Patterns of pyrrolizidine alkaloids in 12 Ethiopian Crotalaria species

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ont matter # 2004 Elsevier Ltd. All rights reserved.

04.03.004

Biochemical Systematics and Ecology 32 (2004) 915–930

www.elsevier.com/locate/biochemsyseco

Patterns of pyrrolizidine alkaloids in 12Ethiopian Crotalaria species

Kaleab Asres a,b, Frank Sporer a, Michael Wink a,�

a Institut fur Pharmazie und Molekulare Biotechnologie, Universitat Heidelberg, Im Neuenheimer Feld

364, 69120 Heidelberg, Germanyb School of Pharmacy, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia

Received 18 March 2003; accepted 20 March 2004

Abstract

Thirty-three alkaloidal extracts prepared from different parts of 12 Ethiopian Crotalariaspecies have been analysed for their alkaloid profiles using GLC–MS. Eleven of the speciesinvestigated namely, C. agatiflora Schweinf. subsp. erlangeri Bak. f., C. albicaulis Franch.,C. axillaris Ait., C. emarginella Vatke, C. fascicularis Polhill, C. gillettii Polhill, C. incanaL. subsp. purpurascens (Lam.) Milne-Redh., C. laburnifolia L. subsp. tenuicarpa Polhill,C. mildabraedi Bak. f., C. phillipsiae Bak. and C. pycnostachya Benth. contain pyrrolizidinealkaloids (PAs). No pyrrolizidine alkaloid could be detected in C. spinosa Hochst. ex Benth.Alkaloid contents ranged between 0.02% dry weight (for the twigs of C. pycnostachya) and2.88% (for the seeds of C. axillaris). The alkaloidal patterns of the various organs of theplants have been determined by capillary GLC and GLC–MS. In addition to the commonlyfound PAs (simple esters and macrocyclic diesters), C. laburnifolia subsp. tenuicarpa andC. phillipsiae were shown to be adept in the biosynthetic elaboration of seco-pyrrolizidinealkaloids. The simple indolizidine alkaloid tashiromine, figured as a major compound inC. emarginella, C. phillipsiae and C. spinosa. It was also detected as a minor component ofC. fascicularis. Altogether 37 alkaloids have been identified by GLC–MS. Most of thesealkaloids were detected in the respective plants for the first time. Prior to our work, someof the alkaloids were not reported in the genus Crotalaria. The majority of the alkaloidsidentified were the potentially toxic macrocyclic diesters with a double bond at 1:2 position.# 2004 Elsevier Ltd. All rights reserved.

Keywords: Crotalaria spp.; Pyrrolizidine; Indolizidine; Alkaloid profiles; Capillary gas chromatography–

mass spectrometry

K. Asres et al. / Biochemical Systematics and Ecology 32 (2004) 915–930916

1. Introduction

Pyrrolizidine alkaloids (PAs) are considered to be important secondary metabo-lites largely on account of their biological activities, which include acute hepato-toxic (Mattocks, 1986), mutagenic (Hirono et al., 1979), carcinogenic (Hirono et al.,1978), teratogenic (Green and Christie, 1961), anticancer properties (Kovach et al.,1979) and neuroactive properties (Schmeller et al., 1997). The cytotoxicity of PAsis due to the their pyrrolic metabolites formed by microsomal bioactivation, notthe original alkaloids themselves (Fig. 1). The biotransformation in the liver createsan alkylating agent that can bind to DNA and proteins. An unsaturated (3-pyrroline)ring containing two hydroxyl groups, each attached to the pyrroline ring via one car-bon atom with at least one of these hydroxyls esterified are the minimum structuralfeatures needed for a PA to be potentially mutagenic (Mattocks, 1986; Cheeke,1994). Plants and some insects which sequester PAs from their food plants, consti-tute the only natural source of this group of alkaloids that cause toxic reactions inman and animals.Although over 300 of these alkaloids are known to occur in nature, the recorded

human toxicity has mainly been caused by a relatively small number of com-pounds. The main sources of these toxic alkaloids are almost all genera of the fam-ily Boraginaceae, the tribes Senecioneae and Eupatorieae of the family Asteraceaeand the genus Crotalaria of the family Fabaceae (Smith and Culvenor, 1981).Among these flowering plants, Crotalaria species are known to cause damage inthe broadest range of tissues in most domestic species. Plants belonging to thisgenus are mainly found in tropics and subtropics of Africa, South America, Asiaand Australia (Culvenor, 1980).In Ethiopia, 85 species of Crotalaria have been recorded (Thulin, 1989). Whilst

about 15 of these species are endemic to the country, the remaining are known tooccur in other tropical countries, mainly in Africa. For example, C. rosenii is so farknown only from the forest margins, secondary scrubs and stream banks in a fewregions of Ethiopia at altitudes between 1350 and 2800 m above sea-level (Tadesse,1991). Similarly, C. gillettii is restricted within the country growing in forest mar-gins, grassland and bushland at altitudes between 1700 and 2800 m. The occur-rence of some species including C. phillipsiae and C. pycnostachya extends beyondthe boundaries of Ethiopia into the adjoining dry bushland of Somalia and Kenyaup to 1500 m. Other species like C. axillaris and C. spinosa have a wider distri-bution covering the whole of tropical Africa, whereas the annual or short-livedperennial shrub, C. incana subsp. incana is native to tropical America and adven-tive in other parts of the tropics (Thulin, 1983).A review of the present literature unveiled that a large number of Ethiopian Cro-

talaria species have not been investigated for their PA content, although it isknown that poisoning by such alkaloids poses severe problem both for humansand animals. Therefore, it was deemed prudent to study the distribution of alka-

917K. Asres et al. / Biochemical Systematics and Ecology 32 (2004) 915–930

Fig. 1. Structures of PAs found in Crotalaria.


loids, if any, in these plants for two major reasons: (i) some Crotalaria species are

used either alone or in combination with other plants in the traditional medical

practices of Ethiopia; (ii) in Ethiopia, where there is a recurrent drought, grazing

animals are often forced to consume these plants which are known to thrive in dry

and arid climates even during periods of drought.In the present study, total alkaloid extracts prepared from various plant organs

of 12 Ethiopian Crotalaria species have been subjected to phytochemical analysis.

Among these, four of the plants namely, C. agatiflora, C. axillaris, C. incana and

C. laburnifolia either collected from other regions or belonging to different sub-

species have been investigated previously for their PA profiles (Smith and Culvenor,

1981). However, no information could be found in the literature on phytochemical

Fig. 1 (continued )


analysis of C. albicaulis, C. emarginella, C. fascicularis, C. gillettii, C. milabraedi,C. phillipsae, C. pycnostachya and C. spinosa.Capillary gas chromatography coupled with mass spectrometry (GLC–MS) has

been shown to be a rapid and most powerful technique for the analysis of complexPAs (Witte et al., 1993; El-Shazly et al., 1996a–d, 1998, 1999, 2003). Identificationof these alkaloids was therefore based on using this technique. Characterisation ofthe compounds was further facilitated by the presence of an extensive library ofmass spectral and GLC retention index (RI) data of authentic PAs at the Heidelberginstitute where this investigation was carried out.

2. Materials and methods

2.1. Plant material

All the plants were collected from south and south-eastern part of Ethiopia byAto Melaku Wondafrash and Dr Kaleab Asres between 23 and 28 October 1996.The plants were identified by Ato Melaku Wondafrash (The National Herbarium,Department of Biology, Addis Ababa University, where voucher specimens weredeposited). Site of collection, altitude and voucher specimen number for each ofthe plants investigated in the present study are given in Table 1.

2.2. Alkaloid extraction

About 10 g (accurately weighed) of each of the plant materials were extractedwith 80% methanol by maceration (3� 24 h). The combined extracts were evapo-rated to dryness under reduced pressure, dissolved in 20 ml of 1 M HCl andfiltered. The filtrate was washed with dichloromethane until the washings werecolourless. The dichloromethane extract gave a negative test with Dragendorffreagent, indicating the absence of alkaoidal salts in the organic solvent. The acidicextract was made alkaline with concentrated ammonia and extracted with dichlor-omethane (6� 25 ml). The latter dichloromethane extracts were combined, dried(Na2SO4), filtered and concentrated to dryness under reduced pressure to yieldalkaloid fraction 1. The residual ammonical solution was made acidic and stirredwith zinc dust for 2 h, centrifuged and decanted. The supernatant was then madealkaline with concentrated ammonia and again extracted with dichloromethane(6� 25 ml) to give alkaloid fraction 2. The two alkaloidal fractions were com-bined to yield the total alkaloid extract, which was calculated as a percentage dryweight.

2.3. GLC–MS analysis

The analyses were carried out on Carlo Erba HRGC 4160 gas chromatographequipped with a fused silica DB1 (30 m� 0:3 mm) column. The capillary columnwas directly coupled to a quadrupole mass spectrometer, Finnigan MAT 4500. EI-


mass spectra were recorded at 40 eV. Condition: injector 250vC; temperature pro-

gramme 120–300vC, 6

vC min�1 and/or 120–300

vC 1.5

vC min�1; split ratio 1:20;

carrier gas He 0.5 bar. Retention indices were calculated using co-chromato-graphed standard hydrocarbons.

3. Results and discussion

The distribution of alkaloids in 12 Crotalaria species has been studied. It wasfound that the yields of alkaloids ranged between 0.03% dry weight for the twigs ofC. pycnostachya and 2.98% for the seeds of C. axillaris (Table 2). Altogether 37alkaloids have been identified by comparing their mass spectra and Kovats reten-

Table 1

Sites of collection, altitude and voucher specimen numbers of Crotalaria species collected in Ethiopia

Plant species C
ollection site Altitude above
sea-level (m)

V

s

oucher

pecimen no.

C. agatiflora Schweinf.

subsp. erlangeri Bak. f.

H

D

arena forest—46 km from

ello Manna town towards

Bale Goba (along roadside)

2460 2
59
C. albicaulis Franch. 7
km from Siro village towards
Filtu (woodland)

1300 2
32
C. axillaris Ait. H
arena Forest—11 km from
Dello Manna town towards


1450 2
56
C. emarginella Vatke 2
7 km from Bedri village towards
Dello Menna town

1050 2
52
C. fascicularis Polhill 3
km North of Wadera towards
Kibre Mengist town

1730 1
92
C. gillettii Polhill H
arena Forest—11 km from


1450 2
57
C. incana L. subsp.

purpurascens (Lam.)

Milne-Redh.

1

w

a

5 km from Bedri village on the

ay to Dello Menna (woodland

nd dry riverside)

1480 2
49
C. laburnifolia L. subsp.

tenuicarpa Polhill

B

M

ehind Mojo Shell Oil station in

ojo town, about 65 km South

of Addis Ababa

1810 1
38
C. mildabraedi Bak. f. H
arena forest- 36 km from


1880 2
58
C. phillipsiae Bak. 1
0 km from siro village towards
Filtu (woodland)

1300 2
37
C. pycnostachya Benth. B
ehind Mojo Shell Oil station in
Mojo town, about 65 km South

of Addis Ababa

1810 1
34
C. spinosa Hochst. ex Benth. 1
1 km from Mojo town towards
Zwai (along roadside)

1650 1
39


T

T

p

P

C

C

C

C

C

C

C

C

C

C

C

C

N

tion indices (Table 3) with those data of authentic PAs available in the IPMB andin some cases with those reported in the literature (El-Shazly et al., 1996a–d, 1998;Toppel et al., 1988; Witte et al., 1993). The identification of some of the minorcomponents must be regarded as tentative, since material was not available to iso-late the compounds and to determine the exact position of hydroxyl groups and/ormethyl groups. As shown in Table 4, all the plants investigated with the exceptionof C. spinosa contain PAs (this result was confirmed with material including seedsfrom a different source).Seed samples of all species showed free PA bases only, whereas other parts con-

tained both PA-N-oxides and free PAs; in C. axillaries and C. gelletti, only free baseswere found throughout. This feature was also seen in extractions carried out with-out maceration but using solid phase extraction, so that a secondary reduction ofPA-N-oxides to the free base during extraction can be ruled out as an explanationfor the patterns of PA-N-oxides and free PA bases.Previously, the seeds of C. axillaris have been reported to contain axillarine and

axillaridine, macrocyclic diesters containing an 11-membered ring of the crota-laran-type. Whilst axillarine was isolated as a major alkaloid, axillaridine wasobtained in a very small amount (Crout, 1969). In the present GLC–MS analysis,axillarine could not be detected in any parts of the plant investigated. However,seven alkaloids including axillaridine and its isomer desoxyaxillarine have beenunequivocally identified. The 12-membered ring containing macrocyclic esterssuch as senecionine and integerrimine, which were not previously reported fromC. axillaris have been detected as minor alkaloids in the twigs and leaves of thisplant. An unknown alkaloid (RI 2305), which has not been reported before andaxillaridine were detected as the main components of the seeds, pods and leaves ofC. axillaris. The absence of axillarine from all the investigated parts of Ethiopian

able 2

otal alkaloid yields in the different botanical parts of Crotalaria species, calculated as percent of dried

lant material

lant
Alkaloid content (%)
Seeds
Pods T wigs L eaves
. agatiflora
1.88 NA 0 .78 1 .50
. albicaulis
NA NA 0 .70 0 .90
. axillaris
2.88 0.29 0 .40 1 .07
. emarginella
NA NA 0 .16 0 .40
. fascicularis
NA NA 0 .41 0 .96
. gillettii
NA NA 0 .11 0 .12
. incana
0.33 0.08 0 .06 0 .28
. laburnifolia
1.91 0.44 0 .20 0 .50
. mildbraedi
NA NA 0 .24 0 .29
. phillipsiae
NA NA 0 .20 0 .72
. pycnostachya
0.14 0.11 0 .03 0 .17
. spinosa
NA 0.22 0 .08 N A
A, not analysed.


Table 3

Alkaloids identified in 12 Ethiopian Crotalaria species by GLC–MS

Alkaloid
RI [M]+ Characteristic ions m=z (relative abundance)
Tashiromine
1285 155 155 (44), 154 (43), 138 (59), 124 (52), 96
(100), 83 (49), 69 (48)

5,6-Dehydro-7,9-dihydroxy-

7H-pyrrolizine-

ethylether

1405
181 181 (29),150 (62), 120 (100),119 (26), 106
(15)

Retronecine
1428 155 155 (23), 138 (19), 111 (55), 94 (18), 80
(100), 68 (15)

Heliotridine
1447 155 155 (22), 111 (55), 94 (16), 80 (100), 68 (14),
53 (11)

9-Isosenecioylretronecinea
1754 237 237 (0), 138 (54), 94 (31), 93 (100), 80 (31),
55 (12), 43 (50)

9-Angeloylretronecine
1795 237 237 (4), 193 (5), 154 (6), 137 (72), 106 (15),
93 (100), 83 (11), 80 (54)

7-Seneciolylretronecine
1805 237 237 (7), 137 (79), 124 (21), 111 (34), 106
(38), 94 (51), 80 (100)

9-Hydro-

xyheptanoylretronecine

2039
283 283 (0), 155 (27), 138 (72), 137 (56), 94 (38),
93 (100), 80 (22)

9-Hydro-

xyisohexenoylretronecinea
2041 267 267 (0), 239 (1), 155 (23), 138 (100), 94 (34),
93 (88), 80 (15)

Rinderine
2152 299 299 (0), 138 (98), 94 (55), 93 (100), 80 (12)
9-Hydroxytigloylretronecine

isomera
2160 253 253 (0), 155 (9), 138 (72), 94 (50), 93 (100),
80 (11)

Monocrotaline
2268 325 325 (0), 236 (55), 136 (46), 120 (100), 119
(64), 93 (36), 80 (12), 43 (69)

Dihydrosenecionine

isomera
2271 337 337 (8), 222 (11), 136 (67), 120 (84), 119
(100), 93 (100)

Senecionine isomera
2283 335 335 (13), 246 (8), 220 (32), 153 (29), 136
(100), 120 (93), 93 (77)

Retroisosenine
2283 335 335 (8), 220 (20), 138 (38), 136 (100), 120
(76), 119 (60), 93 (74)

Senecionine
2292 335 335 (8), 246 (10), 220 (36), 136 (100), 120
(100), 93 (70)

Unknown
2310 339 339 (22), 210 (21), 140 (37), 122 (75), 96
(27), 82 (100)

Nemorensine isomera
2325 337 337 (7), 222 (11), 138 (48), 136 (61), 120
(78), 119 (70), 93 (100)

Methylmonocrotalinea
2351 339 339 (0), 250 (40), 136 (26), 120 (100), 119
(26), 94 (16), 93 (14)

Integerrimine
2352 335 335 (2), 291 (6), 248 (5), 220 (9), 136 (48),
120 (100), 93 (48)

Integerrimine isomera
2370 335 335 (6), 291 (14), 248 (11), 220 (16), 138
(45), 136 (60), 120 (56), 119 (61), 93 (100)

Trichodesmine
2388 353 353 (1), 264 (56), 222 (5), 136 (36), 120
(100), 93 (23), 80 (16), 43 (40)

Axillaridine
2418 353 353 (3), 309 (3), 250 (77), 136 (54), 120
(100), 119 (71), 93 (49)

7-Tigloyl-9-dihydroxy-

senecioylretronecinea
2420 351 351 (0), 335 (6), 291 (14), 248 (11), 220 (16),
138 (45), 136 (60), 120 (56), 119 (61), 93

(100)


C. axillaris could be due to variation in factors such as altitude and climate. It has

been reported that the content of PAs in a given plant is subject to fluctuations

according to the climate, soil conditions and time of harvesting (Danninger et al.,

1983; Hartmann and Zimmer, 1986).The unknown alkaloid (RI 2305) showed a molecular ion at m=z 339 with a

characteristic fragmentation pattern of saturated ester alkaloids (Table 3). It has

been reported that the main fragmentation pathways of macrocyclic diesters leave

the saturated ring of the nucleus intact until a late stage. Hence, the base peak at

m=z 82 is attributed to a saturated necine moiety (Neuner-Jehle et al., 1965). Fur-

thermore, the presence of fragments in the ranges m=z 95–97, 122–123 and 138–140

is indicative of macrocyclic diesters having platynecine moiety (Luthy et al., 1981).

In accord with this, the spectrum of the unknown alkaloid, which possesses promi-

nent peaks at m=z 140, 122 and 96 is most likely that of a macrocyclic diester con-

taining a platynecine base.Seco-pyrrolizidine alkaloids appear to be the main components of C. laburnifolia

subsp. tenuicarpa, a plant whose distribution is restricted to eastern Africa. The

major alkaloids that occur in this plant were shown to be macrocyclic diesters

containing the 12-membered ring of the otonecan-type namely, senkirkine and

hydroxysenkirkine. Previously, these two alkaloids and anacrotine as well as

madurensine, a 13-membered ring containing madurensan-type alkaloid, have been

Axillaridine isomera 2
436 3 53 3 53 (3), 309 (10), 284 (22), 136 (57), 120
(100), 119 (91), 93 (51)

Madurensine 2
440 3 51 3 51 (1), 316 (8), 153 (8), 135 (100), 93 (35),
80 (75), 43 (47)

Anacrotine 2
449 3 51 3 51 (1), 236 (11), 152 (80), 135 (75), 93
(100), 80 (100)

Acetylseneciverninea 2
450 3 77 3 77 (16), 290 (52), 46 (10), 136 (100), 120
(69), 119 (41), 94 (51), 93 (49)

Senkirkine 2
460 3 65 3 65 (1), 266 (10), 250 (6), 168 (24), 153 (41),
80 (38), 43 (100)

Hydroxysenkirkinea 2
476 3 81 3 81 (0), 365 (1), 266 (10), 168 (24), 153 (41),
80 (38), 43 (100)

Jacoline 2
488 3 69 3 69 (0), 140 (42), 136 (21), 120 (100), 119
(77)

Acetylsenecivernine

isomera2
513 3 77 3 77 (7), 290 (52), 246 (9), 136 (100), 120
(67), 119 (69), 94 (63), 93 (77)

Gynuramine 2
516 3 51 3 51 (0), 220 (27), 136 (100), 120 (70), 119
(68), 94 (51), 93 (72), 80 (35)

Retrosine 2
518 3 51 3 51 (8), 246 (6), 220 (25), 138 (30), 120
(100), 119 (75), 80 (37)

Sceleratinea 2
546 3 69 3 69 (0), 120 (100), 119 (30), 93 (25)
Usaramine 2
578 3 51 3 51 (3), 246 (7), 220 (12), 138 (27), 136 (82),
120 (100), 119 (88)

9-Angeloylanacrotinea 2
858 4 33 4 33 (10), 390 (12), 334 (9), 264 (4), 234 (18),
218 (30), 153 (44), 118 (100), 83 (60)

a Tentative identification.

Table4

Alkaloidsidentified

inthevariousorgansofCrotalariaspecies

Constituentalkaloids

RI

Plantspecies

Plantpartsa

Seeds

Pods

Twigs

Leaves

Tashiromine

1285

C.em

arginella

+++

+++

C.fascicularis

++

C.phillipsiae

+++

+++

C.spinosa

+++

tr

5,6-D

ehydro-7,9-dihydroxy-7H-

pyrrolizine-ethylether

1405

C.fascicularis

++

Retronecine

1428

C.pycnostachya

++

trtr

+

Heliotridine

1447

C.axillaris

+++

++

+

9-Isosenecioylretronecine

1754

C.mildbraedii

�tr

9-A

ngeloylretronecine

1795

C.pycnostachya

++

tr�

+

7-Senecioylretronecine

1805

C.pycnostachya

++

tr�

tr

9-H

ydroxyheptanoyl-retronecine

2039

C.albicaulis

�++

9-H

ydroxyisohexenoyl-retronecine

2041

C.albicaulis

+++

Rinderine

2152

C.albicaulis

�+

9-H

ydroxytigloyl-retronecineisomer

2160

C.albicaulis

�+

Monocrotaline

2268

C.albicaulis

�++

Dihydrosenecionineisomer

2271

C.axillaris

++

++

+

C.em

arginella

�+

C.fascicularis

trtr

C.incanasubsp

purpurascens

++

��

�C.phillipsiae

++

Retroisosenine

2283

C.gillettii

++

++

Senecionineisomer

2283

C.agatiflora

subsp.im

perialis

�+

++

C.em

arginella

�+

Senecionine

2292

C.agatiflora

subsp.im

perialis

++

�+

C.axillaris

��

++

C.em

arginella

�+

C.gillettii

++

C.phillipsiae

trtr

Unknown

2310

C.axillaris

+++

++

++

+++


Nem

orensineisomer

2325

C.incanasubsp

purpurascens

++

�tr

�Methylm

onocrotaline

2351

C.fascicularis

�+

Integerrimine

2352

C.agatiflora

subsp.im

perialis

++

+

C.albicaulis

++

+

C.axillaris

��

++

+

C.gillettii

++

+++

C.incanasubsp

purpurascens

++

++

trtr

C.laburnifoliasubsp.tenuicarpa

��

++

+

C.mildbraedii

trtr

C.pycnostachya

tr�

��

Integerrimineisomer

2370

C.gillettii

++

Trichodesmine

2388

C.mildbraedii

++

+++

Axillaridine

2418

C.axillaris

+++

+++

++

++

7-Tiglyl-9-dihydroxy-

senecioylretronecine

2420

C.gillettii

tr�

Axillaridineisomer

2436

C.axillaris

++

+++

�Madurensine

2440

C.agatiflora

subsp.im

perialis

+++

++

++


�++

++

++

Anacrotine

2449

C.agatiflora

subsp.im

perialis

+++

++

++

C.gillettii

�+

C.incanasubsp

purpurascens

+tr

++


�++

++

+++

Acetylsenecivernine

2450

C.gillettii

�tr

Senkirkine

2460


+++

++

trtr

C.phillipsiae

�tr

Hydroxysenkirkine

2476


++

��

-

Jacoline

2488

C.fascicularis

++

Acetylsenecivernineisomer

2513

C.gillettii

�+

Gynuramine

2516

tr�

Retrosine

2518

C.agatiflora

subsp.im

perialis

�++

�Sceleratine

2546

C.fascicularis

trtr

Usaramine

2578

C.agatiflora

subsp.im

perialis

��

+

C.gillettii

++

9-A

ngeloylanacortine

C.agatiflora

subsp.im

perialis

�++

+++

a+++,major;++,interm

ediate;+,minor;tr,trace;�,noalkaloid.



reported from subsp. eldomae (Crout, 1972). Moreover, crotalburnine has been iso-lated from subsp. laburnifolia seeds of Indian origin (Emmanuel and Ghosh, 1964).However, crotalburnine was later shown to be identical with anacrotine (Crout,1972). Although C. laburnifolia has been described as a complex species with fivesubspecies occurring in the African population only (Polhill, 1968), it appears thatthese subspecies are similar with respect to their alkaloid profiles.GLC–MS analysis of C. agatiflora subsp. imperialis indicated that 9-angeloylana-

crotine is the major alkaloid in the leaves, whereas the seeds contain high quan-tities of madurensine. Retrosine, integerrimine, anacrotine, usaramine, senecionineand an isomer of senecionine, all possessing a 12-membered ring structure of thesenecionan-type have also been identified either in one or more parts of the plant.6-Acetylmadurensine, 7-acetylmadurensine, 7-acetyl-cis-madurensine, 6-acetyl-trans-anacrotine and crotafoline previously reported from the above ground parts ofC. agatiflora of unspecified subspecies (Atal et al., 1966; Smith and Culvenor,1981) were absent from all parts of the plant investigated.

C. incana subsp. purpurascens, which is widespread in tropical Africa apparentlycontains isomers of dihydrosenecionine, integerrimine, anacrotine and a nemor-ensine isomer. The isomer of nemorensine was previously detected in Senecio caca-liaster, and S. nemorensis (Asteraceae) (Witte et al., 1993). But, there is no reportin the literature concerning the presence of this alkaloid in Crotalaria species. Inprevious investigations of C. incana, it was reported that the seeds contain inte-gerrimine (Adams and Duuren, 1953) and usaramine (Sawhney and Atal, 1970),whereas the aboveground parts of the plant yielded anacrotine (Mattocks, 1968).In the current analysis, the presence of usaramine in all parts of subsp. purpur-ascens could not be confirmed. Although the subspecies from which usaramine wasisolated was not indicated, it is possible that this alkaloid was obtained fromsubsp. incana, a plant native to America but with wide distribution throughout thetropics.Ten PAs including retroisosenine, acetylsenecivernine, gynuramine and 7-tigloyl-

9-dihydroxysenecioylretronecine have been identified in the straggling-ascendingherb, C. gillettii, a plant known to occur only in Ethiopia. With the exception of7-tigloyl-9-dihydroxysenecioylretronecine which is a diester esterified at C-7 and C-9positions of retronecine, all the other alkaloids detected were macrocyclic diesters.Retroisosenine is macrocyclic diester containing a 13-membered ring, whereas theremaining alkaloids possess a 12-membered ring senecionan-type structure. To ourknowledge some of these alkaloids including acetylsenecivernine and gynuraminehave not been reported from Crotalaria species previously.The alkaloid patterns of C. albicaulis, C. mildabraedi and C. pycnostachya

appear to be similar in that they contain a mixture monoesters and macrocyclicdiesters. Moreover, integerrimine was identified in one or more plant parts of allthe three species. In C. albicaulis most of the alkaloids identified were monoesterswith the necine base esterified at C-9. These alkaloids namely, 9-hydro-xyheptanoylretronecine, 9-hydroxyisohexenoyl-retronecine, an isomer of 9-hydro-xytigloylretronecine and rinderine together with the 11-membered containingcrotalanan-type macrocyclic diester, monocrotaline, were prominent in the leaves.


In C. pycnostachya the necine bases of the monoesters are esterified either at C-7,as in 7-senecioylretronecine or at C-9, as in 9-angeloylretronecine. In C. milda-braedi, 9-isosenecioylretronecine was the only monoester alkaloid detected both inthe twigs and leaves in trace amounts. The major alkaloid identified in this plantwas trichodesmine, a macrocyclic diester having an 11-membered ring of thecrotalanan-type. Although this alkaloid was originally isolated from Trichodesmaincanum (Boraginaceae), it is now known to occur in several species of Crotalaria(Smith and Culvenor, 1981).

C. fascicularis is similar to the Crotalaria species discussed above in that it pro-duces the senecionan-type macrocyclic diesters such as jacoline and sceleratine aswell as the crotalanan-type alkaloid, methylmonocrotaline. However, these alka-loids were detected in trace amounts or as minor components. On the other handC. fascicularis is different from the other plants discussed above with respect to thepresence of the indolizidine alkaloid tashiromine and also 5,6-dehydro-7,9-dihy-droxy-7H-pyrrolizine-ethylether. Both of these alkaloids were detected in the twigsand leaves as minor components.

C. emarginella, and C. phillipsiae displayed similar alkaloid patterns. The simpleindolizidine alkaloid, tashiromine figured as a major alkaloid in the twigs andleaves of both plants. In addition, the common senecionan-type macrocyclicdiesters such as the isomer of dihydrosenecione have been identified in both. How-ever, traces of the seco-pyrrolizidine alkaloid, senkirkine was also detected in theleaves of C. phillipsiae. The alkaloid pattern of C. spinosa was different from all theother species investigated in that no PAs could be detected in it. It appears ratheruncommon for members of Crotalaria species not to produce PAs. However, theindolizidine alkaloid, tashiromine was identified as a major component in the twigsas well as in the pods of the plant. TLC of the alkaloid extracts prepared from thetwigs and pods of C. spinosa indicated the presence of a series of minor alkaloids.However, it was not possible to identify these alkaloids since their mass fragmen-tation pattern was found to be different from those of PAs for which retentionindex and mass spectral data are available within our institute. It is possible thatthese alkaloids are esters of the indolizidine base, tashiromine. Tashiromine(5-hydroxymethyl-trans-indolizidine) is not a very common alkaloid. It was firstisolated from the Japanese leguminous plant, Maackia tashiroi (Ohmiya et al.,1990). Subsequently, its presence in the genus Poecilanthe of the family Leguminosaehas been apparent (Greinwald et al., 1995). It is interesting to note that in bothcases the alkaloid was identified in association with quinolizidine alkaloids. To thebest of our knowledge, this is the first report for the occurrence of tashiromine inthe genus Crotalaria.From the foregoing, it can be seen that with the exception of C. spinosa, all the

plants investigated contain PAs. In Ethiopia, herbs are the mainstay of traditionalmedicine for the majority of the population. Some Crotalaria species are also usedfor the treatment of various diseases either alone or in combination with otherplant drugs. It is therefore possible that drugs containing such herbs have beencausing acute or chronic toxicity although the extent of damage caused by consum-ing such preparations has not been assessed. It has been reported that a single


episode of PA toxicity and possibly a long term low level exposure may lead tocirrhosis of the liver. It was also proved that the most toxic PAs are certain macro-cyclic diesters of heliotridine and retronecine (Mattocks, 1986). The present investi-gation revealed that most of the plants investigated contain macrocyclic diestersbased on retronecine as their major alkaloids. Even those plants like C. laburnifoliawhose major components are modified seco-pyrrolizidine alkaloids such as senkirkinepose health risk since such compounds have been shown to form pyrrolic metaboliteswhich are highly active alkylating agents and entirely responsible for the toxicity ofPAs (Mattocks and White, 1971).In view of the above, it is considered highly essential to create awareness among

the Ethiopian public in general and those responsible for the delivery of health ser-vices in particular, about the possible hazards of consuming such plants for medic-inal purposes.Since Crotalaria covers a large genus with almost 700 species (ILDIS, 2001), it is

too early to speculate on the systematic significance of this present study.Especially noteworthy is the apparent absence of PAs in C. spinosa and the occur-rence of the indolizidine alkaloid tashiromine.

Acknowledgements

One of the authors (K.A.) is most grateful to the Deutscher AkademischerAustauschdienst (DAAD) for their generous financial support. The authors wouldalso like to thank Ato Melaku Wondafrash (Addis Ababa) for collection andidentification of plant material and the referees for helpful comments.

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Documents

Patterns of pyrrolizidine alkaloids in 12 Ethiopian Crotalaria species