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Phenolic Compounds and Bioactive Properties of Ruscus aculeatus L. (Asparagaceae): The Pharmacological Potential of an Underexploited Subshrub

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Joana Patrícia Barreira Rodrigues at Polytechnic Institute of Bragança
Joana Patrícia Barreira Rodrigues
Angela Fernandes at Polytechnic Institute of Bragança
Angela Fernandes
Maria Inês at Polytechnic Institute of Bragança
Maria Inês
Carla Pereira at Polytechnic Institute of Bragança
Carla Pereira
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Abstract and Figures

Ruscus aculeatus L. is a subshrub used in traditional medicine in different parts of the world, namely in Europe and the Iberian Peninsula. According to reported folk knowledge, the aerial parts are mainly used as diuretics and the underground organs are used for the treatment of disorders of the urinary system and as a laxative. In this work, the aerial part and the roots and rhizomes of R. aculeatus were chemically characterized with regard to the content of phenolic compounds and bioactive properties. Aqueous (infusions and decoctions) preparations and hydroethanolic extracts from the two mentioned parts of the plant were prepared. Nine phenolic compounds were detected in all the extracts. Apigenin-C-hexoside-C-pentoside isomer II was the major compound in aqueous extracts and, in the hydroethanolic extract was quercetin-O-deoxyhexoside-hexoside followed by apigenin-C-hexoside-C-pentoside isomer II. All extracts revealed antioxidant activity and potential to inhibit some of the assayed bacteria; aqueous extracts of the aerial part and infusions of roots and rhizomes did not show cytotoxic effects on a non-tumor primary cell culture. This preliminary study provides suggestions of the biological potential associated with the empirical uses and knowledge of this species, in particular its bioactivities.
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Molecules2021,26,1882.https://doi.org/10.3390/molecules26071882www.mdpi.com/journal/molecules
Article
PhenolicCompoundsandBioactivePropertiesofRuscus
aculeatusL.(Asparagaceae):ThePharmacologicalPotentialof
anUnderexploitedSubshrub
JoanaP.B.Rodrigues,ÂngelaFernandes*,MariaInêsDias,CarlaPereira,TâniaC.S.P.Pires,
RicardoC.Calhelha,AnaMariaCarvalho,IsabelC.F.R.FerreiraandLillianBarros*
CentrodeInvestigaçãodeMontanha(CIMO),InstitutoPolitécnicodeBragança,CampusdeSantaApolónia,
5300‐253Bragança,Portugal;joanapbrodrigues@ipb.pt(J.P.B.R.);maria.ines@ipb.pt(M.I.D.);
carlap@ipb.pt(C.P.);tania.pires@ipb.pt(T.C.S.P.P.);calhela@ipb.pt(R.C.C.);anacarv@ipb.pt(A.M.C.);
iferreira@ipb.pt(I.C.F.R.F.)
*Correspondence:afeitor@ipb.pt(Â.F.);lillian@ipb.pt(L.B.);Tel.:+351‐273330904(A.F.);+351‐273330901(L.B.)
Abstract:RuscusaculeatusL.isasubshrubusedintraditionalmedicineindifferentpartsofthe
world,namelyinEuropeandtheIberianPeninsula.Accordingtoreportedfolkknowledge,the
aerialpartsaremainlyusedasdiureticsandtheundergroundorgansareusedforthetreatmentof
disordersoftheurinarysystemandasalaxative.Inthiswork,theaerialpartandtherootsand
rhizomesofR.aculeatuswerechemicallycharacterizedwithregardtothecontentofphenolic
compoundsandbioactiveproperties.Aqueous(infusionsanddecoctions)preparationsand
hydroethanolicextractsfromthetwomentionedpartsoftheplantwereprepared.Ninephenolic
compoundsweredetectedinalltheextracts.Apigenin‐C‐hexoside‐C‐pentosideisomerIIwasthe
majorcompoundinaqueousextractsand,inthehydroethanolicextractwasquercetin‐O‐
deoxyhexoside‐hexosidefollowedbyapigenin‐C‐hexoside‐C‐pentosideisomerII.Allextracts
revealedantioxidantactivityandpotentialtoinhibitsomeoftheassayedbacteria;aqueousextracts
oftheaerialpartandinfusionsofrootsandrhizomesdidnotshowcytotoxiceffectsonanon‐tumor
primarycellculture.Thispreliminarystudyprovidessuggestionsofthebiologicalpotential
associatedwiththeempiricalusesandknowledgeofthisspecies,inparticularitsbioactivities.
Keywords:RuscusaculeatusL.;aerialpart;rootsandrhizomes;phenoliccompounds;bioactivities
1.Introduction
Inadditiontoprovidingoxygen,plantsareasourceofnaturalcompoundsthatare
alsousedbyhumanssincetheyhavearomatic,medicinal,andfoodcapabilities[1].The
useofplantsformedicinalpurposesisbasedonancientknowledge,passeddownfrom
generationtogeneration,andforcenturiestheyhavebeentheonlyresourceintermsof
medical,curative,orpreventivecareformanypopulations[2].Medicinalplantshave
beenandarestillusedinthedevelopmentofnewdrugsforthetreatmentofseveral
diseases.Theirpopularuseismostlybasedonempiricalknowledgeandbeliefs;therefore,
someoftheirtherapeuticapplicationslackscientificfoundation[3].
However,suchspeciesarecalledmedicinalplantsbecausetheyhavetherapeutic
benefits,whichmustbelinkedtoparticularsubstances,namelyactiveingredientsthat
mighthavearecognizedpharmacologicalaction.Thus,itisimportanttoknowwhich
partsoftheplantsaretraditionallyusedformedicinalpurposes,howaretheyprocessed
andapplied,andwhichchemicalcompoundsareresponsiblefortheirtherapeutic
properties.Theincreasedinterestinnewherbalmedicineshasledtothediscoveryofnew
compoundsoftherapeuticinterest,suchassteroids,alkaloids,saponins,terpenoids,and
glycosides[4].
Citation:JoanaP.B.Rodrigues;Fer‐
nandes,Â.;Dias,M.I.;Pereira,C.;
TâniaC.S.P.Pires;Calhelha,R.C.;
Carvalho,A.M.;IsabelC.F.R.
Ferreira;Barros,L.Phenolic
CompoundsandBioactive
PropertiesofRuscusaculeatusL.
(Asparagaceae):The
PharmacologicalPotentialofan
UnderexploitedSubshrub.Molecules
2021,26,1882.https://doi.org/
10.3390/molecules26071882
AcademicEditors:RaffaeleCapasso
andLorenzoDiCesareMannelli
Received:4March2021
Accepted:23March2021
Published:26March2021
Publisher’sNote:MDPIstaysneu‐
tralwithregardtojurisdictional
claimsinpublishedmapsandinsti‐
tutionalaffiliations.
Copyright:©2021bytheauthors.
LicenseeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsandcon‐
ditionsoftheCreativeCommonsAt‐
tribution(CCBY)license(http://cre‐
ativecommons.org/licenses/by/4.0/).
Molecules2021,26,18822of14
Thenatureofaplant‐basedmedicineisdeterminedbythecontentofdifferentactive
components,andthepotentialthateachplanthasinthesecomponentscancontributeto
anexcellenttherapeuticcomplementtoconventionalmedicine[2].Thus,accordingtothe
WorldHealthOrganization(WHO),wildorcultivatedspeciesareusedasmedicinal,both
intraditional/folkmedicineandincomplementarymedicine[5].
RuscusaculeatusL.isaEurasianspeciesoftheMonocotyledongroup,currentlypart
oftheAsparagaceaebotanicalfamilyreportedasamedicinalspeciesinEuropeanfolk
medicine[6–9].Theplantisasmallsubshrub,alwaysgreen,withthickunderground
rhizomes[10].Theempiricalmedicinalusesarerelatedwithaerialpartsareused
empiricallyasdiureticsand,theundergroundparts(rootsandrhizomes)toalleviatethe
symptomsofseveraldisordersofvenousinsufficiency,edema,urinarysystem,
premenstrualsyndrome,andhemorrhoids[8,11].
Fewresearchgroupshavealreadystudiedthisspecies[11–13];thesameauthors
reportthatthemainactiveingredientsfoundinR.aculeatusaresteroidalsaponins
(rucogeninandneoruscogenin),whichareresponsibleforitspharmacologicaleffects;
otherconstituentshavebeenisolated,includingsterols,triterpenes,flavonoids,
coumarins,sparteine,tyramine,andglycolicacid.Moreover,someofthetraditional
applicationsofthespeciesseemrelatedtoparticularcompounds,sincethehighest
concentrationofruscogeninsislocatedintherhizome[11].
ThisworkrepresentsthedetailedcharacterizationofR.aculeatusregardingthe
phenoliccompoundsoftheaerial(laminarstemsandlateralbranches)andthe
underground(rhizomeswithroots)parts.Inaddition,thebioactivepotentialofthe
hydroethanolicandaqueousextractswasalsoassessedintermsoftheirantioxidant,anti‐
tumor,anti‐inflammatory,hepatotoxicity,andantimicrobialproperties.Therefore,this
workintendstocontributetotheknowledgeofthechemicalcompositionofthisspecies
andtorelateittoitsdocumentedempiricaluses.
2.ResultsandDiscussion
Theidentificationofindividualphenoliccompoundswascarriedoutconsidering
theirretentiontimes,wheneverpossibleincomparisonwithcommerciallyavailable
standards,andbothUVandMSspectra.DataobtainedbyHPLC‐DADESI/MSanalysis
(retentiontime,λmax,pseudomolecularion([M−H]−),andmainfragmentionsinMS2),
phenoliccompounds’tentativeidentifications,andrespectivequantificationarepresent
inTable1.
Thestudyrevealedthepresenceofninephenoliccompounds,onecaffeicacid
derivative,andeightflavonoids,namelysixC‐glycosylatedderivativesofapigeninand
twoO‐glycosylatedderivativesofquercetinandkaempferol.
Peak1showedapseudomolecularion[M−H]−atm/z341andMS2fragmentsatm/z
179and135consistentwiththelossofacaffeicacid,therefore,beingtentativelyidentified
ascaffeicacidhexoside.Peaks2/5/7([M−H]−atm/z563)werealltentativelyidentifiedas
apigenin‐C‐hexoside‐C‐pentosideisomersI,II,andIII,respectively.Ontheotherhand,
peaks3/4/6,alsoshowingapseudomolecularionat[M−H]−atm/z563weretentatively
identifiedasapigenin‐C‐pentoside‐C‐hexosideisomersI,II,andIII,respectively.The
differentiationbetweenisomersandsugarpositiontookintoaccountwhatwas
previouslydescribedbysomeauthorsTahiretal.[14]andFerreresetal.[15].Finally,
peaks8([M−H]−atm/z609)and9([M−H]−atm/z593)presentedasingleMS2fragment
atm/z301and285,aglyconesofquercetinandkaempferol,respectively,correspondingto
thelossofa308u(146u+162u,deoxyhexoside+hexosidemoieties,respectively),and
therefore,beingtentativelyidentifiedasquercetin‐O‐deoxyhexoside‐hexosideand
kaempferol‐O‐deoxyhexoside‐hexoside,respectively.
Thehydroethanolicextractsoftheaerialpartpresentedthehighestlevelsofphenolic
compounds(107±3mg/gextract,Figure1),followedbytheaqueousextracts,the
decoction(18±1mg/gextract),andtheinfusion(14.6±0.3mg/gextract).Allextracts
Molecules2021,26,18823of14
performedwithaerialpartsshowedthesamephenolicprofile;however,thesamewasnot
verifiedfortheroots,wherenophenoliccompoundswereidentified.
Luísetal.[16]reporttheexistenceoftwophenoliccompounds,p‐coumaricacidand
quercetininthemethanolicextractsoftheaerialpartofR.aculeatus;thisstudyreportsthe
existenceofotherphenolicacids,namely,gallicacid,vanillicacid,caffeicacid,chlorogenic
acid,syringicacid,ferulicacid,andellagicacid,intheaerialpartsofR.aculeatus.Another
study,Gonçalvesetal.[17]evaluatedtotalphenoliccontent,measuredthroughtheFolin
Ciocalteucolorimetricmethod,andreports121.73μmolGAEg/dw,butaccordingtothe
appliedmethodology,itisnotpossibletomakecomparisonswithourstudy.
Figure1.Phenolicprofileofhydroethanolicextractoftheaerialpart,recordedat280nm(A)and370nm(B).Peak
numberingisindicatedasdefinedinTable1.
Molecules2021,26,18824of14
Table1.Retentiontime(Rt),wavelengthsofmaximumabsorptioninthevisibleregion(λmax),massspectraldata,tentativeidentificationandquantificationofthephenoliccompounds
(mg/gofextract)foundinhydroethanolicextracts,andinfusionanddecoctionpreparationsofR.aculeatus(mean±SD,n=9).
Peaks
Rt
(min)
λmax
(nm)
[M − H]−
(m/z) MS2 (m/z) Tentative Identification Quantification
Hydroethanolic Infusion Decoction
1 5.96 320 341 179 (100), 135 (20) Caffeic acid hexoside 1.42 ± 0.03 a 0.013 ± 0.001c 0.091 ± 0.005 b
2 10.49 334 563 545 (21), 473 (100), 443 (91), 413 (*), 383 (36), 353 (41), 297 (*) Apigenin-C-hexoside-C-pentoside isomer I 2.8 ± 0.1 a 0.446 ± 0.007 c 0.87 ± 0.01 b
3 12.05 334 563 545 (34), 473 (100), 443 (63), 413 (*), 383 (29), 353 (24), 297 (*) Api
g
enin-C-pentoside-C-hexoside isomer I 1.36 ± 0.01 a 0.162 ± 0.005 c 0.45 ± 0.02 b
4 12.82 335 563 545 (29), 473 (100), 443 (79), 413 (*), 383 (32), 353 (29), 297 (*) Apigenin-C-pentoside-C-hexoside isomer II 13.1 ± 0.3 a 3.19 ± 0.08 b 2.96 ± 0.01 c
5 13.15 335 563 545 (17), 473 (69), 443 (100), 413 (*), 383 (19), 353 (23), 297 (*) Apigenin-C-hexoside-C-pentoside isomer II 32 ± 1 a 5.63 ± 0.04 c 7.4 ± 0.3 b
6 14.54 338 563 545 (18), 473 (100), 443 (81), 413 (5), 383 (26), 353 (34), 297 (*) Apigenin-C-pentoside-C-hexoside isomer III 3.7 ± 0.1 a 0.528 ± 0.001 b 0.52 ± 0.01 b
7 14.72 340 563 545 (15), 473 (71), 443 (100), 413 (*), 383 (15), 353 (23), 297 (*) Apigenin-C-hexoside-C-pentoside isomer III 7.1 ± 0.4 a 0.79 ± 0.02 c 1.68 ± 0.02 b
8 16.88 353 609 301 (100) Quercetin-O-deoxyhexoside-hexoside 39 ± 2 a 3.6 ± 0.2 b 4.0 ± 0.2 b
9 20.05 340 593 285 (100) Kaempherol-O-deoxyhexoside-hexoside 6.23 ± 0.05 a 0.175 ± 0.009 c 0.42 ± 0.02 b
Total Phenolic Compounds 107 ± 3 a 14.6 ± 0.3 c 18 ± 1 b
*relativepercentagelessthan5%;calibrationcurvesusedinthequantification:standardcalibrationcurves:caffeicacid(y=388345x+406369,R2=0.99;detectionlimit(LOD)=0.78
μg/mL;quantificationlimit(LOQ)=1.97μg/mL,peak1),apigenin‐6‐C‐glucoside(y=107025x+61531,R2=0.998;LOD=0.19μg/mL;LOQ=0.63μg/mLpeaks2,3,4,5,6,7);quercetin‐
3‐O‐rutinoside(y=13343x+76751,R2=0.999;LOD=0.21μg/mL;LOQ=0.71μg/mL,peaks8and9).Differentlettersinthesamelinemeansignificantdifferences(p<0.05).
Molecules2021,26,18825of14
Theantioxidantactivityofthehydromethanolicandaqueousextractspreparedfrom
theaerialandtheundergroundpartsofR.aculeatusweretestedbytwodifferentinvitro
assays,whichensuredanoverviewoftheantioxidantactivityindifferingsurroundings.
TheresultsoftheTBARSformationinhibitionandOxHLIAassaysarepresentedinTable
2.Asitcanbeseen,significantdifferencesbetweentheevaluatedextractswereobserved.
ThehydroethanolicextractshowedthebestactivityintheTBARSassay,fortheaerialpart
andfortherootsandrhizome,withanEC50of0.28and0.78mg/mL,respectively.Inturn,
thedecoctionextract,bothfromtheaerialpartandrootsandrhizome,showedthelowest
activity,withEC50valuesof0.88and1.55mg/mL,respectively.
Ithasbeendescribedthattheextractionscarriedoutwithahigherpercentageof
waterhavethecapacitytoextractmorepolarcompounds,whereasmethanolor
ethanol:watermixturespresenthigherextractionyields[18].
Theantihemolyticpropertiesoftheextractswereassessedinanexvivoerythrocyte
systembytheOxHLIAassay.Theinfusionoftheaerialpartandthehydroethanolic
extractoftherootsandrhizomepresentedanIC50valueof236and230μg/mL,
respectively,valuesrequiredtoprotecthalfoftheerythrocytepopulationfromthe
hemolyticactioncausedbytheoxidativeagentfor60min.Atthesametime,thedecoction
oftheaerialpartandrootsandrhizomepresentedIC50valuesof427and661μg/mL,
respectively.Theaqueousextractoftheaerialpartonlyrevealedthecapacitytoprevent
thehemolysisfor60min,whilethehydroethanolicextractdidnotrevealantihemolytic
activity.Plantextractsarecomplexmixturesofdifferentantioxidantandnon‐antioxidant
compoundsthatcanactatseverallevelsofcelloxidativedegradationandthisassay,by
usingerythrocytes,offerstestconditionsthatareveryclosetotheinvivosituation[19].
Todate,therearefewstudiesevaluatingtheantioxidantactivityoftheaerialpart
and,tothebestoftheauthors’knowledge,thepresentstudyprovidesafirstreportofthe
antioxidantactivityofrootsandrhizomes.Luísetal.[16]describedthatthemethanolic
extractoftheaerialpartshowedlittleantioxidantactivityintheDPPHassay,withanIC50
valueof171.9mg/L.Otherstudies,usingalsotheaerialpart,reportedthatethylacetate
andbutanolextractsweretheonesthatrevealedthebestactivityfortheDPPHassay,and
obtainedEC50valuesof158and173μg/mL,respectively[20]and,Jakovljevićetal.[12]
obtainedEC50valuesof183μg/mLofethylacetateextracts.
Theloweractivityverifiedinthischemicalassaymayberelatedtothevariationof
compoundsduetodifferentstatesofthegrowingcycle,thegatheringseason,diverse
climate,andlandscapeconditions,aswellastheuseofdifferentsolventsforthe
preparationoftheextracts,sincethesolventhasaninfluenceontheextraction
methodology[21].
Kobus‐Cisowskaetal.[22],inastudyusingAsparagusofficinalisL.,whichbelongsto
thesamefamilyasR.aculeatus,alsoreportedtheantioxidantpotentialofgreen,purple,
andwhitevarieties,withthefirstonerevealingthegreaterantioxidantcapacity,inthe
DPPHassay.AccordingtoJakovljević etal.[12],theantioxidantactivityofplantsis
mainlyassociatedwiththeirbioactivecompounds,especiallyphenolics,flavonols,and
flavonoidsandtheirhighercontent.
Comparedtochemicalmethods(suchasDPPHandreducingpower,amongothers),
thehereinemployedTBARSandOxHLIAmethodsusebiologicaltissues(porcinebrain
andsheeperythrocytes),therefore,beingmethodsthatreproduceandresembleinvivo
conditionswhichmakesthemmorepredictiveassays.
Theresultsobtainedforthefourhumantumorcelllinesandtheprimarycultureof
non‐tumorcellsarepresentinTable2,andareexpressedinvaluesoftheconcentrationof
extractresponsiblefor50%inhibitionofcellgrowth—GI50,inμg/mL.
Itisobservedthat,withtheexceptionoftheinfusionoftheaerialpart(forthelines
MCF7andHepG2)anddecoctionextracts(lineMCF7),alltheremainingextracts
presentedeffectiveresultsintheinhibitionofthetestedcelllines;thehydroethanolic
extractoftheaerialpartrevealedalowerGI50,whichmayberelatedtothehighlevelsof
phenoliccompoundsfoundinthisextract.
Molecules2021,26,18826of14
Theinfusionanddecoctionoftheaerialpart,andtheinfusionoftherootsand
rhizomesdidnotshowcytotoxiceffectsonanon‐tumorprimarycellculture.Inturn,the
hydroethanolicextractsoftheaerialpartandrootsandrhizomes,aswellasthedecoction
ofrootsandrhizomes,presentedtoxicitytowardstheliverprimarycellculture(PLP2).
TheseresultsareinagreementwiththosedescribedbyBassiletal.[23],inwhichthey
exploredtheeffectofethanolicextractonleukemiaacutelymphocytecancerproliferation,
andconcludedthattheR.aculeatusextractisnotagoodcandidateforthedevelopmentof
anti‐tumordrugs,asitdemonstratedacytotoxiceffect,althoughfurtherstudiesare
neededtoconfirmthisstatement.
Chenetal.[24]evaluatedthecytotoxicactivityofOrnithogalumsaundersiae
(Asparagaceae)andfoundthatthecompoundOsaundersiosideCinhibitedspecific
cytotoxicityinrelationtotheMCF‐7celllinewithIC50valuesof0.20μM,similartothe
positivecontrolpaclitaxel.
FewstudiesevaluatethecytotoxicandhepatotoxiceffectsindifferentextractsofR.
aculeatus,makingitdifficulttocomparetheresultswiththeliterature.Additionalstudies
wouldbeessential,possiblyinvolvingthefractionationandidentificationoftheactive
compounds,inordertobetterunderstandtheactionandpotentialofthesetypesof
extractsandcompounds.
Regardingtheanti‐inflammatoryproperties,fortherangeoftestedconcentrations
(upto400μg/mL),intheextractsofinfusionanddecoctionoftheaerialpart,aswellas
theinfusionoftherootsandrhizome,theresultsindicatedtheabsenceofactivityinLPS‐
activatedmurinemacrophagessincenodecreaseofnitricoxidelevelswasobserved
(Table2).
Thehydroethanolicextractoftheaerialpartwastheonethatpresentedaneffective
result,60μg/mLbeingnecessarytopromotethe50%inhibitionofnitricoxideproduction,
whichmayalsoberelatedtothehighpresenceofphenoliccompoundsinthisextract.
Comparingtheaqueousextractsoftheaerialpartwiththoseoftherootsandrhizome,
onlythedecoctionoftherootsandrhizomepresentanti‐inflammatoryactivity(129
μg/mL).Theseresultsareinagreementwiththosedescribedbydifferentauthors,who
demonstratethatplantsoftheAsparagaceaefamilyhaveanti‐inflammatoryproperties
mainlyduetotheexistenceofsteroidalsaponins,primarilyruscogenin[25,26].
Molecules2021,26,18827of14
Table2.Antioxidant,cytotoxicity,hepatotoxicandanti‐inflammatoryactivityofthehydroethanolicextracts,infusionanddecoctionpreparationsofR.aculeatus(mean±SD,n=9).
AerialPartRootsandRhizomesPositiveControl
HydroethanolicInfusionDecoctionHydroethanolicInfusionDecoctionTrolox(μg/mL)
Antioxidantactivity
TBARS(EC50,mg/mL)a0.28±0.01f0.49±0.03e0.88±0.01c0.78±0.04d1.00±0.01b1.55±0.03a5.8±0.6
OxHLIA(IC50
,
μg/mL)b
Δt=60minn.a.236±16c 427±36b230±11c646±33a661±25a21.8±0.2
Δt=120minn.a.n.a.n.a.383±13c1389±48a1198±28b43.5±0.3
Cytotoxicity(GI50,μg/mL)cEllipticine
HeLa 31±4d373±27a270±20b98±6c302±25b111±6c0.9±0.1
NCIH460 70±4d273±15b302±7a51±3e201±17c69±2d.e1.03±0.09
MCF7 70±3c>400>40089±4b350±16a94±2b1.21±0.02
HepG2 72±3d>400260±22b71±2d300±12a168±9c1.10±0.09
Hepatotoxicity(GI50
,
μg/mL)c
PLP2152±8c>400>400179±7b>400265±9a2.3±0.2
Anti‐inflammatoryactivity(EC50μg/mL)dDexamethasone
Productionofnitricoxide(NO)in
RAW264.760±5c>400>400111±4b>400129±5a16±1
n.a.:noactivity.aEC50values:extractconcentrationcorrespondingto50%ofantioxidantactivity.bIC50values:extractconcentrationnecessarytokeep50%oftheerythrocytepopulation
intactfor60and120min.cGI50valuescorrespondtothesampleconcentrationresponsiblefor50%inhibitionofgrowthintumorcellsorinaprimarycultureoflivercells‐PLP2.dEC50
valuescorrespondtotheextractconcentrationachieving50%oftheinhibitionofNO‐production.Differentlettersinthesamelinemeansignificantdifferences(p<0.05).
Molecules2021,26,18828of14
Table3showstheresultsoftheantimicrobialactivityofthehydroethanolicand
aqueousextractsofR.aculeatusagainstthreeGram‐positiveandfiveGram‐negative
multi‐resistantpathogenicstrains,isolatedfromhospitalizedpatients.Ingeneral,all
extractspresentpotentialeffectsagainstthebacterialstrainstestedinthisstudy;Gram‐
positivebacteriapresentlowerMICvaluesand,therefore,superiorsensitivitywhen
comparedwithGram‐negativebacteria.Theinfusionandhydroethanolicextractsofthe
aerialpart,wereeffectiveagainstGram‐positivebacteriawithanMICof10mg/mL.
RegardingtheinhibitionofGram‐negativebacteria,onlytheinfusionand
hydroethanolicextractsoftheaerialpartpresentedanMICof10mg/mL.Thedecoction
oftheaerialpartwaseffectiveagainsttheMRSAstrain—methicillin‐resistantS.aureus,
withanMICof5mg/mL.Infusionoftherootandrhizomepresentthesmallest
antibacterialpotential,withMICandMBCvalueshigherthan20mg/mLforthetested
bacteria.
Accordingtotheauthorsof[20],theethylacetateextractofR.aculeatusdemonstrated
bacteriostaticactivityagainstS.aureusandbactericidalactivityagainstE.coliandP.
aeruginosa.Onestudydemonstratedthattheinfusionandethanolicextractsoftheroots
werelesseffectiveagainstthefungusC.albicans(ATCC1023)[27].Astudyinwhichthe
antimicrobialactivityofthemethanolicextractofleavesofAgavesisalana(Asparagaceae)
wasmeasuredfoundthattheydidnotrevealantimicrobialactivityagainstthe
microorganismsused(S.aureusandK.pneumoniae)[28].
Ingeneral,theextractsevaluatedinthisworkwereeffectiveagainstGram‐positive
bacteria,whichcanbeexplainedbythefactthatthisgroupofmicroorganismshasaless
complexcellwallcomparedtoGram‐negativebacteria.Itshouldbenotedthatinthis
work,themicroorganismswereobtainedfromclinicalisolates,whichoftenhavegreater
resistancetoantibioticscomparedtocommercialstrains.
Molecules2021,26,18829of14
Table3.Antibacterialactivity(MICandMBC,mg/mL)ofthehydroethanolicextracts,infusionanddecoctionpreparationsofR.aculeatus.
AerialPartRootsandRhizomeNegativeControls
HydroethanolicInfusionDecoctionHydroethanolicInfusionDecoctionAmpicillin
(20mg/mL)
Imipenem
(1mg/mL)
Vancomycin
(1mg/mL)
MICMBCMICMBCMICMBCMICMBCMICMBCMICMBCMICMBCMICMBCMICMBC
Gram‐negativebacteria  
Escherichiacoli10>20>20>2020>2020>20>20>2020>20<0.15<0.15<0.0078<0.0078n.t.n.t.
Klebsiellapneumoniae20>2020>2020>20>20>20>20>20>20>201020<0.0078<0.0078n.t.n.t.
Morganellamorganii10>2010>2020>20>20>20>20>20>20>2020>20<0.0078<0.0078n.t.n.t.
Proteusmirabilis20>20>20>20>20>20>20>20>20>20>20>20<015<0.15<0.0078<0.0078n.t.n.t.
Pseudomonasaeruginosa>20>20>20>20>20>20>20>20>20>20>20>20>20>200.51n.t.n.t.
Gram‐positivebacteria 
Enterococcusfaecalis10>2010>2020>2020>2020>20>20>20<0.15<0.15n.t.n.t.<0.0078<0.0078
Listeriamonocytogenes10>2010>2010>20>20>20>20>20>20>20<0.15<0.15<0.0078<0.0078n.t.n.t.
MRSA10>2010>205>20>20>2020>2010>20<0.15<0.15n.t.n.t.0.250.5
MRSA—Methicillin‐resistantStaphylococcusaureus;MIC—minimalinhibitoryconcentration;MBC—minimalbactericidalconcentration;n.t.—nottested.
Molecules2021,26,188210of14
3.MaterialsandMethods
3.1.PlantMaterial
SamplesofR.aculeatuswereharvestedinApril2019insidewoodlandsand
hedgerows,inValpaços,VilaReal,Portugal.Twodistinctpartsoftheplantweregathered,
namelytheaerialpart(cladodesorlaminarstemsandlateralbranches)andthe
undergroundorgans(rhizomeswithroots).Theseplantmaterialswerecleanedofsoil
remains,lyophilized(FreeZone4.5,Labconco,KansasCity,MO,USA),andreducedtoa
finepowderthatwasstoredinsealedplasticbags.
Consideringtheabundanceofthisspeciesatthegrowingsiteanditsstrategyof
reproductionandnaturalregeneration(theseedsarerelativelyabundantanddistributed
bybirds,andtheplantalsopropagatesthroughitsvegetativeparts,rhizomes),the
harvestingofthiswildmaterialwasdoneinasustainablewayanddidnotputwild
populationsatrisk.
3.2.HydroethanolicExtractsandAqueousPreparations
Theplantmaterialwasusedtopreparehydroethanolicextracts,infusions,and
decoctionspreparations.Thepreparation/extractionmethodswereselectedaccordingto
thetraditional/empiricalusesofthedifferentpartsoftheplant[13,20].Hydroethanolic
extractionswereperformedbystirringtheplantmaterial(~3g)with45mLof
ethanol/watersolution(80:20,v/v)underconstantmagneticstirring,atroomtemperature,
for1h.ThepreparationwasfilteredthroughaWhatmanfilterpaperNo.4andtheresidue
wasre‐extractedandthecombinedfiltrateswerethenevaporatedunderpressureat40°C
(rotaryevaporatorBuchiR‐2010,Flawil,Switzerland)andsubsequentlylyophilized.
Approximately3gofplantmaterialwasinfusedwith100mLoffreshlyboiled
distilledwater(heatingplate,VELPscientific),leftasidefor5min,andsubsequently
filteredthroughWhatmanfilterpaperNo4.Theresultingextractswerefrozenand
lyophilized.
Theplantmaterialwasdecoctedbyadding100mLofdistilledwater(~3g),and
boiledfor5min.Subsequently,themixtureswerelefttostandfor5minandthenfiltered
throughWhatmanNo.4paper.Theobtaineddecoctionswerefrozenandlyophilized.
3.3.AnalysisofPhenolicCompounds
Phenoliccompoundswereanalyzedinthehydroethanolicextractsandaqueous
preparations,whichwerere‐dissolvedinethanol/water(80:20,v/v)andwater,
respectively,toafinalconcentrationof10mg/mLandfilteredthought0.22‐μmdisposable
filterdisks.TheanalysiswasperformedinanHPLCsystem(DionexUltimate3000UPLC,
ThermoScientific,SanJose,CA,USA)coupledwithadiode‐arraydetector(DAD,using
280and370nmaspreferredwavelengths)andaLinearIonTrap(LTQXL)mass
spectrometer(MS,ThermoFinnigan,SanJose,CA,USA)equippedwithanelectrospray
ionization(ESI)source.SeparationwasmadeinaWatersSpherisorbS3ODS‐2C18
column(3μm,4.6×150mm;Waters,Milford,MA,USA).Theequipmentandoperating
conditionswerepreviouslydescribedbytheauthors[29]aswellastheidentificationand
quantificationprocedures.Thephenolicstandards(caffeic acid, apigenin-6-C-glucoside,
and quercetin-3-O-rutinoside)wereacquiredfromExtrasynthèse,Genay,France.The
resultswereexpressedasmgpergofextract.
3.4.EvaluationofBioactivePropertiesofExtracts
Theevaluationofthebioactivepotentialoflyophilizedhydroethanolicextractsand
aqueouspreparationswasperformedinvitro.

Molecules2021,26,188211of14
3.4.1.ThiobarbituricAcidReactiveSubstances(TBARS)FormationInhibitionCapacity
Theextractspreparedabovewerere‐dissolvedinwaterandsubjectedtodilutions
from5to0.0390mg/mL.Porcine(Susscrofa)brainswereobtainedfromanimals
slaughteredatofficiallylicensedpremises,dissected,andhomogenizedwithTris‐HCl
buffer(20mmol/L,pH7.4)toproduceabraintissuehomogenate,whichwascentrifuged
at3000gfor10min.Analiquot(100μL)ofthesupernatantwasincubatedwiththe
differentconcentrationsoftheextractsolutions(200μL)inthepresenceofFeSO4(10
mmol/L;100μL)andascorbicacid(0.1mmol/L;100μL)at37°Cfor1h.
Thereactionwasstoppedbytheadditionoftrichloroaceticacid(28g/100μL,500
μL),followedbythiobarbituricacid(TBA,2g/100mL,380μL),andthemixturewasthen
heatedat80°Cfor20min.Aftercentrifugationat3000gfor10mintoremovethe
precipitatedprotein,thecolorintensityofthemalondialdehyde(MDA)‐TBAcomplexin
thesupernatantwasmeasuredat532nm;theinhibitionratio(%)wascalculatedusingthe
followingformula:[(A−B)/A]×100%,whereAandBcorrespondtotheabsorbanceofthe
controlandextractsample,respectively[30].TheresultswereexpressedinEC50values
(mg/mL,sampleconcentrationproviding50%ofantioxidantactivity).Trolox(Sigma‐
Aldrich,St.Louis,MO,USA)wasusedasthepositivecontrol.
3.4.2.OxidativeHemolysisInhibition(OxHLIA)Capacity
Theantihemolyticactivityoftheextractswasevaluatedbytheoxidativehemolysis
inhibitionassay(OxHLIA),usingerythrocytesisolatedfromhealthysheepbloodand
centrifugedat1000gfor5minat10°C.Afterdiscardingtheplasmaandbuffycoats,the
erythrocyteswerefirstwashedwithNaCl(150mM)andthenthreetimeswithphosphate‐
bufferedsaline(PBS,pH7.4).TheerythrocytepelletwasthenresuspendedinPBSto
obtainaconcentrationof2.8%(v/v).Usingaflat‐bottom48‐wellmicroplate,200μLofthe
erythrocytesolutionweremixedwith400μLofPBSsolution(control),antioxidant
extractsdissolvedinPBS,orwater(forcompletehemolysis).Afterpre‐incubationat37°C
for10minwithshaking,2,2′‐azobis(2‐methylpropionamidine)dihydrochloride(AAPH,
160mMinPBS,200μL)wasaddedtoeachwellandtheopticaldensitywasmeasuredat
690nm.Theresultswereexpressedasthedelayedtimeofhemolysis(Δt),whichwas
calculatedaccordingtotheequation:Δt(min)=Ht50(sample)−Ht50(control),whereHt50
isthetime(min)correspondingto50%hemolysis,graphicallyobtainedfromthe
hemolysiscurveofeachantioxidantsampleconcentration.TheΔtvalueswerethen
correlatedwiththeextractconcentrations,andfromthecorrelationobtained,theextract
concentrationabletopromoteaΔthemolysisdelaywascalculated.Troloxwasusedasa
positivecontrol.TheresultsweregivenasIC50values(μg/mL)atΔt60and120min,i.e.,
extractconcentrationrequiredtoprotect50%oftheerythrocytepopulationfromthe
hemolyticactionfor60and120min[19].
3.4.3.CytotoxicityActivity
Thecytotoxicitycapacityoftheextractswasevaluatedusingfourhumantumorcell
lines:MCF‐7(breastadenocarcinoma),NCI‐H460(non‐smallcelllungcancer),HeLa
(cervicalcarcinoma),andHepG2(hepatocellularcarcinoma)fromDSMZ(Leibniz‐Institut
DSMZ‐DeutscheSammlungvonMikroorganismenundZellkulturenGmbH).The
sulforhodamineB(SRB,Sigma‐Aldrich,StLouis,MO,USA)assaywasperformed
accordingtoaprocedurepreviouslydescribedindetailbytheauthors[31].
Thecellgrowthinhibitionwascalculatedaccordingtotheequation:((Abssampleextract
andcells−0.05)/(Abscontrol−0.05)×100).Ellipticine(Sigma‐Aldrich,St.Louis,MO,USA)
wasusedasthepositivecontrol,andtheresultswereexpressedinGI50values(μg/mL),
correspondingtotheextractconcentrationthatprovides50%ofcellgrowthinhibition.

Molecules2021,26,188212of14
3.4.4.HepatotoxicActivity
Hepatotoxicityoftheextractswasevaluatedusingaprimarycellcultureprepared
fromporcineliver(PLP2),whichwaspreparedaccordingtotheprocedureoptimizedand
describedbytheauthors[32].Thetestedconcentrationofbothhydroethanolicand
aqueousextractsrangedfrom400to6.5μg/mL.TheresultsweremeasuredusingtheSRB
methodandwereexpressedasGI50values(concentrationthatinhibits50%ofcellgrowth,
μg/mL).Ellipticinewasusedasthepositivecontrol.
3.4.5.Anti‐InflammatoryActivity
Theanti‐inflammatoryactivityoftheextractswasdeterminedbasedonthenitric
oxide(NO)productionbyamurinemacrophage(RAW264.7)cellline,inducedbythe
additionoflipopolysaccharide(LPS).Thetestedconcentrationofbothhydroethanolicand
aqueousextractsrangedfrom400to6.5μg/mL.NOproductionwasquantifiedbasedon
nitriteconcentrationusingtheGriessReagentSystemkitcontainingsulfanilamide,N‐1‐
naphthylethylenediaminedihydrochloride,andnitritesolutionsfollowingaprocedure
previouslydescribedbytheauthors[33].Dexamethasonewasusedasapositivecontrol
whilenoLPSwasaddedinnegativecontrols.
TheeffectofthetestedextractsinNObasallevelswasalsoassessedbyperforming
theassayintheabsenceofLPS.TheresultswereexpressedasIC50values(μg/mL),
correspondingtotheextractconcentrationproviding50%inhibitionofNOproduction.
3.4.6.AntimicrobialActivity
Theantimicrobialactivityoftheextractswasdeterminedagainstclinicalisolates
obtainedfrompatientshospitalizedintheLocalHealthUnitofBragançaandHospital
CenterofTrás‐os‐MontesandAlto‐DouroVilaReal,followingthemicrodilutionmethod
coupledtotherapidp‐iodonitrotetrazoliumchloride(INT)colorimetricassaydescribed
bytheauthors[34].Thetestedconcentrationofbothhydroethanolicandaqueousextracts
rangedfrom20to0.156mg/mL.
ThetestedmicroorganismsincludedGram‐positive(Enterococcusfaecalis,Listeria
monocytogenes,andmethicillin‐resistantStaphylococcusaureus)andGram‐negativebacteria
(Escherichiacoli,Klebsiellapneumoniae,Morganelamorganii,Proteusmirabilis,and
Pseudomonasaeruginosa).Theminimuminhibitoryconcentration(MIC)andtheminimum
bactericidalconcentration(MBC)wereevaluated,anddifferentantibioticswereusedas
negativecontrol(ampicillinandimipenemforGram‐negativebacteria,andvancomycin
andampicillinforGram‐positivebacteria).Culturebroth(MullerHintonBrothadded
with5%dimethylsulfoxide)inoculatedwitheachbacteriumwasusedasthepositive
control[34].
3.5.StatisticalAnalysis
Allexperimentswerecarriedoutintriplicateandtheresultswereexpressedasmean
±standarddeviation(SD).ThestatisticalanalysiswasperformedusingSPSSv.23.0
softwareforWindows(IBMCorp.,Armonk,NY,USA)andusingtheone‐wayanalysisof
variance(ANOVA),whilethecomparisonofmeanswascarriedoutwiththeTukey’sHSD
test(p<0.05)whensignificantstatisticaldifferencesweredetected.
4.Conclusions
Thisworkaimedtoexplorethebiochemicalcharacterizationofawildplantforwhich
fewstudieshavebeenperformed.Wildplantscanbeconsideredsourcesofbioactivities,
sincetheyhaveseveralcompoundsand,consequently,differentpotentialtherapeutic
action.Thepresentworkallowedtodeterminethephenoliccompoundsinthethree
extractsobtainedfromtheaerialpartandfromtherhizomesandrootsofR.aculeatususing
differentextractionandsolventtechniques,butalsotoevaluatetheirbioactiveproperties.
Molecules2021,26,188213of14
Ingeneral,thisstudyprovidedinnovativeresultsinrelationtothechemical
characterizationandbioactivepropertiesofthislittle‐studiedandexploredwildplant.
However,theseinnovativeresultsarenotenoughtorelatetheempiricaluseswiththe
chemicalcharacteristicsandbioactivepropertiesdemonstratedbytheextractsobtained
fromboththeaerialandtheundergroundparts.Therefore,itwillbeessentialtocontinue
exploringthecompoundsandthementionedactivities,sothatitispossibletocorroborate
andsubstantiatetheuseofthisspeciesintraditionalmedicine.
AuthorContributions:J.P.B.R.:Formalanalysis;Methodology,Investigation,Datacuration,
Writing—originaldraft;Â.F.,M.I.D.,andC.P.:Methodology,Software,Validation,Investigation,
Datacuration,Writing—review&editing;T.C.S.P.P.:MethodologyandR.C.C.:Methodology;
A.M.C.:Conceptualization,Writing—review&editing,Visualization,Supervision;I.C.F.R.F.:
Supervision,Projectadministration;L.B.:Conceptualization,Validation,Investigation,Writing—
review&editing,Visualization,Supervision,Projectadministration,Fundingacquisition.All
authorshavereadandagreedtothepublishedversionofthemanuscript.
Funding:TheauthorsaregratefultotheFoundationforScienceandTechnology(FCT,Portugal)
forfinancialsupportthroughthenationalfundsFCT/MCTEStoCIMO(UIDB/00690/2020)and
nationalfundingbyFCT,P.I.,throughtheinstitutionalscientificemploymentprogramcontractfor
L.Barros,A.Fernandes,M.I.Dias,C.Pereira,andR.C.Calhelha.Theauthorsarealsogratefulto
FEDER‐InterregEspaña‐PortugalprogramforfinancialsupportthroughtheprojectTRANSCoLAB
0612_TRANS_CO_LAB_2_PandtoEuropeanRegionalDevelopmentFund(ERDF)throughthe
RegionalOperationalProgramNorth2020,withinthescopeofProjectNorte‐01‐0145‐FEDER‐
000042:GreenHealth.
ConflictsofInterest:Theauthorsstatenoconflictofinterest.
SampleAvailability:Samplesoftheplantspeciesareavailablefromtheauthors.
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... Вместе с тем наибольшее количество активных веществ, определяющих основные терапевтические эффекты иглицы, содержится в ее корнях. В частности, в них обнаружены биофлавоноиды, сапонозиды и гетерозиды [12][13][14]. ...
... Известно, что большое количество пациентов пожилого возраста с ХЗВ в качестве неблагоприятного коморбидного фона имеют сахарный диабет 2-го типа. Именно у этой категории больных Цикло 3 не только обеспечил снижение частоты и тяжести веноспецифических симптомов, но и продемонстрировал выраженный капилляропротективный эффект [13,26,60]. ...
Fixed-dose combinations in pharmacotherapy of chronic venous diseases
Article
  • Nov 2024
Chronic venous diseases are the most common vascular diseases of the lower extremities, negatively affecting all components of the quality of life in the modern society. Due to their high prevalence, the prevention and treatment of chronic venous diseases are one of the priorities of national healthcare systems in industrially developed countries. In recent years, numerous experimental and clinical studies have uncovered an important role of vascular inflammation and endothelial dysfunction in the pathogenesis of chronic venous diseases, which determine not only a wide range of vein-specific symptoms, but are also the main drivers of pathological remodelling of the vein wall and its valve apparatus, followed by the development of varicose syndrome and more severe chronic venous insufficiency. Modern knowledge of the pathogenesis of chronic venous diseases enables us to identify several priority strategies aimed at managing the basic pathophysiological mechanisms of the development and progression of the disease. Pharmacotherapy with phlebotropic drugs is one of them. Some of these drugs are able to inhibit the vein-specific inflammation, improve the microcirculatory bed regulation, increase the vein tone and contractility and, as a result, have a positive effect on the clinical signs of chronic venous disease. This review presents the mechanism of action of a phlebotropic drug that is new to the Russian Federation. It is a combination of Ruscus aculeatus extract (Ruscus extract) with hesperidin methyl chalcone (HMC) and ascorbic acid. Studies showed a high level of evidence of clinical efficacy and safety of this drug for the treatment of patients with various forms of chronic venous diseases. Due to the unique features of its mechanisms of action, the optimal focus groups for its use may include patients of different age groups with early-stage disease, in which vein-specific symptoms predominate, and patients with chronic venous oedema.
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... In a study by Jakovljević et al. [62], the R. hypoglossum and R. aculeatus extracts were screened for their total phenolic content, which showed significant results. Furthermore, Rodrigues et al. [63] also chemically analyzed the aerial parts, roots, and rhizomes of R. aculeatus. Overall, nine phenolic compounds were detected in the extracts, with major compounds apigenin-C-hexoside-C-pentoside isomer II, quercetin-O-deoxyhexoside-hexoside, and apigenin-C-hexoside-C-pentoside isomer [63]. ...
... Furthermore, Rodrigues et al. [63] also chemically analyzed the aerial parts, roots, and rhizomes of R. aculeatus. Overall, nine phenolic compounds were detected in the extracts, with major compounds apigenin-C-hexoside-C-pentoside isomer II, quercetin-O-deoxyhexoside-hexoside, and apigenin-C-hexoside-C-pentoside isomer [63]. The study revealed that extracts and the latter compounds showed considerable antioxidant, and antibacterial activity, though, did not display cytotoxic effects. ...
Bioactive Compounds and Biological Activities of Ruscus Species
Chapter
  • Mar 2024
The genus Ruscus (Asparagaceae) is comprised of seven species which include R. aculeatus L., R. colchicus Yeo., R. hypoglossum L., R. hypophyllum L., R. Hyrcanus Woronow., R. × microglossus Bertol. and R. streptophyllus Yeo. These perennial, rhizomatous, evergreen shrubs display a native distribution in the Mediterranean, Southern, and Western Europe areas. The rhizomes of the Ruscus species possess anti-inflammatory activity and are regularly used in traditional medicine for the treatment of urinary ailments, abdominal pain, vascular prevention, and skin disease. Ruscus species, especially R. aculeatus L., are a rich source of steroidal saponins which is responsible for their specific biological activities such as antihypertension, anti-hemorrhoids, and anti-venous sufficiency. This chapter aims to provide an updated overview of the phytochemical components and biological activities of Ruscus species to reveal their usage in traditional and modern medicine.
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... RA, also known as butcher's broom, is a medicinal plant native to Mediterranean Europe and Africa. RA extract (RAE) is mainly prepared from the roots of RA and shows medicinal effects against venous insufficiency, oedema, premenstrual syndrome, and haemorrhoids [18]. RAE is generally considered to be a safe medicine which is also used as a cosmetic ingredient [19,20]. ...
Ruscus aculeatus extract promotes RNase 7 expression through ERK activation following inhibition of late-phase autophagy in primary human keratinocytes
Article
Full-text available
Antimicrobial peptides (AMPs) are crucial for protecting human skin from infection. Therefore, the expression levels of beneficial AMPs such as ribonuclease 7 (RNase 7) must be appropriately regulated in healthy human skin. However, there is limited understanding regarding the regulating AMP expression, especially when using applications directly to healthy human skin. Here, we investigated the effects of the extract of Ruscus aculeatus (RAE), a medicinal plant native to Mediterranean Europe and Africa that is known to have a high safety level, on AMP expression in primary human keratinocytes. Treatment with RAE induced RNase 7 expression, which was suppressed by an extracellular signal-regulated kinase (ERK) inhibitor. The autophagic flux assay and the immunofluorescence analysis of microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ and p62 showed that RAE inhibited late-phase autophagy. Moreover, both the inhibition of early-phase autophagy by EX-527, an inhibitor of silent information regulator of transcription 1 (SIRT1) and its enhancement by resveratrol, an activator of SIRT1 inhibited RNase 7 and ERK expression, indicating that autophagosome accumulation is necessary for RAE-induced RNase 7 expression. Additionally, spilacleoside was identified as the active component in RAE. These findings suggest that RAE promotes RNase 7 expression via ERK activation following inhibition of late-phase autophagy in primary human keratinocytes and that this mechanism is a novel method of regulation of AMP expression.
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... Various anti-cancer agents designed on natural compounds have demonstrated multiple functions across different pathways due to their inherent characteristics. For instance, (1) ruscogenin, an anti-inflammatory and antithrombotic steroid found in Ruscus aculeatus (a traditional medicine used as diuretics and treatment for urinary system disorders) [136,137], shows a decreased pattern in regulating MMP2, MMP9, VEGF, and HIF-1α that could suppress the metastasis of hepatocellular carcinoma [138], and (2) plumbagin, an extract from the medicinal plant Plumbago zeylanica (one of the oldest herbs in Ayurveda including functions such as anti-inflammatory and antitumour [139]) can down-regulate tumour cell growth by inhibiting the mTOR/PI3K/Akt pathway and EMT transition in prostate cancer [140], in addition, inducing ROS production for specific cell apoptosis [141,142]. Some advanced computational-based methods (such as chemogenomics, proteochemometric modelling, target fishing, and system pharmacology) are also applied for further investigation [143]. ...
Use of Drug Sensitisers to Improve Therapeutic Index in Cancer
Article
Full-text available
  • Jul 2024
The clinical management of malignant tumours is challenging, often leading to severe adverse effects and death. Drug resistance (DR) antagonises the effectiveness of treatments, and increasing drug dosage can worsen the therapeutic index (TI). Current efforts to overcome DR predominantly involve the use of drug combinations, including applying multiple anti-cancerous drugs, employing drug sensitisers, which are chemical agents that enhance pharmacokinetics (PK), including the targeting of cellular pathways and regulating pertinent membrane transporters. While combining multiple compounds may lead to drug–drug interactions (DDI) or polypharmacy effect, the use of drug sensitisers permits rapid attainment of effective treatment dosages at the disease site to prevent early DR and minimise side effects and will reduce the chance of DDI as lower drug doses are required. This review highlights the essential use of TI in evaluating drug dosage for cancer treatment and discusses the lack of a unified standard for TI within the field. Commonly used benefit–risk assessment criteria are summarised, and the critical exploration of the current use of TI in the pharmaceutical industrial sector is included. Specifically, this review leads to the discussion of drug sensitisers to facilitate improved ratios of effective dose to toxic dose directly in humans. The combination of drug and sensitiser molecules might see additional benefits to rekindle those drugs that failed late-stage clinical trials by the removal of detrimental off-target activities through the use of lower drug doses. Drug combinations and employing drug sensitisers are potential means to combat DR. The evolution of drug combinations and polypharmacy on TI are reviewed. Notably, the novel binary weapon approach is introduced as a new opportunity to improve TI. This review emphasises the urgent need for a criterion to systematically evaluate drug safety and efficiency for practical implementation in the field.
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... Moreover, it has been shown in the literature that Ruscus extract and HMC prevented the elevation of microvascular permeability induced by both LTB 4 and I/R injury [23]. The anti-inflammatory and antioxidant actions of Ruscus extract in our experimental model are in line with studies in vitro [60] and in humans [61]. In addition, other study has demonstrated that the beneficial effect of Ruscus extract on macromolecular leakage, were inhibited by atropine, suggesting that activation of muscarinic receptors was involved in this endothelial barrier function protection [22]. ...
Protective effects of Ruscus extract in combination with ascorbic acid and hesperidine methylchalcone on increased leukocyte-endothelial interaction and macromolecular permeability induced by ischemia reperfusion injury
Article
  • Jul 2024
  • CLIN HEMORHEOL MICRO
BACKGROUND: Despite the well-recognized effectiveness of Ruscus aculetus extract combined or not with ascorbic acid (AA) and hesperidine methyl chalcone (HMC) on ischemia reperfusion (I/R) injury protection, little is known about the contribution of each constituent for this effect. OBJECTIVE: To investigate the effects of AA and HMC combined or not with Ruscus extract on increased macromolecular permeability and leukocyte-endothelium interaction induced by I/R injury. METHODS: Hamsters were treated daily during two weeks with filtered water (placebo), AA (33, 100 and 300 mg/kg/day) and HMC (50, 150 and 450 mg/kg/day) combined or not with Ruscus extract (50, 150 and 450 mg/kg/day). On the day of experiment, the cheek pouch microcirculation underwent 30 min of ischemia, and the number of rolling and adherent leukocytes and leaky sites were evaluated before ischemia and during 45 min of reperfusion. RESULTS: Ruscus extract combined with AA and HMC (Ruscus extract mixture) significantly prevented post-ischemic increase in leukocyte rolling and adhesion and macromolecular permeability compared to placebo and these effects were more prominent than AA and HMC alone on leukocyte adhesion and macromolecular leakage. CONCLUSION: Ruscus extract mixture were more effective than its isolated constituents in protect the hamster cheek pouch microcirculation against I/R injury.
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... Butcher's broom extracts exhibit anti-inflammatory, astringent, and anticoagulatory properties, as well as remarkable anti-elastase activity. R. aculeatus is an important phlebotherapeutic agent which can be used to treat chronic venous insufficiency and vasculitis [3,[13][14][15]. ...
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... Potentiodynamic polarization curves of carbon steel in acidic solution with and without addition of the RAL alcoholic extract. Rodrigues et al. (2021) reported that quercetin-Odeoxyhexoside-hexoside apigenin-C-hexoside-Cpentoside isomer II are the main compounds found in the RAL ethanolic extract. These compounds contain heteroatoms, benzoic ring and double bonds that are crucial for the adsorption, and therefore for the effective protection of the carbon steel samples. ...
Study Of The Performance Of Ruscus Aculeatus Leaves Alcoholic Extract As Corrosion Inhibitor For Carbon Steel In 1 M HCl Solution
Conference Paper
  • Aug 2023
The addition of corrosion inhibitors is one of the most widely used methods for the protection of metals and alloys from corrosion in various environments. Although synthetic organic compounds have been found to be highly effective in mitigating corrosion, they are often toxic, non-biodegradable, and rather expensive. Plant extracts are among the environmentally friendly alternatives to replace organic compounds as corrosion inhibitors. This study aims to investigate the corrosion inhibition efficiency of the Ruscus aculeatus leaves (RAL) alcoholic extract for carbon steel immersed in 1 M HCl solution, using weight loss and potentiodynamic polarization measurements. The optimum concentration of RAL alcoholic extract, i.e. 200 mg/L, gave a corrosion inhibition efficiency of 91.09% at 25 ºC. Potentiodynamic polarization measurements showed that the RAL alcoholic extract acts as a mixed-type inhibitor with a predominant influence on the anodic corrosion reaction. The corrosion inhibition efficiency of the RAL alcoholic extract decreased at an acceptable rate with increasing temperature in the 25-55 ºC range.
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... In this study, we demonstrate that the roots with the highest MAMP content were: The study of plant lignans and phytochemicals, which are thought to be the cause of these effects, has dominated research on the therapeutic benefits of roots up until this point. While effects of these products on " immune boosting' have been reported extensively (e.g Burdock root (Chan et al., 2011;He et al., 2018;Suzuki et al., 2002;Wang and Yang, 1993), Essiac (1998) Kava Kava Root (Minh et al., 2022;Pont-Fernandez et al., 2022), Butchers broom (Masullo et al., 2016;Rodrigues et al., 2021) Dandelion Root Talapphet et al., 2021) and Pleurisy Root (Warashina and Noro, 2010), the plants microbiome has not been investigated in the way that would be most appropriate. Further study into relevance of the microbiome of plant will likely unveil it to be a most significant influencer of immune system response. ...
Functional immune boosters; the herb or its dead microbiome? Antigenic TLR4 agonist MAMPs found in 65 medicinal roots and algae’s
Article
Full-text available
  • Aug 2023
Background: Humans have been consuming medicinal plants (as herbs/ spices) to combat illness for centuries Herbs while ascribing beneficial effects predominantly to the plant/phytochemical constituents, without recognizing Edible microbiome the power of obligatory resident microorganism' communities (MOCs) (live/dead bacteria, fungus, yeast, molds Medicinal microbiome etc.) which remain after industrial microbial reduction methods. Very little is known about the taxonomic Bugs as drugs identity of residual antigenic microbial associated molecular patterns (MAMPs) debris in our botanical over the Immune boosting counter (OTC) products, which if present would be recognized as foreign (non-self) antigenic matter by host pattern recognition receptors (PRRs) provoking a host immune response; this the basis of vaccine adjuvants. As of today, only few research groups have removed the herbal MAMP biomass from herbs, all suggesting that immune activation may not be from the plant but rather its microbial biomass; a hypothesis we corroborate. Purpose: The purpose of this work was to conduct a high through put screening (HTPS) of over 2500 natural plants, OTC botanical supplements and phytochemicals to elucidate those with pro-inflammatory; toll like receptor 4 (TLR4) activating properties in macrophages. Study design: The HTPS was conducted on RAW 264.7 cells vs. lipopolysaccharide (LPS) E. coli 0111:B4, testing iNOS / nitric oxide production (NO2-) as a perimeter endpoint. The data show not a single drug/chemical/ phytochemical and approximately 98 % of botanicals to be immune idle (not effective) with only 65 pro-inflammatory (hits) in a potency range of LPS. Method validation studies eliminated the possibility of false artifact or contamination, and results were cross verified through multiple vendors/ manufacturers/lot numbers by botanical species. Lead botanicals were evaluated for plant concentration of LPS, 1,3:1,6-β-glucan, 1,3:1,4-β-D-glucan and α-glucans; where the former paralleled strength in vitro. LPS was then removed from plants using high-capacity endotoxin poly lysine columns, where bioactivity of LPS null "plant" extracts were lost. The stability of E.Coli 0111:B4 in an acid stomach mimetic model was confirmed. Last, we conducted a reverse culture on aerobic plate counts (APCs) from select hits, with subsequent isolation of gram-negative bacteria (MacConkey agar). Cultures were 1) heat destroyed (retested/ confirming bioactivity) and 2) subject to taxonomical identification by genetic sequencing 18S, ITS1, 5.8 s, ITS2 28S, and 16S. Conclusion: The data show significant gram negative MAMP biomass dominance in A) roots (e.g. echinacea, yucca, burdock, stinging nettle, sarsaparilla, hydrangea, poke, madder, calamus, rhaponticum, pleurisy, aconite etc.) and B) oceanic plants / algae's (e.g. bladderwrack, chlorella, spirulina, kelp, and "OTC Seamoss-blends" (irish moss, bladderwrack, burdock root etc), as well as other random herbs (eg. corn silk, cleavers, watercress, cardamom seed, tribulus, duckweed, puffball, hordeum and pollen). The results show a dominance of gram negative microbes (e.g. Klebsilla aerogenes, Pantoae agglomerans, Cronobacter sakazakii), fungus (Glomeracaea, Ascomycota, Irpex lacteus, Aureobasidium pullulans, Fibroporia albicans, Chlorociboria clavula, Aspergillus_sp JUC-2), with black walnut hull, echinacea and burdock root also containing gram positive microbial strains (Fontibacillus, Paenibacillus, Enterococcus gallinarum, Bromate-reducing bacterium B6 and various strains of Clostridium). Conclusion: This work brings attention to the existence of a functional immune bioactive herbal microbiome, independent from the plant. There is need to further this avenue of research, which should be carried out with consideration as to both positive or negative consequences arising from daily consumption of botanicals highly laden with bioactive MAMPS.
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Traditional medicinal plants in South Tyrol (northern Italy, southern Alps): biodiversity and use
Article
Full-text available
  • Nov 2020
Background Worldwide mountain regions are recognized as hotspots of ethnopharmacologically relevant species diversity. In South Tyrol (Southern Alps, Italy), and due to the region’s high plant diversity and isolated population, a unique traditional botanical knowledge of medicinal plants has flourished, which traces its history back to prehistoric times. However, changes in rural life and culture may threaten this unique biodiversity and cultural heritage. Our study aims to collect and analyze information on native plants used in traditional folk medicine, focusing on the preservation of botanical and cultural diversity. Methods Data were collected through a review of published material that documents traditionally used medicinal plants of South Tyrol in order to capture the total diversity of plants and their usage. We evaluated different parameters, comprising the ethnobotanicity index (EI), ethnophytonomic index (EPI), relative frequency of citation (RFC), red list status, and regional legislation with regard to the plant species. Results A total of 276 species, including 3 mushrooms and 3 lichens, were identified. These belonged to 72 families, most frequently to the Asteraceae, Rosaceae, and Lamiaceae. The most frequently cited species were Hypericum perforatum L., Urtica dioica L., and Plantago lanceolata L. According to 12 ICPC-2 disease categories, the most frequently treated human health symptoms were from the digestive and respiratory systems as well as the skin. A total of 27 species were listed as endangered, of which 16 are not protected and two are now already extinct. Among the 59 predominantly alpine species, 11 species are restricted to the high altitudes of the Alps and may be threatened by global warming. Conclusions Our research revealed that the ethnobotanical richness of South Tyrol is among the highest in Italy and throughout the Alps. Nevertheless, it is evident that biodiversity and traditional knowledge have been heavily eroded. Furthermore, we point out particularly sensitive species that should be reconsidered for stronger protections in legal regulations.
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Spectral data for cholestane glycosides from the bulbs of Ornithogalum saundersiae Baker
Article
Full-text available
  • Aug 2019
Herein, the spectral data, including nuclear magnetic resonance spectroscopy (NMR) and mass spectral data, and gas chromatography data of eight cholestane glycosides from Ornithogalum saundersiae Baker (Asparagaceae) bulbs are described. The data are linked with the article entitled “Structure and bioactivity of cholestane glycosides from the bulbs of Ornithogalum saundersiae Baker” (Chen et al., 2019). Keywords: Ornithogalum saundersiae Baker, Asparagaceae, Cholestane glycosides, Acylated steroidal glycoside