Available online at www.sciencedirect.com

www.elsevier.com/locate/biochemsysecoBiochemical Systematics and Ecology 36 (2008) 399e407

Occurrence of pyrrolizidine alkaloids in threeEthiopian Solanecio species

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

a Department of Pharmacognosy, School of Pharmacy, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopiab Institut fur Pharmazie und Molekulare Biotechnologie, Universitat Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

Received 7 November 2006; accepted 6 October 2007

Abstract

Three Ethiopian Solanecio species, namely Solanecio angulatus (Vahl) C. Jeffrey, Solanecio mannii (Hook. f.) C. Jeffrey, andSolanecio tuberosus (Sch. Bip. ex A. Rich.) C. Jeffrey var. tuberosus were analysed by capillary gas chromatographyemass spec-trometry (GLSeMS) for their pyrrolizidine alkaloid content. All the extracts investigated contain pyrrolizidine alkaloids. Whilstonly traces of alkaloids could be detected in the leaf extract of S. angulatus, the content of alkaloids in the other samples rangedbetween 0.13% dry weight (for the flowers of S. angulatus) and 0.58% (for the tubers of S. tuberosus). Altogether 17 alkaloids weredetected out of which 14 were unambiguously identified by comparing their retention indices, molecular masses and mass fragmen-tation patterns with defined reference data from PAs database or in some cases with reference compounds. The hepatotoxic mac-rocyclic diesters senecionine and retrosine figured as major alkaloids in the flowers of S. angulatus, whereas the platynecine typealkaloid, 7-O-senecioylplatynecine occurred in higher amount than the other alkaloids detected in the leaves of S. mannii. The tu-berous annual herb, S. tuberosus contains eruciflorine as a major alkaloid in the leaves, flowers and tubers. Senecionine figures asone of the major components of the tubers of S. tuberosus. To the best of our knowledge this is the first published report on theoccurrence of pyrrolizidine alkaloids in the genus Solanecio. In addition to the chemotaxonomic significance of the detected alka-loids, a brief remark is made on the findings in the light of the use of these plants as medicinal herbs and/or as nectar or pollen sourcefor the production of honey.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Solanecio angulatus; Solanecio mannii; Solanecio tuberosus; Senecioneae; Pyrrolizidine alkaloids; Capillary gas chromatographye

mass spectrometry

1. Introduction

The genus Solanecio (Asteraceae) contains 17 species confined to tropical Africa, Yemen and Madagascar. Mem-bers of the genus are perennial herbs, shrubs or small soft wooded trees and a few of them are epiphytic (Tadesse,2004). Six species have been reported to occur in Ethiopia. Whilst Solanecio gigas (Vatke) C. Jeffrey, Solanecio hare-nnensis Mesfin and Solanecio tuberosus (Sch. Bip. ex A. Rich.) C. Jeffrey var. pubescens Mesfin are endemic to the

* Corresponding author. Tel.: þ49 62 21 54 48 80; fax: þ49 62 21 54 48 84.

E-mail address: wink@uni-hd.de (M. Wink).

0305-1978/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.bse.2007.10.003

400 K. Asres et al. / Biochemical Systematics and Ecology 36 (2008) 399e407

country, the remaining ones namely, Solanecio angulatus (Vahl) C. Jeffrey, Solanecio mannii (Hook. f.) C. Jeffrey andSolanecio nandensis (S. Moore) C. Jeffrey are known to occur elsewhere (Tadesse, 2004). Members of the genus areclosely related to the plant species of the genera Cacalia, Crassocephalum, Emilia, and Senecio, all of which belong tothe tribe Senecioneae and are known to produce pyrrolizidine alkaloids (PAs) (Cava et al., 1968; Asada et al., 1985;Cheng and Roder, 1986; Mattocks, 1986). However, no previous report could be found in the literature concerning thechemistry of any of the species in the genus Solanecio. The taxonomical similarities between the genera Solanecio andSenecio implied that members of Solanecio, like those of the extensively investigated Senecio species, might besources of pyrrolizidine alkaloids.

It was on the basis of the above supposition that extracts prepared from various plant organs of three EthiopianSolanecio species viz. S. angulatus (syn. Cacalia sonchifolia auct., non L.; Cacalia angulata Vahl; Senecio subscan-dens Hochst. ex A. Rich.; Crassocephalum subscandens; Senecio borjeri DC.), S. mannii (syn. Senecio mannii (Hook.f.); Crassocephalum mannii (Hook. f.) Milne-Rdh.) and S. tuberosus var. tuberosus (syn. Senecio tuberosus Sch. Bip.ex A. Rich.; Cacalia tuberosa Del.; Senecio solanoides Sch. Bip. ex Asch.; Cacalia abyssinica Walp.) were examinedfor their PA content.

S. angulatus is a climbing, creeping or straggling herb, which grows up to 3 m in height. The leaves and stems aresucculent with white florets and orange-yellow stigmas, and they resemble certain members of the tribe Lactuceae butdiffer in having a watery sap, not a milky one (Jeffrey, 1986). It is found in evergreen bushland and along fencesaround houses at altitudes ranging from 1300 m to 2500 m. It also grows in South Africa as the only species repre-senting the genus Solanecio (Pooley, 1998). In Ethiopian traditional medicine chewing of the leaves of S. angulatusis said to give an instant relief of tooth pain. In Tanzania, it is one of the most commonly used plants for the treatmentof dermatological and gastrointestinal problems (Schlage et al., 1999). The flowers are also very good source of pollenand nectar for honeybees (Fichtl and Adi, 1994).

S. mannii is a 2e6 m tall shrub or small tree, the flowers of which contain 6e12 yellow florets. It mainly grows inmontane evergreen forest especially along margins and derived evergreen bushland, particularly in the humid westernand southwestern parts of the country at altitudes up to 2400 m. In Cameroon a handful of young leaves of S. manniimixed with other plants are used as enema for the treatment of epilepsy (Noumi and Fozi, 2003).

S. tuberosus var. tuberosus is an erect perennial herb, which grows up to only 30e60 cm in height. Its florets arebright yellow or orange. Unlike the other members of the genus, it has a tuber, which is brown on the outside andwhitish inside having a peppery smell (Tadesse, 2004). In Ethiopia, both the aerial parts and the tubers are usedfor wound healing and also for stomach problems.

In view of the sensitivity and high resolution power of capillary gas chromatography coupled with mass spectrom-etry (GLCeMS) for the analysis of complex PAs (Witte et al., 1993; El-Shazly et al., 1996, 1998, 1999; Asres et al.,2004), identification of the alkaloids was based on this technique. Characterisation of the individual compounds wasachieved by comparison of their mass spectral and GLC retention indices (RIs) with that of the data of authentic PAsavailable at the Institute in Heidelberg where the investigation was carried out.

2. Materials and methods

2.1. Plant material

All plants were collected by Mr. Melaku Wondafrash and Dr. Kaleab Asres from different parts of Ethiopia andwere identified by the former (the National Herbarium, Department of Biology, Addis Ababa University, wherevoucher specimens are deposited for future reference). Date and site of collection, altitude and voucher specimennumber for each of the 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 extracted (3� 24 h) with 80% of methanol(150 ml) in a shaker. The combined extracts were evaporated to dryness under reduced pressure, dissolved in 40 mlof 1 M HCl and filtered. The filtrate was washed with dichloromethane until the washings were colourless. The di-chloromethane extract gave negative test with Dragendorff’s reagent, indicating the absence of the hydrochloride saltsof the alkaloids in the organic solvent. The acidic extract was stirred with zinc dust for 12 h to convert PA-N-oxides to

Table 1

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

Plant species Collection date Collection site Altitude above

sea-level (m)

Voucher

specimen no.a

S. angulatus 25 June 2006 Western part of Addis Ababa, Gulele area 2500 2758

S. mannii 27 May 2006 Harena Forest e about 31 km from Dello Mena

town towards Bale Goba (along road side)

1800 2724

S. tuberosus var. tuberosus 21 July 2006 Around the city of Bahir Dar about 600 km north

of Addis Ababa

1800 2759

a Voucher specimens were deposited at National Herbarium, Addis Ababa University, Ethiopia.

401K. Asres et al. / Biochemical Systematics and Ecology 36 (2008) 399e407

free PAs, centrifuged and decanted. The supernatant was then made alkaline (pH 9) with concentrated ammonia andapplied onto columns packed with Chem Tube Hydromatrix (Varian, Inc., Palo Alto, CA, USA). The free alkaloid baseswere eluted with dichloromethane (300 ml). The organic solvent was evaporated under reduced pressure to yield thetotal alkaloid extract, which was calculated as a percentage dry weight (Table 2).

2.3. GLCeMS analysis

The analyses were carried out on HewlettePackard gas chromatograph (GC 5890 II, HewlettePackard GmbH, BadHomburg, Germany) equipped with OV-1 bonded capillary column (30 m, i.d.: 250 mm, film thickness: 0.25 mm)(Ohio Valley, Marietta, Ohio, USA). The capillary column was directly coupled to a quadrupole mass spectrometer(SSQ 7000, Thermo-Finnigan, Bremen, Germany). Samples were injected (1 ml) with split mode (split ratio, 1:20).The injector temperature was 250 �C. Helium carrier gas flow rate was 1.0 ml min�1 with a pressure of 14 psi. Thecolumn temperature programme was 100e300 �C, 6 �C min�1 and/or 100e300 �C, 4 �C min�1. All the mass spectrawere recorded with the following condition: filament emission current, 200 mA; electron energy, 70 eV; ion source,175 �C; mass range, 60e150; scan time, 0.5 s. Retention indices were calculated using co-chromatographed standardhydrocarbons (C8eC28).

3. Results and discussion

3.1. S. angulatus

GLCeMS analysis of the reduced alkaloid extracts obtained from the flowers of S. angulatus led to the identifica-tion of retrosine and senecionine. Retrosine occurred as a major compound comprising 82.8%, while senecionine con-stituted the remaining 17.2% of the total PA content of 0.13% dry weight (Table 2). These structurally similaralkaloids are based on the necine base retronecine (unsaturation at 1,2 position) and they both have a 12-memberedmacrocyclic diester as a necic acid moiety (Fig. 1), which is derived from isoleucine.

The only PAs found in detectable levels in the alkaloidal extract of the leaves of S. angulatus were the two mac-rocyclics senecionine and integerrimine. These alkaloids are isomeric and they are also based on retronecine. How-ever, their presence was in trace amounts (<0.001% of the dried plant material). It is of interest to note that only tracesof PAs could be detected in the leaves while the flowers contain significant amount. Hartmann and Toppel (1987) and

Table 2

Total alkaloid yields (calculated as percent of dried plant material) different botanical parts of Solanecio spp.

Plant species Alkaloid content (%)

Leaves Flowers Tuber

S. angulatus tr 0.13 e

S. mannii 0.05 NA e

S. tuberosus var. tuberosus 0.20 0.27 0.58

NA¼ not analysed; tr¼ traces (amounts< 0.001%).

R=H Senecionine

R=OH Retrosine

R=H Integerrimine

R=OH Eruciflorine

Seneciphylline

Jaconine

Erucifoline

OHH

OH

N

Retronecine

RH

N

OH

O

H

O

R

O

HO

O

N

R=OH Platynecine

R=O-Senecionyl

R=O-Tigloyl

OH

O

O

HO

O

N

R

OH

O

O

HO

O

N

O

OH

H

O

O

HO

O

N

Cl

Retroisosenine

Neoplatyphylline

CH3H

OH

OCH3

O

HO

O

N

CH3

H

Bulgarsenine

CH3H

OH

OCH3

O

HO

O

N

CH3

O

CH2OH

OH

OO

N

CH3CH3

OH

O

OO

N

O

CH3

12

13

12

356

7 8

1415

16

2021

7-O-Senecionylplatynecine7-O-Tigloylylplatynecine

19

9

11

12

19

18

O

Jacobine

OH

O

O

HO

O

N

o

Fig. 1. Structures of PAs found in S. angulatus, S. mannii and S. tuberosus.

402 K. Asres et al. / Biochemical Systematics and Ecology 36 (2008) 399e407

403K. Asres et al. / Biochemical Systematics and Ecology 36 (2008) 399e407

Hartmann et al. (1989) reported that in the family Asteraceae PAs are synthesized in the roots as alkaloid N-oxides thatare specifically translocated into shoots via the phloem-path and are channeled to the inflorescences, which are con-sidered to be one of the preferred sites of storage. Therefore, large quantitative differences may exist in the amount ofPAs between organs, which explains the distribution pattern of PAs in the two organs of S. angulatus investigated.There is also a report in the literature, which indicates the presence of up to 30-fold higher concentrations of PAsin the inflorescences of some Senecio species than in the leaves or roots (Hartmann and Zimmer, 1986).

From the alkaloid pattern of S. angulatus, it can be said that the use of leaf extracts of this plant as a tra-ditional medicine for tooth pain, skin disorders or gastrointestinal problems may be safe since they contain onlytraces of PAs. However, intentional incorporation of the flowers or the presence of flowers as contaminants intraditional remedies containing the leaves of S. angulatus may pose a health risk since the flowers contain sig-nificant amount of the potentially hepatotoxic, mutagenic and carcinogenic 1,2-dehydro-pyrrolizidine esteralkaloids.

Moreover, in recent years honey obtained from plants containing PAs have been reported to be a potential threat tohealth (Edgar et al., 2002). In Ethiopia, the flowers of S. angulatus are significant contributors to honey production.The plant flowers almost all year round and honeybees collect pollen and nectar from the flowers frequently. The plantis also considered very valuable for strengthening bee colonies at times when other bee food is scarce (Fichtl and Adi,1994). It is therefore very likely that honey produced from flowers of S. angulatus contain PAs and contributes to ex-posure of consumers to PAs. However, further investigation must be carried out to determine the percentage of PAs inhoney produced by honeybees gathering nectars from flowers of S. angulatus in order to assess the actual health riskposed by such honeys.

3.2. S. mannii

Analysis of the alkaloid extract of the leaves of S. mannii demonstrated the presence of four alkaloids of which twowere unambiguously identified as 7-O-senecioylplatynecine and 7-O-tigloylplatynecine. The mass spectra of thesetwo isomers are indistinguishable; identification was therefore based on RI and comparisons with the spectra of plantsknown to contain these alkaloids. The major component, based on comparison with a known standard, was 7-O-senecioylplatynecine, which comprised 81.9% of the total PA content of 0.05% dry weight (Table 2). 7-O-Tigloylpla-tynecine was also present in a large concentration accounting for 17.1% of the total PA content. The structures of thesealkaloids are based on platynecine as a necine base and a monoester with the C5 acids senecioic acid and tiglic acid asesterifying acids (Fig. 1), respectively. These alkaloids are not as commonly found in the tribe Senecioneae as theretronecine-based alkaloids, although some Senecio species are known to biosynthesize and accumulate them (Mat-tocks, 1986).

Two alkaloids identified as unknown A (RI¼ 3056) and unknown B (RI¼ 3090) were also detected in very lowamounts in the leaf extract of S. mannii. These components accounted for 0.2% and 0.8% of the total PA content, re-spectively. Further structural analysis of these alkaloids was not possible due to their limited occurrence in traceamounts. However, their general structure type can be deduced from their MS fragmentation patterns. The two closelyrelated alkaloids showed molecular ions at m/z 451with a characteristic fragmentation pattern of saturated macrocy-clic ester alkaloids. As shown in Table 3, the characteristic fragments for the retronecine part of retronecine-basedalkaloids 93e95, 119e121, and 136 (120 as a base peak) were shifted to 95, 96, 122, 123 and 138 (82 as a basepeak) in the MS of both unknown A and unknown B (Table 3). These latter fragments are typical for MS of saturatedPAs. Neuner-Jehle et al. (1965) and Rashkes et al. (1978) reported that the main fragmentation pathways for macro-cyclic diesters leave the saturated ring of the nucleus intact until a late stage. Therefore, the base peak at m/z 82 isattributed to a saturated necine moiety. In accord with this, the two unknown alkaloids are most likely macrocyclicdiesters containing platynecine as a necine base.

It is worth noting that all the alkaloids detected in the leaf extract of S. mannii are of the platynecine type whichmeans that they cannot undergo oxidation to pyrrolic metabolites because of the absence of a double bond at 1,2 po-sition. Such alkaloids can be excluded from consideration as potential hepatotoxins; they are not genotoxic nor do theyinduce tumours. Therefore, the use of leaf extracts of S. mannii in traditional medicine may be safe. Furthermore, theuse of this herb as a traditional medicine for the treatment of epilepsy could be attributed to the presence of PAs, whichare known to interact with neuroreceptors (Atal, 1978; Schmeller et al., 1997).

Table 3

Alkaloids identified in S. angulatus, S. mannii and S. tuberosus by GLCeMS

Alkaloid RI [M]þ Characteristic ions m/z (relative abundance)

7-O-Tigloylplatynecine 1823 239 239(1), 194 (7), 156 (28), 139 (90), 138 (30), 108 (13), 82 (100), 55 (58)

7-O-Senecioylplatynecine 1848 239 239(<1), 156 (22), 139 (88), 138 (37), 137 (24), 106 (59),

82 (100), 80 (81), 55 (74)

Senecionine isomer 2283 335 335 (5), 220 (32), 192 (7), 138 (22), 136 (49), 120 (100),

108 (24), 95 (67), 80 (28), 53 (31)

Retroisosenine 2290 335 335 (13), 248 (15), 220 (21), 136 (75), 120 (100), 119 (80),

108 (27), 94 (89), 81 (36), 53 (54)

Senecionine 2307 335 335 (12), 246 (13), 220 (19), 138 (42), 136 (100), 120 (100),

109 (23), 94 (75), 93 (77), 80 (49), 67 (21), 55 (28), 53 (52)

Seneciphylline 2320 333 333 (4), 246 (11), 138 (35), 136 (70), 120 (100), 94 (77),

80 (45), 67 (18), 53 (47)

Bulgarsenine 2353 337 337 (2), 246 (12), 211 (32), 140 (100), 138 (88), 136 (29), 123 (52),

122 (88), 96 (41), 82 (95), 53 (68)

Integerrimine 2362 335 335 (7), 291 (8), 248 (10), 220 (16), 153 (8), 138 (42), 136 (95),

120 (100), 119 (95), 109 (25), 93 (81), 80 (52), 53 (58)

Neoplatyphylline 2387 337 337 (4), 220 (6), 211 (14), 140 (100), 138 (70), 122 (80), 120 (35),

108 (16), 95 (37), 82 (95), 55 (46)

Jacobine 2454 351 351 (6), 236 (7), 208 (18), 194 (24), 136 (35), 120 (100), 95 (60),

93 (57), 80 (33), 55 (27)

Jacobine isomera 2471 351 351 (5), 208 (22), 194 (28), 138 (21), 136 (52), 120 (100), 95 (71),

93 (61), 80 (31), 55 (31)

Erucifoline (Z) 2542 349 349 (10), 218 (4), 138 (19), 136 (79), 120 (100), 106 (18), 94 (83),

80 (46), 53 (47)

Retrosine 2548 351 351 (12), 246 (17), 220 (19), 138 (35), 136 (97), 120 (100), 93 (75),

80 (43), 53 (54)

Jaconine 2584 387 387 (0), 224 (12), 209 (17), 138 (8), 136 (19), 120 (100), 119 (30),

93 (37), 80 (21), 53 (27)

Eruciflorine (E) 2630 351 351 (3), 307 (4), 236 (18), 138 (32), 136 (68), 120 (100), 106 (18),

93 (79), 80 (40), 53 (45)

Unknown A 3056 451 451 (3), 385 (16), 250 (7), 138 (70), 123 (43), 122 (100), 120 (29),

96 (44), 83 (28), 82 (63), 55 (46)

Unknown B 3090 451 451 (3), 385 (25), 252 (7), 147 (11), 138 (85), 123 (28), 122 (100),

120 (23), 96 (49), 95 (43), 82 (81), 80 (16), 55 (44)

a Tentative identification.

404 K. Asres et al. / Biochemical Systematics and Ecology 36 (2008) 399e407

3.3. S. tuberosus var. tuberosus

The tubers, leaves and flowers of S. tuberosus var. tuberosus were found to contain higher percentage of alkaloidswhen compared to the other Solanecio species investigated. Thirteen alkaloids were found in detectable levels in thetubers of S. tuberosus as shown in Table 4. Twelve of these alkaloids were identified unequivocally by comparing theirRIs, molecular masses and mass fragmentation patterns with defined reference data from our PA database or in somecases with reference compounds. The major components were eruciflorine and senecionine, which comprised 39.2%and 29.8% of the total PA content of 0.58% dry weight, respectively. Two other compounds were also present in rel-atively high concentrations, integerrimine and seneciphylline. These components accounted for 11.4% and 7.8% ofthe total PA content, respectively. With the exception of two (neoplatyphylline and bulgarsenine), all the other alka-loids identified in the tuber extract of S. tuberosus as relatively minor compounds were exclusively macrocyclic al-kaloids of the senecionine type containing the necine base retronecine (Fig. 1). They include jacobine, jaconine,senecionine isomer, retroisosenine, erucifoline and retronecine. These are secondary metabolites characteristic forPA containing species of the tribe Senecioneae, which are primarily species that are usually assigned to the genus Se-necio (Pelser et al., 2005). Moreover, all can be regarded as derivatives of the biosynthetic backbone structure sene-cionine (Toppel et al., 1987). In addition to the above alkaloids, a compound with RI¼ 2471 was detected as a minorconstituent comprising 1.9% of the total PA content. The RI and the fragmentation pattern of this alkaloid did notmatch with either the reference data in our PA database or with those obtained from the literature. Its mass spectrum

Table 4

Pyrrolizidine alkaloid (PA) profile for S. angulatus, S. mannii and S. tuberosus var. tuberosus as detected by GLCeMS

Plant Compound RI Alkaloid compositiona (%)

Tubers Leaves Flowers

S. angulatus Senecionine 2307 e tr 17.2

Integerrimine 2362 e tr ND

Retrosine 2548 e ND 82.8

S. mannii 7-O-Tigloylplatynecine 1813 e 17.1 e

7-O-Senecioylplatynecine 1847 e 81.9 e

Unknown A 3056 e 0.2 e

Unknown B 3090 e 0.8 eS. tuberosus var. tuberosus Senecionine isomer 2283 1.4 4.9 16.9

Retroisosenine 2290 0.4 ND ND

Senecionine 2307 29.8 tr 1.3

Seneciphylline 2320 7.83 2.0 ND

Bulgarsenine 2353 2.2 ND ND

Integerrimine 2362 11.4 5.3 9.0

Neoplatyphylline 2387 tr ND ND

Jacobine 2454 3.8 ND ND

Jacobine isomerb 2471 1.9 ND ND

Erucifoline (Z) 2542 0.3 ND ND

Retrosine 2548 0.2 ND ND

Jaconine 2584 1.8 ND ND

Eruciflorine (E) 2630 39.2 87.7 72.3

ND¼Not detected; tr¼ traces (amounts< 0.1%).a Total alkaloid 100%.b Tentative identification.

405K. Asres et al. / Biochemical Systematics and Ecology 36 (2008) 399e407

was identical to that of jacobine, but the former eluted before the latter. It was therefore tentatively identified asa jacobine isomer.

Neoplatyphylline and bulgarsenine were also detected in the tuber extract of S. tuberosus with the former in traceamount and the latter making up only 2% of the total PA present. The two compounds are the only saturated PAs(based on platynecine) that were detected in the sample.

Structurally, the two major alkaloids of the tuber extract of S. tuberosus var. tuberosus eruciflorine and senecionineare 12-membered macrocyclic esters generally classified as senecionine type PAs. The former contains an additionalhydroxyl group at C21 of the necic acid moiety of integerrimine, which is the geometrical isomer of senecionine(Fig. 1). Eruciflorine was originally isolated from Senecio erucifolius (Witte et al., 1992) and later it was identifiedin a few other Senecio species (Pelser et al., 2005). Similarly, Kompis and Santavy (1962) first isolated the minor al-kaloid erucifoline from S. erucifolius, although Sedmera et al. (1972) elucidated the structure years later. These twoalkaloids were thought to be unique for the genus Senecio since evaluation of reference data from previous reports didnot reveal their detection outside members of the genus Senecio.

PAs occur mainly in the three unrelated flowering plant families namely, Asteraceae, Boraginaceae, and Fabaceae.In the case of the former, they are restricted just to the two subtribes Senecioneae and Eupatorieae (Smith andCulvenor, 1981). Because of their wide distribution within these two subtribes, they are of little chemotaxonomic im-portance for distinguishing lower taxa. However, the presence of the rather uncommon alkaloids eruciflorine and eru-cifoline only in the genera Solanecio and Senecio is a strong indication that S. tuberosus is closely related to membersof the genus Senecio; it is interesting to note that Solanecio tuberosus was previously described as Senecio tuberosus.

The leaf and flower extracts of S. tuberosus showed similar alkaloidal patterns to those of the extracts from thetubers. However, the percentage content and the number of alkaloids detected were less than those detected in thetuber samples. The major component identified in both samples was again eruciflorine making up 87.7% and72.3% of their total PA content, respectively. Two other alkaloids were also present in large concentrations, the isomerof senecionine and integerrimine. These components accounted for 4.9%, 16.9% and 5.3%, 9.0% in the leaves andflowers, respectively.

From the foregoing it can be seen that nearly all the alkaloids that occur in S. tuberosus share the common structuralfeatures i.e. 1,2 unsaturation, an esterified allylic hydroxyl group at C9 and an esterified alcoholic hydroxyl group at C7

406 K. Asres et al. / Biochemical Systematics and Ecology 36 (2008) 399e407

that generally make PAs potentially toxic. Thus, all of them can be metabolically activated by cytochrome P450 en-zymes to create pyrrolic intermediates that are highly reactive with biological nucleophiles causing genotoxicity andcancer (Fu et al., 2004).

In conclusion, the close taxonomical similarities between the genus Solanecio and the other genera in the tribeSenecioneae in general and the genus Senecio in particular was further confirmed by the presence of PAs in the threespecies of Solanecio studied. Whilst S. mannii was found to contain exclusively saturated PAs based on platynecinemoiety, macrocyclic esters of retronecine appear to have dominated the alkaloidal extracts of S. angulatus and S.tuberosus. Thus, it would be prudent to regard the latter two plants as potentially hepatotoxic and using them in tra-ditional medicine should therefore be avoided or minimized as far as possible. The current study is the first report ofthe presence of PAs in the genus Solanecio.

Acknowledgements

One of the authors (K.A.) would like to express his utmost gratitude to Deutscher Akademischer Austauschdienst(DAAD) for their generous financial support. The authors would also like to thank Mr. Melaku Wondafrash (AddisAbaba) for collection and identification of plant material.

References

Asada, Y., Shiraishi, M., Takeuchi, T., Osawa, Y., Furuya, T., 1985. Pyrrolizidine alkaloids from Crassocephalum crepidioides. Planta Med. 51,

539e540.

Asres, K., Sporer, F., Wink, M., 2004. Patterns of pyrrolizidine alkaloids in 12 Ethiopian Crotalaria species. Biochem. Syst. Ecol. 32, 915e930.

Atal, C.K., 1978. Semisynthetic derivatives of pyrrolizidine alkaloids of pharmacodynamic importance. Lloydia 41, 315e326.

Cava, M.P., Rao, K.V., Weisbach, J.A., Raffauf, R.F., Douglas, B., 1968. The alkaloids of Cacalia floridana. J. Org. Chem. 33, 3570e3573.

Cheng, D., Roder, T., 1986. Pyrrolizidine alkaloids from Emilia sonchifolia. Planta Med. 52, 484e486.

Edgar, J.A., Roeder, E., Molyneux, R.J., 2002. Honey from plants containing pyrrolizidine alkaloids: a potential threat to health. J. Agric. Food

Chem. 50, 2719e2720.

El-Shazly, A., Sarg, T., Ateya, A., Abdel Aziz, E., Witte, L., Wink, M., 1996. Pyrrolizidine alkaloids of Cynoglossum officinale and Cynoglossum

amabile (Family Boraginaceae). Biochem. Syst. Ecol. 24, 415e421.

El-Shazly, A., El-Domiathy, M., Witte, L., Wink, M., 1998. Pyrrolizidine alkaloids in members of the Boraginaceae from Sinai (Egypt). Biochem.

Syst. Ecol. 26, 619e636.

El-Shazly, A., Abdel-All, M., Tei, A., Wink, M., 1999. Pyrrolizidine alkaloids from Echium rauwolfii and Echium horridum (Boraginaceae). Z.

Naturforsch. 54c, 295e300.

Fichtl, R., Adi, A., 1994. Honeybee Flora of Ethiopia. Margraf Verlag, Weikersheim, Germany.

Fu, P.P., Xia, Q., Lin, G., Chou, M.W., 2004. Pyrrolizidine alkaloids e genotoxicity, metabolism enzymes, metabolic activation, and mechanisms.

Drug Metab. Rev. 36, 1e55.

Hartmann, T., Zimmer, M., 1986. Organ specific distribution and accumulation of pyrrolizidine alkaloids during the life history of two annual

Senecio species. J. Plant Physiol. 122, 67e80.

Hartmann, T., Toppel, G., 1987. Senecionine N-oxide, the primary product of pyrrolizidine alkaloids biosynthesis in the root cultures of Senecio

vulgaris. Phytochemistry 26, 1639e1643.

Hartmann, T., Ehmke, A., Eilert, U., von Borstel, K., Theuring, C., 1989. Sites of synthesis, translocation and accumulation of pyrrolizidine

alkaloids N-oxides in Senecio vulgaris L. Planta 177, 98e107.

Jeffrey, C., 1986. The Senecioneae in east tropical Africa. Kew Bull. 41, 873e943.

Kompis, I., Santavy, F., 1962. Alkaloids of Senecio erucifolius. Collect. Czech. Chem. Commun. 27, 1413e1421.

Mattocks, A.R., 1986. Chemistry and Toxicology of Pyrrolizidine Alkaloids. Academic Press, London.

Neuner-Jehle, N., Nesvadba, H., Spiteller, G., 1965. Application of mass spectrometry to the elucidation of the structure of alkaloids. VI.

Pyrrolizidine alkaloids of Laburnum. Monatsh. Chem. 96, 321e388.

Noumi, E., Fozi, F.L., 2003. Ethnomedical botany of epilepsy treatment in Fong-Tongo village, western province, Cameroon. Pharm. Biol. 41,

330e339.

Pelser, P.B., de Vose, H., Theuring, C., Beuerle, T., Vrieling, K., Hartmann, T., 2005. Frequent gain and loss of pyrrolizidine alkaloids in the

evolution of Senecio section Jacobaea (Asteraceae). Phytochemistry 66, 1285e1295.

Pooley, E., 1998. A Field Guide to Wild Flowers KwaZulu-Natal and the Eastern Region. Natal Flora Publications Trust, Durban, p. 330.

Rashkes, Ya.V., Abdullaev, U.A., Yunusov, S.Yu., 1978. Mass spectra of pyrrolizidine alkaloids. Chem. Nat. Compd. 14, 121e135.

Schlage, C., Mabula, C., Mahunnah, R.L.A., Heinrich, M., 1999. Ethnobotany and preliminary ethnopharmacological evaluation of Wasamba me-

dicinal plants (Tanzania). In: Proceedings of 12 Jahrestagung der Deutschen Gesellschaft fur Tropenokologie e gto 17e19 February 1999,

Poster P-4.18.

Schmeller, T., El-Shazly, A., Wink, M., 1997. Allelochemical activities of pyrrolizidine alkaloids: interactions with neuroreceptors and acetylcho-

line related enzymes. J. Chem. Ecol. 23, 399e416.

407K. Asres et al. / Biochemical Systematics and Ecology 36 (2008) 399e407

Sedmera, P., Klasek, A., Duffield, A.M., Santavy, F., 1972. Pyrrolizidine alkaloids XIX. Structure of erucifoline. Collect. Czech. Chem. Commun.

37, 4112e4119.

Smith, L.W., Culvenor, C.C.J., 1981. Plant sources of hepatotoxic pyrrolizidine alkaloids. J. Nat. Prod. 44, 129e152.

Tadesse, M., 2004. Asteraceae (Compositae). Part 2. In: Hedberg, I., Friis, I., Edwards, S. (Eds.), Flora of Ethiopia and Eritrea, vol. 4. The

National Herbarium, Addis Ababa University, Addis Ababa.

Toppel, G., Witte, L., Riebesehl, B., von Borstel, K., Hartmann, T., 1987. Alkaloid patterns and biosynthetic capacity of root cultures from some

pyrrolizidine alkaloid producing Senecio species. Plant Cell Rep. 6, 466e469.

Witte, L., Ernst, L., Adam, H., Hartmann, T., 1992. Chemotypes of two pyrrolizidine alkaloid-containing Senecio species. Phytochemistry 31,

559e565.

Witte, L., Rubiolo, P., Bicchi, C., Hartmann, T., 1993. Comparative analysis of pyrrolizidine alkaloids from natural sources by gas chromatog-

raphyemass spectrometry. Phytochemistry 32, 187e196.