Comparative Analysis of Amaryllidaceae Alkaloids from Three Lycoris Species

16 Pages·2015·1.41 MB·English

Article
Comparative Analysis of Amaryllidaceae Alkaloids
from Three Lycoris Species
YongqiangTian1,2,ChunyunZhang1andMingquanGuo1,*
Received:8November2015;Accepted:30November2015;Published:7December2015
AcademicEditor:MarcelloIriti
1 Sino-AfricaJointResearchCenter/KeyLaboratoryofPlantGermplasmEnhancementandSpecialty
Agriculture,WuhanBotanicalGarden,ChineseAcademyofSciences,Wuhan430074,China;
[email protected](Y.T.);[email protected](C.Z.)
2 GraduateUniversityofChineseAcademyofSciences,Beijing100049,China
* Correspondence:[email protected];Tel./Fax:[phone]
Abstract: The major active constituents from Amaryllidaceae family were reported to be
Amaryllidaceae alkaloids (AAs), which exhibited a wide spectrum of biological activities, such as
anti-tumor, anti-viral, andacetyl-cholinesterase-inhibitoryactivities. Inordertobetterunderstand
theirpotentialasasourceofbioactiveAAsandthephytochemicalvariationsamongthreedifferent
species of Lycoris herbs, the HPLC fingerprint profiles of Lycoris aurea (L. aurea), L. radiata, and
L. guangxiensis were firstly determined and compared using LC-UV and LC-MS/MS. As a result,
39 peakswereresolvedandidentifiedasAAs,ofwhichninepeakswerefoundincommonforall
these three species, while the other 30 peaks could be revealed as characteristic AAs for L. aurea,
L. radiata and L. guangxiensis, respectively. Thus, these AAs can be used as chemical markers for
theidentificationandqualitycontroloftheseplantspecies. Tofurtherrevealcorrelationsbetween
chemical components and their pharmaceutical activities of these species at the molecular level,
thebioactivitiesofthetotalAAsfromthethreeplantspecieswerealsotestedagainstHepG2cells
with the inhibitory rate at 78.02%, 84.91% and 66.81% for L. aurea, L. radiata and L. guangxiensis,
respectively. Thisstudyfirstlyrevealedthatthethreespeciesunderinvestigationweredifferentnot
onlyinthetypesofAAs, butalsointheircontents, andbothcontributedtotheirpharmacological
distinctions. To the best of our knowledge, the current research provides the most detailed
phytochemicalprofilesofAAsinthesespecies,andoffersvaluableinformationforfuturevaluation
andexploitationofthesemedicinalplants.
Keywords: Amaryllidaceaealkaloids;chemicalfingerprints;chemicalmarker;anti-HepG2activity
1. Introduction
BelongingtotheAmaryllidaceaefamily,plantsofgeneraLycoriswereknownnotonlyfortheir
ornamental value, but also for their medicinal value being used as a medicinal herb for thousands
of years in China [1,2]. It has been well documented that the Amaryllidaceae alkaloids (AAs) were
responsiblefortheirpharmaceuticalactivities,andexhibitedawidespectrumofbiologicalactivities,
such as anti-tumor, anti-malarial, and acetylcholinesterase inhibitory activities [3–8]. In turn, there
has been growing interest in the search for new AAs with better bioactivities from Amaryllidaceae
plants [9]. In the past few years, more and more alkaloids were isolated from the Amaryllidaceae
family,andmostofwhichbelongtogalanthaminetype,lycorinetype,homolycorinetype,tazettine
type and crinine type in terms of chemical structures [10]. Among them, galanthamine and
lycoraminewerereportedtoexhibitgoodactivityagainstAlzheimer’sdisease[7].WhilemoreAAs,
such as lycorine, dihydrolycorine, haemanthamine, pretazettine, pseudolycorine, and narciclasine,
Molecules2015,20,21854–21869;doi:10.3390/molecules201219806 www.mdpi.com/journal/molecules
Molecules2015,20,21854–21869
showed significant activity against a variety of cancer cells either by inhibiting cancer cell growth
mainly through cytostatic effects targeting small RHO GTPases or through the inhibition of protein
synthesisandthesubsequentdisorganizationoftheactincytoskeleton[11–14].Duetotheremarkable
pharmaceutical activities, AAs have led to increasing interest in the search for new resources and
new bioactive components from different species in the Amaryllidaceae family. However, most of
the current research only focused on certain major species on the market, and little work has been
conducted for the comprehensive analysis of AAs from different Lycoris species. Since remarkable
chemical differences have often been found in different species of medicinal plants or even from
differentgeographicorigins,whichsignificantlyaffectedqualityandbioactivities. Inmostcases,the
chemical differences often resulted in pharmacological distinctions. In this context, we set out to
investigateandcomparechemicalfingerprintprofilesofthreeLycorisspecies. Duetothecomplexity
and variety of components in these plant species, it is of primary importance to develop a tailored
analyticalmethodforthecomprehensiveanalysisofAAsfromthesemedicinalplants[15,16].
In the past few decades, a number of analytical methods including GC-MS, LC-MS, and
CE-ESI-IT-MShavebeendevelopedfortheanalysisofAAs[17–23], whichcontributedsignificantly
to the better understanding of AAs from medicinal plants. Due to the high sensitivity and the
accesstotheMSdatabase,GC-MSwasdeemedasaneffectivemethodfortheanalysisofAAsinthe
past[19]. However,itwaslimitedtoAAswithrelativelyhighervolatility,whichwasinappropriate
to some AAs, especially in this work. Compared to GC-MS, LC-MS has been more widely used
in the analysis of alkaloids in various plant sources due to its ability in detecting thermo-unstable
and high-molecular-weight alkaloids in recent years [19,21], and it is thus applied in this study.
While usedfordifferentresearchpurposes,thoseLC-MSmethodsreportedhavelimitationsinboth
theirresolutionandcapacityofprofilingAAs,andcanonlyanalyzeoneoraverylimitednumberof
AAs[23–27]. InordertoconductacomprehensiveanalysisofAAs,andovercometheselimitations,a
moreeffectivemethodisrequiredtocomparefingerprintprofilesofdifferentLycorisspecies. Thus,a
rapid,sensitive,andreliableHPLC-UV/ESI-MS/MSmethodhasbeensuccessfullydevelopedforthe
comparative analysis of the AAs from different Lycoris species, which resulted in the simultaneous
separation and identification of over 30 AAs from different Lycoris species under the optimized
conditions. To the best of our knowledge, the present study is the first report on qualitative and
quantitative assessment of AAs from different Lycoris species, and provides an important clue for
futurevaluationandexploitationofthesemedicinalplants.
2. ResultsandDiscussion
2.1. OptimizationofChromatographicConditions
Previous phytochemical investigations into AAs in Lycoris species revealed its complexity
in chemical structure and the diversity in AAs types, e.g., galanthamine, galanthine, lycorine,
lycoramine, narwedine, tazettine, and haemanthamine [17,18]. This posed significant challenges to
the comprehensive analysis of AAs from different species. Considering the structural differences
of AAs and other reported LC methods, chromatographic conditions, such as wavelength of
maximum absorption, LC columns, mobile phase and mobile phase additives, were systematically
investigated to achieve better separation of AAs. By scanning the samples extracted from different
species, the maximum absorption wavelength at 232 nm was selected for LC-UV analysis in
this study. Based on the column efficiency, and the resultant chromatograms, Phenomenex ODS
column (150ˆ2.00mm, 5 µm, Phenomenex, Torrance, CA, USA) was chosen as a preferred one
over the reverse phase C18 column (Sunfire C18, 150 mm ˆ 4.6 mm, 3.5 µm, Waters, Milford,
MA, USA). By stepwise investigation of mobile phase and additives, including methanol-water,
acetonitrile-water, acetonitrile-0.1% formic acid, acetonitrile-10 mM ammonium acetate, and
acetonitrile-40mMammoniumacetate,thecombinationofacetonitrileand40mMammoniumacetate
waschosenasthemobilephaseandadditivesaccordingtotheirbetterperformanceandresolution
21855
Molecules2015,20,21854–21869
inthegradientelutionat232nm,respectively. ThefinaloptimizedconditionsfortheanalysisofAAs
Molecules 2015, 20, page–page
fromdifferentLycorisspeciesaregiveninSection3.3.1.
22..22.. SSaammppllee AAnnaallyyssiiss aanndd AAmmaarryylllliiddaacceeaaee AAllkkaallooiiddss’’ ((AAAAss)) IIddeennttiifificcaattiioonn
22..22..11.. HHPPLLCC FFiinnggeerrpprriinntt PPrroofifilleess
TToottaall AAAAss eexxttrraacctteedd ffrroomm tthhee bbuullkkss ooff tthhee tthhrreeee LLyyccoorriiss ssppeecciieess wweerree ffuurrtthheerr eennrriicchheedd bbyy OOaassiiss
MMCCXX SSPPEE ccaarrttrriiddggeess,, aanndd ssuubbsseeqquueennttllyy aannaallyyzzeedd iinn ppaarraalllleell oonn aa PPhheennoommeenneexx ccoolluummnn uunnddeerr tthhee
ooppttiimmiizzeedd LLCC ccoonnddiittiioonnss.. AAs sshshoowwnn ini nFiFgiugruer e1a1–ac–, ct,hteh HePHLPCL fCinfignergperripnrtiinntgi npgropfriloefis loefs AofAAs Afrsomfr othme
tthhereteh srpeeecsiepse wcieersew deetreermdeinteerdm. Iint weda.s oIbtsweravsedo bthseartv 3e9d pethaakts w39erpee wakesll rweesorelvwede lulnrdeesor ltvheed giuvnend eHrPtLhCe
gciovnednitHioPnLsC, incodnicdaittiinogn st,hiantd tihcae toinpgtitmhaiztetdh ecoonpdtiimtioiznesd wcoenred iatciohniesvwede rfeoar cthhiee vceodmfpoarrtahtievceo amnpaalyrsaitsi voef
aAnAalsy fsriosmof tAheA tshfrreoem Ltyhceortihsr espeeLcyiecso.r isspecies.

FFiigguurree 11.. HHPPLLCC pprroofifilleess ooff LL.. rraaddiiaattaa ((aa));; LL.. aauurreeaa ((bb)) aanndd LL.. gguuaannggxxiieennssiiss ((cc))..
22..22..22.. IIddeennttiifificcaattiioonn ooff AAAAss
AAccccoorrddiinngg ttoo tthhee HHPPLLCC pprroofifilleess ((FFiigguurree 11)),, 3399 AAAAss wweerree ddeetteecctteedd ffrroomm tthhee tthhrreeee LLyyccoorriiss ssppeecciieess,,
wwhhiicchh wweerree lliisstteedd iinn TTaabbllee 11.. AAmmoonngg tthheemm,, 2299 AAAAss wweerree iiddeennttiififieedd iinn tthhiiss ssttuuddyy bbyy ccoommppaarriinngg tthheeiirr
MMSS//MMSS wwiitthh ththee cocorrreressppoonnddiningg auauththenentitci cstsatnadndaradrds soro prupbulbislhisehde dlitleirteartuatruerse ass asshoshwonw inn TinabTlaeb 1le. T1o.
Tthoe tbheestb oefs touorf konuorwknleodwglee, dsgeve,ense ovfe tnheo fAtAhes (AlaAbsel(eldab aesl e“da”a isn“ Taa”bilne 1T)a wbleer1e )fiwrsetrlye rfierpsotlryterde pino rgteendeirna
gLeynceorrias,L ayncodr fisiv,aen odf tfihveemo (fltahbeemled(l aabs e“lbe”d ians T“abb”lein 1)T wabelree1 f)irwstelyre rfieprsotrlyterde pino trhteed AinmtahreylAlimdaacreyaleli dfaamceialye.
fBaamseildy. onB atsheed idoennttihfeicaidtieonntsi,fi tchaeti ocnhse,mtihcealc shtermucitcuarlesst roufc tthuere 2s9o Af Athse w29ereA Asusmwmearerizseudm imn aFriigzuedre i2n.
FSiignucere d2if.fSeirnecnet tdyipffeesr eonft AtyApse dsiosfpAlaAyesdd disipfflearyeendt fdriafgfemreenntt fpraagthmweanytsp uasthinwga EySsIu-MsinS/gMESS,I t-hMeS s/trMuSct,uthrael
sdtrivuecrtsuirtayl odfi vAerAssit ymoafdAe Athsemira iddeenthtiefiircaidtieonnt ivfiecrayti ocnomveprlyiccaotemdp. lTicoa tseidm.pTloifysi mthpel iifnytethrperienttaetriponre toaft itohne
oofbtthaeinoebdt aMinSe/dMMS Sd/aMtaS fdoart aAfAors,A wAes ,cwlaescsliafisesdifi tehdemth eimntion tfoivfiev gergoruopusp sacaccocordrdiningg ttoo tthheeiirr cchheemmiiccaall
ssttrruuccttuurreess,, ii..ee..,, ggaallaanntthhaammiinnee ttyyppee,, llyyccoorriinnee ttyyppee,, ccrriinniinnee ttyyppee,, hhoommoollyyccoorriinnee ttyyppee aanndd ttaazzeettttiinnee
ttyyppee [[1177,,2244,,2255]].. IInn mmoorree ddeettaaiill,, tthhee rreepprreesseennttaattiivveei inntteerrpprreettaattioionnsso offt htheeM MSS//MMSS ddaattaa ffoorr fifivvee aallkkaallooiidd
ttyyppeess wweerree ddiissccuusssseedd aass bbeellooww..

21856
3
Molecules2015,20,21854–21869
Molecules 2015, 20, page–page

FFiigguurree 22.. CChheemmicicaalls tsrtuructcutruerseso fotfh ethAe AAsAidse nidtiefinetdififerdo mfroLm.a uLr.e aa,uLr.eraa, dLia. taraadniadtaL .agnuda nLg.x gieunasnisg(xNieontseis:
t(hNeonteu:m thbee nrsucmorbreerssp coonrdretsoptohnedp teoa tkhne upmeabke rnsusmhobwerns sinhoFwignu rine 1F)i.gure 1).
TTaabbllee1 1.. PPeeaakkss ddeetteecctteedd aass AAAAss bbyy LLCC--MMSS//MMSS..
Peak Rt PI-MS Alkaloid
MS/MS Data Identification
NPoNe. aok. (min(m)R itn) [M [+MP HI-+]M+H S]+ MS/MSData Identification AlkaloidTyTyppee/R/Reeff..
1 3.22 284 266, 256, 249, 241, 237, 226, 213, 199 1,11-didehydrogalanthamine b GA [17]
2 1 3.613 .22 304 284 2662,8265,6 2,62849, ,225401,, 222377,, 222161,,2 1135,01 99 1,111-1d-idheyhdyrdorxoygavliatntathtianmei nNe-boxide a GACR[1 7[2]7,33]
2 3.61 304 286,268,250,227,211,150 11-hydroxyvittatineN-oxidea CR[27,33]
3 3 3.933 .93 290 290 2722,7 222,252, 52,1271,7 2,21111, ,220011,, 119999,,1 1881,11, 2142,41,1 1919 3-hy3d-hroyxdyrlaotxifyalliautmifainliCumbin C b TAT[3A3] [33]
4 4 4.974 .97 276 276 25285, 82,1291,9 2,21155, ,220011,, 118899,,1 1775,51, 51353 DihDydihroy-ldartiofa-llaiutmifainliCuma inC a TAT[3A3] [33]
5 5 5.715 .71 274 274 25265, 62,2252,5 2,21177, ,220077,, 119999,,1 1997,71, 81181 LatifLaalituimfailniuCmain C a TAT[3A3] [33]
6 5.76 350 332,281,267,255,223,193,180 Unidentified
6 5.76 350 332, 281, 267, 255, 223, 193, 180 2α-HydroxUy-n3-ihdyednrtoif-6ie-Od-
7 6.54 334 316,298,270,267,255,238,173 HO[36]
2αm-eHthyydlordouxlyin-3e-bhydro-6-O-
7 6.54 334 316, 298, 270, 267, 255, 238, 173 HO [36]
8 7.17 274 231,225,213,198,183 N-demethmyle-tghaylalnotdhuamlininee b GA[26,27]
9 9.48 290 272,254,226,149,136,112,68 Dihydrolycorine LY[29–31]
8 10 7.171 0.05 274 306 231,2 2882,52, 7201,32,2 91,9188,9 183 N-Cdreimnaemthabyiln-egaalanthamine CGRA[3 5[]26,27]
9 11 9.481 2.90 290 288 27227, 02,5245,2 ,222262,, 114797,, 114376,,1 1119,29, 568 LDyichoyridnreolycorine LYL[Y1, 1[72]9–31]
101 2 10.0154 .08 306 286 282850, ,227400,, 222269,, 114879 (+)-5,6-deChryidnraomlyacobriinnee a LYC[5R] [35]
13 14.79 290 272,233,215,189 lycoramine GA[1]
11 12.90 288 270, 252, 222, 177, 147, 119, 95 Lycorine LY [1,17]
14 17.40 288 270,231,225,213,198,181 Galanthamine GA[17]
121 5 14.0188 .24 286 306 288,224570,2, 3234,02,2 29,2261, 51,4270 1,189 ly(c+o)r-a5m,6i-ndeeNh-yodxirdoelycorine GA[1L5,Y25 []5]
131 6 14.7292 .04 290 264 2472,71289, ,213636,, 214195,, 113839, 116 Hipplyacdoinreamine LY[G32A] [1]
17 23.23 288 270,255,239,193,162,151,121,108,94 Pluviine LY[16,19]
14 17.40 288 270, 231, 225, 213, 198, 181 Galanthamine GA [17]
18 24.20 262 244,228,219,205,179,165,147,123,98,91 Unidentified
151 9 18.2246 .58 306 302 228884,, 226467,, 225353,,1 9232,91,7 251,514, 52,0110,8 1,8949 lyOcodrualmineine N-oxide HGOA[3 6[]15,25]
162 0 22.0246 .74 264 332 3002,4278,2 1,82964, ,126364,, 124259,, 211333,,1 9191,61 69 AmbHelliipnepaadine CR[1L7Y,3 4[]32]
17 23.23 288 270,255,239, 193,1 62, 151,121, 108, 94 Pluviine LY [16,19]
18 24.20 262 244, 228, 219, 205, 179, 165, 147, 123, 98, 91 Unidentified
19 26.58 302 284, 266, 255, 193, 175, 145, 108, 94 Oduline HO [36]
20 26.74 332 300, 282, 264, 234, 225, 213, 199, 169 Ambelline a CR [17,34]
21857
4
Molecules2015,20,21854–21869
Table1.Cont.
PNeaok. (mRitn) [MPI-+MHS]+ MS/MSData Identification AlkaloidType/Ref.
21 27.33 272 254,242,226,149,136,108 Vittatine CR[17]
22 27.88 318 300,286,268,250,227,209,199,149 Crinamidinea CR[17,33]
23 29.08 332 300,282,275,267,255,243,223,195,124 HippeastrineN-oxide HO[37]
24 29.15 286 255,229,225,197,179,168,58 Narwedinea GA[28]
25 29.64 302 270,259,226,211,196,181,168 Haemanthamine CR[17]
26 30.96 316 298,280,273,239,222,191,126,96,83 hippeastrine HO[36]
27 31.86 332 314,282,253,239,211,223,175,96 2α-Hydroxy-6-O-methyloduline HO[36]
(+)-8,9-methylenedioxylhomolycorine
28 32.82 316 298,280,267,239,207,191,176,160,108,94 HO[5]
N-oxide
29 33.15 318 286,271,267,177 Unidentified
30 33.38 332 300,282,257,251,243,191,163,94, 2-methoxyodulineb HO[36]
31 34.49 334 302,270,259,245,231,217,213,199 3,11-dimethoxy-lycoramineb GA[17]
285,267,256,239,228,
32 35.58 316 Unidentified
207,175,157,129,118
33 36.19 332 300,284,271,251,239,219,191,94,81 Unidentified
34 36.19 266 250,236,222,208,109 Unidentified
314,300,282,271,264,240,
35 36.84 332 Unidentified
224,211,181,153,120,107
36 37.56 346 314,282,253,239,225,211,175,147,96 2α-Methoxy-6-O-methyloduline HO[36]
37 40.20 344 312,280,266,252,195,89 Unidentified
38 41.15 298 270,248,238,212,180 Unidentified
39 44.57 346 288,241,239,211,209,183,168,140,116,94 Unidentified
Abbreviations: GA, galanthamine type; CR, crinine type; TA, tazettine type; HO, homolycorine type; LY,
lycorinetype;a:firstlyreportedinlycorisgenera;b:firstlyreportedinAmaryllidaceaefamily.
2.2.3. IdentificationofGalanthamineTypeAlkaloids
For the identification of galanthamine type alkaloids, the loss of N-methyl and vicinal carbon
atoms was the typically characteristic fragmentation pathways in the MS analysis among AAs.
AccordingtotheMS/MSspectra,sevenpeaks,numberedas1,8,13,14,15,24,and31,wereidentified
andclassifiedintothisgroup.
By comparing the MS/MS data with the authentic standard, peaks 13 and 14 were definitely
identified as lycoramine and galanthamine, respectively [1,17]. For lycoramine (peak 13), several
abundant fragment ions at m/z 272 [M + H ´ H O]+, 233 [M + H ´ C H N]+, and 215
2 3 7
[M+H´H O´C H N]+wereobserved,andanotherfragmentionatm/z189wasattributedtothe
2 3 7
further loss C H (26 Da) from fragment ion at m/z 215. For galanthamine (peak 14), the fragment
2 2
ionatm/z270wasformedbytheneutrallossofH O.ThefurtherlossoftheNvicinalmoietyfrom
2
fragment ion at m/z 270 produced fragment ions at m/z 231, 225, 213 and 198. The fragment ion at
m/z181wasobservedduetothefurtherneutrallossofCH OH(32Da)fromionatm/z213.
3
Figure 3 showed the MS/MS spectrum and the fragmentation pathways of peak 31, in which
several abundant fragments at m/z 231, 213 and 198 were found very similar to galanthamine.
Since peak31showedamolecularionatm/z334([M+H]+)andtwosubsequentfragmentionsat
m/z302([M+H´CH OH]+)andatm/z270([M+H´2CH OH]+),suggestingthepresenceoftwo
3 3
methoxygroups, thusitcanbetentativelyidentifiedas3, 11-dimethoxy-lycoramine[17]. Similarto
MS/MSofgalanthamine,severaltypicalfragmentionsatm/z231,213and198werealsoobservedfor
peak8. Consideringthemolecularionofpeak8atm/z274[M+H]+ (C H NO ),peak8wasthus
16 19 3
tentatively identified as N-demethyl-galanthamine [26,27]. In this way, peak 1 could be tentatively
identifiedas1,11-didehydrogalanthamine,sinceitsmolecularionatm/z284[M+H]+(C H NO ),
19 27 4
andsimilarfragmentionsatm/z213and198tothoseofgalanthaminewereobservedalongwithan
abundantfragmentionatm/z266[17].
In addition, peak 15 was also tentatively identified as lycoramine N-oxide based on the
characteristic fragment ions at m/z 233, 215 and 189 similar to galanthamine and its molecular ion
observed at m/z 306 ([M + H]+), along with some weak fragment ions at m/z 288 and 247 by the
successivelossofH O(18Da)andC H N(53Da)[15,25,26].Asforpeak24,aneutrallossofCH NH
2 3 3 3 2
(31 Da) from the molecular ion at m/z 286 [M + H]+ resulted in a fragment ion at m/z 255, and the
fragment ions at m/z 225, 197 and 179 could be attributed to the successive loss of CH O, CO and
2
H O from fragment ion at m/z 255. Another abundant fragment ion at m/z 229[M+H´C H N]+
2 3 7
21858
Molecules2015,20,21854–21869
Molecules 2015, 20, page–page
wobasse rpvreodbawbalys pprroodbaubcleyd pbryo dtuhcee dlosbsy otfh Ne lomsosieotfy NC3mHo7Nie ty(58C 3DHa7).NTh(u5s8, pDeaak). T2h4u sw,apse taeknt2a4tivwealys
itdenentattiifvieedly aisd neanrtwifieeddinase n[2a8r]w. edine[28].

FFiigguurree 33.. TThhee MMSS//MMSS ssppeeccttrruumm ooff ppeeaakk 3311 ((aa)) aanndd tthhee pprrooppoosseedd ffrraaggmmeennttaattiioonn ppaatthhwwaayyss ((bb))..
2.2.4. Identification of Lycorine Type Alkaloids
2.2.4. IdentificationofLycorineTypeAlkaloids
Through the investigation on fragment pathways of lycorine type alkaloids, the unique
Through the investigation on fragment pathways of lycorine type alkaloids, the unique
structures of this type typically produced characteristic fragment ions via Retro-Diels-Alder (RDA)
structures of this type typically produced characteristic fragment ions via Retro-Diels-Alder (RDA)
cleavages. Based on their MS/MS data, peaks 9, 11, 12, 16, and 17 were identified and classified into
cleavages. BasedontheirMS/MSdata,peaks9,11,12,16,and17wereidentifiedandclassifiedinto
lycorine type alkaloids.
lycorinetypealkaloids.
Among the lycorine type alkaloids identified, both peak 11 and peak 17 exhibited a [M + H]+ ion
at m/z 288. For peak 11, in addition to the RDA cleavages of B and C rings produced two characteristic
fragment ions at m/z 177 [M + H − C6H9NO]+ and 147 [M + H − C7H11NO2]+, the successive loss of H2O,
21859
6
Molecules2015,20,21854–21869
Amongthelycorinetypealkaloidsidentified,bothpeak11andpeak17exhibiteda[M+H]+ion
atm/z288.Forpeak11,inadditiontotheRDAcleavagesofBandCringsproducedtwocharacteristic
fragment ions at m/z 177 [M + H ´ C H NO]+ and 147 [M + H ´ C H NO ]+, the successive
6 9 7 11 2
loss of H O, 2H O and CH O from the parent ion at m/z 288 yielded three fragment ions at m/z
2 2 2
270, 252 and 222, respectively. In reference to the MS/MS data from the authentic standard [1,17],
peak 11 was identified as lycorine. As for peak 17, the loss of C H N (95) and C H O (194)
6 9 11 14 3
from [M + H]+ led to the characteristic fragment ions at m/z 193 and 94 by RDA cleavages and the
fragment ion at m/z 270 was observed due to the loss of H O (18 Da). The further RDA cleavage
2
of fragment at m/z 270 yielded two fragment ions at m/z 255 [M + H ´ H O ´ NH]+ and m/z 239
2
[M+H´H O´CH N]+. Thus,peak17wasfinallyidentifiedaspluviine[16,29]. Inthisway,peak
2 5
9 was also identified based on the typical fragment ions at m/z 272, 254 and 149, which were 2 Da
more than those corresponding ions at m/z 270, 252 and 147 for lycorine. Therefore, peak 9 was
identifiedasdihydrolycorine[26,29–31]. Forpeak12,thelossofH OandC H Ofromthemolecular
2 2 2
ion at m/z 286 [M + H]+ produced abundant fragment ions at m/z 250 [M + H ´ 2H O]+ and 226
2
[M+H´H O´C H O]+ bysuccessiveneutrallosses. Meanwhile,thefragmentionatm/z147was
2 2 2
alsoobservedbythelossofC H NO(121Da)duetoRDAcleavages. Thus,peak12wastentatively
7 7
identifiedas(+)-5,6-dehydrolycorine[5].Inrespecttopeak16,severalabundantfragmentionsatm/z
247,149and166wereproducedmainlyviaRDAcleavages. Asaresult,itwastentativelyidentified
as(+)-5,6-dehydrolycorinebasedonthefragmentpathwaysabove[32].
2.2.5. IdentificationofCrinineTypeAlkaloids
In terms of MS fragmentation pathways of crinine type alkaloids, the abundant fragment ions
observed in the MS/MS spectrum were produced due to both RDA and α-cleavages. As shown
in Table 1, peaks 2, 10, 20, 21, 22 and 25 were classified into crinine type group based on their
characteristicfragmentions.
ComparedtheMS/MSdatawiththosefromauthenticstandards,peak21and25wereidentified
as vittatine and haemanthamine [17]. For peak 21, except for the most intensive and characteristic
fragmention at m/z 136 produced due to the RDA and α-cleavages, the fragment ions at m/z 254,
240,and226wereobservedbysuccessiveneutrallossesfromthemolecularionatm/z272[M+H]+.
Asforpeak25,thecharacteristicfragmentionatm/z259[M+H´43]+ wasproducedbyRDAand
α-cleavages, and the fragment ion at m/z 226 was observed due to the neutral loss of CH OH and
3
furtherdissociationofanHradicalfromthefragmentionatm/z259. Fragmentionsatm/z270,252,
211and181weregeneratedbythesuccessiveneutrallossofCH OH(32Da),H O(18Da),aswellas
3 2
RDAcleavageradicaleliminatingC H N(41Da)andCH O(30Da)fromthemolecularionatm/z
2 3 2
302[M+H]+.
Inregardstopeaks2and22, thesimilarfragmentionsat286, 268, 250and227wereobserved.
Peak2showedamolecularionatm/z304[M+H]+,andadifferentialfragmentionatm/z150was
probablygeneratedbythelossofC H O fromthefragmentionatm/z268. Forpeak22,thelossof
7 2 2
H Ofromthemolecularion[M+H]+ atm/z318producedthefragmentionatm/z300,andthena
2
further loss of H O and CO from fragment ion at m/z 227 resulted in the fragment ions at m/z 209
2
and199,respectively. Basedoninterpretationsofthesefragmentionsabove,peaks2and22couldbe
tentatively identified as 11-hydroxyvittatine N-oxide and crinamidine, respectively [17,27,33]. With
the molecular ion at m/z 332 ([M + H]+), peak 20 exhibited the similar MS/MS spectrum as that
of peak 22 and was identified as ambelline [3,17]. As for peak 10, with the molecular ion at m/z
306 ([M + H]+), its four major fragment ions at m/z 288, 270, 229 and 152 were 2 Da more than
thosecorrespondingfragmentionsatm/z286,268,227and150frompeak2,respectively,alongwith
anothertwodifferentialfragmentionsatm/z215and189probablyproducedduetothecleavageof
Bring. BasedontheseMS/MSdata,peak10wasidentifiedascrinamabinewhichwasreportedfrom
otherplantsinthepast[34].
21860
Molecules2015,20,21854–21869
2.2.6. IdentificationofHomolycorineTypeAlkaloids
As for MS/MS data derived from homolycorine type alkaloids, major fragment ions were
oMbosleecruvlees d201d5u, 2e0,t poagReD–pAagec leavage and the losses of some substituents. Peaks 7, 19, 23, 26, 27, 28, 30
and36wereclassifiedintohomolycorinetypegroupandidentifiedbasedontheirMS/MSdata.
TThhee MMSS//MMSS spspeectcrturumm ofo fpepaeka k7 7wiwthit thheth [eM[ M+ H+]+H io]+n iaot nma/zt 3m3/4z w3a3s4 swhoawsnsh ionw FniguinreF 4igau, arend4 ait,
aconudldit bceo utelndtabteivteelnyt iadtievnetliyfieidd eanst 2ifiαe-dHyadsr2oαx-yH-3y-dhryodxryo--36--hOy-dmreot-h6y-Olo-dmuelitnhey l[o3d5]u blianseed[3 o5n] bthaes epdroopnosthede
pfrraogpmoseendt pfraatghmweanyts pinat Fhiwguayres 4inb.F igure4b.

FFiigguurree 44.. TThhee MMSS//MMSS ssppeeccttrruumm ooff ppeeaakk 77 ((aa)) aanndd tthhee pprrooppoosseedd ffrraaggmmeennttaattiioonn ppaatthhwwaayyss ((bb))..
Peaks 19, 26, 27 and 36, which showed the corresponding [M + H]+ ions at m/z 302, 316, 332 and
Peaks19,26,27and36,whichshowedthecorresponding[M+H]+ionsat m/z302,316,332and
346, respectively, were identified as oduline, hippeastrine and 2α-Hydroxy-6-O-methyloduline,
346, respectively, were identified as oduline, hippeastrine and 2α-Hydroxy-6-O-methyloduline,
2α-Methoxy-6-O-methyloduline by comparing their MS/MS spectra with those reported standards [35].
2α-Methoxy-6-O-methyloduline by comparing their MS/MS spectra with those reported
In the MS/MS spectrum of peak 23, fragment ions at m/z 275, 267, 255, 243 and 223 were produced
standards [35]. In the MS/MS spectrum of peak 23, fragment ions at m/z 275, 267, 255, 243
mainly due to the loss of N vicinal moiety, the fragment ion at m/z 195 was observed because of the
and223wereproducedmainlyduetothelossofNvicinalmoiety,thefragmentionatm/z195was
further loss of CO from the fragment ion at m/z 223, and another fragment ion at m/z 124 was produced
observedbecauseofthefurtherlossofCOfromthefragmentionatm/z223,andanotherfragment
by RDA cleavage. Thus, peak 23 was identified as hippeastrine N-oxide [36].
In regard to peak 28, the abundant fragment ion at m/z 207 was produced by RDA cleavage from
its [M + H]+ ion at m/z 316. Comparing the MS/M21S8 d6a1ta with those of peak 26, the fragment ion at m/z
126 was not observed, suggesting the lack of a hydroxyl at C-2. Thus, peak 28 was tentatively
identified as (+)-8,9-methylenedioxyl-homolycorine N-oxide [5]. For peak 30, the fragment ion at m/z
300 corresponding to the loss of methyl group from its parent ion at m/z 316 [M + H]+ , and another
8
Molecules2015,20,21854–21869
ion at m/z 124 was produced by RDA cleavage. Thus, peak 23 was identified as hippeastrine
N-oxide[36].
Inregardtopeak28,theabundantfragmentionatm/z207wasproducedbyRDAcleavagefrom
its[M+H]+ ionatm/z316. ComparingtheMS/MSdatawiththoseofpeak26,thefragmentionat
m/z 126 was not observed, suggesting the lack of a hydroxyl at C-2. Thus, peak 28 was tentatively
identified as (+)-8,9-methylenedioxyl-homolycorine N-oxide [5]. For peak 30, the fragment ion at
m/z 300 corresponding to the loss of methyl group from its parent ion at m/z 316 [M + H]+ , and
anothMeorlecfurlaesg 2m01e5,n 2t0, ipoange–aptagme /z 191 was observed due to the loss of C H NO (141 Da) produced by
8 15
RDAcleavage. Therefore,peak30wastentativelyidentifiedas2-methoxyoduline[35].
fragment ion at m/z 191 was observed due to the loss of C8H15NO (141 Da) produced by RDA cleavage.
Therefore, peak 30 was tentatively identified as 2-methoxyoduline [35].
2.2.7. IdentificationofTazettineTypeAlkaloids
A2.c2c.7o. rIddienngtifticoattihonei orf MTaSz/eMttinSe dTaytpae aAnlkdaltohiedsp roposed fragmentation patterns, peaks 3, 4 and 5
could be classified as tazettine type alkaloids. The MS/MS spectrum and the proposed fragment
According to their MS/MS data and the proposed fragmentation patterns, peaks 3, 4 and 5 could
pathwbea yclaosfsipfieeadk a5s twazeertetinseh otywpne ainlkaFliogidusr.e T5h.e IMnSt/hMeSM spSe/cMtruSmsp aencdt rtaheo pfrpoepaokse3d afrnadgm5,entht epastahmweays eries
of fraogfm peeankt 5i ownesrea sthmow/zn i2n2 F5i,g2u1re7 ,5.1 I9n9 thaen MdS1/8M1Sw speercetrdae otfe pcteeadk, 3a anndd t5h, ethen esaumtrea lselroiesss eosf forfagHm2eOnt from
thecoiornres sapt omn/zd 2in25g, [2M17,+ 19H9 ]a+ndio 1n8s1 awtemre/ dzet2e9c0teadn, adn2d7 t4hep nreoudturacle ldosfsreasg omf Hen2Ot iforonms athtem c/orzre2s7p2onfodrinpge ak3
andm[M/z +2 H56]+ fioonrsp aeta mk/5z ,2r9e0s panedct 2iv7e4 lpy.roBdyucceodm fpraagrminegntt hioenirs Mat Sm//zM 2S72s pfoerc ptreaakw 3i tahndth mos/ze 2o5f6 tfhoer paueatkh entic
stand5a, rrdesspreecptiovretleyd. ,Bpy ecaokmp3arwinags tihdeeirn MtifiSe/MdSa ssp3e-chtryad wroitxhy ltahtoisfea loiuf mthien auCt,hwenhtiicl estpaenadkar5dsw reapsoirdteedn,t ified
peak 3 was identified as 3-hydroxylatifaliumin C, while peak 5 was identified as Latifaliumin C [33].
as Latifaliumin C [33]. For peak 4, the major fragment ions at m/z 258, 219, 201 and 173 were 2 Da
For peak 4, the major fragment ions at m/z 258, 219, 201 and 173 were 2 Da more than those
morethanthosecorrespondingfragmentionsatm/z256,217,199and171ofpeak5. Inaddition,the
corresponding fragment ions at m/z 256, 217, 199 and 171 of peak 5. In addition, the molecular ion at
molecular ion at m/z 276 of peak 4 also showed 2 Da more than that of peak 5 and was tentatively
m/z 276 of peak 4 also showed 2 Da more than that of peak 5 and was tentatively identified as dihydro-
identifiedasdihydro-latifaliuminC[33].
latifaliumin C [33].

FigurFeig5u.rTe h5e. TMheS /MMS/SMsSp sepcetcrturummo offp peeaakk 55 ((aa)) aanndd ththee pprorpoopsoesde fdrafgramgemnteantitoant ipoanthpwaathyws (aby).s (b).
2.3. Comparison of AAs among the Three Lycoris Species
Based on the identification of AAs from the three Lycoris species above, a comparison of AAs
21862
among different species was conducted, which was depicted in Figure 6a. It can be summarized that
19 alkaloids were found in the sample of L. aurea, while 28 and 18 in L. radiata and L. guangxiensis,
respectively. Of these, nine AAs (peaks numbered with 4, 8, 11, 13, 14, 17, 19, 21 and 22) are the
common components existing in all three species, and some components were found only in two of
9
Molecules2015,20,21854–21869
2.3. ComparisonofAAsamongtheThreeLycorisSpecies
Based on the identification of AAs from the three Lycoris species above, a comparison of AAs
amongdifferentspecieswasconducted,whichwasdepictedinFigure6a. Itcanbesummarizedthat
19 alkaloids were found in the sample of L. aurea, while 28 and 18 in L. radiata and L. guangxiensis,
respectively. Of these, nine AAs (peaks numbered with 4, 8, 11, 13, 14, 17, 19, 21 and 22) are the
Molecules 2015, 20, page–page
commoncomponentsexistinginallthreespecies,andsomecomponentswerefoundonlyintwoof
tthheemm,, wwhhiicchh aarree ffoouurr AAAAss ((ppeeaakkss nnuummbbeerreedd aass 22,, 1155,, 2255,, aanndd 2266)) iinn LL.. rraaddiiaattaa aanndd LL.. aauurreeaa,, ttwwoo AAAAss
((ppeeaakkss nnuummbbeerreedd aass 33 aanndd1 100))i innL L..r raaddiaiattaaa anndd LL.. gguuaannggxxiieennssiiss,, aanndd ttwwoo AAAAss ((ppeeaakkss nnuummbbeerreedd aass 99 aanndd
2244)) iinn LL.. aauurreeaa aanndd LL.. gguuaannggxxiieennssiiss, ,rreessppeecctitviveelyly. .TToo fifinndd ppotoetnentitaila AlAAAs smmarakrekresr fsofro trhteh iedeidnetinfitcifiatciaotnio onr
oqruqaulitayli tcyoncotrnotlr oolf odfidffieffreernetn stpsepceiceise sfrformom ggenenuus sLLyyccoorirsi,s ,uunniqiquuee ppeeaakkss ooff tthheessee tthhrreeee ssppeecciieess wweerree
ssuucccceessssffuullllyy ppiicckkeedd oouutt,, wwhhiicchh aarree ffoouurr AAAAss ((ppeeaakkss nnuummbbeerreedd wwiitthh 55,, 3333,, 3377,, aanndd3388 )) iinn LL.. aauurreeaa,, 1144
AAAAss ((ppeeaakkss nnuummbbeerreedd aass 11,, 66,, 77, ,1122, ,2200, ,232,3 2,72,7 2,82, 82,92,390,,3 302,, 3324,, 3345,, 3a5n,da 3n9d) i3n9 )L.i nraLd.iartaad, iaantad, faonudr fAoAurs
A(pAesak(ps enaukmsnbuemrebde aresd 1a6s, 1168,, 1381,, 3a1n,dan 3d6)3 6in) ingugaunagnxgixeniesniss,i sr,ersepsepcetcitvievleyl.y .BBrireieflflyy, ,tthhee ccrroossssccuuttttiinngg aanndd
iinncclluussiioonn rreellaattiioonnsshhiippss ooff AAAAss ffoouunndd iinn tthheessee tthhrreeee LLyyccoorriiss ssppeecciieess ccaann pprroovviiddee ggrreeaatt kknnoowwlleeddggee oonn
bbootthh qquuaalliittyy ccoonnttrrooll ffoorr tthheessee ssppeecciieess aanndd bbeetttteerr uunnddeerrssttaannddiinngg ooff tthhee ccoorrrreellaattiioonn bbeettwweeeenn AAAAss aanndd
tthheeiirr aassssoocciiaatteedd aaccttiivviittiieess..

Figure 6. The number of AAs (a) and relative contents of five types of AAs (b) detected from L. radiata,
Figure6.ThenumberofAAs(a)andrelativecontentsoffivetypesofAAs(b)detectedfromL.radiata,
L. aurea and L. guangxiensis.
L.aureaandL.guangxiensis.
Since the evaluation of bioactivity of AAs in one plant species depends on both the qualitative
and quantitative knowledge, the quantitative information of the five types of AAs in the three Lycoris
species was then investigated based on their LC2 c1h8r6o3matograms, which was shown in Figure 6b. It can
be seen that galanthamine and lycorine type alkaloids were the two major types of AAs accounting for
55.3% and 19.1% of galanthamine and lycorine type in L. aurea, 40.1% and 26.9% of galanthamine and
lycorine type in L. radiate and 64.4% and 25.6% of galanthamine and lycorine type in L. guangxiensis,
respectively. However, the discrepancies were mainly found in the contents of the other three types
among the three species. In L. aurea, those three types account for about 25.6% from the total AAs, which
are 1.8%, 14.6%, and 9.2% for homolylcorine, crinine, and tazettine types of alkaloids, respectively.
10