Protoanemonin in australian Clematis

Phytochemistry, Vol. 33, No. 5. pp. 1099 1102, 1993 Printedin Great B&am.

003 1 -9422/93 $6.00 + 0.00 Q 1993 PergamonPress Ltd

PROTOANEMONIN IAN

A.

IN AUSTRALIAN

SOUTHWELL

CLEMATIS

DAVID J. TUCKER*

and

Agricultural Institute, Wollongbar, NSW 2477,Australia; *Department of Chemistry, University of New England, Armidale, NSW 2350, Australia (Received 18 November 1992)

Key Word Index-Clematis glycinoides; C. aristata; C. fawcettii; C. gentianoides; C. microphylla; C. microphylla var. leptophyllu; C. pubescens; Ranunculaceae; medicinal plant volatile oil, protoanemonin determination; chemotaxonomy; butenolide.

Abstract-Protoanemonin has been isolated from the Australian ‘Headache Vine’ Clematis glycinoides. Quantitative examination of this and the other six Australian Clematis taxa has shown protoanemonin concentrations of 2.19,0.31, 1.96,0.17,0.87, 1.31 and 1.21% (dry weight) for C. aristata, C. fawcettii, C. gentianoides, C. glycinoides, C. microphylla, C. microphylla var. leptophylla and C. pubescens, respectively.

INTRODUCTION

Clematis glycinoides DC. is an evergreen woody vine growing to a height of 2 m in moist gullies near rainforest on the coastal ranges of eastern Australia and is commonly known as ‘Travellers Joy’ or ‘Headache Vine’ [ 1, 23. The latter name derives from use as a medicinal plant in the treatment of headaches and colds by the inhalation of the vapours emitted from freshly crushed leaves [l-5]. The active constituents, also reported as poisonous to stock [4,5] are however ‘not known’ [l] but ‘would certainly repay investigation’ [2]. The closely related small leaf clematis, C. microphylla, also poisonous to stock [4, 51 has value as a counter-irritant when applied briefly as a poultice but then develops vesicant properties after a few minutes [ 11. In recently published alkaloid and anti-tumour screening results [6], C. aristata and C. microphylla responded negatively to anti-tumour testing, whereas C. glycinoides was screened for alkaloids only. This investigation reports the isolation, identification and quantification of the active constituent in all the Australian Clematis taxa. RESULTS AND DISCUSSION

Extraction uncondensate graphic peak 997). ‘H and

of the steam distilled condensate and the still pot water gave a single gas chromatowith short retention time (Kovats index 13CNMR, EI-mass, CI-mass, IR and UV

4 I

0

0

spectra indicated the methylene butenolide protoanemonin (1) which has been detected in several other Ranunculaceae species including Clematis angustzfolia, C. j7ammula, C. jubata, C. montana, C. recta and C. vitalba [7-91. The instability of this y-lactone was immediately evident with the presence of signals for the dimer (2) as an impurity in the NMR spectra, the precipitation of insoluble dimeric and polymeric solids from solution and the presence of additional peaks in the gas chromatogram of samples exposed to air [S-12]. The ‘H and i3C NMR signals of 1 are shown in Table 1. The ‘H NMR spectrum differs considerably from that published by Hirabayashi and Yokota [12] for protoanemonin in perdeuterotoluene at 0” but is in good agreement with that obtained by Martin et al. [ll] in deuterochloroform. Particularly anomalous is the cis coupling (11 Hz) between H-2 and H-3 reported by Hirabayashi and Yokota which is well removed from the typical range (5-6 Hz) found for similar butenolides [ 13, 141. The assignments of the i3CNMR resonances were based on long-range ’ 3C-1H couplings found from a fully coupled 13C spectrum and from selective INEPT experiments [15] (optimized for Jc,u of 12 Hz). These assignments are different to those of Martin et al. [ 1l] for all but one carbon (C-5). Long-range coupling from H-5E (64.89) and H-5Z (65.20) to the quaternary carbon resonating at 6154.9 establish it as C-4, while the occurrence of three long-range couplings to the resonance at

+z / 0

00

’

=gj*oJ-Jo k

Ho

0

1

2

3 1099

1100

I.A. SOUTHWELL

and D.J.

TUCKER

Table 1. The ‘H and 13CNMR signals and couplings assigned for protoanemonin Carbon

bH

Multiplicity

J,.,, (Hz)

6C

Multiplicity

J,,, (Hz)

GH-LR*

1 2 3

5.5, 0.8 5.5, 1.8, 0.8

13.4, 8.3 184.2, 2.6 178.5, 9.0, 4.3, 4.3

7.38, 6.23

d

169.7 121.7 143.3

dd

7.38 6.23

154.9

m

98.1

t

ddd

4 4.89(E) 5.20(Z)

5

2.5, 0.8 2.5, 1.8

dd dd

*Long-range “C-‘H

dd

ddt

7.38, 6.23 4.89, 5.20 164.8, 164.8

correlations found from selective INEPT experiments [lS] optimized for

J C,H of 12 Hz.

Table 2. Protoanemonin

concentrations in Australian Clematis leaves

Harvest dates

Species

No.

Provenance

C. arutata*

91364 91592 92158 91361 92162 91362 92159 91359 91680 92160 91466 91360 92161 91476

24.6.91 22.10.91 24.2.92 24.6.91 Mt. Dumaresq, Qld 24.2.92 24.6.91 24.2.92 Orford, Tas 24.6.91 2.12.91 Boonah. Qld 24.2.92 Triggs Is., WA 15.8.91 Black Mt. ACT 24.6.91 24.2.92 Manjimup, WA 1.9.91

C. fuwcettiit C. gentianoidest C. glycmoides*

C. microphylla C. microphylla var. leptophyllat C. pubescens

Canberra, ACT

Mean (range) protoanemonin concentration fresh weight (%) dry weight (%)

0.62 (0.16-1.04) 0.05 (0.2-0.7) 0.55 (0.48-0.61)

2.19 (0.38-3.21) 0.31 (0.29-0.33) 1.96 (1.10-2.82)

0.05 (0.0&0.07) 0.21 0.33 (0.1l-0.54) 0.57

0.17 ‘(0.11-0.20) 0.87 1.31 (0.35-2.26) 1.21

*Mean of three collections. tMean of two collections.

6143.3 is only consistent with the assignment of this signal as C-3. An estimate of the relative protoanemonin concentration in all seven Australian Clematis taxa was obtained by solvent extraction and gas chromatographic quantification of leaf material (Table 2). Ranunculin (3), presumably the precursor of protoanemonin (1) as in other Ranunculaceae species 173 gave 1 readily on solvolysis in ethanol. In addition, steam distillation gave yields of 0.12,0.05 and 0.67% protoanemonin from fresh whole plant samples of C. microphylla, C. glycinoides and C. aristata, respectively. These yields were comparable with solvent extraction determinations (Table 2). Spectrophotometric [ 161 and HPLC [8,9,17] methods for protoanemonin determinations are well known but must also be considered estimates due to the volatility and instability of protoanemonin coupled with its formation from precursor ranunculin (3) by crushing, hydrolysing or distilling fresh leaf. Similarly, this solvent extraction-gas chromatography method, while encountering the same difficulties, provides a valid alternative.

This interspecies comparison categorizes the Australian Clematis as poor (C.fawcetti, C. glycinoides), moderate (C. microphylla) or good (C. aristata, C. gentianoides, C. microphylla var. leptophylla, C. pubescens) sources of protoanemonin with the greatest concentrations in C. gentianoides and C. aristata (0.61 and 1.04%, respectively, fresh leaf) 10 times greater than for other Clematis leaves and as high as those for Helleborus niger (0.58%), Ranunculus bulbosus (0.78%) and R. illyricus (0.51%) [8,9, 171. The concentration of protoanemonin in the reproductive organs [17] and the varying levels in leaf [S, 91 suggest that ranunculin (3) may translocate from leaves to flowers. Consequently the Australian Clematis species must be considered an excellent source of protoanemonin, the simplest of the medicinally important y-alkylidenebutenolides. Thus, the species traditionally attributed headache and cold curative properties [l-4] now needs to be evaluated in terms of the broader spectrum antibiotic [8, 17-201, anti-tumour [19], anti-leukaemic [8, 191, sedative [ 111, hypothermic [l 11, antipyretic [ 111, antimutag-

1101

Protoanemonin in Australian Clematis enic [19], synthetic [14] and industrial [12] (including cell culture production [21]) potential of protoanemonin.

EXPERIMENTAL

Plant sources. Clematis glycinoides (H91001) and C. aristata (H91002) were collected from the Grafton to

Armidale Road, 115.8 km from Armidale and herbarium voucher specimens are lodged at the Royal Botanic Gardens Herbarium, Sydney. Clematis microphylla and C. pubescens were collected from Triggs Island (Perth 01535765) and Manjimup-Walpole (01544381) and specimens are lodged at the Western Australian Herbarium. Specimens C. aristata C.8701748 c Canberra, (ACT) provenance], C. fawcettii C.8601966 s Mt. Dumaresq (Queensland) provenance], C. gentianoides I.7231 19 p Orford (Tasmania) provenance], C. glycinoides C.7803328 s Boonah (Queensland) provenance] and C. microphylla var. leptophylla C.8103901 c Black Mountain (ACT) provenance] were all obtained from living collections at the Australian National Botanic Gardens, Canberra. Samples were transported fresh as living material at ambient temperature from collection site to laboratory. Isolation and determination of protoanemonin. Fresh leaf and stem material (523.7 g) was hydrodistilled with dionized H,O (1.0 1) for 3 hr. The condensate was extracted with Et,0 (3 x 30 ml), the organic layer washed with brine (30 ml), dried (Mg S04) and made up to 100 ml with Et,O. This solution (l.Oml) mixed with linalool (1.00 mg) in EtOH (1.0 ml) was injected into a Hewlett Packard 5890 gas chromatograph at approx. 1 ml min- ’ H, flow for determination of protoanemonin concn using an intermediate phase RSL 300 60 m x 0.2 mm i.d. FSOT column from SO”(10 min) at 10” min- ’ using FID detection with a HP7673A Controller and 3393A Integrator. All Clematis species examined showed only one peak with retention index 997 with respect to the n-alkane series. Solvent removal with intentional polymerization gave 0.6757 g of solid polymeric material indicating a response factor of 0.44 for protoanemonin with respect to linalool. This response factor was used for the calculation of protoanemonin yields from steam distillations and concentrations from ethanolic extractions. Replacement of Et,0 as extraction solvent with CDCl, and other solvents gave protoanemonin solns suitable for NMR and other spectroscopic investigations. The instability of protoanemonin has in the past discouraged the publication of meaningful spectral data. Only the UV spectrum in water (LX 259 mm) was consistent with literature spectra [8,11,12]. The NMR (Bruker AC-300) spectra have been discussed, the IR spectrum in CHCl, (Perkin Elmer 681) showed strong absorption at 1795 err-’ higher than values given by Hirabayashi and Yokota [12] (CHCl, solution) and Bellamy [22] (film) but consistent with the effect of an electronegative substituent y to a y-lactone, GCEIMS (20 EV Shimadzu QP 1000 using a 30m x 0.22 mm i.d. DB17 FSOT column) indicated prominent molecular ion ([M]‘96) and (M -28) fragments and GCCIMS (isobutane) also indicated prominent frag-

ments at 96 and 68 rather than the (M + 1) 97 and 69 fragments. For each Clematis specimen, 2-3 g (in 2 or 3 replicates where material was sufficient) of accurately weighed fresh leaf material (or leaf with stem for small leaf species) was extracted with ethanol (20.0 ml) containing linalool (20.0 mg) as int. standard, at 20” for 40 hr. Estimates of the protoanemonin concn were obtained by GC quantification using the previously calculated response factor. The extracts were evaporated at room temperature, dried and weighed so that dry weight concentrations could also be estimated. Acknowledgements-The

authors thank Peter Hardwick, Wilderness Foods, Byron Bay, for drawing ‘Headache Vine’ to our attention; Mark Richardson, Ruth Hallett and Lyn Meredith, Australian National Botanic Gardens and Jim Armstrong and Ray Cranfield, Western Australian Herbarium, for providing Clematis specimens; Rob Black, Chemical Residues Laboratory, Lismore, for GCMS determinations; and Ian Stiff and Vanessa Williams, Wollongbar Agricultural Institute for technical assistance.

REFERENCES

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4. Webb, L. J. (1948) Guide to the Medicinal and Poisonous Plants of Queensland, CSZRO Bulletin No. 232, p. 10, 137. Government Printer, Melbourne. 5. Hurst, E. (1942) Tke Poison Plants of NS W, p. 114. Snelling, Sydney. 6. Collins, D. J., Culvenor, C. C. J., Lamberton, J. A., Loder, J. W. and Price, J. R. (1990) Plants for Medicines: A Chemical and Pharmacological Survey of Plants in the Australian Region, p. 61. CSIRO, East

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9. Bonora, A., Dall’Olio, G., Donini, A. and Bruni, A. (1987) Phytochemistry 26, 2277. 10. Font, J., Gracia, A. and de March, P. (1989) An. Quim. 85, 5.

11. Martin, M. L., Ortiz de Urbina, A. V., Montero, M. J., Carron, R. and san Roman, L. (1988) J. Ethnopharmacol. 24, 185.

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13. Pieken, W. A. and Kozarich, J. W. (1990) J. Org. Chem. 55, 3029.

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I. A. SOUTHWELL and D. J. TUCKER

14. Alonso, D., Orti, J., Branchadell, V., Oliva, A., Ortuno, R. M., Bertran, J. and Font, J. (1990)J. Org. Chem. 55, 3060. 15. Bax, A. (1984) J. Magn. Reson. 57, 314. 16. Mahran, G. H., Hifny Saber, A. and El-Alfy, T. (1968) Planta Med. 16, 323. 17. Bonora, A., Botta, B., Menziani-Andreoli, E. and Bruni, A. (1988) Biochem. Physiol. PJlnnz. 183, 443. 18. Caltrider, P. G. (1967) Protoanemonin. Antibiotics. Vol. 1. Mechanism of Action (Corcoran, J. W. and Hann, F., eds), pp. 671-673. Springer, Berlin.

19. Iovel, I., Goldberg Y. and Shymanska, M. (1990) J. Chem. Sot., Chem. Commun. 1079. 20. Martin, M. L., San Roman, L. and Dominguez, A. (1990) Planta Med. 56, 66. 21. Bonora, A., Poli. F., Fasulo, M. P. and Bruni, A. (1989) Biochem. Physiol. Pjknz. 185, 397. 22. Bellamy, L. J. and Branch, R. F. (1954) J. Chem. Sot. 4491.