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A distinctive flavonoid chemistry for the anomalous genus Biebersteinia
Phytochemistry 56 (2001) 87±91
www.elsevier.com/locate/phytochem
A distinctive ¯avonoid chemistry for the anomalous genus Biebersteinia Jenny Greenham a, Dionyssios D. Vassiliades a, Jerey B. Harborne a,*, Christine A. Williams a, John Eagles b, ReneÂe J. Grayer c, Nigel C. Veitch c a
Department of Botany, The University of Reading, Whiteknights, PO Box 221, Reading RG6 6AS, UK b Institute of Food Research, Colney, Norwich NR4 7UA, UK c Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK Received 8 June 2000; received in revised form 24 August 2000
Abstract Leaf surface extracts of Biebersteinia orphanidis have yielded a complex mixture of ®ve ¯avones with the unusual 5,7-dihydroxy6,8-dimethoxy A ring substitution pattern. They are acerosin, hymenoxin, nevadensin, sudachitin and 5,7,40 -trihydroxy-6,8-dimethoxy¯avone. Also present at the leaf surface are gardenin B, luteolin, apigenin, acacetin and the coumarin umbelliferone. The internal leaf ¯avonoids include the 7-glucosides of apigenin, luteolin and tricetin, together with the 7-rutinosides of apigenin and luteolin. This pro®le diers from those of B. heterostemon and B. odora. It appears that B. orphanidis is as highly distinctive in its ¯avonoid pattern as it is phytogeographically. The data also con®rm the conclusion of other studies, including rbcL and atpB gene sequence analysis, that Biebersteinia is completely unrelated to the Geraniaceae, where it was once placed. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Flavonoids; Biebersteinia; Chemotaxonomy
1. Introduction Biebersteinia Stephan is a genus of ®ve rare, herbaceous species distributed in the mountainous semi-arid areas of central and western Asia and the eastern Mediterranean as far as Greece. Its taxonomic position has been in dispute since its discovery in 1806. In 1841, Endlicher gave it familial status, while Boissier included it ®rst in the Zygophyllaceae and then later (1867) in the Geraniaceae. Subsequent authors, notably Kunth (1931), have followed the latter classi®cation. However, a palynological study by BortenschlaÈger (1967) suggested it was wrongly placed in the Geraniaceae and this was supported by later anatomical (Tutel, 1982), embryological (Kamelina and Komarova, 1990) and chemotaxonomic (Bate-Smith, 1973) studies. A recent phylogenetic analysis of rbcL and atpB gene sequences of B. orphanidis Boiss. (Bakker et al., 1998) indicated that the genus was nested in the Sapindales but with no * Corresponding author. Tel.: +44-118-931-8162; fax: +44-118975 3676.
link to any other clade within this order or within the Geraniales. Phytochemically, the genus has not been analysed in detail. Bate-Smith (1973) in a phenolic survey of Geranium and related genera examined Bierbersteinia multi®da DC. and B. odora Stephan and found that they had completely dierent ¯avonoid patterns from those known for any other genus of the Geraniaceae. He reported myricetin, prodelphinidin and procyanidin from B. odora and a number of unusual unidenti®ed ¯avonoids from both taxa. A later phytochemical paper included a study of the ¯avones (luteolin 7-glucoside and 7-rutinoside) (Omurkamzinova et al., 1991) of B. multi®da. In the Tibetan species, B. heterostemon Maxim. (Zhang et al., 1995) a rare ¯avone, 8-hydroxytricetin 8,40 ,50 -trimethyl ether was reported together with a luteolin C-glucoside, quercetin 7-glucoside and umbelliferone. Biebersteinia orphanidis, which has not been investigated previously for its phenolic constituents, occupies a dierent phytogeographical position from the other four species, i.e. in Anatolian Turkey and Greece.
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00355-1
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J. Greenham et al. / Phytochemistry 56 (2001) 87±91
Therefore, the present ¯avonoid analysis of B. orphanidis was undertaken in the hope it might reveal useful geographic dierences and hence some insight into the evolution within the genus and the taxonomic position of Biebersteinia in the dicotyledons. 2. Results 2.1. Surface ¯avonoids The acetone washings of uncut leaf tissue of B. orphanidis were concentrated, separated and puri®ed by a combination of paper chromatography, TLC and HPLC to give nine ¯avones. Because of the rarity of the plant material and the complexity of the ¯avonoid mixture present, no compound was available in more than milligram amounts. Three were readily identi®ed by direct comparison with authentic markers as luteolin, apigenin and acacetin. Ultraviolet, MS and NMR studies established that the remaining six ¯avones have a dimethoxy±dihydroxy substitution in the A-ring and are variously substituted in the B-ring with either 40 - or 30 ,40 hydroxy/methoxy substituents. One compound (5) has an additional methyl substitution in the A-ring (see below).
short UV, a peak at 277±280 nm and a second peak or shoulder at 294±296 nm. These are exactly matched in the UV spectrum of nevadensin (5,7-dihydroxy-6,8,40 trimethoxy¯avone, 2) but dier in the alternative structures (5,6-dihydroxy- and 5,8-dihydroxy). Similarly in the luteolin series, ¯avones 4±6, there are two short wavelength UV bands at 255 and 278±280 nm. This is almost precisely matched by hymenoxin (5,7-dihydroxy6,8,30 ,40 -tetramethoxy¯avone, 6) but diers in the ¯avones with alternative structures (Table 1). From these results, therefore, it is possible to conclude that 1 is 5,7,40 -trihydroxy-6,8-dimethoxy¯avone, 2 is nevadensin, 3 is gardenin B, 4 is acerosin, 5 is sudachitin and 6 is hymenoxin. Authentic markers of nevadensin and gardenin B were available for comparison and co-TLC and co-HPLC con®rmed the identities of 2 and 3. Other relevant data for 1±6 are collected in Table 2. Additional evidence supporting methoxylation at positions 6- and 8- is present in the mass spectra of 1±6. All the compounds measured have an M-15 fragment ion which is more intense than the molecular ion. This is a characteristic feature of 6- and 8-methoxylated ¯avones (Harborne et al., 1978). Identi®cation of 2 as nevadensin was also con®rmed using 1D 1H, 1D 1H±1H XSROESY and 2D 1H±13C HSQC NMR experiments acquired at 500 MHz. In particular, the location of the three methoxyl substituents was established using the 1D 1H±1H XSROESY experiment, which allows detection of `through-space' 1H±1H ROE connectivities by siteselective excitation of individual resonances in the 1D 1 H NMR spectrum (Gradwell et al., 1997). Application of this procedure to nevadensin has been described Table 1 UV spectral maxima of highly substituted ¯avone methyl ethers Compound
The only outstanding structural problem remaining was the location of the methoxyl substituents in the Aring. Spectral shifts with aluminium chloride in the UV region showed that the 5-hydroxyl was free in all six compounds. Placement of the other free hydroxyl group at position 7 on spectral evidence alone was not possible, because 7-hydroxy¯avones which are methoxylated at adjacent positions (6-, 8- or 6,8-) fail to undergo the normal bathochromic sodium acetate shift that would be expected. Proof that these six ¯avones have the 5,7dihydroxy-6,8-dimethoxy A-ring substitution came from a detailed consideration of the neutral UV spectra (Table 1), when compared with model compounds. The data in Table 1 show that in the apigenin series of ¯avones (compounds 1±3) there are two signals in the
Apigenin-based ¯avones 1 2 nevadensin 3 gardenin B Model ¯avonesa 5,7-dihydroxy-6,8-dimethoxy 5,6-dihydroxy-7,8-dimethoxy 5,8-dihydroxy-6,7-dimethoxy Luteolin-based ¯avones 4 acerosin 5 sudachitin 6 hymenoxin 5,7-dihydroxy-6,8-dimethoxy 5,6-dihydroxy-7,8-dimethoxy 5,8-dihydroxy-6,7-dimethoxy
UV spectral maxima (nm) in MeOHb 278, 296sh, 335 280, 294sh, 333 277, 296, 333 280, 294sh, 333 ±, 297, 331 282, ±, 332 255, 279, 346 255, 278, 348 254, 280, 344 250sh, 279, 336 252, 288, 344 254, 284, 298sh, 341
a Apigenin series: nevadensin, pilosin, pedunculin; luteolin series: hymenoxin, thymonin, leucantho¯avone. These spectral data are based on Grayer et al. (2000), Harborne and Baxter (1999) and references therein, and La Duke (1982). b sh=shoulder.
J. Greenham et al. / Phytochemistry 56 (2001) 87±91
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of which were 59.9, 61.0 and 55.6, respectively, by HSQC. The B-ring was found to be substituted at both C30 and C40 , according to the 1H NMR spectrum, with characteristic resonances at 6.97 (d, J=8.8 Hz, H-50 ), 7.55 (dd, J=8.8, 2.1 Hz, H-60 ) and 7.56 (d, J=2.1 Hz, H-20 ). Site-selective excitation of the methoxyl resonance at 3.89 using 1D XSROESY gave a ROE connectivity to H-20 ( 7.56), indicating that the corresponding methoxyl group must be located at C30 , rather than C40 . The remaining methoxyl groups were assigned to C6 and C8 on the basis of their 1H and 13C chemical shift values, again by analogy with corresponding data for 1 and 2. The identity of 5 was thus con®rmed to be 5,7,40 trihydroxy-6,8,30 -trimethoxy¯avone. All these ¯avones, apart from 1, have been reported before in nature (Harborne and Baxter, 1999) and have largely been found in plants of the Compositae. In general, methylated ¯avones with 5,6,7,8,40 -pentahydroxy or 5,6,7,8,30 ,40 -hexahydroxy substitution are mainly con®ned to plants of the Rutaceae, Labiatae and Compositae. In Biebersteinia, there are two biogenetically related series present. In the apigenin series, there are the three compounds increasing in O-methylation from 1 to 2 to 3. In the luteolin series, there is the so far undetected 5,7,30 ,40 -tetrahydroxy-6,8-dimethoxy¯avone, a putative precursor which on increasing O-methylation has yielded 4 and 5 and eventually 6.
recently, and allows the speci®c assignment of the methoxyl 1H NMR resonances of the methoxyl substituents of 2 at 3.78, 3.87 and 3.88 (in DMSO-d6) to 6-OCH3, 40 -OCH3 and 8-OCH3, respectively (Grayer et al., 2000). The corresponding 13C NMR resonances were obtained by inverse-detection using the HSQC experiment, as 59.7, 55.2 and 60.8, respectively. The amount of 1 available for NMR analysis was less than 0.1 mg, which did not permit use of the 1D XSROESY experiment over a reasonable timescale. However, analysis of the 1D 1H NMR spectrum indicated that the compound contained only two methoxyl substituents ( 3.78 and 3.86). These could be placed in the A-ring, as the chemical shift values of the H-20 ,60 ( 7.92, 2H, d, J=8.9 Hz) and H-30 ,50 ( 6.96, 2H, d, J=8.9 Hz) protons in the B-ring indicated that the 40 -OH group was free (Markham and Geiger, 1994). The chemical shift values of the 13C resonances of the methoxyl groups at 3.78 and 3.86 were obtained as 60.1 and 61.1, respectively, by HSQC. The combined 1H and 13C NMR data indicated assignment of these resonances to 6-OCH3 and 8-OCH3, respectively, by comparison with data for the same substituents in 2. Thus the NMR data for 1 supported its identi®cation as 5,7,40 -trihydroxy-6,8dimethoxy¯avone. In the case of 5, three methoxyl resonances at 3.78, 3.88 and 3.89 were detected in the 1 H NMR spectrum, the corresponding 13C resonances
Table 2 Spectral and chromatographic data for ¯avone methyl ethers 1±6 Mass spectraa
TLCb Rf100
Compound
Structure
M
B-ring
HPLC Rt (min)
1
2
1 2 3 4 5 6
5,7,40 -triOH,6,8-diOMe 5,7-diOH,6,8,40 -triOMe (nevadensin) 5-OH,6,7,8,40 -tetraOMe (gardenin B) 5,7,30 -triOH,6,8,40 -triOMe (acerosin) 5,7,40 -triOH,6,8,30 -triOMe (sudachitin) 5,7-diOH,6,8,30 ,40 -tetraOMe (hymenoxin)
330 344 ± 360 360 374
121 132 ± ± 149 162
7.04 11.94 16.38 8.11 7.37 10.77
16 47 47 31 31 39
66 83 ± 61 61 72
a Five of the six compounds show a fragment ion at 197 a.m.u., corresponding to a dihydroxydimethoxy A-ring. All ®ve showed an M-15 ion uniformly more intense (ca. 117%) than the molecular ion. b TLC system 1, toluene-HOAc (4:1) on silica gel plates. TLC system 2, 50% HOAc on cellulose.
Table 3 Internal ¯avonoids of B. orphanidis leaf Rf (100) a
Compound
Identity
Neutral max
HPLC Rt
BAW
15% HOAc
7 8 9 10 11
Luteolin 7-glucoside Luteolin 7-rutinoside Apigenin 7-glucoside Apigenin 7-rutinoside Tricetin 7-glucoside
255, 267, 350 255, 267, 350 267, 336 267, 336 249, 260, 268, 352
17.87 17.70 21.02 20.50 14.26
44 38 56 57 21
07 24 17 29 09
a
Positive shifts with NaOH, NaOAc, AlCl3, AlCl3±HCl with all compounds and with H3BO3 in the case of 7, 8 and 10.
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J. Greenham et al. / Phytochemistry 56 (2001) 87±91
2.2. Vacuolar ¯avonoids Leaf material of B. orphanidis, after acetone washing to remove surface ¯avonoids, was extracted into 80% methanol. The water-soluble ¯avonoids present were then separated and puri®ed by standard procedures. Identi®cations were based on UV spectral analyses, hydrolyses to aglycone and sugar and, wherever possible, direct comparison by co-PC and co-HPLC with authentic markers. The results for the ®ve major ¯avone glycosides are shown in Table 3. The compounds present are the 7-glucosides of apigenin, luteolin and tricetin (9, 7 and 11) and the 7-rutinoside of apigenin and luteolin (10 and 8). The 7-rutinoside linkage and site of attachment in 8 was con®rmed by 1H NMR measurements and co-chromatography with an authentic sample in our collection. Some free tricetin and free luteolin were present in the internal ¯avonoid extract. Three further ¯avone glycosides were detected, but none was present in sucient quantity for complete characterisation. Two of the three compounds were provisionally recorded as luteolin and tricetin 7-diglucosides, on the basis of acid hydrolysis and chromatographic mobilities. The third compound appeared to be an apigenin 7-glucoside with aliphatic acylation. 3. Discussion The present studies show that B. orphanidis has a highly distinctive ¯avonoid pro®le and uniquely contains a set of six closely related ¯avone methyl ethers, with dihydroxy, dimethoxy A-ring substitution. Related work on B. heterostemon (Zhang et al., 1995) and our own preliminary investigation on the ¯avonoids of B. odora suggest that this pattern in ¯avonoid production is common throughout this small genus. These plants are quite dierent from species in the Geraniaceae, which generally have ¯avonols, condensed and hydrolysable tannins (Bate-Smith, 1973). The chemical data indicate that the earlier positioning of Biebersteinia in the Geraniaceae or Geraniales is misplaced and agree with the conclusions of an rbcL and atpB sequence comparison that Biebersteinia deserves to be placed in its own family. Furthermore, the DNA analyses provide support for a position for Biebersteinia in the Sapindales. What is particularly interesting is that it is placed near to Rutaceae species (Bakker et al., 1998). Such a link was proposed originally by De Candolle (1824). It is now apparent in the ¯avonoid data, since methylated ¯avones with 5,7dihydroxy-6,8-dimethoxy A-ring substitution are quite uncommon in nature but do appear in a number of Rutaceae spp. In fact, sudachitin, here reported for the ®rst time in Biebersteinia, was earlier isolated from
unripe fruit of Citrus sudachii and fruit peel of C. reticulata (Horie et al., 1961). Thus, these chemical results are completely in harmony with the macromolecular phylogeny which places Biebersteinia fairly close to Ruta and Xanthoxylum of the Rutaceae, and within the same order Sapindales.
4. Experimental 4.1. Plant materials Aerial parts of B. orphanidis were collected from the wild in Greece in May 1997 most of which were airdried. It was veri®ed by D. Vassiliades and a herbarium specimen has been deposited in the Herbarium of the University of Athens (ATHU). 4.2. Flavonoid analysis 4.2.1. Lipophilic ¯avonoids Lipophilic ¯avonoids were rinsed from uncut leaf tissue by brie¯y dipping 2 in Me2CO at room temperature. The concentrated extracts were run on silica gel TLC in toluene:HOAc (4:1) and the eluted bands rerun on Whatman 3MM paper in 30% HOAc. Final puri®cation was either on paper in H2O or by CC on Sephadex LH-20. Compounds 1, 2 and 5 were further puri®ed by HPLC before 1H and 13C NMR analysis using a reverse phase Waters Bondapak Phenyl analytical column at 25 C and UV detection at 350 nm on a diode array detector. The solvents used were (A) 2% HOAc and (B) MeOH±HOAc±H2O (18:1:1) using a ¯ow rate of 1 ml/min and a gradient elution programme changing from 40% A/60%B to 100%B over 20 min in a linear mode. Molecular weights were determined by EIMS and LCMS. Known compounds were identi®ed by comparison of UV spectral data and Rf values in six solvents with standard markers and/or literature data. 4.2.2. NMR analyses NMR spectra were recorded using a Varian 500 MHz instrument. All samples studied were dissolved in DMSO-d6 and referenced to the residual solvent resonance at H 2.50 ppm or C 39.50 ppm as appropriate. All NMR experiments were carried out at 37 C. 4.2.2.1. 5,7,40 -Trihydroxy-6,8-dimethoxyflavone (1). 1H NMR (500 MHz, DMSO-d6): 7.92 (2H, d, J=2.1 Hz, H-20 ), 6.96 (2H, d, J=8.9 Hz, H-30 ,50 ), 6.78 (1H, s, H-3), 3.86 (3H, s, 8-OCH3), 3.78 (3H, s, 6-OCH3); 13C NMR (125 MHz, DMSO-d6) (assignment of selected nonquaternary C atoms from HSQC): 61.1 (8-OCH3), 60.1 (6-OCH3).
J. Greenham et al. / Phytochemistry 56 (2001) 87±91
4.2.2.2. 5,7-Dihydroxy-6,8,40 -trimethoxyflavone (Nevadensin) (2). 1H NMR (500 MHz, DMSO-d6): 12.75 (1H, s, 5-OH), 8.02 (2H, d, J=8.8 Hz, H-20 ,60 ), 7.15 (2H, d, J=8.8 Hz, H-30 ,50 ), 6.86 (1H, s, H-3), 3.88 (3H, s, 8-OCH3), 3.87 (3H, s, 40 -OCH3), 3.78 (3H, s, 6-OCH3); 13 C NMR (125 MHz, DMSO-d6) (assignment of nonquaternary C atoms from HSQC): 127.9 (C-20 ,60 ), 114.5 (C-30 ,50 ), 102.9 (C-3), 60.8 (8-OCH3), 59.7 (6OCH3), 55.2 (40 -OCH3). 4.2.2.3. 5,7,40 -Trihydroxy-6,8,30 -trimethoxy¯avone (5) 1 H NMR (500 MHz, DMSO-d6): 7.56 (1H, d, J=8.8 Hz, H-20 ), 7.55 (dd, J=8.8, 2.1 Hz, H-60 ), 6.97 (d, J=8.8 Hz, H-50 ), 6.86 (1H, s, H-3), 3.89 (3H, s, 30 OCH3), 3.88 (3H, s, 8-OCH3), 3.78 (3H, s, 6-OCH3); 13 C NMR (125 MHz, DMSO-d6) (assignment of nonquaternary C atoms from HSQC): 120.1 (C-20 ), 116.0 (C-50 ), 110.0 (C-60 ), 102.6 (C-3), 61.0 (8-OCH3), 59.9 (6OCH3), 55.6 (30 -OCH3). 4.3. Polar ¯avonoids Leaf tissue of B. orphanidis, after removal of the surface ¯avonoids with acetone, was extracted with boiling 80% methanol. The concentrated extract was run on Whatman 3 MM paper in BAW (n-BuOH:HOAc:H2O, 4:1:5, top layer) and the eluted bands separated in 15% HOAc. Further puri®cation was carried out in BAW, CAW (CHCl3:HOAc:H2O, 10:10:1), 15% HOAc and H2O. The known glycosides 7±11 were identi®ed by standard procedures (TLC, HPLC, UV spectral data) including comparison with authentic markers. UV spectra, Rf and HPLC data are shown in Table 3. The HPLC programme for these ¯avone glycosides involved the use of 75% solvent A/25% solvent B to 35% A/65% B over 23 min in a linear gradient. Otherwise, HPLC details are similar to those described for the lipophilic ¯avonoids.
Acknowledgements We thank Professor J.S. Parker, Director of the Cambridge Botanic Garden, for a plant of Biebersteinia odora.
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References Bakker, F.T., Vassiliades, D.D., Morton, C., Savolainen, V., 1998. Phylogenetic relationships of Biebersteinia Stephan (Geraniaceae) inferred from rbcL and atpB seqence comparisons. Botanical Journal of the Linnean Society 127, 149±158. Bate-Smith, E.C., 1973. Chemotaxonomy of geranium. Botanical Journal of the Linnean Society 67, 347±359. Boissier, E., 1867. Biebersteiniae. Flora Orientalis 1, 899±900. BortenschlaÈger, S., 1967. VorlaÈu®ge Mitteilungen zur Pollenmorphologie in der familie der Geraniaceen und ihre systematische Bedeutung. Grana Palynologica 7, 2±3. De Candolle, A.P., 1824. Biebersteinia. In: Prodromus Systematis Naturalis Regni Vegetabilis, Sine Enumerato Contracta Ordinum Generum Specierumque Plantarum, Vol. 1. Argentorati & Londini, Paris, p. 707. Gradwell, M.J., Kogelberg, H., Frenkiel, T.A., 1997. Applying excitation sculpting to construct singly and doubly selective ID NMR experiments. Journal of Magnetic Resonance 124, 267±270. Grayer, R.J., Veitch, N.C., Kite, G.C., Price, A.M., Kokubun, T., 2000. Distribution of 8-oxygenated external ¯avones in the genus Ocimum. Phytochemistry, in preparation Harborne, J.B., Baxter, H., 1999. The Handbook of Natural Flavonoids,, Vol. 1. Wiley, Chichester. Harborne, J.B., Saleh, N.A.M., Smith, D.M., 1978. On the natural occurrence of gossypetin 7- and 8-monomethyl ethers. Phytochemistry 17, 589±591. Horie, T., Masumura, M., Okumura, F.S., 1961. Sudachitin, a new ¯avone pigment of sudachi. Bulletin of the Chemical Society of Japan 34, 1547. Kamelina, O.P., Komarova, V.A., 1990. Embryological characters of the genus Biebersteinia Stephan in relation to its systematic position. Doklady Akademii Nauk Tadzhikskoi SSR 33, 193±195. Kunth, R., 1931. Tribe Biebersteineae. In: Harms, H. (Ed.), Die NatuÈrlichen P¯anzenfamilien. Band 19a. Verlag von Wilhelm Engelmann, Leipzig, Germany. La Duke, J.C., 1982. Flavonoid chemistry and systematics of Tithonia. American Journal of Botany 69, 784±792. Markham, K.R., Geiger, H., 1994. 1H NMR spectroscopy of ¯avonoids and their glycosides in DMSO-d6. In: Harborne, J.B. (Ed.), The Flavonoids, Advances in Research since 1986. Chapman and Hall, London, pp. 441±497. Omurkamzinova, V.B., Maurel, N.D., Bikbulatova, T.N., 1991. Flavonoids from Biebersteinia multi®da. Khimiya Prirodnykh Soedinenii 5, 720±721. Tutel, B., 1982. Comparison of the taxonomy and the leaf anatomy of the genus Biebersteinia with the other genera of Geraniaceae of Turkey, Istanbul Universitesi Fen Fakultesi Mecmuasi. Seri B, 47± 48, 51±87 Zhang, X.F., Hu, B.L., Zhou, B.N., 1995. Studies on the active constituents of Tibetan herb Biebersteinia heterostemon Maxim. Acta Pharmaceutica Sinica 30, 211±214.
www.elsevier.com/locate/phytochem
A distinctive ¯avonoid chemistry for the anomalous genus Biebersteinia Jenny Greenham a, Dionyssios D. Vassiliades a, Jerey B. Harborne a,*, Christine A. Williams a, John Eagles b, ReneÂe J. Grayer c, Nigel C. Veitch c a
Department of Botany, The University of Reading, Whiteknights, PO Box 221, Reading RG6 6AS, UK b Institute of Food Research, Colney, Norwich NR4 7UA, UK c Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK Received 8 June 2000; received in revised form 24 August 2000
Abstract Leaf surface extracts of Biebersteinia orphanidis have yielded a complex mixture of ®ve ¯avones with the unusual 5,7-dihydroxy6,8-dimethoxy A ring substitution pattern. They are acerosin, hymenoxin, nevadensin, sudachitin and 5,7,40 -trihydroxy-6,8-dimethoxy¯avone. Also present at the leaf surface are gardenin B, luteolin, apigenin, acacetin and the coumarin umbelliferone. The internal leaf ¯avonoids include the 7-glucosides of apigenin, luteolin and tricetin, together with the 7-rutinosides of apigenin and luteolin. This pro®le diers from those of B. heterostemon and B. odora. It appears that B. orphanidis is as highly distinctive in its ¯avonoid pattern as it is phytogeographically. The data also con®rm the conclusion of other studies, including rbcL and atpB gene sequence analysis, that Biebersteinia is completely unrelated to the Geraniaceae, where it was once placed. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Flavonoids; Biebersteinia; Chemotaxonomy
1. Introduction Biebersteinia Stephan is a genus of ®ve rare, herbaceous species distributed in the mountainous semi-arid areas of central and western Asia and the eastern Mediterranean as far as Greece. Its taxonomic position has been in dispute since its discovery in 1806. In 1841, Endlicher gave it familial status, while Boissier included it ®rst in the Zygophyllaceae and then later (1867) in the Geraniaceae. Subsequent authors, notably Kunth (1931), have followed the latter classi®cation. However, a palynological study by BortenschlaÈger (1967) suggested it was wrongly placed in the Geraniaceae and this was supported by later anatomical (Tutel, 1982), embryological (Kamelina and Komarova, 1990) and chemotaxonomic (Bate-Smith, 1973) studies. A recent phylogenetic analysis of rbcL and atpB gene sequences of B. orphanidis Boiss. (Bakker et al., 1998) indicated that the genus was nested in the Sapindales but with no * Corresponding author. Tel.: +44-118-931-8162; fax: +44-118975 3676.
link to any other clade within this order or within the Geraniales. Phytochemically, the genus has not been analysed in detail. Bate-Smith (1973) in a phenolic survey of Geranium and related genera examined Bierbersteinia multi®da DC. and B. odora Stephan and found that they had completely dierent ¯avonoid patterns from those known for any other genus of the Geraniaceae. He reported myricetin, prodelphinidin and procyanidin from B. odora and a number of unusual unidenti®ed ¯avonoids from both taxa. A later phytochemical paper included a study of the ¯avones (luteolin 7-glucoside and 7-rutinoside) (Omurkamzinova et al., 1991) of B. multi®da. In the Tibetan species, B. heterostemon Maxim. (Zhang et al., 1995) a rare ¯avone, 8-hydroxytricetin 8,40 ,50 -trimethyl ether was reported together with a luteolin C-glucoside, quercetin 7-glucoside and umbelliferone. Biebersteinia orphanidis, which has not been investigated previously for its phenolic constituents, occupies a dierent phytogeographical position from the other four species, i.e. in Anatolian Turkey and Greece.
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00355-1
88
J. Greenham et al. / Phytochemistry 56 (2001) 87±91
Therefore, the present ¯avonoid analysis of B. orphanidis was undertaken in the hope it might reveal useful geographic dierences and hence some insight into the evolution within the genus and the taxonomic position of Biebersteinia in the dicotyledons. 2. Results 2.1. Surface ¯avonoids The acetone washings of uncut leaf tissue of B. orphanidis were concentrated, separated and puri®ed by a combination of paper chromatography, TLC and HPLC to give nine ¯avones. Because of the rarity of the plant material and the complexity of the ¯avonoid mixture present, no compound was available in more than milligram amounts. Three were readily identi®ed by direct comparison with authentic markers as luteolin, apigenin and acacetin. Ultraviolet, MS and NMR studies established that the remaining six ¯avones have a dimethoxy±dihydroxy substitution in the A-ring and are variously substituted in the B-ring with either 40 - or 30 ,40 hydroxy/methoxy substituents. One compound (5) has an additional methyl substitution in the A-ring (see below).
short UV, a peak at 277±280 nm and a second peak or shoulder at 294±296 nm. These are exactly matched in the UV spectrum of nevadensin (5,7-dihydroxy-6,8,40 trimethoxy¯avone, 2) but dier in the alternative structures (5,6-dihydroxy- and 5,8-dihydroxy). Similarly in the luteolin series, ¯avones 4±6, there are two short wavelength UV bands at 255 and 278±280 nm. This is almost precisely matched by hymenoxin (5,7-dihydroxy6,8,30 ,40 -tetramethoxy¯avone, 6) but diers in the ¯avones with alternative structures (Table 1). From these results, therefore, it is possible to conclude that 1 is 5,7,40 -trihydroxy-6,8-dimethoxy¯avone, 2 is nevadensin, 3 is gardenin B, 4 is acerosin, 5 is sudachitin and 6 is hymenoxin. Authentic markers of nevadensin and gardenin B were available for comparison and co-TLC and co-HPLC con®rmed the identities of 2 and 3. Other relevant data for 1±6 are collected in Table 2. Additional evidence supporting methoxylation at positions 6- and 8- is present in the mass spectra of 1±6. All the compounds measured have an M-15 fragment ion which is more intense than the molecular ion. This is a characteristic feature of 6- and 8-methoxylated ¯avones (Harborne et al., 1978). Identi®cation of 2 as nevadensin was also con®rmed using 1D 1H, 1D 1H±1H XSROESY and 2D 1H±13C HSQC NMR experiments acquired at 500 MHz. In particular, the location of the three methoxyl substituents was established using the 1D 1H±1H XSROESY experiment, which allows detection of `through-space' 1H±1H ROE connectivities by siteselective excitation of individual resonances in the 1D 1 H NMR spectrum (Gradwell et al., 1997). Application of this procedure to nevadensin has been described Table 1 UV spectral maxima of highly substituted ¯avone methyl ethers Compound
The only outstanding structural problem remaining was the location of the methoxyl substituents in the Aring. Spectral shifts with aluminium chloride in the UV region showed that the 5-hydroxyl was free in all six compounds. Placement of the other free hydroxyl group at position 7 on spectral evidence alone was not possible, because 7-hydroxy¯avones which are methoxylated at adjacent positions (6-, 8- or 6,8-) fail to undergo the normal bathochromic sodium acetate shift that would be expected. Proof that these six ¯avones have the 5,7dihydroxy-6,8-dimethoxy A-ring substitution came from a detailed consideration of the neutral UV spectra (Table 1), when compared with model compounds. The data in Table 1 show that in the apigenin series of ¯avones (compounds 1±3) there are two signals in the
Apigenin-based ¯avones 1 2 nevadensin 3 gardenin B Model ¯avonesa 5,7-dihydroxy-6,8-dimethoxy 5,6-dihydroxy-7,8-dimethoxy 5,8-dihydroxy-6,7-dimethoxy Luteolin-based ¯avones 4 acerosin 5 sudachitin 6 hymenoxin 5,7-dihydroxy-6,8-dimethoxy 5,6-dihydroxy-7,8-dimethoxy 5,8-dihydroxy-6,7-dimethoxy
UV spectral maxima (nm) in MeOHb 278, 296sh, 335 280, 294sh, 333 277, 296, 333 280, 294sh, 333 ±, 297, 331 282, ±, 332 255, 279, 346 255, 278, 348 254, 280, 344 250sh, 279, 336 252, 288, 344 254, 284, 298sh, 341
a Apigenin series: nevadensin, pilosin, pedunculin; luteolin series: hymenoxin, thymonin, leucantho¯avone. These spectral data are based on Grayer et al. (2000), Harborne and Baxter (1999) and references therein, and La Duke (1982). b sh=shoulder.
J. Greenham et al. / Phytochemistry 56 (2001) 87±91
89
of which were 59.9, 61.0 and 55.6, respectively, by HSQC. The B-ring was found to be substituted at both C30 and C40 , according to the 1H NMR spectrum, with characteristic resonances at 6.97 (d, J=8.8 Hz, H-50 ), 7.55 (dd, J=8.8, 2.1 Hz, H-60 ) and 7.56 (d, J=2.1 Hz, H-20 ). Site-selective excitation of the methoxyl resonance at 3.89 using 1D XSROESY gave a ROE connectivity to H-20 ( 7.56), indicating that the corresponding methoxyl group must be located at C30 , rather than C40 . The remaining methoxyl groups were assigned to C6 and C8 on the basis of their 1H and 13C chemical shift values, again by analogy with corresponding data for 1 and 2. The identity of 5 was thus con®rmed to be 5,7,40 trihydroxy-6,8,30 -trimethoxy¯avone. All these ¯avones, apart from 1, have been reported before in nature (Harborne and Baxter, 1999) and have largely been found in plants of the Compositae. In general, methylated ¯avones with 5,6,7,8,40 -pentahydroxy or 5,6,7,8,30 ,40 -hexahydroxy substitution are mainly con®ned to plants of the Rutaceae, Labiatae and Compositae. In Biebersteinia, there are two biogenetically related series present. In the apigenin series, there are the three compounds increasing in O-methylation from 1 to 2 to 3. In the luteolin series, there is the so far undetected 5,7,30 ,40 -tetrahydroxy-6,8-dimethoxy¯avone, a putative precursor which on increasing O-methylation has yielded 4 and 5 and eventually 6.
recently, and allows the speci®c assignment of the methoxyl 1H NMR resonances of the methoxyl substituents of 2 at 3.78, 3.87 and 3.88 (in DMSO-d6) to 6-OCH3, 40 -OCH3 and 8-OCH3, respectively (Grayer et al., 2000). The corresponding 13C NMR resonances were obtained by inverse-detection using the HSQC experiment, as 59.7, 55.2 and 60.8, respectively. The amount of 1 available for NMR analysis was less than 0.1 mg, which did not permit use of the 1D XSROESY experiment over a reasonable timescale. However, analysis of the 1D 1H NMR spectrum indicated that the compound contained only two methoxyl substituents ( 3.78 and 3.86). These could be placed in the A-ring, as the chemical shift values of the H-20 ,60 ( 7.92, 2H, d, J=8.9 Hz) and H-30 ,50 ( 6.96, 2H, d, J=8.9 Hz) protons in the B-ring indicated that the 40 -OH group was free (Markham and Geiger, 1994). The chemical shift values of the 13C resonances of the methoxyl groups at 3.78 and 3.86 were obtained as 60.1 and 61.1, respectively, by HSQC. The combined 1H and 13C NMR data indicated assignment of these resonances to 6-OCH3 and 8-OCH3, respectively, by comparison with data for the same substituents in 2. Thus the NMR data for 1 supported its identi®cation as 5,7,40 -trihydroxy-6,8dimethoxy¯avone. In the case of 5, three methoxyl resonances at 3.78, 3.88 and 3.89 were detected in the 1 H NMR spectrum, the corresponding 13C resonances
Table 2 Spectral and chromatographic data for ¯avone methyl ethers 1±6 Mass spectraa
TLCb Rf100
Compound
Structure
M
B-ring
HPLC Rt (min)
1
2
1 2 3 4 5 6
5,7,40 -triOH,6,8-diOMe 5,7-diOH,6,8,40 -triOMe (nevadensin) 5-OH,6,7,8,40 -tetraOMe (gardenin B) 5,7,30 -triOH,6,8,40 -triOMe (acerosin) 5,7,40 -triOH,6,8,30 -triOMe (sudachitin) 5,7-diOH,6,8,30 ,40 -tetraOMe (hymenoxin)
330 344 ± 360 360 374
121 132 ± ± 149 162
7.04 11.94 16.38 8.11 7.37 10.77
16 47 47 31 31 39
66 83 ± 61 61 72
a Five of the six compounds show a fragment ion at 197 a.m.u., corresponding to a dihydroxydimethoxy A-ring. All ®ve showed an M-15 ion uniformly more intense (ca. 117%) than the molecular ion. b TLC system 1, toluene-HOAc (4:1) on silica gel plates. TLC system 2, 50% HOAc on cellulose.
Table 3 Internal ¯avonoids of B. orphanidis leaf Rf (100) a
Compound
Identity
Neutral max
HPLC Rt
BAW
15% HOAc
7 8 9 10 11
Luteolin 7-glucoside Luteolin 7-rutinoside Apigenin 7-glucoside Apigenin 7-rutinoside Tricetin 7-glucoside
255, 267, 350 255, 267, 350 267, 336 267, 336 249, 260, 268, 352
17.87 17.70 21.02 20.50 14.26
44 38 56 57 21
07 24 17 29 09
a
Positive shifts with NaOH, NaOAc, AlCl3, AlCl3±HCl with all compounds and with H3BO3 in the case of 7, 8 and 10.
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J. Greenham et al. / Phytochemistry 56 (2001) 87±91
2.2. Vacuolar ¯avonoids Leaf material of B. orphanidis, after acetone washing to remove surface ¯avonoids, was extracted into 80% methanol. The water-soluble ¯avonoids present were then separated and puri®ed by standard procedures. Identi®cations were based on UV spectral analyses, hydrolyses to aglycone and sugar and, wherever possible, direct comparison by co-PC and co-HPLC with authentic markers. The results for the ®ve major ¯avone glycosides are shown in Table 3. The compounds present are the 7-glucosides of apigenin, luteolin and tricetin (9, 7 and 11) and the 7-rutinoside of apigenin and luteolin (10 and 8). The 7-rutinoside linkage and site of attachment in 8 was con®rmed by 1H NMR measurements and co-chromatography with an authentic sample in our collection. Some free tricetin and free luteolin were present in the internal ¯avonoid extract. Three further ¯avone glycosides were detected, but none was present in sucient quantity for complete characterisation. Two of the three compounds were provisionally recorded as luteolin and tricetin 7-diglucosides, on the basis of acid hydrolysis and chromatographic mobilities. The third compound appeared to be an apigenin 7-glucoside with aliphatic acylation. 3. Discussion The present studies show that B. orphanidis has a highly distinctive ¯avonoid pro®le and uniquely contains a set of six closely related ¯avone methyl ethers, with dihydroxy, dimethoxy A-ring substitution. Related work on B. heterostemon (Zhang et al., 1995) and our own preliminary investigation on the ¯avonoids of B. odora suggest that this pattern in ¯avonoid production is common throughout this small genus. These plants are quite dierent from species in the Geraniaceae, which generally have ¯avonols, condensed and hydrolysable tannins (Bate-Smith, 1973). The chemical data indicate that the earlier positioning of Biebersteinia in the Geraniaceae or Geraniales is misplaced and agree with the conclusions of an rbcL and atpB sequence comparison that Biebersteinia deserves to be placed in its own family. Furthermore, the DNA analyses provide support for a position for Biebersteinia in the Sapindales. What is particularly interesting is that it is placed near to Rutaceae species (Bakker et al., 1998). Such a link was proposed originally by De Candolle (1824). It is now apparent in the ¯avonoid data, since methylated ¯avones with 5,7dihydroxy-6,8-dimethoxy A-ring substitution are quite uncommon in nature but do appear in a number of Rutaceae spp. In fact, sudachitin, here reported for the ®rst time in Biebersteinia, was earlier isolated from
unripe fruit of Citrus sudachii and fruit peel of C. reticulata (Horie et al., 1961). Thus, these chemical results are completely in harmony with the macromolecular phylogeny which places Biebersteinia fairly close to Ruta and Xanthoxylum of the Rutaceae, and within the same order Sapindales.
4. Experimental 4.1. Plant materials Aerial parts of B. orphanidis were collected from the wild in Greece in May 1997 most of which were airdried. It was veri®ed by D. Vassiliades and a herbarium specimen has been deposited in the Herbarium of the University of Athens (ATHU). 4.2. Flavonoid analysis 4.2.1. Lipophilic ¯avonoids Lipophilic ¯avonoids were rinsed from uncut leaf tissue by brie¯y dipping 2 in Me2CO at room temperature. The concentrated extracts were run on silica gel TLC in toluene:HOAc (4:1) and the eluted bands rerun on Whatman 3MM paper in 30% HOAc. Final puri®cation was either on paper in H2O or by CC on Sephadex LH-20. Compounds 1, 2 and 5 were further puri®ed by HPLC before 1H and 13C NMR analysis using a reverse phase Waters Bondapak Phenyl analytical column at 25 C and UV detection at 350 nm on a diode array detector. The solvents used were (A) 2% HOAc and (B) MeOH±HOAc±H2O (18:1:1) using a ¯ow rate of 1 ml/min and a gradient elution programme changing from 40% A/60%B to 100%B over 20 min in a linear mode. Molecular weights were determined by EIMS and LCMS. Known compounds were identi®ed by comparison of UV spectral data and Rf values in six solvents with standard markers and/or literature data. 4.2.2. NMR analyses NMR spectra were recorded using a Varian 500 MHz instrument. All samples studied were dissolved in DMSO-d6 and referenced to the residual solvent resonance at H 2.50 ppm or C 39.50 ppm as appropriate. All NMR experiments were carried out at 37 C. 4.2.2.1. 5,7,40 -Trihydroxy-6,8-dimethoxyflavone (1). 1H NMR (500 MHz, DMSO-d6): 7.92 (2H, d, J=2.1 Hz, H-20 ), 6.96 (2H, d, J=8.9 Hz, H-30 ,50 ), 6.78 (1H, s, H-3), 3.86 (3H, s, 8-OCH3), 3.78 (3H, s, 6-OCH3); 13C NMR (125 MHz, DMSO-d6) (assignment of selected nonquaternary C atoms from HSQC): 61.1 (8-OCH3), 60.1 (6-OCH3).
J. Greenham et al. / Phytochemistry 56 (2001) 87±91
4.2.2.2. 5,7-Dihydroxy-6,8,40 -trimethoxyflavone (Nevadensin) (2). 1H NMR (500 MHz, DMSO-d6): 12.75 (1H, s, 5-OH), 8.02 (2H, d, J=8.8 Hz, H-20 ,60 ), 7.15 (2H, d, J=8.8 Hz, H-30 ,50 ), 6.86 (1H, s, H-3), 3.88 (3H, s, 8-OCH3), 3.87 (3H, s, 40 -OCH3), 3.78 (3H, s, 6-OCH3); 13 C NMR (125 MHz, DMSO-d6) (assignment of nonquaternary C atoms from HSQC): 127.9 (C-20 ,60 ), 114.5 (C-30 ,50 ), 102.9 (C-3), 60.8 (8-OCH3), 59.7 (6OCH3), 55.2 (40 -OCH3). 4.2.2.3. 5,7,40 -Trihydroxy-6,8,30 -trimethoxy¯avone (5) 1 H NMR (500 MHz, DMSO-d6): 7.56 (1H, d, J=8.8 Hz, H-20 ), 7.55 (dd, J=8.8, 2.1 Hz, H-60 ), 6.97 (d, J=8.8 Hz, H-50 ), 6.86 (1H, s, H-3), 3.89 (3H, s, 30 OCH3), 3.88 (3H, s, 8-OCH3), 3.78 (3H, s, 6-OCH3); 13 C NMR (125 MHz, DMSO-d6) (assignment of nonquaternary C atoms from HSQC): 120.1 (C-20 ), 116.0 (C-50 ), 110.0 (C-60 ), 102.6 (C-3), 61.0 (8-OCH3), 59.9 (6OCH3), 55.6 (30 -OCH3). 4.3. Polar ¯avonoids Leaf tissue of B. orphanidis, after removal of the surface ¯avonoids with acetone, was extracted with boiling 80% methanol. The concentrated extract was run on Whatman 3 MM paper in BAW (n-BuOH:HOAc:H2O, 4:1:5, top layer) and the eluted bands separated in 15% HOAc. Further puri®cation was carried out in BAW, CAW (CHCl3:HOAc:H2O, 10:10:1), 15% HOAc and H2O. The known glycosides 7±11 were identi®ed by standard procedures (TLC, HPLC, UV spectral data) including comparison with authentic markers. UV spectra, Rf and HPLC data are shown in Table 3. The HPLC programme for these ¯avone glycosides involved the use of 75% solvent A/25% solvent B to 35% A/65% B over 23 min in a linear gradient. Otherwise, HPLC details are similar to those described for the lipophilic ¯avonoids.
Acknowledgements We thank Professor J.S. Parker, Director of the Cambridge Botanic Garden, for a plant of Biebersteinia odora.
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