Quantitative Determination of Thapsigargins in Roots and Fruits from

Thapsia gymnesica Anita V. Christiansen', Henrik Paalum', ,4 Soren M. Andersen', Antonio Pujadas, and Ulla W. Smitt1 ' Department of Medicinal Chemistry, Section of Pharmacognosy, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark 2 Department of Organic Chemistry, Danish Technical University, DK-2800 Lyngby, Denmark 3 Jardin Botánico de Córdoba, Avda. Linneo, sin, E-14004 Córdoba, Spain ' Address for correspondence

guaianolide type, with unique bioactivity such as activators of a broad number of cells belonging to the immune system (5), as inhibitors of Ca'` pumps in the sat-co- or endoplasmic reticulum

(6) and as tumor promotors (7). Chemotaxonomic studies of the genus Thapsia have shown pronounced phytochemical variations (2-4, 8). Consequently, it would be of interest to investigate T. gymnesica for the presence of thapsigargins. From the roots of Thapsia gymnesica three guaianolides, thapsigargin (1), thapsigargicin (2), and nortrilobolid (3) as well as 6-methoxy-7-geranyloxycoumarin were isolated. Their 3 structures were confirmed by comparing the 'Fl- and ' C-NMR spectra with those of the authentic compounds isolated from Tgarganica (9-1 1). The three compounds 1-3, have previously been found only in T.garganica, whereas 6-methoxy-7-geranyloxycoumarin has been found in all those Thapsia species that contain thapsigargins (3, 12).

Received: January 30, 1997: Revision accepted: May 1, 1997

Abstract: Three sesquiterpene lactones, thapsigargin (1), thapsigargicin (2), and nortrilobolid (3), and 6-methoxy-7geranyloxycoumarin have been isolated from the roots of Thapsia gymnesica (Apiaceae) for the first time. The concentrations of the three thapsigargins in the roots and the fruits have been determined by HPLC analysis.

Thapsia gymnesica Rosselló & Pujadas (Apiaceae) was reported as a new species, growing only on the Spanish islands Mallorca and Minorca, in 1991 (1). The genus Thapsia, Apiaceae, tribe Laserpitiae, has attracted considerable pharmacological interest due to the presence of thapsigargins in some of its species (2-4). Thapsigargins are sesquiterpene lactones of the Planta Medica 63 (1997) 565 -567 Georg Thieme Verlag Stuttgart • New York

The results of the quantitative HPLC analysis of 1-3 in roots and fruits from T. gymnesica are shown in Fig. 1. In the roots 2 occurs in the highest concentration (0.21%), followed by 1 (0.17 %), and 3 (0.03 %) only represents a very small amount. This is opposed to the fruits, where 3 is the main constituent (0.37%), 2 represents a smaller amount (0.18 %), and 1 accounts for the lowest concentration (0.15 %). Pujadas et al. (1) mention that T. garganica and T. gymnesica may have a common origin. However, the plant material studied by Pujadas et al. was later identified as T transtagana Brot. and not T.garganica. Although very similar in morphological characters T. garganica and T. transtagana have been separated based on phytochemical data, e.g., they contain different thapsigargins, and based on their fruit anatomy. Furthermore, T.garganica was found to include two chemotypes,

566

Planta Med. 63 (1997)

Letters

under reduced pressure to a gum (8.7 g), which was partitioned in EtOAc/H 2 O, 1 : 1. The EtOAc phase was evaporated to dryness. The residue (4.0g) was dissolved in CH-2 Cl2, and fractionated on 40g of silica gel 'SC-60A (ICN Biomedicals Art. 04526) under vacuum, using different mixtures of CH1C1 .2 and EtOAc [ 19 : 1 (400 ml, 18:2 (400 ml), 17:3 (400 ml), 16: 4 (200 ml), 14 : 6 (200 ml ), 0 : 1 (200 ml)] as eluents. Thapsigargins were quantitatively extracted with the last four eluents. Separation of the thapsigargins were carried out on a Lobar column, size B (310-25), Lichroprep RP-18 (40-63 mm ), Merck Art. 10625, with MeOH/H 2 0 (5 : 1) as eluent, and by preparative HPLC using Shimadzu (Kyoto, _japan) equipment: a SCL-6A system controller, two LC-6A pumps, a SPD-6AV VV detector, and a C-R3A integrator. Column: Nucleosil 100 C18 (5 Arm), 16 x 250 mm 1. D., eluent: MeOH/H20 (7 : 3) and (8 : 2 ), flow rate: 6 ml/min and UV detection: 230 nm. The above mentioned first fraction eluted with CH2Cl 2 /EtOAc (19:1) was evaporated to dryness. The residue was fractionated by MPLC on 35 g of silica gel TSC-60A (ICN Biomedicals Art. 04526) using an accelerating gradient from heptane (300 ml), CH 2 C1 2 (150 ml), EtOAc (150 ml) to MeOH (150 ml ). Fractions containing a compound (Rf 0.5) showing blue fluorescent (UV 365 nm) on the TLC plate (silica gel 60 F 254 Merck Art. 1.05549, eluent toluene/EtOAc/MeOH, 30:8: 1) were combined. Final purification was performed by a disc chromatotron (1 mm) using heptane/EtOAc (2: 1) with 0.1 5 MeOH as eluent. Identification of the three thapsigargins 1-3 and of 6met hoxy-7-geranylox_ycoumarin was carried out by comparing the data of their 'H- and t3 C6-NMR spectra with those of the authentic compounds isolated from T.garganica (9-11). The NMR spectra were recorded in CDC1 3 using a Bruker AC 500 MHz (compounds 1-3) and a Bruker AC 200 MHz (6methoxy-7-geranylcoumarin) instrument with -f MS as the internal standard. Fig.1 Concentrations (% of dry weight) of thapsigargin (1), thapsigargicin (2), and nortrilobolid (3) in roots and fruits of Thapsia gymnesica. Mean values of five experiments, with relative standard deviations from 1.0 to 3.9%.

one containing 1 as the major constituent and another with 1 in a relatively minor amount (2). Compound 1 was not found to be the major constituent either in the roots or in the fruits of T. gymnesica (Fig. 1). In this respect. T. gymnesica is most similar to the last mentioned chemotype of T. garganica. This indicates a close relationship between these two and may give a possible explanation to the evolution of the two chemotypes recognized within T. gargarr ica.

HPLC analysis of the thapsigargins was performed according to the description in (13). For all three compounds 1-3 the calibration graphs were linear in the concentration range 0-1.0 mg/ml, with correlation coefficients between 0.9991 and 0.9997. in Fig. 1 the results are given, in % of dry weight, as the mean value of five experiments, with relative standard deviations from 1.0 to 3.9 %. Details are described in (14). Acknowledgements We thank Ph. D. student Mikael Rasmussen, Danish Technical University, for recording the NMR spectra of 6-methoxy-7geranyloxycouniarin. References

Materials and Methods

Pujadas, A., Rosselló,.J. A., Barceló, P. (1991 ) Candollea 46, 65--74. Smitt, U. W., Jäger, A. K., Adsersen, A.. Gudiksen, L. (1995) Bot. J.

Roots of Thapsia gymnesica were collected in May 1994 on the Alcudia peninsula, Mallorca. Fruits were collected in July 1995 at Cala Sant Vicens, Mallorca. Voucher specimens are deposited both at the Royal Danish School of Pharmacy and at the Herbarium of the Jardin Botánico, Córdoba (COA).

Lin. Soc. 117,281-292. Smitt, U. W. (1995) Bot. J. Lin. Soc. 118, 367-377. Avato, P., Jacobsen, N., Smitt, U. W. (1992) J. Ess. Oil Res. 4, 467 -473. Christensen, S. B., Andersen, A., Lauridsen, A., Moldt, P., Smitt, U. W. (1992) in: New leads and targets in drug research, Alfred Benzon Symposium 33 (Krogs Gaard-Larsen, P., Christensen, S. B., Kofod, H., eds.), Munksgaard, Copenhagen, pp. 243 -252. Thastrup, O. (1990) Agents and Actions 29, 8-15.

The dried and powdered roots (200g) were extracted with ethanol on an ultrasonic bath, and the extract was evaporated

5

Letters Hakii, H., Fujiki, H., Suganuma, M., Nakayasu, M., Tahira, T., Sugimura, T., Scheuer, P. J., Christensen, S. B. (1986) J. Cancer Res. Cl i n. Oncol. 1 1 1,117-181. De Pascual Teresa, J., Moran. J. R., Hernández, J. M., Grande, M. (1985) Phytochemistr_y 24, 2071 -2074. ' Christensen, S. B., Larsen, I. K., Rasmussen, U., Christophersen, C. (1982) J. Org. Chem. 47, 649-652. "' Smitt, U. W., Christensen, S. B. (1991) Planta Med. 57, 196-197.

Planta Med. 63 (1997)

Krogsgaard-Larsen, P., Sandberg, F. (1970) Acta Chem. Scand. 24, 1113-1114. ' Rasmussen, U., Christensen, S. B., Sandberg, F. (1981) Planta Med. 43, 336-341. '3 Jäger, A. K., Gudiksen, L., Adsersen, A., Smitt, U. W. (1993) J. Chrom. 634, 135-137. '4 Paalum, H., Christiansen, A. V. (1996) Thapsia gymnesica, Thesis, Royal Danish School of Pharmacy, Copenhagen, Denmark.

567