Research Article Monoester-Diterpene Aconitum Alkaloid Metabolism in Human Liver Microsomes ...

25 Pages·2015·2.25 MB·English

HindawiPublishingCorporation
Evidence-BasedComplementaryandAlternativeMedicine
Volume2013,ArticleID941093,24pages
http://dx.doi.org/10.1155/2013/941093
Research Article
Aconitum
Monoester-Diterpene Alkaloid Metabolism in Human
Liver Microsomes: Predominant Role of CYP3A4 and CYP3A5
LingYe,1Xiao-ShanYang,1Lin-linLu,1Wei-YingChen,1ShanZeng,1Tong-MengYan,1
Ling-NaDong,1Xiao-JuanPeng,1JianShi,1andZhong-QiuLiu1,2
1DepartmentofPharmaceutics,SchoolofPharmaceuticalSciences,SouthernMedicalUniversity,Guangzhou,
Guangdong510515,China
2InternationalInstituteforTranslationalChineseMedicine,GuangzhouUniversityofChineseMedicine,Guangzhou,
Guangdong510006,China
CorrespondenceshouldbeaddressedtoZhong-QiuLiu;[email protected]
Received26March2013;Revised23May2013;Accepted23May2013
AcademicEditor:GerhardLitscher
Copyright©2013LingYeetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Aconitum,widelyusedtotreatrheumatoidarthritisforthousandsofyears,isatoxicherbthatcanfrequentlycausefatalcardiac
poisoning.Aconitumtoxicitycouldbedecreasedbyproperlyhydrolyzingdiester-diterpenealkaloidsintomonoester-diterpene
alkaloids.Monoester-diterpenealkaloids,includingbenzoylaconine(BAC),benzoylmesaconine(BMA),andbenzoylhypaconine
(BHA), are the primary active and toxic constituents of processed Aconitum. Cytochrome P450 (CYP) enzymes protect the
human body by functioning as the defense line that limits the invasion of toxicants. Our purposes were to identify the CYP
metabolites of BAC, BMA, and BHA in human liver microsomes and to distinguish which isozymes are responsible for their
metabolismthroughtheuseofchemicalinhibitors,monoclonalantibodies,andcDNA-expressedCYPenzyme.High-resolution
massspectrometrywasusedtocharacterizethemetabolites.Atotalof7,8,and9metabolitesweredetectedforBAC,BMA,andBHA,
respectively.Themainmetabolicpathwaysweredemethylation,dehydrogenation,demethylation-dehydrogenation,hydroxylation
anddidemethylation,whichproducedlesstoxicmetabolitesbydecomposingthegroupresponsibleforthetoxicityoftheparent
compound. Taken together, the results of the chemical inhibitors,monoclonal antibodies, and cDNA-expressed CYPenzymes
experimentsdemonstratedthatCYP3A4andCYP3A5haveessentialfunctionsinthemetabolismofBAC,BMA,andBHA.
1.Introduction So the death of patients suffering from Aconitum toxicities
always is cardiac arrest or respiratory insufficiency. Up to
Aconitum has been used as an essential drug in China and now,thereisnoeffectiveantidotetoAconitumintoxication.
in other East Asian countries for centuries owing to its The initial toxic signs are gastrointestinal including nausea,
extremely excellent effect against rheumatosis and rheuma- vomiting, and diarrhea. This is followed by a sensation of
toid arthritis [1, 2]. It was reported that among 500 known burning,tingling,andnumbnessinthemouthandface,and
prescriptions,thefrequencyofuseoftheAconitumis13.20%, ofburningintheabdomen[9,10].
ranked 9 [3]. Nevertheless, Aconitum is a toxic herb that Therefore,preventionofAconitumtoxicityisakeyissue
can cause fatal cardiac poisoning [4–6] and is reportedly for its safe application. Efflux transporters and metabolic
involvedinsuicidalandhomicidalattempts[6,7].Sincethe enzymes act as the human body’s first line of protection
therapeuticdoseofAconitumisveryclosetoitstoxicdose, by limiting the invasion of toxicants [11]. Investigation on
its poisoning incidents are not uncommon. According to the roles of efflux transporters and metabolic enzymes in
the literature data, in 188 cases of patients with Aconitum, Aconitumismeaningful.
111 patients had varying degrees of toxic reactions [3]. The Aconitum contains high amounts of three highly toxic
toxicological mechanism of Aconitum is widely recognized diester-diterpenealkaloids(DDAs),namely,aconitine(AC),
+
to be associated with voltage-dependent Na channels [8]. mesaconitine (MA), and hypaconitine (HA) [12]. Selling
2 Evidence-BasedComplementaryandAlternativeMedicine
unprocessedAconitumisforbiddenontheChinesemarket. 2.MaterialsandMethods
Withproperprocessing,AC,MA,andHAbecomeexposed
to abundant hydrolysis, transforming them into less toxic 2.1.Materials. BAC,BMA,BHA,andtestosterone(asinter-
monoester-diterpenealkaloids(MDAs),namely,benzoylaco- nalstandard)(purity>98%)werepurchasedfromNanjing
nine (BAC), benzoylmesaconine (BMA), and benzoylhypa- Zelang Medical Technology Co., Ltd. (Nanjing, China).
conine(BHA)[12]. Pooledhumanlivermicrosomes(HLMs,20mg/mL,includ-
ing reductase), 𝛽-nicotinamide adenine dinucleotide phos-
MDAs are the primary active and toxic constituents of
phate (NADP), glucose-6-phosphate (6-P-G), and glucose-
processedAconitum.ThealkaloidcontentinprocessedAconi-
tumisreportedlyasfollows:BMA > HA > BAC > MA > 6-phosphate dehydrogenase (PDH) were purchased from
BD Gentest Corp. (Woburn, MA, USA). cDNA-expressed
AC[13].AsidefromDDAs,MDAsaredetectableinhuman
CYPenzymes(CYP1A2,2C8,2C9,2C19,2D6,2E1,3A4,and
serum and urine after oral intake of Aconitum extract [14].
3A5), chemical inhibitors (fluvoxamine maleate, quercetin,
Moreover,MDAsareevidentintheplasma,liver,andkidney
amiodarone, omeprazole, quinidine, diethyldithiocarbamic
aftertheadministrationofDDAs,indicatingthatDDAsare
acid, and ketoconazole) [22–27], and inhibitory mono-
chemically and biologically hydrolyzed into MDAs [2, 15].
clonal antibodies were also obtained from BD Gentest
BMA could reportedly recover the impaired resistance of
Corp. (Woburn, MA, USA). The monoclonal antibodies
thermally injured mice infected by HSV type 1 or Candida
(mAbs, 10mg/mL protein content) used were mAb-1A2
albicans[16].ThetoxicityofMDAsismuchlessthanthose
(anti-human CYP1A2), mAb-2C8 (anti-human CYP2C8),
of DDAs (the human lethal dose is 1mg to 4mg) [17, 18].
mAb-2C19 (anti-human CYP2C19), mAb-2D6 (anti-human
However, the toxic reaction will still occur in the event of
CYP2D6),mAb-2E1(anti-humanCYP2E1),andmAb-3A4/5
excessiveMDAabsorption[12].Frequentcasesofpoisoning
(anti-human CYP3A4/5). Other chemicals, generally of
after the intake of processed Aconitum are encountered in
reagentgradeorbetter,wereusedasreceived.
clinics.
Previous studies suggested that DDAs could be metab-
olized into less toxic products by CYP enzymes (the major 2.2. Phase I Metabolism of BAC, BMA, and BHA in HLMs.
functionofCYP3A)inthehumanlivermicrosomes[19–21]. Atypicalincubationsystemcontainedpotassiumphosphate
The main metabolic pathways of DDAs include demethy- (50mM,pH7.4),NADP(1.55mM),6-P-G(3.3mM),MgCl2
lation, dehydrogenation, hydroxylation, and didemethyla- (3.3mM),PDH(0.4U/mL),HLMs(0.4mgprotein/mL),and
BAC,BMA,orBHA(10𝜇M)inatotalvolumeof500𝜇L.After
tion [19–21]. Efflux transporters such as P-glycoprotein,
∘
a5minpreincubationat37 C,thereactionswerestartedby
breast cancer resistance protein, and multidrug resistance-
addingNADPH[28].Incubationswerecarriedoutfor90min
associatedprotein2wereinvolvedinthetransportofDDAs
∘
inashakingwaterbath(150rpm)at37 Candwerestopped
[11]. Thus, the toxicity of DDAs is reduced by the effect of
bytheadditionof4mLice-colddichloromethaneand100𝜇L
metabolic enzymes and efflux transporters [11, 19–21]. We
testosterone (200nM, used as an internal standard). The
investigated the role of efflux transporters in the transport
parentdruganditsmetaboliteswereextractedbyvortexing
ofMDAsandfoundthateffluxtransportersdidnotmediate
for 8min, after which they were centrifuged for 15min at
their transport [11]. This result indicates that the invasion
1000rpm. The supernatant was drawn into another tube
ofMDAscouldnotbelimitedbyeffluxtransporters.Efflux
and evaporated to dryness. The residue was dissolved with
transportersandmetabolicenzymesactasthehumanbody’s 50% methanol in water (125𝜇L) and analyzed by liquid
first line of protection by limiting the invasion of toxicants
chromatography with tandem mass spectrometry detection
[11]. However, thus far, the effect of metabolic enzymes on (LC-MS/MS). Control incubations were conducted in the
MDAs is unknown. Therefore, investigating the metabolic absenceofaNADPH-regeneratingsystem.
mechanismofMDAsforclinicalpracticeissignificant. To determine the protein concentration-dependent
We tested phase I and phase II metabolism of MDAs specificity of the enzyme, HLMs at concentrations of 0.2,
∘
(BAC, BMA, and BHA) by human liver microsomes in 0.4,and0.8mg/mLwereincubatedat37 Cfor90mininthe
preliminaryexperiments.NophaseIImetabolitesbutseveral above-describedreactionsystem,respectively.Todetermine
oxidative metabolites of BAC, BMA, or BHA were detected thetime-dependentspecificity,thereactionswereprocessed
∘
in the reaction systems. This study aims to identify the for 30, 60, 90, and 120min at 37 C in the reaction system,
CYP metabolites of BAC, BMA, and BHA in human liver respectively. To determine the substrate concentration-
microsomesaswellastheCYPisozymesresponsiblefortheir dependent specificity, substrates at concentrations of 2.5,
5, 10, 15, and 20𝜇M were incubated in HLMs at 37∘C for
metabolism.High-resolutionmassspectrometrywasusedto
120min,respectively.Eachexperimentwasperformedusing
characterize the structures of their metabolites. Moreover,
threesamples.
chemical inhibitors of specific CYP enzymes, CYP-specific
inhibitorymonoclonalantibodies,andcDNA-expressedCYP
enzymes (CYP1A2, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 2.3. Effect of Chemical Inhibitors on the Metabolism of BAC,
3A5) were used to verify which isozymes mediate in the BMA,andBHAinHLMs. Theselectiveinhibitorswerecho-
metabolismoftheCYPmetabolites.TheseresultsonMDAs sen based on FDA recommendation, and previous reports,
metabolismprovideimportantdataforthesafeclinicaluseof and are listed as follows: fluvoxamine maleate for CYP1A2,
processedAconitum. quercetinforCYP2C8,amiodaroneforCYP2C9,omeprazole
Evidence-BasedComplementaryandAlternativeMedicine 3
forCYP2C19,quinidineforCYP2D6,diethyldithiocarbamic 2.7. Data Analysis. Data are expressed as mean ± SD. One-
acid for CYP2E1 and ketoconazole for CYP3A4/5 [22–27]. way analysis of variance (ANOVA) with or without Tukey-
To obtain the maximum possible inhibition for each of the Kramermultiplecomparisons(posthoc)testswereusedto
isoforms, the final inhibitor concentrations in the reaction evaluate statistical differences. Differences were considered
mixtures were much higher than the reported Ki values. significantwhen𝑃valueswerelessthan0.05.
Theconcentrationofeachinhibitorwas5𝜇M.Theinhibitors
werepreincubatedwithallincubationmixturesfor5minat
3.Results
∘
37 Cbeforetheinitiationofthereactionby theadditionof
BAC, BMA, or BHA [20]. The mixture was then incubated 3.1.IdentificationoftheBAC,BMA,andBHAMetabolitesin
∘
for 120min at 37 C. A comparison was made between the HLMs. Todeterminethebestconditionsforincubation,we
incubations of samples with and without the inhibitors. All optimized the incubation time, protein concentration, and
incubationswerecarriedoutintriplicate. substrateconcentration.Accordingtothelargestformationof
metabolites,thefinalproteinconcentrationwasselectedtobe
0.4mg/mLandtheappropriateincubationtimewas120min.
2.4.EffectofmAbsontheMetabolismofBAC,BMA,andBHA
Meanwhile,forthesubstrateconcentration-dependentstudy,
in HLMs. Immunoinhibition studies were conducted by
theformationsofBAC,BMA,andBHAmetaboliteswereall
incubatingHLMswithvariousamountsofhumanselective
linearatconcentrationsof2.5𝜇Mto20𝜇M,indicatingthat
mAbs (mAb-1A2, mAb-2C8, mAb-2C19, mAb-2D6, mAb-
2E1, and mAb-3A4/5). MAbs (10𝜇L) were incubated with theconcentrationof10𝜇MBAC,BMA,orBHAthatwechose
for this study was suitable for the experiments. Compared
HLMsfor15minonicebeforetheadditionoftheparentdrug
(BAC,BMA,orBHA,10𝜇M)andtheNADPH-regenerating with the negative control (without NADPH-regenerating
∘ system), a total of 7 BAC metabolites, 8 BMA metabolites
system[21].Themixturewasincubatedat37 Cfor120min.
and9BHAmetaboliteswerefoundintheHLMsalongwith
As a control, comparable incubations were performed with
theNADPH-regeneratingsystem(Figure1).Themetabolites
HLMs and Tris buffer (25mM). All incubations were con-
wereidentifiedbytheretentiontimes,thechromatographic
ductedintriplicate.
behaviors,andcharacteristicmassspectrometricfragmenta-
tionfeatures,whicharesummarizedinTable1.
2.5. Phase I Metabolism of BAC, BMA, and BHA in cDNA-
ExpressedCYPEnzymes. TheincubationproceduresofBAC, 3.2. BAC Metabolites in HLMs. BAC, eluted at 3.77min,
+
BMA,orBHAincDNA-expressed CYPenzymeswereper- possessed a pseudo-molecule ion [M+H] at m/z 604.3109,
formedasdescribedforHLMs,exceptthattheconcentration which corresponded to the smart molecular formula of
2 +
oftheenzymeusedwas40pmol/mL[21,29].Theisozymes C32H45NO10. The MS spectrum of [M+H] provided a
used in this study included CYP 1A2, 2C8, 2C9, 2C19, numberofcharacteristicfragmentionsatm/z586,572,554,
2D6,2E1,3A4,and3A5.Allincubationswereperformedin 540,522,and508.
triplicate.
3.3.BACMetabolitesM1andM2. M1andM2wereelutedat
3.38and3.50min,respectively.M1andM2showedapseudo-
2.6.IdentificationandQuantitationoftheMetabolitesbyLC-
+
moleculeion[M+H] atm/z602.2951,whichconfirmedthe
MS/MS. A quadrupole time of flight tandem mass spec-
trometer(Bruker,Daltonics,USA)wasusedtocalculatethe smartmolecularformula(C32H43NO10),indicatingalossof
2Da (2H) from BAC, displaying that they were dehydro-
molecularweightofthemetabolites,whichcouldbededuced
2
throughtheirmolecularformulas.Thequantitativeanalysis genated metabolites of BAC. The MS spectrum of M1 and
procedurewasasfollows.TheUPLCconditionsweresystem, M2 showed fragmentation ions at m/z 584, 570, 552, 538,
Waters Acquity; column, Acquity UPLC BEH C18 column and506,whichwerealso2Dalowerthanthecharacteristic
(50 × 2.1mm, 1.7𝜇m, Waters, Milford, MA, USA); mobile fragment ions of BAC. Thus, M1 and M2 metabolites were
phase A: 0.1% (v/v) formic acid in water; mobile phase B: identifiedasdehydrogenation-BAC.H2groupcouldbetaken
100% acetonitrile; gradient, 0min to 0.5min at 2 to 2% B, offfrompositions2,12,and15.Therefore,theexactpositions
0.5min to 2min at 2% to 20% B, 2min to 4min at 20% to ofdehydrogenationwerenotconfirmed.
40%B,4minto6.5minat40%to70%B,and6minto7min
at70%to2%B;flowrate,0.35mL/min;columntemperature, 3.4.BACMetaboliteM3. M3hadaretentiontimeof3.08min,
50∘C;andinjectionvolume,10𝜇L.TheMS/MSdetectorused and its pseudo-molecule ion [M+H]+ was at m/z 588.2791.
wasaquadrupoletandemmassspectrometer(Waters,USA). Thus, the smart molecular formula was calculated to be
Samples were analyzed using electrospray ionization in the C31H41NO10, which had an observed loss of CH4 group
positive model. The main working parameters were set as compared with BAC. An MS/MS scan of M3 generated
follows: capillary voltage, 3kV; cone voltage, 50V; collision fragmentionsatm/z 570,556,538,524,506,and492,allof
∘
voltage, 30V; source temperature, 120 C; desolvation tem- whichwere16Dalowerthanthefragmentionsoftheparent
∘
perature, 500 C; desolvation gas flow, 600L/Hr; cone gas molecule. Therefore, M3 was identified as demethylated-
flow, 50L/Hr; and collision gas glow, 0.20mL/min. Data dehydrogenated BAC metabolite. Metabolism may have
were collected and analyzed by Waters Quantify software occurredeitherbydemethylationandthendehydrogenation
(Masslynx4.1,Waters,USA). or by dehydrogenation and then demethylation. Another
4 Evidence-BasedComplementaryandAlternativeMedicine
100
Without NADPH 604.4>554.4 100
Without NADPH 602.6>570.6
5.54e7
8.13e6
%) %)
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
100 M2
With NADPH 5.54e7 100
BAC With NADPH 8.13e6
%) %) M1
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
100
Without NADPH 588.4>538.4 100 574.5>542.2
Without NADPH
2.22e6 3.38e4
%) %)
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
100 With NADPH 2.22e6 100 With NADPH 3.38e4
M3 M4
%) %)
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
100 Without NADPH 590.5>540.5 100 Without NADPH 576.4>494.5
3.83e5 2.98e5
%) %)
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
M7
M6 100
100 With NADPH 3.83e5 With NADPH 2.98e5
%) (%)
(
M5
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
(a)
Figure1:Continued.
Evidence-BasedComplementaryandAlternativeMedicine 5
100
100 Without NADPH 588.6>556.7
Without NADPH 590.4>540.4
3.28e6
5.37e7
%) (%)
(
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
100 M2
100 With NADPH 3.28e6
With NADPH 5.37e7
BMA
%) (%)
( M1
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
100
100 Without NADPH 576.7>526.7
Without NADPH 574.5>542.5
1.83e5
1.35e6
%) (%)
(
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
100 M3 100 With NADPH M7 1.83e5
With 1.35e6
NADPH
%) (%)
(
M4 M6
M5
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
100 100 M8
Without NADPH 606.4>542.4 With NADPH 1.95e5
1.95e5
%) %)
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
(b)
Figure1:Continued.
6 Evidence-BasedComplementaryandAlternativeMedicine
100
100 Without NADPH 572.5>540.4
Without NADPH 574.4>542.4 1.37e6
1.26e8
%) %)
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
M1
100
100 BHA With NADPH 1.37e6
With NADPH 1.26e8
M2
%) %)
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
100
100 Without NADPH 558.5>526.6 Without NADPH 560.5>528.5
3.68e6 5.48e5
%) %)
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
M6
M3 100
100 With NADPH 5.48e5
With NADPH 3.68e6
M4
) M5
%) %
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
100
100 Without NADPH 590.5>526.5 Without NADPH 556.7>528.7
4.02e5
1.28e5
%) %)
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
(c)
Figure1:Continued.
Evidence-BasedComplementaryandAlternativeMedicine 7
M9
100 M8 100 With NADPH 4.02e5
With NADPH 1.28e5
%) M7 %)
( (
0 0
2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time Time
(c)
Figure1:RepresentativeselectiveioncurrentchromatogramsoftheBAC(a),BMA(b),andBHA(c)metabolitesinHLMsincubatedwith
10𝜇Moftheparentcompoundfor120minat37∘C.Atotalof7,8,and9metaboliteswereobservedforBAC,BMA,andBHAafterincubation
withHLMsandNADPH,respectively.
dubiouspeakexistedinthesamereactionchannel,whichwas 3.7. BAC Metabolite M7. M7 exhibited a retention time of
+
elutedat4.10min.Itssmartmolecularformulawascalculated 3.45min and had an [M+H] at m/z 576.2805. The smart
tobeC32H45NO9,whichdidnothavesimilarstructurewith molecular formula was confirmed to be C30H41NO10. The
BAC.Meanwhile,thispeakcouldnotonlybedetectedwith diagnosticionsatm/z558,544,526,512,494,and480were
2
NADPH-regenerating system, but also be detected without 28Da(C2H4)lowerthanthefragmentionsintheMS spectra
NADPH-regenerating system (negative control). Therefore, of BAC. Taken together, the results suggested that M7 may
the peak eluted at 4.10min was not the CYP metabolite of eitherbeametaboliteofBACwithalossofethylationonthe
BAC.Wespeculatedthatitwasjusttheendogenoussubstance nitrogenatomoradidemethyl-BAC.
ofthephaseImetabolicsystem.
3.8.BMAMetabolitesinHLMs. Thepeakelutionat3.48min
corresponded to BMA. BMA exhibited a pseudo-molecule
3.5. BAC Metabolite M4. The M4 peak eluted at 3.20min +
ion[M+H] atm/z590.2963inthefull-scanmassspectrum.
+
exhibited a pseudo-molecule ion [M+H] at m/z 574.2791
The smart molecular formula of C31H43NO10 was demon-
(C30H39NO10), which was 30Da (C2H6) lower than that of strated,andtheMS2spectrumof[M+H]+showedfragment
2
BAC, confirming the smart molecular formula. The MS ionsatm/z572,558,540,526,and508.
spectrum of M4 displayed fragment ions at m/z 542, 524,
510,492,and478,andthesefragmentionswereall30Daless
3.9. BMA Metabolites M1 and M2. M1 and M2 were eluted
thanthecorrespondingfragmentsgeneratedbyBAC.There-
at 3.11 and 3.27min, respectively. They showed a pseudo-
fore, M4 was identified as didemethyl-dehydrogen-BAC or moleculeion[M+H]+atm/z588.2802,confirmingthesmart
deethyl-dehydrogen-BAC. By the way, the peak having a molecular formula of C31H41NO10 and a loss of 2Da (2H)
retention time of 4.10min was not the CYP metabolite of from BMA. The MS2 spectrum of M1 and M2 showed
BAC, because of the peak could not only be detected with fragmentationionsatm/z570,556,538,and524,whichwere
NADPH-regenerating system, but also be detected without also2Dalowerthanthecorrespondingfragmentsgenerated
NADPH-regeneratingsystem(negativecontrol). byBMA.Thus,theyaredehydrogenatedmetabolitesofBMA.
A loss of H2 group could occur in positions 2, 12, and 15,
suggestingthattheexactpositionsofdehydrogenationwere
3.6.BACMetabolitesM5andM6. UPLCretentiontimesof
notconfirmed.
3.08 and 3.31min were obtained for M5 and M6, respec-
+
tively. Their pseudo-molecule ion [M+H] was both at m/z
3.10. BMA Metabolites M3 and M4. The UPLC retention
590.2940, and the smart molecular formula was concluded
times of metabolites M3 and M4 were 2.81 and 3.20min,
tobeC31H43NO10,whicheliminatedaCH2 groupfromthe and their pseudo-molecule ion [M+H]+ was both at m/z
2
parentcompound.TheMS spectrumoffragmentionswas
574.2642. Their smart molecular formula was suggested to
seen at m/z 572, 558, 540, 526 and 508, which were 14Da
be C30H39NO10, and a CH4 group was lost compared with
lowerthanthefragmentionsofBAC.Conclusively,M5and BMA. MS/MS scan of M3 and M4 showed fragment ions
M6wereidentifiedasdemethyl-BAC,butthedemethylation at m/z 556, 542, 524, 510, and 492, which were 16Da lower
would occur at positions 1, 6, 16, and 18. Similarly, the thanthatofBMA.Therefore,M3andM4wereidentifiedas
peak eluted at 4.1min was not the CYP metabolite of BAC, demethyl-dehydrogen-BMA.Tospeakof,thepeakselutedat
becausethepeakcouldnotonlybedetectedwithNADPH- 3.9and4.1minwerenottheCYPmetaboliteofBMA,because
regeneratingsystem,butalsobedetectedwithoutNADPH- these peaks could be detected with or without NADPH-
regeneratingsystem(negativecontrol). regeneratingsystem(negativecontrol).
8 Evidence-BasedComplementaryandAlternativeMedicine
Table1:LC-MSndataoftheBAC,BMA,andBHAmetabolitesinHLMswithNADPH.
[M+H]+ Smartformula
Metabolite Majorfragmentions Identification
(𝑚/𝑧) [M]
BAC 604.3109 C H NO 586,572,554,540,522,508 BAC
32 45 10
M1 602.2951 C H NO 584,570,552,538,506 Dehydrogen-BAC
32 43 10
M2 602.2949 C H NO 584,570,552,538,506 Dehydrogen-BAC
32 43 10
M3 588.2791 C H NO 570,556,538,524,506,492 Demethyl-dehydrogen-BAC
31 41 10
M4 574.2638 C H NO 542,524,510,492,478. Didemethyl-dehydrogen-BAC/Deethyl-dehydrogen-BAC
30 39 10
M5 590.2940 C H NO 572,558,540,526,508 Demethyl-BAC
31 43 10
M6 590.2944 C H NO 572,558,540,526,508 Demethyl-BAC
31 43 10
M7 576.2805 C H NO 558,544,526,512,494,480 Deethyl-BAC/didemethyl-BAC
30 41 10
BMA 590.2963 C H NO 572,558,540,526,508 BMA
31 43 10
M1 588.2802 C H NO 570,556,538,524 Dehydrogen-BMA
31 41 10
M2 588.2808 C H NO 570,556,538,524 Dehydrogen-BMA
31 41 10
M3 574.2642 C H NO 556,542,524,510,492 Demethyl-dehydrogen-BMA
30 39 10
M4 574.2647 C H NO 556,542,524,510,492 Demethyl-dehydrogen-BMA
30 39 10
M5 576.2789 C H NO 558,544,526,512,494 Demethyl-BMA
30 41 10
M6 576.2790 C H NO 558,544,526,512,494 Demethyl-BMA
30 41 10
M7 576.2792 C H NO 558,544,526,512,494 Demethyl-BMA
30 41 10
M8 606.2902 C H NO 588,574,556,542,524 Hydroxyl-BMA
30 43 11
BHA 574.3008 C H NO 558,542,524,510,492,478 BHA
31 43 9
M1 572.2857 C H NO 556,540,522,508,490,476 Dehydrogen-BHA
31 41 9
M2 572.2862 C H NO 556,540,522,508,490,476 Dehydrogen-BHA
31 41 9
M3 558.2709 C H NO 542,526,508,494 Demethyl-dehydrogen-BHA
30 39 9
M4 560.2843 C H NO 544,528,510,496,464 Demethyl-BHA
30 41 9
M5 560.2855 C H NO 544,528,510,496,464 Demethyl-BHA
30 41 9
M6 560.2857 C H NO 544,528,510,496,464 Demethyl-BHA
30 41 9
M7 590.2978 C H NO 574,558,540,526,508 Hydroxyl-BHA
31 43 10
M8 590.2959 C H NO 574,558,540,526,508 BMA
31 43 10
M9 556.2547 C H NO 542,528,524,497,465,450 Demethyl-didehydrogen-BHA
30 37 9
2
3.11. BMA Metabolites M5, M6, and M7. UPLC retention was calculated to be C31H43NO9. The MS spectrum of
+
timesof2.82,3.16,and3.41minwereobservedforM5,M6, [M+H] providedanumberoffragmentionsatm/z558,542,
+
and M7, respectively. Their pseudo-molecule ion [M+H] 524,510,492,and478.
was all at m/z 576.2792, and their smart molecular formula
was concluded as C30H41NO10, whic2h lacked a CH2 group 3.14.BHAMetabolitesM1andM2. M1andM2wereelutedat
fromtheparentcompound.TheMS spectrumoffragment
retentiontimesof3.53and3.90min,respectively.Theyboth
ionswasseenatm/z558,544,526,512,and494,whichwere
showedamoleculeionatm/z572.2862andalossofH2group
14DalowerthanthefragmentionsofBMA.Therefore,they 2
from BHA. The MS scan of the two metabolites included
wereestimatedtobedemethyl-BMA.
majorfragmentationionsatm/z556,540,522,508,490,and
476,whichwere2DalowerthanthoseofBHA.Theseresults
3.12. BMA Metabolite M8. M8 had a retention time of indicated that they were dehydrogen-BHA. Similar to BAC
+
3.01minandshowedapseudo-moleculeion[M+H] atm/z and BMA, the exact positions of dehydrogenation of BHA
606.2902. The smart molecular formula was calculated to werenotdetermined.
2 +
be C31H43NO11. The MS spectrum of [M+H] provided a
numberofcharacteristicfragmentionsatm/z588,574,556,
3.15. BHA Metabolite M3. M3 had a retention time of
542, and 524, which were 16Da (O) higher than those of +
3.16min, and its pseudo-molecule ion [M+H] was at m/z
BMA.M8wasthusidentifiedashydroxyl-BMA.
558.2709.Thesmartmolecularformulawascalculatedtobe
C30H39NO9,andalossofCH4groupwasobservedcompared
3.13. BHA Metabolites in HLMs. BHA exhibited a retention with BHA. MS/MS scan of M3 included fragment ions at
+
time of 3.99min, and its pseudo-molecule ion [M+H] m/z 542, 526, 508, and 494, which were 16Da lower than
appeared at m/z 574.3008, whose smart molecular formula the fragment ions of the parent compound. Therefore, M3
Evidence-BasedComplementaryandAlternativeMedicine 9
M1 formation M2 formation
3A4/5 3A4/5
∗ ∗
2E1 2E1
ors 2D6 ors 2D6
bit bit
hi 2C19 hi 2C19
n n
cal i 2C9 ∗ cal i 2C9 ∗
mi mi
e 2C8 e 2C8
h h
C C
1A2 1A2
∗
Control Control
0 50 100 150 0 50 100 150
Control (%) Control (%)
M3 formation M4 formation
3A4/5 3A4/5
∗ ∗
2E1 2E1
ors 2D6 ors 2D6
bit bit
hi 2C19 hi 2C19
n n
al i 2C9 ∗ al i 2C9
mic mic
he 2C8 he 2C8
C C
1A2 1A2
Control Control
0 50 100 150 0 50 100 150
Control (%) Control (%)
M5 formation M6 formation
3A4/5 3A4/5
∗ ∗
2E1 2E1
ors 2D6 ors 2D6
bit bit
hi 2C19 hi 2C19
n n
cal i 2C9 cal i 2C9 ∗
mi mi
e 2C8 e 2C8
h h
C C
1A2 1A2
∗
Control Control
0 50 100 150 0 50 100 150
Control (%) Control (%)
M7 formation
3A4/5
∗
2E1
ors 2D6
bit
hi 2C19
n
al i 2C9
c
mi
e 2C8
h
C
1A2
Control
0 50 100 150
Control (%)
(a)
Figure2:Continued.
10 Evidence-BasedComplementaryandAlternativeMedicine
M1 formation M2 formation
3A4/5 3A4/5
∗ ∗
2E1 2E1
bitors 2D6 ∗ bitors 2D6
nhi 2C19 ∗ nhi 2C19 ∗
cal i 2C9 ∗ cal i 2C9 ∗
mi mi
he 2C8 ∗ he 2C8 ∗
C C
1A2 1A2
∗
Control Control
0 50 100 150 0 50 100 150
Control (%) Control (%)
M3 formation M4 formation
3A4/5 3A4/5
∗ ∗
2E1 2E1
bitors 2D6 ∗ bitors 2D6 ∗
nhi 2C19 ∗ nhi 2C19 ∗
cal i 2C9 ∗ cal i 2C9 ∗
mi mi
he 2C8 ∗ he 2C8 ∗
C C
1A2 1A2
∗
Control Control
0 50 100 150 0 50 100 150
Control (%) Control (%)
M5 formation M6 formation
3A4/5 3A4/5
∗ ∗
2E1 2E1
ors 2D6 ors 2D6
bit bit
nhi 2C19 nhi 2C19 ∗
mical i 2C9 ∗ mical i 2C9 ∗
he 2C8 he 2C8
C C
1A2 1A2
Control Control
0 50 100 150 0 50 100 150
Control (%) Control (%)
M7 formation M8 formation
3A4/5 3A4/5
∗ ∗
2E1 2E1
bitors 2D6 ∗ bitors 2D6 ∗
nhi 2C19 ∗ nhi 2C19 ∗
cal i 2C9 ∗ cal i 2C9 ∗
mi mi
he 2C8 ∗ he 2C8 ∗
C C
1A2 1A2
∗
Control Control
0 50 100 150 0 50 100 150
Control (%) Control (%)
(b)
Figure2:Continued.