Identification of carotenoids and flavonoids in spinach: Effects of growing season, genotype, maturity, and pressurized fluid extraction technologies

Total phenolics and antioxidant capacity (ORAL) of 11 commercial cultivars (CC) and 15 advanced breeding lines (ABL) of spinach were determined over two growing seasons. Flavonoids and carotenoids of fall-grown spinach were also determined. Total phenolics, total flavonoids, carotenoids, and ORAC of 8 CC and 8 ABL at three maturity stages were investigated. The optimal extraction conditions for carotenoids and flavonoids from spinach by Pressurized Liquid Extraction (PLE) were verified. Consecutive PLE of carotenoids and flavonoids were compared to traditional extraction methods. The effects of three Supercritical Fluid Extraction (SFE) factors on carotenoid extraction were determined with a response surface model. Overwinter spinach had higher phenolics and ORAC than fall spinach, indicating that biotic or abiotic stresses influenced phenolic metabolism. Genetics also appeared to affect phenolic metabolism and ORAC in spinach. The ABL with increased disease resistance had higher phenolics, individual and total flavonoids, carotenoids, and ORAC than CC. Plant breeders can select for increased phenolics to increase ORAC in spinach genotypes, or the crops can be grown in different seasons or under certain stress conditions to elevate levels of antioxidants. Medium maturity spinach leaves had higher phenolics and flavonoids than immature and mature leaves. ORAC correlated better with total phenolics than with flavonoids indicating that other phenolic compounds affected the antioxidant activity of spinach. Carotenoids and chlorophylls increased considerably with maturation. The high relationship between chlorophylls and carotenoids suggest that plant breeders can select for dark green color to boost carotenoids. The best solvent and temperature of PLE for carotenoids and flavonoids were acetone at 50°C and 70% ethanol at 80°C, respectively. The large increase in ORAC values at elevated PLE temperatures appeared to be related to the development of brown pigments and/or the release of cell-wall-bound phenolic acids. Consecutive PLE resulted in comparable or greater extraction of carotenoids and flavonoids than solid-liquid and Soxhlet extraction methods. Pressure and ethanol modifier concentration influenced lutein extraction with SFE, whereas beta-carotene extraction was influenced by temperature, pressure, and ethanol concentration. Lutein and beta-carotene extraction from spinach was efficient with ethanol as co-modifier, but flavonoid extraction with SFE was inefficient.