Pyrrolizidine alkaloids from Canarian endemic plants and their biological effects

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Biochemical Systematics and Ecology 36 (2008) 153e166 www.elsevier.com/locate/biochemsyseco

Pyrrolizidine alkaloids from Canarian endemic plants and their biological effects Dulce M. Domı´nguez a, Matı´as Reina a, Arnoldo Santos-Guerra b, Omar Santana c, Teresa Agullo´ c, Carmen Lo´pez-Balboa c, Azucena Gonzalez-Coloma c,* a

Instituto de Productos Naturales y Agrobiologı´a, CSIC, Avda. Astrofı´sico Francisco Sanchez, 3 La Laguna, Tenerife, Spain b Jardı´n de Aclimatacio´n de La Orotava, ICIA, Puerto de la Cruz, Tenerife, Spain c Instituto de Ciencias Agrarias-CCMA, CSIC, Serrano 115 duplicado, Madrid 28006, Spain Received 19 July 2007; accepted 26 August 2007

Abstract Pyrrolizidine alkaloid (PA) producing plants belonging to the Boraginaceae (Echium wildpretti) and Asteraceae (Canariothamnus palmensis, Kleinia neriifolia, Pericallis appendiculata, Pericallis echinata, Pericallis hansenii, Pericallis multiflora, Pericallis steetzii and Senecio bollei) were selected to study their alkaloidal composition and the defensive properties (antifeedant and phytotoxic effects) of their ethanolic and alkaloidal extracts plus their isolated PAs against insects (Spodoptera littoralis, Leptinotarsa decemlineata, the aphids Myzus persicae and Rhopalosiphum padi) and Lactuca sativa seeds. We also tested the selective cytotoxic effects of these PAs on insect-derived Sf9 and mammalian CHO cells. Most of the insect antifeedant effects were found in the ethanolic extracts. The isolated PAs had species- and structure-dependent antifeedant effects and all of them decreased L. sativa radicle growth, suggesting a specific mode of action against insects and a generalized one against plants. Given the relatively low alkaloid content of these species, we assume that their herbivore defenses are not only alkaloid-based. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Canarian endemism; Asteraceae; Boraginaceae; Pyrrolizidine alkaloid; Antifeedant; Cytotoxic; Phytotoxic

1. Introduction Pyrrolizidine alkaloids (PAs) are considered feeding deterrents for insect herbivores (Hartmann, 1999; Hartmann and Ober, 2000; Van Dam et al., 1995). Individual PA patterns have been proposed to be genetically controlled as the result of evolution under selective pressure (Hartmann and Dierich, 1998; Macel et al., 2005), suggesting an evolutionary advantage for the structural diversity of PAs. Pyrrolizidine alkaloids exist in two molecular forms, the free base and the non-toxic N-oxide. Plant PAs are mostly found as N-oxides (Hartmann and Ober, 2000). These are easily reduced in the herbivore gut and absorbed by diffusion as the free base into the hemolymph. The PA free bases are metabolized by the cytochrome P-450 oxidases resulting in their bioactivation via the formation of reactive pyrrole intermediates. A number of insect species from different taxa * Corresponding author. Fax: þ34 915640800. E-mail address: [email protected] (A. Gonzalez-Coloma). 0305-1978/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2007.08.015

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have evolved adaptations to sequester, store and utilize plant PAs against their predators and parasitoids (Hartmann, 1999; Hartmann and Ober, 2000; Eisner et al., 2002). Canary Island flora has great botanical diversity with several PA-producing genera such as Pericallis (the only genus in the tribe Senecioneae endemic to Macaronesia with both woody and herbaceous species), Canariothamnus (endemic Canarian genus previously included in Senecio with three endemic species), Senecio (widely distributed around the world, with three endemic Macaronesian species), and Echium (with 30 endemic Macaronesian species) (Bramwell and Bramwell, 2001; Hansen and Sunding, 1993; Nordenstam, 2006; Panero et al., 1999). As part of a broad study on the chemistry and bioactivity of PA-producing species (Reina et al., 1993, 1995, 1997, 1998, 2001), we have selected several Canarian plants belonging to the Boraginaceae (Echium wildpretti) and Asteraceae families (Canariothamnus palmensis, Kleinia neriifolia, Pericallis appendiculata, Pericallis echinata, Pericallis hansenii, Pericallis multiflora, Pericallis steetzii and Senecio bollei) to study the antifeedant properties of their ethanolic and alkaloidal (N-oxide free) extracts and their isolated PAs (as free bases which are the pro-toxic molecular form) on several insect species with varying feeding adaptations (Spodoptera littoralis, a PA adapted generalist; Leptinotarsa decemlineata, olyphagous feeding on Solanaceae, and the aphids Myzus persicae, a generalist, and Rhopalosiphum padi, a cereal specialist) and their effects on Lactuca sativa germination and radicle growth. We have also tested the selective cytotoxic effects of these PAs to insect-derived Sf9 and mammalian CHO cells. 2. Materials and methods 2.1. General procedures Optical rotations were measured at room temperature on a PerkineElmer 343 Plus polarimeter. IR spectra were measured on a PerkineElmer 1600 spectrophotometer. 1H and 13C NMR spectra were measured on a Bruker AMX 500 MHz spectrometer with pulsed-field gradient in CDCl3 (500 MHz for 1H and 125 MHz for 13C), using this solvent as internal standard. The programs used for DEPT, 1H, COSY, NOESY, HSQC and HMBC experiments were those furnished in the Bruker software. Exact mass measurements and electronic impact mass spectroscopy (EIMS) results were recorded on a Vg-Micromass ZAB-2F instrument at 70 eV, temp 220 . Silica gel (Merck Art. 115111, 107741, 105554, 105715) was used for column chromatography, TLC and preparative TLC. Lichroprep RP-18 (Merck Art. 110625) was used for reverse phase column chromatography. Alkaloids were visualized on TLC with a Dragendorff’s reagent. 2.2. Plant material All plants were collected during the flowering season from their natural habitats in the Canary Islands (Spain). Voucher specimens have been deposited at the herbarium of the Botanical Garden, La Orotava, Tenerife, Spain. C. palmensis (Nees) B. Nord. (¼Senecio palmensis Chr. Sm. in Buch) was collected at Boca Tauce, Tenerife, July 2000 (voucher number ORT 36393). E. wildpretti Pears. & Hook fil. subsp. wildpretti was collected at Cumbres de Fasnia (2100 m), Tenerife, June 1996 (voucher number ORT 32531). K. neriifolia Haw. (¼Senecio kleinia (L.) Less.) was collected in Barranco del Infierno, Tenerife, June 2000 (voucher number ORT 39064). P. appendiculata (L. fil.) B. Nord. was collected at Monte de las Yedras (Anaga), Tenerife, August 2000 (voucher number ORT 36772). P. echinata (L. fil.) B. Nord. was collected at Las Aguas (San Juan de la Rambla), Tenerife, April 1993 (voucher number ORT 32003). P. hansenii (Kunk.) Sund. was collected between Me´riga and Acevin˜os, La Gomera, June 1994 (voucher number ORT 32002). P. multiflora (L’He´r.) B. Nord. was collected at Santa Ursula, Tenerife, May 1999 (voucher number ORT 27594). P. steetzii (Bolle) B. Nord. was collected in Laguna Grande, Parque Nacional de Garajonay, La Gomera, May 1991 (voucher number ORT 33451). S. bollei Sund. et Kunk. s.l. was collected in Riscos de Famara, Lanzarote, April 1996 (voucher number ORT 32501). 2.3. Extraction and isolation Ground dried plant material (above ground plant tissues including stems, leaves and flowers) was extracted in ethanol at room temperature during 72 h (extract yields in Table 3). The crude EtOH extract was treated with a mixture of H2SO4 0.5 M and CH2Cl2 (1:1). The aq. soln. was stirred with Zn dust for 4e6 h (except for E. wildpretti), filtered,

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basified with NH4OH 20% (pH 9), and the alkaloids were extracted with CH2Cl2 and n-butanol to give an N-oxide free alkaloidal extract (extract yields in Table 3). These extracts were chromatographed on a Lichroprep RP-18 column and eluted with CH3CNeMeOHeH2O (80:15:5). Further purification on a Si gel column eluted with CH2Cl2eMeOH (95:5) and/or preparative TLC (20  20 cm, 0.25 mm) over Si gel eluted with CH2Cl2eMeOH (93:7), respectively, afforded erucifoline (2, C. palmensis 1.7  103%), senecionine (3, C. palmensis 1.2  103%; P. echinata 1.4  102%; P. multiflora 1.9  102%), integerrimine (4, P. echinata 1.1  102%; P. multiflora 1.9  102%), seneciphylline (5, C. palmensis 4.0  104%; P. steetzii 5.4  102%; P. appendiculata 4.4  102%; P. multiflora 9.7  103%), spartioidine (6, P. multiflora 2.4  102%), retrorsine (7, P. echinata 1.8  102%; P. multiflora 1.9  102%; P. hansenii 1.9  102%), usaramine (8, P. echinata 3.6  103%; P. multiflora 1.9  102%; P. hansenii 5.6  103%), senecivernine (10, S. bollei 2.4  102%), senkirkine (11, K. neriifolia, 8.6  102%), acetylsenkirkine (12, K. neriifolia, 1.1  102%), echimidine (13, E. wildpretti 3.3  102%), echimidine N-oxide (14, E. wildpretti 3.3  102%) (compound yields expressed as % of ethanolic extract). 2.3.1. Erucifoline (2) 25 1 þ [a]25 D 15 (CHCl3; c, 0.6  10 ) {lit. [a]D 69 (CHCl3)} (Reina et al., 1993); EIMS m/z [M] 349 (13), 304 (3), 290 (2), 278 (3), 262 (1), 218 (3), 183, (2), 136 (100), 120 (95), 106 (9), 93 (66), 80 (31), 67 (10), 53 (21); HREIMS, m/z [M]þ 349.1574, calculated for C18H25NO6. 1H and 13C NMR data identical to these reported (Logie et al., 1994; Roeder, 1990). 2.3.2. Senecionine (3) þ 25 [a]25 D 67.2 (CHCl3; c, 0.054) {lit. [a]D 56 (CHCl3)} (Adams and Gianturco, 1956); EIMS m/z [M] 335 (1), 333 (6), 291 (3), 248 (2), 220 (2), 153 (5), 136 (100), 120 (98), 119 (70), 93 (72), 80 (35), 53 (27); HREIMS, m/z [M]þ 335.1783, calculated for C18H25NO5. 1H and 13C NMR data identical to these reported (Logie et al., 1994; Roeder, 1990). 2.3.3. Integerrimine (4) þ 25 [a]25 D 23.6 (CHCl3; c, 0.12) {lit. [a]D 13.6 (CHCl3)} (Reina et al., 2001); EIMS m/z [M] 335 (6), 291 (13), 248 (11), 220 (18), 153 (9), 136 (100), 120 (95), 106 (16), 93 (90), 80 (31), 67 (12), 53 (24); HREIMS, m/z [M]þ 335, calculated for C18H25NO5. 1H and 13C NMR data identical to these reported (Logie et al., 1994; Roeder, 1990). 2.3.4. Seneciphylline (5) 3 þ 25 [a]25 D 92 (CHCl3; c, 0.86$10 ) {lit. [a]D 134.2 (CHCl3)} (Klasek et al., 1968); EIMS m/z [M] 333 (7), 316 (1), 289 (7), 274 (2), 246 (9), 220 (6), 150 (5), 138 (43), 136 (79), 120 (100), 108 (12), 106 (15), 95 (43), 94 (62), 93 (62), 80 (34), 53 (23); HREIMS, m/z [M]þ 333.1521, calculated for C18H23NO5. 1H and 13C NMR data identical to these reported (Logie et al., 1994; Roeder, 1990). 2.3.5. Spartioidine (6) 25 [a]25 D 74 (EtOH; c, 0.26) {lit. [a]D 83.5 (EtOH)} (Bull et al., 1968). IR (CHCl3) nm ax 3019, 2400, 1716, 1 þ 1652 cm . EIMS m/z [M] 333 (11), 289 (43), 246 (27), 218 (3), 139 (13), 138 (60), 137 (21), 136 (87), 120 (100), 119 (74), 118 (13), 108 (13), 106 (15), 95 (41), 94 (57), 93 (59), 83 (12), 80 (26), 53 (15), 48 (11); HREIMS, m/z [M]þ 333.1543, calculated for C18H25NO5. 1H and 13C NMR data in Table 1. 2.3.6. Retrorsine (7) 25 1 þ [a]25 D 23 (EtOH; c, 1.4$10 ) {lit. [a]D 29.2 (CHCl3)} (Reina et al., 1993). EIMS m/z [M] 351 (20), 320 (6), 292 (5), 246 (11), 220 (26), 138 (40), 136 (100), 121(2), 120 (83), 119 (61), 106 (12), 94 (49), 93 (2), 93 (63), 80 (32), 70 (44), 53 (27); HREIMS, m/z [M]þ 351.1686, calculated for C18H25NO6. 1H and 13C NMR data identical to these reported (Logie et al., 1994; Roeder, 1990). 2.3.7. Usaramine (8) 2 25 [a]25 D þ25 (EtOH; c, 3.2$10 ) {lit. [a]D þ7.1 (EtOH)} (Culvenor and Smith, 1966). IR (CHCl3) nm ax 3360, 2918, 1715, 1647, 1272, 1210, 1153, 928, 754 cm1. EIMS m/z [M]þ 351 (8), 316 (6), 256 (13), 248 (6), 241 (8), 220 (13),

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Table 1 1 H, 13C, COSY, HMQC, and HMBC NMR Data of compound 6 (spartioidine) Proton

d (JHeH in Hz)

COSY

Correlated Carbons HMQC

2 3a 3b 5a 5b 6a 6b 7 8 9a 9b

6.19 3.39 3.97 3.29 2.53 2.36 2.08 4.98 4.34 4.01 5.42

13 14a 14b

3.2 d (16.7) 2.6 d (16.7)

H-14b, H-19a H-14a

18 19a 19b 20 21

1.52 5.18 4.92 6.67 1.74

H-14a e H-21 H-20

a

d (1.1) dd (15.9, 4.8) d (16.1) t (8.3) m dd (13.9, 5.6) m t (3.7) br s d (11.8) d (11.7)

s d d q d

(2.2) (1.9) (7.1) (7.2)

e H-3b, H-2 H-3a H-5b, H-6b H-5a, H-6a, H-6b H-5b, H-6b H-5a, H-5b, H-6a H-8 H-3a, H-3b, H-7 H-9b H-9a

C-1 131.2 s 137.5 d 61.9 t 61.9 t 52.9 t 52.9 t 33.8 t 33.8 t 75.1 d 76.9 d 60.6 t 60.6 t C-11 176.8 s C-12 76.2 s C-13 144.7 s 28.8 t 28.8 t C-15 131.7 s C-16 168.0 s 25.1q 113.6 t 113.6 t 136.4 d 13.6 q

a

HMBC

C-1, C-1, C-1, C-3, C-3, C-5, C-5 C-5 C-1, C-1, C-1,

C-3, C-2, C-2, C-6, C-6 C-7,

C-8 C-5, C-7 C-5, C-7 C-7, C-8 C-8

C-2, C-7 C-2, C-8, C-11 C-2, C-8, C-11

C-12, C-13, C-15, C-16, C-19, C-20 C-12, C-13, C-15, C-16, C-19, C-20

C-11, C-12, C-12, C-13, C-14,

C-12, C-13 C-13, C-14 C-13, C-14 C-14,C-15, C-16, C-21 C-15, C-16, C-20

Multiplicities of 13C NMR were established by DEPT experiment.

136 (100), 120 (97), 93 (80), 80 (31), 67 (82), 53 (27); HREIMS, m/z [M]þ 351.1660, calculated for C18H25NO6. 1H and 13C NMR data identical to these reported (Logie et al., 1994; Roeder, 1990). 2.3.8. Senecivernine (10) 1 25 [a]25 D 15 (CHCl3; c, 0.6  10 ) {lit. [a]D 34.9 (EtOH)} (Roeder et al., 1979). IR (CHCl3) nm ax 3400, 1718, 1654, 1560, 1508, 1458, 1255, 1160, 764 cm1. EIMS m/z [M]þ 335 (6), 291 (10), 248 (12), 218 (2), 153 (53), 136 (84), 120 (100), 93 (68), 81 (54), 55 (46); HREIMS, m/z [M]þ 335.0020, calculated for C18H25NO6. 1H and 13 C NMR data in Table 2. 2.3.9. Senkirkine (11) 25 1 [a]25 D 12.0 (CHCl3; c, 0.58  10 ) {lit. [a]D 6.2 (CHCl3)} (Diaz-Rodriguez et al., 1967); IR (CHCl3) nm ax 1 þ 3400, 1720, 1650, 1612 cm ; EIMS m/z [M] 365 (5), 337 (15), 321 (16), 294 (20), 266 (32), 254 (17), 250 (19), 222 (15), 211 (10), 184 (7), 168 (84), 153 (100), 151 (97), 140 (18), 139 (12), 138 (20), 135 (11), 128 (22), 123 (55), 110 (80), 100 (42), 96 (42), 95 (10), 94 (34), 83 (31), 82 (50); HREIMS, m/z [M]þ 365.1834, calculated for C19H27NO6. 1H and 13C NMR data identical to these reported (Cheng and Roeder, 1986). 2.3.10. Acetylsenkirkine (12) 25 1 þ [a]25 D 10.5 (MeOH; c, 0.86  10 ) {lit. [a]D 34.0 (MeOH)} (Briggs et al., 1966); EIMS m/z [M] 407 (6), 363 (2), 337 (9), 320 (17), 303 (11), 294 (15), 266 (26), 249 (33), 168 (50), 153 (100), 122 (45), 110 (76), 100 (39), 82 (41), 70 (34), 53 (71); HREIMS, m/z [M]þ 407.1906, calculated for C21H29NO7. 1H NMR data identical to those reported (Gonzalez et al., 1986). 13C NMR (CDCl3, 125 MHz): d 133.7 (s, C-1), 138.1 (d, C-2), 58.5 (t, C-3), 53.0 (t, C-5), 36.1 (t, C-6), 78.5 (d, C-7), 192.0 (s, C-8), 64.6 (t, C-9), 171.0 (s, C-11), 83.4 (s, C-12), 40.4 (d, C-13), 37.2 (t, C-14), 131.7 (s, C-15), 166.4 (s, C-16), 21.9 (q, C-18), 11.3 (q, C-19), 137.2 (d, C-20), 15.2 (q, C-21), 40.5 (q, N-CH3), 21.4 (q, eOCOCH3), 169.9 (s, OCOCH3).

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Table 2 1 H, 13C, COSY, HMQC, and HMBC NMR Data of compound 10 (senecivernine) Proton

d (JHeH in Hz)

COSY

Correlated carbon a

HMQC

2 3a 3b 5a 5b 6a 6b 7a 8 9a 9b

6.22 3.98 3.43 3.28 2.54 2.38 2.08 5.06 4.34 4.10 5.53

13 14

1.72 m 2.54 m

H-19 H-20

18 19 21 20a 20b

1.33 0.85 1.08 5.24 5.85

H-13 H-14 H-20b H-20a

a

s (1.1) d (15.9) dd (16.0, 6.3) t (8.4) m dd (13.9, 5.8) m t (3.7) br s d (11.7) d (11.7)

s d (7.1) d (6.9) s s

e H-3b, H-2 H-3a H-5b, H-6b H-5a, H-6a, H-6b H-5b, H-6b H-5a, H-5b, H-6a H-8 H-2, H-7a H-9b H-9a

C-1 131.4 s 136.2 d 62.6 t 62.6 t 53.2 t 53.2 t 34.2 t 34.2 t 75.5 d 77.8 d 60.7 t 60.7 t C-11 178.2 s C-12 77.7 s 40.7 d 35.8 d C-15 147.4 s C-16 168.9 s 26.2 q 5.7 q 12.0 q 120.6 t 120.6 t

HMBC C-3, C-1, C-1, C-7 C-3, C-7 e e e C-1, C-1,

C-8 C-2 C-2, C-5 C-7

C-2, C-11 C-2, C-8, C-11

C-14, C-15, C-19, C-20 C-12, C-15, C-16, C-19, C-20, C-21

C-11, C-12, C-13, C-14, C-14,

C-12, C-13, C-14, C-15, C-16

C-13 C-14 C-15 C-16

Multiplicities of 13C NMR were established by DEPT experiment.

2.3.11. Acetylusaramine (9) Usaramine (8, 2.0 mg) was acetylated with Ac2Oepyridine at room temperature for 24 h. The solvent was evaporated to give a residue of 2.10 mg which was chromatographed on a Si gel column to give 2.0 mg (89.6%) of compound 9. EIMS m/z [M]þ 393 (5), 351 (1), 349 (3), 320 (4), 306 (1), 274 (3), 246 (8), 220 (12), 138 (40), 136 (100), 121 (37), 120 (76), 119 (72), 95 (35), 94 (40), 93 (73), 80 (20) and 55 (17). 1H NMR (CDCl3, 500 MHz): d 0.92 (3H, d, J ¼ 7.0 Hz, H-19), 1.80 (3H, dd, J ¼ 7.0, 1.7 Hz, H-21), 2.05 (3H, s, OCOCH3), 2.23 and 2.0 (1H each, m, H-14a and 14b), 2.15 and 2.47 (1H each, m, H-6a and H-6b), 2.62 and 3.38 (1H each, m, H-5a and H-5b), 3.50 (1H, m, H-3a), 4.04 (1H, d, J ¼ 16.0 Hz, H-3b), 3.69 and 3.83 (1H each, d, J ¼ 11.2 Hz, H-18a and H-18b), 4.22 and 5.47 (1H each, d, J ¼ 11.9 Hz, H-9a and H-9b), 4.45 (1H, br s, H-8), 5.09 (1H, br s, H-7), 6.28 (1H, br s, H-2), 6.61 (1H, q, J ¼ 7.05 Hz, H-20). 13C NMR (CDCl3, 125 MHz): d 131.4 (s, C-1), 137.4 (d, C-2), 62.0 (t, C-3), 53.4 (t, C-5), 34.0 (t, C-6), 75.2 (d, C-7), 77.0 (d, C-8), 61.2 (t, C-9), 176.0 (s, C-11), 79.0 (s, C-12), 37.4 (t, C-13), 29.5 (t, C-14), 133.1 (s, C-15), 168.8 (s, C-16), 68.5 (t, C-18), 12.7 (q, C-19), 136.0 (d, C-20), 14.5 (q, C-21). 2.3.12. Echimidine (13) 25 1 þ [a]25 D þ11.5 (EtOH; c, 3.3  10 ) {lit. [a]D þ13.1 (EtOH)} (Roeder et al., 1991); HREIMS, m/z [M] 1 13 397.212051, calculated for C20H31NO7. H and C NMR data identical to these reported (Roeder et al., 1991). 2.3.13. Echimidine N-oxide (14) 1 H NMR data identical to these reported (Roeder et al., 1991). HMQC 13C RMN (100 MHz, CDCl3): d 132.5 (s, C-1), 122.6 (d, C-2), 77.6 (t, C-3), 69.3 (t, C-5), 32.7 (t, C-6), 72.3 (d, C-7), 93.6 (d, C-8), 60.5 (t, C-9), 174.5 (s, C-10), 85.1 (s, C-11), 70.0 (d, C-12), 18.6 (q, C-13), 73.0 (s, C-14), 24.6 (q, C-15), 26.4 (q, C-16), 165.2 (s, C-17), 126.5 (s, C-18), 140.7 (d, C-19), 15.8 (q, C-20), 20.3 (q, C-21). EIMS m/z, FAB: [M þ 1] 414 (100), 398 (33), 329 (8), 307 (9), 289 (8), 254 (8), 238 (14), 214 (20), 176 (39), 154 (78), 13 (59), 55 (49).

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2.4. Insect bioassays S. littoralis, L. decemlineata and the aphid colonies (M. persicae and R. padi) were reared on artificial diet and their respective host plants (Solanum tuberosum, Capsicum annuum and Hordeum vulgare) and maintained at 22  1  C, >70% relative humidity, and photoperiod of 16L:8D in a growth chamber as described in Reina et al. (2001). 2.4.1. Choice feeding assays These experiments were conducted with sixth-instar S. littoralis larvae and adult L. decemlineata, M. persicae and R. padi (apterous). C. annuum, S. tuberosum or H. vulgare leaf disks/fragments (1.0 cm2) were treated on the upper surface with 10 ml of the test substance. Two treated and two control disks were arranged alternatively on five agarcoated Petri dishes (9.0 cm diameter) with three insects (S. littoralis or L. decemlineata) and allowed to feed in a growth chamber (environmental conditions as described above). Each experiment was repeated three times. Feeding was terminated after the consumption of between 50 and 75% of the control disks. Percent feeding inhibition (%FI) was calculated as described in Reina et al. (2001). For the aphids, 20 2  2 cm boxes with 10 insects each were used for tests and their settling inhibition index (%SI) calculated as described by Gutierrez et al. (1997). Compounds with an FI > 70% were tested in a doseeresponse experiment (dose series between 50.00 and 0.08 mg/cm2) to calculate their relative potency (EC50 values, the effective dose for 50% feeding reduction) which was determined from linear regression analysis (STATGRAPHICS Plus) (%FI on log dose). 2.4.2. Oral cannulation This experiment was performed with pre-weighed newly molted S. littoralis L6 larvae. Each experiment consisted of 20 larvae orally dosed with 40 mg of the test compound in 4 ml of DMSO (treatment) or solvent alone (control) as described in Reina et al. (2001). At the end of the experiments (72 h), larval consumption and growth were calculated on a dry weight basis. An analysis of covariance (ANCOVA1) on biomass gains with initial biomass as covariate (covariate p > 0.05) showed that initial insect weights were similar among all treatments. A second analysis (ANCOVA2) was performed on biomass gains with food consumption as covariate to test for postingestive effects. 2.4.3. Phytotoxicity tests These experiments were conducted with L. sativa var. Carrascoy seeds placed on paper disks (Whatman no. 1, 2.0 cm2) treated with 50 mg/cm2 of the test compound or solvent for the control. The disks were placed in lidded clear plastic boxes (2  2 cm2) lined with 4 g of calibrated sand humidified with 200 ml of deionized water and then placed in a plant growth chamber (25 þ 1  C, >70% relative humidity with a photoperiod of 16:8 h L:D) for 6 days. A total of 100 seeds were used (20 seeds/box, five boxes, Moiteiro et al., 2006). Germination was monitored daily and the radicle length measured at the end of the experiment (20 digitalized radicles randomly selected for each experiment) with the application Image J Version 1.37r, 2006 (http://rsb.info.nih.gov./ij/). A non-parametric analysis (KruskalleWallis Test) was performed on germination and radicle length data. 2.4.4. Cytotoxicity Sf9 cells derived from Spodoptera frugiperda pupal ovarian tissue (European Collection of Cell Cultures, ECCC) and mammalian Chinese hamster ovary cells (CHO, a gift from Dr. Pajares, I.C. Biome´dicas, CSIC) were grown as previously described in Gonzalez-Coloma et al. (2002a). Cell viability was analyzed by an adaptation of the MTT colorimetric assay method (Mossman, 1983). The active compounds were tested in a doseeresponse experiment to calculate their relative potency (LD50 values, the effective dose to give 50% cell viability) which was determined from linear regression analysis (% cell viability on log dose). 3. Results and discussion The alkaloidal and ethanolic extracts (yield and composition) of the selected plant species along with their antifeedant effects and phytotoxicity are shown in Table 3. Alkaloid extract yield ranged between 0.04 and 2.0% of the ethanolic extract. C. palmensis and P. multiflora showed the lowest and the highest alkaloid content. The total alkaloid levels found for these Canarian species (0.01e0.07% of plant dry weight) are lower than those reported for Senecio spp (0.09e0.18% of plant dry weight) (see Gardner et al., 2006), except for P. multiflora (0.43% of plant

Table 3 Yield, alkaloids isolated, insect antifeedant effects and phytotoxic activity (Lactuca sativa germination and radicle length expressed as % control) of Alkaloidal (A) and Ethanolic (E) extracts (100 mg/cm2) from endemic Canarian PA-producing species Yield

Alkaloid

L. decemlineata c

C. palmensis E. wildpretti K. neriifolia P. appendiculata P. echinata P. hansenii P. multiflora P. steetzii S. bollei

A E A E A E A E A E A E A E A E A E

0.04a 35.7b 1.33 5.0 0.49 9.9 0.24 7.7 0.50 9.3 0.29 10.0 1.94 22.4 0.72 10.0 0.46 7.6

2, 3, 5 13, 14 11, 12 5 3, 4, 7, 8 7, 8 3, 4, 5, 6, 7, 8 5 10

S. littoralis c

M. persicae d

R. padi d

d

Germination d

%FI

%FI

%C

%T

%C

%T

nt 85* 96* 79* nt nt nt nt nt 91 nt 89* 73* 82* nt 84* 96* 82*

38 19 <50 0 20 49 37 37 10 36 18 52* 54* 19 7 62* <50 <50

48 61 60 80 55 96 48 87 55 60 57 56 45 78 49 75 68 66

52 40 40 20* 45 4* 52 13* 44 40 43 44 55 22* 51 25* 31* 34*

49 44 54 51 50 67 56 52 48 58 51 51 63 50 47 74 45 79

51 56 46 49 50 33* 44 48 52 42 49 48 37* 50 53 26* 54 21*

H, pe nt, Not tested. *p < 0.05,Wilcoxon Signed Rank Paired Test. a % of the E extract. b % Plant dry weight. c %FI ¼ 1  (T/C)  100, where T and C are the consumption of treated and control leaf disks, respectively. d %C and %T are percent aphids settled on control and treated leaf disks, respectively. e KruskalleWallis Test H and p values. f Median is significantly different from the Control.

24 h

48 h

Radicle length (144 h)

84 97 90 95 88 97 95 100 93 98 92 98 100 97 84 89 35e 95 A: 30.7, <0.01 E: 6.0, >0.05

99 99 98 96 98 99 100 100 100 99 99 100 100 99 99 99 98 100 6.0, >0.05

90 103 87 74f 67f 83f 83f 77f 96 95 99 118 70f 51f 75f 63f 71f 99 E: 103.3, <0.05 A: 34.4, <0.00001

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Plant extract

159

160

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dry weight). Senecio inflorescens may contain 60e80% of total PAs (see Hartmann and Dierich, 1998). Therefore, higher alkaloid concentrations could have been obtained for upper leaves and/or flower extracts rather than from above ground flowering plant extracts (including stems, leaves and flowers) assuming a similar PA distribution for these plant species. The Boraginaceae E. wildpretti had 0.06% alkaloid content, a yield within the range of continental Echium species such as Echium humile (0.025%) (El-Shazly et al., 1996), in contrast to other Canarian Echium species (Echium pininana) with 10 times lower alkaloid content (0.009%) (Roeder et al., 1991). The ratios of Ethanolic (E) to Alkaloidal (A) active extracts were 3:1 for S. littoralis with P. steetzii-E being the most active, 6:1 for M. persicae with K. neriifolia-E, P. appendiculata-E, E. wildpretti-E and P. multiflora-E being the strongest antifeedants, 3:1 for R. padi with S. bollei-E being the most active and 5:6 for L. sativa (radicle length) with P. multiflora-E being the strongest inhibitor (Table 3). Therefore, the ethanolic extracts were in general more active than the alkaloidal ones except for their phytotoxic effects. Considering that the alkaloidal fraction of these ethanolic extracts accounts for a maximum of 2% and that most of the PAs present in these alkaloidal fractions could be in their N-oxide form (Hartmann and Ober, 2000) which has been shown to be less bioactive (Macel et al., 2005; Van Dam et al., 1995), the chemistry of the non-alkaloidal fraction could partially explain their antifeedant effects. Senecio species produce benzofurans, cacalolides and eremophilanolides among other compounds with antifeedant and phytotoxic effects (Reina et al., 2001, 2006; Burguen˜o-Tapia et al., 2007), while C. palmensis accumulates bisabolane and silphinene-type sesquiterpenes which are strong L. decemlineata antifeedants (11e17% yield) (GonzalezColoma et al., 2002b; Reina et al., 2002). Other studies suggest that chemicals present in the non-alkaloidal fraction of Senecio spp can be an important part of their defense (Joshi and Vrieling, 2005). Furthermore, sesquiterpenes and PAs can act synergistically on the feeding behavior of insects as shown for cacalol and seneciphylline on Adenostyles alliariae specialist insects (Ha¨gele and Rowell-Rahier, 2001) further emphasizing the defensive role that these terpenes may play in PA-producing plants. Eight unsaturated macrocyclic diester PAs have been isolated from these alkaloidal extracts, erucifoline (2), senecionine (3), integerrimine (4), seneciphylline (5), spartioidine (6), retrorsine (7), usaramine (8) and senecivernine (10), two unsaturated macrocyclic diester seco-PAs, senkirkine (11) and acetylsenkirkine (12), the open diester echimidine (13) and its N-oxide (14) (Fig. 1). The structures of these alkaloids were elucidated by mono and bidimensional NMR and by comparison with previously reported spectroscopic data. Complete NMR data of spartioidine (6) and senecivernine (10) are shown in Tables 1 and 2. Senecivernine (10) resonance values for C-3 and C-9 have been corrected to 62.6 (t) and 60.7 (t) from previously reported data (Pieters and Vlietinck, 1988) according to an HMQC experiment. Usaramine (8) was acetylated with Ac2OePy to give 18-acetylusaramine (9) for comparison purposes. P. multiflora showed the highest PA diversity (6 PA molecules), followed by P. echinata (5 PA molecules), C. palmensis (3 PA molecules), P. hansenii (2 PA molecules), E. wildpretti, P. appendiculata, S. bollei, K. neriifolia and S. steetzii (1 PA molecule). The antifeedant or phytotoxic effects of these alkaloidal extracts (Table 3) did not correlate with their PA diversity, except for P. multiflora. From among the Asteraceae genera, the most frequently isolated PA was seneciphylline (5) followed by senecionine (3), retrorsine (7), usaramine (8), integerrimine (4) > erucifoline (2), spartioidine (6), senecivernine (10), senkirkine (11) and acetylsenkirkine (12) (Table 3). PAs in the Asteraceae, and specifically in Senecio spp are produced in the roots as senecionine N-oxide and transported via the phloem to the above ground organs (Hartmann et al., 1989) where it is transformed into several related PAs (Hartmann and Dierich, 1998; Pelser et al., 2005). The PA structures found in Canariothamnus, Kleinia and Pericallis (senecionine-related) suggest a similar biosynthetic pattern to Senecio. Further research is needed to pinpoint the specific location and pathways of PA biosynthesis in these genera. The antifeedant effects of the individual alkaloids against several herbivorous insects are shown in Table 4. L. decemlineata responded to a larger number of PAs than S. littoralis. Compounds 3, 5 and 13 were strong antifeedants to L. decemlineata followed by 4 and 6. Spartioidine (6) was a strong antifeedant to S. littoralis, 24 times more potent against this insect than against L. decemlineata. Among the 13 compounds tested, M. persicae settling behavior was affected by five senecionine-related PAs (2, 6, 8, 11 and 12) while R. padi responded to the unrelated ones (1 and 13). However, senecionine (3) and seneciphylline (5) reduced M. persicae survival in artificial diets while senkirkine (11) was inactive (Macel et al., 2005), suggesting different modes of action between the deterrent and toxic effects of these compounds against M. persicae. The antifeedant and toxic effects of the PAs studied here are species- and structure-dependent. The macrocyclic diesters senecionine (3), integerrimine (4), seneciphylline (5) and spartioidine (6) were strong antifeedants to

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HO

HO H Me

Me

20

Me

O

O

O

Me

15

13

O

O

O

H

O

CH2OH O O

O

N

N

1

2

3

HO

Me

HO

Me

O

O

O

N

N

N

4

5

6

HO

OH

Me

H Me O

O

O

H

Me H

N

N

8

9

HO

Me

Me

Me

AcO

H O

H

Me

O

10 O

O

19

6

O 10 HO

9 1

8

2

H

O

O

O O

N

N

Me

Me

11

12 O

O

OH

11 12 13 16 14 OH 15

Me

H

O

O O

H

Me

O

O

17 O 18 7 21

O

O

N

N

H

H O

7

H

OAc O

O

O

20

HO

OH O

H

H HO

O

O O

H

H

O

O

O

O

Me

O CH2

H

H

HO

Me

O CH2

H Me

O

3

5

O

O

2

N

HO

O

1

8

6

O

11 O

7

H

O

12

Me 19 9 H

H

16 O

18 Me

HO

14

21

161

H

O

O H

H

OH

HO OH

N

N 3

5

13

O

14

Fig. 1. Structures of PAs: 1, monocrotaline; 2, erucifoline; 3, senecionine; 4, integerrimine; 5, seneciphylline; 6, spartioidine; 7, retrorsine; 8, usaramine; 9, acetylusaramine; 10, senecivernine; 11, senkirkine; 12, acetylsenkirkine; 13, echimidine and 14, echimidine N-oxide.

L. decemlineata. The lateral chain at C-15 and a-Me and b-OH at C-12 could be structural requirements for this effect. The open diester echimidine (13) lost its antifeedant action in its N-oxide form (14). Other studies have shown that N-oxides are less deterrent to insects than the free bases (Macel et al., 2005; Van Dam et al., 1995). However, integerrimine N-oxide was an effective antifeedant to L. decemlineata (Reina et al., 2001).

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Table 4 Comparative antifeedant effects of PAs on divergent insect species Compound

S. littoralis

L. decemlineata 2

EC50 (mg/cm ) 1 2 3 4 5 6 7 8 9 10 11 12 13 14

b

>50 >50 >50 >100c w50 0.2 (0.02, 0.8) >50b nt nt >50 >50 >50 >50b >50

b

>50 >50 1.3 (0.2, 7.9) 3.4 (1.3, 9.5)c 1,7 (0, 214, 6) 4.8 (1.3, 17.6) >50b nt nt >50 >50 >50 1.4 (0.8, 2.1)b >50

M. persicaea

R. padia

%C

%T

%C

%T

51 69 60 43 50 65 56 70 60 nt 65 65 60 60

49 31* 40 57 50 35* 44 30* 40 nt 35* 35* 40 40

63 56 60 57 58 58 54 60 nt nt 47 58 64 57

37* 43 40 43 42 42 46 40 nt nt 53 42 36* 43

*p < 0.05, Wilcoxon Signed Rank Paired Test; nt, not tested. a As in Table 3 (dose of 50 mg/cm2). b From Gonzalez-Coloma et al. (2002c). c From Reina et al. (2001).

S. littoralis and M. persicae did not respond to a sufficiently large number of structures to obtain any conclusions on structureeactivity relationships. However, minor structural changes resulted in significant activity. The C-19 methylene group (4 vs. 6) in the presence of a Z (cis) configuration of the lateral chain at C-15 (5 vs. 6) resulted in the antifeedant action of spartioidine (6) on S. littoralis (also active on M. persicae). The 12,13-epoxide with a C-19 hydroxymethyl group of erucifoline (2) resulted in the strongest effect on M. persicae settling behavior. Additionally, the acetylation at C-18 of 8 resulted in the postingestive effect of acetylusaramine (9). Similarly, previous results have shown species- and structure-dependent antifeedant effects for macrocyclic (senecionine, 3 and integerrimine, 4) and open PA diesters (echimidine, 13); as well as unsaturated (supinine) and saturated (30 -acetyltrachelanthamine) PA monoesters against L. decemlineata with no effect on S. littoralis (Gonzalez-Coloma et al., 2002b; Reina et al., 1997, 2001). The unsaturated monoesters lycopsamine and europine and the secopyrrolizidine diester otosenine were moderate antifeedants to S. littoralis (Gonzalez-Coloma et al., 2002c; Reina et al., 1995, 1998). Similarly, other unsaturated PA monoesters isolated from the Boraginaceae Anchusa strigosa were antifeedants to Spodoptera exigua and Pieris brassicae with similar potencies while the saturated ones were inactive to these lepidopterans (Siciliano et al., 2005). The results on the antifeedant action of the individual PAs did not correlate with those from their A extracts, except for P. multiflora whose effects are explained by compound 6. We found stronger and lower effects against L. decemlineata and M. persicae, respectively, for the A extracts than these expected from the activity of their major alkaloids, suggesting compound interactions (positive and negative). Previous results have shown that a PA mixture (integerrimine/senecionine N-oxide) was more active than the respective free bases against the spider Nephila clavipes (Silva and Trigo, 2002). Similarly, indications of synergistic deterrent effects have been found for a PA mixture (senecionine/seneciphylline) against S exigua (Macel et al., 2005). Table 5 shows the postingestive effects to S. littoralis and the selective cytotoxicity of the individual PAs. A covariance analysis (ANCOVA1) of food consumption (DI) and biomass gains (DB) with initial larval weight (BI) as covariate (covariate > 0.05) was performed to test for significant effects of the test compounds on these variables. An additional ANOVA analysis and covariate adjustment on DB with DI as covariate (ANCOVA2) was performed for those compounds that significantly reduced DB to understand their postingestive mode of action (antifeedant and/or toxic) (Reina et al., 2001). Monocrotaline (1, included for comparison purposes) and acetylusaramine (9) reduced biomass gains (DB) but not food consumption (DI) of orally injected S. littoralis larvae. Treatment effects did not disappear with covariance adjustment, indicating that they acted as strong postingestive toxins without antifeedant effects. Among the PAs tested, only 10 had moderate cytotoxic effects to mammalian CHO cells, suggesting that the postingestive toxicity of these PAs is not cytotoxic-related.

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Table 5 Consumption (DI) and biomass gain (DB) of orally injected S. littoralis L6 larvae, expressed as percent of the control and cytotoxic effects on S. frugiperda Sf9 and mammalian CHO cells Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14

S. littoralis

Sf9

DB

DI

EC50 (mg/ml)

47a 106 84 89 87 90 90 102 60b 90 116 100 118 102

98 102 84 88 82 97 88 92 106 98 105 88 165 113

>100 nt >100 >100 >100 >100 >100 >100 >100 >100 nt nt nt nt

CHO

>100 nt >100 >100 >100 >100 >100 >100 >100 13.2 (6.5, 26.8) nt nt nt nt

nt, Not tested. a ANCOVA1(initial larval weight as covariate), p < 0.005 for DB, p ¼ 0.86 for DI. ANCOVA2 (DI as covariate), p < 0.00001 for DB. b ANCOVA1(initial larval weight as covariate), p < 0.005 for DB, p ¼ 0.68 for DI. ANCOVA2 (DI as covariate), p < 0.00001 for DB.

Tertiary PAs are deleterious for vertebrates with a microsomal cytochrome P-450 (Fu et al., 2004). Insects with P-450 enzymes involved in xenobiotic metabolism (Brattsten, 1992; Glendinning, 2002) activate PAs and can cause mutagenic effects (Frei et al., 1992). S. littoralis larvae prevent PA poisoning by rapid and efficient excretion of the absorbed tertiary alkaloid (Lindigkeit et al., 1997). However, monocrotaline (1) and acetylusaramine (9) had toxic postingestive effects on S. littoralis larvae. The secopyrrolizidinic diester otosenine also reduced this insect’s growth rate and food consumption (Gonzalez-Coloma et al., 2002b) in contrast with the lack of effect of senkirkine (11) and acetylsenkirkine (12), suggesting that the excretion of some of these compounds is not efficient enough and therefore might undergo bioactivation by the insect’s cytochrome P-450. Similarly, larvae of the non-adapted lepidopteran Philosamia ricini excreted most of the ingested seneciphylline, accumulating only traces in their bodies that resulted in toxic effects (Narberhaus et al., 2005). Fig. 2 shows the effects on L. sativa of the individual alkaloids. Some PAs significantly inhibited seed germination (Dunn’s test, p < 0.05) at 24 h (1, 2, 7, 8, 9, 11, 12), 48 h (1, 11, 12), 72 h, 96 h (1, 11), 120 h and 144 h except for 1 which remained active up to 144 h. Radicle length was moderately inhibited (<50% inhibition) by all these compounds. Some Senecio species are invasive weeds (Senecio vulgaris, Senecio jacobaea, Senecio inaequidens and Senecio madagascariensis) (Gardner et al., 2006; Joshi and Vrieling, 2005; Robinson et al., 2003; Scherber et al., 2003). Their invasion success could be partially explained by the presence of allelopathic compounds (PAs and nonalkaloidal compounds such as cacalol and related sesquiterpenes) (Burguen˜o-Tapia et al., 2007) in tissues such as the roots or seeds where they could play a role in planteplant interactions. Indirect evidence of PA presence in the soil comes from the fact that S. jacobaea PA chemotypes influenced soil-borne fungal communities (Kowalchuk et al., 2006).

4. Conclusions We have studied the antifeedant and phytotoxic properties of ethanolic and alkaloidal extracts of nine PAproducing plant species endemic to the Canary Islands. Most of the insect antifeedant effects were found in the ethanolic extracts containing mainly terpenoids with low amounts of PAs. The isolated PAs had speciesand structure-dependent antifeedant effects and were all phytotoxic suggesting a specific mode of action against insects (neuroreceptor-mediated) and a generalized one against plants (DNA-alkylating, etc). In addition to their antiherbivore action, these alkaloids could play a role in planteplant interactions.

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24h

48h

96h

120h

Radicle length

144h

120

100

Control

80

60

40

20

0

1

2

3

4

5

6

7

8

9

11

12

PAs Fig. 2. Phytotoxic activity of PAs (50 mg/cm2) against Lactuca sativa germination and radicle length (expressed as % control). KruskalleWallis Test H and p values were 29.5, <0.005 for 24 h; 22.5, <0.05 for 48 h; 13.7, 0.24 for 72 h; 13.7, 0.24 for 96 h; 10.6, 0.47 for 120 h; 17.8, 0.08 for 1144 h and 72.4, <0.0001 for radicle length.

Acknowledgements This work was supported by grants CTQ2006-15597-C02-01/PPQ and an I3P-CSIC fellowship to D.M. Domı´nguez. Plant collection was possible thanks to the ‘‘Cabildos’’ of Tenerife, Gomera and Lanzarote. We gratefully acknowledge S. Carlin for language revision. References Adams, R., Gianturco, M., 1956. Senecio alkaloids-the alkaloids of Senecio brasiliensis, fremonti and ambrosioides. J. Am. Chem. Soc. 78, 5315e 5318. Bramwell, D., Bramwell, Z., 2001. Flores silvestres de las Islas Canarias, 4a ed. Rueda, Madrid. Brattsten, L.B., 1992. Metabolic defenses against plant allelochemicals. In: Rosenthal, G.A., Berenbaum, M.R. (Eds.), Herbivores Their Interactions with Secondary Plant Metabolites, vol. 2. Academic Press, San Diego, California, pp. 175e242. Briggs, L.H., Cambie, R.C., C,Y, B.J., O’Donovan, G.M., Russell, R.H., Seelye, R.B., 1966. Collq. Int. N. R. S. (144), 147, C.A. 67, 54302, 1967. Bull, L.B., Culvenor, C.C.J., Dick, A.T., 1968. The Pyrrolizidine Alkaloids. North-Holl, Amsterdam. Burguen˜o-Tapia, E., Gonzalez-Coloma, A., Martı´n-Benito, D., Joseph-Nathan, P., 2007. Antifeedant and phytotoxic activity of cacalolides and eremophilanolides. Z. Naturforsch. C 62, 362e366. Cheng, D.L., Roeder, E., 1986. Pyrrolizidine alkaloids from Emilia sonchifolia. Planta Med. 52, 484e486. Culvenor, C.C.J., Smith, L.W., 1966. Usaramine, a new pyrrolizidine alkaloid from Crotalaria usaramoensis. E.G. Baker. Aust. J. Chem. 19, 2127e2131. Diaz-Rodriguez, F., Gonzalez, A.G., Morales-Mendez, A., 1967. An. Quim. 63, 213e220. Eisner, T., Rossini, C., Gonzales, A., Iyengar, V.K., Siegler, M.V.S., Smedley, S.R., 2002. Paternal investment in egg defence. In: Hilker, M., Meiners, T. (Eds.), Chemoecology of Insect Eggs, Egg Deposition. Blackwell, Oxford, pp. 91e116. El-Shazly, A., Sarg, T., Ateya, A., Abdel Aziz, E., El-Dahmy, S., Witte, L., Wink, M., 1996. Pyrrolizidine and tetrahydroisoquinoline alkaloids from Echium humile. Phytochemistry 42 (1), 225e230. Frei, H., Luthy, J., Brauchli, J., Zweifel, U., Wurgler, F.E., Schlatter, C., 1992. Structure/activity relationships of the genotoxic potencies of sixteen pyrrolizidine alkaloids assayed for the induction of somatic mutation, recombination in wing cells of Drosophila melanogaster. Chem.-biol. Interact. 83, 1e22. Fu, P.P., Xia, Q.S., Lin, G., Chou, M.W., 2004. Pyrrolizidine alkaloids e genotoxicity, metabolism enzymes, metabolic activation, mechanisms. Drug Metab. Rev. 36, 1e55. Gardner, D.R., Thorne, M.S., Molyneux, R.J., Pfister, J.A., Seawright, A.A., 2006. Pyrrolizidine alkaloids in Senecio madagascariensis from Australia, Hawaii, assessment of possible livestock poisoning. Biochem. Syst. Ecol. 34, 736e744.

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