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Comparative studies in phytochemistry and fruit anatomy ofThapsia garganicaandT. transtagana,Apiaceae (Umbelliferae)
Botanical Journal of the Linnean Society (1995), 117: 281–292. With 8 figures
Comparative studies in phytochemistry and fruit anatomy of Thapsia garganica and T. transtagana, Apiaceae (Umbelliferae) ¨ GER, ULLA WAGNER SMITT, ANNE KATHARINA JA ANNE ADSERSEN AND LENE GUDIKSEN Department of Pharmacognosy, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark Received November 1994, accepted for publication January 1995
Thapsia garganica L. and T. transtagana Brot. are classified as synonymous in Flora Europaea. In the present investigation significant differences between the two taxa with regard to fruit anatomy and phytochemistry are demonstrated and they are considered as two separate species. Microscopic studies revealed a distinct difference in number and position of secretory spaces in the pericarp of fruits from the two species and in addition pronounced differences were found in the presence of thapsigargins, the bioactive constituents of the two species. Quantitative HPLC analyses of thapsigargins were carried out on different plant organs from T. garganica and T. transtagana collected from various locations. Thapsigargin, thapsigargicin, nortrilobolid and thapsivillosin I and J were dominant compounds in all organs of T. garganica whereas none of these compounds could be detected in any organ of T. transtagana. On the contrary, thapsitranstagin and trilobolid were main thapsigargins of T. transtagana. T. garganica may include two chemotypes, as trilobolid was detected in some specimens of T. garganica only. As this is the first time trilobolid has been detected in these two species, it was isolated from both and identified by 1H NMR-spectroscopy. ADDITIONAL KEY WORDS:—chemotaxonomy – HPLC analysis – secretory spaces – sesquiterpene lactones – thapsigargins – trilobolid.
CONTENTS Introduction . . . . . . . . Material and methods . . . . . . Plant material . . . . . . . Determination of chromosome numbers . Anatomical investigation . . . . Quantitative HPLC analysis . . . Isolation of thapsigargins . . . . Results . . . . . . . . . Morphological and anatomical characters Phytochemistry . . . . . . Discussion . . . . . . . . . Acknowledgements . . . . . . . References . . . . . . . . .
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INTRODUCTION
An ancient remedy, Resina thapsiae, used against pulmonary diseases, catarrhs and rheumatic pains, was obtained from the roots of the Mediterranean plant 0024–4074/95/040281+12 $08.00/0
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© 1995 The Linnean Society of London
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Thapsia garganica L. (French, 1971; Perrot, 1943–44). The resin, which is located in schizogenous secretory canals in the root bark, provokes a vigorous contact dermatitis expressed as erythema, itching and small vesiculae. As recently as the nineteenth century the resin was used in medicine, especially by the Arabs of north Africa, who named the resin Bou ne´fa (father of health), and the plant, De´rias. The resin of T. garganica and a medical plaster, Sparadrap de thapsia, has been included in the 1937 edition of the French pharmacopoeia. T. garganica has been described under several names and varieties in different floras (Rasmussen, 1979). In Flora Europaea, T. garganica, T. transtagana Brot. and T. decussata Lag. are classified as synonymous (Tutin, 1968). However, T. transtagana, described for the first time in Flora Lusitanica (Brotero, 1804), with Montemor o Novo and Serpa in Portugal cited as habitats, has hirsute leaves, in contrast to T. garganica. The petiole and nerves of the leaves of T. decussata (Lagasca, 1816; Willkomm & Lange, 1880), with habitats in Spain, are similarly described as hirsute. It has previously been shown that plants growing in Portugal identified as T. transtagana Brot. are phytochemically different from T. garganica (Rasmussen, Christensen & Sandberg, 1981). Previous phytochemical investigations of T. garganica have resulted in the isolation of five skin irritating compounds. The main constituents in the roots as well as in the ripe fruits are thapsigargin I and thapsigargicin II, whereas nortrilobolid III, thapsivillosin J IV and thapsivillosin I V were found as minor constituents. None of these compounds were detected in roots of T. transtagana, from which only thapsitranstagin VII has been isolated (Christensen et al., 1984; Smitt & Christensen, 1991). These compounds, named thapsigargins, are all non-cytotoxic histamine liberators (Christensen et al., 1982; Norup, Smitt & Christensen, 1986). Thapsigargin itself, acting as a Ca++-ATPase inhibitor, has become a valuable test compound of great demand in biochemical studies of calcium homeostasis (Christensen et al., 1992), and because it is still available only by isolation from wild growing plants of T. garganica, investigation of the diversity within this species and its possible synonym T. transtagana is of considerable interest. In this paper we present the results of comparative microscopic studies of fruits from T. garganica and T. transtagana as well as the results of quantitative HPLC analyses of thapsigargins in different organs of the two species, each collected at different locations. Furthermore, trilobolid VI, not previously detected in either of the two species, has now been isolated from the roots of T. garganica collected in Italy, and from the ripe fruits of T. transtagana.
MATERIAL AND METHODS
Plant material Unless otherwise cited, plant material of Thapsia garganica and T. transtagana was collected by one of the authors (UWS), in the following locations (see Fig. 1): T. transtagana. PORTUGAL: (1) c. 6 km south of Alter do Cha˜o road no. 245. (2) c. 5 km east of Evora road no. 254. (3) c. 5 km west of Ourique road no. EN 123. (4) c. 2 km east of Santa Luzia road no. 389. (5) Just outside Vilamoura, Algarve.
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Figure 1. Locations where T. transtagana 1–5 (W) and T. garganica 6–12 (R) have been collected.
T. garganica. (6) SPAIN: south end of S. Antonio Abad, Ibiza. (7) ALGERIA: c. 52 km south of Skikda road no. N 3. (8) TUNISIA: c. 7 km east of Beni Khalled road no. C 44. (9) ITALY: c. 7 km east of Carpino, Promontorio Del Gargano (collected by P. Avato). (10) LIBYA: c. 2 km west of Shahhat (collected by F. Sandberg). (11) GREECE: isle of Kalymnos. (12) isle of Samos (collected by F. Sandberg). Plants were at the stage of ripe fruits. Voucher specimens are deposited in the Department of Pharmacognosy, Royal Danish School of Pharmacy, Copenhagen.
Determination of chromosome numbers Chromosome numbers were determined on seedling root tips with the Feulgen squash method, after pretreatment with 0.1% colchicine for 1.5–2 h at 20– 25>C.
Anatomical investigation Cross sections of the middle part of the fruits were made with a razor and embedded in chloral hydrate solution. Microscope: Zeiss Axioplan; Film: Agfaortho 25.
Quantitative HPLC analysis HPLC analyses of thapsigargins were performed as described by Ja¨ger et al. (1993).
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Isolation of thapsigargins Dried, triturated roots or fruits were extracted three times with EtOAc and the combined extracts concentrated in vacuum to produce a brown gum. The gum was dissolved in CH2Cl2 and applied on a dry silica Woelm TSC column. Elution was performed with CH2Cl2 to which increasing amounts of EtOAc (5–100%) were added. Fractions containing thapsigargins were concentrated in vacuum and the residue was rechromatographed twice over silica gel 60 (Merck Art. 7754, 0.063–0.200 mm) with 10% water, using toluene containing increasing amounts of EtOAc, 16–20% and 12.5–20% respectively, as eluents. Final purification was performed by preparative HPLC; Column: Lichrosorb RP 18, 5 mm, 12×250 mm, Eluent: MeOH-H2O 83: 17, Flow 6 ml:min, Detection: 230 nm. Identification was based on comparison of the 1H NMR-spectra with that from authentic material previously isolated in our laboratory. RESULTS
Morphological and anatomical characters The morphological characters of Thapsia garganica are described in Flora Europaea (Tutin, 1968). Based on these characters alone it is hardly possible to distinguish between T. garganica and T. transtagana as leaf morphology is almost identical and the fruits are of the same size. However, in contrast to T. garganica, the leaves of T. transtagana are usually distinctly hairy, being especially hirsute on petiole and nerves, and the lobes are usually broader than those of T. garganica—the outer lobes being 3–10 (–15) mm and 1–5 mm, respectively (Figs 2, 3). The primordial leaves of both species are oval and entire, in contrast to the pinnate primordial leaves of other Thapsia species (Figs 2, 3). The chromosome number of both T. garganica and T. transtagana was determined as 2n 22 (2x). The apiaceous fruits are cremocarps with a characteristic structure. The number and position of the secretory spaces in the pericarp are of significant taxonomic value (Roth, 1977). Thapsia garganica and T. transtagana both contain two types of secretory spaces—vittae and companion canals associated with the vascular bundles. Microscopy of the transverse sections of the fruits from the two species showed a distinct difference in regard to the companion canals (Figs 4, 5). In T. garganica each vascular bundle is accompanied by one companion canal situated on the outer side of the bundle. In T. transtagana, on the contrary, each vascular bundle is accompanied by two companion canals, one on the outer and the other on the inner side of the vascular bundle. Phytochemistry Thapsia garganica L. Two major thapsigargins, I and II, have previously been isolated from the roots of T. garganica together with three minor constituents III, IV and V. Thapsigargins are present as constituents of the resin in all plant organs, although the highest concentrations are found in roots and in fruits (Fig. 6). The results of quantitative HPLC analyses of thapsigargins in roots and fruits of T. garganica collected at locations 6–12 (Fig. 1) are shown in Figure 7. The
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Figure 2. Leaves and fruits of T. garganica. Left: Basal leaf of mature plant. Right: A, Primordial leaf. B, Fourth leaf of plant germinated in greenhouse. C, Ripe fruits of different size.
total concentration of thapsigargins varied from 0.2–1.2% of the dry weight of the roots and from 0.7–1.5% of the ripe fruits. In dry leaves a total concentration of about 0.1% was detected, while in dry stems the total concentration was between 0.1–0.5%. The most notable results of the quantitative HPLC analyses are the relative concentrations of thapsigargins in plants from the different locations. While thapsigargin I, constituting 55–80%, is evidently the major component of plants from location 6, 7, 8 and 10, this does not apply to plants from location 9, 11 and 12, where I constitutes only 20–45%. In the latter plants no particular thapsigargins dominate. We have isolated trilobolid VI, not previously found in T. garganica, from roots collected in Italy location 9. This compound was detected by HPLC in
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Figure 3. Leaves and fruits of T. transtagana. Left: Basal leaf of mature plant. Right: A, Primordial leaf. B, Fourth leaf of plant germinated in greenhouse. C, Ripe fruits of different size.
plants from locations 11 and 12, i.e. it occurs in all plants with low concentration of thapsigargin I. The relative concentrations of the different thapsigargins show the same pattern in all organs of plants at the same location. In dried plant material, thapsigargins are very stable components. An old drug, Radix thapsiae, kept in our department since 1892, had a similar thapsigargin content to that of newly collected roots (Fig. 7). Thapsia transtagana Brot. In previous investigations of T. transtagana, thapsitranstagin VII was isolated from the roots, but until this investigation no thapsigargins have been isolated from the fruits. HPLC analyses of different organs of this species revealed the presence of
CHEMISTRY AND FRUIT ANATOMY IN THAPSIA
Figure 4. Cross section of fruits from T. garganica showing companion canals only on the outside of the vascular bundles. A, Young unripe fruit. B, Ripe fruit. (cf. Fig. 5)
Figure 5. Cross section of fruits from T. transtagana showing companion canals both on the outside and on the inside of the vascular bundles. A, Young unripe fruit. B, Ripe fruit. (cf. Fig. 4)
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Figure 6. Thapsigargins isolated from Thapsia.
several thapsigargins. As in T. garganica the highest concentrations are stored in roots and in fruits. However, total concentrations are generally lower than in T. garganica and there are distinct dissimilarities between the constituents of the two species. Five (I–V) of the six components found in T. garganica were not detected in T. transtagana. HPLC chromatograms of the fractionated extracts from the fruits of T. transtagana showed several peaks, of which the Rt-values of the major ones corresponded to VI, VII, thapsivillosin K VIII and thapsivillosin B IX. These thapsigargins could also be detected in the fractionated root extracts. As VI and VII were the two major constituents of the fruits, they were isolated and identified by 1H NMR. Attempts to isolate VI from the roots did not result in a pure compound. From the 1H NMR-spectrum it is suggested to be a mixture of the two isomers VI and X. Quantitative HPLC analyses of thapsigargins in roots and fruits of T. transtagana collected at five
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Figure 7. Quantitative HPLC analyses of (A) fruits and (B) roots of T. garganica collected at locations 6–12 (see Fig. 1). 1892 indicates the old sample from the year 1892.
different locations in Portugal (Fig. 8) show that the relative concentration of the two major constituents VI and VII varies from place to place. The total concentration of thapsigargins, estimated as VII, is 0.3–0.5% in both roots and fruits, with the exception of fruits from location 1, which contain 0.8%. DISCUSSION
The results of this investigation show that Thapsia garganica and T. transtagana can be distinguished by means of anatomical and phytochemical characters. The most significant difference is the number and position of secretory spaces in the fruits. Before VI was isolated from the roots of T. garganica collected in Italy, it was believed that T. garganica contained only thapsigargins with a butanoic acid esterified to 0–8. Esterification with this acid has not been observed in compounds from T. transtagana. This difference was supported by
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Figure 8. Quantitative HPLC analyses of (A) fruits and (B) roots of T. transtagana collected at locations 1–5 (see Fig. 1). n.i.: not identified.
the detection of two proazulene guaianolides, one with a butanoyl the other with a 2-methylbutanoyl group at 0–8, isolated from T. garganica and T. transtagana respectively (Avato et al., 1993). However, indication of divergent biogenesis of the esterified guaianolides of the two species, is contested by the presence of VI in some plants of T. garganica. As these plants were found to have a fruit anatomy identical to T. garganica (Fig. 4), whereas the phytochemical investigations showed different composition of thapsigargins, we propose the classification of plants from locations 9, 11 and 12 as chemotypes of T. garganica. Thapsia garganica and T. transtagana are easily distinguished from other species of Thapsia due to their bigger fruits and entire linear leaflobes. They must be regarded as separate species as they have different fruit anatomy and composition of secondary metabolic constituents, and separate geographical distributions. T. transtagana is distributed in Portugal, south-west Spain and Morocco, areas where
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T. garganica has not been found. The authors consider that T. decussata may be identical to T. transtagana based on morphological descriptions and habitats. This investigation shows that for the biological test compound I, the highest concentrations (0.6–1.1% by dry weight), are obtained from T. garganica growing at locations 6, 7, 8 and 10. The chemotaxonomy of T. maxima has already been published (Avato, Jacobsen & Smitt, 1992) and the results from the chemotaxonomic studies of T. villosa will be published shortly. Thapsia gymnesica, described as a new species in 1991 (Pujadas, Rosello´ & Barcelo´, 1991), has been found in small populations only on the Balearic Islands, Mallorca and Minorca. It appears to be an intermediate of T. villosa and T. garganica. Its fruits are small as in T. villosa, whereas the leaves are more similar to those of T. garganica. HPLC analyses of the roots and fruits of T. gymnesica show the presence of compounds with the same Rt-values as I, II and III, compounds, which hitherto have been found only in T. garganica. Further studies on T. gymnesica are in preparation.
ACKNOWLEDGEMENTS
We are grateful to Prof. F. Sandberg, Biomedical Centre, Uppsala, Sweden, and to P. Avato, Universita` di Bari, Italy, for the supply of plant material and to S. B. Christensen, Royal Danish School of Pharmacy, Copenhagen, for recording the NMR-spectra. The Bruker AC-200 F instrument was a gift from the Velux Foundation of 1981 and the Torkil Steenbech Foundation.
REFERENCES Avato P, Jacobsen N, Smitt UW. 1992. Chemotaxonomy of Thapsia maxima Miller. Constituents of the essential oil of the fruits. The Journal of Essential Oil Research 4: 467–473. Avato P, Cornett C, Andersen A, Smitt UW, Christensen SB. 1993. Localization of the acyl groups in proazulene guaianolides from Thapsia transtagana Brot. and Thapsia garganica L. Journal of Natural Products 56: 411–415. Brotero FA. 1804. Flora Lusitanica, I. Olissipone, 468. Christensen SB, Larsen IK, Rasmussen U, Christophersen C. 1982. Thapsigargin and thapsigargicin, two histamine liberating sesquiterpene lactones from Thapsia garganica. X-ray analysis of the 7,11-epoxide of thapsigargin. Journal of Organic Chemistry 47: 649–652. Christensen SB, Norup E, Rasmussen U, Madsen JO š. 1984. Structure of histamine releasing guaianolides from Thapsia species. Phytochemistry. 23: 1659–1663. Christensen SB, Andersen A, Lauridsen A, Moldt P, Smitt UW, Thastrup O. 1992. Thapsigargine: A lead to design of drugs with the calcium pump as target. In: Krogsgaard-Larsen P, Christensen SB, Kofod H, eds. New leads and targets in drug research. Alfred Benzon Symposium 33. Copenhagen: Munksgaard, 243–252. French, DH. 1971. Ethnobotany of the Umbelliferae. In: Heywood VH, ed. The biology and chemistry of the Umbelliferae. London: Academic Press, 385–412. Ja¨ger AK, Gudiksen L, Adsersen A, Smitt UW. 1993. High performance liquid chromatography of thapsigargins. Journal of Chromatography 634: 135–137. Lagasca M. 1816. Genera et species plantarum, quae aut novae sunt, aut nondum recte cognoscuntur. Matriti, 12. Norup E, Smitt UW, Christensen SB. 1986. The potencies of thapsigargin and analogues as activators of rat peritoneal mast cells. Planta Medica 4: 251–255. Perrot E. 1943–44. Matieres Premieres usuelles du Regne Vegetal. Paris: Masson, 1630–1632. Pujadas A, Rossello´ JA, Barcelo´ P. 1991. De flora balearica adnotationes (10). Thapsia gymnesica spec. nov. Candollea 46: 65–74.
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Rasmussen, U. 1979. Farmakognostiske underso gelser af slægten Thapsia L. Copenhagen: Unpublished Thesis, Royal Danish School of Pharmacy. Rasmussen U, Christensen SB, Sandberg F. 1981. Phytochemistry of the Genus Thapsia. Planta Medica 43: 336–341. Roth I. 1977. Fruits of Angiosperms. In: Linsbauer K, Tischler G, Pascher A eds. Handbuch der Pflanzenanatomie, Band X, Teil 1, Berlin—Stuttgart: Gebru¨der Borntraeger, 318. Smitt UW, Christensen SB. 1991. Nortrilobolid, a new potent guaianolide secretagogue from Thapsia garganica. Planta Medica 57: 196–197. Tutin TG. 1968. Thapsia L. In: Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walthers SM, Webb DA eds. Flora Europaea, vol. 2. Cambridge: Cambridge University Press, 370. Willkomm M, Lange J. 1880. Prodromus Florae Hispanicae, II. Stuttgart, 27.
Comparative studies in phytochemistry and fruit anatomy of Thapsia garganica and T. transtagana, Apiaceae (Umbelliferae) ¨ GER, ULLA WAGNER SMITT, ANNE KATHARINA JA ANNE ADSERSEN AND LENE GUDIKSEN Department of Pharmacognosy, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark Received November 1994, accepted for publication January 1995
Thapsia garganica L. and T. transtagana Brot. are classified as synonymous in Flora Europaea. In the present investigation significant differences between the two taxa with regard to fruit anatomy and phytochemistry are demonstrated and they are considered as two separate species. Microscopic studies revealed a distinct difference in number and position of secretory spaces in the pericarp of fruits from the two species and in addition pronounced differences were found in the presence of thapsigargins, the bioactive constituents of the two species. Quantitative HPLC analyses of thapsigargins were carried out on different plant organs from T. garganica and T. transtagana collected from various locations. Thapsigargin, thapsigargicin, nortrilobolid and thapsivillosin I and J were dominant compounds in all organs of T. garganica whereas none of these compounds could be detected in any organ of T. transtagana. On the contrary, thapsitranstagin and trilobolid were main thapsigargins of T. transtagana. T. garganica may include two chemotypes, as trilobolid was detected in some specimens of T. garganica only. As this is the first time trilobolid has been detected in these two species, it was isolated from both and identified by 1H NMR-spectroscopy. ADDITIONAL KEY WORDS:—chemotaxonomy – HPLC analysis – secretory spaces – sesquiterpene lactones – thapsigargins – trilobolid.
CONTENTS Introduction . . . . . . . . Material and methods . . . . . . Plant material . . . . . . . Determination of chromosome numbers . Anatomical investigation . . . . Quantitative HPLC analysis . . . Isolation of thapsigargins . . . . Results . . . . . . . . . Morphological and anatomical characters Phytochemistry . . . . . . Discussion . . . . . . . . . Acknowledgements . . . . . . . References . . . . . . . . .
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INTRODUCTION
An ancient remedy, Resina thapsiae, used against pulmonary diseases, catarrhs and rheumatic pains, was obtained from the roots of the Mediterranean plant 0024–4074/95/040281+12 $08.00/0
281
© 1995 The Linnean Society of London
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U. W. SMITT ET AL.
Thapsia garganica L. (French, 1971; Perrot, 1943–44). The resin, which is located in schizogenous secretory canals in the root bark, provokes a vigorous contact dermatitis expressed as erythema, itching and small vesiculae. As recently as the nineteenth century the resin was used in medicine, especially by the Arabs of north Africa, who named the resin Bou ne´fa (father of health), and the plant, De´rias. The resin of T. garganica and a medical plaster, Sparadrap de thapsia, has been included in the 1937 edition of the French pharmacopoeia. T. garganica has been described under several names and varieties in different floras (Rasmussen, 1979). In Flora Europaea, T. garganica, T. transtagana Brot. and T. decussata Lag. are classified as synonymous (Tutin, 1968). However, T. transtagana, described for the first time in Flora Lusitanica (Brotero, 1804), with Montemor o Novo and Serpa in Portugal cited as habitats, has hirsute leaves, in contrast to T. garganica. The petiole and nerves of the leaves of T. decussata (Lagasca, 1816; Willkomm & Lange, 1880), with habitats in Spain, are similarly described as hirsute. It has previously been shown that plants growing in Portugal identified as T. transtagana Brot. are phytochemically different from T. garganica (Rasmussen, Christensen & Sandberg, 1981). Previous phytochemical investigations of T. garganica have resulted in the isolation of five skin irritating compounds. The main constituents in the roots as well as in the ripe fruits are thapsigargin I and thapsigargicin II, whereas nortrilobolid III, thapsivillosin J IV and thapsivillosin I V were found as minor constituents. None of these compounds were detected in roots of T. transtagana, from which only thapsitranstagin VII has been isolated (Christensen et al., 1984; Smitt & Christensen, 1991). These compounds, named thapsigargins, are all non-cytotoxic histamine liberators (Christensen et al., 1982; Norup, Smitt & Christensen, 1986). Thapsigargin itself, acting as a Ca++-ATPase inhibitor, has become a valuable test compound of great demand in biochemical studies of calcium homeostasis (Christensen et al., 1992), and because it is still available only by isolation from wild growing plants of T. garganica, investigation of the diversity within this species and its possible synonym T. transtagana is of considerable interest. In this paper we present the results of comparative microscopic studies of fruits from T. garganica and T. transtagana as well as the results of quantitative HPLC analyses of thapsigargins in different organs of the two species, each collected at different locations. Furthermore, trilobolid VI, not previously detected in either of the two species, has now been isolated from the roots of T. garganica collected in Italy, and from the ripe fruits of T. transtagana.
MATERIAL AND METHODS
Plant material Unless otherwise cited, plant material of Thapsia garganica and T. transtagana was collected by one of the authors (UWS), in the following locations (see Fig. 1): T. transtagana. PORTUGAL: (1) c. 6 km south of Alter do Cha˜o road no. 245. (2) c. 5 km east of Evora road no. 254. (3) c. 5 km west of Ourique road no. EN 123. (4) c. 2 km east of Santa Luzia road no. 389. (5) Just outside Vilamoura, Algarve.
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Figure 1. Locations where T. transtagana 1–5 (W) and T. garganica 6–12 (R) have been collected.
T. garganica. (6) SPAIN: south end of S. Antonio Abad, Ibiza. (7) ALGERIA: c. 52 km south of Skikda road no. N 3. (8) TUNISIA: c. 7 km east of Beni Khalled road no. C 44. (9) ITALY: c. 7 km east of Carpino, Promontorio Del Gargano (collected by P. Avato). (10) LIBYA: c. 2 km west of Shahhat (collected by F. Sandberg). (11) GREECE: isle of Kalymnos. (12) isle of Samos (collected by F. Sandberg). Plants were at the stage of ripe fruits. Voucher specimens are deposited in the Department of Pharmacognosy, Royal Danish School of Pharmacy, Copenhagen.
Determination of chromosome numbers Chromosome numbers were determined on seedling root tips with the Feulgen squash method, after pretreatment with 0.1% colchicine for 1.5–2 h at 20– 25>C.
Anatomical investigation Cross sections of the middle part of the fruits were made with a razor and embedded in chloral hydrate solution. Microscope: Zeiss Axioplan; Film: Agfaortho 25.
Quantitative HPLC analysis HPLC analyses of thapsigargins were performed as described by Ja¨ger et al. (1993).
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Isolation of thapsigargins Dried, triturated roots or fruits were extracted three times with EtOAc and the combined extracts concentrated in vacuum to produce a brown gum. The gum was dissolved in CH2Cl2 and applied on a dry silica Woelm TSC column. Elution was performed with CH2Cl2 to which increasing amounts of EtOAc (5–100%) were added. Fractions containing thapsigargins were concentrated in vacuum and the residue was rechromatographed twice over silica gel 60 (Merck Art. 7754, 0.063–0.200 mm) with 10% water, using toluene containing increasing amounts of EtOAc, 16–20% and 12.5–20% respectively, as eluents. Final purification was performed by preparative HPLC; Column: Lichrosorb RP 18, 5 mm, 12×250 mm, Eluent: MeOH-H2O 83: 17, Flow 6 ml:min, Detection: 230 nm. Identification was based on comparison of the 1H NMR-spectra with that from authentic material previously isolated in our laboratory. RESULTS
Morphological and anatomical characters The morphological characters of Thapsia garganica are described in Flora Europaea (Tutin, 1968). Based on these characters alone it is hardly possible to distinguish between T. garganica and T. transtagana as leaf morphology is almost identical and the fruits are of the same size. However, in contrast to T. garganica, the leaves of T. transtagana are usually distinctly hairy, being especially hirsute on petiole and nerves, and the lobes are usually broader than those of T. garganica—the outer lobes being 3–10 (–15) mm and 1–5 mm, respectively (Figs 2, 3). The primordial leaves of both species are oval and entire, in contrast to the pinnate primordial leaves of other Thapsia species (Figs 2, 3). The chromosome number of both T. garganica and T. transtagana was determined as 2n 22 (2x). The apiaceous fruits are cremocarps with a characteristic structure. The number and position of the secretory spaces in the pericarp are of significant taxonomic value (Roth, 1977). Thapsia garganica and T. transtagana both contain two types of secretory spaces—vittae and companion canals associated with the vascular bundles. Microscopy of the transverse sections of the fruits from the two species showed a distinct difference in regard to the companion canals (Figs 4, 5). In T. garganica each vascular bundle is accompanied by one companion canal situated on the outer side of the bundle. In T. transtagana, on the contrary, each vascular bundle is accompanied by two companion canals, one on the outer and the other on the inner side of the vascular bundle. Phytochemistry Thapsia garganica L. Two major thapsigargins, I and II, have previously been isolated from the roots of T. garganica together with three minor constituents III, IV and V. Thapsigargins are present as constituents of the resin in all plant organs, although the highest concentrations are found in roots and in fruits (Fig. 6). The results of quantitative HPLC analyses of thapsigargins in roots and fruits of T. garganica collected at locations 6–12 (Fig. 1) are shown in Figure 7. The
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Figure 2. Leaves and fruits of T. garganica. Left: Basal leaf of mature plant. Right: A, Primordial leaf. B, Fourth leaf of plant germinated in greenhouse. C, Ripe fruits of different size.
total concentration of thapsigargins varied from 0.2–1.2% of the dry weight of the roots and from 0.7–1.5% of the ripe fruits. In dry leaves a total concentration of about 0.1% was detected, while in dry stems the total concentration was between 0.1–0.5%. The most notable results of the quantitative HPLC analyses are the relative concentrations of thapsigargins in plants from the different locations. While thapsigargin I, constituting 55–80%, is evidently the major component of plants from location 6, 7, 8 and 10, this does not apply to plants from location 9, 11 and 12, where I constitutes only 20–45%. In the latter plants no particular thapsigargins dominate. We have isolated trilobolid VI, not previously found in T. garganica, from roots collected in Italy location 9. This compound was detected by HPLC in
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Figure 3. Leaves and fruits of T. transtagana. Left: Basal leaf of mature plant. Right: A, Primordial leaf. B, Fourth leaf of plant germinated in greenhouse. C, Ripe fruits of different size.
plants from locations 11 and 12, i.e. it occurs in all plants with low concentration of thapsigargin I. The relative concentrations of the different thapsigargins show the same pattern in all organs of plants at the same location. In dried plant material, thapsigargins are very stable components. An old drug, Radix thapsiae, kept in our department since 1892, had a similar thapsigargin content to that of newly collected roots (Fig. 7). Thapsia transtagana Brot. In previous investigations of T. transtagana, thapsitranstagin VII was isolated from the roots, but until this investigation no thapsigargins have been isolated from the fruits. HPLC analyses of different organs of this species revealed the presence of
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Figure 4. Cross section of fruits from T. garganica showing companion canals only on the outside of the vascular bundles. A, Young unripe fruit. B, Ripe fruit. (cf. Fig. 5)
Figure 5. Cross section of fruits from T. transtagana showing companion canals both on the outside and on the inside of the vascular bundles. A, Young unripe fruit. B, Ripe fruit. (cf. Fig. 4)
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Figure 6. Thapsigargins isolated from Thapsia.
several thapsigargins. As in T. garganica the highest concentrations are stored in roots and in fruits. However, total concentrations are generally lower than in T. garganica and there are distinct dissimilarities between the constituents of the two species. Five (I–V) of the six components found in T. garganica were not detected in T. transtagana. HPLC chromatograms of the fractionated extracts from the fruits of T. transtagana showed several peaks, of which the Rt-values of the major ones corresponded to VI, VII, thapsivillosin K VIII and thapsivillosin B IX. These thapsigargins could also be detected in the fractionated root extracts. As VI and VII were the two major constituents of the fruits, they were isolated and identified by 1H NMR. Attempts to isolate VI from the roots did not result in a pure compound. From the 1H NMR-spectrum it is suggested to be a mixture of the two isomers VI and X. Quantitative HPLC analyses of thapsigargins in roots and fruits of T. transtagana collected at five
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Figure 7. Quantitative HPLC analyses of (A) fruits and (B) roots of T. garganica collected at locations 6–12 (see Fig. 1). 1892 indicates the old sample from the year 1892.
different locations in Portugal (Fig. 8) show that the relative concentration of the two major constituents VI and VII varies from place to place. The total concentration of thapsigargins, estimated as VII, is 0.3–0.5% in both roots and fruits, with the exception of fruits from location 1, which contain 0.8%. DISCUSSION
The results of this investigation show that Thapsia garganica and T. transtagana can be distinguished by means of anatomical and phytochemical characters. The most significant difference is the number and position of secretory spaces in the fruits. Before VI was isolated from the roots of T. garganica collected in Italy, it was believed that T. garganica contained only thapsigargins with a butanoic acid esterified to 0–8. Esterification with this acid has not been observed in compounds from T. transtagana. This difference was supported by
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Figure 8. Quantitative HPLC analyses of (A) fruits and (B) roots of T. transtagana collected at locations 1–5 (see Fig. 1). n.i.: not identified.
the detection of two proazulene guaianolides, one with a butanoyl the other with a 2-methylbutanoyl group at 0–8, isolated from T. garganica and T. transtagana respectively (Avato et al., 1993). However, indication of divergent biogenesis of the esterified guaianolides of the two species, is contested by the presence of VI in some plants of T. garganica. As these plants were found to have a fruit anatomy identical to T. garganica (Fig. 4), whereas the phytochemical investigations showed different composition of thapsigargins, we propose the classification of plants from locations 9, 11 and 12 as chemotypes of T. garganica. Thapsia garganica and T. transtagana are easily distinguished from other species of Thapsia due to their bigger fruits and entire linear leaflobes. They must be regarded as separate species as they have different fruit anatomy and composition of secondary metabolic constituents, and separate geographical distributions. T. transtagana is distributed in Portugal, south-west Spain and Morocco, areas where
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T. garganica has not been found. The authors consider that T. decussata may be identical to T. transtagana based on morphological descriptions and habitats. This investigation shows that for the biological test compound I, the highest concentrations (0.6–1.1% by dry weight), are obtained from T. garganica growing at locations 6, 7, 8 and 10. The chemotaxonomy of T. maxima has already been published (Avato, Jacobsen & Smitt, 1992) and the results from the chemotaxonomic studies of T. villosa will be published shortly. Thapsia gymnesica, described as a new species in 1991 (Pujadas, Rosello´ & Barcelo´, 1991), has been found in small populations only on the Balearic Islands, Mallorca and Minorca. It appears to be an intermediate of T. villosa and T. garganica. Its fruits are small as in T. villosa, whereas the leaves are more similar to those of T. garganica. HPLC analyses of the roots and fruits of T. gymnesica show the presence of compounds with the same Rt-values as I, II and III, compounds, which hitherto have been found only in T. garganica. Further studies on T. gymnesica are in preparation.
ACKNOWLEDGEMENTS
We are grateful to Prof. F. Sandberg, Biomedical Centre, Uppsala, Sweden, and to P. Avato, Universita` di Bari, Italy, for the supply of plant material and to S. B. Christensen, Royal Danish School of Pharmacy, Copenhagen, for recording the NMR-spectra. The Bruker AC-200 F instrument was a gift from the Velux Foundation of 1981 and the Torkil Steenbech Foundation.
REFERENCES Avato P, Jacobsen N, Smitt UW. 1992. Chemotaxonomy of Thapsia maxima Miller. Constituents of the essential oil of the fruits. The Journal of Essential Oil Research 4: 467–473. Avato P, Cornett C, Andersen A, Smitt UW, Christensen SB. 1993. Localization of the acyl groups in proazulene guaianolides from Thapsia transtagana Brot. and Thapsia garganica L. Journal of Natural Products 56: 411–415. Brotero FA. 1804. Flora Lusitanica, I. Olissipone, 468. Christensen SB, Larsen IK, Rasmussen U, Christophersen C. 1982. Thapsigargin and thapsigargicin, two histamine liberating sesquiterpene lactones from Thapsia garganica. X-ray analysis of the 7,11-epoxide of thapsigargin. Journal of Organic Chemistry 47: 649–652. Christensen SB, Norup E, Rasmussen U, Madsen JO š. 1984. Structure of histamine releasing guaianolides from Thapsia species. Phytochemistry. 23: 1659–1663. Christensen SB, Andersen A, Lauridsen A, Moldt P, Smitt UW, Thastrup O. 1992. Thapsigargine: A lead to design of drugs with the calcium pump as target. In: Krogsgaard-Larsen P, Christensen SB, Kofod H, eds. New leads and targets in drug research. Alfred Benzon Symposium 33. Copenhagen: Munksgaard, 243–252. French, DH. 1971. Ethnobotany of the Umbelliferae. In: Heywood VH, ed. The biology and chemistry of the Umbelliferae. London: Academic Press, 385–412. Ja¨ger AK, Gudiksen L, Adsersen A, Smitt UW. 1993. High performance liquid chromatography of thapsigargins. Journal of Chromatography 634: 135–137. Lagasca M. 1816. Genera et species plantarum, quae aut novae sunt, aut nondum recte cognoscuntur. Matriti, 12. Norup E, Smitt UW, Christensen SB. 1986. The potencies of thapsigargin and analogues as activators of rat peritoneal mast cells. Planta Medica 4: 251–255. Perrot E. 1943–44. Matieres Premieres usuelles du Regne Vegetal. Paris: Masson, 1630–1632. Pujadas A, Rossello´ JA, Barcelo´ P. 1991. De flora balearica adnotationes (10). Thapsia gymnesica spec. nov. Candollea 46: 65–74.
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Rasmussen, U. 1979. Farmakognostiske underso gelser af slægten Thapsia L. Copenhagen: Unpublished Thesis, Royal Danish School of Pharmacy. Rasmussen U, Christensen SB, Sandberg F. 1981. Phytochemistry of the Genus Thapsia. Planta Medica 43: 336–341. Roth I. 1977. Fruits of Angiosperms. In: Linsbauer K, Tischler G, Pascher A eds. Handbuch der Pflanzenanatomie, Band X, Teil 1, Berlin—Stuttgart: Gebru¨der Borntraeger, 318. Smitt UW, Christensen SB. 1991. Nortrilobolid, a new potent guaianolide secretagogue from Thapsia garganica. Planta Medica 57: 196–197. Tutin TG. 1968. Thapsia L. In: Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walthers SM, Webb DA eds. Flora Europaea, vol. 2. Cambridge: Cambridge University Press, 370. Willkomm M, Lange J. 1880. Prodromus Florae Hispanicae, II. Stuttgart, 27.