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A chemotaxonomic investigation of Thapsia villosa L., Apiaceae (Umbelliferae)
of the Linnean
BotanicalJournal
Society (1995),
7 19: 367-377.
With
5 figures
A chemotaxonomic investigation of Thapsia villosa L., Apiaceae (Umbelliferae) ULLA
WAGNER
SMITT
Department of Pharmacognosy, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2 700 Copenhagen, Denmark ReceivedJune
7995, acceptedforpublication
August 1995
Chemotaxonomic investigations of %@a villosa L., Apiaceae, have resulted in a division of the species into five different types, representing two distinctly different groups. Thapsigargins, the guaianolide sesquiterpene lactones with unique bioactivity, are present only in one of these groups, which includes two morphologically identical types, with different chromosome numbers, 2n=44 [4x] and 2n=66 [GX]. Besides thapsigargins, 6-methoxy-7geranyloxycoumarin and phenylpropanoids are characteristic constituents of the roots, and limonene and methyl eugenol are the two major constituents of the fruit essential oil. The other group with clearly different morphological characters includes three types with the chromosome numbers 2n=22 [xl, 2n=22 [2x] and 2n=44 [4x]. Different sesquiterpenes are the characteristic constituents of their roots, tovarol and hydroindene derivatives being present in all three types, whereas guaiane esters have been detected in only one of the diploid types. Geranyl acetate is the major constituent of the fruit essential oil of all three types within this group. C2 I!195 The
Linnean
ADDITIONAL sesquiterpenes
Society
of London
KEY WORDS:-chemotypes - sesquiterpene lactones - thapsigargins.
chromosome
number
ploidy
-
CONTENTS Introduction . . . Material and methods . Plant material. . . Chromosome numbers . TLC analysis of fruit volatiles TLC analysis of root extracts Results . . . . . Description of plants . . Phytochemistry . . Discussion . . Acknowledgements . References . . . .
. . .
. . .
. .
. . . .
. . . .
. . . .
367 369 369 370 370 370 370 370 373 376 377 373
. .
. . .
. .
.
.
INTRODUCTION
The resin from some species of the genus ZXapsia (Apiaceae, Laserpitiae) provokes a vigorous contact dermatitis. This reaction is due to the presence of different guaianolide sesquiterpene lactones, named thapsigargins, which induce a non-cytotoxic histamine release (Rasmussen, Christensen & Sandberg, 0024-4074/95/012367+11$12.00/0
367
8 1995 The Lirmean
Society
of London
368
U. W. SMIlT
1978; Rasmussen, 1979). One of the most potent compounds, thapsigargin, has been isolated from T. gargunica L. (Norup, Smitt & Christensen, 1986). Thapsiga,+ is a specific inhibitor of certain Ca*+-ATPases, and has become an important tool in biochemical studies of calcium homeostasis and cell activation (Christensen et al, 1992). The unique bioactivity of thapsigargins has contributed to the pharmacological importance of the genus 27upsia; the present chemotaxonomic study of i? uiUo.su was initiated when it was discovered that only some plants within this species contained thapsigargins (Rasmussen, Christensen & Sandberg, 1981). napsia villosa is one of three species within the genus according to Flora Europaea (Tutin, 1968): i? gurgunicu and T maxima Miller are the other two. In the same flora, T. transtuguna Brot. is classified as synonymous to T gurgunica. However, it has been shown that the two species have different anatomy of the fruits and contain different thapsigargins @mitt et al., 1995). T. maxima was found to include two morphological types, I and II, neither of which contain thapsigargins (Avato, Jacobsen & Smitt, 1992). Two species T. minorHoffgg. et Link (Hab.-Portugal: Beira transmontane, Beira centrale, Beira littorale, Beira mei-idionale, centre littoral and Alemtejo littoral) (Rouy, 1895a) and T. Zuciniuta Rouy (Hab.-Spain: prov. de Valencia: sierra de San-Filipe de Jativa) have previously been described as separate from T. villosu (Rouy, 189513). A number of varieties of T. villosu has also been described (Rasmussen, 1979). A variety of different sesquiterpenes and sesquiterpene lactones, along with other compounds, have been isolated from T. uillosu. This has disclosed a pronounced phytochemical heterogeneity within the species, and caused confusion in the naming of the investigated plants. The following thapsigargins, thapsivillosin A, B, C, D, E, F, G, H and trilobolid, have been isolated from the roots of some specimens of T. villosa together with 6-methoxy-7-geranyloxy-coumarin (Rasmussen, Christensen & Sandberg, 198 1; Christensen et aZ., 1984). Th ap sivillosin K, thapsitranstagin, four tovarol derivatives and further phenylpropanoids were isolated from T. uillosa L. var. uillosu (De Pascual Teresa et aZ., 1985a-c). Later it was reported that thapsigargins are found only in polyploid plants with the chromosome numbers 2n = 44 [4x] or 2n = 66 [6x] and that in some specimens of the hexaploid plants the amount of thapsigargins may be reduced and replaced by slovanolides, another type of guaianolide sesquiterpene lactone (Smitt et al, 199Oa). In the fruit, essential oils from these polyploid plants, methyl eugenol and limonene, are the two major constituents (Smitt et aZ., 1993). Of the above mentioned compounds only tovarol derivatives have also been detected in the roots from specimens of T. dlosa L. var. minor (De Pascual Teresa et aZ., 1985c; De Pascual Teresa et cd., 1986a). The most characteristic components of these plants and of diploid, 2n = 22 [2x], specimens of T. villosu are some hydroindene sesquiterpenes, named thapsans (Lemmich et al., 1984; De Pascual Teresa et aZ., 1986b, c; Smitt et al., 1990b). Whereas different guaiane esters were found only in some of the diploid specimens of T. doss (Lemmich et aZ., 1991). Also in contrast to the first mentioned polyploid plants, the main constituent of the fruit essential oil from the diploid plants is geranyl acetate (Smitt et cd., 1993). In .the present paper, the morphological characters and the chromosome numbers of five different types of T. villosu will be presented and correlated to the
CHEMOTAXONOMY
OF
369
ThYPSZA VELOSA
a
zy;
(ISmationr
of the different
types
of
Thapsiu villosa. Type
1 (O),
2 (0).
3 (o),
4 (A)
results of TLC analysis of root extracts and fruit volatiles of the five types, collected at different locations. Furthermore, the results of this chemotaxonomic evaluation of T. uiha will be discussed in relation to the chemotaxonomy of the whole genus. MATERIAL
AND
METHODS
Plant material Plant material of Thapsia villosa was collected by the author in the following locations (Fig. 1 first two figures indicate year of infection, last two are reference numbers for that year): Type 7 (2n = 22); PORTUGAL: 81.19, c. 13 km north of Loule road no. 396; 88.06, c. 6 km east of Castalo on the road to Setubal; 88.17, Sierra de Ossa, c. 14 km south of Estremoz road no. 381; 88.30, c. 20 km north of Monchique road no. 266. Type 2 (2-z = 22): PORTUGAL: 88.02, c. 5 km from Capo Espicel on the road no. 379 from Santana; 81.04, c. 4 km west of Tavira road no. E.N.270; 88.13, near the ruined castle south of Elvas; SPAIN: 87.12, c. 7 km from El Burgo road no. C 344 from Ronda; 87.36, c. 34 km north of Malaga road no. N 321; FRANCE: 90.01, c. 12 km from Frejus road no. N 7; 90.03,9 km north of Se Maxime road no. D 25. Type 3 (2n = 44): PORTUGAL:
88.26, c. 7 km west of Colares road no. 247.
Type 4 (2n = 44): PORTUGAL: 81.01, Praia da Falesia, Algarve; 8 1.02, near the beach c. 2 km east of Quarteria; 88.14, in vicinity of the ruined castle south of Elvas; 88.16, Sierra de Ossa, c. 7 km south of Estremoz road no. 381; 88.23, Forto do Oitavos near Capo Raso; 88.24, Capo de Rota; SPAIN: 87.05, c. 12 km west of Guadix road no. 324; 87.11, c. 7 km before El Burgo road no. C 344 from
370
U. W. SMI-IT
Ronda; 93.01, near the coast road along lake L’Albufera, south of Valencia; 93.02, c. 2 km south of Baiieres road no. C-3313 from Alcoy; 90.14, c. 2 km west of Calogne at the road from Romanya de la Selva. FRANCE: 90.13, between Canetplage and St. Cyprien-plage, east of Perpignan. Type 5 (2n = 66): PORTUGAL: 88.01, Capo Espichel; 88.09, c. 6 km south of Alter do Chao road no. 245,88.3 1 c. 15 km north of Monchique road no. 266; 8723, just before Aldeira d,o Bispo by the road from Nave; SPAIN: 87.03, c. 4 km south of Berja road no. C 3.5 1 to Adra; 87.15, c. 3 km north of Alhaurin de la Torre road no. 344 to Coin; 87.17, near Villaharta road no. N 432 to Cordoba; 87.19, c. 7 km from Cristobal, road no. 515 from Sequeros; 87.25, c. 1 km north of Villar de1 Ciervo; 87.26, c. 4 km south of Villamayor; 87.27, c. 15 km west of Salamanca road no. 517; 87.29, c. 1 km north of El Barranco road no. 403. Chromosome numbers Chromosome numbers were determined on seedling root tips with conventional Feulgen squash method, after pretreatment with 0.1% colchicine 1.5-2 h at 20-25°C. TLC analysis ofjkit
the for
uolatiles
Two or three fruits were triturated with 100 mg of silica containing 10% water, and the volatiles applied to a TLC plate (DC-Alufolien Silica gel 60 FYs.,, Merck art. 5554) in a TAS-oven (Stahl, 1970). The plate was eluted with hexan-ether 8 : 2, and visualized by spraying with vanillin-sulphuric acid and heating at 100°C for 5 min. Geranyl acetate and methyl eugenol were isolated from the essential oil of type 2 and type 4, respectively, following the method described by Kubeczka (1973). The IR spectra were identified with those of authentic material. Rf-values for geranyl acetate and methyl eugenol are 0.63 and 0.35, respectively. TLC analysis of root extracts Three hundred mg pulverized root was extracted with 3 ml EtOAc in an ultrasonic bath for 1 h. After centrifugation, 20 111of the supernatant was applied to a TLC plate (DC-Alufolien Silica gel 60 FZ5.&,Merck Art. 5554). Elution was performed in three different systems: CH,Cl,-EtOAc 19 : 1, toluen-EtOAc-MeOH 30 : 8 : 1 and hexan-EtOAc 7 : 2. The plates were visualized in UV-254 nm and W-365 nm as well as by spraying with 4N H,SO,, and heating for 5 min at 110°C. Compounds were identified by comparing the Rf values with those of reference material isolated previously in our laboratory. RESULTS
Description ofplants The investigated plants were classified as Thapsia villosa based on descriptions in Flora Europaea. However, based on morphological characters, chromosome number and secondary metabolites, the plants were divided into two distinctly
CHEMOTAXONOMY TAIU.
1. Chemotaxonomic
OF data
of the
number
Morphological characters
Characteristic constituents
2n= Height of plants (cm) Number of rays Size of fruits (mm) Width/length
of roots
Main constituents of fruit volatiles
1
types
Group Type
371
of
villosu
7kp~iu
1 2
Type
3
22
22
44
30-80 6-10 6-8 8-10
40-100 10-30 6-8 8-10
40-100 6-16 6-8 8-12
Type
+ +
Limonene Methyl eugenol Geranyl acetate
+ c: only
characteristic
+ + +
100-150 20-30 8-12 10-15
+ +
+
Group 4
44
+
Thapsigargins” Slovanolides” Phenylpropanoids 6-Methoxy-7geranyloxycoumarin Tovarol derivativesb Thapsans” Guaiane esters”
a: sesquiterpene lactones; b: sesquiterpenes; De Pascual Teresa el al. (1985c).
KKL.O.SA
different
Type Chromosome
TWSU
2 Type
5
66 100-150 20-30 8-12 IO-15
+ +
+ +c + +
+d
+d
+ +
+ +
+ for some
specimens;
d: according
to
different groups, group 1 including three types and group 2 including two types. The chemotaxonomic data are summarized in Table 1 and the morphological characters of the five types are shown in Figures 2-5. Group 7 includes the following three types: Type 1 (Fig. 2A, B) contains diploid plants (272 = 22 (2x)), 30-80 cm high, with 6-10 rays; the fruits are 6-8 mm wide and 8-10 mm long. These plants fit the description and illustration of T. minor Hoffgg. et Link (Rouy 1895). Type 2 (Fig. 2C, D) contains diploid plants as does type 1 and with fruits of similar size, but they are usually taller, 40-100 cm, and with bigger leaves. The umbel is hemispherical to spherical with lo-30 rays. Type 2 corresponds to the description and illustration of T Zaciniata Rouy (Rouy, 1895), except that the leaves, although hairy, cannot be described as woolly. Type 3 (Fig. 3) shows morphological characters similar to both types 1 and 2. However, the chromosome number is 2n = 44 (4x), plants are 40-100 cm high, with 6-16 rays and the-fruits are 6-8 mm wide and 8-12 mm long. Group 2 includes types 4 and 5 (Figs 4, 5), which are polyploids, with the chromosome numbers 272 = 44 (4x) and 2n = 66 (6x), respectively. Significant morphological characters distinguish these tetra- and hexaploid plants from those of group 1, whereas within the group they possess similar characters. Types 4 and 5 are taller, lOO-150(200) cm, and more stout. The leaves are larger and with broader lobes. Within both types there is pronounced variation in leaf morphology, as seen in Figures 4 and 5. The leaves are more similar to those of T. maxima (Avato, Jacobsen & Smitt, 1992) than to the other types of T. villosa. The umbel is globular with lo-40 (mostly 20-30) rays. The fruits are 8-12 mm wide and lo-15 mm long. T. villosa is distributed in south-east France, Spain (except the Balearic Islands), Portugal and north west Africa. Types 4 and 5 were the two most often found in Spain and Portugal. Types 1 and 3 were found only in Portugal.
372
u. w. SMITT
Figure 2. Thupiu vi&m 2n = 22 (2x). The two upper specimens are type 1 collected same location, 88.17, at different stages, flowering and fruiting. The two lower pictures type 2 collected at different locations, 88.02 and 88.13.
at the show
CHEMOTAXONOMY
OF ntQpsLQ VZLLOSA
373
Figure 3. Thupsia villosa 2n = 44 (4x), flowering plant and basal leaf of type 3, 88-26.
Phytochemzdy The five different types of Thapsia villosa were collected from the locations shown in Figure 1, and constituents of roots and fruits analysed by TLC. Also, in regard to secondary metabolites, distinct differences separate the two groups, comprising types l-3 and types 4 and 5. Group 7 TLC analysis of root extracts showed, as the dominant spot, the presence of 80-feruloyltovarol in all samples. Hydroindene sesquiterpenes (thapsans) were also present in all samples, and form a complex pattern of spots. Variations were observed both between as well as within the three types. The most prominent differenee was that guaiol and the three guaiane esters, [4S, 5S, 7S, KS]-8-pcoumaroyloxy1 (lo)-gu aien-1 l-01, [4S, 5S, 7S, 8S]-8-feruloyloxy-1( lo)--guaien-1 l01 and [4S, 5S, 7S, 8S]-8-senecioyloxy-1 (lo)-gu aien-1 l-01, could be detected in all samples within type 2, but not in any samples of types 1 and 3. The main constituents of the fruit essential oils were analysed by application of the volatiles on a TLC plate in a TAS-oven. Geranyl acetate was the major component of all samples within this group. Group 2 TLC analysis of the root extracts showed a pattern very different from group 1. The characteristic constituents were thapsigargins, 6-methoxy-7-geranyloxycoumarin and phenylpropanoids. Some variations were found in the content of thapsigargins. Trilobolid, which was not detected in most of the samples, occurred
374
U. W. SMI-M-
i -1
Figure 4. Thapsia different locations, . *
morphology.
villa. sa 2n = 44 (4x). Spheric cal umbel and leaves of type 4 collected at A and B: X8-14, C: 88.2 !3 and D: 87.05, showing variations in leaf
CHEMOTAXONOMY
Figure X8.09,
OF
THAPSLA
37.5
WUOSA
5. Thapsia uillosu 2n = 66 (6x). Leaves from type 5 collected at different B: 87-29, C: 88.01 and D: 87.03, showing variations in leaf morphology.
locations,
A:
376
U. W. SMIT-I’
as the main thapsigargin in two samples, 90.13 and 90.14. Slovanolides were detected only in minor amounts in most samples; however, in two samples (88.09 and 87.15) of the hexaploid plants they occurred in high amounts, while the amounts of thapsigargins were comparatively less. TLC analysis of the fruit volatiles also showed a significant variation between groups 1 and 2, as methyl eugenol was the most prominent spot in all samples of group 2. A total GC/MS analysis of the essential oil from all types will be presented elsewhere. It was interesting to discover that a similar analysis of the major constituents of essential oils could be undertaken on fruits from herbarium specimens that were more than 100 years old.
DISCUSSION
Th& heterogeneity within Thupsia villosu may be explained by the different chromosome numbers of the plants. However, this investigation has shown that, although pronounced phytochemical differences are found between the two diploid types of group 1 and the two polyploid types of group 2, the ploidy level seems to have a minor effect on the secondary metabolism of the different cytotypes within each group. Type 1 corresponds to the description and illustration of i? minor (Rouy, 1895a) and has also been found only in Portugal. Type 3, which was found only at one location in Portugal may be a polyploid originating from type 1. On the evidence of the description and the illustration of T. Zuciniuta (Rouy, 1895b), type 2 may be this species. However, the leaves are not as hairy, as described for T luciniatu, and the habitat of T. Zaciniuta, Provence de Valencia, Spain, corresponds to that of a very hairy variety of type 5 (Fig. 5D). A chemotaxonomic study of T. maxima has recently been published (Avato et aZ., 1992). Two phenotypes were described and the major components of the essential oil of both types were limonene (27-34%) and methyl eugenol (59-63%). Both with regard to the constituents of the fruit essential oil and to the morphology of the leaves, there is a great resemblance between T. maxima and the two polyploid types, 4 and 5, of T. villosa, which also contain limonene and methyl eugenol as the main constituents, together composing 80-90% of the oil (Smitt et aZ., 1993). Sesquiterpene lactones have been detected in T maximu, but not of the thapsigargin type. Therefore, types 4 and 5 of T. villosa should be regarded as a separate taxon, of two cytological types, and most probably originating from T. maxima. The types from which the compounds under discussion have previously been isolated in our laboratory by U. W. Smitt (formerly U. Rasmussan) or by P. Avato, are apparent from the locations where they were collected. T. garganicu and T transtuguna are easily distinguished from the other i’Xap.siu species due to their larger fruits and entire linear leaf lobes, whereas, the newly described species, T gymnesicu Rossello & Pujadas appears to be an intermediate with fruits of the same size as T. villosa and T. maxima, but with leaves more similar to those of T gurganicu and T transtuguna (Pujadas, Rossello & Barcelo, 1991). The results of the present and previous chemotaxonomic investigations of Zhpsiu, species will hopefully lead to a much needed taxonomic revision of the genus Thapsia.
CHEMOTAXONOMY
OF Zi%l?SL4
UOSA
377
ACKNOWLEDGEMENTS
The author is most grateful to Prof. F. Sandberg, Biomedical Centre, Uppsala, Sweden, who was the initiator of this project and to Prof. N. Jacobsen, Department of Botany, Royal Veterinary and Agricultural University, Copenhagen, Denmark, for fruitful discussions and advice during the work. The plant collecting was supporting financially by the Danish Natural Science Research Council, H. C. Orsteds Foundation and by the Danish State Biotechnology Program. REFERENCES Avato P, Jacobsen N, Smitt UW. 1992. Chemotaxonomy of Thapsia maxima Miller. Constituents of the essential oil of the fruits. Zke Journal ofEssential Oil Research 4: 467-473. Christensen SB, NONP E, Rasmussen U, 0gaard Madsen J. 1984. Structure of histamine releasing guaianolides from Thapsa species. Phytochemistty 23: 1659-1663. Christensen SB, Andersen A, Lauridsen A, Moldt P, Smitt UW, Thastmp 0.1992. Thapsigargin: A lead to design of drugs with the calcium pump as target. In: Krogsgaard-Larsen P, Christensen SB, Kofod, H, eds. New lea& and targets in drug research. Alfred Benzon Symposium 33. Copenhagen: Munksgaard, 243-252. De Pascual Teresa J, De Pascual M, Arias A, Hem%ndezJM, MortinJR, Grande M. 1985a. Helmanticine, a phenylpropanoid from Thapsia villosa. Phytochemistty 24: 1773- 1778. De Pascual TeresaJ, Mor&m Hem&.ndezJM, Grande M. 198513. Phenylpropanoids and other derivatives from Thapsia villosa. Phytochembtty 24: 2071-2074. De Pascual Teresa J, Mor&n JR, Hem&ndez JM, Grande M. 1985c Tovarol and other germacrane derivatives from K’uzpsia villosa. Phytochemistv 24: 1779-l 783. De Pascual Teresa J, Mor&nJR, Hem%ndezJM, Grande M. 1986a. 12-Hydroxytovarol and derivatives from Thapsia villosa var. minor. Phytochemistry 25: 1167-l 170. De Pascual Teresa J, MortinJR, Fem&ndez A, Grande M. 1986b. Hemiacetalic thapsane derivatives from Thapsia villosa var. minor. Phytochemistry 25: 703-709. De Pascual Teresa J, Mor&n.nJR, Fem%ndez A, Grande M. 1986c Non-acetalic thapsane sesquiterpenoids from lkzpsia villosa var. minor. Phylochemislty 25: 117 l-l 174. Kubeczka KH. 1973. Pre-separation of essential oils and similar complex substance mixtures for GC-analysis by means of modified dry-column chromatography. Chromalographia 6, no. 2: 106-108. Lemmich E, Jensen B, Rasmussen U. 1984. (8R,lSS)-8-Angeloylthapsan-14-01, a sesquiterpene with a novel carbon skeleton, from Thapsia villosa. Phylochemisby 23: 809-8 11. Lemmich E, Smitt UW, Jensen JS, Christensen SB. 1991. G uaiane esters from napsia villosa. Phytochem&y 30: 2987-2990. Nomp E, Smitt UW, Christensen SB. 1986. The potencies of thapsigargin and analogues as activators of rat peritonial mast cells. Planta Medica 4: 25 l-255. Pujadas A, RosseIl6 AJ, Ba.rcel6 P. 1991. De flora balearica adnotationes (10). spec. nov. Candollea 46: 657.5. .-. Rasmussen U, Christensen SB, Sandberg F. 1978. Thapsigargin and thapsigargicin, two new histamine liberators from nabsia parpanica L. Acla Pharmaceutics Sue&a 15: 133-140. Rasmussen U. 1979. ‘F&sognostiske underssgelser of skegten Thapsia L.. Copenhagen: Unpublished Thesis, Royal Danish School of Pharmacy. Rasmussen U, Christensen SB, Sandberg F. 1981. Phytochemistry of the genus Thapsia. Plaata Medica 43: 336-343. Rouy G. 1895a. Illurtrationes Piantarum Europe Rariorum, Fas. I, Paris, 37. . Rouy G. 189513. Illustraliones Plan&urn Europe Rariorum, Fas. I, Paris, 142. Smitt UW, Comett C, Andersen A, Christensen SB, Avato P. 1990% New proazulene guaianolides from Thajka villosa. Journal ofNatural Products53: 1479-1484. Smitt UW, Comett C, NONP E, Christensen SB. 1990b. Novel hydroindene sesquiterpenes from Thapsia villosa. Phytochemislry 29: 873-875. Smitt UW, Avato P, Hvolby K, Jacobsen U. 1993. Constitution of the essential oils of %psia villosa. Planta Medica 59 (Suppl. Issue): A 603. Smitt UW,Jiiger AK, Adsersen A, Gudiksen L. 1995. Comparative studies in phytochemistry and tit anatomy of Thapsia garganica and T. lranrtagana, Apiaceae (Umbelliferae). Botanical Journal of the Linnean Society 117: 281-292. Stahl E. 1970. Chromalographische und mikrospopicche analyse von drop. Stuttgart: Gustav Fischer Verlag, 2226. Tutin TG. 1968. Thapsia L. In: Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walters SM, Webb DA, eds. Flora Europaea, vol. 2. Cambridge: Cambridge University Press, 370.
BotanicalJournal
Society (1995),
7 19: 367-377.
With
5 figures
A chemotaxonomic investigation of Thapsia villosa L., Apiaceae (Umbelliferae) ULLA
WAGNER
SMITT
Department of Pharmacognosy, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2 700 Copenhagen, Denmark ReceivedJune
7995, acceptedforpublication
August 1995
Chemotaxonomic investigations of %@a villosa L., Apiaceae, have resulted in a division of the species into five different types, representing two distinctly different groups. Thapsigargins, the guaianolide sesquiterpene lactones with unique bioactivity, are present only in one of these groups, which includes two morphologically identical types, with different chromosome numbers, 2n=44 [4x] and 2n=66 [GX]. Besides thapsigargins, 6-methoxy-7geranyloxycoumarin and phenylpropanoids are characteristic constituents of the roots, and limonene and methyl eugenol are the two major constituents of the fruit essential oil. The other group with clearly different morphological characters includes three types with the chromosome numbers 2n=22 [xl, 2n=22 [2x] and 2n=44 [4x]. Different sesquiterpenes are the characteristic constituents of their roots, tovarol and hydroindene derivatives being present in all three types, whereas guaiane esters have been detected in only one of the diploid types. Geranyl acetate is the major constituent of the fruit essential oil of all three types within this group. C2 I!195 The
Linnean
ADDITIONAL sesquiterpenes
Society
of London
KEY WORDS:-chemotypes - sesquiterpene lactones - thapsigargins.
chromosome
number
ploidy
-
CONTENTS Introduction . . . Material and methods . Plant material. . . Chromosome numbers . TLC analysis of fruit volatiles TLC analysis of root extracts Results . . . . . Description of plants . . Phytochemistry . . Discussion . . Acknowledgements . References . . . .
. . .
. . .
. .
. . . .
. . . .
. . . .
367 369 369 370 370 370 370 370 373 376 377 373
. .
. . .
. .
.
.
INTRODUCTION
The resin from some species of the genus ZXapsia (Apiaceae, Laserpitiae) provokes a vigorous contact dermatitis. This reaction is due to the presence of different guaianolide sesquiterpene lactones, named thapsigargins, which induce a non-cytotoxic histamine release (Rasmussen, Christensen & Sandberg, 0024-4074/95/012367+11$12.00/0
367
8 1995 The Lirmean
Society
of London
368
U. W. SMIlT
1978; Rasmussen, 1979). One of the most potent compounds, thapsigargin, has been isolated from T. gargunica L. (Norup, Smitt & Christensen, 1986). Thapsiga,+ is a specific inhibitor of certain Ca*+-ATPases, and has become an important tool in biochemical studies of calcium homeostasis and cell activation (Christensen et al, 1992). The unique bioactivity of thapsigargins has contributed to the pharmacological importance of the genus 27upsia; the present chemotaxonomic study of i? uiUo.su was initiated when it was discovered that only some plants within this species contained thapsigargins (Rasmussen, Christensen & Sandberg, 1981). napsia villosa is one of three species within the genus according to Flora Europaea (Tutin, 1968): i? gurgunicu and T maxima Miller are the other two. In the same flora, T. transtuguna Brot. is classified as synonymous to T gurgunica. However, it has been shown that the two species have different anatomy of the fruits and contain different thapsigargins @mitt et al., 1995). T. maxima was found to include two morphological types, I and II, neither of which contain thapsigargins (Avato, Jacobsen & Smitt, 1992). Two species T. minorHoffgg. et Link (Hab.-Portugal: Beira transmontane, Beira centrale, Beira littorale, Beira mei-idionale, centre littoral and Alemtejo littoral) (Rouy, 1895a) and T. Zuciniuta Rouy (Hab.-Spain: prov. de Valencia: sierra de San-Filipe de Jativa) have previously been described as separate from T. villosu (Rouy, 189513). A number of varieties of T. villosu has also been described (Rasmussen, 1979). A variety of different sesquiterpenes and sesquiterpene lactones, along with other compounds, have been isolated from T. uillosu. This has disclosed a pronounced phytochemical heterogeneity within the species, and caused confusion in the naming of the investigated plants. The following thapsigargins, thapsivillosin A, B, C, D, E, F, G, H and trilobolid, have been isolated from the roots of some specimens of T. villosa together with 6-methoxy-7-geranyloxy-coumarin (Rasmussen, Christensen & Sandberg, 198 1; Christensen et aZ., 1984). Th ap sivillosin K, thapsitranstagin, four tovarol derivatives and further phenylpropanoids were isolated from T. uillosa L. var. uillosu (De Pascual Teresa et aZ., 1985a-c). Later it was reported that thapsigargins are found only in polyploid plants with the chromosome numbers 2n = 44 [4x] or 2n = 66 [6x] and that in some specimens of the hexaploid plants the amount of thapsigargins may be reduced and replaced by slovanolides, another type of guaianolide sesquiterpene lactone (Smitt et al, 199Oa). In the fruit, essential oils from these polyploid plants, methyl eugenol and limonene, are the two major constituents (Smitt et aZ., 1993). Of the above mentioned compounds only tovarol derivatives have also been detected in the roots from specimens of T. dlosa L. var. minor (De Pascual Teresa et aZ., 1985c; De Pascual Teresa et cd., 1986a). The most characteristic components of these plants and of diploid, 2n = 22 [2x], specimens of T. villosu are some hydroindene sesquiterpenes, named thapsans (Lemmich et al., 1984; De Pascual Teresa et aZ., 1986b, c; Smitt et al., 1990b). Whereas different guaiane esters were found only in some of the diploid specimens of T. doss (Lemmich et aZ., 1991). Also in contrast to the first mentioned polyploid plants, the main constituent of the fruit essential oil from the diploid plants is geranyl acetate (Smitt et cd., 1993). In .the present paper, the morphological characters and the chromosome numbers of five different types of T. villosu will be presented and correlated to the
CHEMOTAXONOMY
OF
369
ThYPSZA VELOSA
a
zy;
(ISmationr
of the different
types
of
Thapsiu villosa. Type
1 (O),
2 (0).
3 (o),
4 (A)
results of TLC analysis of root extracts and fruit volatiles of the five types, collected at different locations. Furthermore, the results of this chemotaxonomic evaluation of T. uiha will be discussed in relation to the chemotaxonomy of the whole genus. MATERIAL
AND
METHODS
Plant material Plant material of Thapsia villosa was collected by the author in the following locations (Fig. 1 first two figures indicate year of infection, last two are reference numbers for that year): Type 7 (2n = 22); PORTUGAL: 81.19, c. 13 km north of Loule road no. 396; 88.06, c. 6 km east of Castalo on the road to Setubal; 88.17, Sierra de Ossa, c. 14 km south of Estremoz road no. 381; 88.30, c. 20 km north of Monchique road no. 266. Type 2 (2-z = 22): PORTUGAL: 88.02, c. 5 km from Capo Espicel on the road no. 379 from Santana; 81.04, c. 4 km west of Tavira road no. E.N.270; 88.13, near the ruined castle south of Elvas; SPAIN: 87.12, c. 7 km from El Burgo road no. C 344 from Ronda; 87.36, c. 34 km north of Malaga road no. N 321; FRANCE: 90.01, c. 12 km from Frejus road no. N 7; 90.03,9 km north of Se Maxime road no. D 25. Type 3 (2n = 44): PORTUGAL:
88.26, c. 7 km west of Colares road no. 247.
Type 4 (2n = 44): PORTUGAL: 81.01, Praia da Falesia, Algarve; 8 1.02, near the beach c. 2 km east of Quarteria; 88.14, in vicinity of the ruined castle south of Elvas; 88.16, Sierra de Ossa, c. 7 km south of Estremoz road no. 381; 88.23, Forto do Oitavos near Capo Raso; 88.24, Capo de Rota; SPAIN: 87.05, c. 12 km west of Guadix road no. 324; 87.11, c. 7 km before El Burgo road no. C 344 from
370
U. W. SMI-IT
Ronda; 93.01, near the coast road along lake L’Albufera, south of Valencia; 93.02, c. 2 km south of Baiieres road no. C-3313 from Alcoy; 90.14, c. 2 km west of Calogne at the road from Romanya de la Selva. FRANCE: 90.13, between Canetplage and St. Cyprien-plage, east of Perpignan. Type 5 (2n = 66): PORTUGAL: 88.01, Capo Espichel; 88.09, c. 6 km south of Alter do Chao road no. 245,88.3 1 c. 15 km north of Monchique road no. 266; 8723, just before Aldeira d,o Bispo by the road from Nave; SPAIN: 87.03, c. 4 km south of Berja road no. C 3.5 1 to Adra; 87.15, c. 3 km north of Alhaurin de la Torre road no. 344 to Coin; 87.17, near Villaharta road no. N 432 to Cordoba; 87.19, c. 7 km from Cristobal, road no. 515 from Sequeros; 87.25, c. 1 km north of Villar de1 Ciervo; 87.26, c. 4 km south of Villamayor; 87.27, c. 15 km west of Salamanca road no. 517; 87.29, c. 1 km north of El Barranco road no. 403. Chromosome numbers Chromosome numbers were determined on seedling root tips with conventional Feulgen squash method, after pretreatment with 0.1% colchicine 1.5-2 h at 20-25°C. TLC analysis ofjkit
the for
uolatiles
Two or three fruits were triturated with 100 mg of silica containing 10% water, and the volatiles applied to a TLC plate (DC-Alufolien Silica gel 60 FYs.,, Merck art. 5554) in a TAS-oven (Stahl, 1970). The plate was eluted with hexan-ether 8 : 2, and visualized by spraying with vanillin-sulphuric acid and heating at 100°C for 5 min. Geranyl acetate and methyl eugenol were isolated from the essential oil of type 2 and type 4, respectively, following the method described by Kubeczka (1973). The IR spectra were identified with those of authentic material. Rf-values for geranyl acetate and methyl eugenol are 0.63 and 0.35, respectively. TLC analysis of root extracts Three hundred mg pulverized root was extracted with 3 ml EtOAc in an ultrasonic bath for 1 h. After centrifugation, 20 111of the supernatant was applied to a TLC plate (DC-Alufolien Silica gel 60 FZ5.&,Merck Art. 5554). Elution was performed in three different systems: CH,Cl,-EtOAc 19 : 1, toluen-EtOAc-MeOH 30 : 8 : 1 and hexan-EtOAc 7 : 2. The plates were visualized in UV-254 nm and W-365 nm as well as by spraying with 4N H,SO,, and heating for 5 min at 110°C. Compounds were identified by comparing the Rf values with those of reference material isolated previously in our laboratory. RESULTS
Description ofplants The investigated plants were classified as Thapsia villosa based on descriptions in Flora Europaea. However, based on morphological characters, chromosome number and secondary metabolites, the plants were divided into two distinctly
CHEMOTAXONOMY TAIU.
1. Chemotaxonomic
OF data
of the
number
Morphological characters
Characteristic constituents
2n= Height of plants (cm) Number of rays Size of fruits (mm) Width/length
of roots
Main constituents of fruit volatiles
1
types
Group Type
371
of
villosu
7kp~iu
1 2
Type
3
22
22
44
30-80 6-10 6-8 8-10
40-100 10-30 6-8 8-10
40-100 6-16 6-8 8-12
Type
+ +
Limonene Methyl eugenol Geranyl acetate
+ c: only
characteristic
+ + +
100-150 20-30 8-12 10-15
+ +
+
Group 4
44
+
Thapsigargins” Slovanolides” Phenylpropanoids 6-Methoxy-7geranyloxycoumarin Tovarol derivativesb Thapsans” Guaiane esters”
a: sesquiterpene lactones; b: sesquiterpenes; De Pascual Teresa el al. (1985c).
KKL.O.SA
different
Type Chromosome
TWSU
2 Type
5
66 100-150 20-30 8-12 IO-15
+ +
+ +c + +
+d
+d
+ +
+ +
+ for some
specimens;
d: according
to
different groups, group 1 including three types and group 2 including two types. The chemotaxonomic data are summarized in Table 1 and the morphological characters of the five types are shown in Figures 2-5. Group 7 includes the following three types: Type 1 (Fig. 2A, B) contains diploid plants (272 = 22 (2x)), 30-80 cm high, with 6-10 rays; the fruits are 6-8 mm wide and 8-10 mm long. These plants fit the description and illustration of T. minor Hoffgg. et Link (Rouy 1895). Type 2 (Fig. 2C, D) contains diploid plants as does type 1 and with fruits of similar size, but they are usually taller, 40-100 cm, and with bigger leaves. The umbel is hemispherical to spherical with lo-30 rays. Type 2 corresponds to the description and illustration of T Zaciniata Rouy (Rouy, 1895), except that the leaves, although hairy, cannot be described as woolly. Type 3 (Fig. 3) shows morphological characters similar to both types 1 and 2. However, the chromosome number is 2n = 44 (4x), plants are 40-100 cm high, with 6-16 rays and the-fruits are 6-8 mm wide and 8-12 mm long. Group 2 includes types 4 and 5 (Figs 4, 5), which are polyploids, with the chromosome numbers 272 = 44 (4x) and 2n = 66 (6x), respectively. Significant morphological characters distinguish these tetra- and hexaploid plants from those of group 1, whereas within the group they possess similar characters. Types 4 and 5 are taller, lOO-150(200) cm, and more stout. The leaves are larger and with broader lobes. Within both types there is pronounced variation in leaf morphology, as seen in Figures 4 and 5. The leaves are more similar to those of T. maxima (Avato, Jacobsen & Smitt, 1992) than to the other types of T. villosa. The umbel is globular with lo-40 (mostly 20-30) rays. The fruits are 8-12 mm wide and lo-15 mm long. T. villosa is distributed in south-east France, Spain (except the Balearic Islands), Portugal and north west Africa. Types 4 and 5 were the two most often found in Spain and Portugal. Types 1 and 3 were found only in Portugal.
372
u. w. SMITT
Figure 2. Thupiu vi&m 2n = 22 (2x). The two upper specimens are type 1 collected same location, 88.17, at different stages, flowering and fruiting. The two lower pictures type 2 collected at different locations, 88.02 and 88.13.
at the show
CHEMOTAXONOMY
OF ntQpsLQ VZLLOSA
373
Figure 3. Thupsia villosa 2n = 44 (4x), flowering plant and basal leaf of type 3, 88-26.
Phytochemzdy The five different types of Thapsia villosa were collected from the locations shown in Figure 1, and constituents of roots and fruits analysed by TLC. Also, in regard to secondary metabolites, distinct differences separate the two groups, comprising types l-3 and types 4 and 5. Group 7 TLC analysis of root extracts showed, as the dominant spot, the presence of 80-feruloyltovarol in all samples. Hydroindene sesquiterpenes (thapsans) were also present in all samples, and form a complex pattern of spots. Variations were observed both between as well as within the three types. The most prominent differenee was that guaiol and the three guaiane esters, [4S, 5S, 7S, KS]-8-pcoumaroyloxy1 (lo)-gu aien-1 l-01, [4S, 5S, 7S, 8S]-8-feruloyloxy-1( lo)--guaien-1 l01 and [4S, 5S, 7S, 8S]-8-senecioyloxy-1 (lo)-gu aien-1 l-01, could be detected in all samples within type 2, but not in any samples of types 1 and 3. The main constituents of the fruit essential oils were analysed by application of the volatiles on a TLC plate in a TAS-oven. Geranyl acetate was the major component of all samples within this group. Group 2 TLC analysis of the root extracts showed a pattern very different from group 1. The characteristic constituents were thapsigargins, 6-methoxy-7-geranyloxycoumarin and phenylpropanoids. Some variations were found in the content of thapsigargins. Trilobolid, which was not detected in most of the samples, occurred
374
U. W. SMI-M-
i -1
Figure 4. Thapsia different locations, . *
morphology.
villa. sa 2n = 44 (4x). Spheric cal umbel and leaves of type 4 collected at A and B: X8-14, C: 88.2 !3 and D: 87.05, showing variations in leaf
CHEMOTAXONOMY
Figure X8.09,
OF
THAPSLA
37.5
WUOSA
5. Thapsia uillosu 2n = 66 (6x). Leaves from type 5 collected at different B: 87-29, C: 88.01 and D: 87.03, showing variations in leaf morphology.
locations,
A:
376
U. W. SMIT-I’
as the main thapsigargin in two samples, 90.13 and 90.14. Slovanolides were detected only in minor amounts in most samples; however, in two samples (88.09 and 87.15) of the hexaploid plants they occurred in high amounts, while the amounts of thapsigargins were comparatively less. TLC analysis of the fruit volatiles also showed a significant variation between groups 1 and 2, as methyl eugenol was the most prominent spot in all samples of group 2. A total GC/MS analysis of the essential oil from all types will be presented elsewhere. It was interesting to discover that a similar analysis of the major constituents of essential oils could be undertaken on fruits from herbarium specimens that were more than 100 years old.
DISCUSSION
Th& heterogeneity within Thupsia villosu may be explained by the different chromosome numbers of the plants. However, this investigation has shown that, although pronounced phytochemical differences are found between the two diploid types of group 1 and the two polyploid types of group 2, the ploidy level seems to have a minor effect on the secondary metabolism of the different cytotypes within each group. Type 1 corresponds to the description and illustration of i? minor (Rouy, 1895a) and has also been found only in Portugal. Type 3, which was found only at one location in Portugal may be a polyploid originating from type 1. On the evidence of the description and the illustration of T. Zuciniuta (Rouy, 1895b), type 2 may be this species. However, the leaves are not as hairy, as described for T luciniatu, and the habitat of T. Zaciniuta, Provence de Valencia, Spain, corresponds to that of a very hairy variety of type 5 (Fig. 5D). A chemotaxonomic study of T. maxima has recently been published (Avato et aZ., 1992). Two phenotypes were described and the major components of the essential oil of both types were limonene (27-34%) and methyl eugenol (59-63%). Both with regard to the constituents of the fruit essential oil and to the morphology of the leaves, there is a great resemblance between T. maxima and the two polyploid types, 4 and 5, of T. villosa, which also contain limonene and methyl eugenol as the main constituents, together composing 80-90% of the oil (Smitt et aZ., 1993). Sesquiterpene lactones have been detected in T maximu, but not of the thapsigargin type. Therefore, types 4 and 5 of T. villosa should be regarded as a separate taxon, of two cytological types, and most probably originating from T. maxima. The types from which the compounds under discussion have previously been isolated in our laboratory by U. W. Smitt (formerly U. Rasmussan) or by P. Avato, are apparent from the locations where they were collected. T. garganicu and T transtuguna are easily distinguished from the other i’Xap.siu species due to their larger fruits and entire linear leaf lobes, whereas, the newly described species, T gymnesicu Rossello & Pujadas appears to be an intermediate with fruits of the same size as T. villosa and T. maxima, but with leaves more similar to those of T gurganicu and T transtuguna (Pujadas, Rossello & Barcelo, 1991). The results of the present and previous chemotaxonomic investigations of Zhpsiu, species will hopefully lead to a much needed taxonomic revision of the genus Thapsia.
CHEMOTAXONOMY
OF Zi%l?SL4
UOSA
377
ACKNOWLEDGEMENTS
The author is most grateful to Prof. F. Sandberg, Biomedical Centre, Uppsala, Sweden, who was the initiator of this project and to Prof. N. Jacobsen, Department of Botany, Royal Veterinary and Agricultural University, Copenhagen, Denmark, for fruitful discussions and advice during the work. The plant collecting was supporting financially by the Danish Natural Science Research Council, H. C. Orsteds Foundation and by the Danish State Biotechnology Program. REFERENCES Avato P, Jacobsen N, Smitt UW. 1992. Chemotaxonomy of Thapsia maxima Miller. Constituents of the essential oil of the fruits. Zke Journal ofEssential Oil Research 4: 467-473. Christensen SB, NONP E, Rasmussen U, 0gaard Madsen J. 1984. Structure of histamine releasing guaianolides from Thapsa species. Phytochemistty 23: 1659-1663. Christensen SB, Andersen A, Lauridsen A, Moldt P, Smitt UW, Thastmp 0.1992. Thapsigargin: A lead to design of drugs with the calcium pump as target. In: Krogsgaard-Larsen P, Christensen SB, Kofod, H, eds. New lea& and targets in drug research. Alfred Benzon Symposium 33. Copenhagen: Munksgaard, 243-252. De Pascual Teresa J, De Pascual M, Arias A, Hem%ndezJM, MortinJR, Grande M. 1985a. Helmanticine, a phenylpropanoid from Thapsia villosa. Phytochemistty 24: 1773- 1778. De Pascual TeresaJ, Mor&m Hem&.ndezJM, Grande M. 198513. Phenylpropanoids and other derivatives from Thapsia villosa. Phytochembtty 24: 2071-2074. De Pascual Teresa J, Mor&n JR, Hem&ndez JM, Grande M. 1985c Tovarol and other germacrane derivatives from K’uzpsia villosa. Phytochemistv 24: 1779-l 783. De Pascual Teresa J, Mor&nJR, Hem%ndezJM, Grande M. 1986a. 12-Hydroxytovarol and derivatives from Thapsia villosa var. minor. Phytochemistry 25: 1167-l 170. De Pascual Teresa J, MortinJR, Fem&ndez A, Grande M. 1986b. Hemiacetalic thapsane derivatives from Thapsia villosa var. minor. Phytochemistry 25: 703-709. De Pascual Teresa J, Mor&n.nJR, Fem%ndez A, Grande M. 1986c Non-acetalic thapsane sesquiterpenoids from lkzpsia villosa var. minor. Phylochemislty 25: 117 l-l 174. Kubeczka KH. 1973. Pre-separation of essential oils and similar complex substance mixtures for GC-analysis by means of modified dry-column chromatography. Chromalographia 6, no. 2: 106-108. Lemmich E, Jensen B, Rasmussen U. 1984. (8R,lSS)-8-Angeloylthapsan-14-01, a sesquiterpene with a novel carbon skeleton, from Thapsia villosa. Phylochemisby 23: 809-8 11. Lemmich E, Smitt UW, Jensen JS, Christensen SB. 1991. G uaiane esters from napsia villosa. Phytochem&y 30: 2987-2990. Nomp E, Smitt UW, Christensen SB. 1986. The potencies of thapsigargin and analogues as activators of rat peritonial mast cells. Planta Medica 4: 25 l-255. Pujadas A, RosseIl6 AJ, Ba.rcel6 P. 1991. De flora balearica adnotationes (10). spec. nov. Candollea 46: 657.5. .-. Rasmussen U, Christensen SB, Sandberg F. 1978. Thapsigargin and thapsigargicin, two new histamine liberators from nabsia parpanica L. Acla Pharmaceutics Sue&a 15: 133-140. Rasmussen U. 1979. ‘F&sognostiske underssgelser of skegten Thapsia L.. Copenhagen: Unpublished Thesis, Royal Danish School of Pharmacy. Rasmussen U, Christensen SB, Sandberg F. 1981. Phytochemistry of the genus Thapsia. Plaata Medica 43: 336-343. Rouy G. 1895a. Illurtrationes Piantarum Europe Rariorum, Fas. I, Paris, 37. . Rouy G. 189513. Illustraliones Plan&urn Europe Rariorum, Fas. I, Paris, 142. Smitt UW, Comett C, Andersen A, Christensen SB, Avato P. 1990% New proazulene guaianolides from Thajka villosa. Journal ofNatural Products53: 1479-1484. Smitt UW, Comett C, NONP E, Christensen SB. 1990b. Novel hydroindene sesquiterpenes from Thapsia villosa. Phytochemislry 29: 873-875. Smitt UW, Avato P, Hvolby K, Jacobsen U. 1993. Constitution of the essential oils of %psia villosa. Planta Medica 59 (Suppl. Issue): A 603. Smitt UW,Jiiger AK, Adsersen A, Gudiksen L. 1995. Comparative studies in phytochemistry and tit anatomy of Thapsia garganica and T. lranrtagana, Apiaceae (Umbelliferae). Botanical Journal of the Linnean Society 117: 281-292. Stahl E. 1970. Chromalographische und mikrospopicche analyse von drop. Stuttgart: Gustav Fischer Verlag, 2226. Tutin TG. 1968. Thapsia L. In: Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walters SM, Webb DA, eds. Flora Europaea, vol. 2. Cambridge: Cambridge University Press, 370.