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Cytochemistry, Vol. 37, No. 3, pp. 635-639, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. Allrightsreserved 0031-9422/94 S7.00+0.00

GIBBERELLIN-LIKE ACTIVITY OF SOME TETRACYCLIC DITERPENOIDS FROM ELAEOSELINUM SPECIES AND THEIR DERIVATIVES NIEVES VILLALOBOS, LUISA MARTÍN, MARÍA J. MACÍAS,* BALBINO MANCHEÑO* and MANUEL G R A N D E * !

Departamento de Fisiologia Vegetal, Facultad de Biología; 'Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad de Salamanca, E-37008 Salamanca, Spain (Received in revised form 11 April 1994) Key Word Index—Elaeoselinum; Umbelliferae; plant growth activity; phytohormones; tetracyclic diterpenoids; kaurene, beyerene and atisene derivatives.

Abstract—The growth-regulatory activity of 13 tetracyclic diterpenoids with kaurane, beyerane and atisane skeletons isolated from Elaeoselinum species (Umbelliferae) has been tested in six bioassays and their activities were compared with that shown by gibberellic acid, GA 3 . Seven of the tested substances showed an activity similar or greater than that displayed by GA 3 at concentrations 1 to 10 /ig m l - 1 . The most active compounds are eni-15a-angeloyloxykaur-16ene-3/?-yl acetate, methyl enf-18-angeloyloxykaur-16-ene-19-oate, methyl enf-beyer-15-ene-19-oate, methyl eni-14jStygloyloxybeyer-15-ene-19-oate (methyl elasclepiate) and eni-beyer-15-ene-14/?,19-diol.

lettuce hypocotyl assay [17, 18], dwarf pea stem growth assay [19], dwarf rice assay [20], barley endosperm assay [21], cucumber hypocotyl assay [22] and Rumex leaf senescence assay [23], established to test the growth activity of the gibberellins.

INTRODUCTION

The most common secondary metabolites present in Umbelliferae are coumarins, monoterpenoids, sesquiterpenoids and aromatic derivatives found in the essential oils. Sesquiterpene lactones, phenylpropanoids, polyacetylenes and flavonoids are also usually present but as minor components [1, 2]. Diterpenoids are quite uncommon in these plants: some tetracyclic diterpenoids structurally related to kaurane, beyerane and atisane, have been reported in Elaeoselinum gummiferum [3, 4], E. foetidum [5, 6], E. tenuifolium (=Distichoselinum tenuifolium) [7, 8] and E. asclepium subsp. asclepium [9, 10]. From E. asclepium subsp. millefolium we have recently also isolated some kaurane derivatives [11, 12]. Kaurane diterpenoids are known to be biological precursors of gibberellins [13]. Therefore we considered whether the new diterpenoid metabolites available from the umbellifers as mentioned above, might have some growth regulating activity, either by themselves or as precursors of gibberellins. Gibberellin-like activity was in fact previously observed in other kaurane derivatives [14,15]. In this paper we report the activity of the natural diterpenoids 1, 3, 5, 7, 9, 11,12 and 13 we have isolated from Distichoselinum tenuifolium, Elaeoselinum asclepium and E. gummiferum and their derivatives 2, 4, 6, 8 and 10 prepared by acetylation, esterification or saponification of the natural compounds. To determine the activity of these substances as growth regulators we have applied the following bioassays [16]:

fAuthor to whom correspondence should be addressed.

RESULTS

The results of the lettuce hypocotyl bioassay for the tetracyclic diterpenoids and the reference GA 3 are shown in Fig. la. To evaluate the bioactivity of compounds 1-13 compared with GA3, the plants were measured after 3 days, which is the normal practice in this assay [17], and also after five days according to Hoad et al. [18]. Among the tested substances, compounds 2, 4, 6, 8, 11 and 13 showed after three and five days of germination, an activity similar or greater than that observed for gibberellic acid. The higher activity was shown at concentrations from 1 to 10 fig ml"*. However, in this and the remaining bioassays, a saturation effect was observed for some substances at 10 ¡ig m l - *. In this bioassay the maximum activity was observed for compound 2. Intact pea plants in the dwarf pea stem growth assay [19] were particularly sensitive to the substances 2,4,6,8, 10, 11 and 13 and the activity (Fig. lb) was in all cases greater than that of the reference. The optimum activity was reached at a concentration of 1 /ig m l - 1 for compounds 6,8 and 10 and at 10 \ig ml" x for compounds 2,4, 11 and 13. In the dwarf rice bioassay the length of the second leaf sheath was measured [20] after three days as usual. Compounds 2, 4, 6, 8,10,11 and 13 (Fig. lc), showed an

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Fig. 1. Activities of compounds 1-13 and the reference substance GA3 for concentrations 0, 10~3, 10~ 2 ,10" 1 ,1 and 10 fig ml" 1 in the following bioassays: (a) Arctic King lettuce hypocotyl assay [y = hypocotyl length in mm, after three days (— O—) andfivedays (— •—)]; (b) Progress N° 9 dwarf pea stem growth assay (y = stem length in mm, after three days); (c) Tan-ginbozu dwarf rice assay (y = second leaf sheath length in mm, after three days); (d) Barley aleurone a-amylase assay (y=a-amylase released in fig); (e) Cucumber hypocotyl assay (y=hypocoptyl length in mm, after three days), and (f) Rumex leaf senescence assay (y = optical density at 665 urn,). Values are means of four samples: mean s.e. are usually lower than +5%.

activity greater than that shown by GA 3 . In this case compounds 5 and 7 also showed at a concentration of 10 fig m l - 1 an activity slightly lower than that observed for gibberellic acid. However, when the assay was allowed to continue to day 6 and the third leaf sheath measured [18], the above mentioned substances showed more activity than GA 3 at the same concentration. The

optimum concentration in this bioassay was 1 fig ml ' for compounds 6 and 10 and 10 fig m l - 1 for 2,4,8,11 and 13. In the barley endosperm bioassay [21] the induced production of a-amylase was evaluated by the analysis of reducing sugars. In this case (Fig. Id) the compounds 2,4, 6,8 and 11 at 10 fig m l " l were more active than GA 3 and

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Fig. 2. Activities of the substances 1-13 and GA3 for a concentration of 1 ¡ig ml"' in the tested bioassays: lettuce hypocotyl assay (—•—), dwarf pea stem growth assay (—O—), dwarfriceassay (—O—), barley endosperm assay (—A—), cucumber hypocotyl assay (—D—), and Rumex leaf senescence assay (—•—). All activities are expressed as a percentage of the reference substance, GA3> after subtraction of the blank values. The average value for the six bioassays are connected through a dotted Une ( ).

also the compounds 10 and 13 at 1 ng m l - í showed the same or higher activity than the reference. Compounds 10 and 11 showed saturation effect at 10 fig m l - 1 . The results of the cucumber hypocotyl bioassay [22] are shown in Fig. le. Hypocotyls were measured three days after sample application. Compounds 2 and 4 are much more active than GA 3 at a concentration of 1 ng m l - 1 but 4 showed a saturation effect at 10/¿g m l - 1 . Compound 6 showed an activity similar to that of GA 3 and compounds 8, 10, and 11 were also slightly more active than the reference substances, producing an optimum activity at 1 /xg m l - 1 . In the Rumex leaf senescence bioassay chlorophyll retention was evaluated [23] and as can be deduced from Fig. If, several compounds produce similar or higher effects than GA 3 . The most active compounds were again 2, 4 and 8 for a concentration of 1 fig m l - 1 but compounds 6,10 and 11 also displayed more activity than the reference. Compounds 7 and 13 showed activities quite similar to those of GA 3 . To summarize the activities induced by the tested substances, Fig. 2 shows the activities of the substances 1-13 at a concentration of 1 mg m l - 1 (below the saturation effect appeared) as compared to GA 3 (1 mg m l - 1 , 100%) and the control solution (0%).

DISCUSSION

The observed results for the six bioassays are quite consistent and thus allow the substances to be classified into two groups, one including the substances with an activity similar or higher than that displayed by the reference substance (compounds 2, 4,6, 8,10,11 and 13) and the other consisting of substances less active than GA 3 (remaining substances). Comparing the activities and the structural characteristics of the studied substances, it seems that the

following structural features are important in determining the relative activity. (a) The substances with a free hydroxyl group at C-3
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our knowledge of the relationship between the structure of the diterpenoids and their growth-regulating activity.

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Acknowledgements—We would like to thank the Junta de Castilla y León for financial support (Project 0740/90), the P.H.E.T. for a grant to M.J.M. and to J. Ashurst for her help.

EXPERIMENTAL REFERENCES

The tested compounds were: eni-15cc-angeloyloxykaur-16-ene-3/?-ol (1), its 3/?-acetyl derivative (2), ent1. Heywood, V. H. (1971) The Biology and Chemistry of the Umbelliferae. Academic Press, London. 18-angeloyloxykaur-16-ene-19-oic acid (3), its methyl ester (4), eHí-lSa-angeloyloxy-ló/J.n-epoxykauran-Sjg-ol 2. Hegnauer, R. (1990) Chemotaxonomie der Pflanzen. Vol. 9, p. 663. Birkhauser, Stuttgart. (5), and its 3/J-acetyl derivative (6), ent-beyer-15-ene-19oic acid (7), its methyl ester (8), ent-14/?-tygloyloxybeyer3. Pinar, M., Rodríguez, B. and Alemany, A. (1978) Phytochemistry 17, 1637. 15-ene-19-oic acid (elasclepic acid, 9), its methyl ester (10), enr-beyer-15-ene-14/J, 19-diol (11), eni-atis-16-ene-19-oic 4. Rodriguez, B. and Pinar, M. (1979) Phytochemistry acid (12), and en£-7a-angeloyloxyatis-16-ene-19-oic acid 18, 891. (gummiferolic acid, 13). Compounds 1 and 5 were isolated 5. Pinar, M., Rico, M., Pascual, C. and Fernández, B. from Distichoselinum tenuifolium (Lag.) Garcia Martin & (1983) Phytochemistry 22, 2775. Silvestre [7], compounds 3,7,9,11 and 12 from Elaeosel6. Pinar, M. and Galán, M. P. (1986) J. Nat. Prod. 49, inum asclepium (L) Bertol subsp. asclepium (L.) [9,10] and 334. compound 13 from E. gummiferum (Desf.) Tutin [3]. The 7. Grande, M., Segura, M. and Mancheño, B. (1986) remaining substances were obtained from the respective P D F J. Nat. Prod. 49, 259. natural compounds by acetylation (Ac20—pyridine), 8. Grande, M., Macias, M. J., Mancheño, B., Segura, M. methylation (CH 2 N 2 -Et 2 0) or hydrolysis ( K O H - P D F and Zarzo, A. (1991) J. Nat. Prod. 54, 866. MeOH) and their preparation, physical constants and 9. Grande, M., Mancheño, B. and Sánchez, M. J. (1989) spectral data were described in the articles of the respect- P D F Phytochemistry 28, 1955. ive natural precursors as well as in ref. [11]. 10. Grande, M., Mancheño, B. and Sánchez, M. J. (1991) The biological activity of the compound under study PDF Phytochemistry 30, 1977. was compared to that of gibberellic acid (GA3, reference 11. Grande, M., Moran, J. R., Maclas, M. J. and compound), in the following bioassays: (1) lettuce hypo- P D F Mancheño, B. (1993) Phytochem. Anal. 4, 19. cotyl (Lactuca sativa cv Arctic King); the procedure used 12. Maclas, M. J., Rico, F. and Grande, M. (1994) (in [18], was a modification of the Frankland and Wareing preparation). method [17]. (2) Intact dwarf pea stem (Pisum sativum cv 13. Sembdener, G., Gross, D., Liebisch, H.-W. and Progress N°9), using the Kohler and Lang method [19]. Schneider, G. (1980) in Encyclopedia of Plant Physi(3) Dwarf rice (Oryza sativa cv Tan-ginbozu), using a ology (Pirson, A. and Zimmermann, M. H., eds), Vol. modification of the Murakami method [20]: the seeds 9, p. 281. Springer, Berlin. were placed in a wet germination tray for 48 hr under 14. Cross, B. E., Stewart, J. C. and Stoddard (1970) continuous illumination at 32-35° and then were sowed Phytochemistry 9, 1065. in vermiculite under the same conditions. After 4 days, 15. Becker, H. and Kempf, T. (1976) Z. Pflanzenphysiol. 0.5 /d of test solns was applied in the first leaf of the 80, 87. coleoptyl and 3 days later the length of the first and 16. Takahashi, N., Yamaguchji, I. and Yamane, H. (1986) second leaf sheath were measured for each seedling. (4) in Chemistry of Plant Hormones (Takahashi, N., ed.), Induction of the amylase activity in barley endosperm pp. 57-151. CRC Press, Boca Ratón, FL. (Hordeum vulgare cv Himalaya) was measured by a 17. Frankland, B. and Wareing, P. F. (1960) Nature modification of the method of Jones and Varner [21]: (London) 185, 255. three de-embryonated half seeds and 1 p.1 of the test solns 18. Hoad, G. V , Phinney, B. O., Sponsel, V. M. and were incubated for 48 hr at 27° and then the filtered solns MacMillan, J. (1981) Phytochemistry 20, 703. were analysed for reducing sugars according to the 19. Kohler, D. and Lang, A. (1963) Plant Physiol. 38, 555. Somogyi [24] and Nelson [25] method. (5) The cucumber 20. Murakami, Y. (1968) Bot. Mag. (Tokyo) 81, 334. hypocotyl (Cucumis sativus cv Perfection Ridge) assay 21. Jones, R. L. and Varner, J. E. (1967) Planta 72, 155. was performed as described by Brian et al. [22]. (6) The 22. Brian, P. W., Hemming, H. G. and Lowe, D. (1964) dock leaf senescence (Rumex obtusifolius) assayed was Ann. Bot. (London) 28, 369. that described by Hoad and Kuo [23]. 23. Hoad, G. V. and Kuo, C. C. (1970) Can. J. Botany 48, In these 6 plant bioassays each compound was tested in 1323. replicates of 4 at 10~ 3 ,10" 2 ,10" \ 1 and 10 /¿g ml" 1 . 24. Somogyi, J. M. (1952) J. Biol. Chem. 195, 19. Distilled water was used as ctontrol. The mean standard 25. Nelson, N. J. (1957) in Methods in Enzymology error of each measurement were usually lower than (Colowick, S. P. and Kaplan, N., eds), p. 85. Academic ±5%. Press, New York.