Ontogenetic variation of biologically active natural products in Ageratina adenophora

Phyrochemisrry, Vol. 29, No. 2, pp. 453457. Printed in Great Britain.

1990.

003 l-9422/90 $3.00 + 0.00 Q 1990 Pergamon Press plc

ONTOGENETIC VARIATION OF BIOLOGICALLY ACTIVE PRODUCTS IN AGERATINA ADENOPHORA PETER

Institut

fiir Pharmazeutische

PROKSCH,

VICTOR

WRAY,*

MURRAY

B. ISMAN~

and

NATURAL

INES RAHAUS

TU Braunschweig, MendelssohnstraBe. 1, D-3300 Braunschweig, F.R.G.; * Gesellschaft fiir Weg 1, D-3300 Braunschweig, F.R.G.; tDepartment of Plant Science, University of British Columbia, Vancouver, B.C., Canada V6T 2A2

Biologie,

Biotechnologische Forschung mbH, Mascherodet

(Receked

Key Word Index--Ayeratina variation; chemical defence.

adenophora;

12 June 1989)

Asteraceae;

chromenes;

sesquiterpenes;

chlorogenic

acid; ontogenetic

Abstract-The accumulation of major natural products (chromenes, sesquiterpenes, chlorogenic acid) in different leaves of seedlings of Ageratina adenophora was analysed during the first three months of seedling development. Chromenes were found to be confined to leaves at nodes l-8 (leaves at node 1 being the cotyledons) whereas they were lacking in all leaves analysed from higher nodes. The major sesquiterpene as well as chlorogenic acid were, in contrast to the chromenes, hardly detectable in cotyledons and in primary leaves but were increasingly accumulated in leaves at higher nodes resulting in a chemical dichotomy during seedling development. The latter compounds were also present in leaves from lo-month-old plants. The sesquiterpene derivative exhibited contact toxicity and growth retarding activity against larvae of a noctuid species. The physiological aspects of this dichotomy in natural product accumulation during seedling development as well as its possible implications for the chemical defence of A. adenophora are discussed.

INTRODUCTION

Plants are known to produce an astounding complexity of natural products not matched by any other living organisms Cl]. Our knowledge on the distribution of natural products in plants, however, is still largely based on phytochemical screenings conducted only at a certain moment of a plants ontogeny. The static picture thus obtained cannot readily be expected to reflect the phytochemical identity of a plant throughout its whole development. Numerous examples have demonstrated that the accumulation of certain natural products may be correlated only with one distinct developmental stage of a plant (for a compilation of references see [2]) or with distinct organs. For example, the toxic non-protein amino acid canavanine is only detected in seeds of plants from the Leguminosae whereas during seed germination canavanine is catabolysed and the nitrogen utilized for anabolic processes [3]. Essential oils provide vivid examples for different chemical compositions present in different organs of one plant [4]. More complex changes of natural product accumulation in regard to developmental processes, however, that involve more than one group of compounds have by comparison hardly been studied even though, considering the phytochemical complexity of plants, they can be expected to provide a deeper insight into correlations between secondary metabolism and morphological differentiation. Ageratina adenophora (Asteraceae, tribe Eupatorieae) provides an interesting example for correlations between secondary metabolism and seedling development. In a previous study on chromene metabolism in seedlings of A. adenophora [S] we obtained preliminary evidence 453

suggesting that chromenes accumulated largely in young seedlings whereas in older plants analysed they were missing. We have now performed a detailed analysis of the accumulation of major natural products in different leaves of A. adenophora over the first three months of seedling development. The compounds analysed included chromenes, a cadinene sesquiterpene derivative and chlorogenic acid.

RESULTS

Natural product accumulation in young seedlings

Young seedlings of A. adenophora accumulated three chromene derivatives (l-3). The chromenes were structurally closely related and differed only by the nature of their substituent at C-7. Encecalin (3) was the major chromene except in young expanding leaves, followed by demethylencecalin (2) and demethoxyencecalin (1) which was always a minor component. In this study the chromenes l-3 were quantified jointly and are reported as a group of compounds for the various leaves analysed. The occurrence of chromenes in leaves of seedlings of A. adenophora was found to be restricted to those leaves that were present at nodes l-8 (Fig. la) with leaves at node 1 being the cotyledons. In leaves at the upper nodes investigated (nodes 9-12) no chromenes could be detected. The accumulation of chromenes between the various leaves studied was that of an optimum curve with the highest amounts of chromenes found in leaves at nodes 4 and 5 (maxima of 0.7 and 0.9 pmol/leaf respectively). In leaves at nodes l-1 the chromenes were present throughout the

P. PROKSCH et al.

454 0

=I ; Am R

I R= H 2 R=OH 3

R=OCH,

chlorogenic acid were found in leaves at node 9 and node 10 (10 pmol/leaf and 8.75 nmol/leaf, respectively). In those leaves that were studied throughout their whole ontogeny the absolute amounts of chlorogenic acid remained stable through leaf development (e.g. leaves at nodes 2 and 3). The highest concentrations of chlorogenic acid were observed in very young leaves of nodes 612 with 640 pmol/g fr. wt. Natural product accumulation

in mature plants

For comparison of natural product accumulation during the first three months of seedling ontogeny leaves from the upper four nodes present at the shoot tips of lomonth-old plants of A. adenophora were analysed for compounds l--5. As expected the chromene derivatives l-3 were absent. The amounts of the sesquiterpene derivative 4 in the leaves studied ranged from 0.17 pmol/ leaf at the shoot tip to 4.4 pmol/leaf at the lower nodes (Table 1). The amounts of chlorogenic acid (5) were found to range between 2,6~mol/leaf at the shoot tip and 3.4 pmol/leaf at the lower nodes (Table 2). Contact toxicity and growth retarding activity qf natural products from A. adenophora against insects total leaf development until leaf fall. The absolute amounts of chromenes present in the respective leaves were reached early in the differentiation and growth phase of the leaves and showed no major changes during further leaf development. In leaves at nodes 5-8, however, the chromenes were detected only during the early developmental stages analysed whereas the absolute amounts present decreased and were subsequently undetectable. The highest concentrations of chromenes from all leaves analysed were found in cotyledons and primary leaves with up to 30 pmol/g fr. wt. Here as in all other leaves studied the maximum concentrations were present in very young leaves and declined during further leaf development. In leaves from higher nodes the chromene concentrations steadily decreased to ca 0.5-S pmol/g fr. wt in leaves at nodes 6-8. The sesquiterpene derivative 4 showed a very different distribution in regard to the various leaves studied by comparison with the chromenes. If present only trace amounts of 4 could be detected in cotyledons or primary leaves (leaves at node 1 and 2, respectively) whereas starting with leaves at node 3 increasingly larger amounts of the sesquiterpene were found to accumulate (Fig. lb). The highest amounts of 4 (close to 3 pmol/leaf) found during seedling development were present in leaves at node 9. In those leaves that were studied throughout their whole ontogeny (leaves at nodes 14) the absolute amounts of compound 4 accumulated showed no major changes during leaf development and remained stable. The highest concentrations of compound 4 were reached during early ontogenetic stages of leaves at nodes 6-12 with up to 3-4 pmol/g fr. wt. In leaves at the lower nodes only minor concentrations of 4 (0.2-2 pmol/g) were found to accumulate. The distribution of chlorogenic acid (5) in the various leaves studied (Fig. lc) showed similarities to that of 4. Whereas in leaves at the first and second node analysed only small or trace amounts of chlorogenic acid were detected increasingly larger amounts of 5 were accumulated in leaves present at the higher nodes. Maxima of

The major sesquiterpene (4) from leaves of seedlings of A. adenophora was analysed for contact toxicity, as well as for growth retarding effects, on two species of herbivo-

Table 1. Abundance of sesquiterpene (4) in leaves from mature plants from A.

adenophora Abundance of compound 4 (~tmol~leaf ) Leaves Leaves Leaves Leaves

at at at at

node 4 node 3 node 2 node I

0.17 0.54 4.4 3.6

Leaves were harvested from IOmonth-old plants. Leaves at node 4 are from the shoot tips, leaves at node 3 from the next lower node.

Table 2. Abundance of chlorogenic acid (5) in leaves from mature plants of .4. adrnophora .Abundance of compound 5 l,umol ‘leaf) Leaves Leaves Leaves Leaves

at at at at

node node node node

4 3 2 I

2.6 3.4 I .O 1.6

Leaves were harvested from IOmonth-old plants. Leaves at node 4 are from the shoot tips. leaves at node 3 from the next lower node.

455

1

0.5

0B

3 12

7

18

29

58

42

GROWTH

TIME

76

(OA’fS)

0C 12

7

lb

29

-4i

GROWTH

Fig. 1. Accumulation of natural products A. adenophora: (A) accumulation of chromenes of chlorogenic acid (5). Growth time is given leaves

TIME

- 58 (DAYS)

in leaves from different nodes during seedling development of (I-3) (B) accumulation of major sesquiterpene (4), (C) accumulation in days following sowing of achenes, leaves at node 1 =cotyledons, at node 2 = primary leaves.

456

P. PROKSCH et al.

Table 3. Dietary chronic growth bioassay (nochoice) of compound 4 against neonate larvae of Peridroma saucia

60 k

Quantity

J

of

4 (pmol/g fr wt of diet)

Larval wt (mg) (mean + s.d.)

Larval wt % of control

0 (control) 1.2 2.4 3.6

62.0 48.5 39.1 24.3

100 78 63 39

i F & &

7.6 8.8 10.5 5.9

ED 5. =

3 pmol/g fr. wt of diet. The experiment was designed as a no-choice test. Larval weight was determined after nine days of feeding. The number of larvae for each group was 30.

rous insects. The insects employed for the experiments included neonate larvae of the variegated cutworm Peridromu saucia (Noctuidae) and of the migratory grasshopper Melanopous sanguinipes (Acrididae). The contact toxicity of 4 was studied using the residue contact bioassay where the compound is coated as a thin film on the inner surface of glass vials. Survival of neonate Peridroma larvae in this bioassay was dose-dependent (Fig. 2). The LD,, of 4 at 24 hr following treatment was 0.28 pmol/vial (equivalent to 1.49 pg/cm’). The LDsO ofencecalin (3) was previously shown to be 0.22 pmol/vial [6]. When the sesquiterpene 4 was bioassayed in the residue contact bioassay for contact toxicity against neonate larvae of M. sunguinipes the I-D,, at 48 hr following treatment was (equivalent to observed at 178 nmol/nymph 41.3 Lcginymph). The toxicity ofencecalin against neonate larvae of M. sunguinipes following topical application was earlier shown to be more pronounced (66 nmol/nymph) [7]. Following addition of the sesquiterpene to an artificial diet and feeding of neonate larvae of P. suucia for nine days a 50% growth inhibition (relative to controls) was monitored at a concentration of 3 prnol of 4ig fr. wt of diet (Table 3). The EDGE of encecalin was idenrical to that of 4 for neonate larvae of P. saucia under the same conditions [6]. Chlorogenic acid (5) was shown previously to inhibit early larval growth of the fruitworm He/i&is zeu (Noctuidae) when added to artificial diets (ED,, = 6 Ltmol/g fr. wt) [Xl. DISCUSSION

Our study revealed a striking difference of natural products with regard to different leaves analysed during seedling development of A. udenophara. Chromene derivatives were only present in leaves from nodes l-8 and could not be detected in leaves present at node 9 or above (Fig. la). The sesquiterpene derivative 4 as well as chlorogenic acid, by comparison were hardly detectable in cotyledons or primary leaves (nodes 1 and 2), whereas they were increasingly accumulated in each following pair of leaves analysed (Fig. 2b and c). Young seedlings were thus characterized by the presence of chromene derivatives whereas older plants lacked chromenes but showed an enhanced accumulation of the sesquiterpene and of chlorogenic acid. Leaves from lo-month-old plants likewise yielded only the latter compounds but lacked chromene derivatives (Tables 1 and 2).

60

0

Fig. 2. Survival (24 hr) of neonate P. sau~iu larvae in glass vials coated with the sesquiterpene 4. Survival of control larvae is set at 100%.

In leaves at nodes l-4 the chromenes were detected throughout the whole life cycle of the leaves. The absolute amounts of chromenes present were reached early and showed no major changes during further leaf development. Thus, it is assumed that chromene biosynthesis is correlated with early leaf development and no major degradation or bioconversion of chromenes to other products take place in older leaves. In leaves at nodes 5-S chromenes were detected only in young developmental stages and decreased during leaf development. The absolute amounts of chromenes present were usually smaller compared to those in leaves at nodes l--1 and did not remain stable but declined during leaf development. Biosynthesis of chromenes thus seems to decrease in leaves at higher nodes whereas degradation or turnover of chromenes to other unknown products seem more pronounced than in younger leaves. Translocation within the plant as an alternative explanation for the decline of chromenes in leaves at nodes 5-8 seems less likely since neither stems nor roots were found to contain appreciable amounts of chromenes when compared to leaves. Recently we showed that C-acetylchromenes, as well as the related benzofuran derivatives, originate biogenetitally from the phenylpropanoid pathway with phenylalanine and cinnamic acid as precursors ([9] and unpublished results). The C-acetyl substituent of the compounds arises from a degradation of the propanoid side chain. The chromenes are thus biogenetically related to chlorogenic acid. The inversed accumulation of chromenes vs chlorogenic acid in leaves from lower nodes compared to leaves from higher nodes of seedlings of A. udenophoru is interesting in this respect as it may reflect an ontogenetically regulated switch-over between different branches of phenylpropanoid metabolism. The significance of many natural products for the fitness and survival of plants has been demonstrated in numerous cases and is now generally accepted [lo, I I]. The majority of studies directed towards the chemical defense of plants, however, focussed only on one group of compounds present in the respective plants at the time of conducting the experiment. This approach does not pay proper attention to the complexity of natural products from different classes of compounds co-occurring in plants that may influence each other. It furthermore neglects the fact that the accumulation of certain natural products and hence their possible significance for the chemical defense of plants may be correlated only with a certain period of time of the plants total ontogeny as shown in this study.

Ontogenetic variation in Ageratina The different natural products analysed in our study are all biologically active in terms of exhibiting contact toxicity and/or growth retarding activity towards different herbivorous insects at concentrations below those found in seedlings of A. adenophora. The data suggest that, at least under experimental conditions, each of the three types of natural products analysed during seedling development (chromenes, sesquiterpene, chlorogenic acid) has the potential to act as a chemical barrier against herbivorous insects and possibly other herbivores. In leaves of young seedlings chromenes may be the most important compounds in terms of chemical defence whereas in older plants the sesquiterpene and chlorogenic acid may take over the role as protective chemicals. Our study on the ontogenetic changes in natural product accumulation in seedlings of A. adenophora thus underlines the importance of a broad rather than a narrow phytochemical approach when studying chemical defence mechanisms of plants. It furthermore points to possible ecological advantages of ontogenetically correlated changes in the patterns of natural products as insects (or other herbivores) invading a young seedling of A. adenophora will be confronted with a different set of natural products than those confronting an older plant. This chemical variation can be a considerable obstacle both to host plant recognition (chemical triggers will change with time) as well as to the adaptation of herbivores to toxic plant natural products since different types of secondary chemicals will have to be tolerated at different times of the plants ontogeny. EXPERIMENTAL

Seedlings of A. adenophora (Spreng.) R. M. King and H. Robinson were grown from achenes in the green house. For each ontogenetic analysis 10 seedlings were harvested in duplicate. Leaves from the same node of the 10 seedlings were pooled and extracted with MeOH. Natural product analysis was performed by HPLC. The HPLC apparatus has been described before [9]. The sepn column was Nova Pak C,,, 5 pm, 15 cm x 0.4 cm i.d. The compounds were sepd by injecting known amounts of the MeOH extracts (1: 1 dil. with H,O) and applying a linear gradient. The gradient was from 100% A (10% MeCN, 90% H,O containing 1% H,PO,) to 100% B (60% MeCN, 40% H,O containing 1% H,PO,) in 15 min followed by 10 min isocratically at 100% B. The flow was at 1 ml/min. UV-Detection was at 340 nm for chlorogenic acid and at 240 nm for the chromenes and sesquiterpene. Quantification was achieved by the ext. std method using previously isolated and purified compounds. Natural products were extracted from bulk samples of A. adenophora leaves with MeOH. Sesquiterpenes and chromenes were isolated by silica gel CC with CH,CI, as solvent containing increasing amounts of MeOH. Similar frs were combined and

457

chromatographed on a Sephadex LH-20 column with MeOH as solvent. Sometimes final purification was achieved by low-bar CC employing a RP-8 Lichroprep column (40-63 pm, Merck) and MeOH -H,O (4: 1) as eluting solvent. Chlorogenic acid was isolated by CC on Polyamide SC-6 (Macherey & Nagel). Elution was started with H,O followed by addition of increasing amounts of MeOH. Final purification was again by Sephadex LH-20 CC. All compounds were identified based on their spectral data (NMR and MS). Compounds l-4 have previously been described for A. adenophora [S]. Contact toxicity was assessed by coating the inner walls and bottom of 20 ml glass scintillation vials with the test compound [6]. After evapn of the carrier solvent five neonate larvae were placed in each vial and left undisturbed for 4 hr at which time food (diet for P. saucia and wheat blades or M. sanguinipes) was placed in the vial. The vials were then held at 27” for 24 hr at which time surviving larvae were counted. Each treatment consisted of 10 vials with 5 larvae/vial. For determination of growth retarding activity the compounds were added to artificial diet [6]. After complete evapn of the carrier solvent neonate larvae were added to treated diet and larval wt was measured after 9 days of feeding. Acknowledgements-Financial support of this project by a grant of the DFG (to P.P.) by an operating grant from NSERC (to M.I.) and by a collaborative research grant of NATO (to P.P. and M.B.I.) is gratefully acknowledged. We wish to thank Nancy Brard for technical assistance. REFERENCES M. (1984) Secondary Metabolism in Microorganisms, Plants, and Animals. Springer, Berlin. 2. Wiermann, R. (1981) in The Biochemistry of Plants (Conn, 1. Luckner,

E. E., ed.), Vol. 7, p. 86. Academic Press, New York. 3. Rosenthal, G. A. (1982) Plant Nonproteine Amino and Amino Acids. Academic Press, New York. 4. Wagner, H. (1982) Pharmazeutische Biologic, Vol. 2. Gustav Fischer, Stuttgart. 5. Proksch, P., Palmer, J. and Hartmann, T. (1986) Planta 169, 130. 6. Isman, M. B. and Proksch, P. (1985) Phytochemistry 24, 1949. 7. Isman, M. B., Yan, J.-Y. and Proksch, P. (1986) Naturwissenschaften 73, 500. 8. Isman, M. B. and Duffey, S. S. (1982) J. Am. Hart. Sci. 107,

167. 9. Siebertz, R., Proksch, P., Wray, V. and Witte, L. (1989) Phytochemistry 28, 789. 10. Harborne, J. B. (1982) Introduction to Ecological Biochemistry. Academic Press, New York. 11. Rosenthal, G. A. and Janzen, D. H. (1979) Herbivores. Their Interaction with Secondary Plant Metabolites. Academic

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