Antibacterial Derivatives of Marine Algae

23 Pages·2016·0.28 MB·English

marine drugs
Review
Antibacterial Derivatives of Marine Algae:
An Overview of Pharmacological Mechanisms
and Applications
EmerShannonandNissreenAbu-Ghannam*
SchoolofFoodScienceandEnvironmentalHealth,CollegeofSciencesandHealth,
DublinInstituteofTechnology,CathalBrughaStreet,DublinD01HV58,Ireland;[email protected]
* Correspondence:[email protected];Tel.:[phone]
AcademicEditors:TracyJohnMincerandDavidC.Rowley
Received:24February2016;Accepted:15April2016;Published:22April2016
Abstract: Themarineenvironmentishometoataxonomicallydiverseecosystem. Organismssuch
asalgae,molluscs,sponges,corals,andtunicateshaveevolvedtosurvivethehighconcentrations
of infectious and surface-fouling bacteria that are indigenous to ocean waters. Both macroalgae
(seaweeds) and microalgae (diatoms) contain pharmacologically active compounds such as
phlorotannins,fattyacids,polysaccharides,peptides,andterpeneswhichcombatbacterialinvasion.
Theresistanceofpathogenicbacteriatoexistingantibioticshasbecomeaglobalepidemic. Marine
algae derivatives have shown promise as candidates in novel, antibacterial drug discovery. The
efficacyofthesecompounds,theirmechanismofaction,applicationsasantibiotics,disinfectants,and
inhibitorsoffoodbornepathogenicandspoilagebacteriaarereviewedinthisarticle.
Keywords: marine antibacterial; seaweeds; micro-algae; nutraceuticals; antibiotic-resistance;
foodpreservation;disinfectants;allelopathy
1. Introduction
The search for bioactive compounds from marine organisms in recent decades has produced
an abundance of extracts with pharmaceutical and industrial applications. In 2013 alone, over
onethousandpharmacologicallyactivecompoundsofmarineoriginwerecharacterisedworldwide,
with potential efficacy against cancer, viruses, bacteria, fungi, hypertension, high cholesterol and
otherdiseases. Marineorganismscommonlytargetedforscreeningincludemacroandmicroalgae,
cnidarians, phytoplankton, molluscs, sponges, corals, tunicates, and bryozoans. Much of this
research has concerned the identification of antimicrobial agents; particularly compounds active
againstpathogenicbacteria[1,2]. Theglobalepidemicofbacterialresistancetoexistingantibiotics
such as β-lactams and quinolones has prompted the search for naturally occurring candidate
drugs, anti-foulingagents, andfoodpreservationagentsfromterrestrialandmarinesources. The
evolutionofantibacterialbiomoleculesalongsidebacteriaovermillionsofyearsmayprovidethem
with the potential to overcome strains such as methicillin resistant Staphylococcus aureus (MRSA)
and fluoroquinolone-resistant Pseudomonas [3]. Antibacterials from the marine environment in
particularhavebeenstudied. Microandmacroalgae,suchasdiatomsandseaweeds,havedeveloped
indigenous systems to combat pathogenic bacteria and other microbes, ubiquitous to the ocean
environment. This review focuses on antibacterial compounds derived from marine algae, their
mechanism of pharmacological action, and outlines their current and potential applications as
antibiotics,disinfectants,andinhibitorsoffoodbornepathogenicandspoilagebacteria.
Mar.Drugs2016,14,81;doi:10.3390/md14040081 www.mdpi.com/journal/marinedrugs
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2. AntibacterialCompoundsinMarineAlgaeandTheirFunctionalGroups
Algaehavetraditionallybeenusedasfood,orforhydrocolloidssuchasalginateandcarrageenan.
However, modern screening methods have identified antibacterial compounds in the secondary
metabolitesofalgalclassessuchasthePhaeophyceae(brown),Rhodophyceae(red),Chlorophyceae
(green), Chrysophyceae (golden) and Bacillariophyceae (diatoms) [4–8]. Functional groups with
antibacterialactivityinthesecompoundsincludephlorotannins,fattyacids,polysaccharides,peptides,
terpenes,polyacetylenes,sterols,indolealkaloids,aromaticorganicacids,shikimicacid,polyketides,
hydroquinones,alcohols,aldehydes,ketones,andhalogenatedfuranones,alkanes,andalkenes[9–11].
Theecologicalfunctionofsomeofthesesecondarymetabolitesinalgaeisnotyetfullyunderstood.
Sincetheyarenotrequiredfornormalgrowthorreproduction,adaptiontoconditionstowhichthey
areexposedintheoceanisproposedtobethemainfactorcontrollingtheiroccurrence. Impediments
toalgalsurvivalincludegrazingbyherbivores,competitionforspacefromotherorganisms,thallus
injury,andbiofilmformation. Algaearealsoexposedtoosmoticstress;andhighlevelsofUVlight,
oxygen,andsalinity. Inaddition,anaverageofonemillionbacterialcellsarepresentineachmillilitre
ofseawater[12]. Someofthechemicalsproducedbyalgaetocombatthesestresseshaveantibacterial
potential,andmanyhavehighbiologicalactivityduetothedilutingeffectofseawaterandtheharsh
environmentinwhichtheylive[13].
AlgalAllelopathy
Thebiologicalphenomenonofallelopathyinterrestrialandmarinehabitatscontributestothe
productionofpotentiallyantibacterialcompounds. Allelochemicalsarereleasedbymanyalgaeinto
theirenvironment. Thesesecondarymetabolitesexertbeneficialordetrimentaleffectsoncompeting
organisms,theendresultofwhichoffersacompetitiveadvantagetothealga. Sessilealgaesuchas
seaweedsaremorepronetobacterialbiofilmformationandconsumptionbyinvertebratescompared
tomotile,singlecellalgae. Allelochemicalshavebeenidentifiedinseveralspecieswhichaidalgaein
competitionforanchoringspaceonrocksandtheseabed. Manyofthesecompoundsaretoxic,with
applicationsincancerchemotherapy,andarecurrentlybeingscreenedforantibacterialpotential[14].
Vieira et al. [15] characterised three new lobophorenols (C polyunsaturated alcohols) in
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the brown seaweed genus, Lobophora, which had significant allelopathic effects against the coral,
Acroporamuricata,causingbleachingandnecrosis. Similarly,twoacetylatedditerpenesreportedby
Rasheretal.[16]inthegreenalga,Chlorodesmisfastigiata,andtwololiolidederivativesfromtheredalga,
Galaxaurafilamentosa,actedaspotentallelochemicalsagainstcorals. Thelipophiliccompoundswere
secretedfromthallussurfacesandinhibitedphotosynthesisinthecorals,causingbleaching,andin
somecases,death.Akainicacidanalogue,domoicacid,producedbythediatomgenus,Pseudo-nitzschia,
actsasanintraandinter-speciesallelochemical,andisresponsiblefortoxicalgalblooms. Domoic
acidisanexcitotoxicaminoacidthataffectsthecentralnervoussysteminvertebratessuchasshrimp,
andcompetitivelyinhibitsthegrowthofotherPseudo-nitzschiaspecies[17,18]. Similarly,ovatoxins
andpalytoxinsproducedbythetoxicdinoflagellateprotozoan,Ostreopsis,[19]actasallelochemical
deterrentstoneighbouringvertebrates, andcancausealgalblooms. Accoronietal.[20]examined
the allelopathic interactions between Ostreopsis cf. ovata and three seaweeds, Dictyota dichotoma,
Rhodymeniapseudopalmata,andUlvarigidathatcompeteforspaceinthesamehabitat. Freshanddried
thalliofDictyotadichotomaandRhodymeniapseudopalmatawerefoundtosignificantlyinhibitthegrowth
of Ostreopsis ovata. The compounds responsible are currently being elucidated and have potential
applicationsinthepreventionofalgalblooms,andasantibacterials.
Phlorotanninsandterpenesinalgaealsoactasallelochemicaldeterrentstoherbivores,inhibitors
of bacterial biofilm formation, and as antioxidants against UV damage [21–23]. Phlorotannins are
composedofpolyphenolicphloroglucinolunitsandoccurinPhaeophyceae. Theyhavetheabilityto
bindwithandprecipitateproteinsinthesamewaytanninsdointerrestrialplants. Thismakesthe
algaemoreastringentandlesspalatabletograzersasitbindswithproteinsinthemouth[24–26].
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Some seaweeds, such as the green alga, Caulerpa cylindracea, have developed immunity to
severalpathogenicepiphyticVibriospeciesthatliveontheirthallussurface. Asymbioticallelopathic
relationshipwiththebacteriahasbeenproposedbyRizzoetal.[7],wherethepresenceoftheVibrio
actually contributes to successful algal reproduction, allowing the two organisms to function as a
holobiont[27]. FoodborneillnesscausedbyVibrioandotherpathogenicbacteriaisanincreasingglobal
issue. Isolationofthecompoundsandthemechanismresponsibleforalgalimmunitytothesebacteria
could produce useful therapeutics and sanitisers. The harnessing and bioengineering of recently
characterisedallelochemicalsrepresentsapotentialareaofnewmarineantibacterials.
3. MechanismsofPharmacologicalActionandPotentialApplications
Bactericidal and bacteriostatic compounds were first isolated from algae when chloroform
and benzene fatty acid extracts of chlorellin, from Chlorella vulgaris, were found to inhibit
Pseudomonasaeruginosa,Staphylococcusaureus,Streptococcuspyogenes,andBacillussubtilis[28].Chlorellin
was not suitable for large scale commercial use, however it heralded further research into algal
antimicrobialinhibitorsingenerasuchasScenedesmus[29]. Asdiscussed,severalchemicalfunctional
groupsinalgae,suchasphlorotannins,fattyacids,peptides,terpenes,polysaccharides,polyacetylenes,
sterols,indolealkaloids,aromaticorganicacids,shikimicacid,polyketides,hydroquinones,alcohols,
aldehydes, ketones, and halogenated furanones have been reported as bacterial inhibitors. The
mechanism of pharmacological action for some remains uncertain, however, methods of bacterial
inhibitionemployedbythefollowingfunctionalgroupshavebeenproposed.
3.1. Phlorotannins
Phlorotanninsoccurinfucosangranulescalledphysodeswithinalgalcellsandcomprise1%–15%
ofthethallusdrymass[30]. Theantibacterialactivityofphlorotanninshasbeenreportedtobedue
toinhibitionofoxidativephosphorylation,andtheirabilitytobindwithbacterialproteinssuchas
enzymes and cell membranes, causing cell lysis. The phenolic aromatic rings and OH groups of
thephloroglucinolunitsbindtothe-NHgroupsofbacterialproteinsbyH-bondandhydrophobic
interactions[31,32].KameiandIsnansetyo[33]reportedthatthebacteriolyticactivityofphloroglucinol
compoundsagainstVibriospeciesincreasedwhentertiarystructuressuchasmethyl-oracetyl-vinyl
werepresent. However,agreaterminimuminhibitoryconcentration(MIC)wasrequiredtopenetrate
theGram-negativespecies,Vibrioparahaemolyticus,comparedtotheGram-positiveMRSA.Thisisthe
caseformostantibacterials,duetophysiologicaldifferencesinβ-lactamasemechanisms,andtheless
penetrablenatureoftheouterlipopolysaccharidemembraneofGram-negativespeciesincomparison
tothepeptidoglycanGram-positivelayer[34].
Wei et al. [35] reported that low molecular weight phlorotannins extracted from
Sargassum thunbergii damaged the cell membrane and cell wall of Vibrio parahaemolyticus, causing
cytoplasm leakage and deconstruction of membrane permeability. The study suggested that low
molecular weight phlorotannins from algae could potentially be used in food safety control and
aquaculturaldrugs. Leeetal.[36]testedarangeofsolventextractsfromthebrownseaweed,Arame
(Eisenia bicyclis) against antibiotic resistant Propionibacterium-related acne. A phlorofucofuroeckol
compound(phlorotanninwithanalcoholsubstituent)exhibitedthemostpotentantibacterialactivity
with an MIC of 32 µg/mL, while also significantly reversing the resistance of Propionibacterium to
erythromycinandlincomycin. Thesameresearchgrouptestedtheactivityofphlorofucofuroeckol
fromEiseniabicyclisagainstMRSA.PhlorofucofuroeckolsuppressedmecI,mecR1,andmecAgene
expression in the resistant Staphylococcus aureus cells. These three genes regulate the expression
of methicillin resistance in bacteria. This resulted in suppression of penicillin-binding protein 2a
production,whichisconsideredthemainmechanismbywhichMRSAstrainsresistmethicillin[37,38].
Phlorotanninsandtheirderivativesofferapotentiallyusefulsourceofnaturalantibacterialagentsfor
foodandmedicalapplications.
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3.2. FattyAcids
Algal free fatty acids have been reported to act as inhibitors of the electron transport
chain and normal oxidative phosphorylation in bacterial cell membranes. This interferes
with adenosine triphosphate energy transfer, and inhibits enzymes such as bacterial enoyl-acyl
carrier protein reductase, necessary for the synthesis of fatty acids within the bacterial cell.
Lysis of the cell, and formation of peroxidation and auto-oxidation degradation products then
occurs [39–42]. El Shafay et al. [43] identified the antibacterial fatty acids, cyclopentaneacetic acid,
and10,13-octadeadienoicacidasprincipalcomponentsofethanolextractedSargassumvulgareand
diethyletherextractedSargassumfusiforme. Transmissionelectronmicroscopywasusedtomeasure
themorphologicalchangesinStaphylococcusaureusandKlebsiellapneumoniaecellstreatedwiththese
brownseaweedextracts. Thecellwallsofbothbacteriawereperforated,resultinginruptureofthe
cellwall,cytoplasmicleakage,shrinkingoftheprotoplasm,cytoplasmicvacuolation,scatteringof
chromatin,distortionoftheoutercellshape,anddecreasedcellsize. Cˇermáketal.[44]reportedthe
antibacterial long-chain fatty acids in the green microalga Planktochlorella nurekis to be significant
inhibitorsofCampylobacterjejuni,Escherichiacoli,Salmonellaentericavar. Enteritidis,Salmonellaenterica
var. Infantis,Arcobacterbutzleri,andLactobacillusjohnsoniiusingasuspensionconcentrationrangeof
0.75–6mg/mL.Thestudyproposedthatgreenmicroalgaecouldbeusedasanalternativetoin-feed
antibioticstopreventdiseaseinlivestockandpoultryandtomaintainthemicrobialsafetyofanimal
productsinthehumanfoodchain.
3.3. Polysaccharides
Polysaccharidesarecomposedofrepeatingmonosaccharideunitslinkedbyglycosidicbonds.
Theyfunctionprimarilyasstructuralstoragecompoundsinplantsandalgae. Algalpolysaccharides
and sulphated polysaccharides have been used successfully for pharmaceutical and dietary
applications. Theirmechanismofantibacterialactionisproposedtobeduetoglycoprotein-receptors
presentonthecell-surfaceofpolysaccharideswhichbindwithcompoundsinthebacterialcellwall,
cytoplasmicmembrane,andDNA.Thisresultsinincreasedpermeabilityofthecytoplasmicmembrane,
protein leakage, and binding of bacterial DNA [45–47]. Polysaccharides, such as fucoidan and
laminarin, have been successfully used in drug delivery as oral antibiotics to inhibit the growth
ofStaphylococcusaureusandEscherichiacoli;andtopreventtheadhesionofHelicobacterpyloribiofilms
ingastricmucosa. Theyhavealsobeenincorporatedintofoodasadietarysupplementtoimprovethe
immuneresponseoffarmedfish,andreducesusceptibilitytoPiscirickettsiasalmonisinfection[48–51].
Kadametal.[48]reportedasignificantinhibitionofStaphylococcusaureus,Listeriamonocytogenes,
EscherichiacoliandSalmonellatyphimuriumgrowthwithultrasoundassistedextractionoflaminarin
fromtheIrishbrownseaweedsAscophyllumnodosumandLaminariahyperboreausing0.1Mhydrochloric
acid. AbouZeidetal.[52]demonstratedthathotandcoldwater-extractedpolysaccharidesfromthe
redseaweedPterocladiacapillaceaandbrownseaweedDictyopterismembranaceainhibitthegrowthof
Gram-positiveBacilluscereusandStaphylococcusaureus,andGram-negativePseudomonasfluorescensand
Escherichia coli in disc diffusion assays. In the case of Staphylococcus aureus, cold water-extracted
Pterocladia capillacea had an activity equivalent to 56.8% of the antibiotic standard, ampicillin.
Vijayabaskaretal.[53]foundthatsulphatedpolysaccharidesextractedfromSargassumswartziiinhibited
both Gram-positive and Gram-negative bacteria in ten human pathogenic strains. In the case of
Escherichiacoli,thepolysaccharideextractwasmorepotentinthediscdiffusionassaythananampicillin
antibioticstandard.
3.4. ProteinsandPeptides
The antimicrobial activity of amino acids in the form of short-chain peptides, or larger, more
complexproteins,hasbeendemonstratedinanumberofrecentstudies.Theamphipathicconformation
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ofpeptidesenablesthemtobindwithpolarandnon-polarsitesonbacterialcytoplasmicmembranes,
therebyinterferingwithcellularprocessesandpropagation[54–56].
Lectins,forexample,areadiversegroupofproteinsthatoccurinanimals,plants,algae,bacteria,
andviruses[57]. Inhumans,theyhavemultiplebiologicalfunctionsincludingcarbohydrate-binding,
celladhesion,blood-proteinregulation,andimmunedefence[58]. Theabilityoflectinstoselectively
bindwithlipopolysaccharides,β-glucans,andpeptidoglycansonthecellsurfaceofbacteriaaffords
thembactericidalproperties,asnormalcellprocessessuchasnutrientuptakeareblocked[59]. Several
functionalgroupsareinvolved. Hydrogenbondsareformedbetweenpolarmoietiesofaminoacidsin
the lectins and the hydroxyl groups of polysaccharides, coupled with van der Waals interactions,
and packing of hydrophobic polysaccharide regions against amino acid aromatic groups in the
lectins[60–62]. Holandaetal.[63]evaluatedtheinhibitoryeffectoflectinextractsfromtheredalga
SolieriafiliformisagainstGram-negativeandGram-positivepathogenicbacteria. Ataconcentration
of1000µg/mL,theextractinhibitedgrowthoftheGram-negativespeciesPseudomonasaeruginosa,
Enterobacteraerogenes,Serratiamarcescens,Salmonellatyphi,Klebsiellapneumoniae,andProteusspecies.
Thebindingoflectinwithmannanwasbelievedtobethemethodbywhichgrowthwasinhibited.
Mannanisalinearpolymerofthesaccharidemonomermannose,andoccursonthecellsurfaceof
Gram-negativebacteria. Mannanactsasahaptenuponbindingwiththelargelectinmolecule,eliciting
an immune response. However, no inhibition of growth was observed against the Gram-positive
StaphylococcusaureusandBacillussubtilis,possiblyduetounsuitablelectin-polysaccharidebinding
sitesonthecellsurfacesofthesespecies[64].
Enzymatichydrolysisisthemostcommonmethodusedtoisolatebioactivepeptidesfromparent
proteinsformedicalapplications,thusavoidingtoxicsolventresidues. Thebioactivityofindividual
peptidehydrolysatesisoftendistinctfrom,orgreaterthanthatoftheoriginalprotein. Somerecently
characterisedpeptideshavebeentermedcrypteins,duetotheircryptic,orhidden,bioactivity,and
areacurrentareaofnoveltherapeuticstudy[65]. Marinealgaearegoodcandidatesfortheminingof
suchpeptidesastheycontainahighproportionofdiverseproteins[66,67].
Beaulieu et al. [68] extracted antibacterial peptides (>10 kDa mass) from the brown seaweed
Saccharinalongicrurisbyenzymatichydrolysiswithtrypsin. Liquidchromatography-tandemmass
spectrometryidentifiedthesub-fractionsaspeptideprecursorstoproteinssimilartoubiquitin,leucine,
histone,andaribosomalstructure,whichformpartoftheinnateimmunedefenceoftheseaweed.
MaximumspecificgrowthrateofthefoodspoilagebacteriumStaphylococcusaureuswassignificantly
inhibitedbythehydrolysateatconcentrationsof0.31mg/mLto2.5mg/mLmakingitapotential
agentforfoodpreservation.
3.5. Terpenes
Terpenesarecompoundscomposedofrepeatingisopreneunits,oftenwithsubstituentgroups.
Terpenes with pharmacological activity have been successfully extracted from terrestrial plants.
Forexample,paclitaxel(ortaxol)fromPacificyewtreesisusedincancertreatment,andArtemisinin
from the plant Artemisia annua is an anti-malarial drug [69,70]. A number of terpene compounds
from algae, such as diterpene-benzoate bromophycolides, have been found to inhibit bacterial
growth. Laneetal.[71]extractedbromophycolides(diterpene-benzoatemacrolides)fromtheFijian
red alga Callophycus serratus with water, methanol, and dichloromethane. Extracts significantly
inhibitedMRSAandvancomycin-resistantEnterococcusfaecium,withmaximalinhibitoryconcentration
(IC ) values of 1.4 µM and 5.8 µM respectively. Their findings suggested that the mechanism of
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antibacterialactionwasduetothehydrophobicityandconformationalrigidityofthetetrahydropyran
structure. Rodriguesetal.[72]useddichloromethanetoisolatesphaeranebromoditerpenes,including
a previously uncharacterised, rare dactylomelane called sphaerodactylomelol, from the red alga
Sphaerococcuscoronopifolius. TheextractswerefoundtoinhibitEscherichiacoli,Pseudomonasaeruginosa,
Staphylococcus aureus, and Candida albicans. The greatest antibacterial was observed against
Staphylococcusaureus,withanIC valueof6.35µM.Etahirietal.[73]isolatedbromosphaeroneand
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12S-hydroxybromosphaerodiolfromthesamealga,Sphaerococcuscoronopifolius. Bromosphaeroneand
12S-hydroxybromosphaerodiolinhibitedStaphylococcusaureuswithMICvaluesof0.104µg/mLand
0.146µg/mLrespectively. Itwasproposedthattheantibacterialactivityofbromosphaeronewasdue
toitsamphipathicstructureofthreepolaralcoholgroups,withnon-polaraliphaticcarbonandbromine
atomsontheoppositesideofthemolecule,enablingittobindwithbacterialcellmembranes[13,73].
Xanthophylls,suchasluteinandastaxanthin,aretetraterpeneoxygen-containingcompoundsthat
functionaslightharvestingpigmentsinplantsandalgae. Theyhavebeenextensivelydocumented
for their antioxidant activity, but are also effective against bacteria. Fucoxanthin is a xanthophyll
that occurs predominantly in brown algae (Phaeophyceae), diatoms (Bacillariophyceae), and at
lowerconcentrationsingoldenalgae(Chrysophyceae)andRaphidophyceae[74–78]. Rajauriaand
Abu-Ghannam[79]extractedfucoxanthinfromtheIrishbrownseaweedHimanthaliaelongatausing
diethylether,n-hexane,andchloroform,andpurifiedthecrudeextractusingpreparativeTLC.Indisc
diffusion assays, the purified extract was shown to be a potent inhibitor of Listeria monocytogenes,
withaninhibitionzoneof10.27mmataconcentrationof1mg/mL(25µg/disc). Theextractwas
98.4%aseffectiveasananalyticalgradefucoxanthinstandard(inhibitionzone10.89mm). Similarly,
Deyab and Abou-Dobara [80] extracted fucoxanthin from the brown seaweed Turbinaria triquetra,
the green Ulva lactuca, and the red Laurencia obtusa with chloroform and methanol. Extracts
were purified by silica column chromatography and identified by nuclear magnetic resonance
spectroscopy. Turbinariatriquetrashowedthegreatestbacterialinhibition,followedbyLaurenciaobtusa.
Ulvalactucaextractshadsignificantlylowerantibacterialactivity. ZonesofinhibitionforEscherichiacoli,
Bacilluscereus,Bacillussubtilis,Klebsiellapneumoniae,Staphylococcusaureus,andPseudomonasaeruginosa
ranged from 4.0 mm to 7.0 mm (100 µg/mL extract/disc) and in the case of some Gram-negative
species,wereequivalenttoantibioticstandards.
3.6. Chrysophaentins
Plaza et al. [81] isolated eight compounds from the chrysophyte alga Chrysophaeum taylori
usinghexane,chloroform,andmethanol. Thecompoundswereofanewchemicalstructuralclass
which they named chrysophaentins, which consisted of two polyhydroxylated, polyhalogenated
ω,ω1-diarylbutene units connected via two ether bonds. The chrysophaentins exerted powerful
inhibition invitro of MRSA (MIC = 1.5 µg/mL), vancomycin-resistant Enterococcus faecium
(MIC = 2.9 µg/mL), and multidrug-resistant Staphylococcus aureus (MIC = 1.3 µg/mL). The
pharmacological mechanism of action of chrysophaentin is proposed to be unlike any existing
antibacterialagent. Thefunctionalgroupswithinchrysophaentinactasenzymeinhibitorsbybinding
withguanosinetriphosphataseinbacterialcells. ThispreventsthesynthesisofaproteincalledFtsZ
(filamentingtemperature-sensitivemutantZ),requiredforbacterialcelldivision. Thedevelopmentof
antibioticsandotherantibacterialproductsfromchrysophaentincontinuestobeanimportantareaof
marinepharmacologicalinvestigation[82,83].
3.7. Lactones
Lactones are a chemical class of cyclic esters, which includes furanones. The Australian
red seaweed Delisea pulchra has been studied for its ability to remain free of surface bacterial
colonisation. HalogenatedfuranoneextractsfromDeliseapulchrahavebeenusedaseffectivesurface
sanitisersinthepreventionofPseudomonasaeruginosabiofilmformation. Thishalogenatedfuranone
also inhibits quorum sensing mechanisms by interfering with bacterial inter-cell communication.
In order for bacteria to express specific genes during quorum sensing, signalling molecules
called acyl-homoserine lactones (AHLs) are required, as well as luminescence transcriptional
activator (LuxR) regulatory proteins. The furanone extract from Delisea pulchra competes with
AHL for the LuxR receptor site, thereby inhibiting virulence factor production and pathogenesis
in Pseudomonas aeruginosa [84,85]. Ren et al. [86] found a similar inhibition of quorum sensing
in Escherichia coli with a (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone extract from
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Deliseapulchra. QuorumsensinginEscherichiacoliwasinhibitedbyblockingS-ribosylhomocysteine
lyase(LuxS)mediatedAI-2signalling. Thisinfluencesgenesandproteinsinvolvedinthenormal
production of flagellar synthesis, motility, and chemotaxis in the bacterium. Manefield et al. [87]
identifiedanothermechanismofinhibitionexertedbyahalogenatedfuranonefromDeliseapulchra.
The bacterium, Erwinia carotovora, produces carbapenem as a virulence factor during quorum
sensing. Acommerciallyavailable4-bromo-5-(bromomethylene)-3-(11-hydroxybutyl)-2(5H)-furanone
wasfoundtoinhibitcarbapenemproductioninErwiniacarotovorabydisruptingthe3-oxo-C6-HSL
dependentexpressionofthecarABCDEFGHoperon.Castilloetal.[88]alsoreportedthatacommercially
produced furanone, similar to the Delisea pulchra extract, was effective against Gram-negative
Campylobacterjejuni. Whencombinedwithepigallocatechingallatefromgreenteaandacitricacid
extract,AI-2activity,bacterialmotility,andbiofilmformationwassignificantlydecreased.
Considering the efficacy of the Delisea pulchra extract against the Gram-negative
Pseudomonas aeruginosa and Escherichia coli invitro, it is not unreasonable to propose that it could
alsoinhibitGram-negativeCampylobacterjejuni. Algalfuranonesandotherlactonesmayhavepotential
asalternativestosyntheticsurfacesanitisers,andantibiotics. CampylobacterjejuniandEscherichiacoli
aretwooftheleadingcausesoffoodpoisoningworldwide,andhavedevelopedresistancetomany
traditional antibiotics [89]. The demonstrated ability of algal furanone extracts to prevent biofilm
formationmaybeusefulinthetreatmentofPseudomonasaeruginosainfection,whichischaracterised
bytheformationofamucoidfilminthelungsofcysticfibrosissufferers. Pseudomonasaeruginosais
alsoanincreasingrisktotheimmunocompromised,suchasprematurebabies,andlong-termhospital
patients[90].
Algalpolysaccharides,fattyacids,peptides,proteins,phlorotannins,terpenes,chrysophaentins,
andlactonesmakegoodcandidatesforantibiotics,andincorporationintohumanfoodproductsfor
safetyandpreservationastheyareedible,non-toxic,andinexpensive. Furtherresearch,including
invivotoxicologystudies,intotheseantibacterialextractscouldproduceveryusefulfoodpreservation
andpharmaceuticalproducts.
4. EffectofExtractionMethodologyonAntibacterialPotency
4.1. EnvironmentalInfluences
Anumberoffactorsinfluencetheefficacyofalgalantibacterialcompoundsandshouldbetaken
into consideration when comparing extraction methods. Natural factors such as ontogenetic and
environmental influences, seasonal variations, ocean temperature, salinity, and pollutants have a
profoundinfluenceonthecontentandpotencyofalgalbioactives. Forexample,thealginate,mannitol,
laminarin,andpolyphenoliccontentsofLaminariadigitata,Laminariahyperborea,Saccharinalatissima,
andAlariaesculentaareknowntovarysignificantlythroughouttheyear. InLaminariaspeciessampled
offthewestScottishcoast,polyphenolshavebeenreportedtoaccumulatewiththeon-setofthegrowth
phasebetweenMayandJulyandreachtheirlowestlevelsinOctober[91,92]. Bioactivecompounds
arepresentatfluctuatingconcentrationsduringdifferentstagesofthealgallifecycle. Inaddition,
distributionvariesineachdiscretepartofthethallus,i.e.,holdfast,blade,andstipe[93]. Samplingof
eachsectionofthethallus,andanalysisofthesamespeciesatdifferentpointsthroughouttheyear
providesmoreaccuracy.
4.2. ExtractionMethodandSolvent
The means by which algal antibacterial compounds are extracted from raw biomass impacts
theirpharmacologicalefficacy. Theextractionmethodandsolventchoiceinfluencethephysicaland
chemicalpropertiesoftheextract. Variousextractionprotocolsfortherecoveryofantibacterialsfrom
marinealgaehavebeenreported.Someinvolvefreezedrying,Soxhletextraction,andtheuseoforganic
solvents, enzymes, or bacterial fermentation. Maceration, often with liquid nitrogen, is generally
requiredforalgae,inordertoreleasecellcomponentsfromthetough,polysaccharide-richthallusof
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seaweeds,andthesilica-boundcellwallsofmicroalgaldiatoms. Thisexposesthemtotheextraction
solvent. Protocolsemploydifferentextractiontemperatures,times,pHranges,andconcentrations. All
thesevariablesinfluencetheefficacyofthefinalantibacterialproduct.
4.2.1. SolventPolarity
Thepolarityoftheextractionsolventprofoundlyaffectsthemannerinwhichtheextractwill
interactwithfunctionalgroupsonbacterialsurfaces. Forexample,dependingonthepolarityofthe
solvent used, functional groups extracted may be polar hydrophilic or non-polar lipophilic. This
influences the ability of the algal extract to act upon the cell membrane and other components of
Gram-positive and Gram-negative bacteria. In a recent study, Moorthi and Balasubramanian [94]
comparedacetone,methanol,andchloroformSoxhletextractionintheevaluationoftheantibacterial
efficacy of the brown seaweed Sargassum muticum harvested in India. Disc diffusion assays were
used to measure its efficacy against foodborne human pathogenic bacteria Salmonella paratyphi,
Staphylococcusepidermis,Enterobacteraerogenus,Klebsiellapneumoniae,Shigellafleschneri,Proteusvulgaris,
MRSA,andSalmonellatyphimurium. Atconcentrationsof30µL/mL,acetoneextractsweresignificantly
morepotentthanchloroformormethanolextractsagainstSalmonellaparatyphi,Salmonellatyphimurium,
Staphylococcus aureus and Shigella fleschneri. Chemical analysis of the Sargassum muticum acetone
extractsshowedthepresenceoftannins. Theeffectivenessofacetonefortheextractionofantibacterials
fromSargassumwightiiandCaulerpascalpelliformishaspreviouslybeenreported,andattributedtothe
abilityofpolaracetonetobindwiththehydrophilicphlorotanninsinseaweedwhich,asdiscussed,
havepotentantibacterialactivity[95,96].
Since lipophilic compounds in algae also have antibacterial activity, non-polar solvents such
ashexane, orsupercriticalcarbondioxidearealsocommonlyemployed. Mendiolaetal.[97]used
supercriticalCO for60min,at40˝C,400atmtoextractlipidsfromthemicroalgaChaetocerosmuelleri.
2
ThefreefattyacidsextractedsignificantlyinhibitedEscherichiacoliandStaphylococcusaureusgrowth.
Unlike most organic solvents, supercritical CO has the benefit of being environmentally friendly
2
sinceitisgaseousatroomtemperatureandpressure,leavingnotraceintheextracts. Solvent-free
antibacterialextractssuchasthesehavepotentialasfoodpreservationingredients.
Extraction yield is also impacted by solvent choice, which in turn affects efficacy of the
antibacterial. Rajauriaetal.[98]reporteddifferentcombinationsofmethanolandwater(20%–80%)
tohaveasignificanteffectontheyieldofantimicrobialandantioxidantpolyphenoliccompounds
from the Irish brown seaweed Himanthalia elongata. The greatest yield (6.8%) was achieved using
60%methanol,comparedtothelowestyield(1.2%)using100%methanol. Discdiffusionandbroth
dilution tests showed the 60% methanol extract at a concentration of 60 mg/mL to be the most
potentinhibitorofGram-positiveListeriamonocytogenesandEnterococcusfaecalis;andGram-negative
Pseudomonas aeruginosa and Salmonella abony. The Himanthalia elongata extract had greater, or at
least equal, zones of inhibition against all bacteria compared to the synthetic food preservatives
sodium nitrite and sodium benzoate. Cox et al. [5] also found that the antibacterial activity of
polyphenolic seaweed extracts was solvent-dependent. Methanol was determined to be the most
effective for the brown seaweeds Himanthalia elongata, Laminaria digitata, and Saccharina latissima;
while acetone and ethanol were more effective for the green species Enteromorpha spirulina; and
the red species Palmaria palmata and Chondrus crispus. Antibacterial activity was tested on food
spoilage (Enterococcus faecalis and Pseudomonas aeruginosa) and pathogenic (Listeria monocytogenes
andSalmonellaabony)bacteriausingthemicrowellplatemethod. Usingmethanolonly, greenand
redseaweedextractshadsignificantlylowerantibacterialactivitythanthebrownspecies,however
theirpotencyincreasedsignificantlywhenethanolandacetonewereusedassolvents. Inthecase
of Chondrus crispus, Enterococcus faecalis inhibition increased from 39% to 100% using ethanol and
acetone. MethanolextractsofHimanthaliaelongatashowed100%inhibitionofListeriamonocytogenes,
Enterococcusfaecalis,andSalmonellaabony,whichwas,onaverage,4.34%greaterthanthatofsodium
nitriteandsodiumbenzoate. LaminariadigitataalsoinhibitedListeriamonocytogenesby100%.
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Sincethestructureandsolubilityofalgalantibacterialcompoundsvariessogreatly,usingaseries
ofsolventswithanincrementalrangeofpolaritiesandconcentrationswouldensuretheextractionof
polarandnon-polarfractions.
4.2.2. FoodGradeSolvents
Solvent choice also influences potential applications of algal antibacterial extracts in terms of
humanandanimalconsumption. Manyorganicsolventssuchasmethanolanddimethylsulphoxide
(DMSO)producehighextractionyields,butleavetoxiclevelsofresidue. Anumberoffood-grade
solventshavebeenapprovedforuseintheextractionofbioactivesforconsumption. Since1966,The
FoodChemicalsCodexhasbeenrecognisedinternationallyastheauthorityforagreedstandardsin
ingredients,preservatives,extractionsolvents,enzymes,andbacteriathatarepermissibleforfood
use[99]. TheEuropeanUnionalsoissuesDirectivesonfood-approvedsolventsystemsandresidual
limits,whichareadministeredbytheEuropeanFoodSafetyAuthority. Forexample,Commission
Directive2010/67/EUsetsoutthemaximumacceptableresiduesforacetone,hexane,andethanolic
extractsofrosemary. Theacceptableconcentrationsaregivenasnotmorethan500mg/kgacetone;
25mg/kghexane;and500mg/kgethanol. TheDirectivealsorecommendsfurtherpurificationofthe
extractwithactivecarbonand/ormoleculardistillation[100]. Alternatively,solventsthataregenerally
recognisedassafe(GRAS)underSections201and409oftheU.S.FederalFood,Drug,andCosmeticAct
canbeutilised,withnoissuesoftoxicity[101]. Forexample,inarecentstudy,Boisvertetal.[102]used
pressurisedliquidextractionwithapproved,food-gradeethanoltoextracthighyieldsofantibacterial
andantioxidant-richcompoundsfromthreespeciesofseaweed,Ascophyllumnodosum,Ulvalactuca,and
Saccharinalongicruris,fromtheStLawrenceEstuary,Canada. Antibacterialactivitywastestedagainst
threefoodspoilagebacteria,Micrococcusluteus,Escherichiacoli,andBrochothrixthermosphacta,usingthe
microtitermethod. ThegreatestinhibitionofEscherichiacoli(69.5%),Micrococcusluteus(61.4%),and
Brochothrixthermosphacta(21.4%)wasexertedbyUlvalactucaextractsataconcentrationof500µg/mL.
The greater potency of the Ulva lactuca compared to the other species was thought to be due to a
highermineralconcentrationofmetalssuchascopper,zinc,silverandmercury,whichhaveinnate
antibacterialactivity. Thischoiceoffoodgradesolventandgreenextractionmethodexemplifiessome
oftheapproachesthatcanbeusedinthedevelopmentofsafealgalantibacterials.
4.3. NovelExtractionTechnologies
Aside from traditional solid-liquid extraction, a number of novel technologies have been
developedwiththeaimofimprovingyields,minimisingdamagetoanalytes,reducingsolventwaste,
andconservingenergyandtime. Thesecanhavesignificanteffectsontheyieldandpotencyofalgal
antibacterialextracts. Technologiesincludeextractionassistedbymicrowaves,pulsedelectricfields,
pressurisedsolvents,supercriticalfluids,ultrasound,andenzymes[103]. Thelatterthreetechnologies
aremostapplicabletoalgalextraction,andareoutlinedhere.
4.3.1. SupercriticalFluidExtraction
When brought to a pressure and temperature above its critical point, a fluid or gas becomes
supercritical. Whileinthisstate,itpossessesthephysicalpropertiesofagasandcaneffusethrough
solids;butcanalsoactasaliquidsolvent[104]. Thesepropertiesarehighlyusefulfortheextraction
of bioactive compounds from organic tissues. Carbon dioxide is most commonly used for food
and pharmaceutical applications as it completely dissipates after extraction at room temperature.
Coffee, for example, is decaffeinated using supercritical CO . As discussed in Section 4.2.1,
2
Mendiolaetal.[97]usedCO supercriticalfluidextractiontoisolatelipidswithantibacterialactivity
2
from the microalga Chaetoceros muelleri. A highly significant increase in antibacterial activity was
reportedusingthismethodincomparisontotraditionalmethodologies. Tocomparetheefficiencyof
theirmethod,aliquid-liquidDMSOflaskextractionofrawChaetocerosmuelleriwasprepared. Abroth
microdilutionmethodwasusedtoevaluatetheMICvalueofeachextractagainstEscherichiacoliand
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Staphylococcusaureus. Thesupercriticalfluidextractionsexertedthreetimesmorebacterialinhibition
thantheDMSOextract. Despitetheincreasedenergydemandsofsupercriticalfluidextraction,itoffers
agreenchemicalalternativetosolventbasedmethodsforantibacterialagentsdestinedforfoodand
pharmaceuticaluses.
4.3.2. UltrasoundAssistedExtraction
Ultrasound or ultrasonic technology uses high intensity sound waves to penetrate a liquid,
producingalternatinghighandlowpressurecycles. Duringlow-pressure,high-intensityultrasonic
wavescreatetinyvacuumbubbles. Whenthehigh-pressurecyclebeginstheyimplode,orcavitate,
releasing liquid jets at extremely high velocity. This high shear force ruptures the cells of organic
tissue,releasingtheircontents[105]. Thishasapositiveeffectontheyieldandpotencyofbioactive
extracts. Kadam et al. [48] compared the efficiency of ultrasound assisted and solid-liquid flask
extractionoflaminarinfromtheIrishbrownseaweedsLaminariahyperboreaandAscophyllumnodosum.
Two solvents were compared for both extraction methods; water, and 0.1 M hydrochloric acid.
Asdiscussed,laminarinisanalgalpolysaccharidewithantibacterialactivity. Thepercentageyield
oflaminarinwassignificantlygreaterinbothwaterandHClultrasoundassistedextractsforboth
seaweeds. Laminariahyperboreaultrasound/HClyieldwas6.240%,comparedtoonly3.254%fromthe
flask/HClextraction.Theultrasoundassistedyieldwas47.85%greater.Similarly,Ascophyllumnodosum
ultrasound/HClyieldwas5.822%,comparedto4.304%fromtheflask/HClextraction. Theultrasound
assisted yield was 26.08% greater. The ultrasound assisted water extracts of Laminaria hyperborea
(5.975%)andAscophyllumnodosum(5.290%)werealsogreaterthanthewaterflaskextractions(4.362%
and 4.599% respectively). Aside from the substantial increase in laminarin yields, the ultrasound
processwasalsofarmoreenergyefficient,requiringonly15minina750Wultrasonicprocessor,atan
amplitudelevelof60%;comparedto2.5hat70˝Cforsolid-liquidextraction. Ultrasoundassisted
extractionisasuitabletechnologyforderivingantibacterialcompoundsfromalgae,asitobviatesthe
needforhightemperatures,toxicsolvents,andprolongedextractiontimes. Italsofractionatesthe
hardy,complexcellwallsofalgae[106].
4.3.3. EnzymeAssistedExtraction
Extractionthroughtheapplicationofenzymes,suchascellulases,hasthepotentialtoincrease
the yield and safety of algal antibacterials. The presence of branched, sulphated, or complex
polysaccharides,suchasalginateandlaminarin, inalgalcellwallslimitstheefficiencyofclassical
extractionmethods[11,107]. Food-gradedigestiveenzymes,suchasproteasesandcarbohydrases,
couldaidinthedegradationofcellwallmatrices,thusreleasingcellcomponents.Todate,littlehasbeen
publishedontheuseofenzymesfortheextractionofantibacterialcompoundsfromalgaespecifically.
However,intheextractionofalgalbioactivessuchasantioxidants,polysaccharides,carotenoidsand
polyphenols,theuseofenzymeshasshownsignificantpotentialasaviablealternative,oradditionto,
puresolventmethods[108–111]. Sinceantioxidants,polysaccharides,carotenoids,andpolyphenols,
have proven antibacterial activity, it is expected that their enzymatic extracts could also be used
to inhibit bacteria. For example, there is an implied correlation between antioxidant activity and
antibacterialactivity,sincealgalantioxidantcompounds,likefucoxanthin,havebeenshowntobe
potentantibacterials. Enzymeassistedextractioncouldpotentiallyincreaseextractionyieldsofalgal
antibacterials. Bioactiveyieldsfromterrestrialplants,suchasthecarotenoidlycopenefromtomatoes,
havebeenincreasedbyenzymolysis. Zuorroetal.[112]reportedaneighteenfoldincreaseinlycopene
yieldfromtomatoprocessingwasteinpectinaseandcellulasepre-treatedsamples,comparedtohexane
extractionalone.IntheextractionoffucoxanthinfromUndariapinnatifida,Billakantietal.[113]reported
a9.3%increaseinfucoxanthinyieldusinganenzymepre-treatmentofalginatelyase,followedby
dimethyletherandethanolextraction,comparedtonon-enzymetreated. Optimumparametersfor
alginatelyasepre-treatmentwere37˝C,for2h,pH6.2,5%(w/v)solids,with0.05wt%enzyme.