Boldine and its antioxidant or health-promoting properties

17 Pages·2005·0.18 MB·English

Boldine and its antioxidant or health-promoting properties
Peter O’Briena, Catalina Carrasco-Pozob, Herna´n Speiskyb,c,∗
aGraduateDepartmentofPharmaceuticalSciences,FacultyofPharmacy,UniversityofToronto,Toronto,Ont.,Canada
bMicronutrientsUnit,NutritionandFoodTechnologyInstitute,UniversityofChile,ElL´ıbano5524,Macul,
P.O.Box138-11,Santiago,Chile
cPharmacologicalandToxicologicalChemistryDepartment,FacultyofChemicalandPharmaceuticalSciences,
UniversityofChile,Santiago,Chile
Abstract
Theincreasingrecognitionoftheparticipationoffreeradical-mediatedoxidativeeventsintheinitiationand/orprogressionof
cardiovascular,tumoural,inflammatoryandneurodegenerativedisorders,hasgivenrisetothesearchfornewantioxidantmolecules.
Animportantsourceofsuchmoleculeshasbeenplantsforwhichthereisanethno-culturalbaseforhealthpromotion.Animportant
exampleofthisisboldo(PeumusboldusMol.),achileantreewhoseleaveshavebeentraditionallyemployedinfolkmedicineandis
nowwidelyrecognizedasaherbalremedybyanumberofpharmacopoeias.Boldoleavesarerichinseveralaporphine-likealkaloids,
ofwhichboldineisthemostabundantone.Researchconductedduringtheearly1990sledtothediscoverythatboldineisoneofthe
mostpotentnaturalantioxidants.Promptedbythelatter,alargeandincreasingnumberofstudiesemerged,whichhavefocusedon
characterizingsomeofthepharmacologicalpropertiesthatmayarisefromthefreeradical-scavengingpropertiesofboldine.The
presentreviewattemptstoexhaustivelycoveranddiscusssuchstudies,placingparticularattentiononresearchconductedduring
thelastdecade.Mechanisticaspectsandstructure–activitydataarediscussed.Thereviewencompassespharmacologicalactions,
whicharisefromitsantioxidantproperties(e.g.,cyto-protective,anti-tumourpromoting,anti-inflammatory,anti-diabeticandanti-
atherogenicactions),aswellasthosethatdonotseemtobeassociatedwithsuchactivity(e.g.,vasorelaxing,anti-trypanocidal,
immuno-andneuro-modulator,cholagogicand/orcholereticactions).Basedonthepharmacologicalandtoxicologicaldatanow
available,furtherresearchneedsandrecommendationsaresuggestedtodefinetheactualpotentialofboldineforitsuseinhumans.
Keywords: Boldine;Antioxidants;Freeradicals;Oxidativestress;Aporphine-likealkaloids;Boldo
Contents
1. Introduction................................................................................................... 2
1.1. Occurrenceandchemistryofboldine,derivativesandcongeners............................................. 2
1.2. Antioxidantactivityofboldine............................................................................ 3
1.3. Antioxidantactivityofboldineandofstructurallyrelatedsubstances:structure–activityrelationships............ 6
1.4. Pharmacologicalpropertiesofboldineandrelatedsubstancesassociatedwiththeirantioxidantactivity.......... 7
∗ Correspondingauthor.Tel.:[phone];fax:[phone].
E-mailaddress:[email protected](H.Speisky).
2 P.O’Brienetal.
1.5. Pharmacologicalpropertiesofboldinewhicharenotnecessarilyassociatedwithitsantioxidantactivity......... 11
1.6. Toxicologicalstudiesandsafetyconcernsonboldine...................................................... 12
2. Finalremarksandfutureresearchneeds........................................................................ 13
References.................................................................................................. 15
1. Introduction Mapucheverbs“weltum”(tosproutagain)or“volitum”
(to put out new roots), which may refer to this fea-
(S)-2,9-Dihydroxy-1,10-dimethoxy-aporphine (Bol- ture.Theindicationsfortheuseofboldoareextremely
dine) and other related aporphine alkaloids have been broad in range and just as unsubstantiated. According
shown to behave as potent antioxidants in a num- topharmacopoeiasandtreatisesdealingwithmedicinal
berofexperimentalmodels.Pharmacologicalactivities, plants, boldo extracts have been used for the treatment
such as cyto-protective, anti-tumour promoting, anti- ofheadache,earache,rheumatism,“nervousweakness”,
inflammatory, antipyretic and antiplatelet, have been dropsy,dyspepsia,menstrualpain,urinarytractinflam-
associatedwiththeabilityofboldinetoscavengehighly mation and has also been claimed to be a sedative and
reactive free radicals. The occurrence and chemistry mild hypnotic [1,2]. Boldo leaves contain between 0.4
of boldine and closely related congeners will first be and 0.5% of at least 17 different alkaloids belonging
briefly recalled. The present review will address and tothelargebenzylisoquinoline-derivedfamily[3].Bol-
discuss results from studies conducted mostly over the dineisthemajoralkaloidasitaccountsfor12–19%of
last decade on the antioxidant activity of boldine, and the total alkaloid content [4]. Recently, Quezada et al.
whenrelevant,ofsomestructurallyrelatedcompounds, [5]estimatedboldinecontentinboldoleavesas0.14%,
expanding into the main pharmacological properties, avalue,whichissimilartothe0.12%valuepreviously
whicharisefromsuchactivity.Inaddition,itwillattempt reportedbyus[6].Boldoleavescontaintannins,essen-
tocoverdatasupportingpharmacologicalpropertiesof tialoils(mainlyascaridoleandcineole)andflavonoids,
thesesubstances,whicharenotnecessarilyrelatedtoits ofwhichcatechinwasrecentlyreportedtobethemost
antioxidantactivity. abundantone[5,7].Sincethecontentsofcatechin,gal-
lic acid and tannic acid in boldo are much higher than
1.1. Occurrenceandchemistryofboldine, that of boldine, the former compounds appear to con-
derivativesandcongeners tributebyagreaterextenttothetotalantioxidantcapacity
ofboldoinfusions[5,7].Boldobarkcontainsunusually
Boldine (Table 1), is the major leaf and bark alka- highconcentrationsofboldine,withyieldsofupto6%
loid of the Chilean boldo tree. The boldo tree (Peu- based on dry weight [8]. Besides boldine, boldo con-
mus boldus Molina, Monimiaceae) grows abundantly tains, although in much smaller amounts, several other
in the more humid ecosystems of the Mediterranean structurally related alkaloids, amongst which glaucine,
climatic region of central Chile and extends into the an O-dimethylated form of boldine (Table 1) and the
northern half of the much rainier Chilean lake district, unsaturatedboldineanalogue6a,7-didehydroboldineare
◦ ◦
between33 and39 Southlatitude.Thename“boldo” found [2] (Fig. 1). Amongst the non-aporphinoid alka-
or “boldu” is presumably derived from the indigenous loids of boldo, the benzyltetrahydroisoquinoline (R)-
Table1
Structureofaporphines
Name 1 2 3 6 9 10 11
Boldine OCH3 OH H CH3 OH OCH3 H
Glaucine OCH3 OCH3 H CH3 OCH3 OCH3 H
Isoboldine OH OCH3 H CH3 OH OCH3 H
Bulbocapnine –O–CH2–O– H CH3 H OCH3 OH
Anonaine –O–CH2–O– H H H H H
Apomorphine H H H CH3 H OH OH
3-Bromo-boldine OCH3 OH Br CH3 OH OCH3 H
3-Iodo-boldine OCH3 OH I CH3 OH OCH3 H
N-methylglaucinium OCH3 OCH3 H (CH3)2 OCH3 OCH3 H
N-methylboldinium OCH3 OH H (CH3)2 OH OCH3 H
P.O’Brienetal.
Table2
Structureofbenzylisoquinolines
Name 2 4 5 6 7
Reticuline CH3 OCH3 OH OCH3 OH
Coclaurine H OH H OCH3 OH
Laudanosine CH3 OCH3 OCH3 OCH3 OCH3
Laudanosoline CH3 OH OH OH OH
substancesofpotentialtherapeuticutility.Ofparticular
interestaspossiblesourcesofantioxidantsweremedic-
inal plants traditionally used to treat conditions, which
are related to oxidative stress, such as rheumatism and
inflammatoryliverdiseases[12,13].Thus,thepossibil-
ities of preventing or retarding the deleterious effects
associated with excessive production of reactive oxy-
genspecies(ROS),bytheuseofpreviouslyunexplored
groups of natural products seemed attractive as a sub-
jectofresearch.Thisendeavourwasfurtherstimulated
Fig.1. 6a,7-Didehydroboldine.
bytheunsubstantiatedbeliefthattheirnaturalcharacter
reticuline(Table2)isacommonbiosyntheticprecursor wouldimplyinnocuousnessandbyincreasingevidence
of 1,2,9,10- and 1,2,10,11-tetraoxygenated aporphines. of the expression of some forms of toxicity both, after
Boldobarkhasalsoaconsiderableamountof(R)-and acuteandprolongedexposuretohighdosesofsynthetic
(S)-coclaurines (Table 2), which are common precur- antioxidants like butylated hydroxytoluene (BHT) and
sors of benzylisoquinoline-derived alkaloids. Although butylated hydroxyanisole (BHA) [14,15]. Conversely,
presentasaminorconstituent,boldinealsooccursina the ready availability of flavonoids appeared then as a
number of other species of the Monimiaceae, Magno- potentialalternativetothesyntheticcompounds.How-
liaceaeandLauraceae,alongwithmanyotheraporphi- ever,itwassoonshownthatsomeofthesenaturalsub-
noids. stances too might pose risks in terms of mutagenicity
Theanalyticaldetectionandquantificationofboldine, (vide infra) and that limitations to their in vivo use are
early relied on the use of colorimetric, paper elec- imposedbytheirloworalbioavailability,unfavourable
trophoresis,voltammetric,thinlayerandgaschromato- pharmacokineticsandunknowntoxicityconcerns[16].
graphicmethods.Basedonthetwoabsorptionmaxima Ourowninterestinboldinearosefromtheobserva-
(282and303nm)ofboldine,anHPLCmethodcoupled tionofstructuralanalogiesbetweenthisnaturalproduct
toaUVdetectorwassubsequentlydevelopedbyPiettaet and recognized antioxidant substances, namely struc-
al.[9]tobeappliedtopharmaceuticalpreparationsand turesfeaturingphenolichydroxylgroups.Suchinterest
byourlaboratory[10]toassayboldineinplasmaandin wasfurtherpromptedbythefactthatnowadaysboldine-
otherbiologicalfluids,makingitpossibletoinitiatephar- containing herbal teas are widely consumed in South
macokineticstudies.Usingthelattermethod,weshowed Americaandboldoleavesarebeingcontinuallyexported
that upon its oral administration to rats (50–75mg/kg) to some European countries for further pharmaceuti-
boldinewasrapidlyabsorbedandpreferentiallyconcen- calprocessingtoboldine-containingconcentrates.Most
tratedintheliver[11]. well-knownnaturalandsyntheticphenolicantioxidants
arestericallyhinderedphenols,sothatthefreeradicals
1.2. Antioxidantactivityofboldine generated by hydrogen abstraction from the OH group
(Fig.2,reactionB)arenotonlythermodynamically,but
Research on natural antioxidants, prompted by the alsokineticallystableor“persistent”,i.e.,theirpropen-
growinginterestinfreeradical-inducedbiologicaldam- sity to react with the biological substrates they protect
age,intensifiedinthe1980sand1990spartlyasaresult (reactionC)issubstantiallylowerthanthatofthe“par-
of the perceived need to screen endangered floras for ent”freeradical(reactionA).
4 P.O’Brienetal.
•
[23]thatboldineactsasaveryefficientHO scavenger.
Such an ability was subsequently confirmed by Jang
et al. [24] and by Youn et al. [25]. The latter investi-
gators observed that boldine also inhibited nitric oxide
production by the mitochondria. Yet, in contrast with
Fig. 2. Free radical (R•); biological substrate (L–H, i.e., lipid); the suggestion by R´ıos et al. [20], boldine was found
substrate-derived free radical (L•); phenolic antioxidant (PhOH); to either not react with superoxide radicals [23] or to
antioxidant-derivedfreeradical(PhO•).
react very poorly with these species [24]. The lack of
superoxide-scavengingpropertiesofboldinemaybein
However, it was our early observations of the very fact regarded as a possibly advantageous feature since
potentantioxidantactivityofboldineinbiological[17] suchradicalsarephysiologicallygeneratedbycellsand
andabiotic[18]systemsthatencouragedustosystem- may constitute important intra- and intercellular medi-
aticallyexploreitsactualpotentialasanantioxidantand ators of essential biological processes. Kringstein and
(cid:3)
cyto-protective agent. 2,2-Azobis[2-amidinopropane] Cederbaum[26]reportedthatboldinepreventsthenon-
(AAPH) is a water-soluble azo-compound whose ther- enzymaticperoxidationofmicrosomallipidsinitiatedby
maldecompositiongeneratesalkylperoxylradicals.We Fe2+ ortheenzyme-catalyzedperoxidationinitiatedby
showed initially that low concentrations of boldine Fe3+–ATP with NADPH or NADH as cofactors or ini-
(IC =from5×10−6 to15×10−6M)effectivelypro- tiated by CCl plus NADPH as cofactor. Interestingly,
50 4
tected red blood cell plasma membranes against the boldine was shown to prevent lipid peroxidation with-
AAPH-induced increase in antioxidant-sensitive O outinhibitingenzymes,suchasCYP450oritsreductase
2
uptake [17]. The latter parameter is considered as an or by diverting electrons away from the peroxidative
indexoftherateatwhichO reactswithpolyunsaturated process [26]. It was subsequently confirmed that bol-
2
fattyacid(PUFA)-derivedfreeradicals,propagatingthe dinecompletelyinhibitedNAD(P)H/Fe3+–ATP-induced
oxidationofmembranelipids.Noteworthy,insuchasys- humanlivermicrosomallipidperoxidation(K =5(cid:1)M)
i
temtheantioxidant(anti-propagationorchain-breaking) andCYP2E1inactivation[26].Microsomallipidperoxi-
efficiencyofboldine(K =13(cid:1)M)wasfoundtobesim- dationcausedtheinactivationofnumerousendoplasmic
i
ilar to that reported for the widely used bioflavonoid reticularenzymesincludingCYP450.Interestingly,bol-
(+)-cyanidanol-3 and about 30-fold greater than that dine also effectively prevented the occurrence of lipid
of silybin, a mixture of lignano-flavonoids available peroxidationinmicrosomalmembranesincubatedwith
fortherapeuticuseasanantioxidant[19].Boldinewas CCl in the presence of NADPH, but failed to protect
4
alsodemonstratedtoeffectivelyinhibitthespontaneous the CYP2E1 from undergoing the inactivation induced
autoxidationofbrainmembranelipids(assessedbypro- by this halogenated hydrocarbon [26]. CCl -mediated
4
ductionofthiobarbituricreactivesubstancesorTBARS, inactivationofCYP2E1may,therefore,beindependent
O uptake and chemiluminescence) with an apparent ofCCl -mediatedperoxidation.CCl maydamagepro-
2 4 4
K of about 19–30(cid:1)M [17]. Autoxidation of lipids is teinsviainitiallygeneratedelectrophilictrichloromethyl
i
•
a process that mainly reflects the overall rate of tran- (CCl ) radicals. Furthermore, CYP2E1 is indeed an
3
sition metal-dependent decomposition of lipid perox- excellentcatalystforCCl metabolismandinactivation
4
•
ides, both, initially occurring and those formed during ofCYP2E1byCCl maywellresultfromcovalentbind-
3
the incubation conditions. Shortly before, R´ıos et al. ingtotheprotein,therebyescapingtheprotectiveeffect
[20] observed that some phenolic benzylisoquinoline exertedbyboldineonotherfreeradical-mediatedevents
alkaloids inhibited Fe2+/cysteine-induced lipid perox- triggeredbyCCl •.Theabilityofboldineisnotlimited
3
idation (as TBARS production) of rat liver microso- totheinhibitionoftheperoxidationdependentonelec-
mal membranes. Later, using the same experimental tron transfer, as it also protects against the NAD(P)H-
model,theseinvestigatorsmadesimilarobservationson independent oxidation of the membranes initiated by
a broader structural range of benzylisoquinoline alka- t-butyl-hydroperoxide(t-BOOH)decomposition[23].
loids,includingboldineandglaucine[21].Subsequently, Recently, Kubinova et al. [27] reported that when
the same authors found that some of these alkaloids added to mouse liver microsomes, boldine inhibited
alsoprotectdeoxyriboseagainstdegradationinducedby CYP1A-dependent7-ethoxyresorufinO-de-ethylaseand
Fe3+–EDTAinthepresenceofhydrogenperoxide,pre- CYP3A-dependenttestosterone6-(cid:2)-hydroxylaseactiv-
•
sumably, acting as hydroxyl radical (HO ) scavengers ities(enzymeswhichbioactivatepromutagensandpro-
[22].Infact,instudiesaimedatelucidatingthemecha- carcinogens) and observed that 24h after its addition
nismoftheantioxidantactionofboldine,weestablished to a mouse hepatoma Hepa-1 cell line, it stimulated
P.O’Brienetal.
glutathione S-transferase activity. These findings sug- efficiencyinpreventingorretardingrancidity,theiruse
gest the possibility that, in addition to its free radical- as preservatives for human consumption has become
scavengingactivity,boldinecouldalsoprotectsomevital increasingly questioned on the basis of their already
cell components not only by decreasing the metabolic mentionedallegedtoxicity[14,15].Naturalantioxidants,
activation of potentially toxic xenobiotics but also by inturn,haveenjoyedanincreasingrecognitionandpop-
increasing their removal. More studies are needed, ularity during the last decade. The industrial use of
however, to assess the actual chemoprotective poten- naturalantioxidantshasremainedlimited,however,pos-
tial of boldine to act intracellularly through apparently sibly due to their often higher cost and their relatively
antioxidant-independentmechanisms. loweractivity.Regardingthefoodpreservativepotential
Although many substances have been found to be of boldine, studies conducted by us revealed that it is
effective inhibitors of microsomal lipid peroxidation, particularly efficient in protecting PUFA. Boldine was
somefeaturesoftheantioxidantactivityofboldinemay foundtoprotectfishoilagainstspontaneousshort-and
distinguishitfromothernaturalorsyntheticantioxidants long-termO -dependentthermalperoxidation,actingat
2
commonlyusedinfoodsorasreferencecompounds.In concentrations markedly lower than those required by
◦
fact,unlikeagents,suchaspropylgallateorphyticacid, otherantioxidants.Infishoil,long-term(36days,25 C)
whichchelateironandenhanceitsautoxidation,boldine oxidation,boldinedisplaysanactivitysimilartothatof
wasshowntoinhibitiron-mediatedperoxidationatcon- quercetinandatwotothreetimesgreateractivitythan
centrations10timeslowerthanthatoftheironcatalyst (cid:3)-tocopherol,BHAorBHT[18].Boldinealsoshoweda
[23]. remarkablygreaterpotencythanthelattertestedantioxi-
Inadditiontolipids,proteinshavebeenincreasingly dantsinprotectingfishoilagainstFe2+-inducedrancidity
recognized as major biological targets of free radicals. [18].Morerecently,itwasalsofoundtobemoreefficient
In fact, their oxidative damage is regarded of impor- thanquercetinandBHTinprotectingbullfrogoilagainst
tance in the aging process and in the ethiogenesis of heat-inducedoxidation[30]andstabilizingn-3-PUFAof
cataracts,atherosclerosisandseveralinflammatorydis- sardineoilagainstthermaloxidationwithanefficiency
eases. When enzymes are targeted, the oxidative mod- greater than that of BHA, BHT and (cid:3)-tocopherol, but
ification is generally evidenced as carbonyl group for- similartothatofquercetin[31].
mationattheprotein’sstructureandisoftenassociated Undermostconditionsalkoxyl-andperoxyl-freerad-
with a loss of catalytic activity. Lysozyme is particu- icals play a crucial role in the initiation of heat- and
larly, susceptible to peroxyl radical-mediated inactiva- metal-induced oxidation of abiotic substrates. How-
tion.Inearlystudies,wedemonstratedtheeffectiveness ever, in the presence of UV radiation, singlet oxygen
of boldine at protecting this enzyme against inactiva- [O (1(cid:1) )] is a major initiating species in foodstuff
2 g
tioninducedbyAAPH-derivedperoxylradicals[17,28]. deterioration. Boldine-containing boldo infusions were
More recently, we showed that such effect is closely reported to effectively protect tryptophan molecules
associated with a boldine-mediated protection against from undergoing O (1(cid:1) )-dependent oxidation [32].
2 g
theoxidationofcertaintryptophanresiduesbyperoxyl Subsequently,Zanoccoetal.[33]foundthatboldineand
radicals,whichcausedlysozymeinactivation[29].Inter- glaucinewereexcellentquenchersofexcitedO ,partic-
2
estingly, the enzyme inactivation and tryptophan loss ularly,inpolarsolvents.Kineticstudiesofthereactions
preventedbyboldinewerefoundtobemechanistically between these aporphines and O (1(cid:1) ) suggest that a
2 g
dissociablefromtheAAPH-inducedcarbonylgroupfor- chargetransfercomplexbeformedinitiallybetweenthe
mation.Thus,itwouldseemthattheantioxidantproper- aromatic ring and O and that such complex can later
2
tiesofboldinecompriseaprotection,notonlyoflipids eithertransfertheelectronback,givingground-stateO
2
but also of protein targets, which, at least in the case and the alkaloids or proceed to unidentified products
oflysozyme,translatesintopreventingtheoxidationof [33]. Thus, the ability of boldine (and possibly other
certain amino acids involved in the enzyme’s catalytic aporphines) to quench O (1(cid:1) ) may also contribute to
2 g
activity. protectfoodstuffsagainstoxidativedeterioration.
Inadditiontotheapparentadvantagesofboldineover Intensive ultraviolet radiation, in addition to affect-
otherantioxidantsinbiologicalsystems,thisalkaloidhas ing foodstuff, can also promote harmful effects onto
emergedasalsodisplayingpotentiallyusefulproperties the skin of humans. UV light may cause a mild skin
in abiotic systems. Most oil- and fat-containing foods burn, skin cell DNA damage and premature skin age-
undergo oxidative rancidity in the absence of added ingpossiblyresultinginskincancer.Sincefreeradicals
antioxidants,bothduringandafterfoodstuffpreparation. are recognized to participate in either the aetiology or
Although synthetic antioxidants have a demonstrated thedevelopmentofmostUV-inducedskinlesions[34],
6 P.O’Brienetal.
substances displaying free radical-scavenging capacity of an ortho-semiquinone anion. However, the observa-
havestemmedaspotentiallyinterestingphoto-protective tionthatglaucine,theO-dimethylatedboldinederivative,
orphoto-preventiveagents.Interestingly,inadditionto retains most of the parent molecule’s activity suggests
itsantioxidantproperties,theboldinemoleculehastwo that the presence of hydroxyl groups can add to, but
majorabsorptionpeaks,at280and302nm[9,10].The is not essential for conserving the antioxidant activity
latterwouldconferboldineaUVlight-filteringproperty oftheseaporphineanalogues.Usingtheauto-oxidation
relevant to a photo-protective action. In fact, Hidalgo ofbrainhomogenates,Casselsetal.[28]confirmedthe
et al. [35] showed boldine to be photo-unstable when apomorphine molecule as the most active antioxidant
irradiatedatwavelengthsupto300nmandtodisplaya amongstthetestedaporphines,buttheactivityofboldine
photo-protector effect against UV-B both in vitro and andglaucinesuggeststhatO-methylationcausesonlya
in vivo in mice. The photo-protection was evidenced marginal decrease of activity. A slightly lower activity
through the prevention of the UV-induced increase in ofglaucinerelativetoboldinewasreportedbyUbedaet
skin temperature of the rodents. More recently, Ran- al. [22] using the Fe3+–EDTA–H O -induced deoxyri-
2 2
canetal.[36]investigatedthephoto-filteringproperties bose degradation assay (hydroxyl radical-mediated).
of boldine in humans. These authors observed that the Glaucine had only half of the activity of boldine using
applicationofboldine(25mM)ontoa12cm2areaofthe the Fe2+/H O /ascorbic acid-induced liver microsomal
2 2
backofvolunteersprotectedtheirskinagainsterythema lipidperoxidationassay(hydroxylradical-mediated)and
formationtoanextentslightlylowerthanthatofacom- theAAPH-inducedlossoflysozymeactivityassay[37].
mercial sun cream for which a UV-protection factor of The latter authors studied also the effect of halogena-
5wasinformed.Inthesamestudy,itwasobservedthat tion(withbromooriodine)ofboldineattheC-3posi-
theinvitroirradiationofhumanTlymphocytesthrough tion(Table1)andobservedthatsuchmodificationdoes
a thin boldine-containing solution protected these cells notaffectlyzozymeprotection,butitalmostdoublethe
againstlossofviabilitywithapotencyevengreaterthan antioxidanteffectivenessonthemicrosomallipidperox-
that shown by octylmethoxycinnamate, a UV-B refer- idationassay.Theresultsseeninthelatterassayarein
encefilter. linewiththerecognizedimportanceofthelipophilicity
inchain-breakinglipidperoxidationwithinmembranes.
1.3. Antioxidantactivityofboldineandof Ontheearlystructure–activityresearchbyMart´ınez
structurallyrelatedsubstances:structure–activity et al. [21], it was suggested that, in the absence
relationships of phenolic groups, aporphines could be easily oxi-
dized to dehydro- (actually 6a,7-didehydro) and oxo-
After the initial observations by R´ıos et al. [20] (infact,6-nor-7-oxo-4,5,6,6a-tetradehydro)aporphines.
that some phenolic benzylisoquinolines could be able Thus, based on the latter, Cassels et al. [28] suggested
to interfere peroxidative processes, several studies that in non-phenolic aporphines, the benzylic C–6a–H
addressedthefreeradical-scavengingand/orantioxidant bond may be the initial point of free radical attack
properties of a larger number of isoquinoline-related and that the neighbouring nitrogen lone pair would
alkaloids. Using a chemically induced (Fe2+–cysteine) stronglycontributetothestabilizationoftheintermedi-
microsomallipidperoxidationassay,Mart´ınezetal.[21] ateradicalbyfurtherextendingelectrondelocalization.
found that, amongst several aporphines tested, boldine Supporting the latter, Cassels et al. [28] showed that
(IC =20(cid:1)M) displayed almost twice as much activ- compared to glaucine, N-methylglaucinium (Table 1),
50
ity as isoboldine (Table 1), suggesting the importance an analogue in which the nitrogen is protonated, is
of the presence of a hydroxyl group in the C1 position completely devoid of activity (in the lysozyme activ-
ofaporphinestructures.Similarly,thegreateractivityof ity protection assay). Milia´n et al. [38] reported that
theaporphinebulbocapninerelativetoanonaine(IC of N-methylboldinium (Table 1) is only half as active as
50
12.5(cid:1)Mversus27(cid:1)M)couldbeexplainedintermsof boldineinscavengingreactiveoxygenspeciesgenerated
the importance of the presence of a hydroxyl group in by a hypoxanthine–xanthine oxidase system. Thus, the
position the C11 (Table 1). The same authors reported stabilization provided by the nitrogen lone pair would
that the dihydroxy-containing apomorphine molecule be essential for conserving the free radical-scavenging
(Table1)exhibitedthehighestactivity(IC =3.3(cid:1)M), activityseeninboth,thephenolicaswellasinthenon-
50
suggestingthatthepresenceofacatecholwithhydroxyl phenolic aporphines. With regard to the importance of
groups at the C10 and C11 positions would be an the piperidine ring in the aporphine structure, it seems
important feature towards the high activity displayed that, at least for boldine, the presence of such ring
by most aporphines, possibly endowing the formation would not be essential for manifesting such activity.
P.O’Brienetal.
Table3
Structureofphenanthrene
Name 1 3 7
N-allylsecoboldine CH2–CH CH2 OH OH
Secoboldine H OH OH
N-methylsecoboldine CH3 OH OH
N-methylsecoboldinium (CH3)2 OH OH
O-dimethylated-N-methylsecoboldinium (CH3)2 OCH3 OCH3
In fact, N-allylsecoboldine (Table 3), a phenanthrenic zylisoquinolines. Noteworthy, laudanosoline, despite
structural analogue of boldine in which the nitrogen presentingtwocathecols,showsonlyhalftheactivityof
atom is part of a side chain (N-allyl derivated), has apormorphine,whichexhibitsonlyoneofsuchmoieties.
beenreportedtoexhibitinterestingantioxidantsproper- About the latter, Cassels et al. [28] postulated that the
ties[39].Likeboldine[17,23],N-allylsecoboldinescav- superiorabilityofaporphinestotrapfreeradicalsisasso-
engeshydroxylandperoxylradicalsandefficiently,pre- ciatedwithanincreasedspindelocalizationofphenoxy
vents lipid peroxidation in rat brain homogenates and radicals in the biphenyl system. The resulting benzylic
erythrocyte membranes. Although, there are no stud- freeradicals,byanalogywithphenoxyradicals,would
ies aimed at addressing the relative activity of boldine presumably be better stabilized in aporphines, as com-
andN-allylsecoboldine,Milia´netal.[38]comparedthe paredtotheirbenzylisoquinolines-derivedcounterparts,
ability of aporphines with that of the corresponding byextendedconjugationacrosstheaporphinebiphenyl
equivalents with a phenanthrene skeleton for scaveng- system.
ingROSgeneratedbyhypoxanthine–xanthineoxidase.
Theseinvestigatorsfoundthatboldineislessactivethan 1.4. Pharmacologicalpropertiesofboldineand
anyofthetestedphenolic(C3–OHandC7–OH)phenan- relatedsubstancesassociatedwiththeirantioxidant
threne analogues: secoboldine, N-methylsecoboldine activity
and N-methylsecoboldinium (N-dimethylsecoboldine)
(Table 3). The higher activity reported for phenolic- Herewereviewevidenceshowingtheabilityofbol-
phenanthrenicrelativetophenolic-aporphinicmolecules dinetolimitexperimentallyinducedprocessesrelevant
may reside on the existence of a third benzylic ring topathophysiologicalconditionsinwhichfreeradicals
which may confer a greater capacity to delocalize the and/orperoxidationproductsareinvolvedasintracellu-
phenoxy free radical to the former. Nonetheless, the lardamagemediators.
samegroupobservedthatboldinewasstillmoreactive Compelling in vitro and in vivo evidence impli-
thanthenon-phenolicphenanthrene,O-dimethylated-N- cates free radicals as major initiators and/or mediators
methylsecoboldinium (Table 3) and that the presence of biochemical events leading to cell damage [13,40].
offreeHOgroupswasanabsoluterequirementforthe Stemming from the free radical-scavenging ability of
activityofphenanthrenederivatives. boldine, the pursuit of its pharmacological potential
Ontheotherhand,instructure–activitystudiescon- has included establishing its effectiveness in protect-
ductedonbenzylisoquinolinealkaloids,Mart´ınezetal. ing cells against oxidative and lytic damage. Erythro-
[21] found that reticuline and laudanosine (analogues cytes are particularly susceptible to undergo oxidative
oftheaporphinesisoboldineandglaucine,respectively) stress. AAPH has been shown to induce in intact iso-
(Table 2), showed very poor activity. However, lau- latederythrocytesextensiveperoxidationofmembrane
danosoline(Table2),abicathecolmolecule,wasfound components (e.g., lipids and proteins) and to promote
to exhibit the greatest activity amongst all tested ben- structural alterations, which lead to the rupturing the
zylisoquinolines (IC50=6.8(cid:1)M). Thus, it appears that erythrocytemembraneandtothesubsequentleakageof
O-methylation substantially affects the activity of ben- hemoglobin [41]. Using a rat erythrocyte suspension,
zylisoquinolines. The presence of at least one or more we showed that boldine cytoprotects against cell lysis
catecholsisacommonfeatureamongstthemostactive induced by AAPH [42]. Boldine prevented, time- and
moleculeswithinboth,thetheaporphinesandtheben- concentration-dependently,theAAPH-inducedleakage
8 P.O’Brienetal.
of hemoglobin into the medium. Such cytoprotection inthecontrolmice[37],itsfailuretopromoteneuropro-
wasobservedwhethertheantioxidantwasaddedtothe tection against MPTP reflects that the concentration of
erythrocyte suspension 1h prior to or simultaneously boldineatthestriatummightbeenoughtomodifycate-
withtheazo-compound,suggestingthatboldinewasnei- cholaminemetabolismbutnottocounteracttheoxidative
thermetabolizednoroxidizedduringsuchpreincubation stringencyinducedbyMPP+atsuchsite.Futurestudies
period[42]. aimedatassessingtheneuroprotectivepotentialofbol-
Themembranepermeabilitytransitionofmitochon- dineshouldestablishitspropertytoreactwithMPP+in
driaisknownasacentraleventinthecourseofavariety relevantinvitromodelsandshouldaddressinvivothe
of toxic and oxidative forms of cell injury as well as abilityofboldinetocrosstheblood–brainbarrier.
apoptosis induced by xenobiotics or drugs [43]. Open- EarlyworkbyLanhersetal.[44]showedthatmicro-
ing of the mitochondrial permeability transition pore molar concentrations of boldine protected isolated rat
has been shown to induce depolarization of the trans- hepatocytesagainstdamageinducedby t-BOOH.Sub-
membranepotential,releaseofsmallsolutes,releaseof sequently, they showed that lower concentrations of
Ca2+ and cytochrome c, osmotic swelling and loss of boldinewashighlyeffectiveinprotectingratlivermicro-
oxidative phosphorylation. The oxidation of dopamine somal membranes against lipid peroxidation induced
(DA) and 6-hydroxy-dopamine (6-OH-DA) in neurons by t-BOOH [23]. Boldine, however, did not prevent
induces free radical formation, inhibition of mitochon- theearly(within60s)andsuddendecline(by50%)of
drialrespiratorychainandopeningofthemitochondrial reduced glutathione (GSH) and the equivalent increase
transitionpore,whichisinhibitedbyoxidantscavengers. inoxidizedglutathione(GSSG)[45]attributedtoaGSH-
Based on the latter, Youn et al. [25] examined the pro- peroxidase-catalyzedreactionwitht-BOOHratherthan
tective effect of boldine on catecholamine (DA and 6- anon-enzymaticreactionwitht-BOOH-derivedfreerad-
OHDA)-inducedbrainmitochondriadysfunctionandon icals.Infact,thelevelsofbothGSHandGSSGrecovered
PC12celldeath.Atconcentrations(10–100(cid:1)M),which tonear-basallevelsafter20min,eitherinthepresenceor
did not affect mitochondrial permeability, boldine sub- absenceofboldine,indicatingthattheprotectiveactions
stantiallydecreasedtheeffectofcatecholamine(CA)on ofboldinedonotdependonthepreventionofchanges
mitochondria swelling and attenuated the alteration of in GSH/GSSG concentrations. On the other hand, the
mitochondrialmembranepotential.Atthesameconcen- delayedadditionofboldine(10or20minafter)tocells
trations,boldineattenuatedtheCA-induceddecreasein incubatedwitht-BOOH,whileeffectivelyblockingany
thioredoxinreductaseactivityandincreaseinthioloxi- furtherincreaseinTBARS,totallyfailedtopreventthe
dationandat100(cid:1)Malsodecreasedthemitochondrial subsequent peroxide-induced loss of cell viability, and
release of cytochrome c. Consistent with such actions, infact,wasassociatedwithincreasedcelldeath.Thus,
Younetal.[25]demonstratedthatboldine(10–100(cid:1)M) itispossiblethattheearlyincreaseinlipidperoxidation
decreasedDA-induceddeathinPC12cells(asassessed productsissufficienttoeithersignalorassurethecontin-
bytheMTTassayandcaspase-3activity).Theauthors uance of other events, whether mechanistically related
[25]concludedthat,sinceboldineeffectivelyreactswith or not, leading to cell death. Conversely, the preincu-
•
HO radicals,itsabilitytoattenuateCA-induceddamage bation of hepatocytes with boldine during 150min, at
of brain mitochondria and to decrease the DA-induced which time no boldine could be detected either intra-
apoptosis and cell death would reside on its radical- orextracellularly,preventedlipidperoxidationandwas
scavengingproperties.Whiletheinvitroobservationsby as effective at protecting cells against the subsequent
Younetal.[25]suggestapotentialforboldineasneuro- addition of t-BOOH as was the simultaneous addition
protectiveagent,subsequentworkbyLoghinetal.[37] of boldine and t-BOOH to cells preincubated during
conductedinvivoofferednosupportforsuchcontention. 150minundercontrolconditions.Onthebasisofsuch
Thus,wheninjected(s.c.)inmiceatadoseof40mg/kg, observations,wesuggestthatearlyexposuretoboldine
boldinewasfoundtobeineffectiveinprotectingagainst may trigger events resulting in cytoprotection, perhaps
the neurotoxic action of 1-methyl-4-phenyl-1,2,3,6- involving the formation of boldine-derived metabolites
tetrahydropyridine (MPTP) [37]. The latter compound withsimilarantioxidantproperties[45].
generatesindopaminergicneuronsanactivemetabolite In addition to t-BOOH, halogenated hydrocarbons
(1-methyl-4-phenyl-pyridinium,MPP+),whichinhibits have been widely used as paradigmatic hepatotoxic
themitochondrialelectrontransportchainandinducesa agents [46]. Using carbon tetrachloride as experimen-
syndromecloselyresemblingParkinson’sdisease.Since tal hepatotoxin in vivo, boldine has also been evalu-
atthesamedosing,boldinewaseffectivetoincreasestri- ated as a cytoprotectant. Work conducted by Lanhers
atallevelsofDOPACandHVAandtheHVA/DAratio etal.[44]showedthatboldinepreventedCCl -induced
4
P.O’Brienetal.
hepatitis in mice. The latter authors reported that bol- tive species are also produced in substantial amounts
dine,giveni.p.atadoseof10mg/kgpartiallyprotected during inflammatory phenomena in association with
miceagainsthepatotoxicity(asassessedbyanincrease leucocytes infiltration [40]. Consequently, antioxidant
in plasma GPT) when administered 30min before the molecules, which may interfere with ROS generation
halocarbon.Thefactthattheprotectionaffordedbybol- may also display anti-inflammatory properties. Using
dinewasonlypartial(lessthan50%)maybeinterpreted the carrageenan-induced paw oedema assay, Lanhers
as an indication that the dose of boldine used by Lan- et al. [44] showed that the administration of a puri-
hersetal.[44]wasinsufficient,eventhoughthedoseof fiedboldoleafextracttoratshadananti-inflammatory
CCl usedinthisstudy(0.03mL/kg)wasfarbelowthat effect that they could not reproduce when boldine was
4
employedbyotherinvestigators[46].Thepartialprotec- administered(10or20mg/kg;i.p.)instead.Subsequent
tiveeffectofboldinemayrelatetotheinvitroobservation work by us [47], however, demonstrated that boldine
byKringsteinandCederbaum[26]thatboldineprotects is very effective (ED =34mg/kg; p.o.) in reducing
50
livermicrosomalmembranesagainstCCl -inducedlipid carrageenan-inducedpawoedemainguineapigsandin
4
peroxidationbutfailstoprotectagainstCYP2E1destruc- preventing (60mg/kg; p.o.) the increase in rectal tem-
tion.Presumably,beforethecytochromeinactivationis perature seen in rabbits treated with bacterial pyrogen.
•
completed,CCI radicalsunderformationbindscova- Theseanti-inflammatoryandantipyreticeffectsofbol-
3
lentlytoanarrayofmacromoleculeswhosefunctionsare dineweremechanisticallysupportedbytheobservation
metabolically key in defining the hepatocyte integrity. thatuponitsadditiontoarataortalringpreparation,bol-
In the latter framework, a partial degree of cytoprotec- dineeffectivelyinhibitedprostaglandinbiosynthesis(as
tion would be expected. On the other hand, it is also 6-keto-PGF). The exact mechanisms by which boldine
necessarytoconsiderthefactthatforanantioxidantto inhibitsPGsynthesisandtherelationshipbetweensuch
protect against tissue damage induced by a (peroxidiz- actionanditsanti-inflammatoryandantipyreticeffects
ing) xenobiotic it is necessary that both substances (or remainstobeinvestigated.
thoseunderlyingtheiraction)shouldbepresentsimul- On the other hand, enhanced generation of reactive
taneously,forenoughtimeandatthesamecellularand oxygenandnitrogenmetabolitesarealsoimplicatedin
subcellularlocus.Itisalsoarequirementthattheantiox- tissue injury observed in several inflammatory bowel
idant be present at its site of action in concentrations diseases(IBD).Thus,antioxidantscouldfunctioname-
relevant to its cyto-protective action. Such conditions liorating and/or preventing the inflammation and cyto-
can often be met in vitro, but achieving them in vivo toxicityseeninexperimentalIBDmodels.Basedonthe
is much more complex. Pharmacokinetic studies con- antioxidantandanti-inflammatorypropertiesofboldine
ducted by us on tissue boldine concentrations suggest [44,47] Gotteland et al. [48] studied in vivo the effect
that for the doses used in the above-referred hepato- of boldine in a rat model of IBD in which the colonic
protection experiments, boldine might be expected to damagewasinducedbytheintrarectaladministrationof
haveattainpotentiallyeffectiveantioxidative(andpos- aceticacid.Theseinvestigatorsfoundthatboldine,given
sibly cyto-protective) concentrations in the liver [11]. as a single dose (100mg/kg) intrarectally and 30min
However, it must be kept in mind that the clearance before acetic acid, prevented oedema formation and
rate of boldine from plasma is extremely high. From affordedsubstantialprotectionagainstthemacroscopic
our kinetic studies, the half-life of boldine in plasma and histologic injury. The mucosa of boldine-treated
was estimated to be around 30min. Thus, in order to animalsexhibitedasubstantialreductioninmyeloperox-
ensure the continued presence of boldine during the idaseactivity,suggestingthattheantioxidantloweredthe
first8–12hpost-administrationofthehepatotoxicagent, extent of neutrophils infiltration into the acid-exposed
period during which the serum enzyme levels had not tissue. Noteworthy, recently Milia´n et al. [38] reported
increased significantly but some damaging intracellu- thatlowmicromolarconcentrationofboldinearehighly
lar events had been presumably already triggered, it effectiveinpreventingROSproductionbyhumanPMNs
would probably be necessary to have the antioxidant inducedbythebacterialchemotacticpeptide,N-formyl-
administeredrepeatedly.Futureassessmentsofnewbol- methionylleucyl-phenylalanine(f-MLP).Thus,theanti-
dineanalogues—withstructuralmodificationsleadingto inflammatory effect of boldine seen in the IBD model
longer half-lives—might merit a more detailed in vivo [48],couldrelatetoantioxidantactionsthatwork,indi-
evaluationoftheirpotentialhepatoprotectiveusefulness. rectlyvialoweringthepresenceofROS-producingcells
ROS are involved in the cycloxygenase- and in the affected tissue and directly, via decreasing ROS
lipoxygenase-mediated conversion of arachidonic acid productionbythesamecells.Futureassessmentsofthe
intopro-inflammatoryintermediatesandthatthesereac- potentialusefulnessofboldineinIBD,shouldconsider
10 P.O’Brienetal.
otherexperimentalmodelsofchronicinflammationand obtainedfromLDL-deficientmicefedsimultaneouslya
usingalsootherroutesofboldineadministration. highfatatherogenicdietandboldine(p.o.1or5mg/day)
Evidencesupportingthehypothesisthatanincreased during 12 weeks. Whole plasma lipids isolated from
oxidativecouldbeinvolvedinthepathogenesisandpro- boldine-fed (LDLR−/−) mice were considerably less
gression of diabetic tissue damage has prompted the susceptible to in vitro oxidation compared to that of
experimental and clinical evaluation of the potential controlanimals.Onthesameinvivoatherogenicstudy
of antioxidants in the prevention and/or treatment of protocol, boldine was shown to significantly decrease
diabetes. In the single study available addressing the theareasoftheaorticatheroscleroticlesions,by22and
usefulness of boldine as anti-diabetic agent, Jang et al. 44% for the 1 and 5mg/day 12-week dosing, respec-
[24]investigatedtheeffectofboldineinpreventingthe tively. The study by Santanam et al. [50] shows that
damage induced by streptozotocin (STZ) in rats. The repeatedly administered, boldine effectively protects
administrationofSTZtoratsinducesdamagetopancre- LDLR−/−micefromdevelopingatheroscleroticlesions.
aticbetacellsandresultsindiabetesbymechanismsthat Sinceboldineexertedsucheffectwithoutalteringplasma
arenotclearlyunderstood,butthatinvolveanincreased cholesterol,triglycerides,LDLandHDLlevels,itsanti-
formationofhydroxylradicalsandotherROSinassocia- atherogeniceffectsmaywellbeattributedtoitsantioxi-
tionwiththepriorgenerationand/orsubsequentdecayof dantproperties.
highlyreactiveSTZcarboniumradicals[49].Oxidative Plateletactivationseeninhypercholesterolemicindi-
alterations within the mitochondria, believed to con- viduals is often associated with the synthesis of pro-
tributetothedysfunctionsseenindiabetes,arepartially inflammatorycytokinesandwiththereleaseofamyriad
replicated in the STZ-induced diabetes model [49]. As ofchemokines,whichcontributetothedevelopmentand
reportedbyJangetal.[24],boldine(givenatadoseof progression of atherosclerotic plaques. In the context
100mg/(kgday)indrinkingwaterduring8weeks)sig- of testing the potential antiplatelet usefulness of apor-
nificantlypreventedtheelevationofbloodglucoselevels phineantioxidants,Tengetal.[51]foundthatinplatelet
andtheweightlossinducedbySTZandattenuatedthe obtainedfromrabbits,boldineinhibitedtheaggregation
elevationofcarbonylsandmalondialdehydelevelsseen induced by arachidonic acid and collagen, but not that
inpancreasmitochondria.Theantioxidanteffectsofbol- inducedbyplatelet-activatingfactor(PAF),thrombinor
dine, were not evident, however, for carbonyls in liver athromboxaneanalogueU46619.Theyconcludedthat
mitochondrianorformalondialdehydeinmitochondria the antiplatelet effect of boldine mainly resulted from
from kidney. Interestingly, Jang et al. [24] observed itsinhibitionofthromboxaneA formationfromarachi-
2
that in vitro boldine inhibited ROS production by iso- donicacid.Itremainstobeestablishedwhetherthisin
lated liver mitochondria when treated with antimycin vitro inhibitory platelet aggregation ability of boldine
c, a respiratory inhibitor. In vivo boldine normalised also contributed to its in vivo anti-atherogenic proper-
the elevated Mn–SOD and GSH-peroxidase activity in tiesrecentlyshownbySantanametal.[50].
mitochondria of the pancreas of STZ-treated animals. Oxidative stress is also implicated in the molec-
These results suggest that boldine, by suppressing the ular mechanism of tumour promotion by inducing a
oxidativestressexistinginthepancreasofSTZ-treated down-regulation of cellular gap junctions (GJIC). The
animals,opensuptheexplorationoftheuseofboldine latter structures play a key role in the control of cell
anditsantioxidant-relatedanaloguesinthetreatmentof growth, development and differentiation. The down-
diabetes-associatedfreeradicaloverproduction. regulationofGJICinducedbysubstances,suchas12-O-
Atherosclerosis involves three processes, oxidation, tetradecanoylphorbol-13-acetate(TPA),occursinasso-
inflammationandhypercholesterolemia.Inviewofthe ciationwithanincreasedproductionofoxidantswithin
previously established antioxidant [17,23,28,42] and cells.Sincethelattercanleadtoarapidclonalexpansion
anti-inflammatory[47]propertiesofboldine,Santanam ofinitiatedcells,invitromodelsofgapjunctionalfunc-
et al. [50] studied the in vitro effect of boldine on the tioncanbeusedtoscreenthepotentialanti-tumourpro-
oxidizability of human LDL (assessed by diene con- motingpropertiesofanantioxidant.Huetal.[52]tested
jugate formation) and its ex vivo effect on the oxidiz- theefficaciesofboldineanditsO-dimethylatedglaucine
abilityofwholeplasmaobtainedfromboldine-fedand toinhibittheTPA-induceddown-regulationofGJICin
control LDL receptor-knockout (LDLR−/−) mice. In WB-F344 rat liver epithelial cells. These investigators
vitro, boldine inhibited human LDL oxidation induced showed that the co-incubation of these cells with TPA
by copper (5(cid:1)M) in a concentration-dependent man- inthepresenceoftheseaporphinespreventedthedown-
ner(0.5–2.5(cid:1)M).Theauthorsalsoinvestigatedcopper- regulation of GJIC in a concentration-dependent man-
induced diene conjugate formation ex vivo in plasma ner.At50(cid:1)Mconcentrationeach,boldineandglaucine