Breeding Buckwheat (Fagopyrum esculentum Moench) for Flavonoids

Transcription

1 Proceedings o/the 9th International Symposium on Buckwheat. Prague 2004 Breeding Buckwheat (Fagopyrum esculentum Moench) for Flavonoids Carolin OlschHiger 1, Dieter Treutte~, Friedrich J. Zeller 1 Technical University ofmunich, JDivision ofplantbreeding andapplied Genetics, 2Institute offruit Science, Freising- Weihenstephan, Germany Corresponding author: caroun. oelschlaeger@web.de ABSTRACT The composition of hydroxycinnamic acids, flavonols and flavanols were estimated in hulls and dehulled seeds of buckwheat breeding lines. It was found that the accumulation of the diverse phenolic groups is independent from each other and the biosynthesis is separately regulated in hulls and seeds. These are prerequisites for breeding buckwheat lines with optimised flavonoid composition for various functional purposes. Keywords: buckwheat, breeding lines, flavonoids, HPLC, chemical reaction detection INTRODUCTION Flavonoids are a group of secondary compounds which are widespread in the plant kingdom (FEUCHT and 'TREUTTER 1989) and exhibit significant biological activity for the plant itself and for human consumption (LATTANZIO 2003, GABOR 1986). Lots of different structures of flavonoids occur in nature, but their qualitative composition varies in plant species. The actual demand for functional nutrition requires the supply of flavonoid rich foods. Buckwheat seeds are a valuable source providing a range of secondary compounds with high antioxidative capacity (QUETTIER-DELEU et al. 2000, TIAN et al. 2002). Among these, the flavonol rutin (quercetin 3-rutinoside) is the most widely studied (HAGELS 1996, DIETRYCH-SZOSTAK and OLESZEK 1999, KREFT et al. 1999). Moreover, flavan 3-ols with derivatives of catechin and epicatechin were identified as bioactive components of buckwheat seeds (WATANABE 1998). The polymeric procyanidins, however, reduce the digestibility of proteins (EGGUM et al. 1981, IKEDA et al. 1991). All these flavonoids are derived from the amino acid phenylalanine and their biosynthesis passes through several common enzymatic steps (HELLER and FORKMANN 1993). Breeding of buckwheat for flavonoids should consider the branched flavonoid pathway leading to different structures. This paper presents a breeding program with the aim to develop buckwheat with optimised flavonoid composition in the seeds. This quality aspect is furthermore coupled to breeding for high yield, which is realised by introducing self-fertility from the wild type F. homotropicum (ZELLER 2001). MATERIAL AND METHODS From different breeding lines of buckwheat (Fagopyrum esculentum Moench) the grains were divided into hulls and de-hulled seeds. Both type of tissues were separately extracted with 80 % aqueous methanol and subjected to HPLC. The HPLC equipment used consisted of an autosampler (Gilson-Abimed Mode11231), of two pumps (Kontron Mode11422), a diode array detector (Bio Tek Kontron 540). For post column derivatisation a further analytical HPLC pump (Gynkotek Modell 300 C) and a VISdetector (640 nm, Kontron Detektor 432) were used. The phenolic compounds were separated according to MAYR et at. (1995) on a column (250 x 4 mm I.D.) prepacked with Hypersil ODS, 3 ~m particle size, following a stepwise gradient using mixtures of 5% formic acid (A) and methanol gradient grade (B) with a flow rate of 0.5 ml/min. The gradient profile used is: 0-5 min, isocratic, 5% B in A; 5-15 min, 5 10% B in A; min, isocratic, 10% B in A, min, 10-15% B in A, min,

2 Proceedings ofthe 9th International Symposium on Buckwheat, Prague 2004 isocratic, 15% B in A, min, % B in A, min, isocratic, 20% B in A, min, 20-25% B in A, min, 25-30% B in A, min, 30-40% B in A, min, 40-50% B in A, min, 50-90% B in A. Hydroxycinnamic acids and flavonols were detected at 280 nm whereas the flavan 3-ols were estimated at 640nm after post column derivatisation with p-dimethyl-aminocinnamic aldehyde (DMACA; TREUTTER, 1989). Flavone was used as internal standard for quantitative analyses of crude extracts. The peaks were identified according to their IN-absorbance, their chromatographic behaviour on reversed phase chromatography (HPLC) and thin layer chromatography (TLC) in comparison to authentic standards. The flavanols were quantified as epicatechin, the hydroxycinnamic acids as ch10rogenic acid and flavonols were calculated as rutin which were available as standards. RESULTS AND DISCUSSION The HPLC-analysis of seeds and hulls allowed the detection and quantification of flavonols and flavanols as well as their precursor metabolites, the hydroxycinnamic acids. Fig. la shows a survey profile detected at 280 nm where all phenolic compounds give signals. This allows the quantification of hydroxycinnamic acids and flavonols. The chromatogram (Fig. 1b) obtained by post column derivatisation selects the flavanols. The varying quantitative distribution ofhydroxycinnamic acids, flavono1s and flavanols (Fig. 2) in twelve selected inbred lines shows that the accumulation of the diverse phenolic groups is independent from each other. Line 10, for instance, has the capacity for the accumulation of high amounts of all the three groups mentioned. This is in contrast to line 1 with generally low amounts. Line 6 produces the highest level of flavanols but Less amount of flavonols. It must further be considered that there is a tissue specific regulation. The hulls of lines 7-12 exhibit high quantities of flavonols which are hardly detectable in the corresponding seeds. The reverse was found for flavanols which predominate in the seeds of most lines. An exception is line 10 with higher amounts offlavanols in the hull than in the seed. These results outline the need of in depth studies ofthe heritability of the biosynthetic pathways leading to the different groups of flavonoids and determining their accumulation both in hulls and seed. The diversity of flavonoid composition occurring in the breeding lines is promising for obtaining buckwheat progenies with optimised flavonoid contents for varying purposes in our breeding programme. These, however, must be combined with other phenotypic characters guaranteeing high agricultural quality. 675

3 Proceedings ofthe 9th International Symposium on Buckwheat, Prague 2004 Fig. la-c HPLC profiles ofbuckwbeat seeds (line No.3 in fig. 2) with detection at 280nm (a), with chemical reaction detection (b) and 360 urn (c). Flavanols (No. 1-12), rutin (13), hyciroxycinnamic acid (14), flavone (internal standard, 15) a) 14 ls b) ", ,- -,- --, Q 20 c) ~ I \.I ~ 0,.. :io

4 P1-oceedings ofthe 9th International Symposium on Buckwheat, Prague 2004 Fig. 2 Concentrations ofhydroxycinnamic acids, flavonols and flavanols in seed and hulls of buc~heatbreedinglines Hydroxyc;lnnam Ie acids 677

5 Proceedings a/the 9th International Symposium on Buckwheat, Prague 2004 ACKNOWLEDGMENTS The authors are grateful to the Arthur and Aenne Feindt-Stiftung for financial support. REFERENCES DIETRYCH-SZOSTAK D., OLESZEK W. (1999): Effect of processing on the flavonoid content in buckwheat (Fagopyrum esculentum Moench) Grain. J. Agric. Food Chern. 47: EGGUM 8., KREFT I., JARVORNIK B. (1981): Chemical composition and protein quality of buckwheat (Fagopyrurn esculenturn Moench). Qual. Plant Foods Hum. Nutr. 30; FEUCHT W., TREUTTER D. (1989): Phenolische Naturstoffe. Obst- und Gartenbauverlag, MUnchen. GABOR M. (1986): Anti-inflammatory and anti-allergic properties of flavonoids. In: CODY, V., MIDDLETON Eo, HARBORNE J.B. (Eds.) Plant Flavonoids in Biology and Medicine. Biochemical, Pharmacological, and Structure-Activity Relationships. Alan R. Liss, Inc., , BAGELS H. (1996): Analytische, pharmazeutische, phytochemische sowie inter- und intraindividuelle Untersuchungen zu Fagopyrum- Arten. Studie zur Pharmakokinetik des Rutins. Diss. FU Berlin. HELLER W., FORKMANN, G. (1993): Biosynthesis of flavonoids. In: HARBORNE, lb., The Flavonoids, Chapman and Hall, London. IKEDA K., SKAGUCHI, T., KUSANO, T. and YASUMOTO, K. (1991): Endogenous factors affetcting protein digestibility in buckwheat. Cereal Chern. 68: KREFT S., KNAPP, M., KREFT, I. (1999): Extraction ofrutin from buckwheat (Fagopyrurn esculenturn Moench) seeds and determination by capillary electrophoresis. 1. Agr. Food Chern, 47: LATTANZIO V. (2003): Bioactive Polypheno1s: their role in quality and storability of fruit and vegetables. 1. Applied Botany 77: MAYR U, TREUTIER D., SANTOS-BUELGA e., BAUER H., FEUCHT W. (1995): Developmental changes in the phenol concentrations of 'olden Delicious' apple fruits and leaves. Phytochemistry 38: QUETTIER-DELEU C. GRESSIER B" VASEUR J., DINE T., BRUNET e., LUYCKX M., CAZIN M., CAZIN le., BAILLEUL F., TROTIN F. (2000): Phenolic compounds and antioxidant activities of buckwheat (Fagopyrum esculentum Moench) hulls and flour. 1. Ethnopharmacol. 72: TIAN Q., LI D., PATIL B.S. (2002): Identification and detennination of flavonoids in buckwheat (Fagopyrurn esculentum Moench, Polygonaceae) by high-performance liquid chromatography with electrospray ionisation mass spectrometry and photodiode array ultraviolet detection. Phytochem. Anal. 13: TREUTTER D. (1998): Chemical reaction detetion of catechitis and proanthocyanidins with p dimethylarninocinnamaldehyde. J. Chromatogr. 467: WATANABE M. (1989): Catechins as antioxidants from buckwheat (Fagopyrurn esculenturn Moench) groats. 1. Agric, Food Chern. 46: ZELLER F.l (2001): Buckwheat (Fagopyrum esculenturn Moench): Utilization, genetics, breeding. Bodenkultur 52: , 678