Pyrrolizidine alkaloids in Chromolaena odorata. Chemical and chemoecological aspects

Pergamoa

PhytochemLclry.

Vol. 35. No. 3. pp. 615

619. 1994

Copyrighl 0 1994 Elmvicr Science Ltd inGreat Britain. All rights revrvcd

Prinled

0031

PYRROLIZIDINE

ALKALOIDS IN CHROMOLAENA ODORATA. AND CHEMOECOLOGICAL ASPECTS

9422/M %.oo+O.M

CHEMICAL

ANDREASBILLER, MICHAEL BOPPRB,* LUDGER WIITE and THOMASHARTMANN lnstitut fiir Pharmazeutische

lForstzoologisches

Biologie der Technischen lnstitut der UniversitPt

Universitiit,

Mendelssohnstr.

Freiburg, Bextoldstralk

1, D-38106 Braunschweig,

Germany;

17, D-79098 Freiburg i.Br., Germany

(Receic;ed 1 August 1993)

Key Word Index-Chronwlaena

odorata; Eupatoriae; Asteraceae; Zonocerus variegatus; Orthoptera;

Pyrgomorphidae; pyrrolizidine alkaloids; hopper attractants; phagostimulants.

pharmacophagy;

anti-herbivore

allelochemicals;

grass-

Abstract-The tropical weed Chromolaenu odorata contains the N-oxides of five pyrrohzidine alkaloids (PAS): 7- and 9-angeloylretronecine, intermedine, rinderine and 3’-acetylrinderine. Highest concentrations occur in roots and mature flower heads, while leaves and stems are almost devoid of alkaloids, and no PAS are present in nectar. The polyphagous grasshopper Zonocerus uariegatus sequesters intermedine and rinderine from Chromolaena flowers and transforms up to 20% of these PAS into lycopsamine and echinatine, respectively, by inversion of configuration at C-3’. Pure rinderine injected into the haemolymph of Zonocerus is partly converted into intermedine, indicating inversion of configuration at C-7.

INTRODUCTION Chromolaena odorata

King and Robinson 1970 (Eupatorium odoratum L.) (Asteraceae: Eupatoriae), the Siam weed, is a perennial pioneering shrub native to the tropical Americas. Following its introduction into India, C. odorata has invaded south-east Asia from where it has been exported to Africa eventually. In its new habitats it spreads quickly, forms dense thickets and seriously interferes not only with natural vegetation, but also with forestry, pasture and plantation crops. It has become a major weed in large parts of tropical Asia and in humid African zones, and it continues to spread in areas below 1000 m altitude and with annual rainfall exceeding 1000 mm (for general accounts on C. odorata see refs 11-31). Previous chemical analyses have revealed sesquiterpene lactones, triterpenes and flavonoids [4]. It is not known if these compounds are responsible for the apparently effective protection of C. odorata from herbivory. Although usually avoided by vertebrates, Chromolaena has been reported to poison cattle [S], and its use to fertilize rice fields resulted in killing of fish 163. Only a few phytophagous insects feed on Chromolaena in Asia and Africa [3]. In its original habitats, some 240 species of arthropods are listed to occur on this plant [7, 83; however, the majority are polyphagous and it is very doubtful that C. odorata serves as a real host plant for any of them. The leaf-feeding moth Pareuchates pseudoinsulata Rego Barros (Lepidoptera: Arctiidae) is reported to be specific on C. odorata and has been successfully estab615

lished as a biological control agent against the weed on Guam [9]. In western Africa, dry-season populations of the polyphagous grasshopper Zonocerus uariegatus (L.) (Orthop tera: Pyrgomorphidae) have increased seemingly in coincidence with the introduction and successive spread of C. odorata [lo], and this insect has reached pest status because of its damage to crops. Strikingly, C. odorata is no proper host for Zonocerus which, however, is attracted to and eagerly consumes flowers of C. odorata [ 1l-143. Considering: (i) this observation, (ii) the finding that Z. elegans Thunb. is attracted to plants containing pyrrolizidine alkaloids (PAS) as well as to pure PAS [ 1S], and (iii) the occurrence of PAS in various Eupatoriae [l&18], suggested that C. odorata is a PA-containing plant, and that Zonocerus might enjoy a non-nutritional relation to C. odorata because of PAS which are beneficial for these insects’ fitness [ 19, 203. The present study investigates the occurrence and tissue-specific distribution of PAS in C. odorata and their sequestration by Z. variegatus in order to contribute to chemoecological studies on herbivore_Chromolaena relationships, particularly on the peculiar relation of Zonocerus to Chromolaena.

RESULTSAND DISCUSSION Five PA monoesters are abundantly present in alkaloid extracts of C. odorata (Table 1). all genuinely occurring as N-oxides. The dominating alkaloids found in roots and

616

A. BILLER et al.

Table

1. GC-MS

analysis of the composition

of PAS in roots and inflorescenses of Chromolaena odornra

and Zonocerus uariegatus

-.

WI’

.-.

C. odorota .-..

2. cariegalus

--.

.- .-.. - -..

Roots

Flower heads

Whole insects*

“/oi

%t

%t

Alkaloid

(m/z)

RI

(n = 3)

(n=6)

1 2

7-Angeloylretronecine 9-Angeloylretronecine

237 237

1787 I797

4-5 IS-21

tr l-2

_-

3

Supinine

283

1978

tr 2633

4-9

12-33

4549

7688

4

lntermedine

299

2130

5

Lycopsamine

299

2145

6

Rinderinc

299

2152

7

Echinatine

299

8 -.

3’-Acetylrinderine ._.~~

341 .-.

Total

. ~..

.___

5-9

2172

_

2220 .~ ..--

*Sample tRclative

1 fr.

3-21

7-12 ._


_-

.--.-

1.5-2.8

wt)

of dry-season sixth-instar abundance

l-4

I .03

PAS (pmol g- ‘dry wt) (pm01 g-

4741

1.3-5.3 larvae (n = 2) and adults (n = 2).

(total PAS= 100%); tr = traces.

inflorescences are the N-oxides of rinderine (6) and intermedine (4). representing 09-esters of the necine bases heliotridine and retronecine, respectively, with (+)trachelanthic acid (Fig. 1). Rinderine is accompanied by the N-oxide of its 3’-0-acetyl ester (8), the O’- and 09esters of retronecine with angelica acid (1 and 2) were found in addition, particularly in the roots. Supinine (3), an alkaloid known to be a biosynthetic precursor of (4) in Eupatorium (T. Hartmann and P. Hiilsmeyer, unpublished results), was frequently detected in trace amounts in root extracts. The alkaloid pattern is representative for species of the Eupatoriae (Asteraceae) and certain genera of the Boraginaceae [16-18, cf. 21, 221; 8 was recently discovered in Heliotropium transalpinum (Boraginaceae) ~231. While high PA concentrations are found in both roots and inflorescences, only trace amounts are detectable in the vegetative parts of the shoots (Table 2); the tiny amounts of PAS in foliage indicate that avoidance of Chromolaena by herbivores is not due to these chemicals. Detailed analyses of inflorescences at different developmental stages revealed that the occurrence of PAS is restricted to the Bore& (Table 2). There is a steady increase in PA concentration, reaching a maximum in florets that are fully open and exhibit their two-lobed stigmata. Within the florets almost half of the PAS are associated with the ovaries where they seem to be kept and transferred into the achenes during seed development. A preferred storage of PAS in the inflorescences was also observed in other Eupatorium species [21,22] and in Senecio [24]. The comparatively high concentrations of PAS in the florets of Chromoluena may suggest that PAS occur in the nectar as has been assumed for Eupatorium species [21, 223, and documented for honey from Senecio jacoboea [25] and Echium plantagineum [26]. Since it is technically impossible to obtain pure nectar samples from

o-

07

5 Fig. found

1. Structures of the major pyrrolizidinc in Chromolaena

spectively: intermedine

odoraru

alkaloid

and Zonocerus

(4). lycopsamine echinatine

N-oxides

wriegarus,

(S), rinderine

re-

(6) and

(7).

the tiny, capillary-like tubular florets of Chromoloena, we fed a day-flying moth, Euchromia lethe, known to pharmacophagously take up and store PAS (M. Bopprt? and J. A. Edgar, unpublished results), exclusively with Chromolaena flowers and investigated body extracts to indirectly assess if the nectar contains PAS. PA analysis of five specimens revealed the absence of even traces of Chromoluena PAS in the bodies of the insects. Also, PAS could not be detected in ‘Chromoluena honey’ (detection iimit: 0.2 nmol g- ’ honey) commercially available in Thailand. Finding corresponding amounts of PAS in ovaries of the florets and in achenes (Table 2) indicates that PAS associated with the ovaries are retained during seed

617

Alkaloids in Chromolaena odorata Table 2. Total PAS found in roots, shoots and inflorescenses of Chromolaena odorata as well as in different developmental stages of the infloresccnses Total PAS (pmol)

Plant organ Roots Stems Leaves leaf blades petioles Flower heads* whole flower heads stigmata corolla plus styles and anthers ovaries receptaculum plus bracts Achenes Flower heads with florets not yet visible (buds) becoming visible open (stigmata not visible) open (stigmata exposed) open (stigmata withered)

Concentration g-‘dry wt

Amount per 100 flower heads

1.03 0.08 < 0.002 0.11 2.44 0.68 2.05 9.85 < 0.002 2.43

3.29 0.07 1.78 1st < 0.002 1.50

0.48 0.65 2.02 I .92 1.67

0.20 0.27 1.89 2.68 2.48

*Developmental stage: florets open (stigmata exposed).

maturation. These results corroborate biological observations: PA-containing flowers are generally avoided by insects which are not specialized to utilize PAS [27, cf. 281, but all kinds of nectar-feeders have been observed visiting Chromolaena flowers in %nin (M. Bopprt and 0. W. Fischer, unpublished results). Furthermore, specific attraction of Zonocerus grasshoppers preferably to fully open flowers of Chromolaena as well as to its roots (but not to its leaves) [14] is also in coincidence with our analytical findings. Extracts of Z. uariegatus collected in the field during the dry-season were found to contain four PAS in all stages and in both sexes, as well as in eggs [20]. The PA pattern, however, differs considerably from the pattern found in inflorescences of Chromolaena (Table 1). As in the plant, 6 and 4 are the major PAS found in insects; however, the angeloylretronecines (1 and 2) and, except for traces, the 3’-acetyl ester of 6 are absent. Instead, two new PAS are detectable which were identified as lycopsamine (5) and echinatine (7), the 3’-S configurated stereoisomers of 4 and 6, which are definitively absent from Chromolaena. Proof that these compounds are produced in the insect from respective plant PAS by inversion of configuration at C-3’ (i.e. conversion of (+)-trachelanthic acid into (-)-viridifloric acid) and not taken up from other plants comes from feeding experiments with PAS extracted and purified from C. odorata roots. If this mixture was orally fed to Zonocerus that had been reared on PA-free plants, 5 and 7 were formed. Injection of pure 6 into the haemolymph of Zonocerus did not result in the formation of 7, but small amounts (ca 5% of PAS in the insect) of 4 could be detected, indicating an inversion of PHYTO 35:3-F

configuratioh of C-7, i.e. conversion of the necine base heliotridine into retronecine. Because both necines are present in the PAS of C. odorata, this effect would not be noticed in the feeding experiments; it may, however, explain the relatively high proportion of 4 and the reduced level of 6 in extracts of Zonocerus in comparison to those of the PA source (Table 1). The observation that 6 is not converted into 7 in the haemolymph suggests that the gut might be involved in this transformation. The ability to catalyse the inversion of configuration at C-7 is already known from the arctiid moth Creatonotos transiens which converts (7S)-heliotrine into (7R)-heliotrine in the course of biosynthesis of its male pheromone (7R)-hydroxydanaidal [29-311. Inversions of configuration at both C-7 and C-3’ have been observed in the arctiid Hyalurga syma feeding on Heliotropism transalpinum [23], and different Brazilian Ithomiinae feeding as adults on flowers of certain Eupatorium species (J. R. Trigo, personal communication). In any case, the observed inversions of configuration were directed exclusively from 7s to 7R (i.e. heliotridine into retronecine) and from 3’R to 3’S (i.e. ( + )-trachelantic acid into (-)-viridifloric acid), respectively. It is remarkable that these reactions are found in systematically unrelated PA storing insects, e.g. an orthopteran and several lepidopterans. The mechanism as well as the importance of these reactions should receive further attention. EXPERIMENTAL Plant material. Samples of C. odorata were collected in a teak plantation near Bohicon, Republic de tinin, West

618

A. BILLER

Africa, in December 1990 and March 1991, i.e. at the beginning and end of the flowering period of the weed. The plant material was either air-dried or preserved in MeOH. For detailed analyses of organ and tissue specific PA-distribution quantified samples of defined developmental stages of flower heads were prepared. Roots (3 g), leaves (10 g) and 100 flower heads per sample were investigated. To examine variations in PA-content, 600 flower heads (in groups of 100) with florets fully opened (stigmata exposed) were analysed. Insects. Euchromia lethe Fabr. (Lepidoptera: Ctenuchiidae) which had been reared on PA-free artificial diet (according to ref. [32]) were kept in cages and given access to freshly cut inflorescences of C. odorata 2-3 times for 7 days. The moths readily fed at the flowers; no additional food was provided to ensure that the insects took up much nectar from the flowers. After 2 days the moths were killed, preserved in MeOH and analysed for PA sequestration. For analyses on sequestration of PAS, Z. variegatus L. (Orthoptera: Pyrgomorphidae) were collected in the field in Benin at the same sites as material of C. odorata. Specimens reared from eggs on PA-free plants in the laboratory were used for feeding and injection experiments; adults were individually fed with fibre-glass discs (Whatman GF/B 2.1 cm) impregnated with 1 mg of crude MeOH extract of roots of C. odorata, reduced and purified as described by ref. [33]; 5 ~1 of Ringer’s solution (after Pringle) containing 0.5 mg of pure rinderine was injected in indoor-raised adults which were killed after 3 days and preserved in MeOH. Alkaloid extraction. Plant material was extracted using method B according to ref. [33] with the modification that instead of 6 ml, 12 ml CH,CI, g- I Extrelut was used to quantitatively elute the rather polar tertiary PAS. In the case of MeOH preserved material the MeOH was removed by evapn prior to aq. extraction. Grasshoppers were ground individually in 3-6 ml hot (80”) MeOH with quartz sand in a mortar for 10min; after centrifugation the residue was again extracted with hot MeOH and centrifuged. The supernatants-and in the case of MeOH-preserved insects the supematants plus the MeOH used for preservation-were combined and after evapn under red. pres. the residue was dissolved in ca 5 ml 0.25 M H,SO, and extracted x 3 with 5-10 ml Et,0 each to remove fatty acids and their methyl esters which would interfere with CC and GC-MS analysis. The remaining acidic aq. soln was further processed as the plant material (above). The same extraction procedure was used for Euchromia except that cold acidic MeOH (1% HCI) was used instead of hot MeOH. Extraction of Cromolaena honey followed the procedure described by Culvenor et al. [26]; in an additional test 10 g honey was dissolved in 100 ml 0.25 M H,S04 and processed in the same way as plant material (above). Capillary GC and GC-MS analysis. Analyses were performed using method II described in ref. [33]. Identification of PAS. The 4 stereoisomers 4-7, were identified by their identical MS fragmentation patterns, but characteristic RI values in comparison to authentic

et al.

samples [33 3. The presence of indicine (RI 2 126) the 5th isomer known to occur in nature, in C. odorata or Zonocerus could be excluded. Compounds 1 and 2 were identified by comparison of their RIs and MS fragmentation patterns to synthetic ref. compounds [33]. Alkaloid 8 was identified in addition to its RI, [M]’ and fragmentation [23] by formation of 6 after acid hydrolysis. Acknowledgements-The study was financially supported by grants of the BMFT through the Gesellschaft fur Technische Zusammenarbeit (GTZ) to M.B. and by grants of the Deutsche Forschungsgemeinschaft to T.H. We are greatly indebted to the Project Benino-Allemand, Porto-Novo, for logistic support during the field work, to Ottmar Fischer and Martin Baumgart for skilful assistance and valuable discussions, and to Dr Jose Roberto Trigo (Campinas, Brazil) for providing rinderine. Chromolaena honey was kindly provided by B. Napompeth (Bangkok). REFERENCES

1. Cruttwell McFadgen, R. E. (1989) Plant Prof. Quart. 4, 3. 2. Ambika, S. R. and Jayachandra (1990) Chromolaena odorata Newsletter (Guam) 3, 8. 3. Boppre, M. and Ladenburger, U. (1994) Chromolaena odorata King and Robinson 1970 (Eupatorium odoratum Linnaeus 1759)--A Comprehensive Treatise of an Introduced Weed in the Asian and African Tropics. GTZ, Eschborn/Getmany (in press).

4. Arene, E. O., Pettit, G. R. and Ode, R. H. (1978) Lloydia 41, 186. 5. Anonymous (1973) Pasture Newsletter 1, 1. 6. Litzenberger, S. C. and Lip, H. T. (1961) Agronomy J. 53, 321. 7. Cruttwell, R. E. (1974) Techn. Bull. Comm. Inst. Biol. Contr. 17, 87. 8. Cruttwell McFadgen, R. E. (1988) Chromolaena odorata Newsletter (Guam) 2, 5. 9. Seibert, T. F. (1989) Entomophaga 34, 531. 10. Toye, S. A. (1974) Rev. Zool. Afr. 88, 205. 1I. Modder, W. W. D. (1984) Bull. Ent. Res. 74, 239. 12. Modder, W. W. D. (1984) Insect Sci. Appl. 5, 527. 13. Modder, W. W. D. (1986) Nigerian Field 51, 41. 14. BopprC, M. and Fischer, 0. W. (1994) in BiologicalIntegrated Control of Grasshoppers and Locusts: New Research Activities (Krall, S. and Wilps, H., eds)

GTZ, Eschbom/Getmany (in press). 15. Boppre, M., Wickler, W. and Seibt, U. (1984) Entomol. Exp. Appl. 35, 7 13. 16. Bull, L. B.. Culvenor, C. C. J. and Dick, A. T. (1968) The Pyrrolizidine Alkaloids. North-Holland, Amsterdam. 17. Mattocks, A. R. (1986) Chemistry and Toxicology of Pyrrolizidine Alkaloids. Academic Press, London. 18. Rizk, A.-F. M. (1990) Naturally Occurring Pyrrolizidine Alkaloids. CRC Press, Boca Raton. 19. Boppre. M. (1991) in Proceedings of the Second Internutional

Workshop on Biological Control of

Alkaloids

20.

21. 22. 23. 24. 25.

in

Chromolaena

Chromolaena odorata (Muniappan, R. and Ferrar, R., eds), pp. 153-157. ORSTOM and SEAMEO BITROP, Bogor, Indonesia. Bopprt, M., Biller, A., Fischer, 0. W. and Hartmann, T. (1992) in Proc. 8th Intern. Symp. Insect-Plant Relationships (Menken, S. B. J., Visser, J. H. and Harrewijn, P., eds), pp. 89-90. Kluwer Academic, Dordrecht. Brown, K. S. (1984) Rev. Brad Biol. 44, 435. Brown, K. S. (1984) Nature 309, 707. Trigo, J. R., Witte, L., Brown, K. S. Jr, Hartmann, T. and Barata, L. E. S. (1993) J. Gem. Ecol. 19, 669. Hartmann, T. and Zimmer, M. (1986) .I. P/ant Physiol. 122, 67. Deinzer, M. L., Thomson, P. H., Burgett, D. M. and Isaacson, D. L. (1977) Science 195,497.

odorata

619

26. Culvenor, C. C. J., Edgar, J. A. and Smith, L. W. (1993) .I. Agric. Food C/rem. 32, 187. 27. Boppre, M. (1990) J. Gem. Ecol. 16, 165. 28. Masters, A. R. (1991) J. Gem. Ecol. 17, 195. 29. Bell, T. W., Boppre, M., Schneider, D. and Meinwald, J. (1984) Experientiu 40, 713. 30. Wink, M., Schneider, D. and Witte, L. (1988) 2. Naturforsch. 43e, 737. 31. Schu1.q S., Francke, W., Boppre, M., Eisner, T. and Meinwald, J. (1993) Proc. Natn. Acad. Sci. U.S.A. 90, 6834. 32. Bergomaz, R. and Boppri, M. (1986) J. Lep. Sot. 40,

131. 33. Witte, L., Rubiolo, P., Bicchi, C. and Hartmann, (1993) Phytochemistry 32, 187.

T.