figure 1

Peaks (A B C and D) presented in all chromatogramseluted in the retention time from 29 to 36min are suggestive


of flavonoid derivativesThese resultats are in accordancewithliterature data [25]

Peak B (119877119905= 31453) was identified as astilbinThis iden-

tificationwas possible because of the comparison between theabsorption spectra of the EE HE and HE + astilbin whosemaximum absorption was 2903 nm

The hump E (11987711990539ndash48) seems to be related to con-

densed tannins observed in this work on TLC analysisOnly monomers and oligomers up to tetramers of catechinsand proanthocyanidins can be separated and detected as adefined peak [26 27] Polymeric catechins present in manyplant materials do not result in well defined peaks Theyallow a drift to happen in the baseline and the formation ofcharacteristic humps in HPLC chromatograms

33 Antibacterial Activity MIC values of Hymenaea stigono-carpa extracts fractions and antimicrobial agents againstStaphylococcus aureus and Enterococcus faecalis strains arelisted in Table 2

Among all microorganisms the Staphylococcus aureusstrains showed to be more sensitive to H stigonocarpaextracts and fractions Gram-negative bacilli showed resis-tance to all extracts and fractions evaluated (MIC ge1024120583gmL) This resistance may be due to the presence oflipopolysaccharide which would have blocked the interac-tion between phytochemicals present in the extract and theouter membrane of the microorganism [28]

The EE HE and AF showed the strongest antistaphy-lococcal activity whose MIC values ranged from 64 to256120583gmL When these extracts were compared no statisti-cal difference was observed

According to Sartoratto et al [29] a strong activity is forMIC values between 50 and 500 120583gmL moderate activityfor MIC values between 600 and 1500 120583gmL and weakactivity for MIC values above 1500 120583gmL [29] In compari-son to literature data the HE AE EE EAF and AF whoseMIC ranged from 64 to 512 120583gmL have a strong antimi-crobial activity against the Staphylococcus aureus strainstested

High phenol contents were found in the EE HE and AFThese compounds can be directly related to antimicrobialactivity These results corroborate with those described byCowan 1999 [30] and Okoro et al [31] who attribute apositive correlation between tannin or flavonoid contents andantimicrobial activity

Tannins are compounds that when in the aqueoussystem form colloidal solutions and their antimicrobialactivity is due to binding to proteins and adhesins inhibitingenzymes the complexation with the cell wall and metalions and disruption of the plasmatic membrane Howeverthe flavonoids have the ability to complex with protein andbacterial cells forming irreversible complexes mainly withnucleophilic amino acids [30]

The action of the phenolic compounds present in thehydroalcoholic extract can be visualized by electron trans-mission microscopy (TEM) on S aureus ATCC 33591 as inFigure 2

34 Ultrastructural Alterations The cytological alterationscaused by hydroalcoholic extract at 12 MIC included par-tially digested cell-wall fragments exfoliated from the bac-terial surface exposition of the patches in the cytoplasmicmembrane (Figures 2(d)-2(e)) the absence of the cell wall ordetachment from the cytoplasmic membrane (Figure 2(e))the thickening of the outer membrane TO (Figure 2(e)) andabnormal septa (Figures 2(d) 2(f) 2(i) 2(k) and 2(l)) whichappears to have been caused by some dysfunction during thecell division

The appearance of a thickening of the outer membrane(TO) and a condensation of the ribonucleus (CR) intobacteria indicate defensive mechanisms to maintain theosmotic pressure after hydroalcoholic extract action Bacteriaalso produce more peptidoglycan to protect themselves anddevelopmesossomes (ME) implicated in the cellular respira-tion [32]

The failure to insert the peptidoglycan caused in theouter membrane the hole formation appearances (PA) (Fig-ures 2(d) 2(e) and 2(f)) This alteration was caused tothe difference in osmotic pressure between the outer andinner surroundings of the bacteria If this event was to beprolonged it would have caused bacterial lysis [33]

Theminimal inhibitory concentration (256120583gmL) of thehydroalcoholic extract was able to provoke alterations in sev-eral cell structures Figures 2(g)ndash2(l) showed cell membranesand cell walls being disrupted and damaged resulting in arelease of cell materials into the cytoplasm (Figures 2(g) and2(h)) Alterations in the intracellular components such asnucleic acids proteins aggregated and branched ribosomesas well as the presence of multiple septa were visualized inFigures from 2(i) to 2(l) The term of DNA coagulated effects(DC) means that the function of DNA gyrase was stoppedfrom fulfilling its main core in supercoiling the DNA duringreplication

Tannins main compounds present in hydroalcoholicextract are able to bind to the membrane to create connec-tions between the double layers and initiate their interactionand adhesionThis process is accompanied by the decrease ofthe membrane potential and interlayer spacing

In this study the cytological changes observed by TEM atdifferent concentrations of the hydroalcoholic extract fromHymenaea stigonocarpa stem barks are supporting evidenceof its antibacterial action against Staphylococcus aureus

4 Conclusion

The results obtained in this study indicate that theHymenaeastigonocarpa stem barks have flavonoids and tannins andthese compounds are possibly the reason for the antimicro-bial activity against Gram-positive cocci

Hymenaea stigonocarpa might be an interesting alterna-tive therapy for infectious diseases caused bymultidrug resis-tant microorganisms In addition more studies includingtoxicity tested in vivo need to be conducted on this plantbefore therapeutic treatments are implemented


Table2Minim

alinhibitory

concentrations

ofthee

xtractsa

ndfractio

nsfro

mHym

enaeastigonocarpaagainstG

ram-positive

cocci

Gram-positive

cocci

Orig

inMinim

alinhibitory

concentrations

(120583gmL)

Resistancep

heno

type

CEEA

EEE

AE

HE

AF

EAF

OXA

TEI

Staphylococcus

aureus

ATCC

25293

ATCC

512

512

256

512

128

128

128

012

mdashCon

trolstrain

Staphylococcus

aureus

ATCC

33591

ATCC

1024

512

256

512

256

256

512

64mdash

Con

trolstrain

Staphylococcus

aureus

LFBM

05Hem

oculture

1024

512

256

512

256

125

512

32mdash

AMPAZIC

FOP

ENStaphylococcus

aureus

LFBM

11ND

512

512

128

128

128

128

256

10mdash

PEN

Staphylococcus

aureus

LFBM

13Va

ginalsecretio

n512

512

512

512

128

64512

10mdash

PEN

Staphylococcus

aureus

LFBM

15Sputum

512

512

128

512

6464

256

012

mdashPE

NG

ETE

RIC

LIStaphylococcus

aureus

LFBM

16Hem

oculture

512

512

256

512

256

125

256

012

mdashAMPAZIC

FOP

ENStaphylococcus

aureus

LFBM

25Hem

oculture

512

512

256

512

256

256

256

256

mdashPE

NG

ETE

RIR

IFC

LIStaphylococcus

aureus

LFBM

26Hem

oculture

512

512

256

512

6464

512

256

mdashAMC

AMPCF

OC

IPStaphylococcus

aureus

LFBM

28Trachealsecretion

1024

512

256

512

256

128

512

16mdash

AMC

CIPCF

OG

ETStaphylococcus

aureus

LFBM

29Hem

oculture

1024

512

256

512

256

256

512

80

mdashAMC

AMPCF

OP

ENStaphylococcus

aureus

LFBM

30Trachealsecretion

1024

512

256

512

256

256

512

16mdash

AMC

AMPCF

OC

IPEn

terococcus

faecalisLF

BM02

Hem

oculture

1024

512

256

256

256

256

512

mdash80

CIPPE

NE

RIIMITE

TEn

terococcus

faecalisLF

BM07

Hem

oculture

1024

512

256

512

512

256

512

mdash80

CIPPE

NE

RIIMITE

TEn

terococcus

faecalisLF

BM08

Urin

e1024

512

256

512

256

256

512

mdash025

Non

eantibiotic

Enterococcus

faecalisLF

BM18

Hem

oculture

1024

512

256

512

256

256

512

mdash40

CIPPE

NE

RIIMITE

TAT

CCA

merican

Type

Cultu

reCollection

CEcyclohexanicextractEA

Eethylacetate

extractEE

ethanolicextractAE

aqueou

sextractH

Ehydroalcoh

olicextractAF

aqueou

sfraction

EAF

ethylacetate

fractio

nOXAo

xacillin

AMP

ampicillin

AZIazithromycinC

FOcefoxitin

CIP

ciprofl

oxacinG

ETgentamicinP

ENp

enicillinC

LIclin

damycinE

RIerythromycinIMIim

ipenem

RIFrifampin

TET

tetracyclin

eLF

BML

aboratorio

deFisio

logiae

Bioq

uımicad

eMicroorganism

osN

Dn

otdeterm

ined


OMPM

R

(a)

OM

S

R

(b)

PM

ROM

(c)

ME

PA

PAST

TO

(d)

PA

(e)

ME

PA

PAST

(f)

CR

DOM

ST

ADS

(g)

GC

(h)

CR

ST

ADS

(i)

JJJJJ

CD

(j)

CRST

ME

(k)

CR

ST

ADS

(l)

Figure 2 Electron transmission microscope images taken of S aureus ATCC-33591 cells not treated (a)ndash(c) show distinct OM outermembrane PM plasmamembrane R ribonucleus and S septa of the bacteria Treatedwith hydroalcoholic extract ofHymenaea stigonocarpain half ofminimum inhibitory concentration (d)ndash(f) show indentation and hole formation appearances (PA) mesossomes (M) thickening ofthe outermembrane (TO) and septum thickness distribution (ST) Treatedwithminimum inhibitory concentration (g)ndash(k) show condensingof the ribonucleus (CR) detachment of outer membrane (DOM) thickness distribution septa (ST) and aberrant division septa (ADS) ghostcells (GC) mesossomes (ME) and coagulated DNA (CD) Magnification of (a) (b) (c) (e) (j) and (k) is 30000x and Magnification of (d)(f) (g) (h) and (l) are 65000x

Conflict of Interests

There was no conflict of interests regarding the publication ofthis paper

Acknowledgment

The authors thank the financial support and infrastructureprovided by Universidade Federal de Pernambuco (UFPE)which made this study possible

References

[1] A P MacGowan ldquoClinical implications of antimicrobial resis-tance for therapyrdquo Journal of Antimicrobial Chemotherapy vol62 no 2 pp ii105ndashii114 2008

[2] N Brusselaers D Vogelaers and S Blot ldquoThe rising problemof antimicrobial resistance in the intensive care unitrdquo Annals ofIntensive Careno vol 1 pp 1ndash7 2011

[3] I H Spicknall B Foxman C F Marrs and J N Eisenberg ldquoAmodeling framework for the evolution and spread of antibioticresistance literature review and model categorizationrdquo Ameri-can Journal of Epidemiology vol 14 pp 1ndash13 2013


[4] P R Marinho A P B Moreira F L P C Pellegrino et alldquoMarine Pseudomonas putida a potential source of antimicro-bial substances against antibiotic-resistant bacteriardquo Memoriasdo Instituto Oswaldo Cruz vol 104 no 5 pp 678ndash682 2009

[5] B B Mishra and V K Tiwari ldquoNatural products an evolvingrole in future drug discoveryrdquo European Journal of MedicinalChemistry vol 46 no 10 pp 4769ndash4807 2011

[6] M R F Lima E C P A Ximenes J S Luna and A E GSantrsquoAna ldquoThe antibiotic activity of some Brazilian medicinalplantsrdquo Revista Brasileira de Farmacognosia vol 16 no 3 pp300ndash306 2006

[7] A G Batista E A Esteves N A V Dessimoni-Pinto L GOliveira S T Pires and R C Santana ldquoChemical compositionof jatoba-do-cerrado (Hymenaea stigonocarpaMart) flour andits effect on growth of ratsrdquo Alimentos e Nutricao Araraquaravol 2 pp 173ndash180 2011

[8] T S M Grandi J A Trindade M J F Pinto L L Ferreira andA C Catella ldquoPlantas medicinais deMinas Gerais BrasilrdquoActaBotanica Brasilica vol 3 no 2 pp 185ndash224 1989

[9] G M Vila Verde J R Paula and D M Carneiro ldquoLevan-tamento etnobotanico das plantas medicinais do cerrado uti-lizadas pela populacao deMossamedes (GO)rdquoRevista Brasileirade Farmacognosia vol 13 pp 64ndash66 2003

[10] I G C Bieski F Rios Santos R M De Oliveira et alldquoEthnopharmacology ofmedicinal plants of the pantanal region(Mato Grosso Brazil)rdquo Evidence-Based Complementary andAlternative Medicine vol 2012 Article ID 272749 36 pages2012

[11] J H Langenheim and G D Hall ldquoSesquiterpene Deterrence ofa Leaf-Tying Lepidopteran Stenoma ferrocanella onHymenaeastigonocarpa in Central Brazilrdquo Biochemical Systematics andEcology vol 11 no 1 pp 29ndash36 1983

[12] T G Matuda and F M Netto ldquoCaracterizacao quımica parcialda semente de jatoba-do-cerradordquo Ciencia e Tecnologia deAlimentos vol 25 pp 353ndash357 2005

[13] P R Orsi F Bonamin J A Severi et al ldquoHymenaea stigono-carpaMart ex Hayne A Brazilian medicinal plant with gastricand duodenal anti-ulcer and antidiarrheal effects in experimen-tal rodent modelsrdquo Journal of Ethnopharmacology vol 143 pp81ndash90 2012

[14] H Wagner and S Bladt Plant Drug Analysis Thin LayerChromatography Atlas Springer Berlin Germany 1996

[15] J B Harbone Phytochemical Methods A Guide to ModernTechniques of Plant Analysis Chapman amp Hall London UK1998

[16] European Pharmacopoeia Determination of Tannins in HerbalDrugs 2814 Council of Europe Strasbourg France 5th edi-tion 2005

[17] L M da Costa V A dos Santos D T Ohana E S Lima M deM Pereira and T P de Souza ldquoTechnological development ofaqueous extracts from Calycophyllum spruceanum (mulateiro)using factorial designrdquo Brazilian Journal of Pharmacognosy vol21 no 1 pp 181ndash186 2011

[18] Clinical and Laboratory Standards Institute CLSI PerformanceStandards for Antimicrobial Susceptibility Testing PA Publica-tion M100-S22 Wayne Pa USA 2012

[19] L SOliveira A L BD Santana CAMaranhao et al ldquoNaturalresistance of five woods to Phanerochaete chrysosporium degra-dationrdquo International Biodeterioration and Biodegradation vol64 no 8 pp 711ndash715 2010

[20] J H Langenheim Y Lee and S S Martin ldquoAn evolutionaryand ecological perspective of Amazonian Hylaea species ofHymenaea (Leguminosae Caesalpinioideae)rdquoActa Amazonicavol 1 pp 5ndash38 1973

[21] E Carneiro J B Calixto F Delle Monache and R A YunesldquoIsolation chemical identification and pharmacological eval-uation of eucryphin astilbin and engelitin obtained from thebark of Hymenaea martianardquo International Journal of Pharma-cognosy vol 31 no 1 pp 38ndash46 1993

[22] M Ishibashi H Oda M Mitamura et al ldquoCasein kinase IIinhibitors isolated from two Brazilian plants Hymenaea parv-ifolia andWulffia baccatardquo Bioorganic and Medicinal ChemistryLetters vol 9 no 15 pp 2157ndash2160 1999

[23] G R Pettit Y Meng C A Stevenson et al ldquoIsolation andstructure of palstatin from the amazon treeHymeneae palustrisrdquoJournal of Natural Products vol 66 no 2 pp 259ndash262 2003

[24] CMO Simoes E P Schenkel G Gosmann J C PMello L AMentz and P R Petrovick Farmacognosia da planta aomedica-mento Editora da UFRGSUFSC Porto AlegreFlorianopolisBrazil 2nd edition 2000

[25] T J Mabry K R Markham and M B ThomasThe SystematicIdentification of Flavonoids Springer Berlin Germany 1970

[26] O M Andersen and K R Markham Flavonoids ChemistryBiochemistry and Applications Taylor amp Francis Boca RatonFla USA 2006

[27] T Yokozawa E J Cho C H Park and J H Kim ldquoProtectiveeffect of proanthocyanidin against diabetic oxidative stressrdquoEvidence-Based Complementary and Alternative Medicine vol2012 Article ID 623879 7 pages 2012

[28] H Ikigai T Nakae Y Hara and T Shimamura ldquoBactericidalcatechins damage the lipid bilayerrdquo Biochimica et BiophysicaActa vol 1147 no 1 pp 132ndash136 1993

[29] A Sartoratto A L M Machado C Delarmelina G MFigueira M C T Duarte and V L G Rehder ldquoCompositionand antimicrobial activity of essential oils from aromatic plantsused in Brazilrdquo Brazilian Journal of Microbiology vol 35 no 4pp 275ndash280 2004

[30] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999

[31] I O Okoro A Osagie and E O Asibor ldquoAntioxidant andantimicrobial activities of polyphenols from ethnomedicinalplants of Nigeriardquo African Journal of Biotechnology vol 9 no20 pp 2989ndash2993 2010

[32] L Santhana Raj H L Hing O Baharudin et al ldquoMesosomesare a definite event in antibiotic-treated Staphylococcus aureusATCC 25923rdquo Tropical Biomedicine vol 24 no 1 pp 105ndash1092007

[33] G C Schito ldquoThe importance of the development of antibioticresistance in Staphylococcus aureusrdquo Clinical Microbiology andInfection vol 12 no 1 pp 3ndash8 2006

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


metabolites and are promising sources of new therapeuticagents including antimicrobials [5]

Approximately 60 to 80 of the worldrsquos populationstill relies on traditional medicines for treatment of severalillnesses In Brazil plants are used as naturalmedicinemainlyby low-income populations and contribute significantly toprimary health care [6]

Hymenaea stigonocarpa Mart ex Hayne (Fabaceae) isa medicinal plant found in the Brazilian savannah andpopularly known as ldquoJatoba-do-cerradordquo [7] Its stem barksare widely used in infusion or decoction to treat stomachpain asthma bronchitis ulcers diarrhea flu and cough [8ndash10]

Phytochemical studies ofHymenaea stigonocarpa showedthe presence of terpenes and sesquiterpenes fatty acidsflavonoids and tannins These metabolites are recognized byits biological activities [11ndash13]

Little research has focused on the pharmacological activ-ity from Hymenaea stigonocarpa stem barks

The antidiarrheal activity and healing properties of theH stigonocarpa stem bark methanolic extract were recentlydemonstrated on experimental gastric and duodenal ulcers[13] However studies on the antimicrobial activity fromHymenaea stigonocarpa stem barks have not yet been pub-lished

Based on this report the aim of the present work was toperform a phytochemical and antibacterial study of extractsand fractions from the Hymenaea stigonocarpa stem barkto establish the fingerprint profile by HPLC-DAD and toidentify the ultrastructural alterations caused by the hydroal-coholic extract on Staphylococcus aureusmethicillin resistantstrain

2 Material and Methods

21 Plant Material Stem barks fromHymenaea stigonocarpaMart ex Hayne were collected in Camocim de Sao Felixcity Pernambuco Brazil (latitude 8∘211015840300010158401015840S and longitude35∘471015840567010158401015840W) A voucher specimen was deposited in theherbariumDardano de Andrade Lima-Instituto Agronomicode Pernambuco (IPA) and registered under number 53563

22 Extracts Preparation The stem barks were air-driedpowdered using a hammer mill (Tigre ASN5) and stored inamber bottles at room temperature Serial exhaustive mac-eration 50 g (1 2 wv) using cyclohexane (CE) ethyl acetate(EAE) ethanol (EE) and water (AE) was performed Ahydroalcoholic extract (HE) was also obtained bymacerationof 60 g (1 2 wv) in ethanolwater (1 1) The hydroalcoholicextract dry residue (5 g) was partitioned (1 1) with water andethyl acetate to obtain the aqueous (AF) and ethyl acetate(EAF) fractions All extracts and fractions were filteredthrough a Whatman No 1 paper filter and evaporated at50∘C under reduced pressure except the aqueous extract andfraction which were lyophilized

23 Phytochemical Screening The phytochemical screen-ing was carried out by thin layer chromatography (TLC)

on silica gel (Merck Germany) following the proceduresdescribed by Wagner and Harborne [14 15] 15120583L aliquotsof each extract solution (05wv) were applied to evaluatethe presence of monoterpenes sesquiterpenes triterpenessteroids alkaloids iridoids coumarins saponins hydrolyzedand condensed tannins phenylpropanoids flavonoids andsugars

231 Total Phenol Contents Total of phenol contents (TPC)was performed in accordance with Folin-Ciocalteu methodas described in the European Pharmacopoeia [16]

The extracts and fractions of the Hymenaea stigonocarpastem bark were solubilized in ethanol (10) to obtain afinal concentration of 05mgmL Five milliliters of eachsolution was diluted to 25mL with distilled water Fromthese solutions 2mL was transferred to a 25mL volumetricflask containing distilled water (10mL) and added to 1mL ofundiluted Folin-Ciocalteu reagent so the volume was madeto 25mL with 10 aqueous Na

2CO3(wv) The blend was

left at room temperature for 30min Then absorbance of thesamples was read at 760 nm in an UVVis spectrophotometer(Evolution 60S Thermo Scientific) Distilled water was usedas a blank

TPC was determined from the extrapolation of thecalibration curve (119910 = 01037119909+00024 1198772 = 09992) whichwas obtained from gallic acid (Sigma Chemical Co St LouisUSA) solution (20ndash80 120583gmL)

The determination of total phenol contents was carriedout in triplicate The TPC was expressed as milligrams ofgallic acid equivalents (GAE) per gram of dried samples

232 Total Tannin Contents In order to determine the totaltannins casein (100mg)was added to 10mLof each extract orfraction solution and homogenized for 60min These blendswere filtered through a Whatman No 1 paper filter Theprocedure for the determination of tanninswas similar to thatdescribed above

The phenol contents in the nontannin fractions werecalculated by standard curve of gallic acid The total tannincontent was calculated as follows total tannins = totalphenols minus nontannin phenols [17]

24 High Performance Liquid Chromatography AnalysisChromatographic fingerprints of Hymenaea stigonocarpastem bark extracts and fractions that present the highesttannin contents and antibacterial activity (EE HE and AF)were obtained by high performance liquid chromatographycoupled with diode array detection (HPLC-DAD)

Extracts and fractions were solubilized in methanol toobtain solutions at the 400120583gmL concentration Astilbinsolution was used as a standard at a concentration of25 120583gmL Then these solutions were filtered through a020120583m polytetrafluoroethylene membrane (Chromafil) andstored in amber vials

Samples analysis was performed on a HPLC-DAD device(Ultimate 3000 Thermo Scientific) equipped with binarypump HPG-3x00RS 3000 (RS) diode array detector andACC-3000 autosampler





























minus100

200

400

600

190 250 300 350 400

()

(nm)

1983

2902

(A)


400

1984

1912 2905

minus100

200

400

600

190 250 300 350

()

(nm)

(B)


1975

19312939

minus100

200

400

600

190 250 300 350 400

()

(nm)

(C)


1910 2943

minus100

200

400

600

190 250 300 350 400

()

(nm)

(D)

HE + astilbin

HE

EE

AF

Mill

iabs

orba

nce u

nits

(mAU

)

00 50 100 150 200 250Time (min)

A

A

AE

E

E

E

B

BC

C

CD

D

D

B

300 350 400 450 500minus100

minus50

0

50

120WVL280 nm



























4 Conclusion




Table2Minim

alinhibitory

concentrations

ofthee

xtractsa

ndfractio

nsfro

mHym


ram-positive

cocci

Gram-positive

cocci

Orig

inMinim

alinhibitory

concentrations

(120583gmL)

Resistancep

heno

type

CEEA

EEE

AE

HE

AF

EAF

OXA

TEI

Staphylococcus

aureus

ATCC

25293

ATCC

512

512

256

512

128

128

128

012

mdashCon

trolstrain

Staphylococcus

aureus

ATCC

33591

ATCC

1024

512

256

512

256

256

512

64mdash

Con

trolstrain

Staphylococcus

aureus

LFBM

05Hem

oculture

1024

512

256

512

256

125

512

32mdash

AMPAZIC

FOP

ENStaphylococcus

aureus

LFBM

11ND

512

512

128

128

128

128

256

10mdash

PEN

Staphylococcus

aureus

LFBM

13Va

ginalsecretio

n512

512

512

512

128

64512

10mdash

PEN

Staphylococcus

aureus

LFBM

15Sputum

512

512

128

512

6464

256

012

mdashPE

NG

ETE

RIC

LIStaphylococcus

aureus

LFBM

16Hem

oculture

512

512

256

512

256

125

256

012

mdashAMPAZIC

FOP

ENStaphylococcus

aureus

LFBM

25Hem

oculture

512

512

256

512

256

256

256

256

mdashPE

NG

ETE

RIR

IFC

LIStaphylococcus

aureus

LFBM

26Hem

oculture

512

512

256

512

6464

512

256

mdashAMC

AMPCF

OC

IPStaphylococcus

aureus

LFBM

28Trachealsecretion

1024

512

256

512

256

128

512

16mdash

AMC

CIPCF

OG

ETStaphylococcus

aureus

LFBM

29Hem

oculture

1024

512

256

512

256

256

512

80

mdashAMC

AMPCF

OP

ENStaphylococcus

aureus

LFBM

30Trachealsecretion

1024

512

256

512

256

256

512

16mdash

AMC

AMPCF

OC

IPEn

terococcus

faecalisLF

BM02

Hem

oculture

1024

512

256

256

256

256

512

mdash80

CIPPE

NE

RIIMITE

TEn

terococcus

faecalisLF

BM07

Hem

oculture

1024

512

256

512

512

256

512

mdash80

CIPPE

NE

RIIMITE

TEn

terococcus

faecalisLF

BM08

Urin

e1024

512

256

512

256

256

512

mdash025

Non

eantibiotic

Enterococcus

faecalisLF

BM18

Hem

oculture

1024

512

256

512

256

256

512

mdash40

CIPPE

NE

RIIMITE

TAT

CCA

merican

Type

Cultu

reCollection


Eethylacetate

extractEE

ethanolicextractAE

aqueou

sextractH

Ehydroalcoh

olicextractAF

aqueou

sfraction

EAF

ethylacetate

fractio

nOXAo

xacillin

AMP

ampicillin

AZIazithromycinC

FOcefoxitin

CIP

ciprofl

oxacinG

ETgentamicinP

ENp

enicillinC

LIclin

damycinE

RIerythromycinIMIim

ipenem

RIFrifampin

TET

tetracyclin

eLF

BML

aboratorio

deFisio

logiae

Bioq

uımicad

eMicroorganism

osN

Dn

otdeterm

ined


OMPM

R

(a)

OM

S

R

(b)

PM

ROM

(c)

ME

PA

PAST

TO

(d)

PA

(e)

ME

PA

PAST

(f)

CR

DOM

ST

ADS

(g)

GC

(h)

CR

ST

ADS

(i)

JJJJJ

CD

(j)

CRST

ME

(k)

CR

ST

ADS

(l)




Acknowledgment


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























minus100

200

400

600

190 250 300 350 400

()

(nm)

1983

2902

(A)


400

1984

1912 2905

minus100

200

400

600

190 250 300 350

()

(nm)

(B)


1975

19312939

minus100

200

400

600

190 250 300 350 400

()

(nm)

(C)


1910 2943

minus100

200

400

600

190 250 300 350 400

()

(nm)

(D)

HE + astilbin

HE

EE

AF

Mill

iabs

orba

nce u

nits

(mAU

)

00 50 100 150 200 250Time (min)

A

A

AE

E

E

E

B

BC

C

CD

D

D

B

300 350 400 450 500minus100

minus50

0

50

120WVL280 nm



























4 Conclusion




Table2Minim

alinhibitory

concentrations

ofthee

xtractsa

ndfractio

nsfro

mHym


ram-positive

cocci

Gram-positive

cocci

Orig

inMinim

alinhibitory

concentrations

(120583gmL)

Resistancep

heno

type

CEEA

EEE

AE

HE

AF

EAF

OXA

TEI

Staphylococcus

aureus

ATCC

25293

ATCC

512

512

256

512

128

128

128

012

mdashCon

trolstrain

Staphylococcus

aureus

ATCC

33591

ATCC

1024

512

256

512

256

256

512

64mdash

Con

trolstrain

Staphylococcus

aureus

LFBM

05Hem

oculture

1024

512

256

512

256

125

512

32mdash

AMPAZIC

FOP

ENStaphylococcus

aureus

LFBM

11ND

512

512

128

128

128

128

256

10mdash

PEN

Staphylococcus

aureus

LFBM

13Va

ginalsecretio

n512

512

512

512

128

64512

10mdash

PEN

Staphylococcus

aureus

LFBM

15Sputum

512

512

128

512

6464

256

012

mdashPE

NG

ETE

RIC

LIStaphylococcus

aureus

LFBM

16Hem

oculture

512

512

256

512

256

125

256

012

mdashAMPAZIC

FOP

ENStaphylococcus

aureus

LFBM

25Hem

oculture

512

512

256

512

256

256

256

256

mdashPE

NG

ETE

RIR

IFC

LIStaphylococcus

aureus

LFBM

26Hem

oculture

512

512

256

512

6464

512

256

mdashAMC

AMPCF

OC

IPStaphylococcus

aureus

LFBM

28Trachealsecretion

1024

512

256

512

256

128

512

16mdash

AMC

CIPCF

OG

ETStaphylococcus

aureus

LFBM

29Hem

oculture

1024

512

256

512

256

256

512

80

mdashAMC

AMPCF

OP

ENStaphylococcus

aureus

LFBM

30Trachealsecretion

1024

512

256

512

256

256

512

16mdash

AMC

AMPCF

OC

IPEn

terococcus

faecalisLF

BM02

Hem

oculture

1024

512

256

256

256

256

512

mdash80

CIPPE

NE

RIIMITE

TEn

terococcus

faecalisLF

BM07

Hem

oculture

1024

512

256

512

512

256

512

mdash80

CIPPE

NE

RIIMITE

TEn

terococcus

faecalisLF

BM08

Urin

e1024

512

256

512

256

256

512

mdash025

Non

eantibiotic

Enterococcus

faecalisLF

BM18

Hem

oculture

1024

512

256

512

256

256

512

mdash40

CIPPE

NE

RIIMITE

TAT

CCA

merican

Type

Cultu

reCollection


Eethylacetate

extractEE

ethanolicextractAE

aqueou

sextractH

Ehydroalcoh

olicextractAF

aqueou

sfraction

EAF

ethylacetate

fractio

nOXAo

xacillin

AMP

ampicillin

AZIazithromycinC

FOcefoxitin

CIP

ciprofl

oxacinG

ETgentamicinP

ENp

enicillinC

LIclin

damycinE

RIerythromycinIMIim

ipenem

RIFrifampin

TET

tetracyclin

eLF

BML

aboratorio

deFisio

logiae

Bioq

uımicad

eMicroorganism

osN

Dn

otdeterm

ined


OMPM

R

(a)

OM

S

R

(b)

PM

ROM

(c)

ME

PA

PAST

TO

(d)

PA

(e)

ME

PA

PAST

(f)

CR

DOM

ST

ADS

(g)

GC

(h)

CR

ST

ADS

(i)

JJJJJ

CD

(j)

CRST

ME

(k)

CR

ST

ADS

(l)




Acknowledgment


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















4 Conclusion




Table2Minim

alinhibitory

concentrations

ofthee

xtractsa

ndfractio

nsfro

mHym


ram-positive

cocci

Gram-positive

cocci

Orig

inMinim

alinhibitory

concentrations

(120583gmL)

Resistancep

heno

type

CEEA

EEE

AE

HE

AF

EAF

OXA

TEI

Staphylococcus

aureus

ATCC

25293

ATCC

512

512

256

512

128

128

128

012

mdashCon

trolstrain

Staphylococcus

aureus

ATCC

33591

ATCC

1024

512

256

512

256

256

512

64mdash

Con

trolstrain

Staphylococcus

aureus

LFBM

05Hem

oculture

1024

512

256

512

256

125

512

32mdash

AMPAZIC

FOP

ENStaphylococcus

aureus

LFBM

11ND

512

512

128

128

128

128

256

10mdash

PEN

Staphylococcus

aureus

LFBM

13Va

ginalsecretio

n512

512

512

512

128

64512

10mdash

PEN

Staphylococcus

aureus

LFBM

15Sputum

512

512

128

512

6464

256

012

mdashPE

NG

ETE

RIC

LIStaphylococcus

aureus

LFBM

16Hem

oculture

512

512

256

512

256

125

256

012

mdashAMPAZIC

FOP

ENStaphylococcus

aureus

LFBM

25Hem

oculture

512

512

256

512

256

256

256

256

mdashPE

NG

ETE

RIR

IFC

LIStaphylococcus

aureus

LFBM

26Hem

oculture

512

512

256

512

6464

512

256

mdashAMC

AMPCF

OC

IPStaphylococcus

aureus

LFBM

28Trachealsecretion

1024

512

256

512

256

128

512

16mdash

AMC

CIPCF

OG

ETStaphylococcus

aureus

LFBM

29Hem

oculture

1024

512

256

512

256

256

512

80

mdashAMC

AMPCF

OP

ENStaphylococcus

aureus

LFBM

30Trachealsecretion

1024

512

256

512

256

256

512

16mdash

AMC

AMPCF

OC

IPEn

terococcus

faecalisLF

BM02

Hem

oculture

1024

512

256

256

256

256

512

mdash80

CIPPE

NE

RIIMITE

TEn

terococcus

faecalisLF

BM07

Hem

oculture

1024

512

256

512

512

256

512

mdash80

CIPPE

NE

RIIMITE

TEn

terococcus

faecalisLF

BM08

Urin

e1024

512

256

512

256

256

512

mdash025

Non

eantibiotic

Enterococcus

faecalisLF

BM18

Hem

oculture

1024

512

256

512

256

256

512

mdash40

CIPPE

NE

RIIMITE

TAT

CCA

merican

Type

Cultu

reCollection


Eethylacetate

extractEE

ethanolicextractAE

aqueou

sextractH

Ehydroalcoh

olicextractAF

aqueou

sfraction

EAF

ethylacetate

fractio

nOXAo

xacillin

AMP

ampicillin

AZIazithromycinC

FOcefoxitin

CIP

ciprofl

oxacinG

ETgentamicinP

ENp

enicillinC

LIclin

damycinE

RIerythromycinIMIim

ipenem

RIFrifampin

TET

tetracyclin

eLF

BML

aboratorio

deFisio

logiae

Bioq

uımicad

eMicroorganism

osN

Dn

otdeterm

ined


OMPM

R

(a)

OM

S

R

(b)

PM

ROM

(c)

ME

PA

PAST

TO

(d)

PA

(e)

ME

PA

PAST

(f)

CR

DOM

ST

ADS

(g)

GC

(h)

CR

ST

ADS

(i)

JJJJJ

CD

(j)

CRST

ME

(k)

CR

ST

ADS

(l)




Acknowledgment


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Minim

alinhibitory

concentrations

ofthee

xtractsa

ndfractio

nsfro

mHym


ram-positive

cocci

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Acknowledgment


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


OMPM

R

(a)

OM

S

R

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TO

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ADS

(l)




Acknowledgment


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

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