Humulus lupulus L. as a Natural Source of Functional Biomolecules

Abstract

Hops (Humulus lupulus L.) are used traditionally in the brewing industry to confer bitterness, aroma, and flavor to beer. However, in recent years, it has been reported that female inflorescences contain a huge variety of bioactive compounds. Due to the growing interest of the consumers by natural ingredients, intense research has been carried out in the last years to find new sources of functional molecules. This review collects the works about the bioactive potential of hops with applications in the food, pharmaceutical, or cosmetic industries. Moreover, an overview of the main extraction technologies to recover biomolecules from hops is shown. Bioactivities of hop extracts such as antibacterial, antifungal, cardioprotective, antioxidant, anti-inflammatory, anticarcinogenic, and antiviral are also summarized. It can be concluded that hops present a high potential of bioactive ingredients with high quality that can be used as preservative agents in fresh foods, extending their shelf life, and they can be incorporated in cosmetic formulation for skincare as well.

Full text

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Appl.Sci.2020,10,5074;doi:10.3390/app10155074www.mdpi.com/journal/applsci
Review
HumuluslupulusL.asaNaturalSource
ofFunctionalBiomolecules
GonzaloAstray
1
,PatriciaGullón
2
,BeatrizGullón
3
,PauloE.S.Munekata
4
andJoséM.Lorenzo
4,5,
*
1
DepartmentofPhysicalChemistry,FacultyofScience,UniversityofVigo(CampusOurense),AsLagoas,
32004Ourense,Spain;gastray@uvigo.es
2
NutritionandBromatologyGroup,DepartmentofAnalyticalandFoodChemistry,FacultyofFoodScience
andTechnology,UniversityofVigo,OurenseCampus,32004Ourense,Spain;pgullon@uvigo.es
3
DepartmentofChemicalEngineering,FacultyofScience,UniversityofVigo(CampusOurense),AsLagoas,
32004Ourense,Spain;bgullon@uvigo.es
4
CentroTecnológicodelaCarnedeGalicia,RúaGaliciaNº4,ParqueTecnológicodeGalicia,SanCibraodasViñas,
32900Ourense,Spain;paulosichetti@ceteca.net
5
ÁreadeTecnologíadelosAlimentos,FacultaddeCienciasdeOurense,UniversidaddeVigo,
32004Ourense,Spain
*Correspondence:jmlorenzo@ceteca.net
Received:30June2020;Accepted:21July2020;Published:23July2020
Abstract:Hops(HumuluslupulusL.)areusedtraditionallyinthebrewingindustrytoconfer
bitterness,aroma,andflavortobeer.However,inrecentyears,ithasbeenreportedthatfemale
inflorescencescontainahugevarietyofbioactivecompounds.Duetothegrowinginterestofthe
consumersbynaturalingredients,intenseresearchhasbeencarriedoutinthelastyearstofindnew
sourcesoffunctionalmolecules.Thisreviewcollectstheworksaboutthebioactivepotentialofhops
withapplicationsinthefood,pharmaceutical,orcosmeticindustries.Moreover,anoverviewofthe
mainextractiontechnologiestorecoverbiomoleculesfromhopsisshown.Bioactivitiesofhop
extractssuchasantibacterial,antifungal,cardioprotective,antioxidant,anti‐inflammatory,
anticarcinogenic,andantiviralarealsosummarized.Itcanbeconcludedthathopspresentahigh
potentialofbioactiveingredientswithhighqualitythatcanbeusedaspreservativeagentsinfresh
foods,extendingtheirshelflife,andtheycanbeincorporatedincosmeticformulationforskincare
aswell.
Keywords:hop;emergingextractiontechnologies;bioactivities;functionalmolecules

1.Introduction
Thecommonhopplant(HumuluslupulusL.)isahardyanddioeciousvinewhoseaerialpartis
herbaceousandannual,whiletherootstockisperennial,anditcancreateadventitiousrootsevery
year[1].ItisaspeciesofthegenusHumulusthatbelongstotheCannabaceaefamilywhosegenus
origincouldbeChina[2].ItcanbefoundprincipallyinEuropeanandinWesternAsiadeciduous
forestsandthickets,andinotherzoneareaswithatemperateclimate[3].Hopswerecultivatedin
Babylonaround200A.D.[4],andtheyhavebeeninongoinguseforcenturies(evenamillennium)
mainlyasabeeringredient[5].IntheEuropeanUnion,themainproducerisGermany,registering
for2018aproductionof41,792tonsforatotalof57,239tonsfromthememberstates[6].Thehop
conesarethemostutilizedportionofthisplant[2];nevertheless,therearepartssuchastheyoung
shootsorasparagusthatcanbeeatenindifferentMediterraneancountries[7].
Inthelastdecades,thebiologicalbenefitsofplants,traditionallyusedinfolkand/ortraditional
medicine,havebeenexploredbythescientificworld[5,8].Inthissense,besidestheiruseinthe

Appl.Sci.2020,10,50742of18
brewingindustry,traditionallyhopconeshavebeenappliedwithmedicalpurposesforthecontrol
ofthespasms,anxiety,fever,inflammation,activationofthegastricfunction,andthetreatmentof
sleepingdisorders,amongothers[2,9].Indeed,hopspresentnumerousbenefitsforhumanhealth
duetotheirantibacterial,antifungal,cardioprotective,antioxidant,anti‐inflammatory,
anticarcinogenic,andantiviralbioactivities[2,5,9–13].
Thefemaleinflorescences(calledhopconesorsimplyhops)aswellasotherpartsoftheplant
(leaves,stems,andrhizomes)arerichindifferentbiologicallyactivemoleculesaspolyphenolic
compoundsandacylphloroglucides,whichareresponsibleforthedifferenthealth‐promotingeffects
andbioactivities[3,9].Thelupulinglands,whichpresentayellow‐greencolor,arelocatedonthehop
umbel(intheinnerandouterbracteoles)andcontainbitterresinsandaromasubstances[1].The
secondarymetabolitesofthefemaleinflorescences(presentedinthelupulinglands)canbedivided
intothreegroups:(1)thehopresins,(2)thehopoil,and(3)thehoppolyphenols[14].Thelevelsof
thearomaticessentialoilandthebitterhopα‐andβ‐acidsdependonseveralfactorssuchasthe
varietyorripeningstageandevenonclimatologicalconditions[15].Themainuseofhops
worldwide,around97%,isusedforbrewingpurposes[16]toaddbitterness,aroma,andtastetothe
beer.
Takingtheaboveintoaccount,andthecurrenttrendofsearchingnaturalcompoundswithnovel
biologicalproperties,thewealthofthehopinbioactivecompoundsmakesitanexcellentsourceto
extractbiologicallyactivemoleculeswithmultipleapplicationsinthealimentary,pharmaceutical,
andnutraceuticalindustries[12,15,17–20].Thishaspromotedintenseresearchinthefieldofbioactive
compoundsfromhopsthatallowsontheonehandachievinghighyieldsandontheotherhand
keepingthebioactivitiesoftheextractedcompounds.Forthis,theselectionofadequateextraction
technologyaswellastheappropriatesolventisfundamental.
Inthiscontext,manypublicationsrelatedtotheisolationoftheactivecompoundshavebeen
developedoverthepastdecades[5].Severalisolationtechnologiesarewellestablishedusing
conventional(organic)solvents[5,21];nevertheless,theirresiduallevelsmustbecontrolled[21],
becausetheycanremaininthefinalproductandhavedetrimentalhealtheffects[5].Accordingto
Marriott[21],EUlegislationandorganiccertificationbodiesestablishtheallowedextractionsolvents,
theirmaximumresiduelevels,andfurtherrestrictions,respectively.Toovercometheshortcomings
oftheconventionalsolvents[22],eco‐friendlysolventssuchasnaturaldeepeutecticonesrepresenta
saferalternative,andtheirusehasincreasedinthelastdecades[23].Moreover,theuseofemerging
technologiessuchas(1)ultrasound‐assistedextraction(UAE),(2)microwave‐assistedextraction
(MAE),(3)pressurizedliquidextraction(PLE),and/or(4)supercriticalfluidextraction(SFE)has
increasedinthelastdecade[24–28].Theseapproachesopennewalternativesinthedesignofthe
extractionprocessofmoreefficientofbiocompoundsfromnaturalbiomasswithintheframeworkof
GreenChemistry[23].
Inlightoftheaboveandencouragedbythegrowinginterestinhopsduetotheirsignificant
potentialasasourceofbioactivecompounds,thisreviewcollectsthestudiesabouttheuseofhops
asanaturalsourceofthesebiocompounds.Aspectsrelatedtothedifferentcurrentextraction
methodsappliedintherecoveryofphytochemicalsfromhopaswellastheevaluationoftheir
differentbioactivitiesarereviewed.Moreover,theapplicationsofthesecompoundsindifferentfields
suchaspharmaceutical,nutraceutical,andcosmeticarealsosummarized.
2.MainComponents
Thehopconeshavedifferentcomponents,suchas(1)resins,(2)essentialoils,(3)proteins,and
(4)polyphenols,amongothers[16,29,30].Theclassificationofhopconecompoundsisshownin
Figure1.Someofthesephytochemicalsarebrieflydescribednext.

Appl.Sci.2020,10,50743of18

Figure1.Abriefclassificationofhopsconecompoundsbasedonthereviewedliterature[16,29,31–
36].
2.1.HopResin
Theresinsarehopplantsecondarymetabolitesthatcanbesolubilizedincoldmethanoland
diethylether[14,16].Thetotalresinscanbedividedintotwokinds:softandhardresins[1,14,16,37],
andtheyarecharacterizedbytheirsolubilityandinsolubilityinhexane,respectively[14,16].
AccordingtoAlmagueretal.[16],thesoftresincontentinwholehopconesishighcomparedtothe
hardresin;infact,softresinsrangewithin10–25%,whilethehardresinscomprisebetween3%and
5%ofthetotalweightofdriedhops.Thesoftresinsareformedbytwodifferentbitteracids:α‐acids
(between3%and17%)andβ‐acids(between2%and7%)[1].Thereisathirdgroup,uncharacterized
components,thattogetherwithβ‐acidsformtheβ‐fraction[16,38].Theα‐acidshavefivecomponents:
humulone,cohumulone,adhumulone,prehumulone,andposthumulone[1,15].Thefirstthreeare
themajority,andtheprehumuloneandposthumulonearetheminority[15].Inthesameway,β‐acid
hasanotherfivehomologs(lupulones)[1,15].Dependingonthehopvariety,cultivationconditions,
andclimate,thecontentofthesesubstanceshomologsmayvarygreatly[5].Moreover,thehopresins
couldsufferfromdifferentchangesinthestoragewiththeoxidationstartoftheα‐acidsandβ‐acids;
duetothisprocess,theseacidswoulddecreaseduringthestorage[16].Itiscommonlyacceptedthat
hardresinscanfromtheoxidationofthesoftresins(althoughitisnotyetwelldefined)[16].During
thetraditionalbrewingprocess,rawhopsareaddedtotheboilingwort,andα‐acidsareisomerized
toiso‐α‐acids[15],whicharethemaincompoundsresponsibleforthebittertasteofbeer[15,39].
2.2.HopOil
Essentialoilsaresecondarymetabolitesexudedinthelupulinglands[14,16],andtheyarecalled
essentialsbecausetheyprovidethehopswiththeirdistinctivesmellandtransfertheiraromaand
flavortobeer[16].Essentialoilsaccountfor0.5–3%ofthedriedhops,withover400different
compoundsidentifiedinthehopoilfraction[16].Theircomponentswouldbedividedintothree
hugegroups:(1)hydrocarbons,(2)oxygenated,and(3)sulfur[5,16].Accordingtothebibliography
compiledbyAlmagueretal.[16],differentfamiliesofmoleculessuchasaliphatic,monoterpenes,
andsesquiterpenesarepresentinthefirstgroup,thesecondgroupcontainsalcohols,aldehydes,
acids,andesters,amongothers,andfinally,thesulfurgroupisconstitutedbythioestersandcyclic
terpenoidsulfides,amongothers.

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Thehydrocarbonfractionisthemostabundant(between50%and80%oftotaloil)andthemost
abundantcompoundsfromthisfractionaremonoterpenesα‐andβ‐pineneandmyrcene,among
others[12].Accordingtotheauthors[12],theoxygenatedfractionrepresents30%ofthetotaloil,
beingacomplexmixtureofdifferentcompoundssuchasaldehydesorketones,amongothers.The
maincompoundsstudiedfromthisfractionarelinaloolorgeraniol,amongothers[12].Thesulfur
fractionispresentinsmallquantitiesintheessentialoilofhops(upto1%).Thesecompoundshave
potentaromasandlowodorthresholds,sotheyplayakeyroleintheoverallflavorofbeer[12,16].
2.3.HopPolyphenols
Polyphenolsareawidegroupofbiologicallyactivesecondarymetabolites[14,16]whosecontent
andprofiledependonthehopvarietyaswellasthedifferentclimaticconditionsofcultivation[3,16].
Theconebractpresentsthegreatestcontentofpolyphenolsinthehopplant,andtheircontentinthe
conecanfluctuatebetween4%and14%(drymatter)[40].Thehoppolyphenolscanbegroupedinto
(1)flavonols,(2)flavan‐3‐ols,(3)phenoliccarboxylicacids,and(4)otherpolyphenoliccompounds
[16,36].Themostabundantflavonols,accordingtoitscontent,arequercetinandkaempferol,and
amongthedominantflavan‐3‐ols,(+)‐catechin,(−)‐epicatechin,and(+)‐gallocatechinstandout,as
wellastheirpolymersproanthocyanidinsandcondensedtannins[12,16].Ferulicacidisthemost
representativecompoundofthegroupofphenoliccarboxylicacids[12,16].Otherphenolic
compoundsthatareuniquetohopinflorescencesaremultifidolglucosides(phloroglucinol
derivativeswithprenylsidechains)andprenylflavonoids(xanthohumol,isoxanthohumol,
desmethylxanthohumol,and6‐ and8‐prenylnaringenin)[5,12].Numerouseffortshavebeen
developedtoproducehopextractswithhighpolyphenoliccontentduetotheirpossibleuseasnatural
additives(antioxidantand/orantimicrobial)inindustrialapplications[5].
3.ExtractionTechniques
Theextractionefficiencyoftheplethoraofphytochemicalsthatcanbeobtainedfromhopsis
highlydependentontheextractiontechnologyapplied.Thesemethodsmustbefast,versatile,easy
touse,environmentallyfriendly,cost‐effective,andbeabletobothextractwithhighyieldsandkeep
thequalityofthetargetcompounds[28].Theextractiontechniquesusedfortherecoveryofbioactive
compoundsrangefrom(1)theconventionalmethodstoinnovativetechnologiessuchas(2)
ultrasound‐assistedextraction,(3)microwave‐assistedextraction,(4)pressurizedliquidextraction,
and(5)supercriticalfluidextraction,includingtheuseofgreensolventsasdeepeutecticsolvents
(DES).
3.1.ConventionalMethods
Solid–liquidextraction(SLE)andsteamdistillationhavebeenappliedforextractingbioactive
compoundsfromhops.Forthispurpose,severalorganicsolventssuchasethanol,methanol,
methylenechloride,ethylacetate,acetone,hexane,andtheirmixtureswithwaterhavebeenused.
Theextractionefficiencyisgreatlyaffectedbythetypeandpolarityofthesolvent,theexperimental
conditionsoftimeandtemperature,andtheextractionnumberofcycles[41].Severalphytochemicals
havebeenextractedfromhopsusingconventionalmethods.Forinstance,hydrodistillationwas
appliedtoextractessentialoilsfromtheconepowder,obtainingayieldof6.3mL/kgofdrycones
[10].Usinggaschromatographycoupledtomassspectrometry(GC‐MS/MS),theauthorsidentified
thepresenceof16compounds,ofwhichmyrcene,trans‐caryophyllene,andα‐humulenewerethe
threemaincomponentsofhopsessentialoil[10].Inanotherstudy,Jeliazkovaetal.[42]evaluatedthe
yieldandcompoundprofileofessentialoilextractedfromwholehopconesviasteamdistillation
usingasequentialelution.Theresultsindicatedthat83.2%oftheoilwasextractedduringthefirst
hour,incomparisonwithcontrolperformedduring4‐hnoninterrupteddistillation[42].
Furthermore,theprofileofelutedcompoundswasalsosignificantlydifferent,observingthat
monoterpeneswereelutedbeforesesquiterpenes.

Appl.Sci.2020,10,50745of18
Theeffectoffourorganicsolventswithdifferentpolarities(ethylenechloride,acetone,ethyl
acetate,andmethanol)ontherecoveryofflavonoidsfromspenthopswasstudiedbyBartmańskaet
al.[43].Accordingtotheirresults,theincreaseofpolarityoftheextractantimprovedtheefficacyof
extraction,obtainingthehighestyieldwithmethanol(92.95g/kg).Theauthorsfoundthatthemost
abundantextractcompoundwasxanthohumol,andtheyindicatedthatthehighestefficiencyand
selectivitytorecoverthiscompoundfromspenthopswasethylacetate(3.51g/kg)andthesolvent,
whichledtothelowestextractionyieldbeingmethylenechloride(1.33g/kg)[43].Inhydroalcoholic
extractsobtainedbymacerationfromhopcones,twomaingroupsofcompoundswereidentifiedby
reversephaseHPLC‐UV:prenylatedchalconesandacylphloroglucinolderivatives[10].
Prencipeetal.[44]indicatedthatthedynamicmacerationofhopswithMeOH–HCOOH(99:1,
v/v)ledtothebestresultintermsofhopcomponentsrecovery,specificallyprenylflavonoidsand
bitteracids,incomparisonwithothermethodsincludingheat‐refluxextraction,microwave,or
ultrasound.
Althoughtheapplicationoforganicsolventsiswidespreadinindustrialextractionprocesses,
theiremploymentpresentssomeinconveniencesrelatedtohealthriskandenvironmentalpollution,
limitingtheiruseintherecoveryofbioactivecompounds[27].Duetothis,anewmethodofsolvents
generationknownasdeepeutecticsolvents(DESs)hasbeenappliedinthelastdecadeasapromising
greenalternativesubstitutionforconventionalorganicsolventsintherecoveryofbiomolecules[27].
Inthisline,Lakkaetal.[23]testedtheabilityofaeutecticmixturecomposedofglycerolandL‐alanine
toextractpolyphenolsfromhops.Inthisstudy,theauthorsappliedaresponsesurfacemethodology
(RSM)basedonaBox–Behnkendesignfortheoptimizationofprocessparameters(DES
concentrationinaqueousmixtures(CDES:55–85%w/w),liquid‐to‐solidratio(LSR:20–60mL/g),and
speedofstirring(SS:200–800rpm)).Undertheoptimizedextractionconditions(CDES=85%(w/w),
LSR=59mL/gandSS=688rpm),atheoreticallyoptimalyieldoftotalpolyphenolsof118.97mggallic
acidequivalents(GAE)/gdmwasobtained[23].
3.2.EmergingExtractionTechnologies
Theefficientrecoveryofvaluablecompoundsfromdifferentbioresourceslargelydependson
thetechnologyusedfortheirextraction.Conventionalextractionmethodspresentvarious
disadvantagesassociatedwiththehighconsumptionofsolvents,prolongedextractiontimes,and
degradationofthermosensitivebiomolecules.Toavoidoperationalhazardsandthepresenceof
harmfulsolventtracesintheextractmaterial,novelalternativeextractiontechniqueshavebeen
exploredtomeetthedemandofgreenerprocessestoextractandfractionatethedifferenthop
valuablecomponents(eventovalorizethehop‐exhaustedsolid)[15].
3.2.1.Ultrasound‐AssistedExtraction(UAE)
Ultrasoundhasbeenidentifiedforitspotentialuseinthephytopharmaceuticalextraction
industry[45].TheUAEsystemisanuncomplicated,economical,andefficientalternativetothe
extractionmethodsusedtraditionallythatcanimproveextractionyield,extractionrates,orthe
recoveryofheat‐sensitivecompounds[46].Thiskindoftechniqueuseshigh‐frequencywavesthat
promotetheformationofbubblesinthemedium,leadingtotheformationofthecavitation
phenomenon[47].Theimplosionofthesebubblesdisruptsthecellularstructureandfacilitatesthe
diffusionofthesolventintothecellularplanttissue,whichincreasesmasstransferandconsequently
improvestheextraction[47].TheefficiencyoftheUAEisinfluencedbyvariousoperatingparameters
suchasthetypeandconcentrationofthesolvent,sonicationtime,temperature,LSR,ultrasonic
power,andfrequency,whichmustbeoptimizedtoreachhighextractionyields[48].Togivean
example,Almeidaetal.[49]appliedaCentralCompositeRotationalDesign(CCRD)tooptimizethe
recoveryofphytochemicalswithantioxidantcapacityfromBrazilianhops.Theauthorsevaluatedthe
effectofthreeextractionparameters:temperature(33–67°C),EtOHconcentration(43–77%),andLSR
(17–33mL/g)onthetotalphenoliccontent(TPC).Accordingtotheresultsofregressionanalysis,the
LSRwastheparameterthatshowedagreaterinfluenceonTPCoftheextracts,whilethetemperature
andtheconcentrationofethanolhadalessereffect.Underoptimizedextractionconditions(52°C,

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49%ethylicalcoholandLSRof34mL/g),aTPCof33.93mgGAE/gforBrazilianhopswasobtained.
Moreover,theauthorscomparedBrazilianhopextractswiththoseobtainedwiththesamevarietyof
hopsgrownintheUSA.TheresultsshowedthattheBrazilianhopsshowedahighercontentof
phenolsandflavonoidsandbetterantioxidantpotentialanalyzedbytheABTS(2,2’‐azino‐di(3‐
ethylbenzothiazoline‐6‐suslfonicacid)andDPPH(α,α‐Diphenyl‐‐picrylhydrazyl)testscomparedto
theUSAhops.
Inanotherstudy,theUAEhasbeenappliedwithsuccessfortheextractionofdiversetypesof
biocompoundsfromhops.Forexample,Muzykiewiczetal.[3]usedthistechniquewithdifferent
extractants(methyl,ethyl,andisopropylalcohol)underthreedifferentconcentrationstoassessthe
totalphenoliccontentandtheantioxidantactivity.Thisresearchwascarriedoutusingfreshhop
leavesfromdifferentharvestedyears(2017and2018).Processparameterswerefixedat40kHz
duringdifferenttimes(15,30,and60min)atroomtemperatureandunderdifferentsolvent
concentrations(40%,70%and96–99.5%(v/v)).Theauthorsreportedthattheextracts(fromyoung
hopleavesharvestedatthestartingvegetation)presentedahighantioxidantactivity,andthis
antioxidantpotentialwasinfluencedbydifferentfactors(includingthesolventtypeorextraction
time)andconcludedthatthemosteffectiveprocesswasusingundilutedmethanolduringonehour
ofultrasound‐assistedextraction.Theantioxidantscontentdependsontheharvestingtimeandcan
bealsoinfluencedbyclimaticconditions,amongotherenvironmentalelements[3].
3.2.2.Microwave‐AssistedExtraction(MAE)
Inthelastdecade,theuseofmicrowave‐assistedextraction(MAE)torecoveractivemolecules
fromseveralnaturalsources,includinghops,hasalsobeenaddressed.Thisextractiontechniqueis
basedontheapplicationofmicrowaveenergy,whichisconvertedintoheatmainlythroughtwo
mechanisms,whichareionicconductionanddipolerotation[48].Thisprocessincreasesthepressure
andtemperatureinsidethecellmatrix,whichcausestheruptureofthecellstructure,improvingthe
releaseofthedesiredcompounds.MAEhasbeenrecognizedasapromisinggreentechniqueover
conventionalextractionmethodssinceitpresentsvariousadvantages,including(1)shorterextraction
time,(2)minorenergyinput,(3)decreasedsolventconsumption,(4)easeofoperation,and(5)a
minimumdegradationofbioactivecompounds[28,48].
Tyśkiewiczetal.[50]exploredtheuseofmicrowave‐assistedhydrodistillation(MAHD)to
extractessentialoilsfromhopsscCO2(sc:supercritical)extract,andtheresultswerecomparedwith
thoseobtainedusingconventionalhydrodistillation(HD).Underoptimizedconditions(335W
microwavepoweratLSRof8:3),MAHDyielded3.77%ofessentialoilsinanextractiontimeof30
min,whilewithconventionalHD,only1.90%wasreachedusingalongertime(276min).Theresults
ofquantitativeanalysesof‐myrceneandα‐humuleneoftheobtainedoilbyMAHDwere77.36%
and9.47%,respectively.
3.2.3.PressurizedMethods
Anotherinterestingtechniqueisthepressurizedliquidextraction(PLE),whichisalsonamed
acceleratedsolventextraction(ASE)orpressurizedhotwaterextraction(PHWE)whenwaterisused
[51];also,pressurizedsolventextraction(PSE)presentsimportantbenefitscomparedtoconventional
extractions[52].
Formatoetal.[53]studiedtheefficiencyofcyclicallypressurizedsolid–liquidextractionusinga
NaviglioExtractorfortherecoveryofacidiccompoundsfromhopsflowersincomparisonwith
supercriticalfluidextraction(SFE).Theresultsshowedthatthecyclicallypressurizedsolid–liquid
extractionwasmoreeffectivefortheextractionofαacidsandiso‐α‐acids,whileSFEexhibiteda
greaterpotentialfortheisolationofβacids.Theauthorsconcludedthatbothtechnologiescanbe
usedtoobtainhighyieldsofhopsextracts,confirmingthepossibilityofadjustingtheexperimental
parametersofbothmethodstomakeitselectiveforspecifickindsofcompounds.
Gil‐Ramírez[51]assessedthesuitabilityofPHWEfortheselectiveextractionofisoxanthohumol
(IX)againstxanthohumol(XN)fromhops.Waterextractionwasperformedusing1500psiat150°C
andanextractiontimeof30min(5minpercyclefor6cycles).Ethanolextractionat150°Cduring30

Appl.Sci.2020,10,50747of18
min(5minpercyclefor6cycles)wascarriedoutforcomparativepurposes.Besides,sequential
extractionswereperformedusingasolventinincreasingpolarityorder(hexane,ethanol,andwater).
Basedontheresults,PHWEshowedhighselectivitytowardisoxanthohumolagainstxanthohumol
(2.34mg/gand0.11mg/g,respectively,witharatioIX/XNof21)incomparisonwithPLEusingEtOH
asasolvent,afterusinghexane(5.15mg/gand2.57mg/g,respectively,withaIX/XNratioof2)[51].
PSEhasbeenproposedtoextractα‐acidsandβ‐acidsfromhopsandhopproducts[52].The
authorsstudiedthesamplepreparationmethodinfluenceandtheparametersthataffectthe
extractionefficiencytofindanalternativemethodtotheEBC7.7extractionmethod(usedforbitter
acidsdeterminationinhopproducts)tosavetimeandfacilitatethelaboriousextraction.Themost
importantparameterswereoptimized(1)temperature,(2)extractionsolventtype,(3)processof
samplepreparation,and(4)numberofcycles.ThePSEmethodsavedsolvent,itwaslessarduous
andtime‐consuming,andaccordingtothestatisticalevaluationcarriedoutbytheauthors,the
developedPSEprocesscanbeconsideredcomparabletotheEBC7.7extractionmethod.
3.2.4.SupercriticalFluidExtraction(SFE)
Anotheremergingtechnologythathasbeenadvantageouslypositionedforthegreenextraction
ofheat‐sensitivebiocompoundsissupercriticalfluidextraction(SFE).CO2iswidelyusedforSFEdue
toitbeinglowtoxic,non‐flammable,inexpensive(comparedtoorganicsolvents),andrecyclable.
AccordingtoHrnčičetal.[5],newopportunitiesusingunconventionalsupercriticalsolventshave
beenappearedsuchasSF6ornoblegases(ortheirmixtures),althoughsupercriticalCO2remainsas
themoreusedsolventfortheseoperations.However,CO2isintrinsicallynon‐polar;thus,toimprove
theextractionofpolarcompoundssuchasphenoliccompounds,itrequirestheadditionofpolar
cosolvents(modifiers)suchasethanolormethanol[28].Formatoetal.[53]evaluatedtheextractive
efficiencyofSFE‐CO2,withorwithoutacosolvent,toextracttheacidiccompoundscontainedinhops
flowers.TheresultsrevealedthattheuseofsupercriticalCO2withcosolventimprovedthe
performanceofαacids(28.3%)comparedtopureCO2(21.5%).Incontrast,thehighestyieldinβacids
(46.2%)wasfoundintheextractsobtainedwithsupercriticalCO2versus37.5%forSFE‐CO2with
ethanol.
SFEhasbeenappliedsuccessfullyfortheselectiveisolationofbitteracidsfromtwohopcultivars
(HallertauMagnumandHerkules)[54].Theauthorsidentified,byHPLCanalysis,thepresenceof
twomaingroupsofmolecules:α‐acids(55.2w/wand46.9w/wintheHerkulesandHallertau
Magnumhops,respectively)andβ‐acids(18.3w/wand22.9w/w,respectively)[54].Cohumulone
accounted38.7%ofα‐acidsinHerkuleshopsand24.9%inHallertauMagnumhops,andthe
colupulonewasthemajorcomponentofβ‐acids,reaching57.2%and44.2%inHerkulesandHallertau
Magnum,respectively[54].
Tovalorizethespenthops,Jackowskietal.[55]proposedtheiruseasarawmaterialforthe
recoveryofxanthohumol.Under80°Cand850bar,ayieldof1.23%ofxanthohumolwasobtained.
Somestudiesabouttheextractiontechnologiesofdifferentphytochemicalsfromhopsare
presentedinTable1.


Appl.Sci.2020,10,50748of18
Table1.Extractiontechnologiesforobtainingactivemoleculesfromhops.
MatrixTargetCompoundMethodExtractionConditionsOutcomesReference
Hops
Essentialoil
(desmethylxanthohumol,
xanthohumol,co‐humulone,
lupulone,co‐lupulone,and
lupulone)
Maceration
Extractionwithethanol:H2O(9:1)with3
cyclesof2handafullnightinstirringin
thedark
Fractionationoftheactivefractionwas
carriedoutusingaliquid/liquidextraction
withmethylenechlorideinproportion
CH2Cl2/H2O(5:5)
Essentialoilyield:6.3
mL/kgofdrycones
Identificationof16
compounds,thethree
majorcompoundsbeing
myrcene,trans‐
caryophyllene,andα‐
humulene
[10]
Wholehop
cones
Essentialoil(monoterpenesand
sequiterpenes)
Steam
distillationSequentialelutionat8distillationtime
Essentialoilyield:83.2%
(thefirsthour),9.6%(the
secondhour),and7.2%
(secondhalfofthe
distillation)
Chemicalprofile:
Monoterpenesarethefirst
tobeelutedand
sequiterpeneswerelater
eluted
[42]
SpenthopsFlavonoids(xanthohumol)SLE
Extractionwithmethylenechloride,
acetone,ethylacetate,andmethanolfor24
honarotaryshakerandLSR4:1(mL/g)
Yield:92.95g/kg
(methanol);38.57g/kg
(ethylacetate);29.82g/kg
(acetone);26.01g/kg
(methylenechloride)
Xanthohumol:3.51g/kg
(ethylacetate);2.97g/kg
(acetone),2.94g/kg
(methanol);1.33g/kg
(methylenechloride)
[43]
HopsPrenylflavonoidsandbitteracids
(prenylphloroglucinols)
Dynamic
maceration
ExtractionwithMeOH–HCOOH(99:1,
v/v),atroomtemperaturefor30minunder
magneticstirringusinganLSRof20mL/g
≈17.5mg/gbitteracidsand
≈1.4mg/g
prenylflavonoids
[44]
HopsPolyphenolsSLE
ExtractionwithDESbasedonglyceroland
L‐alaninefor150min,at50°Cinanoil
bath.Optimalconditionsofextraction:
CDES=85%(w/w),LSR=59mL/g,andSS=
688rpm
Yield:118.97mgGAE/of
drymass[23]
HopleavesPolyphenolsUAE
Extractionwithmethyl,ethyl,and
isopropylalcoholatdifferent
concentrations(40%,70%,and96–99.5%
(v/v))usingafrequencyof40kHzfor15,
30,and60minatroomtemperature
TPC:0.51–6.60mgGA/g
rawmaterial(collectedin
2017)and0.02–6.22GA/g
rawmaterial(collectedin
2018)
[3]
HopextractsEssentialoils(β‐myrceneandα‐
humulene)MAHD335Wmicrowavepowerfor30minusing
anLSRof8:3(usingwaterassolvent)
Yield:3.77%;β‐myrcene:
77.36%;α‐humulene:
9.47%
[50]
HopflowersAcidiccompounds(αacids,isoα
acids,βacids)
PLEwitha
Naviglio
Extractor
Sampleweight:21g;solvent:ethylalcohol;
staticphase:2min;dynamicphase:5
cycleswith12secofstoppiston;total
cycles:360(24h)
αacids:50.2%;isoαacids:
9.3%;βacids:16.3%[53]
Hops
(pellets)
Isoxanthohumoland
xanthohumolPHWE/PLE
PHWE:1500psiat150°Candextraction
timeof30min(5minbycycle‐6cycles‐)
PLE:EtOHasasolvent,afterusinghexane
at150°Cfor20mineachextraction
PHWE:2.34mg/gof
isoxanthohumoland0.11
mg/gofxanthohumol
PLE:5.15mg/gof
isoxanthohumoland2.57
mg/gofxanthohumol
[51]
Hopandhop
productsα‐andβ‐acidsPSE
PSEoptimalconditions:numberofcycles3
(5mineach),staticmode,80°C,15MPa,
solvent:methanol‐diethylether(1:1),inert
matrix:seasand(50to70μm),solvent
rinsing:20sec,nitrogenblowdown:2min,
amountofsample:1.5gofgroundhop
conesorpelletsor0.3gofhopextract
Yieldsbetween96.8%and
102.7%[52]
HopflowersAcidiccompounds(αacids,β
acids)SFE
SFE‐CO2(SFE‐I)andSFE‐CO2withethanol
(SFE‐II):350barat35°C,astaticperiodof
10min,andadynamicphaseof260min
SFE‐I:21.5%αacids;46.2%
βacids
SFE‐II:28.3%αacids;
37.5%βacids
[53]
Hoppellets
(Herkules
and
Hallertau
Magnum)
Acidiccompounds(α‐acids
(cohumulone),β‐acids
(colupulone))
SFESFE‐CO2wascarriedoutatapressureof
29MPa,50°Cduring4h
H.lupulus‘Herkules’:
55.2%ofα‐acids(38.7%of
cohumulone);18.3%ofβ‐
acids(57.2%colupulone)
H.lupulus‘Hallertau
Magnum’:46.9%ofα‐acids
(24.9%ofcohumulone);
22.9%ofβ‐acids(44.2%
colupulone)
[54]
SpenthopsXanthohumolSFESFE‐CO2wascarriedoutatapressureof
850barat80°C
1.23%ofyieldof
xanthohumol [55]
DES:deepeutecticsolvents;LSR:liquid‐to‐solidratio;SFE:supercriticalfluidextraction;SLE:solid–
liquidextraction;UAE:ultrasound‐assistedextraction;MAHD:microwave‐assisted
hydrodistillation;PSE:pressurizedsolventextraction;GAE:gallicacidequivalents;IX:
isoxanthohumol;XN:xanthohumol;PLE:pressurizedliquidextraction;PHWE:pressurizedhotwater
extraction;SS:speedofstirring;CDES:concentration.

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4.BiologicalActivitiesofHopCompounds
Asmentionedpreviously,hopcontainsseveralbioactivemoleculesthatexhibitmultiple
therapeuticproperties,suchas(1)antioxidant,(2)antimicrobial,(3)antifungal,(4)antiviral,(5)anti‐
inflammatory,and(6)anticancer,interalia[5,12,53].
4.1.AntioxidantActivity
Severalofthecompoundsidentifiedinhopsandhopproductshavebeeninvestigatedfortheir
antioxidantproperties.Theantioxidantpotentialofhopextractsdependsonvariousfactors
includingthetypeofassayusedtodeterminetheantioxidantactivity,theextractionsolvent,aswell
asthecultivarofhop[43,49,56].Forexample,Kobus‐Cisowskaetal.[56]evaluatedtheantioxidant
potentialofhopconeextractsfromthreedifferentcultivars(Magnum,Lubelski,andMarynka)
measuredbyDPPHandABTSmethods.Theaqueousextractsofthethreecultivarspresentedthe
highestDPPHvalues;however,theethanolicextractsexhibitedahighervalueofantioxidantactivity
bytheABTSassay.
Alonso‐Estebanetal.[2]testedtheantioxidantcapacityofmethanolicextractfromhopseeds
throughdifferentinvitroassays,namelytheDPPH,reducingpower,β‐carotenebleachinginhibition,
andthiobarbituricacidreactivesubstances(TBARS)formationinhibition.Thisextracthadagreater
inhibitoryeffectonthegenerationofTBARSfromtheexvivodecompositionofcertainlipid
peroxidationproducts(withanEC50valueof128μg/mL),butitexhibitedtheworstbehaviorforthe
β‐carotenebleachinginhibitioncapacity(withanEC50valueof1330μg/mL).Thelatterindicatesthat
thehopextractislessactivetoneutralizethelinoleichydroperoxylradicalsformedinvitrofromthe
oxidationoflinoleicacid.Theauthorsattributedtheantioxidantpotentialmainlytothepresenceof
(+)‐catechinand(−)‐epicatechininhopseedextract.
Inapreviousstudy,Abrametal.[57]foundthatethanolicextractsofhopconespresentedupto
ca.5‐foldmoreDPPHactivitycomparedtothatobtainedintheleafextracts.Onthecontrary,thebest
activitymeasuredbytheferricreducingantioxidantpower(FRAP)trialwasfoundintheleaf
extracts.Inanotherstudy,Gerhauseretal.reportedthatthexanthohumolpresentinthehopsshowed
anantioxidantactivitythatwas8.9and2.9timeshigherthanthereferencecompound6‐hydroxy‐
2,5,7,8‐tetramethylchroman‐2‐carboxylicacid(TROLOX)inscavenginghydroxylandperoxyl
radicals,respectively[58].
InastudycarriedoutbyLiuetal.[59],thehydroxylradicalscavengingactivityofdifferenthops
fractions(hopbitteracids,isomerizedhopbitteracids,andothers)wasevaluated.Theresults
indicatedthatallthecompoundsshowedantioxidantpotentialatdifferentlevels,ofwhichtheα‐
acidswerethosethatexhibitedalowerEC50.Humulone,lupulone,andsometerpenes(linalool,β‐
pineneandγ‐terpinene,β‐farnesene)fromhopessentialoilshavealsobeenreportedfortheir
antioxidantpotential[12].
4.2.AntimicrobialActivity
Hopshavetraditionallybeenusedinbeerasanaturalpreservativebecauseoftheirhighcontent
ofbitteracidsandpolyphenols,whichinhibitthegrowthofabroadspectrumofmicroorganisms.
Pilnaetal.[54]studiedtheantimicrobialpotentialofextractsoftwohopvarieties.Allextractsshowed
antimicrobialactivityagainstallpathogenicbacteriatestedatminimuminhibitoryconcentrations
(MICs)intherangeof8–512μg/mL.Gram‐positivestrains(MICs8–64μg/mL)weremoresensitive
thanGram‐negativestrains(MICs≥32μg/mL)andyeast(MIC=512μg/mL).Theauthorsidentified
α‐andβ‐acidsasthemainsubstancesinvolvedintheinhibitoryeffectsofhopextracts.Humulinic
acid(derivedfromiso‐α‐acids)hasalsobeenreportedfortheirantimicrobialactivityagainst
Lactobacillusbrevis[60].
Today,thereisaglobalconcerntofindnewactiveagentstocombatmicrobialresistanceto
antibiotics[61].Inthissense,methicillin‐resistantStaphylococcusaureus(MRSA)strainscause
differentpathologiesthataredifficulttotreatduetotheirvirulence,resistancetoalmostalluseful
antibiotics,aswellastheformationofpersistentbiofilms[61].Plant‐derivednaturalcompounds,

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characterizedbythepresenceofawidespectrumofactivebiomolecules,havebeenstudiedas
potentialantimicrobialagentsthatareusefultotreatseveraldiseasescausedbymultidrug‐resistant
microorganisms.AccordingtoBartmánskaetal.[43],sevenflavonoids,amongthemtwonatural
(α,β‐dihydroxanthohumoland8‐prenylnaringenin)flavonoidsidentifiedinspenthopsextracts,
showedremarkableantimicrobialactivityagainstmethicillin‐sensitive(MSSA)andresistant
Staphylococcusaureus(MRSA)strains.Lupulone,xanthohumol,anddesmethylxanthohumolalso
exhibitedstrongantimicrobialactivityagainstMRSAaswellastheabilitytoinhibitbiofilmformation
[61].SimilarfindingswerealsofoundbyBogdanovaetal.[62],whoreportedthathumulone,
lupulone,andxanthohumolhadstrongantimicrobialandantibiofilmactivityagainstMRSAstrains.
Inthisresearch,theauthorsindicatedthatlupuloneexhibitedthestrongesteffect,whichwas
followedbyxanthohumol.Ethanolicextractsofhopsalsoexhibitedanantimycobacterialeffecton
rifampin‐sensitiveandresistantstrainsofMycobacteriumtuberculosis[63].
Someauthorshavealsodemonstratedtheantifungalactivityofhopextracts.Bocquetetal.[10]
testedcrudeextractsofdifferentpartsofhop(leaves,stems,rhizomes,andfemalecones)andthe
essentialoilofhopsagainstZymoseptoriatritici.Allextractsandessentialoilshowedantifungal
activity,althoughonlyfemaleconesandtheessentialoilexhibitedavisibleactivity,observinga
reductionof85%and100%inthegrowthofZ.triticiat1.25g/L,respectively.Theauthorsattributed
theantifungalactivityofhopconeextractstothepresenceofdesmethylxanthohumolandco‐
humuloneandconcludedthatthecombinationofhopsessentialoilwithsyntheticfungicidescould
beanappropriatestrategytoreducethedoseofconventionalfungicidesincropprotection.Similarly,
Alonso‐Estebanetal.[2]alsoreportedtheantifungalactivityofhopseedextract.Theresults
indicatedthatthisextracthadaremarkableantifungaleffect,whichwasevenbetterthanthepositive
control,againstfungiofthegenusPenicillium.
4.3.EffectsonSpecificDiseases
4.3.1.Anti‐InflammatoryActivity
Inflammationisabiologicalresponseoftheorganismtoinjuryorinfection[12].However,
accordingtotheauthors,anexcessiveinflammatoryreactionisassociatedwithcertaindiseasessuch
ascancerorischemicheartdisease,amongothers.Bitteracidsandpolyphenoliccompoundsfrom
hopshavebeenidentifiedaspromisingmoleculestoinhibitinflammatoryprocesses.Bothmolecules
actonthenuclearfactorkappaB(NF‐kB)thatisinvolvedintheexpressionofpro‐inflammatory
genessuchastumornecrosisfactoralpha(TNF‐α),induciblenitricoxidesynthase(iNOS),kinases,
cyclooxygenases1and2(COX‐1andCOX‐2),andanamountinterleukins[12,64].Severalinvitro
andinvivostudiesevidencedthattheanti‐inflammatoryeffectsofhopsareprimarilyattributedto
humulone,xanthohumol,and8‐prenylnaringenin[12,64].Forinstance,Leeetal.[65]reportedthat
thetopicalapplicationofhumuloneinhibited12‐O‐tetradecanoylphorbol‐13acetate(TPA)‐induced
COX‐2expressionthroughtheregulationofnuclearfactor‐kB.Humulonealsoreducestheexpression
ofvariouskinasesrelatedtotheinflammatoryresponse[66].Xanthohumoland8‐prenylnaringenin
alsoshowedastronganti‐inflammatoryeffectviatheinactivationofNF‐kcinmicroglialcelllines
[67].Ithasalsobeenstudiedthattheisoxanthohumolreducedinflammatoryfactorsincludingtumor
necrosisfactoralpha(TNF‐α)andnuclearfactorkappaBinhumanaorticsmoothmusclecellsand
humanumbilicalveinendothelialcells[68].Inbothanimalmodelsandhumaninterventiontrials,
isohumuloneshavebeeneffectiveinthetreatmentofinflammatoryarthritis[69].
4.3.2.Cancer‐RelatedActivities
Cancer,animportantcausesofdeathinthe21stcentury,supposesforthehealthsystemsan
importanteconomicimpact,duetothehighcostofitstreatment[12].Forthisreason,itisnecessary
todevelopnewsubstanceswithanticancerpotentialthatcanprevent,stop,orreversethedisease
progression,whichisthereasonwhythesesubstanceshavebeenthefocusofalargenumberof
scientificstudies[12].Inthiscontext,ithasbeenreportedthatsomeofthebioactivecompounds
presentinhopshaveanticancerproperties.Forinstance,xanthohumolhasbeenidentifiedasanew

Appl.Sci.2020,10,507411of18
agentforthetreatmentofdifferenttypesofcancer[12,64].Yongetal.[11]demonstratedthat
xanthohumolhadanapoptoticeffectonthehumanalveolaradenocarcinomacelllineinadose‐and
time‐dependentmanner.Recently,Roehreretal.[13]evaluatedtheantiproliferativeeffectof
xanthohumol,xanthohumolC,andcrudehopextractonhumanbreastcancercells.Accordingtothe
authors,after2daysofincubation,xanthohumolCpresentedthestrongestgrowthinhibitionwith
anIC50of4.18μM,incomparisonwithcrudehopextract(8.84μM)andxanthohumol(12.25μM).
Lupulone,aβ‐acidpresentinhopextracts,displayedanticanceractivityonprostatecancercellsvia
theinductionofcaspase8‐dependentcelldeath[70].
4.3.3.OtherBiologicalActivities
Otherbioactivitiesattributedtohopcomponentsincludeneuroprotective,antidiabetic,and
cardioprotectiveeffects.Xanthohumolpromotedneuronalrecoveryinratswithintracerebral
hemorrhage[71],anditalsoshowedaprotectiveeffectonthebraindamageinducedbyaging[72].
Thiscompoundalsohastheabilitytoinhibitadipogenesissoitcanbeusedinthepreventionof
obesity.Inastudyinmicefedwithahigh‐fatdiet,thesupplementationwith2%or5%hopextract
reducedtheincreaseinbodyandadiposetissueweightandimprovedtheglucoseintolerance[73].
Adietrichinxanthohumoland8‐prenylnaringeninimprovesmetabolicdisordersrelatedtotype‐2
diabetesbymodulationoftheglucoseandlipidpathways[74].
Table2summarizesseveralbiologicalactivitiesofdifferentbiocompoundsextractedfromhop
andthemainresultsfound.
Table2.Biologicalactivitiesofcompoundsextractedfromhops.
ResponsibleCompound MethodologyOutcomesReference
AntioxidantActivity
PhenolicAcidsandflavonolsDPPHandABTS
DPPHrangedfrom3.50mmolTrolox/gdwfor
Marynkavarietyethanolextractto4.75mmolTrolox/g
dwforMagnumvarietywaterextract.ABTSvaried
from1.32mmolTrolox/gdwforMagnumvariety
waterextractto2.43mmolTrolox/gdwforMarynka
varietyethanolextract
[56]
(+)‐catechinand(−)‐epicatechin
DPPH,reductionpower,
β
‐
carotenebleachinginhibition
capacity,TBARS
EC50values:DPPH:505μg/mL;Reductionpower:530
μg/mL;β‐Carotenebleachinginhibitioncapacity:1330
μg/mL;TBARS:128μg/mL
[2]

PhenoliccompoundsDPPHandFRAPDPPHEC50:0.070mg/mL;FRAP:reductionof0.117
mL/μgofferricionsin25min[57]
XanthohumolORAC
TheantioxidantactivitydeterminedbytheORAC
assaywashigherthanthereferencecompound
TROLOX
[58]
QuercetinandisoquercetinDPPHandABTSDPPHEC50:3.91μL/mL;ABTSEC50:21.29μL/mL[49]
Bitteracids,isomerizedhop
bitteracids,hopoil,and
hexahydro‐β‐acids
Hydroxylradicalscavenging
activity
EC50:α‐acid:0.21mg/mL;β‐acid:0.96mg/mL;
Dihydro‐iso‐α‐acid:1.36mg/mL;tetrahydro‐iso‐α‐acid
1.77mg/mL;hexahydro‐iso‐α‐acid1.40mg/mL;
hexahydro‐β‐acid0.50mg/mL;oil0.18mg/mL
[59]
AntimicrobialActivity
α‐andβ‐acidsMicrodilutionmethod
Gram‐positivebacteria:MICs8–64μg/mL;Gram‐
negativebacteria:MICs≥32μg/mL;yeast:MIC=512
μg/mL
[54]
Flavonoidsamongthemtwo
natural(α,β‐
dihydroxanthohumoland8‐
prenylnaringenin)
MicrodilutionmethodGrowthinhibitionofMSSAandMRSAatMIC80values
of5–50μg/mL[43]
HumulinicacidMicrodilutionmethodInhibitionofLactobacillusbrevisatMIC<10μM[60]
Lupulone,xanthohumol,and
desmethylxanthohumolMicrodilutionmethod
AntimicrobialeffectagainstMSSAandMRSAwith
MICsvaluesof0.6–1.2μg/mLforlupulone,9.8–19.5
μg/mLforxanthohumoland19.5–39μg/mLfor
desmethylxanthohumol
Xanthohumoltotallyinhibitedthebiofilmformationat
theMICvalue.Desmethylxanthohumolandlupulone
inhibitedthebiofilmformationatsub‐inhibitory
concentrations
[61]
Humulone,lupulone,and
xanthohumolMicrodilutionmethod
MICsvalues:7.5–30μg/mLforhumulone,0.5–4μg/mL
forlupulone,2–4μg/mLforxanthohumol
MBCsvalues:30–125μg/mLforhumulone,1–15μg/mL
forlupulone,2–7.5μg/mLforxanthohumol
[62]

Appl.Sci.2020,10,507412of18
90%reductioninbiofilmformationusing2–7.5μg/mLof
lupulone,15–30μg/mLofxanthohumoland30–250
μg/mLofhumulone
Desmethylxanthohumolandco‐
humuloneSpottingmethod
AntifungalactivityagainstZymoseptoriatritici.
IC50values:0.36g/Lforessentialoiland0.73g/Lforcone
extracts
[10]
(+)‐catechinand(−)‐epicatechinMicrodilutionmethod
MIC:0.15mg/mLforPenicilliumochrochloron,0.075
mg/mLforPenicilliumfuniculosum,0.15mg/mLfor
Penicilliumverrucosumvar.cyclopium
MBC:0.30mg/mLforPenicilliumochrochloron,0.15
mg/mLforPenicilliumfuniculosum,0.30mg/mLfor
Penicilliumverrucosumvar.cyclopium
[2]
Anti‐InflammatoryActivity
Humulone
Mouseskinstimulatedwiththe
tumorpromoter12‐O‐
tetradecanoylphorbol‐13‐
acetate(TPA)
Humuloneat10μmolinhibitedTPA‐inducedCOX‐2
expressionthroughtheregulationofnuclearfactor‐kB[65]
Isoxanthohumol
Humanumbilicalvein
endothelialcells(HUVEC)and
humanaorticsmoothmuscle
cells(HASMC)
Isoxanthohumolatadoseof10μmolreducedin
HASMCtheTNF‐αby26%andnuclearfactorkappaB
by24%;inHUVEC,thedecreasewas40%forTNF‐α
and42%fornuclearfactorkappaB
[68]
AnticancerActivity
XanthohumolSulforhodamineBassayInducedcelldeathinhumanalveolaradenocarcinoma
cellline[11]
Xanthohumol,xanthohumolC,
andcrudehopextract
CellTiter96AqueousNon‐
RadioactiveCellProliferation
AssayfromPromega
Antiproliferativeeffectsonbreastcancercells[13]
LupuloneMTTAssay
Anticancerpotentialon2prostatecancercelllines(PC3
andDU145cells).IC50was5μMforbothcelllinesafter3
daysoftreatment
[70]

HopextractMTTAssayAntiproliferativeeffectsonhumanhepatoma
carcinomaatdosesof0.6–1mg/mL[75]
OtherBioactivities
Xanthohumol
Intracerebralhemorrhage
modelwasinducedby
intrastriatalinjectionof
bacterialcollagenase
Reducedthehemorrhagicinjuryandpromotethe
neuronalrecovery[71]
Xanthohumol
Senescence‐acceleratedprone
malemice
(SAMP8)
Preventstheexpressionofbraindamageinducedby
aging[72]
Hopextract
MaleC57BL/6Jmice(4weeks
old)feda
high‐fatdiet
Inhibitedtheincreasingbodyandadiposetissueweight,
adiposecelldiameter,andliverlipids,andimproved
glucosetoleranceinducedbyahigh‐fatdiet
[73]
Xanthohumolor8‐
prenylnaringenin
Type2diabetesmellitus
(T2DM)micemodel
Improvesmetabolicdysfunctionsassociatedwith
diabetes:bodyweightgain;decreasedglycemia,
triglyceride,cholesterolandalkalinephosphataselevels;
andimprovedinsulinsensitivity
[74]
ORAC:oxygenradicalabsorbancecapacity;TBARS:thiobarbituricacidreactivesubstances;MIC:
minimuminhibitoryconcentration;MBC:minimumbactericidalconcentration;MSSA:methicillin
sensitivestrainsStaphylococcus;MRSA:methicillinresistantstrainsStaphylococcus;TPA:12‐O‐
tetradecanoylphorbol‐13‐acetate;MTT:3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium
bromide.
5.CurrentApplicationsofFunctionalMoleculesfromHop
Takingintoaccountthepropertiesofthehopextracts,inthelastyears,theyhavebeenusedas
preservativeagentsindifferentfoodproducts.Inthecaseoffreshmeatandmeatproducts,plant
extractspreventtheiroxidativeandmicrobialdeterioration,extendingtheirshelf‐lifeandsafetyas
wellasconferringfunctionalproperties[76–78].Forexample,Krameretal.[18]studiedthe
antimicrobialeffectinvitroofseveralhopextractsagainstL.monocytogenes,Staphylococcusaureus,
Salmonellaenterica,andEscherichiacoliinamodelmeatmarinadeandonmarinatedporktenderloins.
TheresultsobtainedbytheseauthorsdemonstratedthattheGram‐positivebacteriawerehighly
inhibitedbyhopextractsduetoitscontentinβ‐acid,butinthecaseofextractscontainingα‐acid,the
inhibitionwaslower;incontrast,Gram‐negativebacteriaexhibitedahighresistanceagainstall
evaluatedhopextracts.
Inanotherwork,Villalobos‐Delgadoetal.[79]evaluatedtheeffectoftheincorporationofhop
infusionorpowderinrawandcookedlambpatties,overtheoxidativestabilityoflipids,proteins,

Appl.Sci.2020,10,507413of18
and/orcolorundertwoscenarios:refrigeratedorfrozenconditions,aswellasintheirsensory
acceptability.Theseauthorsconcludedthattheuseofhoppowdershowedhigherantioxidant
activitythanhopinfusioninlambpatties.Moreover,thelipidandproteinoxidationofcookedpatties
underrefrigeratedconditionswasreduced.However,thesensorialacceptancebytheconsumers
decreasedduetochangesinflavorwhenhoppowderwasadded.
Hopextractsfindalsoapplicationasnaturalingredientstoextendtheshelflifeofbreaddueto
theirantifungalproperties.Forinstance,Nionellietal.[19]formulatedwheatbreadwithhopextracts
andassessedtheireffectsonitsshelflife,aswellasonitsrheologicalandsensoryfeatures.They
reportedthatthehopextractexhibitedantifungalpropertiesagainstAspergillusparasiticus,Penicillium
carneum,Penicilliumpolonicum,Penicilliumpaneum,Penicilliumchermesinum,Aspergillusniger,and
Penicilliumroqueforti.Moreover,theseauthorsisolatedlacticacidbacteriafromhopsandselected
threeforsourdoughfermentationforbreadmakingwithhopextracts.Theauthorsconcludedthat
theadditionofhopextractsincreasedtheantioxidantactivityandtheconcentrationofphenols.
Moreover,theuseofhop‐sourdoughwithorwithouttheincorporationofhopextracttoelaborate
breaddelayedthegrowthoffungiuntil14days,extendingitsshelflife.Therheologicalandsensory
propertieswerenotaffected.
Incosmetics,hopsareusedinbathlotions,amongothers[80].Vogtetal.[17]usedsupercritical
CO2extractsofhopconestoformulateshowergels,andtheresultsobtainedshowedthattheir
additionenhancedtheirskin‐conditioningpropertiesduetotheircontentinbioactiveingredients.
Moreover,theformulationscontainingtheextractofhopconespresentcompoundstotreatoiland
dandruffinhair.Theuseofhopextractinhaircosmeticsissupportedbyitsantifungaland
antiseborrheicpropertiesthatdecreaseitsbrittleness,nourishit,giveitshine,increaseitsstrength,
andpreventitsloss[17].
6.Conclusions
Traditionally,hopshavebeenusedinbrewing,butrecentstudieshaverevealedtheirwealthin
bioactivemoleculeswithahugerangeoftherapeuticproperties.Thishasencouragedintense
researchinthedevelopmentofasustainableprocessthatuseseco‐friendlysolventscombinedwith
intensificationtechnologiesthatguaranteehighyieldsandextractswithhighqualityfromhops.In
fact,inthisreview,severalworksbasedonthesetechnologiesdemonstratetheirsuitabilitytorecover
extractsthatarebiologicallyactive.Amyriadofpropertiesrelatedtothehopextractshavebeen
evaluated,amongthemantibacterial,antifungal,cardioprotective,antioxidant,anti‐inflammatory,
anticarcinogenic,andantiviral.Therefore,hopsareshapingupasanexcellentandalternativesource
ofbioactivecompoundswithpotentialgreatacceptabilitybytheconsumersthatincreasingly
demandnaturalingredientswithhealthyproperties.Moreover,theextractsfromhopsfind
applicationsinthealimentary,cosmetic,nutraceutical,andpharmaceuticalsector,openingnew
alternativesintheformulationofdifferentcommoditiesbeyondtheirconventionaluses.However,
duetothehugevarietyofbiomoleculespresentinthehopextracts,itisnecessarytoinvestigatetheir
interactionwithothercomponentsofthematrixwheretheyareincorporated,aswellastheir
bioavailabilitywhenareingestedaspartofafoodorusedatatopical