Effect of Meteorological Conditions on Flavonoids in Portuguese Landrace Varieties of Onion

Transcription

1 Effect of Meteorological Conditions on Flavonoids in Portuguese Landrace Varieties of Onion A.S. Rodrigues and M.S. García-Falcón and J. Simal-Gándara D.P.F Almeida PORTUGAL UNIVERSIDADE DE VIGO SPAIN CBQF, Escola Superior de Biotecnologia, UNIVERSIDADE CATÓLICA PORTUGUESA PORTUGAL th International ISHS Symposium on Edible Alliaceae (ISEA) October 7 Onion Consumption & Production in Portugal Consumption (per capita/year) Word: 8 kg Europe: kg Portugal: kg third vegetable more consumed, after potato and brassicas Production Portugal 9 t Mainly in Northwest of Portugal Póvoa de Varzim Landrace varieties - - Branca da Póvoa (white onion) - Vermelha da Póvoa (red onion) Crop Production Yearbook,

2 Plant material Allium cepa L. Vermelha da Póvoa (red onion) Branca da Póvoa (white onion) Open air cultivation sowing Transplanting - March Growing Harvesting - July Curing in field ~ days Packaging

3 Methodology Extraction and analysis of flavonoids bulbs/variety g edible portion methanol:formic acid:water (MFW; ::; v:v:v) + tertbutylhydroquinone (TBHQ; g L - ) (stability during extraction and injection period avoid extract deterioration) Acid and alkaline hydrolysis- flavonoids aglycones and deacylated anthocyanins HPLC analysis* spectral analysis Population onions Vermelha da Póvoa Branca da Póvoa * Solvent extraction MFW: Methanol:Formic:Water (::; v:v:v) + TBHQ: antioxidant tert-butylhydroquinone ( g/l) sample n = g/onion bulb ml (MFW+TBHQ) Shaking (up and down) h. ultra-turrax Extract fraction Centrifugation min. ml (MFW+TBHQ) Shaking (up and down) h. Extract fraction Centrifugation min. ml (MFW+TBHQ) Extract fraction final extraction ( ml MFW) (diluted 6 ml) Agitation (up and down) min. Filtration in vacuum HPLC/DAD ( µl) + ml (MFW+TBHQ) Wash the filter

4 Instrumental and conditions parameters - HPLC analysis* Guard column Analytical column Pelliguard LC-8 ( x.6 mm d.i. µm) Waters Symmetry C 8 (mm x.6 mm d.i. µm) Columns temperature Injected volume µl Mobile phase Regimen ºC - best resolution and retention time stability A ( % formic acid in water):b (methanol) Gradient elution Flow rate ml.min - Detection wavelength 6 nm (flavonols); nm (anthocyanins) Instrumental and conditions parameters - HPLC analysis* Detection wavelength 6 nm (flavonols); nm (anthocyanins) Banda II: -6 nm Banda I: -8 nm Banda II: 7-8 Banda I: - nm 6 nm (flavonols) nm (anthocyanins)

5 mau mau Identification: Flavonols and Anthocyanins 8 6 Detector -Scan-6 nm CRP MFWExt 8Jul Colh MetSR. quercetin 7, -diglucoside. quercetin, -diglucoside. isorhamnetin, -diglucoside. quercetin -glucoside. quercetin -glucoside 6. isorhamnetin -glucoside 8 Detector -Scan- nm CRP MFWExt 8Jul Colh MetSR 6. cyanidin -glucoside. cyanidin -laminaribioside. cyanidin -(6 -malonylglucoside). cyanidin (6 -malonyl-laminaribioside). cyanidin dimalonylaminaribioside+peonidin - malonylglucoside a b Fig.- Chromatograms of the red onion extract: a) flavonols (6 nm); b) anthocyanins ( nm). Identification Flavonols-aglycones: Acid hydrolysis 8 Detector -Scan-6 nm CBH8B MFW colhsept. quercetin 7, -diglucoside. quercetin, -diglucoside. isorhamnetin, -diglucoside. quercetin -glucoside. quercetin -glucoside 6. isorhamnetin -glucoside 6 6 Fig.. Chromatogram of the white onion extract (6 nm), before acid hydrolysis.

6 mau mau Identification Flavonols-aglycones: Acid hydrolysis Detector -Scan-6 nm CBH 8 MFW hidrolisada quercetin aglycone 8 6 isorhamnetin aglycone Fig.. Chromatogram of the white onion extract (6 nm), after acid hydrolysis. Identification Anthocyanins-aglycones: Acid hydrolysis 8 Detector -Scan- nm CRconHid ml HCl 6Fev cyanidin aglycone peonidin aglycone Fig.. Chromatogram of the red onion extract ( nm), after acid hydrolysis. 6

7 mau mau Identification Anthocyanins-deacylated: Alkaline hydrolysis 8 Detector -Scan- nm CRE C8 Jun. cyanidin -glucoside. cyanidin -laminaribioside. peonidin -glucoside (NQ). cyanidin -(6 -malonylglucoside). cyanidin (6 -malonyl-laminaribioside) 6. cyanidin dimalonylaminaribioside+peonidin - malonylglucoside Fig.. Chromatogram of the red onion extract ( nm), before alkaline hydrolysis. Identification Anthocyanins-deacylated: Alkaline hydrolysis Detector -Scan- nm CRE hidrolisada C8 Jun. cyanidin -glucoside. cyanidin -laminaribioside. peonidin -glucoside (NQ) Fig. 6. Chromatogram of the red onion extract ( nm), after alkaline hydrolysis (deacylated anthocyanins). 7

8 Anthocyanins content (mg kg - FW) Individual Flavonols (mg kg - FW) Individual Flavonols (mg kg - FW) Results: flavonols content * Fig. 7. Individual flavonols content in a) white onion and b) red onion. () quercetin 7, -diglucoside, () quercetin, -diglucoside, () isorhamnetin, -diglucoside, () quercetin -glucoside, () quercetin -glucoside and (6) isorhamnetin -glucoside. * account for 9 to 9% of the total flavonol content in each variety Results: anthocyanins content ND ND ND ND ND ND total Fig. 8. Individual and total anthocyanins content in red onion. () cyanidin -glucoside, () cyanidin - laminaribioside, *() cyanidin -(6 -malonylglucoside), () cyanidin -(6 -malonyl-laminaribioside), () cyanidin -dimalonylaminaribioside + peonidin -malonylglucoside, (ND) not detectable. * representing 9 to 8 % of total anthocyanins 8

9 Individual Flavonols (mg kg Results: flavonols content - FW) white onion red onion Fig. 9. Total flavonols content in portuguese onion landrace varieties. Total flavonol content: higher in the red variety vermelha da Póvoa (more than twice) higher in, a very dry and hot season varied by 68% and % among years for the red and white onions, respectively Results: Effect of meteorological conditions Table. Total Flavonols in onion varieties and climatic conditions in each of three years. Total Rainfall-R, Mean Air Temperature-T, Mean Soil Temperature ( cm)-st, Mean Air Relative Humidity-RH, Total Global Radiation-GR and Mean Soil Water Content-SWC for the growth period from March through July -6 Year Total flavonols (mg kg - FW) Meteorological Conditions White Red R (mm) T (ºC) ST (ºC) RH (%) GR (kj m - ) SWC (%)

10 Discussion Higher levels of flavonoids observed in - probably related to the higher global radiation and lower rainfall during the growing season. These meteorological conditions can enhance secondary metabolism, favouring the synthesis of flavonols In contrast, in the year with the lowest soil and air temperatures, higher relative humidity and higher soil water availability, onions accumulated less flavonols. The red onion variety did not accumulate detectable amounts of anthocyanins in 6- lowest global radiation and highest air and soil temperatures. A common environmental cue for anthocyanin induction is solar radiation, which both produces energy through photosynthesis to drive anthocyanin biosynthesis and provides external signals for regulating developmental processes (Neill, ). Cold temperatures have been shown to induce anthocyanin synthesis in many plant species (Chalker-Scott, 999; Hasegawa et al., ), and it has been suggested by Christie et al. (99) that cold regulation genes are involved in their synthesis. Conclusion Our results show that genetic and meteorological factors, particularly water deficit, temperature and radiation, have a marked influence on the content of flavonoids in onion bulbs

11 Tank you! PORTUGAL UNIVERSIDADE DE VIGO SPAIN CBQF, Escola Superior de Biotecnologia, UNIVERSIDADE CATÓLICA PORTUGUESA PORTUGAL IDENTIFICACIÓN DE ANTOCIANINAS PEONIDIN CYANIDIN GLUCOSE LAMINARIBIOSE ANTOCIANINAS R R R λ max. A / A. λ max. (%) t.r. Cianidina -glucósido glucosa H Cianidina -laminaribiósido laminaribiósa H H.6 Peonidina--glucósido glucosa H -.7 Cianidina -(6 - malonilglucósido) Cianidina -(6 -malonil laminaribiósido) Cianidina-(,6 dimalonil laminaribiósido) Peonidina--malonilglucósido glucosa laminaribiosa laminaribiosa glucosa Ácido Malónico Ácido Malónico Ácido Malónico Ácido Malónico H Ácido Malónico %: Cyanidin o Peonidin

12 mau mau mau Detector -Scan- nm CRE C8 Jun Detector -Scan- nm CRE hidrolisada C8 Jun IDENTIFICACIÓN DE ANTOCIANINAS HIDRÓLISIS ACIDA.- Cianidina aglicon.- Peonidina aglicon Area Cebolla Vermelha da Póvoa.- Cianidina -glucósido.- Cianidina -laminaribiósido.- Peonidina -glucósido.- Cianidina (6 malonilglucósido).- Cianidina (6 malonillaminaribiósido) 6.- Peonidina -malonilglucósido + Cianidina -dimalonillaminaribiósido 6 HIDRÓLISIS ALCALINA Ácido aromático.- Cianidina -glucósido.- Cianidina -laminaribiósido.- Peonidina -glucósido Formas aciladas Ácido alifático IDENTIFICACIÓN DE FLAVONOLES R R R R λ max Quercetina 7, -diglucosido H Glu H Glu /66 Quercetina, -diglucosido Glu Glu H H / Isorhamnetin, - diglucósido? Glu Glu Me H / Quercetina - glucosido Glu H H H 6/ Quercetina -glucosido H Glu H H /6 Isorhamnetin -glucósido? H Glu Me H /66 Detector -Scan-6 nm CBP UV- 8Jul Quercetina-7, -diglucósido.- Quercetina-, -diglucósido.- Quercetina--glucósido.- Isoharmnetin-, -diglucósido.- Quercetina- -glucósido 6.- Isoharmnetin- -glucósido.- Quercetina aglicon.- Isorhamnetin aglicon 6 6 8

13 mg / Kg m.s. IDENTIFICACIÓN DE FLAVONOLES: AUTOLISIS ENZIMÁTICA FLAVONOLES 6 TIEMPO(horas) Quercetina-,'-diglucósido Quercetina-'-monoglucósido Quercetina Quercetina--glucosidasa Quercetina- -glucosidasa CONDICIONES CROMATOGRÁFICAS: FASE MÓVIL Columna Waters Symmetry C 8 (mm x,6 mm d.i. µm) Precolumna Volumen de inyección Pelliguard LC-8 ( x,6 mm d.i. µm) µl Temperatura ºC Fase móvil Régimen Flujo Metanol (A): Ácido fórmico % (B) Gradiente - min: : -: 8: -:: 6 -: : (Lavabo) 6-: : (Acondicionam.), ml/min Distribución de antocianinas en función del ph del medio (Zamora Marín, ) (F: catión flavilio (rojo), B: Base carbinol y C: Chalcona (incoloras), Q: Base quinoidal (azul) Base quinoidal (Q) (azul) ph Catión flavilio (F) (rojo) λ detección -6 nm λ cuantificación Flavonoles: 6 nm (Quercetina) Antocianinas: nm (Cianidina) Chalcona (C)(amarilla) Pseudo base carbinol (B) (incoloro)