Flavonoid Quantification in Onion by Spectrophotometric and High Performance Liquid Chromatography Analysis

Direct spectrophotometric determination of quercetin content in onions (Allium cepa L.) was investigated as a possible alternative to high-performance liquid chromatography (HPLC) analysis. Quercetin content in five onion varieties was monitored at 362 nm and quantified using simple spectrophotometric and HPLC methods. HPLC revealed that 3,4'-Qdg and 4'-Qmg comprised up to 93% of total flavonol content detected in the studied varieties. These major quercetin conjugates combined (3,4'-Qdg + 4'-Qmg) and total flavonol conjugates quantified by HPLC correlated closely with spectrophotometer values. Correlation coefficients were 0.96 (P < 0.0001) for 3,4'-Qdg + 4'-Qmg and 0.97 (P < 0.0001) for total flavonol conjugates in onion. Simple spectrophotometric procedure proved to be a valid, efficient, and cost-effective method for the quantification of total quercetin in onion. Chemical names used: quercetin-3,4'-Odiglucoside (3,4'-Qdg); quercetin-4'-O-glucoside (4'-Qmg). al., 1995; Price and Rhodes, 1997; Rhodes and Price, 1996). Evaluation of quercetin content in onion is of interest to plant breeders, food technologists and nutritionists alike to assess breeding lines for genetic selection purposes or monitor levels during storage or processing. This interest has underscored the need for rapid, efficient quantification methods. HPLC methods, although more precise, are costly and require a trained operator. Simple spectrophotometric assays allow more rapid analysis (Mayhew et al., 1984; Lin and Wagner, 1994). We address here the precision of spectrophotometer values as a reflection of total quercetin, which represents the majority of flavonol content in onion (Price and Rhodes, 1997). We compared values obtained with simple spectrophotometer readings to the cumulative amounts of individual flavonols identified through HPLC analysis. Materials and Methods Plant material. Five long-day, storage-type onion varieties consisting of one red skinned variety purchased from a local grocery store (Albertsons, Lubbock, Texas, presumed to be of one variety) and four yellow skinned varieties: Rio Rita, RNX 10968, Predator (Rio Colorado Seed Co., Bakersfield, Calif.) and Tamara (Bejo Zaden Seed Co., Warmenhuizen, Netherlands) were used in this study. Ten bulbs were selected randomly from each of the five varieties and stored at 4 °C until sampled. Sample preparation. Over the course of 10 d, one bulb from each of the five varieties was selected randomly each day for quercetin extraction. Outer, inedible portions from bulbs were removed. Onions were quartered; one quarter section was weighed then frozen in liquid nitrogen. Frozen tissue was ground to a fine powder using a coffee grinder (Braun, Boston). Extraction. Quercetin was extracted from 20-g ground onion powder in 80 mL of 80% EtOH (Patil et al., 1995) by filtering twice through number 8 (Fisher Scientific, Houston) and grade 42 (Whatman; Clifton, N.J.) filter paper. Filtrate was collected in 2-mL Eppendorf tubes and placed into storage at –20 °C until analysis. Phytochemicals are biologically active compounds present in edible foods that when ingested, have the potential to prevent or delay the onset of disease (as reviewed by Guhr and Lachance, 1997). Flavonoids are a specific class of phenolic plant phytochemicals represented by over 5000 compounds, subdivided into 13 categories that include anthocyanidins, catechins, flavonols, and flavones (as reviewed by Croft, 1998). The flavonol quercetin has shown much promise as an antioxidant agent, imparting a protective effect in reducing the risk of developing cardiovascular disease (Hertog and Hollman, 1996; Keli et al., 1996; Knekt et al., 1996), and certain types of cancer (Knekt et al., 1997; Leighton et al., 1992). High concentrations of quercetin occur in onion (Allium cepa L.) (Hertog et al., 1992; Leighton et al., 1992; Price and Rhodes, 1997), and the amounts vary with bulb color and type (Bilyk et al., 1984; Patil et al., 1995). Three predominant forms of quercetin in onion are quercetin aglycone, quercetin-3,4'-O-diglucoside and quercetin-4'-O-glucoside (Leighton et al., 1992; Price and Rhodes, 1997) (Fig. 1). Quantification of flavonoid compounds often employ methods of separation combined with ultraviolet (UV) detection (Harborne, 1984; Jurd, 1962; Mabry et al., 1970; Price and Rhodes, 1997; Seikel, 1962). In onions, concentrations of quercetin aglycone isolated by TLC (thin layer chromatography) have been estimated spectrophotometrically using VO2 (vanadyl) metal ions as a shift reagent (Kaushal et al., 1978). These techniques have largely been replaced by high-performance liquid chromatography (HPLC), which has become one of the most important tools in the separation and identification of flavonoids from raw plant extracts. Indeed HPLC has been used for the quantification of quercetin in onions by several research teams (Bilyk et al., 1984; Crozier et al., 1997; Hertog et al., 1992; Patil et Received for publication 22 Nov. 2000. Accepted for publication 28 Aug. 2001. This paper is a portion of a thesis submitted by Kevin Lombard to fulfill partial requirement for a MS degree. Mention of a trademark, proprietary products, or vendors does not constitute a guarantee or warranty of the product by Texas Tech Univ. and does not imply its approval to the exclusion of other products or vendors that also may be suitable. We thank Dan Krieg, Paul Paré, Susan San Francisco, the Institute for Biotechnology, Bob Wyatt, and Andy Herring for their assistance. Dept. of Plant and Soil Sciences, Texas Tech Univ., Lubbock, TX 79409. 2 Rio Colorado Seeds, 3239 Shafter Road, Bakersfield, CA 93313. Current address: Institut National d’Horticulture, 2 rue Le Nôtre, 49045, Angers, France. Fig. 1. Quercetin compounds measured. Redrawn in part from Price and Rhodes (1997). Compound R1 R2 isoquercitrin (quercetin-3-O-glucoside) H glucose quertetin aglycone H H quercetin-3, 4'-O-diglucoside glucose glucose quercetin-4'-O-glucoside glucose H 7