Quantitative Determination of Alkaloids in Lotus Flower

17 Pages·2016·1.65 MB·English

molecules
Article
Quantitative Determination of Alkaloids in Lotus
Flower (Flower Buds of Nelumbo nucifera) and
Their Melanogenesis Inhibitory Activity
ToshioMorikawa1,2,†,NiichiroKitagawa1,3,†,GenzohTanabe4,KiyofumiNinomiya1,2,
ShuheiOkugawa1,3,ChiakiMotai1,3,IyoriKamei1,MasayukiYoshikawa1,I-JungLee5
andOsamuMuraoka1,2,4,*
1 PharmaceuticalResearchandTechnologyInstitute,KindaiUniversity,3-4-1Kowakae,Higashi-osaka,
Osaka577-8502,Japan;[email protected](T.M.);[email protected](N.K.);
[email protected](K.N.);[email protected](S.O.);
[email protected](C.M.);[email protected](I.K.);
[email protected](M.Y.)
2 AntiagingCenter,KindaiUniversity,3-4-1Kowakae,Higashi-osaka,Osaka577-8502,Japan
3 KoshiroCompanyLtd.,2-5-8Doshomachi,Chuo-ku,Osaka541-0045,Japan
4 FacultyofPharmacy,KindaiUniversity,3-4-1Kowakae,Higashi-osaka,Osaka577-8502,Japan;
[email protected]
5 NationalResearchInstituteofChineseMedicine,155-1,Sec.2,LinongSt.,BeitouDistrict,Taipei11221,
Taiwan;[email protected]
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
† Theseauthorscontributedequallytothiswork.
AcademicEditor:MichaelWink
Received:5July2016;Accepted:12July2016;Published:19July2016
Abstract: A quantitative analytical method for five aporphine alkaloids, nuciferine (1),
nornuciferine (2), N-methylasimilobine (3), asimilobine (4), and pronuciferine (5), and five
benzylisoquinolinealkaloids,armepavine(6),norarmepavine(7),N-methylcoclaurine(8),coclaurine
(9),andnorjuziphine(10),identifiedastheconstituentsresponsibleforthemelanogenesisinhibitory
activityoftheextractsoflotusflowers(theflowerbudsofNelumbonucifera),hasbeendevelopedusing
liquidchromatography-massspectrometry. Theoptimumconditionsforseparationanddetectionof
these10alkaloidswereachievedonaπNAPcolumn,areversed-phasecolumnwithnaphthylethyl
group-bondedsilicapackingmaterial,withCH CN–0.2%aqueousaceticacidasthemobilephaseand
3
usingmassspectrometryequippedwithapositive-modeelectrosprayionizationsource.Accordingto
theprotocolestablished,distributionsofthese10alkaloidsinthepetal,receptacle,andstamenparts,
whichwereseparatedfromthewholeflower,wereexamined. Asexpected,excellentcorrelations
wereobservedbetweenthetotalalkaloidcontentandmelanogenesisinhibitoryactivity. Among
theactivealkaloids,nornuciferine(2)wasfoundtogiveacarbamatesalt(211)viaformationofan
unstablecarbamicacid(21)byabsorptionofcarbondioxidefromtheair.
Keywords: lotus flower; Nelumbo nucifera; melanogenesis inhibitor; nuciferine; nornuciferine;
quantitativeanalysis;carbamatesalt
1. Introduction
A Nymphaeaceae plant Nelumbo nucifera Gaertn. (common name “lotus” in English) is
extensivelycultivatedinEasternAsiancountries[1–3]. Allpartsofthisplant,includingtheleaves,
stamens, flowers, seeds, and rhizomes, have been used as traditional medicines or vegetables for
thousands of years [2–4]. The lotus flower, the flower buds of N. nucifera, has been used for the
treatment of vomiting blood, bleeding caused by internal and external injuries, and various skin
Molecules2016,21,930;doi:10.3390/molecules21070930 www.mdpi.com/journal/molecules
Molecules2016,21,930 2of17
diseases,andalsoasasedativeandananti-inflammatoryagentintraditionalAsianmedicines[2].
In the course of our studies on the bioactive constituents from the flower buds of N. nucifera,
we have isolated several alkaloids, e.g., nuciferine (1), nornuciferine (2), N-methylasimilobine (3),
asimilobine (4), pronuciferine (5), and armepavine (6), with melanogenesis inhibitory activities in
theophylline-stimulatedmurineB16melanoma4A5cells[2]. Asaresultoftheincreasinginterest
in lotus flower as a possible cosmetic for skin whitening, there is a strong demand for efficient
quality control measurements to ensure the authenticity and content of the active constituents in
such products, and to verify the labeled claims. In this paper, we propose a simple, rapid, and
precise analytical method for liquid chromatography-mass spectrometry (LC-MS) simultaneous
quantitativedeterminationoffiveaporphinealkaloids(1–5)andfivebenzylisoquinolinealkaloids,(6),
norarmepavine(7),N-methylcoclaurine(8),coclaurine(9),andnorjuziphine(10),usingaone-step
samplepreparationprocedure.
2. ResultsandDiscussion
2.1. IsolationofPrincipalAlkaloids(1–10)fromLotusFlower
To obtain the principal alkaloids (1–10), an isolation procedure from this plant material was
newlydevelopedinthisstudybymodifyingthepreviouslyreportedmethod[2]. Thus,driedflower
budsofN.nuciferawereextractedwithmethanolunderrefluxtoobtainamethanolextract(9.22%
from the dried material). The methanol extract was partitioned into a mixture of EtOAc and 3%
aqueous tartaric acid (1:1, v/v) to furnish an acidic EtOAc-soluble fraction (2.88%) and an acidic
aqueoussolution. ThepHoftheaqueoussolutionwasadjustedto9withsaturatedaqueousNa CO
2 3
andthenextractedwithCHCl toobtainaCHCl -solublefraction(0.97%). Theaqueouslayerwas
3 3
furtherextractedwithn-BuOHtoobtainann-BuOH-solublefraction(0.62%). AsshowninTable1,
themethanolextractwasfoundtoinhibittheophylline-stimulatedmelanogenesis(IC =5.6µg/mL)
50
withoutcytotoxicity(cellviabilityinthe3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide
(MTT)assay: 103.3˘7.1%)at100µg/mL.Throughbioassay-guidedseparation,theCHCl -soluble
3
fraction(IC =0.37µg/mL)wasfoundtobemoreactivethantheEtOAcn-BuOH-solublefractions
50
(IC =11.1and13.7µg/mL,respectively).
50
Table 1. Inhibitory effects of the methanol extract and its fractions from lotus flower on
theophylline-stimulatedmelanogenesisandviabilityinB164A5cells.
Treatmenta Inhibition(%) IC50
0µg/mL 3µg/mL 10µg/mL 30µg/mL 100µg/mL (µg/mL)
0.0˘1.3 28.3˘3.7 68.9˘3.1** 96.4˘3.1** 97.0˘3.3**
MeOHext. 5.6
(100.0˘9.3) (110.9˘2.7) (124.2˘5.1) (126.5˘5.3) (103.3˘7.1)
0.0˘6.1 11.0˘1.7 51.5˘5.3** 83.0˘2.9** 100.9˘3.4**
EtOAc-solublefraction 11.1
(100.0˘7.2) (99.3˘9.8) (106.8˘5.1) (110.9˘4.2) (118.3˘10.4)
0.0˘13.3 17.6˘7.0 35.9˘7.1 72.3˘4.7** 94.7˘3.5**
n-BuOH-solublefraction 13.7
(100.0˘11.2) (104.6˘11.6) (104.9˘2.6) (114.8˘13.9) (104.3˘3.8)
Inhibition(%) IC50
Treatment
0µg/mL 0.1µg/mL 0.3µg/mL 1µg/mL 3µg/mL (µg/mL)
0.0˘1.7 19.8˘6.8 35.6˘6.6** 79.2˘2.3** 107.2˘3.0**
CHCl3-solublefraction (100.0˘3.5) (101.5˘6.1) (103.1˘6.1) (114.7˘4.6) (107.4˘9.5) 0.37
Eachvaluerepresentsthemean˘S.E.M.(n=4);asterisksdenotesignificantdifferencesfromthecontrolgroup,
**p<0.01;aBioassay-guidedseparationstudywascarriedoutusingtheflowerbudsofN.nuciferaoriginating
inThailand(NN-1).
TheactiveCHCl -solublefractionwassubjectedtonormal-phasesilicagel,reversed-phaseODS
3
columnchromatography,andfinallyHPLCtofurnishnuciferine(1,0.1028%)[2,5,6],nornuciferine
(2, 0.0821%) [2,5,6], N-methylasimilobine (3, 0.0094%) [2,7], asimilobine (4, 0.0345%) [2,6,8],
pronuciferine(5,0.0195%)[2,9],armepavine(6,0.0170%)[2,10],norarmepavine(7,0.0616%)[10,11],
Molecules2016,21,930 3of17
N-methylcoclaurine(8,0.0053%)[12],coclaurine(9,0.0042%)[10,13],andnorjuziphine(10,0003%)[14]
(MMFoiolgelecucuurleeless 12 20)0.1166, ,2 211, ,9 93300 33 o of f1 177

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Molecules2016,21,930 4of17
Table2.Extractionefficientlyofalkaloids(1–10)fromlotusflower.
Contents(mg/ginDryMaterial)a
ExtractionMethod ExtractionYield(%) Total
1 2 3 4 5 6 7 8 9 10
Methanol,reflux 15.0 1.76(100) 1.75(100) 0.07(100) 0.63(100) 0.69(100) 0.83(100) 1.45(100) 5.73(100) 1.30(100) 0.75(100) 14.96(100)
50%Methanol,reflux 25.3 1.09(62) 1.35(77) 0.05(71) 0.50(79) 0.61(88) 0.78(94) 1.35(93) 3.79(66) 0.94(73) 0.56(75) 11.02(74)
H2O,reflux 23.1 0.24(14) 0.35(20) n.d.b 0.21(33) 0.18(26) 0.38(45) 0.78(54) 2.57(45) 0.66(51) 0.29(38) 5.66(38)
Methanol,sonication 9.6 0.88(50) 1.11(64) 0.03(44) 0.39(62) 0.33(48) 0.47(56) 0.97(67) 2.77(48) 0.70(54) 0.42(56) 8.07(54)
50%Methanol,sonication 22.0 0.98(56) 1.27(73) 0.04(58) 0.49(78) 0.47(69) 0.80(96) 1.38(95) 3.93(69) 0.97(75) 0.59(79) 10.92(73)
H2O,sonication 19.3 0.14(8) 0.21(12) n.d.b 0.12(20) 0.08(11) 0.25(30) 0.53(37) 1.91(33) 0.48(37) 0.19(26) 3.91(26)
ExtractionefficientlywastestedusingNN-1(lossofdrying10.33%);arelativevalue(%)againstthecontentobtainedbymethanolunderrefluxisgiveninparentheses;blessthanthe
quantitationlimit.
Table3.Linearities,detectionandquantitationlimits,andprecisionsforalkaloids(1–10)inlotusflower.
Precisionc(RSD,%)
Analyte RegressionEquationa CorrelationCoefficient DetectionLimitb(ng) QuantitationLimitb(ng)
Intra-Day Inter-Day
Nuciferine(1) y=7477635x´1302 0.9998 0.17 0.51 0.25 0.59
Nornuciferine(2) y=2698708x´10941 1.0000 0.71 2.16 0.79 0.43
N-Methylasimilobine(3) y=7054297x+243961 0.9996 0.32 0.99 1.36 1.40
Asimilobine(4) y=2076494x´36021 0.9999 0.70 2.13 0.63 0.57
Pronuciferine(5) y=3522995x+101328 0.9998 0.73 2.18 0.95 1.08
Armepavine(6) y=2076494x´36021 0.9999 0.32 0.97 0.68 1.10
Norarmepavine(7) y=1998354x´15296 0.9999 0.81 2.47 0.54 0.73
N-Methylcoclaurine(8) y=1595194x+53314 0.9999 0.90 2.71 0.59 0.86
Coclaurine(9) y=1878370x+16838 0.9999 0.44 1.33 0.98 0.39
Norjuziphine(10) y=1745634x+15240 1.0000 0.88 2.65 0.64 0.66
aIntheregressionequation,xistheconcentrationoftheanalytesolution(µg/mL),andyisthepeakareaoftheanalyte;bvaluesaretheamountoftheanalyteinjectedon-columnand
cprecisionoftheanalyticalmethodweretestedusingthemethanolextractofNN-1(n=5).
Molecules2016,21,930 5of17
Table4.Recoveriesforalkaloids(1–10)fromlotusflower.
Recoverya(%)
Add(µg/mL)
1 2 3 4 5 6 7 8 9 10
10 98.7˘0.6 101.4˘0.7 95.3˘1.0 99.0˘0.9 93.2˘0.7 98.5˘0.3 104.0˘0.7 94.7˘1.4 97.4˘0.2 97.4˘1.0
15 92.3˘0.4 101.7˘0.2 97.5˘0.7 101.8˘0.1 98.1˘0.5 102.2˘0.6 99.4˘1.1 99.8˘1.6 104.0˘0.2 100.2˘0.8
20 98.4˘0.1 105.8˘0.7 101.2˘0.8 102.0˘0.7 95.9˘0.8 105.1˘0.6 99.9˘0.9 96.3˘1.1 105.8˘0.3 95.3˘0.8
aTherecoveryratesweredeterminedbyaddinganalytesofthreedifferentconcentrations(10,15,and20µg/mL)tothesamplesolution;recoveriesspikedwiththemethanolextractof
NN-1(each400µg/mL,n=3).
Table5.Contentsofalkaloids(1–10)inthemethanolextractsfromlotusflower.
Lossof Extraction Contents(mg/ginDryMaterial)a
SampleNo. Part Dryinga(%) Yieldb(%) 1 2 3 4 5 6 7 8 9 10 Total
NN-1 wholeflowers 10.3 15.0 1.76 1.75 0.07 0.63 0.69 0.83 1.45 5.73 1.30 0.75 14.96
NN-2 petals 8.6 16.6 1.99 2.41 0.07 1.22 0.62 1.02 1.74 5.85 1.60 0.52 17.04
NN-3 receptacles 10.0 8.6 0.06 0.07 n.d.c 0.01 0.01 0.01 0.01 0.20 0.04 n.d.c 0.41
NN-4 stamens 8.6 20.0 0.58 0.68 n.d.c 0.25 0.23 0.47 0.64 2.88 0.60 0.32 6.65
NN-5 wholeflowers 8.1 16.3 0.56 0.27 n.d.c 0.04 n.d.c 0.23 0.34 1.74 0.19 0.16 3.53
NN-6 petals 7.3 19.0 0.80 0.34 n.d.c n.d.c 0.01 0.36 0.52 3.14 0.32 0.27 5.76
NN-7 receptacles 9.2 7.4 0.27 0.53 n.d.c n.d.c 0.05 0.03 0.03 0.74 0.27 n.d.c 1.92
NN-8 stamens 7.9 15.5 0.01 n.d.c n.d.c n.d.c n.d.c n.d.c n.d.c 0.03 0.01 0.03 0.08
aEachpowderedsamplewasdriedat105˝Cfor8h;beachpowderedsamplewasextractedtwotimeswithmethanolunderrefluxfor120minandclessthanthequantitationlimit.
Molecules2016,21,930 6of17
Prior to analysis, extraction conditions were examined to optimize the extracts1 quality in
association with the contents of the alkaloids (1–10). The extraction efficacies were compared for
threesolventsystems(methanol,50%aqueousmethanol,andwater)undertwodifferentconditions
(refluxfor120minorsonicationfor30min,eachtwice). AsshowninTable2,“refluxinmethanol”
affordedthehighestcontentsoftheactivealkaloids(1–10). Therefore,alltheanalyticalsampleswere
preparedbyemployingthemethod“refluxinmethanolfor120min”.
Some analytical parameters, such as linearity and limit of quantitation of the developed
method,wereevaluatedasshowninTable3. Thecalibrationcurvewaslinearintherangestudied
(0.5–50µg/mL)showingacorrelationcoefficient(R2)ofgreaterthan0.9996foreachconstituent.Linear
regressionequationsoftheircalibrationcurvesforeachconstituentaredescribedinTable3,whereyis
thepeakareaandxistheconcentrationoftheanalyte.Thedetectionandquantitationlimitswereestimated
tobe0.17–0.90and0.51–2.65ng,respectively,indicatingsufficientsensitivityofthismethod.Therelative
standarddeviation(RSD)valueswere0.25%–1.36%forintra-dayand0.39%–1.40%forinter-dayassays.
AccuracywasdeterminedinrecoveryexperimentsusingthemethanolextractofNN-1. Asshownin
Table4,recoveryratesof92.3%–105.8%wereobtained,withRSDvaluesoflowerthan1.6%.
According to the protocol thus established, contents of the alkaloids (1–10) collected in two
different regions (NN-1 in Thailand; NN-5 in Taiwan) were measured. The assay was found to
bereproducible,precise,andreadilyapplicabletothequalityevaluationoflotusflower1sextracts.
As shown in Table 5, N-methylcoclaurine (8, NN-1: 5.73 mg/g in dry material; NN-5: 2.88 mg/g)
wastherichestconstituentamongthealkaloids(1–10). ThetotalalkaloidcontentintheThai(NN-1:
14.96 mg/g) and Taiwanese (NN-5: 3.53 mg/g) samples were quite different. However, a more
extensivestudywouldberequiredtoconfirmthatthisresultwasduetodifferencesbetweenregions.
Tocharacterizethedistributionofthealkaloids(1–10)intheflower, thewholeflowerparts(NN-1
andNN-5)wereseparatedintopetals(NN-2andNN-6),receptacles(NN-3andNN-7),andstamens
(NN-4andNN-8);then,quantitativeanalysisofeachseparatedsamplewasperformed. Itwasfound
thatthealkaloids(1–10)weremainlycontainedinthepetalpart.Furthermore,otherpartsofthelotus
plant(e.g.,leaf(NN-9),fruit(NN-10and11),andembryoparts(NN-12),whichareusedfortraditional
medicines)werealsoexamined.Itwasfoundthatthetotalalkaloidcontentoftheleaf(NN-9:1.20mg/g),
fruit(NN-10andNN-11:eachlessthanthequantitationlimit),andembryoparts(NN-12:0.64mg/g)of
N.nuciferawerelowerthanthoseoftheflowerbuds(NN-1andNN-5)(TableS1).
2.3. AmmoniumCarbamateSalt(211)FormationfromtheFreeAlkaloid(2)
The gradual transformation of one of the alkaloids isolated in this study, nornuciferine (2),
intoahighlypolarmaterial211wasobserved,when2wasexposedtotheatmosphereindeuterated
chloroform (CDCl ) at room temperature. After three weeks of standing in CDCl , compound 211
3 3
was obtained as a main product (Figure 3). As summarized in Table 6, 1H- and 13C-NMR spectra
of211 suggestedthattherearetwokindsofpartsderivedfromthenornuciferineframeworkinthe
structure of 211. Thus, with respect to one of the nornuciferine parts, five carbons α and β to the
nitrogenatomappearedaspairsignals[δ : 29.9/30.2(C-4),41.8/44.4(C-5),54.9/55.8(C-6a),33.9/35.9
C
(C-7), 124.8/124.9 (C-11c)] in the 13C-NMR spectrum of 211. Additionally, a pair of signals, which
correspondedtoanamidetypecarbonylcarbon,wasalsoobservedatδ 157.5and160.1. Thechemical
C
shiftofthesignalssuggestedthatthenitrogenatomof2wasfunctionalizedasacarbamateanionby
theCO uptakefromtheatmosphere. Ontheotherhand,inthe1H-NMRspectrumof211 adownfield
2
shift owing to the ammonium ion formation was observed with respect to the signals due to C-51
methylene (at δ 3.24 and 3.87) and C-6a1 methine (at δ 4.29) protons of the other nornuciferine
H H
partsascomparedwiththoseof2[δ : 3.01and3.40(H -5),3.85(H-6a)]. Twobroadsinglets,which
H 2
appeared at the highly-deshielded regions (δ 9.96 and 10.84), were due to acidic protons, which
H
alsosupporttheammoniumionstructuredepictedinFigure3. Theanticipatedstructureof21 was
strongly supported by the IR spectrum, which showed N+–H and C=O stretching absorptions at
2720–2500and1721cm´1,respectively. Moreover,thepositiveionpartof211 wasdetectedasaNa
Molecules2016,21,930 7of17
adductionsignalatm/z282.1483[M´C H NO ]+ (calcdforC H O ,282.1489)inthepositive
19 18 4 18 20 2
ESImode;however,asignalduetothecarbamategroupwasnotdetectedinthenegativeESImode.
Fortunately,thenegativeionpartof211couldbeconfirmedasthecorrespondingmethylcarbamate
2a. Thus, 211 easily gave a 1:1 mixture of methyl carbamate 2a and original amine 2 by treatment
withmethanolatroomtemperature(FigureS1). AsshowninTable6,compound2ashowedsimilar
13C-NMRspectroscopicpropertiestothoseof211 and/or2,exceptforasinglet(δ 52.6)duetothe
C
methylcarbonoftheNCO CH moiety,whichwasconfirmedbythecorrelationbetweenthesingletat
2 3
δ 3.76duetothemethylprotonsandasingletatδ 156.0duetocarbonylcarbonintheHMBCof2a.
H C
InthepositiveESIMSof2a,aquasimolecularionpeakwasobservedatm/z362.1361[M+Na]+(calced
forC H NO Na,362.1363).
20 21 4
Table6.1H-(800MHz)and13C-(200MHz)NMRdatafor211,2a,andoriginalalkaloid2inCDCl .
3
211(anionpart) 211(cationpart)
Position Position
δH(JinHz) δC δH(JinHz) δC
1 146.0a 11 146.2a
2 152.5b 21 153.7b
3 6.69(s) 111.4 31 6.67(s) 111.4
3a 128.7 3a1 125.7
2.74(brd,ca.15) 2.95(dd,3.8,16.8)
4 29.9,30.2 41 25.5
2.98(brd,ca.15) 3.62(ddd,5.7,13.5,16.8)
3.20–3.35(m) 3.24(brddd-like,ca.13.5,13.5,13.5)
5 41.8,44.4 51 41.4
4.62(brd,ca.12.5) 3.87(brdd-like,ca.5.7,13.5)
6a 4.92(brd,ca.13) 54.9,55.8 6a1 4.29(brdd-like,ca.13.5,13.5) 53.0
2.86–2.90(m) 3.38(dd,13.5,13.5)
7 33.9,35.9 71 34.0
3.02–3.14(m) 3.45(dd,4.5,13.5)
7a 135.7 7a1 133.1
8 7.24–7.30(m) 128.0c 81 7.24–7.30(m) 128.3c
9 7.24–7.30(m) 127.4c 91 7.24–7.30(m) 128.2c
10 7.35(m) 127.3c 101 7.35(m) 127.8c
11 8.44(brs-like) 128.5c 111 8.41(d,7.9) 128.6c
11a 131.3 11a1 131.3
11b 127.5d 11b1 127.0d
124.8,
11c 11c1 121.4
124.9
1-OCH3 3.667e(s) 60.3f 11-OCH3 3.673e(s) 60.0f
2-OCH3 3.90g(s) 56.0h 21-OCH3 3.91g(s) 55.9h
157.5, 9.96(brddd-like,ca.13.5,13.5,13.5)
N-COO 160.1 NH2 10.84(brd-like,ca.13.5)
2a 2
Position Position
δH(JinHz) δC δH(JinHz) δC
1 145.7 1 145.3
2 152.1 2 152.2
3 6.67(s) 111.5 3 6.65(s) 111.8
3a 126.1 3a 128.5
2.65(brd,ca.15) 2.71(d,13.1)
4 30.3 4 28.9
2.88(m) 3.05(m)
3.00(brdd,ca.11,13) 3.01(m)
5 38.8 5 43.0
4.73(brd,ca.13) 3.40(brq,ca.6)
6a 4.46(brs) 51.4 6a 3.85(brdd,ca.5,14) 53.5
2.86(m) 2.77(t,13.8)
7 35.2 7 37.3
2.98(dd,12.8,15.8) 2.87(dd,4.6,13.8)
7a 136.8 7a 135.9
8 7.25(dd,1.6,7.8) 128.2 8 7.24(m) 127.8
9 7.27(brdd,ca.8,8) 127.5 9 7.21(ddd,1.1,7.1,7.1) 127.4
10 7.32(ddd,1.6,7.8,8.0) 126.9 10 7.30(m) 127.0
11 8.44(brd,ca.8) 128.3 11 8.39(brd,ca.8) 128.4
11a 131.7 11a 132.1
11b 127.6 11b 126.5
11c 129.7 11c 128.7
1-OCH3 3.66(s) 60.2 1-OCH3 3.67(s) 60.2
2-OCH3 3.90(s) 56.0 2-OCH3 3.88(s) 55.9
N-COO 156.0
N-CO2CH3 3.76(s) 52.6
a–hMaybeinterchangeable.
Molecules2016,21,930 8of17
Molecules 2016, 21, 930 8 of 17
Molecules 2016, 21, 930 8 of 17

FigFuigreur3e. 3C. hCehmemicaiclatlr tarnasnfsofromrmaatitoionno offn noorrnnuucciiffeerriinnee ((22)) iinnttoo iittss aammmmoonniuiumm cacarbrbamamataet esaslat l(t2(′′2) 1a1)nadn tdo to
methyl carbamate (2a).
mFiegtuhryel 3c.a Crbhaemmaitceal( 2traa).nsformation of nornuciferine (2) into its ammonium carbamate salt (2′′) and to
methyl carbamate (2a).
It is well known that ammonia, primary amines, or secondary amines (A) absorb CO2 to
Itiswellknownthatammonia,primaryamines,orsecondaryamines(A)absorbCO totransform
transform into the corresponding carbamic acids (B), which easily react with the origina2l amines to
intotIht eisc owrreelsl pkonnodwinng tchaartb aammimc aocniidas, (pBr)i,mwahryic hameainsielsy, roera cstewcointhdathrye oamriginineas l(aAm) inaebssotrobp CroOd2u ctoe
produce stable carbamic acid ammonium salts and (C) as shown in Figure 4 [15–24]. Therefore, it is
transform into the corresponding carbamic acids (B), which easily react with the original amines to
stablecarbamicacidammoniumsaltsand(C)asshowninFigure4[15–24]. Therefore,itisreasonable
reasonable to anticipate that the product 2′′ forms via an acid-base reaction between original amine
tporoadnuticceip satateblteh acatrtbhaempirco adcuidct a2m11mfoornmiusmvi asaalnts aacnidd -(bCa)s easr esahcotwionn ibne Ftwigeuerne 4o r[i1g5i–n2a4l]a. mThineree2foarne,d ita ins
2 and an unstable carbamic acid (2′), which was obtained by the CO2 absorption reaction with the
urenansstiotarnboalgebelcnea atrotbo aammn toiicfc i2ap.c aidte (t2h1a),t wthhei cphrowdauscto b2′t′a fionremdsb vyiath aenC aOci2d-abbassoer pretiaocntiorena bcetitowneweni tohritghiennali tarmogienne
a2t oanmdo afn2 .unstable carbamic acid (2′), which was obtainedR 1by the CO2 absorption reaction with the
nitrogen atom of 2.
HN
R1
R1NH CO2 R1N-CO2H HNR2 R1N-CO2– R1
RR1N2HA CO2 RR12N-CBO2H R2 RR21N-CCOH2–2+NR2R1
R1, R2R =2 H,A alkyl, aryl R2(unsBtable) R2 (stabCleH)2+NR 2
R1, R2 F= iHgu, arelk y4l., Tahryel report(eudn sretaabclteiv)ity of amines with CO2.
(stable)
2.4. Effects of the Hydrochlorides of These Alkaloids (1–10) and 2a on Theophylline-Stimulated Melanogenesis
FFiigguurree 44.. TThhee rreeppoorrtteedd rreeaaccttiivviittyy ooff aammiinneess wwiitthh CCOO2..
Inhibitory Activity 2
22..44.. EEffffeeTcctotss coolffa trthhifeey HH thyyedd rreoofccfhihcllaoocrryiidd oeessf oothff eTT hheeessstaee bAAlillskkhaaelloodiid dqssu ((a11n––t11i00ta))t aaivnnedd a22naaa oolynns TTishh oeeoof pp1hh0yy allllliiknnaeel--oSSittdiimms (uu1ll–aa1ttee0dd) aMMs eeall aaqnnuooaggleeinntyee ssiiss
IInnhhciiobbniitttoorrroyyl AAfoccrtt iivvloiitttyyu s flower, correlations between the total alkaloid contents and the melanogenesis
inhibitory activities of the corresponding extracts were examined. Previously, we have reported the
TToo ccllaarriiffyy tthhee eefffificcaaccyy ooff tthhee eessttaabblliisshheedd qquuaannttiittaattiivvee aannaallyyssiiss ooff 1100 aallkkaallooiiddss ((11––1100)) aass aa qquuaalliittyy
ccoonnmttrreoolalln ffooogrre nllooesttuiusss i nflfhlooiwbwiteeorrr,,y c coaocrrtrrieevlliaatitteiisoo nnossf fbbreeeettww neeueecnnif ettrhhineee tto(o1tt,aa lIlC aa5l0l kk=aa ll1oo5ii.dd8 cµcooMnn)tt,ee nnnottssrn aaunncdidfe rtthihneee mm(2e,e ll6aa2nn.9oo ggµeeMnne)e,ss iiss
N-methylasimilobine (3, 14.5 µM), asimilobine (4, >100 µM), pronuciferine (5, 47.9 µM), and armepavine
iinnhhiibbiittoorryy aaccttiivviittiieess ooff ththee cocorrrersepspoonnddiningg exetxrtarcatcst swwereer eexeaxmaimneinde. dP.rePvrieovuisolyu,s lwy,e whaevhea rveeporertpeodr ttehde
(6, 25.6 µM) [2]. Since the hydrochlorides of these alkaloids (1–10), which were of higher purity and
tmheesltaamnboielgiltaeynn eothsgaiesn n ietnhshoiissbei itnoorfh yit bhaietc otcirovyritrieeasscp tooivnf idtfiirenesge fnorfueecf irfaeelrkeianlneo uid(c1si,,f eIwCrie5nr0 ee= u (1s15e,d.8 I fCµo5rM0 t)h,= en so1tr5an.n8udcaµirfMder is)n,aemn (po2lr,e ns6u 2oc.f9i f tehµreMi n)e,
(N2-,m62e.t9hyµlMas)i,mNil-ombienthe y(3la, s1i4m.5i lµoMbi)n, eas(i3m,1il4o.b5inµeM (4),, a>s1i0m0 iµloMbi)n, per(o4n,u>c1i0fe0rµinMe ()5,,p 4r7o.n9 uµcMife),r ianned( a5r,m47e.p9aµvMin)e,
present quantitative analysis, the melanogenesis inhibitory activities of these hydrochloride salts were
a(6n, d25a.r6m µeMpa) v[2in].e S(i6n,ce2 5t.h6e µhMyd)r[o2c]h.loSrinidcees tohfe thheysder oaclkhallooriiddse s(1o–f10th), ewsehiaclhk awloeirdes o(f1 h–1ig0h),erw phuicrhityw aenrde
newly examined. It was found that aporphine alkaloids, nuciferine (1, IC50 = 7.1 µM) and nornuciferine
ostfabhiilgithye rthpaun rtihtyosaen odf stthaeb icloitryretshpaonndthinogs efroefe tahlekaclooridress, pwoenrde inugsedfr efeora ltkhael ostiadnsd, awrder esaumspeldesf oorf tthhee
(2, 3.9 µM), and benzylisoquinoline alkaloids, armepavine (6, 6.5 µM), norarmepavine (7, 7.5 µM),
sptraensdenatr dqusaanmtiptalteisveo fanthaleyspisr,e tsheen tmqeulaannotigteantievseis ainnhaliybistiosr,yt haectimvietileasn oofg ethneessei shyindhriobcihtolorryidaec tsiavlittsi ewseoref
N-methylcoclaurine (8, 6.5 µM), and coclaurine (9, 3.9 µM), were found to show relatively strong
tnheewinselhyih beyixtdoarmroyi cnahceltdoiv.r iiIdtti eewss awasl ifttoshuwonuedtr enthonateat wbalpleyo creypxthoaitmnoexi niacel kedfa.fleoIctitdws saa, tns tufhoceiu feenfrdfiencteht i(av1te, aIcCpoon50rc =pe nh7t.i1rna eµtiMoanlk) (aaTlnoadibd lneso ,7rn)n.u uTcchiiffeeesrreii nnee
((12,, a3IlCk.9a5 l0µoiM=ds)7 , .w1aneµdreM bm)enaoznreyd lipnsoootqreunnuitn ctoihflaeinnrie n aaerlbk(u2at,lion3i .d9(sIC,µ a5M0r m=), e1ap7na4dv µibnMeen )(,z 6ya, l6ics.o5o mqµumMine)ro, clniianollreya arumlkseeapdloa imvdisen,leaa n(ro7m,g e7en.p5ea sµvisMi n)e,
(N6-,mi6n.he5tihbµyiMtlocr)o, ucnlsaoeurdra iarnmse a e (pp8,oa sv6ii.tn5iv eeµ( Mc7o,)n7, t.r5aonµld M[ 2c5)o–,c2Nl7a-]um. rAienmteho yn(l9cg,o t3ch.le9am uµr, Mi2n ae),n (wd8, e96r s.e5h ofµowMuen)d,d ae sntopd escchioaoclwllya usrtrerilonanetigv( 9ea,lcy3ti .v9sittrµyoM,n g),
winehwriebhiiftcoohur ysnh daocwttoievdsit himeoswo rwer ietthlhaaotniuv t4e 0nl yotitmsatbreosle ng gcryeiatnotehtroi bxthiitcao ner yftfheaacctt tsio vaf itta itrehuseb wuetfiifnteh. coPtuirvetevn icoooutnaslbcyel,e ntthcreya ttnoioattnoux r(iaTclalyebf-lofeec cc7tu)s.r raTinthgte hsee
eaflfkaeapcltooirivpdehs icnwoen eacrleekn amltoraiodtri, eo4 n,5p-(odTtiaedbnelhte ytd7h)ra.ongT uaharedbsiusectiainnlek a((IIlCCo5i50d0 =s= 4 w.71e 7µr4Me µm) Misoor)le,a tape doc tfoermonmtmt Hhearocnrniasalcrlhybu ucuhtiisane od(bI lCimq5ue0el,a =hna1os7 gb4eeneµneMs i)s,
ainchroiembpiotmortree rudcs i[ae2ld8ly ]a. usT soae tpdhoems bieteilvsatne o ocfgo oenuntrre osklin s[o2iwn5–hle2ibd7ig]t.eo A,r cmuosmoenpdgoa utshnaedmsp o2, s2ai ntaidnv ed9 c9ao rsneh ttrohowel em[2do5 es–ts2 pp7e]o.cteiAanmltl ymo snetglraontnhogegm eanc,et2isviasint yd,
9wshihnicohhwi bseihtdoorewss epwdei tchmiainoll ryteh sitsthr coalnna sg4s 0ao cftt inimvaitetusy r,agwlr epharitcoehdr ustchhtoasn.w ethdamt oofr eartuhbanut4in0. tPimreevsiogurselayt,e rthteh annattuhraatlloyf-oacrcuubruritning.
Papreovrpiohuinsley a,ltkhaelonida,t 4u,r5a-dlliyd-eohcycudrrroignugadaipsocirnpeh (iInCe50 a=l k4a.7l oµidM,) 4is,5o-ldatiedde hfryodmro Hgourandscihscuicnheia (oIbCli5q0ue=, h4a.7s bµeMen)

reported [28]. To the best of our knowledge, compounds 2 and 9 are the most potent melanogenesis
inhibitors within this class of natural products.
Molecules2016,21,930 9of17
isolatedfromHornschuchiaoblique,hasbeenreported[28]. Tothebestofourknowledge,compounds2
and9arethemostpotentmelanogenesisinhibitorswithinthisclassofnaturalproducts.
Table7.Inhibitoryeffectsofthealkaloids(1–10)and2aontheophylline-stimulatedmelanogenesisand
viabilityinB164A5cells.
Treatment Inhibition(%) IC50
0µM 3µM 10µM 30µM 100µM (µM) (µg/mL)b
Nuciferine¨HCl(1)a 0.0˘4.0 28.3˘4.2** 57.8˘2.3** 89.7˘1.7** 91.8˘2.6** 7.1 2.4
(100.0˘2.6) (112.1˘2.6) (112.1˘2.5) (100.9˘5.8) (45.9˘3.3#)
Nornuciferine¨HCl(2)a 0.0˘3.4 44.6˘3.4** 70.6˘3.6** 94.3˘3.2** — 3.9 1.2
(100.0˘4.9) (99.5˘8.2) (93.9˘4.1) (70.6˘4.8#) (1.9˘0.1#)
N-Methylasimilobine¨HCl(3)a (1000.0.0˘˘43.7.9) (936..36˘˘22..96) (1190.05.4˘˘2.34.0**) (6131.94.3˘˘5.92.4**) (8597..87˘˘21..13*#*) 43.1 13.7
Asimilobine¨HCl(4)a (1000.0.0˘˘85.1.7) (1966..25˘˘172.5.8) (3918..99˘˘21.02.0**) 8(776.2.6˘˘36.3.3*)* (13.7˘—2.2#) 11.3 3.4
Pronuciferine¨HCl(5)a 0.0˘10.7 23.1˘3.4 18.8˘1.2 37.2˘2.6** 88.4˘3.7** 47.1 16.3
(100.0˘4.5) (104.2˘3.4) (97.4˘0.7) (98.4˘1.8) (89.1˘9.4)
Armepavine¨HCl(6)a 0.0˘6.0 33.9˘0.8** 58.5˘7.1** 81.8˘3.0** 97.4˘0.4** 6.5 3.4
(100.0˘2.5) (104.0˘3.3) (104.0˘7.7) (104.0˘7.2) (78.1˘2.0)
Norarmepavine¨HCl(7)a (1000.0.0˘˘32.1.6) 3(826.6.4˘˘44.4.0*)* 5(831.5.2˘˘84.5.0*)* 8(813.6.3˘˘12.4.2*)* (9608..58˘˘11..24*#*) 7.5 2.5
N-Methylcoclaurine¨HCl(8)a 0.0˘5.4 38.6˘2.4** 55.7˘3.4** 74.7˘2.0** — 6.5 2.2
(100.0˘3.0) (97.1˘1.5) (92.8˘4.1) (96.4˘4.2) —
Coclaurine¨HCl(9)a (1000.0.0˘˘22.9.9) 4(957.6.0˘˘77.7.7*)* 6(956.4.2˘˘25.5.0*)* 8(826.4.5˘˘37.5.7*)* (6583..01˘˘68..43*#*) 3.9 1.3
Norjuziphine¨HCl(10)a (1000.0.0˘˘55.4.3) (1988.5.9˘˘46.0.0*) 3(961.8.3˘˘44.5.9*)* 9(843.4.9˘˘28.0.3*)* (15076..00˘˘22..40#*)* 14.4 4.6
0.0˘7.3 13.1˘8.9 43.1˘7.8** 54.5˘4.3** —
2a 19.9 6.7
(100.0˘4.7) (109.8˘4.3) (127.5˘4.8) (129.1˘2.8) —
Treatment Inhibition(%) IC50
0µM 30µM 100µM 300µM 1000µM (µM) (µg/mL)
0.0˘1.4 20.4˘0.5 38.1˘0.9** 61.5˘0.6** 83.7˘0.5**
Arbutin[25–27] (100.0˘2.1) (82.4˘3.0) (78.1˘1.9) (79.8˘2.2) (53.1˘1.8#) 174 47.4
Eachvaluerepresentsthemean˘S.E.M.(n=4);asterisksdenotesignificantdifferencesfromthecontrolgroup,
*p<0.05,**p<0.01;#cytotoxiceffectswereobserved,andvaluesinparenthesesindicatecellviability(%)in
MTTassay;commercialarubutinwaspurchasedfromNakalaiTesqueInc.,(Kyoto,Japan);aeachalkaloidwas
evaluatedbyitshydrochloridesalt;beachIC50valuewasconvertedtoµg/mLofcorrespondingfreealkaloid.
2.5. EffectsonMushroomTyrosinase
Tocharacterizethemodeofactionofmelanogenesisinhibitoryactivitiesofthealkaloids,inhibitory
effectson(i)enzymatictyrosinaseactivityand(ii)expressionsoftyrosinase-relatedproteins(TRPs)
e.g.,tyrosinase,TRP-1,andTRP-2wereexamined.
A copper-containing enzyme tyrosinase is a key enzyme in melanin biosynthesis involved
in determining the color of skin and hair. It catalyzes oxidation of both L-tyrosine and L-DOPA,
followinganotheroxidationof L-DOPAtodopaquinoneand,finally,oxidativepolymerizationvia
several dopaquinone derivatives to yield melanin. Tyrosinase inhibitors are being clinically used
forthetreatmentofseveraldermatologicaldisordersassociatedwithmelaninhyperpigmentation.
Thetyrosinaseinhibitorkojicacidiscommonlyusedasanadditiveincosmeticsforskinwhitening
and/ordepigmentation[25–27].AsshowninTable8,noneofthealkaloidsshowedinhibitoryactivities
when using both L-tyrosine and L-DOPA as substrates. This suggests that tyrosinase inhibition is
barelyinvolvedinthemechanismsofactionofthesemelanogenesisinhibitors.
Molecules2016,21,930 10of17
Table8.Effectsonactivityoftyrosinasefrommushroom.
Inhibition(%)
Substrate:Treatment L-Tyrosine L-DOPA
0µM 10µM 100µM 0µM 10µM 100µM
Nuciferine¨HCl(1)a 0.0˘3.2 6.5˘3.3 30.4˘1.9** 0.0˘2.9 4.4˘3.8 11.0˘3.4
Nornuciferine¨HCl(2)a 0.0˘2.6 1.0˘0.7 14.2˘1.5** 0.0˘3.1 15.4˘4.1 8.7˘4.1
N-Methylasimilobine¨HCl(3)a 0.0˘3.6 7.2˘7.3 0.8˘3.5 0.0˘1.5 2.3˘1.5 3.1˘3.9
Asimilobine¨HCl(4)a 0.0˘1.8 10.5˘2.5* 14.0˘1.2** 0.0˘3.3 3.2˘2.4 5.7˘3.0
Pronuciferine¨HCl(5)a 0.0˘5.3 ´0.4˘2.4 ´4.4˘8.1 0.0˘2.0 6.5˘2.0 5.3˘5.2
Armepavine¨HCl(6)a 0.0˘4.4 ´1.9˘0.9 40.2˘3.7** 0.0˘0.5 4.5˘0.8 2.9˘1.2
Norarmepavine¨HCl(7)a 0.0˘1.8 ´1.7˘1.5 23.3˘1.6** 0.0˘2.7 ´1.3˘1.4 1.5˘1.5
N-Methylcoclaurine¨HCl(8)a 0.0˘5.2 ´5.0˘5.7 15.3˘4.3 0.0˘2.3 6.1˘1.4 7.4˘0.8
Coclaurine¨HCl(9)a 0.0˘2.3 9.0˘0.7* 35.1˘1.7** 0.0˘2.5 ´4.9˘1.1 4.4˘1.1
Norjuziphine¨HCl(10)a 0.0˘2.5 5.1˘1.1 27.4˘3.0** 0.0˘2.0 1.8˘0.7 22.3˘3.6**
2a 0.0˘1.5 5.3˘1.7 2.9˘0.7 0.0˘2.7 3.2˘1.1 6.6˘2.3
Substrate:L-Tyrosine Inhibition(%)
Treatment 0µM 10µM 30µM 100µM 300µM IC50(µM)
Kojicacid[25–27] 0.0˘2.4 12.2˘3.3 46.4˘2.6** 66.5˘2.1** 96.8˘0.9** 43.6
Substrate:L-DOPA Inhibition(%)
Treatment 0µM 10µM 30µM 100µM 300µM IC50(µM)
Kojicacid[25–27] 0.0˘0.9 22.3˘2.1** 50.6˘0.6** 78.2˘0.7** 89.3˘0.3** 29.6
Eachvaluerepresentsthemean˘S.E.M.(n=4); asterisksdenotesignificantdifferencesfromthecontrol
group,*p<0.05,**p<0.01;commercialkojicacidwaspurchasedfromNakalaiTesqueInc.,(Kyoto,Japan);
aeachalkaloidwasevaluatedbyitshydrochloridesalt.
2.6. EffectsonExpressionofTyrosinase,TRP-1,andTRP-2
The TRP enzyme family (tyrosinase, TRP-1, and TRP-2) catalyze the major steps in melanin
synthesis[25–27]. Toclarifythemechanismsofactionoftheseactiveconstituents,weexaminedthe
effects of the principal alkaloids (1, 2, 6, 7, and 9) on expression of mRNAs for tyrosinase, TRP-1,
andTRP-2inB16melanoma4A5cells. Exceptforcompound7,thesuppressiontendencyofmRNA
expressionfortyrosinaseinthesealkaloidswasobservedaspresentedinTable9.
Table9.Effectsof1,2,6,7,and9onexpressionoftyrosinase,TRP-1,andTRP-2mRNAinB164A5cells.
TyrosinasemRNA/-actinmRNA
Treatment
0µM 3µM 10µM
Nuciferine¨HCl(1)a 1.00˘0.15 0.59˘0.03* 0.45˘0.05*
Nornuciferine¨HCl(2)a 1.00˘0.19 0.76˘0.05 0.51˘0.10
Armepavine¨HCl(6)a 1.00˘0.14 0.86˘0.13 0.74˘0.02
Norarmepavine¨HCl(7)a 1.00˘0.24 0.81˘0.08 1.00˘0.11
Coclaurine¨HCl(9)a 1.00˘0.14 0.82˘0.21 0.52˘0.05
TRP-1mRNA/-actinmRNA
Treatment
0µM 3µM 10µM
Nuciferine¨HCl(1)a 1.00˘0.12 1.18˘0.17 1.18˘0.25
Nornuciferine¨HCl(2)a 1.00˘0.10 1.21˘0.18 1.15˘0.19
Armepavine¨HCl(6)a 1.00˘0.22 0.98˘0.32 0.83˘0.15
Norarmepavine¨HCl(7)a 1.00˘0.09 0.87˘0.22 1.03˘0.25
Coclaurine¨HCl(9)a 1.00˘0.24 0.51˘0.08 0.66˘0.13
TRP-2mRNA/-actinmRNA
Treatment
0µM 3µM 10µM
Nuciferine¨HCl(1)a 1.00˘0.16 1.33˘0.35 0.87˘0.16
Nornuciferine¨HCl(2)a 1.00˘0.12 0.92˘0.20 1.36˘0.09
Armepavine¨HCl(6)a 1.00˘0.05 1.07˘0.22 0.81˘0.22
Norarmepavine¨HCl(7)a 1.00˘0.18 0.80˘0.24 0.77˘0.14
Coclaurine¨HCl(9)a 1.00˘0.11 1.02˘0.22 1.07˘0.07
Eachvaluerepresentsthemean˘S.E.M.(n=3);asterisksdenotesignificantdifferencesfromthecontrolgroup,
*p<0.05;aeachalkaloidwasevaluatedbyitshydrochloridesalt.
2.7. CorrelationbetweentheMelanogenesisInhibitoryActivityandTotalContentsofAlkaloids(1–10)inLotus
FlowerExtracts
The inhibitory effects of the methanol extracts of the lotus flowers (NN-1–8) on
theophylline-stimulatedmelanogenesiswereexamined. Asaresult,theIC valuesweredetected
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