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The major flavonoids in the leaves of Milium effusum L.
BiochemicalSystematicsand Ecology,Voi. 17, No. 3, pp. 197-198,1989. Printed in GreatBritain.
0305-1978/89$3.00+ 0.00 © 1989PergamonPressplc.
The Major Flavonoids in the Leaves of Milium effusum L. RUTH J. MOULTON and STEPHEN J. WHITTLE Life Sciences Department, Goldsmiths' College London, Rachel McMillan Building, Creek Road, Depfford SE8 5BU, UK
Key Word Index--Milium effusum; Gramineae; flavonoids; flavonol glycosides; chemosystematics. Abstract--Specimens from natural populations of Milium effusum were analysed for their flavonoid contents. Four major flavonoids were commonly found and these were identified as quercetin 3-O-rutinoside, quercetin 3-O-glucoside, kaempferol 3-O-rutinoside and kaempfeml 3-O-glucoside. In addition, most populations showed some other flavonoids which were present in trace amounts. Milium is rare amongst the grasses in having flavonols as the major leaf flavonoids.
Introduction Milium effusum is a perennial grass found in woodlands and scrubland, usually on moist base-rich soils. It has a wide distribution, being found throughout Europe, except for Greece and Portugal, as well as in temperate Asia and in North America. It exists both as a diploid (2n=14) and as a tetraploid (2n=28), [1]: the tetraploid being the more common. In the only extensive flavonoid survey of the Gramineae, where about one-fifth of the known genera were examined, Harborne and Williams [2] showed that the major flavonoids were tricin and flavone C-glycosides based on apigenin and luteolin. By contrast, apigenin and luteolin O-glycosides were comparatively rare, as were the O-glycosides of the flavonols, kaempferol and quercetin. Milium was placed in the tribe Milieae [1] but has recently been assigned to the tribe Stipeae [3]. Harborne and Williams [2] examined some species of one genus of this tribe, Stipa, and found that it conformed to the flavonoid pattern found in most grasses. This study forms part of a detailed investigation of the evolutionary relationships and speciation processes in Milium. It looked at the flavonoids present in a number of natural populations of IV/. effusurn, all having the cytotype 2n=28 [4].
(Received 4 January 1989)
Results Two-dimensional thin-layer chromatograms on cellulose of aqueous methanolic extracts of M. effusum showed four major flavonoid spots, F1, F2, F3 and F4. Their Rf values in BAW and 15% HOAc, respectively, were F1, 0.47/0.54; F2, 0.58/ 0.61; F3, 0.56/0.30 and F4, 0.70/0.39. Separation of these flavonoids was achieved on silica gel TLC plates using the solvent EFAW (Rf values; F1, 0.30; F2, 0.40; F3, 0.58 and F4, 0.65). These were purified on cellulose TLC plates with 15% HOAc as solvent (Rf values: F1, 0.55; F2, 0.64; F3, 0.30 and F4, 0.42). Subsequent analysis of the flavonoids was carried out by chemical and spectroscopic means and a positive identification of each of the flavonoids was achieved. They are based upon quercetin and kaempferol and are all glycosides linked at position three. Following acid hydrolysis, F1 and F3 yielded quercetin and F2 and F4 gave kaempferol, the sugars released were rhamnose and glucose from F1 and F2 and glucose from F3 and F4. The main glycoside, F1, is quercetin-3-O-rutinoside with kaempferol-3-O-rutinoside (F2), quercetin-3-Oglucoside (F3) and kaempferol-3-O-glucoside (F4) being present in decreasing amounts (relative amounts F1, 50%; F2, 21%; F3, 18% and F4, 11%). In addition to F1, F2, F3 and F4, other minor flavonoids were also found in some samples but at concentrations too low for identification. Acid hydrolysis of the extract yielded only quercetin and kaempferol and it assumed that the trace flavonoids are also glyco197
198
sides of these flavonols. There was no evidence of C-glycosides being present. Discussion The finding that flavonols constitute the major flavonoids in the leaves of this grass is of interest since Harborne and Williams [2] found only one species (Rottboellia exaltata) in which flavonols replaced flavones as the major flavonoid conponent. Indeed, in their survey of 247 species of Gramineae, only 6% were found to contain quercetin and kaempferol. The major components were tricin-O-glycosides and apigenin, and luteolin-C-glycosides. Flavonol glycosides are, therefore, uncommon as leaf constituents, although they may occur widely in other tissues such as pollen. Ceska and Styles [5] identified a series of 10 flavonol glycosides in maize pollen, these being glycosides of quercetin, isorhamnetin and kaempferol. Milium effusurn belongs to the genus Milium. Tutin et al. [1 ] placed Miliurn in the tribe Milieae and also placed the genus Zingeria in the same tribe. However, Clayton and Renvoize [3] assigned Milium to the tribe Stipeae, whilst assigning Zingeria to the tribe Aveneae. Harborne and Williams [2] reported that three species in the genus Stipa contained tricin and flavone C-glycosides with no flavonols present. However, Saleh et aL [6] isolated possible acylated derivatives of kaempferol and quercetin from leaves of Stipa lemmoni~ Flavonols are generally considered a primitive feature and are replaced in more advanced groups by flavones [7]. We are continuing this study by analysing other populations of M. effusum, together with members of closely related species. It would be of interest if other species shared this unusual flavonoid pattern. Experimental Seeds were obtained from Botanic Gardens in Europe including those at the Martin Luther University, Halle-Wittenburg, DDR, the University of Lund, Sweden and the Louis Pasteur University, Strasbourg, France. Seeds were subsequently germinated and individual plants grown in pots outdoors. Leaves were harvested in late summer. The material was chopped, dried and extracted successively with 90% MeOH
RUTHJ. MOULTONAND STEPHENJ. WHITTLE and 50% MeOH. Standard procedures for the identification of flavonoids were applied [8-10]. Two-dimensional chromatograms of the methanolic extracts were run on cellulose (Merck TLC layers 5577) in BAW and 15% HOAc. The flavonoids were initially separated by one-way TLC on silica gel 60 (Merck plates 5721) using the solvent EFAW (ethyl acetate-formic acid-acetic acid-water; 100:11:11:27 [11] and purified by oneway TLC on cellulose (Merck plates 5516) using 15% acetic acid. Acid hydrolysis was carried out with 2 M HCI. Aglycones were identified by co-chromatography. Sugars were identified by GLC of their TMS ethers using a Perkin-Elmer Chromatograph F33 with a 100-200 2 M × 4 mm column containing 3% OV-1 on chromasorb WHP. Chemical structures of the flavonol glycosides were elucidated using UV, 1H NMR, lzc NMR and also TLC and HPLC co-chromatography. UV spectra were recorded in a Pye-Unicam SP 8-100 UV-Vis spectrophotometer. HPLC separations were performed using a PyeUnicam LC with a Spherisorb C8 5p. 250X4.6 mm column. A gradient programme was used at room temperature: Solvent A=MeOH-HOAc-H20; 30:5:56, Solvent B=MeOH; Solvent A was modified by B at a rate of 3.67% rain-1. The flow rate was set at 2 ml min -1 and the detection wavelength was 340 nm; the retention times relative to rutin were 1.00 (F1), 1.05 (F2), 1.21 (F3) and 1.25 (F4). ~H NMR and "~zcNMR spectra were recorded on a BrL~kerWH400 pulsed FT spectrometer using a 45° pulse angle at 400.13 MHz for ~H and 100.62 MHz for 13C; samples were dissolved in DMSO-de with TMS as an internal standard.
Acknowledgements--Wethank Professor J. B. Harbome and Dr Christine Williams for their advice in the initial stages of this project, Matthew Ma for his technical assistance with GLC and HPLC, Peter Haycock of the University of London (Queen Mary College) LILIRS WH-400 NMR Service for running the NMR analyses, Dr Sandra M. Thomas for providing the Milium seeds and Brenda Whittle for cultivating the plants.
References 1. Tutin, T. G., Heywood, V. H., Burges, N. A., Moore, D. M., Valentine, D. H., Waiters, S. M., Webb, D. A., Chater, A. O. and Richardson, I. B.K. (1980) Flora Europaea, Vol. 5. Cambridge University Press, Cambridge. 2. Harborne, J. B. and Williams, C. A. (1976) Biochem. Syst. Ecol. 4, 267. 3. Clayton, W. D. and Renvoize, S. A. (1986) Genera Graminum; Grasses of the Wodd, HMSO, London. 4. Bennett, S. T. (1988) Personal communication. 5. Ceska, O. and Styles, E. D. (1984) Phy,tochemistry23, 1822. 6. Saleh, N. A. M., Bohm, B. A. and Maze, J. R. (1971) Phytochemism/10, 490. 7. Harborne, J. B. (1977) Biochem. Syst. Ecol. 5, 7. 8. Harborne, J. B. (1987) ComparaEve Biochemistry of the Flavonoids. Academic Press, London. 9. Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970) The Systema~'c Iden~'ficatJonof Flavonoids. Springer, New York. 10. Markham, K. R. (1982) Techniques of Flavonoid Identification. Academic Press, London. 11. Wagner, H., Bladt, S. and Zgainski, E. M. (translated by Scott, A.) (1984) Plant Drug Analysis. Springer, Berlin.
0305-1978/89$3.00+ 0.00 © 1989PergamonPressplc.
The Major Flavonoids in the Leaves of Milium effusum L. RUTH J. MOULTON and STEPHEN J. WHITTLE Life Sciences Department, Goldsmiths' College London, Rachel McMillan Building, Creek Road, Depfford SE8 5BU, UK
Key Word Index--Milium effusum; Gramineae; flavonoids; flavonol glycosides; chemosystematics. Abstract--Specimens from natural populations of Milium effusum were analysed for their flavonoid contents. Four major flavonoids were commonly found and these were identified as quercetin 3-O-rutinoside, quercetin 3-O-glucoside, kaempferol 3-O-rutinoside and kaempfeml 3-O-glucoside. In addition, most populations showed some other flavonoids which were present in trace amounts. Milium is rare amongst the grasses in having flavonols as the major leaf flavonoids.
Introduction Milium effusum is a perennial grass found in woodlands and scrubland, usually on moist base-rich soils. It has a wide distribution, being found throughout Europe, except for Greece and Portugal, as well as in temperate Asia and in North America. It exists both as a diploid (2n=14) and as a tetraploid (2n=28), [1]: the tetraploid being the more common. In the only extensive flavonoid survey of the Gramineae, where about one-fifth of the known genera were examined, Harborne and Williams [2] showed that the major flavonoids were tricin and flavone C-glycosides based on apigenin and luteolin. By contrast, apigenin and luteolin O-glycosides were comparatively rare, as were the O-glycosides of the flavonols, kaempferol and quercetin. Milium was placed in the tribe Milieae [1] but has recently been assigned to the tribe Stipeae [3]. Harborne and Williams [2] examined some species of one genus of this tribe, Stipa, and found that it conformed to the flavonoid pattern found in most grasses. This study forms part of a detailed investigation of the evolutionary relationships and speciation processes in Milium. It looked at the flavonoids present in a number of natural populations of IV/. effusurn, all having the cytotype 2n=28 [4].
(Received 4 January 1989)
Results Two-dimensional thin-layer chromatograms on cellulose of aqueous methanolic extracts of M. effusum showed four major flavonoid spots, F1, F2, F3 and F4. Their Rf values in BAW and 15% HOAc, respectively, were F1, 0.47/0.54; F2, 0.58/ 0.61; F3, 0.56/0.30 and F4, 0.70/0.39. Separation of these flavonoids was achieved on silica gel TLC plates using the solvent EFAW (Rf values; F1, 0.30; F2, 0.40; F3, 0.58 and F4, 0.65). These were purified on cellulose TLC plates with 15% HOAc as solvent (Rf values: F1, 0.55; F2, 0.64; F3, 0.30 and F4, 0.42). Subsequent analysis of the flavonoids was carried out by chemical and spectroscopic means and a positive identification of each of the flavonoids was achieved. They are based upon quercetin and kaempferol and are all glycosides linked at position three. Following acid hydrolysis, F1 and F3 yielded quercetin and F2 and F4 gave kaempferol, the sugars released were rhamnose and glucose from F1 and F2 and glucose from F3 and F4. The main glycoside, F1, is quercetin-3-O-rutinoside with kaempferol-3-O-rutinoside (F2), quercetin-3-Oglucoside (F3) and kaempferol-3-O-glucoside (F4) being present in decreasing amounts (relative amounts F1, 50%; F2, 21%; F3, 18% and F4, 11%). In addition to F1, F2, F3 and F4, other minor flavonoids were also found in some samples but at concentrations too low for identification. Acid hydrolysis of the extract yielded only quercetin and kaempferol and it assumed that the trace flavonoids are also glyco197
198
sides of these flavonols. There was no evidence of C-glycosides being present. Discussion The finding that flavonols constitute the major flavonoids in the leaves of this grass is of interest since Harborne and Williams [2] found only one species (Rottboellia exaltata) in which flavonols replaced flavones as the major flavonoid conponent. Indeed, in their survey of 247 species of Gramineae, only 6% were found to contain quercetin and kaempferol. The major components were tricin-O-glycosides and apigenin, and luteolin-C-glycosides. Flavonol glycosides are, therefore, uncommon as leaf constituents, although they may occur widely in other tissues such as pollen. Ceska and Styles [5] identified a series of 10 flavonol glycosides in maize pollen, these being glycosides of quercetin, isorhamnetin and kaempferol. Milium effusurn belongs to the genus Milium. Tutin et al. [1 ] placed Miliurn in the tribe Milieae and also placed the genus Zingeria in the same tribe. However, Clayton and Renvoize [3] assigned Milium to the tribe Stipeae, whilst assigning Zingeria to the tribe Aveneae. Harborne and Williams [2] reported that three species in the genus Stipa contained tricin and flavone C-glycosides with no flavonols present. However, Saleh et aL [6] isolated possible acylated derivatives of kaempferol and quercetin from leaves of Stipa lemmoni~ Flavonols are generally considered a primitive feature and are replaced in more advanced groups by flavones [7]. We are continuing this study by analysing other populations of M. effusum, together with members of closely related species. It would be of interest if other species shared this unusual flavonoid pattern. Experimental Seeds were obtained from Botanic Gardens in Europe including those at the Martin Luther University, Halle-Wittenburg, DDR, the University of Lund, Sweden and the Louis Pasteur University, Strasbourg, France. Seeds were subsequently germinated and individual plants grown in pots outdoors. Leaves were harvested in late summer. The material was chopped, dried and extracted successively with 90% MeOH
RUTHJ. MOULTONAND STEPHENJ. WHITTLE and 50% MeOH. Standard procedures for the identification of flavonoids were applied [8-10]. Two-dimensional chromatograms of the methanolic extracts were run on cellulose (Merck TLC layers 5577) in BAW and 15% HOAc. The flavonoids were initially separated by one-way TLC on silica gel 60 (Merck plates 5721) using the solvent EFAW (ethyl acetate-formic acid-acetic acid-water; 100:11:11:27 [11] and purified by oneway TLC on cellulose (Merck plates 5516) using 15% acetic acid. Acid hydrolysis was carried out with 2 M HCI. Aglycones were identified by co-chromatography. Sugars were identified by GLC of their TMS ethers using a Perkin-Elmer Chromatograph F33 with a 100-200 2 M × 4 mm column containing 3% OV-1 on chromasorb WHP. Chemical structures of the flavonol glycosides were elucidated using UV, 1H NMR, lzc NMR and also TLC and HPLC co-chromatography. UV spectra were recorded in a Pye-Unicam SP 8-100 UV-Vis spectrophotometer. HPLC separations were performed using a PyeUnicam LC with a Spherisorb C8 5p. 250X4.6 mm column. A gradient programme was used at room temperature: Solvent A=MeOH-HOAc-H20; 30:5:56, Solvent B=MeOH; Solvent A was modified by B at a rate of 3.67% rain-1. The flow rate was set at 2 ml min -1 and the detection wavelength was 340 nm; the retention times relative to rutin were 1.00 (F1), 1.05 (F2), 1.21 (F3) and 1.25 (F4). ~H NMR and "~zcNMR spectra were recorded on a BrL~kerWH400 pulsed FT spectrometer using a 45° pulse angle at 400.13 MHz for ~H and 100.62 MHz for 13C; samples were dissolved in DMSO-de with TMS as an internal standard.
Acknowledgements--Wethank Professor J. B. Harbome and Dr Christine Williams for their advice in the initial stages of this project, Matthew Ma for his technical assistance with GLC and HPLC, Peter Haycock of the University of London (Queen Mary College) LILIRS WH-400 NMR Service for running the NMR analyses, Dr Sandra M. Thomas for providing the Milium seeds and Brenda Whittle for cultivating the plants.
References 1. Tutin, T. G., Heywood, V. H., Burges, N. A., Moore, D. M., Valentine, D. H., Waiters, S. M., Webb, D. A., Chater, A. O. and Richardson, I. B.K. (1980) Flora Europaea, Vol. 5. Cambridge University Press, Cambridge. 2. Harborne, J. B. and Williams, C. A. (1976) Biochem. Syst. Ecol. 4, 267. 3. Clayton, W. D. and Renvoize, S. A. (1986) Genera Graminum; Grasses of the Wodd, HMSO, London. 4. Bennett, S. T. (1988) Personal communication. 5. Ceska, O. and Styles, E. D. (1984) Phy,tochemistry23, 1822. 6. Saleh, N. A. M., Bohm, B. A. and Maze, J. R. (1971) Phytochemism/10, 490. 7. Harborne, J. B. (1977) Biochem. Syst. Ecol. 5, 7. 8. Harborne, J. B. (1987) ComparaEve Biochemistry of the Flavonoids. Academic Press, London. 9. Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970) The Systema~'c Iden~'ficatJonof Flavonoids. Springer, New York. 10. Markham, K. R. (1982) Techniques of Flavonoid Identification. Academic Press, London. 11. Wagner, H., Bladt, S. and Zgainski, E. M. (translated by Scott, A.) (1984) Plant Drug Analysis. Springer, Berlin.