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Chemotaxonomic relevance of sesquiterpenes within the Achillea millefolium group
Biochemical Systematics and Ecology 27 (1999) 437—444
Chemotaxonomic relevance of sesquiterpenes within the Achillea millefolium group Wolfgang Kubelka*, Ulrike Kastner, Sabine Glasl, Johannes Saukel, Johann Jurenitsch Institute of Pharmacognosy, University of Vienna, Pharmaziezentrum, Althanstra}e 14, A-1090 Wien, Austria
Abstract The chemotaxonomic relevance of sesquiterpenoids within the Achillea millefolium group has been evaluated by means of morphological, anatomical, cytological and phytochemical data. The sesquiterpene patterns of Achillea setacea Waldst. & Kit, Achillea asplenifolia Vent., Achillea roseoalba Ehrend., Achillea collina Becker, Achillea ceretanica Sennen, Achillea pratensis Saukel & La¨nger, Achillea distans subsp. styriaca Saukel in edit., Achillea millefolium L. and Achillea pannonica Scheele are described and shown to be characteristic for each species. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Achillea millefolium; Asteraceae; Compositae; Sesquiterpenoids; Chemotaxonomy
1. Introduction Achillea millefolium s.l. (Asteraceae) is widely spread over the Northern hemisphere represented by a polyploid complex of species and subspecies. According to the classification of populations in Central Europe (Saukel and La¨nger, 1992a, b) the Achillea millefolium group is actually divided into 12 well-defined species, which are characterized by morphological, anatomical and caryological features. However, high biodiversity and naturally occurring hybrids obviously complicate the clear definition of plant individuals. The relative ontogenetic stability of the essential oil composition as additional tool for the differentiation of species has been reported (Kastner et al., 1992) and quantitative differences in the flavonoid pattern (Valant, 1978; Krenn, 1997)
* Corresponding author. Tel.: 0043 1 31336 8066; fax: 0043 1 31336 772; e-mail: pharmakognosie @univie.ac.at 0305-1978/99/$ — See front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S0 30 5 - 19 7 8( 9 8 )00 1 00 -8
438
W. Kubelka et al./Biochemical Systematics and Ecology 27 (1999) 437—444
and in the content of nitrogen containing compounds (Mehlfu¨hrer et al., 1997) are described. Hegnauer (1989) claimed the polymorphism and polytypism of sesquiterpenes within the Achillea millefolium group worthwhile for further investigations. The present knowledge about these compounds in combination with results of crossing experiments (Vetter et al., 1997), cell culture breeding (Wawrosch et al., 1997) and DNA-analysis (Wallner et al., 1996) on different species justify the assumption, that these pharmacologically relevant substances also serve as helpful chemotaxonomic markers. This survey is based on the recent taxonomic classification and includes the investigation of more than 1000 plant individuals by means of TLC and HPLC.
2. Material and methods 2.1. Sample collection Above ground parts of air-dried flowering plants of nine species belonging to the A. millefolium group were collected throughout Central Europe (Austria, France, Germany, Hungary, Italy, Slovakia). Vouchers are deposited in the Herbarium of the Institute of Pharmacognosy, University of Vienna. The identification of plant material is based on morphological and anatomical data (e.g. proportion of ligulate florets and leaf pinnae) and caryological methods (Vetter et al., 1996). 2.2. Sample preparation After isolation and identification of sesquiterpenes from the defined species individual plants were examined for their sesquiterpene patterns. 100 mg of air-dried flowering heads were extracted with dichloromethane (10% w/v) in an ultrasonic bath for 5 min at room temperature. This solution was directly used for thin layer chromatography (TLC), but had to be concentrated for high-pressure liquid chromatography (HPLC): after evaporating the dichloromethane the residue was redissolved in 500 ll methanol 50% ready for injection. 2.3. Sesquiterpene analysis 2.3.1. TLC Plates: silica gel 60 Merck, F254 (0.25 mm) 5;5 cm, mobile phase: dichloromethane-aceton (9#1, v/v), detection: EP-reagent (water 20 g, acetic acid 100% 50 g, phosphoric acid 85% 5 g, dimethylaminobenzaldehyde 250 mg; Stahl, 1967) after heating the plate at 120°C (blue coloured spots for proazulenes, fluorescence quenching zones for matricarine derivatives) and anisaldehyde-H SO -reagent (Huber and Fro¨hlke, 1972) after heating at 140°C (different colours for the respective sesquiterpenes).
W. Kubelka et al./Biochemical Systematics and Ecology 27 (1999) 437—444
439
2.3.2. HPLC-UV/»IS Stationary phase: reversed phase material (spherisorb RP8, 5 lm, 250;4.6 mm), detection at UV 220 nm and 255 nm, mobile phase: flat methanol—water gradient beginning at 20% methanol up to 80% methanol in 180 min (rate"0.33%/min, flow"1.0 ml/min, room temperature). On-line recorded UV-spectra and retention times were used for identification criteria.
3. Results The composition of the individual compounds is summarized at the species level in Table 1. It has to be emphasized, that TLC and HPLC analyses were carried out on all samples, offering the opportunity to compare the reproducibility and reliability of these test systems. Results of TLC corresponded perfectly to those of HPLC. Whereas TLC analysis allowed a quick but only semiquantitative glimpse at the sesquiterpenes, the HPLC method was suitable also for quantitative analysis, especially useful comparing species with similar sesquiterpene patterns. Sesquiterpenes within the Achillea millefolium group can be subdivided into different classes based on their carbon skeletons (guaianolides, eudesmanolides, longipinenes, germacrane derivatives). Superimposed on each skeleton — more or less significant for the species — is a set of substituents, which seems to be rather narrow for Achillea, limited to the acyl-residues of acetic, tiglic and angelic acid. Skeletal classes and substitutional features both display taxonomically useful patterns of distribution within the following species. On the diploid level (A. setacea (Zitterl-Eglseer et al., 1991), A. asplenifolia (Kastner et al., 1992; Schro¨der et al., 1994) and A. roseoalba (Kastner et al., 1991a, b; Schro¨der et al., 1994)) sesquiterpene lactones from the guaianolide-type are dominant. Whereas A. setacea does not show any proazulenes (azulenogenic guaianolides), the proazulene-pattern of A. roseoalba and A. asplenifolia seems rather similar, showing only quantitive differences with a surplus of angelic acid derivatives for A. asplenifolia and otherwise of tiglic acid derivatives for A. roseoalba, respectively. This also applies to the 3-oxa-guaianolides, quantitatively far less important components within these two species. Proazulenes and 3-oxa-guaianolides in the same manner characterize also the spectrum of the tetraploid species of A. collina (Kastner et al., 1991a, b; 1994; Schro¨der et al., 1994) and A. ceretanica (Glasl et al., 1997, 1998). In case of the latter the azulenogenic guaianolides reach considerably higher concentrations than in A. collina including a further guaianolide, which seems to be a specific marker for A. ceretanica. In contrast to these species marked by a guaianolide skeleton, the tetraploid species A. pratensis (Saukel and La¨nger, 1992c) is characterized by eudesmanolides and therefore separates clearly from proazulene containing species. The longipinenederivatives isolated from the tetraploid A. distans subsp. styriaca ("Achillea millefolium Typ DIS A; Saukel, 1994; Kastner et al., 1996) are rather exotic among the sesquiterpenes indicating that this species might have passed a separate way during the ontogenesis.
2n
2n
2n
4n
2n 4n
4n
4n
A. setacea
A. asplenifolia
A. roseoalba
A. collina
A. ceretanica
A. pratensis
A. distans
8n
A. pannonica
p.: Ploidy, r.a.: Relative amount.
6n
A. millefolium
subsp. styriaca
p
Species
8a-angeloxy-artabsin 1 8a-tigloxy-artabsin 2 achillicin 3 8a-acetoxy-2-hydroxy-1,5a, 6b, 11bH-guaia-3,10(14)-dien-12,6-olide 8 2a,8a-dihydroxy-1,5a, 6b, 11bH-guaia-3,10(14)-dien-12,6-olide 9
8a-angeloxy-artabsin 1 8a-tigloxy-artabsin 2 achillicin 3 7,8-guaianolides 4 8-desacetyl-4-epi-matricin 5 8-desacetyl-8-tigloyl-4-epi-matricin 6 8a-angeloxy-artabsin 1 8a-tigloxy-artabsin 2 achillicin 3 8-desacetyl-8-tigloyl-matricin 7 8a-angeloxy-artabsin 1 8a-tigloxy-artabsin 2 achillicin 3 8-desacetyl-8-tigloyl-4-epi-matricin 6
Proazulenes
## # # ### # # ## # # ## # ### ## ###
matricarin 17 8-desacetyl-matricarin 18 tauremisin 19 arglanin 20 hydroperoxy-arglanin 21 4-epi-arglanin 22 santamarin 23 tauremisin 19 5b-tigloyl-achillifolin 24 a-longipin-2-en-1-on 25 7a-hydroxy- a-longipin-2-en-1-on 26 8a-angeloxyartabsin-1,4-endoperoxide 27 8a-tigloxyartabsin-1,4-endoperoxide 28 germacrane derivative 29
#
### ### ## #
# # # # # #
r.a.
# # # # # # # # # # # #
rupicolin A 10 rupicolin B 11 11,13-dehydro-desacetylmatricarin 12 8a-angeloxy-3-oxa-artabsin 13 8a-tigloxy-3-oxa-artabsin 14 3-oxa-achillicin 15
Non azulenogenic sesquiterpenes
8a-angeloxy-3-oxa-artabsin 13 8a-tigloxy-3-oxa-artabsin 14 3-oxa-achillicin 15 5a- hydroxy-8-desacetyl-8-tigloyl-matricarin 16 8a-angeloxy-3-oxa-artabsin 13 8a-tigloxy-3-oxa-artabsin 14 3-oxa-achillicin 15 matricarin 17 8-desacetyl-matricarin 18 8a-angeloxy-3-oxa-artabsin 13 8a-tigloxy-3-oxa-artabsin 14 3-oxa-achillicin 15
### ## # ## # # ## ### # # ### ### ## #
r.a.
Table 1 Distribution of sesquiterpenes within nine different species of the Achillea millefolium group (for structures 1—29 see Fig. 1—3)
440 W. Kubelka et al./Biochemical Systematics and Ecology 27 (1999) 437—444
W. Kubelka et al./Biochemical Systematics and Ecology 27 (1999) 437—444
441
Fig. 1. Guaianolides in Achillea taxa (see Table 1).
At the hexaploid level the taxonomic relevance of sequiterpenes still lacks sufficient phytochemical and morphological information and is complicated by the biodiversity and heterogeneity of plant material. As far as described by now, guaianolide-endoperoxides were isolated from the hexaploid A. millefolium (Ru¨cker et al., 1991), and germacrane derivatives dominate the octoploid A. pannonica (Wurzinger, 1995).
442
W. Kubelka et al./Biochemical Systematics and Ecology 27 (1999) 437—444
Fig. 2. Eudesmanolides in Achillea taxa (see Table 1).
Fig. 3. Longipinenones and germacrane-derivatives in Achillea taxa (see Table 1).
4. Discussion Even though the present knowledge about the distribution of sesquiterpenes within the A. millefolium group is not yet complete, distinct carbon skeletons specify the respective species. While valid data are available for the diploid and tetraploid level, the sesquiterpene pattern reported for taxa of higher ploidy seems more variable. This might partly be due to the difficulties in plant identification, the consecutive lack of well founded pharmacognostic studies, but also the high biodiversity of taxa at this ploidy level. Nevertheless, sesquiterpenes serve as an excellent chemotaxonomic tool offering the possibility to combine taxonomic matters with a maximum of information about pharmacologically relevant substances. The described sesquiterpene patterns are significant for each species and not affected by environmental or climatic conditions. Even several steps via micropropagation have no influence on sesquiterpene production and crossing experiments show the inheritance of genetically coded patterns of sesquiterpenes throughout several generations.
W. Kubelka et al./Biochemical Systematics and Ecology 27 (1999) 437—444
443
According to the generally accepted biogenetic pathway of sesquiterpenoids (Croteau, 1986) the biogenesis of either guaianolides or eudesmanolides might be controlled by a distinct gene locus favouring the respective cyclisation from position 1 to 5 (guaianolide) or from 10 to 5 (eudesmanolide). Comparing the chemotaxonomic relevance of sesquiterpenes to the essential oil composition the expression of proazulenes appears associated to relatively high amounts of {-pinene and sabinene, while species free of proazulenes rather favour cineole and camphor. Whether this is a matter of coincidence rather than an expression of genetically based correlation should be further investigated. However, the sesquiterpene pattern is strictly linked to the genotype and is a part of the phenotype equal to morphology, ploidy and other phytochemical characteristics. References Croteau, R., 1986. Biosynthesis of monoterpenes and sesquiterpenes of the essential oils. In: Craker, L. E., Simon, J. E. (Eds.), Spices and Medicinal Plants: Recent Advances in Botany, Horticulture and Pharmacology. vol. 1. Oryx Press, Phoenix AZ, pp. 81—133. Glasl, S., Kastner, U., Werner, I., Wawrosch, Ch., Schubert-Zsilavecz, M., Jurenitsch, J., Kubelka, W., 1997. Sesquiterpenoids of a tetraploid clone of Achillea ceretanica Sennen. Pharm. Pharmacol. Lett. 7, 119—120. Glasl, S., Presser, A., Werner, I., Wawrosch, Ch., Kastner, U., Jurenitsch, J., Haslinger, E., Kubelka, W., 1998. A. ceretanica Sennen. Two proazulenes for A. ceretanica Sennen. Phytochemistry, in press. Glasl, S., Toperzer, G., Kastner, U., Jurenitsch, J., Baumann, A., Kubelka, W., 1991. 8-Desacetylmatricarin aus Achillea collina Becker. Sci. Pharm. 62, 112. Hegnauer, R., 1989. Chemotaxonomie der Pflanzen, Band VIII. 301. Huber, W., Fro¨hlke, F., 1972. A new spray-reagent for the detection and quantitative estimation of peroxides. Chromatographia 5, 256—257. Kastner, U., Jurenitsch, J., Glasl, S., Baumann, A., Robien, W., Kubelka, W., 1991. Three unusual 3-oxa-guaianolides from Achillea roseo-alba Ehrend. and Achillea collina Becker. Pharm. Pharmacol. Lett. 1, 53—54. Kastner, U., Jurenitsch, J., Glasl, S., Baumann, A., Robien, W., Kubelka, W., 1991. The major proazulenes from Achillea roseo-alba Ehrend. Pharm. Pharmacol. Lett. 1, 55—56. Kastner, U., Jurenitsch, J., Glasl, S., Baumann, A., Robien, W., Kubelka, W., 1992. Proazulenes from Achillea asplenifolia. Phytochemistry 31, 4361—4362. Kastner, U., Jurenitsch, J., Lehner, S., Baumann, A., Robien, W., Kubelka, W., 1991. The major proazulenes from Achillea collina Becker: a revision of structure. Pharm. Pharmacol. Lett. 1, 27—28. Kastner, U., Saukel, J., Zitterl-Eglseer, K., La¨nger, R., Reznicek, G., Jurenitsch, J., Kubelka, W., 1992. A®therisches O®l-ein zusa¨tzliches Merkmal fu¨r die Charakterisierung der mitteleuropa¨ischen Taxa der Achillea millefolium Gruppe. Sci. Pharm. 60, 87—99. Krenn, L., 1997. Flavonoide verschiedener Schafgarben-Taxa. Workshop Achillea, Vienna, Drogenreport, in press. Mehlfu¨hrer, M., Troll, K., Jurenitsch, J., Auer, H., Kubelka, W., 1997. Betaines and free Proline within the Achillea millefolium group. Phytochemistry 44, 1067—1069. Saukel, J., 1994. Achillea millefolium-Gruppe. In: Adler W., Oswald K., Fischer R. (Eds.), Exkursionsflora von O$ sterreich. Verlag Eugen Ulmer, Stuttgart. Wien, pp. 813—821. Saukel, J., La¨nger, R., 1992a. Die Achillea millefolium Gruppe (Asteraceae) in Mitteleuropa, 1. Phyton 31, 185—207. Saukel, J., La¨nger, R., 1992b. Die Achillea millefolium Gruppe (Asteraceae) in Mitteleuropa, 2. Phyton 32, 47—78. Saukel, J., La¨nger, R., 1992c. Achillea pratensis Saukel & La¨nger, spec. nova, eine tetraploide Sippe des Achillea millefolium-Komplexes. Phyton 32, 159—172.
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Schro¨der, H., Kastner, U., Gargula, K., Budesinsky, M., Haslinger, E., Jurenitsch, J., Kubelka, W., 1994. Artabsin and 3-oxa-artabsin derivatives from Achillea millefolium species. Phytochemistry 36, 1449—1451. Stahl, E., 1967. Du( nnschicht-Chromatographie. Springer, Berlin, p. 825. Valant, K., 1978. Charakteristische Flavonoidglykoside und verwandtschaftliche Gliederung der Gattung Achillea. Naturwissenschaften 65, 437—438. Vetter, S., Franz, Ch., Glasl, S., Kastner, U., Saukel, J., Jurenitsch J., 1997. Inheritance of sesquiterpene lactone types within the Achillea millefolium complex (Compositae). Plant Breeding 116, 79—82. FWF project PO9341-BIO. Vetter, S., Lambrou, M., Franz, Ch., Ehrendorfer, F., 1996. Cytogenetics of experimental hybrids within the Achillea millefolium polyploid complex (yarrow). Caryologia 49, 1—12. Wallner, E., Weising, K., Rompf, R., Kahl, G., Kopp, B., 1996. Oligonucleotide fingerprinting and RAPD analysis of Achillea species: Characterization and long-term monitoring of micropropagated clones. Plant Cell Reports 15, 647—652. Wawrosch, Ch., Kopp, B., Sto¨ckl, J., Glasl, S., Kubelka, W., 1997. In vitro propagation of tetraploid Achillea ceretanica Sennen. Pharm. Pharmacol. Lett. 7, 116—118. Wurzinger, G., 1995. Isolierung von Peroxiden und weiteren Terpenoiden aus Achillea pannonica. Diplomarbeit, Universita¨t Wien. Zitterl-Eglseer, K., Jurenitsch, J., Korhammer, S., Haslinger, E., Sosa, S., Della Loggia, R., Kubelka, W., Franz, Ch., 1991. Planta Med. 57, 444—446.
Chemotaxonomic relevance of sesquiterpenes within the Achillea millefolium group Wolfgang Kubelka*, Ulrike Kastner, Sabine Glasl, Johannes Saukel, Johann Jurenitsch Institute of Pharmacognosy, University of Vienna, Pharmaziezentrum, Althanstra}e 14, A-1090 Wien, Austria
Abstract The chemotaxonomic relevance of sesquiterpenoids within the Achillea millefolium group has been evaluated by means of morphological, anatomical, cytological and phytochemical data. The sesquiterpene patterns of Achillea setacea Waldst. & Kit, Achillea asplenifolia Vent., Achillea roseoalba Ehrend., Achillea collina Becker, Achillea ceretanica Sennen, Achillea pratensis Saukel & La¨nger, Achillea distans subsp. styriaca Saukel in edit., Achillea millefolium L. and Achillea pannonica Scheele are described and shown to be characteristic for each species. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Achillea millefolium; Asteraceae; Compositae; Sesquiterpenoids; Chemotaxonomy
1. Introduction Achillea millefolium s.l. (Asteraceae) is widely spread over the Northern hemisphere represented by a polyploid complex of species and subspecies. According to the classification of populations in Central Europe (Saukel and La¨nger, 1992a, b) the Achillea millefolium group is actually divided into 12 well-defined species, which are characterized by morphological, anatomical and caryological features. However, high biodiversity and naturally occurring hybrids obviously complicate the clear definition of plant individuals. The relative ontogenetic stability of the essential oil composition as additional tool for the differentiation of species has been reported (Kastner et al., 1992) and quantitative differences in the flavonoid pattern (Valant, 1978; Krenn, 1997)
* Corresponding author. Tel.: 0043 1 31336 8066; fax: 0043 1 31336 772; e-mail: pharmakognosie @univie.ac.at 0305-1978/99/$ — See front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S0 30 5 - 19 7 8( 9 8 )00 1 00 -8
438
W. Kubelka et al./Biochemical Systematics and Ecology 27 (1999) 437—444
and in the content of nitrogen containing compounds (Mehlfu¨hrer et al., 1997) are described. Hegnauer (1989) claimed the polymorphism and polytypism of sesquiterpenes within the Achillea millefolium group worthwhile for further investigations. The present knowledge about these compounds in combination with results of crossing experiments (Vetter et al., 1997), cell culture breeding (Wawrosch et al., 1997) and DNA-analysis (Wallner et al., 1996) on different species justify the assumption, that these pharmacologically relevant substances also serve as helpful chemotaxonomic markers. This survey is based on the recent taxonomic classification and includes the investigation of more than 1000 plant individuals by means of TLC and HPLC.
2. Material and methods 2.1. Sample collection Above ground parts of air-dried flowering plants of nine species belonging to the A. millefolium group were collected throughout Central Europe (Austria, France, Germany, Hungary, Italy, Slovakia). Vouchers are deposited in the Herbarium of the Institute of Pharmacognosy, University of Vienna. The identification of plant material is based on morphological and anatomical data (e.g. proportion of ligulate florets and leaf pinnae) and caryological methods (Vetter et al., 1996). 2.2. Sample preparation After isolation and identification of sesquiterpenes from the defined species individual plants were examined for their sesquiterpene patterns. 100 mg of air-dried flowering heads were extracted with dichloromethane (10% w/v) in an ultrasonic bath for 5 min at room temperature. This solution was directly used for thin layer chromatography (TLC), but had to be concentrated for high-pressure liquid chromatography (HPLC): after evaporating the dichloromethane the residue was redissolved in 500 ll methanol 50% ready for injection. 2.3. Sesquiterpene analysis 2.3.1. TLC Plates: silica gel 60 Merck, F254 (0.25 mm) 5;5 cm, mobile phase: dichloromethane-aceton (9#1, v/v), detection: EP-reagent (water 20 g, acetic acid 100% 50 g, phosphoric acid 85% 5 g, dimethylaminobenzaldehyde 250 mg; Stahl, 1967) after heating the plate at 120°C (blue coloured spots for proazulenes, fluorescence quenching zones for matricarine derivatives) and anisaldehyde-H SO -reagent (Huber and Fro¨hlke, 1972) after heating at 140°C (different colours for the respective sesquiterpenes).
W. Kubelka et al./Biochemical Systematics and Ecology 27 (1999) 437—444
439
2.3.2. HPLC-UV/»IS Stationary phase: reversed phase material (spherisorb RP8, 5 lm, 250;4.6 mm), detection at UV 220 nm and 255 nm, mobile phase: flat methanol—water gradient beginning at 20% methanol up to 80% methanol in 180 min (rate"0.33%/min, flow"1.0 ml/min, room temperature). On-line recorded UV-spectra and retention times were used for identification criteria.
3. Results The composition of the individual compounds is summarized at the species level in Table 1. It has to be emphasized, that TLC and HPLC analyses were carried out on all samples, offering the opportunity to compare the reproducibility and reliability of these test systems. Results of TLC corresponded perfectly to those of HPLC. Whereas TLC analysis allowed a quick but only semiquantitative glimpse at the sesquiterpenes, the HPLC method was suitable also for quantitative analysis, especially useful comparing species with similar sesquiterpene patterns. Sesquiterpenes within the Achillea millefolium group can be subdivided into different classes based on their carbon skeletons (guaianolides, eudesmanolides, longipinenes, germacrane derivatives). Superimposed on each skeleton — more or less significant for the species — is a set of substituents, which seems to be rather narrow for Achillea, limited to the acyl-residues of acetic, tiglic and angelic acid. Skeletal classes and substitutional features both display taxonomically useful patterns of distribution within the following species. On the diploid level (A. setacea (Zitterl-Eglseer et al., 1991), A. asplenifolia (Kastner et al., 1992; Schro¨der et al., 1994) and A. roseoalba (Kastner et al., 1991a, b; Schro¨der et al., 1994)) sesquiterpene lactones from the guaianolide-type are dominant. Whereas A. setacea does not show any proazulenes (azulenogenic guaianolides), the proazulene-pattern of A. roseoalba and A. asplenifolia seems rather similar, showing only quantitive differences with a surplus of angelic acid derivatives for A. asplenifolia and otherwise of tiglic acid derivatives for A. roseoalba, respectively. This also applies to the 3-oxa-guaianolides, quantitatively far less important components within these two species. Proazulenes and 3-oxa-guaianolides in the same manner characterize also the spectrum of the tetraploid species of A. collina (Kastner et al., 1991a, b; 1994; Schro¨der et al., 1994) and A. ceretanica (Glasl et al., 1997, 1998). In case of the latter the azulenogenic guaianolides reach considerably higher concentrations than in A. collina including a further guaianolide, which seems to be a specific marker for A. ceretanica. In contrast to these species marked by a guaianolide skeleton, the tetraploid species A. pratensis (Saukel and La¨nger, 1992c) is characterized by eudesmanolides and therefore separates clearly from proazulene containing species. The longipinenederivatives isolated from the tetraploid A. distans subsp. styriaca ("Achillea millefolium Typ DIS A; Saukel, 1994; Kastner et al., 1996) are rather exotic among the sesquiterpenes indicating that this species might have passed a separate way during the ontogenesis.
2n
2n
2n
4n
2n 4n
4n
4n
A. setacea
A. asplenifolia
A. roseoalba
A. collina
A. ceretanica
A. pratensis
A. distans
8n
A. pannonica
p.: Ploidy, r.a.: Relative amount.
6n
A. millefolium
subsp. styriaca
p
Species
8a-angeloxy-artabsin 1 8a-tigloxy-artabsin 2 achillicin 3 8a-acetoxy-2-hydroxy-1,5a, 6b, 11bH-guaia-3,10(14)-dien-12,6-olide 8 2a,8a-dihydroxy-1,5a, 6b, 11bH-guaia-3,10(14)-dien-12,6-olide 9
8a-angeloxy-artabsin 1 8a-tigloxy-artabsin 2 achillicin 3 7,8-guaianolides 4 8-desacetyl-4-epi-matricin 5 8-desacetyl-8-tigloyl-4-epi-matricin 6 8a-angeloxy-artabsin 1 8a-tigloxy-artabsin 2 achillicin 3 8-desacetyl-8-tigloyl-matricin 7 8a-angeloxy-artabsin 1 8a-tigloxy-artabsin 2 achillicin 3 8-desacetyl-8-tigloyl-4-epi-matricin 6
Proazulenes
## # # ### # # ## # # ## # ### ## ###
matricarin 17 8-desacetyl-matricarin 18 tauremisin 19 arglanin 20 hydroperoxy-arglanin 21 4-epi-arglanin 22 santamarin 23 tauremisin 19 5b-tigloyl-achillifolin 24 a-longipin-2-en-1-on 25 7a-hydroxy- a-longipin-2-en-1-on 26 8a-angeloxyartabsin-1,4-endoperoxide 27 8a-tigloxyartabsin-1,4-endoperoxide 28 germacrane derivative 29
#
### ### ## #
# # # # # #
r.a.
# # # # # # # # # # # #
rupicolin A 10 rupicolin B 11 11,13-dehydro-desacetylmatricarin 12 8a-angeloxy-3-oxa-artabsin 13 8a-tigloxy-3-oxa-artabsin 14 3-oxa-achillicin 15
Non azulenogenic sesquiterpenes
8a-angeloxy-3-oxa-artabsin 13 8a-tigloxy-3-oxa-artabsin 14 3-oxa-achillicin 15 5a- hydroxy-8-desacetyl-8-tigloyl-matricarin 16 8a-angeloxy-3-oxa-artabsin 13 8a-tigloxy-3-oxa-artabsin 14 3-oxa-achillicin 15 matricarin 17 8-desacetyl-matricarin 18 8a-angeloxy-3-oxa-artabsin 13 8a-tigloxy-3-oxa-artabsin 14 3-oxa-achillicin 15
### ## # ## # # ## ### # # ### ### ## #
r.a.
Table 1 Distribution of sesquiterpenes within nine different species of the Achillea millefolium group (for structures 1—29 see Fig. 1—3)
440 W. Kubelka et al./Biochemical Systematics and Ecology 27 (1999) 437—444
W. Kubelka et al./Biochemical Systematics and Ecology 27 (1999) 437—444
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Fig. 1. Guaianolides in Achillea taxa (see Table 1).
At the hexaploid level the taxonomic relevance of sequiterpenes still lacks sufficient phytochemical and morphological information and is complicated by the biodiversity and heterogeneity of plant material. As far as described by now, guaianolide-endoperoxides were isolated from the hexaploid A. millefolium (Ru¨cker et al., 1991), and germacrane derivatives dominate the octoploid A. pannonica (Wurzinger, 1995).
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Fig. 2. Eudesmanolides in Achillea taxa (see Table 1).
Fig. 3. Longipinenones and germacrane-derivatives in Achillea taxa (see Table 1).
4. Discussion Even though the present knowledge about the distribution of sesquiterpenes within the A. millefolium group is not yet complete, distinct carbon skeletons specify the respective species. While valid data are available for the diploid and tetraploid level, the sesquiterpene pattern reported for taxa of higher ploidy seems more variable. This might partly be due to the difficulties in plant identification, the consecutive lack of well founded pharmacognostic studies, but also the high biodiversity of taxa at this ploidy level. Nevertheless, sesquiterpenes serve as an excellent chemotaxonomic tool offering the possibility to combine taxonomic matters with a maximum of information about pharmacologically relevant substances. The described sesquiterpene patterns are significant for each species and not affected by environmental or climatic conditions. Even several steps via micropropagation have no influence on sesquiterpene production and crossing experiments show the inheritance of genetically coded patterns of sesquiterpenes throughout several generations.
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According to the generally accepted biogenetic pathway of sesquiterpenoids (Croteau, 1986) the biogenesis of either guaianolides or eudesmanolides might be controlled by a distinct gene locus favouring the respective cyclisation from position 1 to 5 (guaianolide) or from 10 to 5 (eudesmanolide). Comparing the chemotaxonomic relevance of sesquiterpenes to the essential oil composition the expression of proazulenes appears associated to relatively high amounts of {-pinene and sabinene, while species free of proazulenes rather favour cineole and camphor. Whether this is a matter of coincidence rather than an expression of genetically based correlation should be further investigated. However, the sesquiterpene pattern is strictly linked to the genotype and is a part of the phenotype equal to morphology, ploidy and other phytochemical characteristics. References Croteau, R., 1986. Biosynthesis of monoterpenes and sesquiterpenes of the essential oils. In: Craker, L. E., Simon, J. E. (Eds.), Spices and Medicinal Plants: Recent Advances in Botany, Horticulture and Pharmacology. vol. 1. Oryx Press, Phoenix AZ, pp. 81—133. Glasl, S., Kastner, U., Werner, I., Wawrosch, Ch., Schubert-Zsilavecz, M., Jurenitsch, J., Kubelka, W., 1997. Sesquiterpenoids of a tetraploid clone of Achillea ceretanica Sennen. Pharm. Pharmacol. Lett. 7, 119—120. Glasl, S., Presser, A., Werner, I., Wawrosch, Ch., Kastner, U., Jurenitsch, J., Haslinger, E., Kubelka, W., 1998. A. ceretanica Sennen. Two proazulenes for A. ceretanica Sennen. Phytochemistry, in press. Glasl, S., Toperzer, G., Kastner, U., Jurenitsch, J., Baumann, A., Kubelka, W., 1991. 8-Desacetylmatricarin aus Achillea collina Becker. Sci. Pharm. 62, 112. Hegnauer, R., 1989. Chemotaxonomie der Pflanzen, Band VIII. 301. Huber, W., Fro¨hlke, F., 1972. A new spray-reagent for the detection and quantitative estimation of peroxides. Chromatographia 5, 256—257. Kastner, U., Jurenitsch, J., Glasl, S., Baumann, A., Robien, W., Kubelka, W., 1991. Three unusual 3-oxa-guaianolides from Achillea roseo-alba Ehrend. and Achillea collina Becker. Pharm. Pharmacol. Lett. 1, 53—54. Kastner, U., Jurenitsch, J., Glasl, S., Baumann, A., Robien, W., Kubelka, W., 1991. The major proazulenes from Achillea roseo-alba Ehrend. Pharm. Pharmacol. Lett. 1, 55—56. Kastner, U., Jurenitsch, J., Glasl, S., Baumann, A., Robien, W., Kubelka, W., 1992. Proazulenes from Achillea asplenifolia. Phytochemistry 31, 4361—4362. Kastner, U., Jurenitsch, J., Lehner, S., Baumann, A., Robien, W., Kubelka, W., 1991. The major proazulenes from Achillea collina Becker: a revision of structure. Pharm. Pharmacol. Lett. 1, 27—28. Kastner, U., Saukel, J., Zitterl-Eglseer, K., La¨nger, R., Reznicek, G., Jurenitsch, J., Kubelka, W., 1992. A®therisches O®l-ein zusa¨tzliches Merkmal fu¨r die Charakterisierung der mitteleuropa¨ischen Taxa der Achillea millefolium Gruppe. Sci. Pharm. 60, 87—99. Krenn, L., 1997. Flavonoide verschiedener Schafgarben-Taxa. Workshop Achillea, Vienna, Drogenreport, in press. Mehlfu¨hrer, M., Troll, K., Jurenitsch, J., Auer, H., Kubelka, W., 1997. Betaines and free Proline within the Achillea millefolium group. Phytochemistry 44, 1067—1069. Saukel, J., 1994. Achillea millefolium-Gruppe. In: Adler W., Oswald K., Fischer R. (Eds.), Exkursionsflora von O$ sterreich. Verlag Eugen Ulmer, Stuttgart. Wien, pp. 813—821. Saukel, J., La¨nger, R., 1992a. Die Achillea millefolium Gruppe (Asteraceae) in Mitteleuropa, 1. Phyton 31, 185—207. Saukel, J., La¨nger, R., 1992b. Die Achillea millefolium Gruppe (Asteraceae) in Mitteleuropa, 2. Phyton 32, 47—78. Saukel, J., La¨nger, R., 1992c. Achillea pratensis Saukel & La¨nger, spec. nova, eine tetraploide Sippe des Achillea millefolium-Komplexes. Phyton 32, 159—172.
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