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Bellissaponins BA1 and BA2, acylated saponins from Bellis perennis
Phytochemistry, Vol. 30,No. 2, pp. 627631, 1991 Printed in Gnat Britain.
0031.-9422/91 $3.00+0.00 (c> 1991Pergamon Press plc
BELLISSAPONINS BA, AND BA,, ACYLATED FROM BELLIS PERENNZS THOMAS
SCH~PKE,
VICTOR
WRAY,*
BEATE RZAZEWSKA
and
SAPONINS
KARL
HILLERt
Humboldt-Universitlt Berlin, Fachbereich Pharmazie, Goethestrasse 54, Berlin, G.D.R.-1120; *Gesellschaft fiir Biotechnologische Forschung mbH, Maschercder Weg 1, D-3300 Braunschweig, F.R.G. (Received in revisedform 18 June 1990) Key Word Index-Bellis
perennis; Asteraceae; triterpenoid saponins; bcllissaponins BA, and BA,.
Abstract-Two novel acylated triterpenoid saponins were isolated from Bellis perennis. The structures of these have been elucidated as 3-O-a-L-rhamnopyranosyland 3-O-~-D-glucopyranosyl-2~,3~,16a,23-tetrahydroxyolean-l2-en28-oic acid-28-0-a-L-rhamnopyranosyk 1+3)-/?-D-xylopyranosyl( 1+4)-a-L-rhamnopyranosyl( 1+2)-[E-buta-Zenoic acid (1+4)]-B-D-fucopyranoside. by means of high field 1D and 2DNMR spectroscopic methods without recourse to derivatization or comparison with previous data.
INTRODUCTION
Bellis perennis, the common daisy, is a plant that is native
to almost the whole of Europe. It has been used in folk medicine in the treatment of various diseases, e.g. rheumatism, and as an expectorant [ 1,2]. In a previous paper [3] we reported the structure elucidation of two deacylated triterpenoid saponins, bellissaponins 1 (3) and 2 (4) obtained from Bellis perennis. The present paper describes the isolation and structure elucidation of bellissaponins BA, (1) and BA, (2), two new acylated saponins, obtained from the same source after partial deacylation of the genuine ester saponin mixture. Although a considerable volume of NMR data exists for the triterpenoid saponins, in particular r3C chemical shift data, the complete assignment of their’H spectra has been reported infrequently [4]. Normally there is an incomplete assignment of the NMR data for the aglycone and, only after derivatization, complete assignment of the sugar signals [S, 63. In studies presented here modern 1D and 2DNMR techniques have been used and the results of these are described in some detail as these have allowed a complete assignment of both the ‘H and “C NMR data of underivatized material without recourse to comparisons with previous data. In particular the usefulness for the structural elucidation of triterpene saponins of direct and long-range ‘H-detected tH-i3C shifts, correlations at the highest magnetic field available (14 Tesla, 600 MHz ‘H) will be described. These techniques not only allow ‘H and i3C assignments but provide unambiguous information about the structure of the aglycone and about the nature, position of attachment and sequence of the various substituents in the molecule.
after separation on SephadexR LH-20 and treatment with 1% aq. KOH. The two major polar compounds have been characterized as bellissaponins 1 and 2 (compounds 3 and 4) by TLC. Flash-chromatography on silica gel using chloroform-methanol-water (15 : 10: 2) and CC on reversed phase silica gel using methanol-water (3: 2) afforded bellissaponin BA, (1) and repeated flash chromatography on silica gel using chlorofornmethanol-water mixtures gave bellissaponin BA, (2). Mineral acid hydrolysis with HCl vapour at 100” of both 1 and 2 gave polygalacic acid (2/?,3/3,16a,23-tetrahydroxyolean-12-en-28-oic acid) as their common aglycone and L-rhamnose, D-xylOSe and D-fuCOSe as sugar components. In addition 2 yielded D-glucose. Alkaline hydrolysis of 1 and 2 performed with 5% KOH (1 hr, 100’) afforded two prosapogenins P-BA, (5) and P-BA, (6) isolated by preparative TLC. On mineral acid hydrolysis only L-rhamnose and D-ghXSe, respectively, were. obtained as free sugars, indicating that the remaining sugar components are bound acyl-glycosidically. The FAB mass spectrum of 5 gave an ion [M -HIat m/z 649 and an ion [M - 146-H]at m/z
R' 1 OH OH
FHlOH
RESULTS AND DISCUSSION OH
Extraction of the roots and rhizomes of B. perennis (Experimental), afforded a mixture of four major saponins i)H 6H R'- /+/CO-
tAuthor to whom correspondence should be addressed. 627
Saponins from Bellis perennis plants contain saponins esterified with carboxylic acids. Unfortunately it is not possible to compare them further as the position of the ester group in the latter has not been characterized. It is of special interest to note that saponins possessing in part the same structure as bellissaponins have been isolated from the monocotyledonous plant Crocosimn crocosmiijora [9, lo]. Crocosmiosides A-D are B-D-xylopyranosyl-( 1+4)-a+rhamnopyranosyl(l-2)- j?-D-fucopyranosides of polygalacic acid carrying rhamnose or apiose in position 4 of the xylose moiety or apiose in position 3 of the xylose moiety and esterified in position 4 of the fucose moiety similar to 1 and 2. In contrast to bellissaponins they possess different oxygenated octadecanoic acid residues as the acyl moieties.
EXPERIMENTAL
NMR techniques. 1D and 2D ‘H and 13CNMR spectra were
recorded at ambient temp. on either 600 (1D: ‘H and “C spectra, 2D: COSY, ‘H-detected one-bond [S] and multiplebond 13C multiple-quantum coherence spectra [7], HMQC and HMBC respectively) or 300 MHz (1D: ‘H and 13C spectra) spectrometers locked to the major deuterium of the solvent, CD,OD. The value of the delay to optimise one-bond correlations in the HMQC spectrum and suppress them in the HMBC spectrum was 3.45 msec and the evolution delay for long range couplings in the latter was set to 70 msec. All 1D and 2D spectra were recorded using standard software and data manipulation of the 2D spectra were performed on a Bruker Aspect X32 Data station. All chemical shifts are given in ppm relative to TMS and couplings in Hz. Spray reagents. Anisaldehyde-H,SO, (saponins, sapogenins), thymol-H,SO, (sugars), H,O (prep. TLC of prosapogenins). Plant material. The plant material was collected in Berlin and Warsaw (Poland) in May 1988. Aerial and underground parts were separated, dried at S&70” and ground. Extraction and separation. Dried and ground roots and rhizomes (600 g) of B. perennis were refluxed with 80% MeOH ( x 2 for 1 hr). The extracts were combined, evapd, dissolved in H,O and defatted with CHCl, (H,O-CHCl, 1: 1). The defatted extract was partitioned twice with n-BuOH and the n-BuOH layer was evapd to dryness. The dark brown residue was dissolved in a small amount of MeOH and dropped into a lOfold amount of Et,0 affording 4OSg (6.75%) of a glycoside mixt. as a light brown powder. This mixt. (40 g) was subjected to Sephadex’ LH-20 (Pharmacia, Uppsala, Sweden) chromatography using MeOH as mobile phase. The major saponin fraction was hydrolysed with 1% aq. KOH for 1 hr at room temp. to give a mixt. consisting of 4 major compounds. The reaction mixt. was acidified (pH 5) and extracted with n-BuOH. Flash chromatography of the n-BuOH extract (pressure:
631
0.5 kpcm-‘) on silica gel (Merck, particle size do.063 mm) using CHCl,-MeOH-H,O (15: 10:2) as mobile phase afforded 1 (451.1 mg) and 2 (520.2 mg) with slight impurities. Compound 1 was further purified by flash chromatography on silica gel using CHCl,-MeOH-H,0(17: 10:2) and by CC on silanized silica gel (Merck, particle size 0.063-0.200 mm) giving 47.3 mg of 1 (mp 222-225”, TLC: R, 0.67, CHCl,-MeOH-H,O, 15: 10:2). Compound 2 was further purified by flash chromatography on silica gel using CHCl,-MeOH-H,O (17: 10:2) giving 126.6 mg 2 (mp 223-225”. TLC: R, 0.64, CHCl,-MeOH-H,O, 15 : 10: 2). Isolation ofprosupogenins. Compounds 1 and 2 (50 mg) were hydrolysed with 5% aq. KOH for 2 hr at loo”. The reaction mixt. was acidified (pH 5), extracted with n-BuOH and subjected to prep. TLC on silica gel using CHCl,-MeOH-H,O (50: 10: 2) as mobile phase. The frs were evapd to dryness, dissolved in a small amount of MeOH and filtered through Sephadex= LH-20 to give ca 5 mg of 5 and 6, respectively. Acid hydrolysis. Acid hydrolysis and TLC determination of the aglycone and sugar constituents of 1,2,5 and 6 was performed according to ref. [ll] at loo”. TLC: Silica gel using CHCl,-MeOH (9:1, detection of the aglycones) and nBuOH-H,O-EtOH (100: 10:2, upper layer, detection of sugars). Acknowledgement-We thank H. Dirks (GBF, Braunschweig) for recording the FABMS of compounds 1 and 2, Prof. Dr. G. Dube (AdW, ZIPC, Berlin) for the FABMS of compounds 5 and 6, and C. Kakoschke (GBF, Braunschweig) for some NMR spectra.
UEFERENCES 1. Kroeber, L. (1981) Pharm. Zentralhalle 72, 745. 2. Hagers Handbuch der pharmazutischen Praxis (1982) Vol. III, p. 376. Springer, Berlin. 3. Hiller, K., Schiipke, T., Wray, V. and Schulten, H.-R. (1988) Pharmazie 43,840. 4. Babadjamian, A., Elias, R., Fame, R., Vidal-Olivier, E. and Balansard G. (1988) Spectrosc. Letters 21, 565.
5. Massiot, G., Lavaud, C., Guillaume, D., Le. Men-Olivier, L. and Van Binst G. (1986) J. Chem. Sot., Chem. Commun. 1485. 6. Massiot, G., Lavard, C, Le Men-Olivier, L., Van Binst, G., Miller, S. P. F. and Fales, H. M. (1988) J. Chem. Sot., Perkin Trans I 3071. 7. Summers, M. F., Marzilli, L. G. and Bax, A. (1986) J. Am. Chem. Sot. lO& 4285. 8. Cavanagh, J., Hunter, C. A., Jones, D. N. M., Keeler, J. and Sanders, J. K. M. (1988) Magn. Reson. Chem. 26, 867. 9. Furuya, T., Ueoka, T. and Asada, Y. (1987) Tennen Yuki Kagobutsu Toronkai Keen Yoshishu 29, 592. 10. Furuya, T., Ueoka, T. and Asada, Y. (1989) Chem Pharm. Bull. 36, 444. 11. Kartnig, T. and Wegschaider, 0. (1971) .I. Chromatogr. 61, 375.
0031.-9422/91 $3.00+0.00 (c> 1991Pergamon Press plc
BELLISSAPONINS BA, AND BA,, ACYLATED FROM BELLIS PERENNZS THOMAS
SCH~PKE,
VICTOR
WRAY,*
BEATE RZAZEWSKA
and
SAPONINS
KARL
HILLERt
Humboldt-Universitlt Berlin, Fachbereich Pharmazie, Goethestrasse 54, Berlin, G.D.R.-1120; *Gesellschaft fiir Biotechnologische Forschung mbH, Maschercder Weg 1, D-3300 Braunschweig, F.R.G. (Received in revisedform 18 June 1990) Key Word Index-Bellis
perennis; Asteraceae; triterpenoid saponins; bcllissaponins BA, and BA,.
Abstract-Two novel acylated triterpenoid saponins were isolated from Bellis perennis. The structures of these have been elucidated as 3-O-a-L-rhamnopyranosyland 3-O-~-D-glucopyranosyl-2~,3~,16a,23-tetrahydroxyolean-l2-en28-oic acid-28-0-a-L-rhamnopyranosyk 1+3)-/?-D-xylopyranosyl( 1+4)-a-L-rhamnopyranosyl( 1+2)-[E-buta-Zenoic acid (1+4)]-B-D-fucopyranoside. by means of high field 1D and 2DNMR spectroscopic methods without recourse to derivatization or comparison with previous data.
INTRODUCTION
Bellis perennis, the common daisy, is a plant that is native
to almost the whole of Europe. It has been used in folk medicine in the treatment of various diseases, e.g. rheumatism, and as an expectorant [ 1,2]. In a previous paper [3] we reported the structure elucidation of two deacylated triterpenoid saponins, bellissaponins 1 (3) and 2 (4) obtained from Bellis perennis. The present paper describes the isolation and structure elucidation of bellissaponins BA, (1) and BA, (2), two new acylated saponins, obtained from the same source after partial deacylation of the genuine ester saponin mixture. Although a considerable volume of NMR data exists for the triterpenoid saponins, in particular r3C chemical shift data, the complete assignment of their’H spectra has been reported infrequently [4]. Normally there is an incomplete assignment of the NMR data for the aglycone and, only after derivatization, complete assignment of the sugar signals [S, 63. In studies presented here modern 1D and 2DNMR techniques have been used and the results of these are described in some detail as these have allowed a complete assignment of both the ‘H and “C NMR data of underivatized material without recourse to comparisons with previous data. In particular the usefulness for the structural elucidation of triterpene saponins of direct and long-range ‘H-detected tH-i3C shifts, correlations at the highest magnetic field available (14 Tesla, 600 MHz ‘H) will be described. These techniques not only allow ‘H and i3C assignments but provide unambiguous information about the structure of the aglycone and about the nature, position of attachment and sequence of the various substituents in the molecule.
after separation on SephadexR LH-20 and treatment with 1% aq. KOH. The two major polar compounds have been characterized as bellissaponins 1 and 2 (compounds 3 and 4) by TLC. Flash-chromatography on silica gel using chloroform-methanol-water (15 : 10: 2) and CC on reversed phase silica gel using methanol-water (3: 2) afforded bellissaponin BA, (1) and repeated flash chromatography on silica gel using chlorofornmethanol-water mixtures gave bellissaponin BA, (2). Mineral acid hydrolysis with HCl vapour at 100” of both 1 and 2 gave polygalacic acid (2/?,3/3,16a,23-tetrahydroxyolean-12-en-28-oic acid) as their common aglycone and L-rhamnose, D-xylOSe and D-fuCOSe as sugar components. In addition 2 yielded D-glucose. Alkaline hydrolysis of 1 and 2 performed with 5% KOH (1 hr, 100’) afforded two prosapogenins P-BA, (5) and P-BA, (6) isolated by preparative TLC. On mineral acid hydrolysis only L-rhamnose and D-ghXSe, respectively, were. obtained as free sugars, indicating that the remaining sugar components are bound acyl-glycosidically. The FAB mass spectrum of 5 gave an ion [M -HIat m/z 649 and an ion [M - 146-H]at m/z
R' 1 OH OH
FHlOH
RESULTS AND DISCUSSION OH
Extraction of the roots and rhizomes of B. perennis (Experimental), afforded a mixture of four major saponins i)H 6H R'- /+/CO-
tAuthor to whom correspondence should be addressed. 627
Saponins from Bellis perennis plants contain saponins esterified with carboxylic acids. Unfortunately it is not possible to compare them further as the position of the ester group in the latter has not been characterized. It is of special interest to note that saponins possessing in part the same structure as bellissaponins have been isolated from the monocotyledonous plant Crocosimn crocosmiijora [9, lo]. Crocosmiosides A-D are B-D-xylopyranosyl-( 1+4)-a+rhamnopyranosyl(l-2)- j?-D-fucopyranosides of polygalacic acid carrying rhamnose or apiose in position 4 of the xylose moiety or apiose in position 3 of the xylose moiety and esterified in position 4 of the fucose moiety similar to 1 and 2. In contrast to bellissaponins they possess different oxygenated octadecanoic acid residues as the acyl moieties.
EXPERIMENTAL
NMR techniques. 1D and 2D ‘H and 13CNMR spectra were
recorded at ambient temp. on either 600 (1D: ‘H and “C spectra, 2D: COSY, ‘H-detected one-bond [S] and multiplebond 13C multiple-quantum coherence spectra [7], HMQC and HMBC respectively) or 300 MHz (1D: ‘H and 13C spectra) spectrometers locked to the major deuterium of the solvent, CD,OD. The value of the delay to optimise one-bond correlations in the HMQC spectrum and suppress them in the HMBC spectrum was 3.45 msec and the evolution delay for long range couplings in the latter was set to 70 msec. All 1D and 2D spectra were recorded using standard software and data manipulation of the 2D spectra were performed on a Bruker Aspect X32 Data station. All chemical shifts are given in ppm relative to TMS and couplings in Hz. Spray reagents. Anisaldehyde-H,SO, (saponins, sapogenins), thymol-H,SO, (sugars), H,O (prep. TLC of prosapogenins). Plant material. The plant material was collected in Berlin and Warsaw (Poland) in May 1988. Aerial and underground parts were separated, dried at S&70” and ground. Extraction and separation. Dried and ground roots and rhizomes (600 g) of B. perennis were refluxed with 80% MeOH ( x 2 for 1 hr). The extracts were combined, evapd, dissolved in H,O and defatted with CHCl, (H,O-CHCl, 1: 1). The defatted extract was partitioned twice with n-BuOH and the n-BuOH layer was evapd to dryness. The dark brown residue was dissolved in a small amount of MeOH and dropped into a lOfold amount of Et,0 affording 4OSg (6.75%) of a glycoside mixt. as a light brown powder. This mixt. (40 g) was subjected to Sephadex’ LH-20 (Pharmacia, Uppsala, Sweden) chromatography using MeOH as mobile phase. The major saponin fraction was hydrolysed with 1% aq. KOH for 1 hr at room temp. to give a mixt. consisting of 4 major compounds. The reaction mixt. was acidified (pH 5) and extracted with n-BuOH. Flash chromatography of the n-BuOH extract (pressure:
631
0.5 kpcm-‘) on silica gel (Merck, particle size do.063 mm) using CHCl,-MeOH-H,O (15: 10:2) as mobile phase afforded 1 (451.1 mg) and 2 (520.2 mg) with slight impurities. Compound 1 was further purified by flash chromatography on silica gel using CHCl,-MeOH-H,0(17: 10:2) and by CC on silanized silica gel (Merck, particle size 0.063-0.200 mm) giving 47.3 mg of 1 (mp 222-225”, TLC: R, 0.67, CHCl,-MeOH-H,O, 15: 10:2). Compound 2 was further purified by flash chromatography on silica gel using CHCl,-MeOH-H,O (17: 10:2) giving 126.6 mg 2 (mp 223-225”. TLC: R, 0.64, CHCl,-MeOH-H,O, 15 : 10: 2). Isolation ofprosupogenins. Compounds 1 and 2 (50 mg) were hydrolysed with 5% aq. KOH for 2 hr at loo”. The reaction mixt. was acidified (pH 5), extracted with n-BuOH and subjected to prep. TLC on silica gel using CHCl,-MeOH-H,O (50: 10: 2) as mobile phase. The frs were evapd to dryness, dissolved in a small amount of MeOH and filtered through Sephadex= LH-20 to give ca 5 mg of 5 and 6, respectively. Acid hydrolysis. Acid hydrolysis and TLC determination of the aglycone and sugar constituents of 1,2,5 and 6 was performed according to ref. [ll] at loo”. TLC: Silica gel using CHCl,-MeOH (9:1, detection of the aglycones) and nBuOH-H,O-EtOH (100: 10:2, upper layer, detection of sugars). Acknowledgement-We thank H. Dirks (GBF, Braunschweig) for recording the FABMS of compounds 1 and 2, Prof. Dr. G. Dube (AdW, ZIPC, Berlin) for the FABMS of compounds 5 and 6, and C. Kakoschke (GBF, Braunschweig) for some NMR spectra.
UEFERENCES 1. Kroeber, L. (1981) Pharm. Zentralhalle 72, 745. 2. Hagers Handbuch der pharmazutischen Praxis (1982) Vol. III, p. 376. Springer, Berlin. 3. Hiller, K., Schiipke, T., Wray, V. and Schulten, H.-R. (1988) Pharmazie 43,840. 4. Babadjamian, A., Elias, R., Fame, R., Vidal-Olivier, E. and Balansard G. (1988) Spectrosc. Letters 21, 565.
5. Massiot, G., Lavaud, C., Guillaume, D., Le. Men-Olivier, L. and Van Binst G. (1986) J. Chem. Sot., Chem. Commun. 1485. 6. Massiot, G., Lavard, C, Le Men-Olivier, L., Van Binst, G., Miller, S. P. F. and Fales, H. M. (1988) J. Chem. Sot., Perkin Trans I 3071. 7. Summers, M. F., Marzilli, L. G. and Bax, A. (1986) J. Am. Chem. Sot. lO& 4285. 8. Cavanagh, J., Hunter, C. A., Jones, D. N. M., Keeler, J. and Sanders, J. K. M. (1988) Magn. Reson. Chem. 26, 867. 9. Furuya, T., Ueoka, T. and Asada, Y. (1987) Tennen Yuki Kagobutsu Toronkai Keen Yoshishu 29, 592. 10. Furuya, T., Ueoka, T. and Asada, Y. (1989) Chem Pharm. Bull. 36, 444. 11. Kartnig, T. and Wegschaider, 0. (1971) .I. Chromatogr. 61, 375.