Thiocarbamates from Moringa oleifera Virulent and...

Research ArticleThiocarbamates fromMoringa oleifera Seeds Bioactive againstVirulent and Multidrug-Resistant Vibrio Species

Renata Albuquerque Costa1 Oscarina Viana de Sousa2 Ernesto Hofer3 Jair Mafezoli4

Francisco Geraldo Barbosa4 and Regine Helena Silva dos Fernandes Vieira2

1Federal University of Ceara Fortaleza CE Brazil2Sea Sciences Institute Federal University of Ceara Fortaleza CE Brazil3Oswaldo Cruz Institute Rio de Janeiro RJ Brazil4Department of Organic and Inorganic Chemistry Federal University of Ceara Fortaleza CE Brazil

Correspondence should be addressed to Renata Albuquerque Costa renataalbuqgmailcom

Received 31 March 2017 Accepted 31 May 2017 Published 9 July 2017

Academic Editor Veronica Lazar

Copyright copy 2017 Renata Albuquerque Costa 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

Prospect of antibacterial agents may provide an alternative therapy for diseases caused by multidrug-resistant bacteria This studyaimed to evaluate the in vitro bioactivity of Moringa oleifera seed extracts against 100 vibrios isolated from the marine shrimpLitopenaeus vannamei Ethanol extracts at low (MOS-E) and hot (MOS-ES) temperature are shown to be bioactive against 92and 90 of the strains respectively The most efficient Minimum Inhibitory Concentration (MIC) levels of MOS-E and MOS-ESagainst a high percentage of strains were 32 120583gmLminus1 Bioguided screening of bioactive compounds showed that the ethyl acetatefraction from both extracts was the only one that showed antibacterial activity Vibriocidal substances niazirine and niazimicinewere isolated from the aforementioned fraction through chromatographic fractionation

1 Introduction

Bacteria ofVibrio genus are ubiquitous in themarine environ-ment and are part of the indigenous microbiota of marineinvertebrates Some species are recognized as human patho-gens often associated with diseases such as cholera and acutegastroenteritis [1 2] Vibrios are also seen as opportunisticpathogens of cultured aquatic organisms which is one ofthe reasons for observing the use of antibiotics in shrimpcultivation

Furthermore inappropriate use of antimicrobial drugs inaquaculture has been associated with negative environmentalimpacts selection of bacterial populations resistant to drugs[3 4] and contamination of adjacent ecosystems to cultureponds

Thus detection of antibacterial activity in higher plantsagainst vibrios with virulent antimicrobial-resistant pro-files is of utmost importance In the present research dueto its high medicinal activity [5] the vibriocidal capacityof the angiosperm Moringa oleifera was investigated The

antimicrobial effect of Moringa has been researched sincethe 1980s and seems to be related to some specific com-ponents including pterygospermin and Moringa glycosidesas well as 4-(120572-L-rhamnosyloxy)-benzyl isothiocyanate and4-(120572-L-rhamnosyloxy)-phenyl-acetonitrile which act espe-cially against Bacillus subtilis Mycobacterium phlei Serra-tia marcescens Escherichia coli Pseudomonas aeruginosaShigella sp and Streptococcus sp [6]

Despite the extensive scientific evidence of the bioactivityof Moringa against bacteria [7ndash10] studies on its effectsagainst vibrios are still incipient Thus this research aimedto evaluate the bioactive potential from extracts of Moringaseeds against vibrios with virulent and multidrug antimicro-bial profile

2 Material and Methods

21 Origin of the Strains One hundred Vibrio sp strainsisolated from the hemolymph of Litopenaeus vannameishrimpwere used all of which belonged to the bacteriological

HindawiBioMed Research InternationalVolume 2017 Article ID 7963747 6 pageshttpsdoiorg10115520177963747

2 BioMed Research International

collection of the Laboratory of Environmental and FisheryMicrobiology at Sea Sciences Institute (LABOMAR-UFC-Brazil) The 100 strains were phenotypically identified as Vnavarrensis (119899 = 53) V brasiliensis (119899 = 15) V para-haemolyticus (119899 = 10) V xuii (119899 = 8) V coralliilyticus (119899 =5) V cholerae (119899 = 4) V neptunis (119899 = 2) V alginolyticus(119899 = 1)V diazotrophicus (119899 = 1) andV vulnificus B3 (119899 = 1)[11] The enzymatic profile and antimicrobial resistance wereused as a criterion for selection [12]

22 BotanicalMaterial Moringa oleifera seeds were collectedfrom two specimens grown in a campus of the FederalUniversity of Ceara (Pici Fortaleza Ceara) Separation fromthe fruit (pod) removal of husks and posterior packing inplastic polyethylene bags followed the material collection

23 Moringa oleifera Extracts All extraction procedureswere performed in the Department of Organic and InorganicChemistry at Federal University of Ceara (UFC) Part of thecrushed seeds of M oleifera (110 g) was subjected to threeextractions with 300mL cold hexane (PA) at 24 h intervalsAfter filtration and evaporation of the solvent under reducedpressure in a rotary evaporator 1536 g of an extract of fluidand yellowish appearance called MOS-H was obtained Theresulting cake was subjected to three cold extractions with300mL ethanol (PA) in 24 h intervals After filtration andevaporation of the solvent under reduced pressure in a rotaryevaporator 1164 g of an extract of fluid and dark appearancecalledMOS-Ewas obtained 139 g of crushedM oleifera seedswas used for hot extraction in a Soxhlet apparatuswith 800mlof hexane (PA) for 48 h After filtration and evaporation of thesolvent under reduced pressure in a rotary evaporator 2829 gof an extract of fluid and yellowish aspect calledMOS-HSwasobtained Another extraction was carried out with 800ml ofethanol (PA) for 48 h After filtration and evaporation of thesolvent under reduced pressure in a rotary evaporator 1366 gof a pasty and dark-colored appearance called MOS-ES wasobtained

24 In Vitro Susceptibility Testing of Moringa oleifera ExtractsSusceptibility of Vibrio sp strains to the four extracts types(MOS-H MOS-E MOS-HS and MOS-ES) was assessedusing the disk diffusion method (DDM) and by MinimumInhibitory Concentration (MIC) [13] In order to proceedwith the DDM paper discs (6mm) containing 100 120583L of eachextract were applied in triplicate on Mueller-Hinton platespreviously seeded with bacterial cultures (108UFCmLminus1)As negative and positive Gram control strains of V para-haemolyticus IOC and Staphylococcus aureus ATCC 25923respectively were used For MIC determination macrodilu-tion technique in Mueller-Hinton broth containing 1 NaClwas used Concentrations of 4 8 16 32 and 64120583gmLminus1 weretested using the MOS-E oils (cold extraction with ethanol)and MOS-ES (hot extraction with ethanol) in comparison toisolates susceptible to crude extracts in the DDM test

25 Chromatographic Fractionation of MOS-E and ES-MOSandObtainingActive Fraction Ethyl Acetate (MOS-ESA) Partof the MOS-E extract (31 g) was adsorbed onto 73 g of silica

gel and chromatographed on 525 g of silica gel in opencolumn (Oslash 50 cm) Elution was done in order of increas-ing polarity with dichloromethane (1200ml) (MOS-ED)ethyl acetate (800mL) (MOS-EA) and methanol (600mL)(MOS-EM) Solvents were evaporated under reduced pres-sure in a rotary evaporator yielding the following massand yields MOS-ED 19208mg 6196 MOS-EA 2316mg747 MOS 6608mg 2131 MOS-ES extract (26 g) wasadsorbed onto 39 g of silica gel and chromatographed on483 g of silica gel in open column (Oslash 50 cm) Elution wasdone in order of increasing polarity with dichloromethane(500ml) (MOS ESD) ethyl acetate (700ml) (MOS-ESA) andmethanol (600ml) (MOS-ESM) Solvents were evaporatedunder reduced pressure in a rotary evaporator yielding thefollowing mass and yields MOS-ED 348mg 133 MOS-EA 3353mg 1289 MOS 1978mg 7607 All fractionswere subjected to antimicrobial activity test by disk diffusionmethod The bioactive fraction was subjected to chromato-graphic fractionation by High-Performance Liquid Chro-matography (HPLC) in order to isolate its active principles

26 Chromatographic Fractionation of the Ethyl Acetate Frac-tion (MOS-ESA) by High-Performance Liquid Chromatogra-phy (HPLC) and Isolation of Active Substances Part of theMOE-ESA active fraction (285mg) was analyzed by HPLC ina chromatograph Shimadzu (UFLCmodel) equipped with aUV-Vis detector with diode array (model SPD-M20A) Sepa-ration was performed in reverse phase conditions in semi-preparative column (C-185 120583m) with isocratic elution usingMeOHH

2O (1 1) with a 472mLminminus1 flow Chromato-

graphic fractionation of the ethyl acetatersquos fraction from theMOS-ES fixed oil resulted in the detection (at 284 nm) andisolation of three main substances (Figure 1) which wereobtained as whitish amorphous solids the compound relatedto the peak 1 (233m 119905

119903= 499min) was called MOS-ES-1

related to peak 2 (40mg 119905119903= 706min) was called MOS-ES-

2 and related to peak 3 (651mg 119905119903= 1745min) was called

MOS-ES-3 Isolated substances (S1 and S3) had their struc-tures determined by the analysis of Nuclear Magnetic Res-onance (NMR) and Infrared (IR) spectral data and also bycomparison to other findings described in the literature [14 15]

3 Results

31 Disk Diffusion Test From all the 100 strains tested onlyfive were resistant to MOS-E fixed oil on the other hand36 had its growth inhibited with average inhibition zonesranging from 13 to 15mm (Table 1) When comparing theoils MOS-ES tests demonstrated inhibitory bacterial ratesomewhat lower since most strains (119899 = 37) had inhibitionsindicated by halos in the range 10 to 12mm (Table 2)In addition seven strains were resistant to the MOS-ESThe larger inhibition halo (22 to 24mm) was observedon MOS-E test against a V navarrensis strain (Table 1)Both extracts showed antibacterial effect against the Gram-positive (Staphylococcus aureus) and Gram-negative (Vibrioparahaemolyticus) controls (Tables 1 and 2) MOS-H andMOS-HS extracts showed no bioactivity against any of theisolates (119899 = 100)

BioMed Research International 3

Table 1 Distribution according to the average inhibition zone (mm) of the number of strains inhibited by cold ethanol extract ofMoringaoleifera seeds (MOS-E)

Vibrio species 119899Average inhibition zone (mm)

22ndash24 19ndash21 16ndash18 13ndash15 10ndash12 7ndash9 0V navarrensis 53 1 2 5 27 15 3 mdashV brasiliensis 15 mdash 1 2 4 2 5 1V parahaemolyticus 10 mdash mdash mdash mdash mdash 9 1V xuii 8 mdash mdash mdash 1 mdash 5 2V coralliilyticus 5 mdash mdash 2 1 1 1 mdashV cholerae 4 mdash mdash mdash 2 1 1 mdashV neptunis 2 mdash mdash 1 mdash 1 mdash mdashV alginolyticus 1 mdash mdash mdash 1 mdash mdash mdashV diazotrophicus 1 mdash 1 mdash mdash mdash mdash mdashV vulnificus B3 1 mdash mdash mdash mdash mdash mdash 1V parahaemolyticus IOCa 1 mdash mdash 1 mdash mdash mdash mdashS aureus ATCC25923b 1 mdash 1 mdash mdash mdash mdash mdashTotal 102 1 5 11 36 20 24 5119899 number of isolates a standard strain used as Gram-negative control b standard strain used as Gram-positive control

123 45

67 8

9

10

11 12

13

14

15

(mAU

)

(Min)

MOS-ES1

MOS-ES2

MOS-ES3

2017515012510075502500

0

250

500

Figure 1 HPLC chromatogram analysis of an active fraction ofMOS-ESA and isolation of MOS-ES-1 MOS-ES-2 and MOS-ES-3

32 Minimum Inhibitory Concentration (MIC) MIC levels oftheMOS-E show that 83 (902) of the strains were inhibitedin the presence of a 32 120583gmLminus1 concentration Levels of 816 and 64 120583gmLminus1 were able to inhibit 1 6 and 2 strainsrespectively Also MIC levels of the MOS-ES able to inhibitthe highest percentage of strains (119899 = 88 978) were that of32 120583gmLminus1 On the other hand only 2 (22) of the strainswere inhibited by a MOS-ES MIC of 16 120583gmL

33 Bioactivity of the Fractions Study of the bioactivityof dichloromethane (CH

2Cl2) ethyl acetate (EtOAc) and

methanol (MeOH) fractions ofMOS-E andMOS-ES revealedthat only EA fractions from both extracts showed antimicro-bial activity (Table 3) Ethyl acetate fractions were selectedand subjected to chromatographic fractionation by HPLC inorder to isolate bioactive substances

34 Identification of Individual Substances From the MOS-EA fraction three substances were identified S1 (MOS-ES-1) S2 (MOS-ES-2) and S3 (MOS-ES-3) Due to the smallamount obtained from MOS-ES-2 substance (S2) it wasimpossible to obtain spectroscopic data enough for a struc-tural characterization Those two substances derived from

MOE-EA fraction were also obtained in small amounts andidentified based only byThin Layer Chromatography (TLC)Furthermore the drain of this fraction yielded 23mg ofunidentified less polar substances

35 Structural Characterization of MOS-ES-1 (S1) and MOS-ES-3 (S3) S1 was isolated as a white amorphous solidand presented 129ndash132∘C [M + Na]+ = 3021012 RMN 1H(500MHz CD

3OD) 120575 728 (2H d J = 86Hz) 707 (2H d J =

86Hz) 543 (1H d J = 14Hz) 384 (1H dd J = 34 e 95Hz)382 (2H s) 382 (2Hm) 362 (1Hm) 346 (1H t J = 95Hz)121 (3H d J = 62Hz) RMN 13C (125MHz CD

3OD) 120575 1574

(C-1) 1181 (C-2 e C-6) 1302 (C-3 e C-5) 1258 (C-4) 227 (C-7) 1198 (C-8) 999 (C-11015840) 720 (C-21015840) 720 (C-31015840) 738 (C-41015840)707 (C-51015840) 180 (C-61015840) IV ]maxcm

minus1 (KBr) 3411 (]O-H) 2948(]-C-H) 2250 (]CequivN) 1611ndash1513 (]C=C) 1227 (]C-O)

Analysis of spectral data and the comparison with infor-mation in the literature [15] were used to characterize S1 asthe 4-[(120572-L-rhamnosyloxy)benzyl] nitrile or niazirine

S3 was also isolated as a white amorphous solid and pre-sented 130ndash133∘C [M + Cl]minus = 3920951 RMN 1H (500MHzCD3OD) 120575 725 (2H d J = 87Hz) 701 (2H d J = 87Hz)

540 (1H d J = 18Hz) 463 (2H s) 446 (2H q J = 71Hz)399 (1H dd J = 18 e 34Hz) 383 (1H dd J = 34 e 95Hz)362 (1Hm) 345 (1H t J = 95Hz) 129 (3H t J = 71Hz) 121(3H d J = 62Hz) RMN 13C (125MHz CD

3OD) 120575 1571 (C-

1) 1176 (C-2 e C-6) 1301 (C-3 e C-5) 1331 (C-4) 490 (C-7)1923 (C-8) 669 (C-9) 147 (C-10) 1002 (C-11015840) 721 (C-21015840)723 (C-31015840) 739 (C-41015840) 706 (C-51015840) 180 (C-61015840) IV ]maxcm

minus1

(KBr) 3405 (]O-H) 2948 (]-C-H) 1611ndash1513 (]C=C) 1233 (]C=S)Analysis of spectral data and the comparison with infor-

mation in the literature [14] were used to characterize S3as O-ethyl-4-[(120572-L-rhamnosyloxy)benzyl] thiocarbamate orniazimicine

36 Evaluation of Antibacterial Activity of the Isolated Sub-stances Bioactivity results of S1 and S3 substances against ten


Table 2 Distribution according to the average inhibition zone (mm) of the number of strains inhibited by hot ethanol extract of Moringaoleifera (MOS-ES)


19ndash21 16ndash18 13ndash15 10ndash12 7ndash9 0V navarrensis 53 1 6 14 26 5 1V brasiliensis 15 1 2 2 4 3 3V parahaemolyticus 10 mdash mdash mdash mdash 9 1V xuii 8 mdash mdash mdash 1 7 mdashV coralliilyticus 5 mdash mdash 1 3 1 mdashV cholerae 4 mdash mdash 1 1 1 1V neptunis 2 mdash mdash mdash 2 mdash mdashV alginolyticus 1 mdash mdash 1 mdash mdash mdashV diazotrophicus 1 mdash 1 mdash mdash mdash mdashV vulnificus B3 1 mdash mdash mdash mdash mdash 1V parahaemolyticus IOCa 1 mdash 1 mdash mdash mdash mdashS aureus ATCC25923b 1 mdash 1 mdash mdash mdash mdashTotal 102 2 11 19 37 26 7119899 number of isolates a standard strain used as Gram-negative control b standard strain used as Gram-positive control

Table 3 Average inhibition halos of ethyl acetate (EtOAc) fractionsof ethanolic extracts ofMoringa oleifera seeds extracted cold (MOS-E) and hot (MOS-ES) with ethanol against ten Vibrio sp strainsisolated from the hemolymph of Litopenaeus vannamei

Strain Vibrio species EtOAc (MOS-E) EtOAc (MOS-ES)1 V coralliilyticus 1451 plusmn 007 1210 plusmn 008

7 V alginolyticus 1265 plusmn 006 1052 plusmn 045

13 V navarrensis 1571 plusmn 021 1403 plusmn 007

35 V diazotrophicus 1714 plusmn 004 1658 plusmn 042


42 V xuii 1239 plusmn 024 1180 plusmn 025

46 V parahaemolyticus 853 plusmn 015 766 plusmn 012

89 V neptunis 1854 plusmn 042 1118 plusmn 009

97 V cholerae 846 plusmn 009 732 plusmn 010

98 V brasiliensis 1501 plusmn 005 1461 plusmn 015

Vibrio sp strains are summarized in Table 4 It is possible toattest in a comparison between substances 1 and 3 (S1 and S3)using the size of the inhibition zone as a criterion a greaterantibacterial efficiency of S3 against all strains

4 Discussion

Studies on the bioactive properties ofMoringa seeds highlightmultiple uses of this phanerogam for example turbidityremoval of contaminated water by coagulation [16] biosorp-tion of heavy metals in effluents [17] anti-inflammatory [18]and antibacterial activity against S aureus and E coli [19]However bioactivity against vibrios has not been widelyresearched Thus the high inhibition level of both extracts(95 for MOS-E 93 for MOS-ES) (Tables 1 and 2) againstantimicrobial-resistant vibrios with virulence factors mustbe stressed This vibriocidal activity is congruent with thefindings of Vieira et al (2010) who investigated antibacterialactivity of ethanol extracts of Moringa seeds and found

inhibition zones ranging from 26 to 295mm against classicalV cholerae 569B

Vibriocidal activity of aerial parts of M oleifera wasreported byPeixoto et al [20]The authors tested the bioactiv-ity of its extracts against standard V parahaemolyticus strainand found average inhibition halos of 219 and 207mm forethanol and aqueous extracts respectively

Moringa seed extracts have also been used in tests againststandard strain of V cholerae Atieno et al [21] observed thebioactivity of hexane and methanolic extracts of M oleiferaand M stenopetala seeds against Salmonella ser Typhi Ecoli and V cholerae For the species of Vibrio the authorsreported inhibition halo sizes of 222 and 138mm for hexaneandmethanolic extracts ofM oleifera respectivelyThe afore-mentioned data support the assertion that Moringa seedshave vibriocidal potential however it cannot be comparedto the ones presented in the present study since antibacterialactivity in leaf extracts was not detected (MOS-H and MOS-HS)

Despite the occurrence of compounds with antibacterialactivity in different parts ofMoringa is being reported in thescientific literature since the early 1980s [22] their use forepizootic purposes has been little exploredThe data obtainedby the disk diffusion test and the results of the MinimumInhibitory Concentration (MIC) serve as evidence of thehigh antibacterial potential of ethanol extracts ofMOS-E andMOS-ES against vibrios

Satisfactory results in disk diffusion tests and the defini-tion ofMIC levels were pivotal for the decision of carrying outcomplementary studies starting with the bioguided screen-ing of bioactive compoundsThe selective antimicrobial effectof Moringarsquos crude extract fractions was also verified byNantachit [23] The author noted that the dichloromethanefraction was active against E coli

Guevara et al [24] reported similar structures describedin this study namely O-ethyl-4-(120572-L-rhamnosyloxy) ben-zyl carbamates 4(120572-L-rhamnosyloxy)-benzyl isothiocyanate


Table 4 Average size of inhibition halos from substances 1 (S1) and 3 (S3) isolated from fractions of ethyl acetate (EtOAc) of Moringaseed in cold extraction with ethanol (MOS-E) and in hot extraction with ethanol (MOS-ES) against ten Vibrio sp strains isolated from thehemolymph of Litopenaeus vannamei

Strains Species Fraction of EtOAc MOS-ESS1 S2 S3

1 V coralliilyticus 811 plusmn 009 nt 1404 plusmn 002

7 V alginolyticus 832 plusmn 002 nt 1598 plusmn 009

13 V navarrensis 976 plusmn 043 nt 1989 plusmn 011

35 V diazotrophicus 742 plusmn 024 nt 2121 plusmn 024


42 V xuii 876 plusmn 029 nt 2033 plusmn 006

46 V parahaemolyticus 766 plusmn 045 nt 990 plusmn 007

89 V neptunis 714 plusmn 011 nt 2011 plusmn 003

97 V cholerae 730 plusmn 027 nt 1015 plusmn 018

98 V brasiliensis 799 plusmn 002 nt 1875 plusmn 006

nt not tested

niazimicine 3-O-(61015840-O-oleoyl-120573-D-glucopyranosyl)-120573-sito-sterol 120573-sitosterol-3-O-120573-D-glucopyranoside niazirine 120573-sitosterol and glycerol-1-(9-octadecanoate) by studying theethanol extracts ofMoringa seedsThe authorsmentioned thebioactivity potential of niazimicine and niazirine Further-more Gilani et al [25] isolated four bioactive compoundsout of the bioguided fractionation of ethanolic extracts ofM oleifera leaves and also found compatible substances withthose described in the present study niazinine A niazinineB niazinine niazimicine and niazinine A + B

In this context Padla et al [10] isolated 4-(alpha-L-Rhamnosyloxy)benzyl isothiocyanate and 4-(41015840-O-acetyl-alpha-L-rhamnosyloxy)-benzyl isothiocyanate fromMoringaoleifera seeds and demonstrated that both substances werebioactive against Gram-positive bacteria Staphylococcusaureus Staphylococcus epidermidis and Bacillus subtilis Inthe present study we isolated from Moringa seeds the sub-stances (1) 4-[(120572-L-rhamnosyloxy)benzyl]nitrile (niazirine)and (2) O-ethyl-4-[(120572-L-rhamnosyloxy)benzyl] thiocarba-mate (niazimicine)

The compounds O-ethyl-p-hydroxybenzene carbamateand O-methyl-4-[(210158403101584041015840-tri-O-acetyl-120572-L-rhamnosylox-y)benzyl] thiocarbamatemust also be added to the previouslymentioned substances as isolates from leaf extracts of Moleifera [26 27] The addition of methanol or ethanol toisothiocyanate is considered a pathway to the synthesis ofthiocarbamates glycosides inMoringa [26]

Since the 1990s biological activities of pharmacologicalinterest concerning nitriles isolated fromMoringa have beendescribed and its antitumor [28] and antihypertensive [14]functions are noteworthy along its use in the prevention ofcarcinogenesis [24]

The presence of niazimicine in Moringa seeds is oftencited as a strong antitumor factor It can be used as a prophyl-actic or therapeutic measure while treating HSV-1 infections[29]

Although this is not the first description of carbamateglycosides in constituent parts of the M oleifera speciesits unexploited vibriocidal potential must be highlighted

In the present study niazirine and niazimicine showedhigh antibacterial efficiency against vibrios with phenotypicprofiles compatible to the presence of virulence factors (exo-enzymes and 120573-hemolysis producers) cross-resistance to 120573-lactams andmonomultiresistance to antibioticsThese find-ings suggest a new class vibriocidal compounds Moreoverthe results are consistent with the demand for new alter-natives to antibacterial drugs in order to mitigate the impactcaused by the indiscriminate use of antimicrobials in aqua-culture

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The first author received a Doctoral Scholarship Grantfrom the National Foundation for Coordenacao de Aper-feicoamento de Pessoal de Nıvel Superior (CAPES)

References

[1] S M Rashed N A Hasan M Alam et al ldquoVibrio cholerae O1with reduced susceptibility to ciprofloxacin and azithromycinisolated from a rural coastal area of Bangladeshrdquo Frontiers inMicrobiology vol 8 2017

[2] M OrsquoRyan R Vidal F Del Canto J C Salazar and DMonteroldquoVaccines for viral and bacterial pathogens causing acutegastroenteritis part i overview vaccines for enteric viruses andVibrio cholerardquo Human Vaccines and Immunotherapeutics vol11 no 3 pp 584ndash600 2015

[3] E O Igbinosa ldquoDetection and antimicrobial resistance of vibrioisolates in aquaculture environments implications for publichealthrdquo Microbial Drug Resistance vol 22 no 3 pp 238ndash2452016

[4] W Xiong Y Sun T Zhang et al ldquoAntibiotics antibiotic resist-ance genes and bacterial community composition in freshwater aquaculture environment in Chinardquo Microbial Ecologyvol 70 no 2 pp 425ndash432 2015


[5] J K Dzotam F K Touani and V Kuete ldquoAntibacterial andantibiotic-modifying activities of three food plants (Xantho-soma mafaffa Lam Moringa oleifera (L) Schott and Passifloraedulis Sims) against multidrug-resistant (MDR) Gram-negativebacteriardquo BMC Complementary and Alternative Medicine vol16 no 1 article 9 2016

[6] S A A Jahn H A Musnad and H Burgstaller ldquoThe treethat purifies water cultivating multipurpose Moringaceae inthe Sudanrdquo Unasylva vol 38 no 152 pp 23ndash28 1986

[7] M C Moura T H Napoleao M C Coriolano P M G PaivaR C B Q Figueiredo and L C B B Coelho ldquoWater-solubleMoringa oleifera lectin interferes with growth survival and cellpermeability of corrosive and pathogenic bacteriardquo Journal ofApplied Microbiology vol 119 no 3 pp 666ndash676 2015

[8] J GOnsare andD S Arora ldquoAntibiofilmpotential of flavonoidsextracted from Moringa oleifera seed coat against Staphylococ-cus aureus Pseudomonas aeruginosa and Candida albicansrdquoJournal of AppliedMicrobiology vol 118 no 2 pp 313ndash325 2015

[9] A RNdhlala RMulaudzi BNcubeHAAbdelgadir C PDuPlooy and J V Staden ldquoAntioxidant antimicrobial and phyto-chemical variations in thirteenMoringa oleifera lam CultivarsrdquoMolecules vol 19 no 7 pp 10480ndash10494 2014

[10] E P Padla L T Solis R M Levida C-C Shen and CY Ragasa ldquoAntimicrobial isothiocyanates from the seeds ofMoringa oleifera Lamrdquo Zeitschrift fur Naturforschung Section Cvol 67 no 11-12 pp 557ndash564 2012

[11] R Albuquerque-Costa R Lima-Araujo and R H Silva dosFernandes-Vieira ldquoPhenotyping of vibrios isolated frommarineshrimp hemolymphrdquo Ciencias Marinas vol 39 no 3 pp 317ndash321 2013

[12] R A Costa R L Araujo and R H S dos Fernandes VieiraldquoEnzymatic activity of vibrios isolated from the hemolymph ofcultured Litopenaeus vannamei shrimprdquo Journal fur Verbrauch-erschutz und Lebensmittelsicherheit vol 8 no 4 pp 307ndash3112013

[13] CLSI Performance Standards for Antimicrobial SusceptibilityTesting Twentieth Informational Supplement vol 1 Wayne PaUSA Clinical and Laboratory Standards Institute 2010

[14] S Faizi B S Siddiqui R Saleem S Siddiqui K Aftab and A-U Gilani ldquoIsolation and structure elucidation of novel hypo-tensive agents niazinin A niazinin B niazimicin and niazi-minin A + B fromMoringa oleifera the first naturally occurringthiocarbamatesrdquo Journal of the Chemical Society Perkin Trans-actions 1 no 23 pp 3237ndash3241 1992

[15] S Faizi B S Siddiqui R Saleem S Siddiqui K Aftab andA-UHGilani ldquoIsolation and structure elucidation of newnitrile andmustard oil glycosides fromMoringa oleifera and their effect onblood pressurerdquo Journal of Natural Products vol 57 no 9 pp1256ndash1261 1994

[16] M Golestanbagh I S Ahamad A Idris and R Yunus ldquoEffectof storage of shelled Moringa oleifera seeds from reaping timeon turbidity removalrdquo Journal of Water and Health vol 9 no 3pp 597ndash602 2011

[17] C S T Araujo V N Alves H C Rezende et al ldquoCharacteriza-tion and use ofMoringa oleifera seeds as biosorbent for remov-ing metal ions from aqueous effluentsrdquoWater Science and Tech-nology vol 62 no 9 pp 2198ndash2203 2010

[18] M L Pereira H D De Oliveira J T A De Oliveira et alldquoPurification of a chitin-binding protein fromMoringa oleiferaseeds with potential to relieve pain and inflammationrdquo Proteinand Peptide Letters vol 18 no 11 pp 1078ndash1085 2011

[19] R S Ferreira T H Napoleao A F S Santos et al ldquoCoagulantand antibacterial activities of the water-soluble seed lectin fromMoringa oleiferardquo Letters in Applied Microbiology vol 53 no 2pp 186ndash192 2011

[20] J R O Peixoto G C Silva R A Costa et al ldquoIn vitro anti-bacterial effect of aqueous and ethanolicMoringa leaf extractsrdquoAsian Pacific Journal of Tropical Medicine vol 4 no 3 pp 201ndash204 2011

[21] W Atieno SWagai A Peter and J Ogur ldquoAntibacterial activityof Moringa oleifera and Moringa stenopetala methanol andn-hexane seed extracts onbacteria implicated in water bornediseasesrdquo African Journal of Microbiology Research vol 5 pp153ndash157 2011

[22] U Eilert BWolters and A Nahrstedt ldquoThe antibiotic principleof seeds of Moringa oleifera and Moringa Stenopetalardquo PlantaMedica vol 42 no 1 pp 55ndash61 1981

[23] K Nantachit ldquoAntibacterial activity of the capsules ofMoringaoleifera Lamk (Moringaceae)rdquo CMU Journal vol 5 no 3 pp365ndash368 2006

[24] A P Guevara C Vargas H Sakurai et al ldquoAn antitumor pro-moter fromMoringa oleifera LamrdquoMutation Research vol 440no 2 pp 181ndash188 1999

[25] A H Gilani K Aftab A Suria et al ldquoPharmacological studieson hypotensive and spasmolytic activities of pure compoundsfromMoringa oleiferardquo Phytotherapy Research vol 8 no 2 pp87ndash91 1994

[26] S Faizi B S Siddiqui R Saleem S Siddiqui K Aftab andA-UGilani ldquoFully acetylated carbamate and hypotensive thiocarba-mate glycosides fromMoringa oleiferardquo Phytochemistry vol 38no 4 pp 957ndash963 1995

[27] S Faizi B S Siddiqui R Saleem F Noor and S Husnain ldquoIso-lation and structure elucidation of a novel glycoside niazidinfrom the pods ofMoringa oleiferardquo Journal of Natural Productsvol 60 no 12 pp 1317ndash1321 1997

[28] A Murakami Y Kitazono S Jiwajinda K Koshimizu andH Ohigashi ldquoNiaziminin a thiocarbamate from the leavesof Moringa oleifera holds a strict structural requirement forinhibition of tumor-promoter-induced epstein-barr virus acti-vationrdquo Planta Medica vol 64 no 4 pp 319ndash323 1998

[29] F Anwar S Latif M Ashraf and A H Gilani ldquoMoringaoleifera a food plant with multiple medicinal usesrdquo Phytother-apy Research vol 21 no 1 pp 17ndash25 2007

Submit your manuscripts athttpswwwhindawicom

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International Journal of

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collection of the Laboratory of Environmental and FisheryMicrobiology at Sea Sciences Institute (LABOMAR-UFC-Brazil) The 100 strains were phenotypically identified as Vnavarrensis (119899 = 53) V brasiliensis (119899 = 15) V para-haemolyticus (119899 = 10) V xuii (119899 = 8) V coralliilyticus (119899 =5) V cholerae (119899 = 4) V neptunis (119899 = 2) V alginolyticus(119899 = 1)V diazotrophicus (119899 = 1) andV vulnificus B3 (119899 = 1)[11] The enzymatic profile and antimicrobial resistance wereused as a criterion for selection [12]

22 BotanicalMaterial Moringa oleifera seeds were collectedfrom two specimens grown in a campus of the FederalUniversity of Ceara (Pici Fortaleza Ceara) Separation fromthe fruit (pod) removal of husks and posterior packing inplastic polyethylene bags followed the material collection

23 Moringa oleifera Extracts All extraction procedureswere performed in the Department of Organic and InorganicChemistry at Federal University of Ceara (UFC) Part of thecrushed seeds of M oleifera (110 g) was subjected to threeextractions with 300mL cold hexane (PA) at 24 h intervalsAfter filtration and evaporation of the solvent under reducedpressure in a rotary evaporator 1536 g of an extract of fluidand yellowish appearance called MOS-H was obtained Theresulting cake was subjected to three cold extractions with300mL ethanol (PA) in 24 h intervals After filtration andevaporation of the solvent under reduced pressure in a rotaryevaporator 1164 g of an extract of fluid and dark appearancecalledMOS-Ewas obtained 139 g of crushedM oleifera seedswas used for hot extraction in a Soxhlet apparatuswith 800mlof hexane (PA) for 48 h After filtration and evaporation of thesolvent under reduced pressure in a rotary evaporator 2829 gof an extract of fluid and yellowish aspect calledMOS-HSwasobtained Another extraction was carried out with 800ml ofethanol (PA) for 48 h After filtration and evaporation of thesolvent under reduced pressure in a rotary evaporator 1366 gof a pasty and dark-colored appearance called MOS-ES wasobtained

24 In Vitro Susceptibility Testing of Moringa oleifera ExtractsSusceptibility of Vibrio sp strains to the four extracts types(MOS-H MOS-E MOS-HS and MOS-ES) was assessedusing the disk diffusion method (DDM) and by MinimumInhibitory Concentration (MIC) [13] In order to proceedwith the DDM paper discs (6mm) containing 100 120583L of eachextract were applied in triplicate on Mueller-Hinton platespreviously seeded with bacterial cultures (108UFCmLminus1)As negative and positive Gram control strains of V para-haemolyticus IOC and Staphylococcus aureus ATCC 25923respectively were used For MIC determination macrodilu-tion technique in Mueller-Hinton broth containing 1 NaClwas used Concentrations of 4 8 16 32 and 64120583gmLminus1 weretested using the MOS-E oils (cold extraction with ethanol)and MOS-ES (hot extraction with ethanol) in comparison toisolates susceptible to crude extracts in the DDM test

25 Chromatographic Fractionation of MOS-E and ES-MOSandObtainingActive Fraction Ethyl Acetate (MOS-ESA) Partof the MOS-E extract (31 g) was adsorbed onto 73 g of silica

gel and chromatographed on 525 g of silica gel in opencolumn (Oslash 50 cm) Elution was done in order of increas-ing polarity with dichloromethane (1200ml) (MOS-ED)ethyl acetate (800mL) (MOS-EA) and methanol (600mL)(MOS-EM) Solvents were evaporated under reduced pres-sure in a rotary evaporator yielding the following massand yields MOS-ED 19208mg 6196 MOS-EA 2316mg747 MOS 6608mg 2131 MOS-ES extract (26 g) wasadsorbed onto 39 g of silica gel and chromatographed on483 g of silica gel in open column (Oslash 50 cm) Elution wasdone in order of increasing polarity with dichloromethane(500ml) (MOS ESD) ethyl acetate (700ml) (MOS-ESA) andmethanol (600ml) (MOS-ESM) Solvents were evaporatedunder reduced pressure in a rotary evaporator yielding thefollowing mass and yields MOS-ED 348mg 133 MOS-EA 3353mg 1289 MOS 1978mg 7607 All fractionswere subjected to antimicrobial activity test by disk diffusionmethod The bioactive fraction was subjected to chromato-graphic fractionation by High-Performance Liquid Chro-matography (HPLC) in order to isolate its active principles

26 Chromatographic Fractionation of the Ethyl Acetate Frac-tion (MOS-ESA) by High-Performance Liquid Chromatogra-phy (HPLC) and Isolation of Active Substances Part of theMOE-ESA active fraction (285mg) was analyzed by HPLC ina chromatograph Shimadzu (UFLCmodel) equipped with aUV-Vis detector with diode array (model SPD-M20A) Sepa-ration was performed in reverse phase conditions in semi-preparative column (C-185 120583m) with isocratic elution usingMeOHH

2O (1 1) with a 472mLminminus1 flow Chromato-

graphic fractionation of the ethyl acetatersquos fraction from theMOS-ES fixed oil resulted in the detection (at 284 nm) andisolation of three main substances (Figure 1) which wereobtained as whitish amorphous solids the compound relatedto the peak 1 (233m 119905

119903= 499min) was called MOS-ES-1

related to peak 2 (40mg 119905119903= 706min) was called MOS-ES-

2 and related to peak 3 (651mg 119905119903= 1745min) was called

MOS-ES-3 Isolated substances (S1 and S3) had their struc-tures determined by the analysis of Nuclear Magnetic Res-onance (NMR) and Infrared (IR) spectral data and also bycomparison to other findings described in the literature [14 15]

3 Results

31 Disk Diffusion Test From all the 100 strains tested onlyfive were resistant to MOS-E fixed oil on the other hand36 had its growth inhibited with average inhibition zonesranging from 13 to 15mm (Table 1) When comparing theoils MOS-ES tests demonstrated inhibitory bacterial ratesomewhat lower since most strains (119899 = 37) had inhibitionsindicated by halos in the range 10 to 12mm (Table 2)In addition seven strains were resistant to the MOS-ESThe larger inhibition halo (22 to 24mm) was observedon MOS-E test against a V navarrensis strain (Table 1)Both extracts showed antibacterial effect against the Gram-positive (Staphylococcus aureus) and Gram-negative (Vibrioparahaemolyticus) controls (Tables 1 and 2) MOS-H andMOS-HS extracts showed no bioactivity against any of theisolates (119899 = 100)





123 45

67 8

9

10

11 12

13

14

15

(mAU

)

(Min)

MOS-ES1

MOS-ES2

MOS-ES3

2017515012510075502500

0

250

500









3OD) 120575 728 (2H d J = 86Hz) 707 (2H d J =


3OD) 120575 1574






3OD) 120575 1571 (C-


minus1




















4 Discussion





















nt not tested










Acknowledgments


References






































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





123 45

67 8

9

10

11 12

13

14

15

(mAU

)

(Min)

MOS-ES1

MOS-ES2

MOS-ES3

2017515012510075502500

0

250

500









3OD) 120575 728 (2H d J = 86Hz) 707 (2H d J =


3OD) 120575 1574






3OD) 120575 1571 (C-


minus1




















4 Discussion





















nt not tested










Acknowledgments


References






































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















4 Discussion





















nt not tested










Acknowledgments


References






































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














nt not tested










Acknowledgments


References






































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Thiocarbamates from Moringa oleifera Virulent and...

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