* CorrespNaturals, BBarcelona,E‐mail: jau

PHYTOTHERAPY RESEARCHPhytother. Res. 25: 1686–1692 (2011)Published online 25 March 2011 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/ptr.3468

Copyright

Antiproliferative Alkaloids fromCrinum zeylanicum

Strahil Berkov,1,2 Stefania Romani,3 Maria Herrera,4 Francesc Viladomat,1 Carles Codina,1Georgi Momekov,5 Iliana Ionkova5 and Jaume Bastida1*1Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n,08028 Barcelona, Catalonia, Spain2AgroBioInstitute, 8 Dragan Tzankov Blvd, 1164 ‐ Sofia, Bulgaria3Departament de Quimica Farmacèutica, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona,Catalonia, Spain4Universidad de Camagüey, Facultad de Química, Departamento de Química, Circunvalación Norte Km 5½, Camagüey, Cuba5Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str., 1000, Sofia, Bulgaria

Crinum zeylanicum is used in folk medicine as a rubefacient in rheumatism, a treatment for malaria or as apoison. Complex alkaloid profiles in C. zeylanicum plant organs were revealed by GC‐MS analysis, includingseveral bioactive compounds. Crinine, lycorine, 11‐O‐acetoxyambelline, ambelline, 6‐hydroxybuphanidrine and6‐ethoxybuphanidrine (an artefact of the isolation procedure) were isolated. Crinine, 6‐hydroxybuphanidrineand 6‐ethoxybuphanidrine showed antiproliferative effects against human tumor cell lines, crinine being themost active (IC50 14.04μM against HL‐60/Dox). The latter compound induced apoptosis in a dose‐dependentmanner in HL‐60 and MDA‐MB‐231 cell lines. Structure‐activity relationships in the studied moleculesindicated that the hydrogenation of the double bond at C1‐C2 leads to a loss of activity, whereas substitutions atC6, C8 and C11 affect their cytotoxicity. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: Crinum zeylanicum; Amaryllidaceae alkaloids; apoptosis; cytotoxicity.

INTRODUCTION

The genus Crinum L. is the only pantropical genus ofthe Amaryllidaceae, with species occurring in Africa,America, Asia and Australia (Meerow et al., 2003).Crinum zeylanicum L. is native to western India and SriLanka where it is used as a folk medicine under the nameof Goda‐manel (Yakandawala and Samarakoon, 2006)as a rubefacient in rheumatism and remedy for earache(Tsuda et al., 1984). The plant is also used to treat malariain the Dominican Republic and as a poison in theMoluccas (Fennell and Staden, 2001). Aqueous andethanol extracts from the plant have shown a hightoxicity against molluscan intermediate hosts of schisto-somiasis and fascioliasis (Chifundera et al., 1993).Significant efforts have been focused on the discovery of

compounds with selective cytotoxicity against cancer cellsin natural sources including the amaryllidaceous genusCrinum (Fennell and Staden, 2001). Some anticanceragents cause cell death by interfering with the processes ofthe cell cycle (Dirsch et al., 2002) and others cause celldeath by apoptosis (McNulty et al., 2009), which plays animportant role in the balance between cell replication andcell death.Amaryllidaceae alkaloids of different structuraltypes, such as pancratistatine (McLachlan et al., 2005),lycorine (Liu et al., 2004), haemanthamine and crinamine(McNulty et al., 2009) have shown prominent cytotoxicand apoptotic‐inducing activities.

ondence to: Dr Jaume Bastida, Departament de Productesiologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat deAv. Joan XXIII s/n, 08028 Barcelona, Spain.mebastida@ub.edu

© 2011 John Wiley & Sons, Ltd.

Ten alkaloids have been reported for C. zeylanicum,namely crinidine (synonymous with crinine), flexinine,6‐hydroxypowelline, zeylamine, lycorine, hamayne,3‐acetylhamayne, crinamine, 6‐hydroxycrinamine and6‐methoxycrinamine (Fales et al., 1959; Tsuda et al., 1984;Doepke et al., 1986; Trimiño et al., 1988).

As part of our ongoing studies on Amaryllidaceaeplants, the aim of the present work onC. zeylanicum is, onthe one hand, to re‐investigate its alkaloid composition insearch of bioactive compounds, and on the other, to studythe antiproliferative activity of some isolated compoundsfrom this plant.

MATERIALS AND METHODS

General procedures. CC and VLC were carried out withsilica‐gel (40–63 and 6–36μm mesh, respectively, SDS,France) and preparative TLC with silica‐gel pre‐coatedplates 60 F254 (SDS, France). Fractions were monitoredby TLC (UV light and Dragendorff’s reagent). The CDspectra were recorded on a Jacso‐810 spectropolarimeterin MeOH. The UV spectra were recorded on a HitachiU‐2000 spectrophotometer. The NMR spectra were runon a Gemini 300MHz, Mercury 400MHz or Varian VXR500MHz, using CDCl3 as a solvent and TMS as aninternal standard. Chemical shifts were reported in δ units(ppm) and coupling constants (J) in Hz. EIMS wereobtainedon aGC‐MSHewlett‐Packard 6890+MSD5975.HR‐ESI‐MS spectra were recorded on a LC/MSD‐TOF(2006) mass spectrometer (Agilent Technologies) bydirect injection of the compounds dissolved in H2O:CH3CN (1:1).

Received 01 September 2010Revised 08 February 2011

Accepted 09 February 2011

1687ALKALOIDS FROM CRINUM ZEYLANICUM

Plant material. Plants from Crinum zeylanicum (L) grow-ing in the vicinity of Camagüey, province of Camagüey,Cuba, were collected in the spring of 2008 during theflowering period. Dr Isidro Mendez identified the plantsand a voucher specimen (HIPC10465)was deposited in theHerbarium of the Universidad de Ciencias Pedagógicas,José Martí, Camagüey, Cuba.

Alkaloid extraction and isolation. Freshbulbs (ca 0.9 kg),leaves (ca 0.7 kg), flowers (ca 0.4 kg), fruits (ca 0.5 kg),stems (ca 0.5 kg) and fruit peduncles (ca 0.5 kg) werecrushed and extracted separately with EtOH (3×1.5L,72 h each). The solvents were evaporated under reducedpressure and the extracts were suspended in 200mLof 2% H2SO4 and filtered. The neutral material wasremoved by extraction with Et2O (3×200mL). Afterbasification of the solutions with 20%NH4OH (pH 9–10),the alkaloids were extracted with EtOAc (3× 250mL).The alkaloid extracts were subjected to GC‐MS analysisand combined after comparing the alkaloid profiles. Twomain alkaloid mixtures, A (1.27 g from aerial parts) and B(0.62 g from the bulbs) were obtained and subjected toCCand VLC, respectively. The alkaloids were eluted withEtOAc/n‐hexane (9:1) and EtOAc gradually enrichedwith MeOH to give six main fractions (I–VI, combinedaccording to their TLC profiles) from each mixture.Lycorine (24, 21mg) was crystallized in MeOH fromfraction V of mixture A. The rest of the alkaloids wereisolated by preparative TLC from fractions of mixturesAandBas follows: 6‐ethoxybuphanidrine 16 (15mg) fromfraction I (EtOAc/MeOH/NH4OH, 9:1:0.1) of extractA, 6‐hydroxybuphanidrine 28 (17mg) from fractions II,11‐O‐acetylambelline 26 (3mg) from fractions III,(EtOAc/MeOH/NH4OH, 9:1:0.1) ambelline 27 (5mg)from fractions IV and crinine 7 (3mg) from fractions VI(EtOAc/MeOH/NH4OH, 4:1:0.1) of both mixtures.

GC‐MS analysis and alkaloid identification. GC‐MSanalyses were performed on a Hewlett Packard 6890+/MSD 5975 instrument (Hewlett Packard, Palo Alto, CA,USA) operating in EI mode at 70 eV. An HP‐5 MS col-umn (30m×0.25 mm×0.25μm) was used. The temper-ature program was: 100–180 °C at 15 °C×min−1, 180–300at 5 °C×min−1 and 10min hold at 300 °C. The injector tem-perature was 250 °C. The flow rate of carrier gas (helium)was 0.8mL×min−1. The split ratio was 1:20. 1μL of thesolution was injected.The alkaloids were identified by comparing their MS

spectra andRI (Kovats retention index) values with thoseof previously isolated standards identified by otherspectroscopic methods (NMR, UV, CD), comparing themass spectral fragmentation of the compounds with thoseof standard reference spectra from the NIST 05 database,and comparing the obtained spectra with those reportedin the literature, or with closely related standardcompounds (Table 1). TheMS spectra were deconvolutedbyAMDIS 2.64 software (NIST) before their comparisonwith the databases and literature. Kovats retentionindexes (RI) of the compounds were recorded with astandard calibration n‐hydrocarbon mixture (C9‐C36)using AMDIS 2.64 software (NIST).The isolated compounds 7, 26 (Viladomat et al., 1995),

24 (Berkov et al., 2009), 27 and 28 (Nair et al., 2005) wereidentified by direct comparison of their chromatographicand spectroscopic properties (TLC, GC‐MS, CD and

Copyright © 2011 John Wiley & Sons, Ltd.

1H‐NMR) with those of authentic compounds isolatedin our laboratory.

6‐Ethoxybuphanidrine (16).CD [Θ]λ20: [Θ]253 +1467, [Θ]

285 ‐1946. HR‐ESI‐MS: m/z 360.1802 (calcd 360.1805 forC20H26NO5).

1H‐NMR (300MHz, CDCl3): δ 1.26 (3H, t,J=7.2Hz, ‐CH2CH3), 1.59 (1H, ddd, J=13.5, 13.5, 4.2Hz,H‐4β), 1.80–1.89 (2H, m, H‐11exo and H‐11endo), 1.98(lH, ddt, J=13.8, 4.2, 1.8Hz, H‐4Θ), 2.69 (lH, ddd, J=13.5,8.7, 6.6Hz, H‐12endo), 3.28 (lH, ddd, 13.8, 9.6, 4.8Hz, H‐12exo), 3.33 (3H, s, ‐OCH3), 3.72 (1H, dd, J=9.3, 7.2Hz, ‐CH2ACH3), 3.75 (lH, dd, 13.8, 4.5Hz, H‐4a), 3.84 (1H, m,H‐3), 3.97 (3H, s, 7‐OCH3), 4.02 (1H, dd, J=9.3, 7.2Hz, ‐CH2BCH3), 4.77 (1H, s, H‐6), 5.84–5.90 (2H, 2d, J=1.2Hz,OCH2O), 5.95 (1H, ddd, 10.2, 5.1, 1.2Hz, H‐2), 6.54 (lH, d,J=7.2Hz, H‐2), 6.57 (lH, s, H‐10). 13CNMR (50MHz,CDCl3): δ 15.6 (q, ‐CH2CH3), 28.8 (t, C‐4), 41.2 (t, C‐11), 44.2 (s, C‐10b), 47.6 (t, C‐12), 3.33 (q, 3‐OCH3), 56.4(d, C‐4a), 59.7 (q, 7‐OCH3), 64.0 (t, ‐CH2CH3). 72.3 (d, C‐3), 91.9(d, C‐6), 97.1 (d, C‐10), 100.8 (t, OCH2O), 119.2 (s,C‐6a), 125.6 (d, C‐2), 132.4 (d, C‐1), 140.0 (s, C‐10a), 142.9(s, C‐7), 149.3 (s, C‐9).

Copies of the original spectra of all isolated com-pounds are obtainable from the corresponding author.

Cell lines and culture conditions. The pharmacologicalstudy was carried out on a panel of human tumor cell linespurchased from the German Collection of Microorgan-isms and Cell Cultures (DSMZ GmbH, Braunschweig,Germany). The tested lines, namely HL‐60 (acutemyeloid leukemia), HL‐60/Dox (multi‐drug resistantsubline), SKW‐3 (a KE‐37 derivative, T‐cell leukemia),HT‐29 (a human colon adenocarcinoma cell line) andMDA‐MB‐231 (ER‐negative breast carcinoma) originatefrom solid tumors and leukemias. The cells weremaintained at 37 °C in an incubator ‘BB 16‐FunctionLine’ Heraeus (Kendro, Hanau, Germany) with humidi-fied atmosphere and 5% CO2. Cells were kept in the logphase by supplementation with fresh growth medium twoor three times aweek. The growthmedium for all cell lineswas 90% RPMI‐1640, supplemented with L‐glutamineand 10% FBS. The solid tumor cell lines (MDA‐MB andHT‐29) were reset by hot trypsinization two times perweek.

Cytotoxicity assessment (MTT‐dye reduction assay).The cell viability, after continuous exposure to the testedextracts was assessed using the standard MTT‐dye(Mosmann, 1983) with small modifications (Konstantinovet al., 1999). For the MTT‐assay exponentially growingcells were seeded in 96‐well flat‐bottomed microplates.After 24h incubation at 37 °C they were exposed tovarious concentrations of the tested extracts or thereference drug topotecan (Hycamptin®) for 72h. Foreach concentration a set of at least 8 wells was used. Afterthe treatment, 10μL MTT solution (10mg/mL in PBS)aliquots were added to each well. The microplates werefurther incubated for 4 h at 37 °C and theMTT–formazancrystals formed were dissolved through addition of100μL/well 5% formic acid (in 2‐propanol). The absorp-tion was read on a microprocessor‐controlled LabeximLMR‐1 microplate reader at 580 nm.

Apoptosis assay. The extent of apoptosis was examinedusing a commercially available ‘Cell‐death detection’ELISA kit (Roche Applied Science). This method allowssemi‐quantitative determination of the histone‐associated

Phytother. Res. 25: 1686–1692 (2011)

1688 S. BERKOV ET AL.

mono‐ and oligonucleosomal DNA‐fragments character-istic of apoptosis using ‘sandwich’ ELISA. Exponentiallygrowing cells were exposed to varying concentrations ofthe tested compounds and thereafter cytosolic fractionsof 1 × 104 cells per group (treated or untreated) served asan antigen source in a sandwich ELISA, using a primaryanti‐histone antibody‐coated microplate and a secondaryperoxidase‐conjugated anti‐DNA antibody. The photo-metric immunoassay for histone‐associated DNAfragments was executed according to the manufac-turers’ instructions at 405 nm, using an ELISA reader(Labexim LMR‐1). The results are expressed as theoligonucleosome enrichment factor (representing a ratiobetween the absorption in the treated vs the untreatedcontrol samples).

Data processing and statistics. The cell survival datawerenormalized as a percentage of the untreated control (set as100% viability), fitted to sigmoidal dose–response curvesand the corresponding IC50 values (concentrationscausing 50% suppression of cellular viability) were

NMe

OOR1

R2O

(3) R1=H, R2=Me(4) R1=H, R2=H(11) R1=Ac, R2=H

R

R

(5(7(1(1(1(1(2(2(2

N

O

O9

N

O

O17

HN

HO

MeO

HO 18

R4

R3

NH2

HO

1

Figure 1. Structures of the alkaloids identified in Crinum zeylanicum.

Copyright © 2011 John Wiley & Sons, Ltd.

calculated using non‐linear regression analysis (GraphPadPrizm Software for PC). The statistical processing ofbiological data included the Student’s t‐test wherebyvalues of p≤ 0.05 were considered as statistically sig-nificant. In addition, the resistance indices as a relativemerit for the level of resistance in HL‐60/Dox weredetermined as the ratio between the IC50 in themulti‐drugresistant HL‐60/Dox and the corresponding IC50 in thesensitive parent line HL‐60.

RESULTS AND DISCUSSION

Alkaloids of C. zeylanicum

As a strategy to search for interesting or potentially newbioactive compounds, aGC‐MSanalysis of different plantorgans from C. zeylanicum was carried out. Twenty‐nineAmaryllidaceae alkaloids, mainly galanthamine, crinineand lycorine types aswell as tyramine‐type protoalkaloids,

N

R2

R3R4

5O

6O

OR1

H

) R1=Me, R2, R3, R4=H, R5+R6=CH2

) R1, R2, R3, R4=H, R5+R6=CH2

0) R1, R2, R3, R4=H, R5=H, R6=CH3

5) R1=Me, R2, R3=H, R4=OMe, R5+R6=CH2

6) R1=Me, R2=H, R3=OEt, R4=OMe, R5+R6=CH2

9) R1, R2, R3=H, R4=OMe, R5+R6=CH2

6) R1=Me, R2=OAc, R3=H, R4=OMe, R5+R6=CH27) R1=Me, R2=OH, R3=H, R4=OMe, R5+R6=CH28) R1=Me, R2=H, R3=OH, R4=OMe, R5+R6=CH2

N

R2

R1

(13) R1=H, R2=OH, R3=H, R4=Me(24) R1, R2=OH, R3+R4=CH2

N

OMe

OMe

O

O

O

25

11

12

3

4

4a

66a

78

910

10a10b

12

H

H

H

O

O

Phytother. Res. 25: 1686–1692 (2011)

Table1.

Alkaloids

detected

inCrinu

mzeylan

icum

byGC‐M

Spresen

tedas

ape

rcen

tage

oftotalioncurren

t(T

IC)

Alkaloid

Rt

M+

Cha

racteristicions

(m/z)

Fruits

Fruit

pedu

ncles

Leav

esStems

Flow

ers

Bulbs

Tyramine(1)a

7.47

137(26)

120(3)108(100)107(90)91(9)77(35)65(5)30(96)

0.1

A1(2)

18.55

253(56)

252(100)250(9)225(24)224(37)166(16)165(12)128(8)113(7)

>0.1

Galan

tham

ine(3)b

21.73

287(87)

286(100)270(44)216(43)174(36)73(63)58(86)57(47)45(69)

>0.1

San

guinine(4)b

22.09

273(100)

272(81)256(20)216(13)202(34)165(14)160(41)152(13)115(16)

0.7

3.1

7.9

2.1

Bup

hanisine

(5)a

22.26

285(100)

270(31)254(33)215(80)201(23)157(18)128(20)115(28)

3.7

2.1

1.9

7.6

2.7

A2(6)

22.66

287(100)

272(45)256(41)232(18)218(20)217(83)203(25)174(21)157(22)

0.4

0.1

Crinine

(7)b

23.05

271(100)

228(21)199(61)187(54)173(18)128(18)115(19)

6.1

1.9

35

10.8

A3(8)

23.19

313(100)

312(48)284(40)270(36)256(25)255(23)228(40)98(67)70(29)

0.2

Anh

ydrolyco

rine

(9)a

23.40

251(43)

250(100)192(10)191(10)190(4)163(3)124(6)96(6)

0.1

0.4

Mac

owine(10)b

23.46

273(100)

230(18)201(88)189(48)175(19)174(14)157(14)128(16)56(20)

0.6

6.5

0.2

3‐O

‐Ace

tylsan

guinine(11)b

23.65

315(47)

314(17)256(100)254(34)212(21)202(20)197(16)165(19)96(33)

0.6

2.9

1.8

4.8

13.8

7.4

A4(12)

23.98

287(100)

286(47)230(99)215(19)214(21)202(28)201(32)189(29)188(22)

0.1

Norpluv

iine(13)a

24.10

273(71)

272(41)254(48)242(8)230(7)229(48)228(100)129(7)94(6)

>0.1

A5(14)

24.41

301(100)

228(33)187(23)175(84)173(18)159(28)143(37)128(17)115(43)

1.6

Bup

hanidrine(15)b

25.14

315(100)

300(30)284(33)260(31)245(53)231(21)228(19)215(16)202(16)

10.1

6‐Ethox

ybup

hanidrine(16)b

25.53

359(9)

315(14)314(38)304(16)288(19)287(100)272(25)259(43)258

(15)254(18)227(13)

7.6

10.9

23.9

12.5

0.8

11,12‐Deh

ydroan

hydrolyc

orine(17)c

24.93

289(8)

288(11)250(10)249(59)248(100)230(9)191(9)190(23)163(7)95(15)

2.3

3.1

1.1

3.8

2.5

0.1

O‐M

ethy

lnorbe

lladine

(18)c

25.24

273(3)

166(28)137(100)138(9)122(6)107(5)94(4)77(3)

0.2

Pow

ellin

e(19)a

25.82

301(100)

282(16)258(35)230(22)229(81)217(45)203(22)202(18)115(29)

2.0

0.2

A6(20)

25.85

345(66)

330(66)314(35)301(46)287(83)286(100)261(81)255(87)229(57)59(49)

4.6

A7(21)b

26.12

317(−)

302(17)286(18)270(19)255(24)246(17)231(41)214(18)201(18)115(19)

0.2

A8(22)

26.29

331(23)

299(25)288(32)287(100)270(17)259(15)255(39)253(14)229(15)

8.4

11.2

A9(23)

26.57

331(100)

273(58)255(100)165(15)151(11)240(18)203(17)139(30)128(17)

0.3

Lyco

rine

(24)b

26.60

287(25)

286(16)268(20)251(60)250(19)228(10)227(65)226(100)147(9)119(6)

16.4

10.0

0.4

0.4

26.6

1.0

Und

ulatine(25)a

26.62

331(100)

300(10)286(15)258(35)205(69)203(31)189(41)173(40)

4.2

10.9

4.9

4.7

1.0

11‐O

‐Ace

tylambe

lline

(26)b

27.10

373(52)

331(100)314(32)300(25)258(54)254(36)218(30)217(29)205(59)203(30)

8.9

10.8

13.4

1.1

Ambe

lline

(27)b

27.40

331(86)

299(42)287(100)260(74)257(53)255(65)254(41)241(43)239(45)211

(49)

45.8

60.2

32.6

52.0

16.0

6‐H

ydroxy

buph

anidrine

(28)b

27.54

331(50)

277(16)276(100)261(28)218(20)217(21)216(18)115(26)56(42)45(18)

0.1

16.0

0.1

29.5

A10(29)

28.41

317(96)

299(35)269(36)262(36)245(47)244(100)23(71)216(44)215(42)

0.2

Iden

tifica

tion

:aNIST05da

taba

se;bco

‐chrom

atog

raph

ywithisolated

stan

dard;cliteratureda

ta.

1689ALKALOIDS FROM CRINUM ZEYLANICUM

Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. 25: 1686–1692 (2011)

1690 S. BERKOV ET AL.

were detected (Figure 1, Tables 1 and 2). Nineteen ofthem were identified as indicated in Table 1. Theconfiguration of the 5,10b‐ethano bridge in the crininealkaloids was tentatively assigned by analogy withthose of the isolated compounds. The plant organsdisplayed different alkaloid profiles, dominated bycrinine‐type alkaloids. Fruits, fruit stems and flowersshowed relatively high amounts of lycorine‐typecompounds and flowers of galanthamine‐type com-pounds. Many of the detected compounds, includingsome of the major alkaloids, have been poorly or notstudied with respect to their bioactivity.Fractionation of extracts from the aerial parts

and bulbs resulted in the isolation of six alkaloids:crinine (7), 6‐ethoxybuphanidrine (16), lycorine (24),11‐O‐acetoxyambelline (26), ambelline (27) and6‐hydroxybupanidrine (28). In contrast to previous stud-ies, all the compounds of the crinane series displayedβ configuration of their 5,10b‐ethano bridge after CDanalysis (De Angelis and Wildman, 1969).6‐Ethoxybuphanidrine (16), which has been reported

only once as an artificially obtained compound from6‐hydroxybuphanidrine (Slabaugh and Wildman, 1971),was isolated as an amorphous compound. A GC‐MSanalysis of a solution of 6‐hydroxybuphanidrine in absoluteethanol left at room temperature for a week showed thepresenceof 16, indicating that it ismost probably anartefactof the isolation procedure and should be considered as6‐hydroxybuphanidrine. Structural determinationof16wasachievedby 2Dhomo‐ andheteronuclear experiments, andCDandHR‐MSspectroscopy. In theMaterial andMethodssection we provide for the first time a full description of 1Hand 13CNMR spectra of this bioactivemolecule.With exceptions of crinine (7) and lycorine (24), the rest

of the alkaloids are reported for the first time for thisspecies. These results confirm our previous observationson the geographic diversity of the Amaryllidaceae

Table 2. Distribution of alkaloid types in the plapercentage of total ion current (TIC)

Alkaloid type Fruits Fruitpeduncles

Lea

Galanthamine 1.3 2.9 1Crinine 78.4 84.0 96Lycorine 18.7 13.1 1TyramineUnknown 1.6 ‐

Table 3. Cytotoxic activity of the tested Ama(MTT‐dye reduction assay)

Compound

SKW‐3 HL‐60

7 16.95±6.56 20.86±1.5916 59.43±9.47 >100.0027 >100.00 >100.0028 45.03±3.21 95.95±11.5Elwesine >100.00 >100.00Topotecan 0.27±0.07 0.17±0.04

Copyright © 2011 John Wiley & Sons, Ltd.

alkaloids within the species (Torras‐Claveria et al., 2010;Berkov et al., 2011).

Cytotoxicity assays

Plants biosynthesize and accumulate bioactive com-pounds as a defence reaction against pathogenic organ-isms and herbivores. The galanthamine‐type alkaloidsfound inC. zeylanicum, galanthamine (3), sanguinine (4)and 3‐O‐acetylsanguinine (11), are AChE inhibitors,sanguinine being even ten times more active than theapproved drug galanthamine (López et al., 2002). Theiraccumulation in the reproductive organs can be relatedwith the insecticidal activity of the AChE inhibitors(Houghton et al., 2006). Lycorine (24), inhibiting the invivo growth of various tumor cells as well as the synthesisof DNA and proteins, also exhibits insect antifeedant,antifungal and antiviral activities (McNulty et al., 2009).

Compounds of the crinane series have been shown toexhibit cytotoxic activity. Haemanthamine and crinamine(‐5,10b‐ethano bridged) have been reported as signifi-cantly more potent apoptosis‐inducing compoundsthan β‐bridged alkaloids (McNulty et al., 2007). Onlyβ‐bridged compounds were isolated from C. zeylonicum.Literature data indicate that ambelline (27) has a weak(IC50 > 50μM) antiproliferative activity in HeLa andVero (Evidente et al., 2009) and in Molt4 and HepG2cell lines (Weniger , 1995) and strong inhibitory activityagainst the murine P‐388 lymphocytic leukemia (ED1.6μg/mL, equivalent to 4.8μM, Pettit et al., 1984).11‐O‐Acetylambelline (26) has shown cytotoxic activityagainst BL6 mouse melanoma cells (Campbell et al.,1998). Although crinine (7) is a common alkaloid in thegenus, data on its antiproliferative activity are scanty.A moderate ability to induce apoptosis in rat liverhepatoma (5123tc) and in human embryonic kidney

nt organs of C. zeylanicum presented as a

ves Stems Flowers Bulbs

.8 7.9 21.7 9.3

.6 87.9 49.2 88.5

.6 4.2 29.1 1.50.3

‐ ‐ ‐ 0.4

ryllidaceae alkaloids after 72 h exposure

IC50 (μM)

Hl‐60/DOX MDA‐MB‐231

14.04±1.34 68.11±7.7895.22±11.08 >100.00>100.00 >100.00

9 76.69±6.99 >100.00>100.00 >100.000.24±0.06 0.35±0.11

Phytother. Res. 25: 1686–1692 (2011)

Table 4. Proapoptotic activity of crinine (7)

Exposure Enrichment factor

HL‐60¼ IC50 (24h) 1.87±0.22a

½ IC50 (24h) 2.06±0.31a

IC50 (24h) 2.45±0.31b

MDA‐MB‐231¼ IC50 (24h) 1.35±0.14a

½ IC50 (24h) 1.82±0.21a

IC50 (24h) 2.07±0.24a

ap ≤0.05 vs the solvent‐treated control group.bp ≤0.01 vs the solvent‐treated control group.

1691ALKALOIDS FROM CRINUM ZEYLANICUM

(HEK‐293t) cells (McNulty et al., 2007) has been found forcrinine. Its ‐5,10b‐ethano bridged isomer, vittatine, hasshown moderate cytotoxic activity against HT29 colonadenocarcinoma (IC50 21.9μg/mL or 80.8μM), H460 lungcarcinoma (IC50 15.9μg/mL or 58.6μM) and RXF393renal cell carcinoma (IC50 29.6μg/mL or 109.1μM, Silvaet al., 2008) To the best of our knowledge, there are nodata on the bioactivity of 6‐hydroxybuphanidrine (28) andits derivative 6‐ethoxybuphanidrine (16).The tested alkaloids crinine (7), ambelline (27),

6‐hydroxybuphandrine (28) and 6‐ethoxybuphandrine(16) exerted concentration‐dependent cytotoxicity inthe panel of human tumor cell lines; the correspondingIC50 values are summarized in Table 3. Throughout thetested cell lines, compound 7 proved to be the mostactive cytotoxic agent with low micromolar IC50 values.Alkaloids 16 and 28 were significantly less active,whereas compound 27 failed to induce a 50% decreaseof cellular viability in all cell lines under investigation.The juxtaposition of the chemosensitivity among thedifferent cell lines shows that the leukemic cell lines aregenerally more sensitive than the breast cancer‐derivedMDA‐MB‐231. Interestingly, alkaloids 7, 16 and 28 wereactually more active against the multi‐drug resistant celllineHL‐60/dox than the parent chemosensitive lineHL‐60.Structure–activity relationships in the studiedmolecules

indicated that the hydrogenation of the double bond atC1‐C2 of crinine (7), as in elwesine (1,2‐dihydrocrinine),led to a loss of activity. Elwesine, used in this study, waspreviously isolated in our laboratory from Leucojumaestivum subsp. pulchelum and its structure was deter-mined byMS,CD, 1H, 13C‐NMR, 2D‐NMR.Substitutionsat C6, C8 and C11 led to a decrease of cytotoxicity. Thesubstituents on C6 affected the antiproliferative activity.Ambelline (27), with a hydroxyl group at C11 and nosubstituent at C6, showed no activity, whereas itspositional isomer, 6‐hydroxybuphanidrine (28), showedmoderate activity. However, the substitution of thehydroxyl group at C6 of compound 28 with an ethoxylgroup (compound 16) led to a decrease of cytotoxicity.In order to elucidate the mechanisms underlying the

observed cytotoxicity, the proapoptotic activity of 7(Table 4) was evaluated, using a commercially availableELISA‐kit to determine oligonucleosomal DNA frag-mentation, which is a key hallmark feature of apoptosis.Exponentially growing HL‐60 and MDA‐MB‐231 cellswere exposed to equi‐effective concentrations of thetested alkaloids for 24 h, and thereafter the cytosoliclevels of histone‐associated DNA fragments wereestimated and expressed as the corresponding enrich-ment factors (summarized in Table 4) and established

Copyright © 2011 John Wiley & Sons, Ltd.

using a commercially available ‘Cell death detection’ELISA‐kit. Alkaloid 7 caused concentration‐dependentfragmentation of genomic DNA. The more pronouncedinduction of apoptosis in HL‐60 vs MDA‐MB‐231 isconsistent with the higher chemosensitivity of the for-mer cell line in the MTT‐assay. These findings clearlysuggest that the induction of apoptotic cell death has animportant role for the cytotoxicity of the tested alkaloid.

The results from this study revealed a complex alkaloidprofile in the organs of C. zeylanicum, including manybioactive compounds. Some of the tested Amaryllidaceaealkaloids exert antiproliferative effects against humantumor cell lines. In view of its low micromolar IC50, theestablished collateral sensitivity of a drug‐resistant cell lineand the encountered propensity to induce apoptotic celldeath even at sub‐effective concentrations, crinine couldbe regarded as a prospective candidate for further onco‐pharmacological investigations. The structure–activityrelationship in the tested crinine‐type alkaloids, namelythe affects of substituents on C2, C3, C6 and C11, indicatesthat the chemical diversity of this group should be studiedin more detail for antiproliferative and apoptotic activities.

Acknowledgements

Financial support from Agencia Española de Cooperación conIberoamerica (AECI A/010053/07), Ministry of Education, Youthand Science, Sofia, Bulgaria (D002‐128/2008 ‐ I. Ionkova) andGeneralitat de Catalonia (2009 SGR1060) are acknowledged.

Confl ict of Interest

The authors have declared that there is no conflict of interest.

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