Published: May 12, 2011 r2011 American Chemical Society 5927 dx.doi.org/10.1021/jf104798n | J. Agric. Food Chem. 2011, 59, 5927–5934 ARTICLE pubs.acs.org/JAFC Isolation and Caenorhabditis elegans Lifespan Assay of Flavonoids from Onion You-Lin Xue, †,‡ Tomoyuki Ahiko, † Takuya Miyakawa, † Hisako Amino, § Fangyu Hu, † Kazuo Furihata, † Kiyoshi Kita, § Takuji Shirasawa, #,^ Yoriko Sawano, † and Masaru Tanokura* ,† † Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan ‡ College of Light Industry, Liaoning University, 66 Chongshan Middle Road, Shenyang 110036, People's Republic of China § Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan # Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan ^ Graduate School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan ABSTRACT: The main flavonoids were isolated from three selected onion cultivars. Three phenolic compounds were obtained by reverse-phase HPLC, and their structures were elucidated by multiple NMR measurements. There were two known compounds, quercetin and quercetin 3 0 -O-β-D-glucopyranoside (Q3 0 G), and one novel compound, quercetin 3-O-β-D-glucopyranoside-(4f1)- β-D-glucopyranoside (Q3M), which was identified in onion for the first time. These flavonoids were found to be more abundant in the onion peel than in the flesh or core. Their antioxidative activities were tested using the DPPH method, and their antiaging activities were evaluated using a Caenorhabditis elegans lifespan assay. No direct correlation was found between antioxidative activity and antiaging activity. Quercetin showed the highest antioxidative activity, whereas Q3M showed the strongest antiaging activity among these flavonoids, which might be related to its high hydrophilicity. KEYWORDS: Allium cepa L., flavonoids, quercetin, antioxidative activity, lifespan assay, Caenorhabditis elegans ’ INTRODUCTION The common onion (Allium cepa L.) has been part of the diet of almost every civilization in world history. In 2008, the annual production of onions was an estimated 67 million tons (http:// faostat.fao.org), and the main areas of production were China, India, the United States, Pakistan, Turkey, and Russia. Numerous valuable phytonutrients have been found in the onion, including flavonoids, fructo-oligosaccharides (FOS), and thiosulfinates and other sulfur compounds. 1 In recent years, flavonoids have received continuous attention due to accumulating evidence of their beneficial properties on human health. For example, flavonoids have been linked to a reduction in the incidence of diseases such as inflammation, heart diseases, and cancer, which are closely con- nected to the generation of reactive oxygen species in the body. 24 The free radical theory of aging is one of the most popular modern aging theories and is based on the assumption that aging is caused by lifelong accumulation of partially unrepaired oxida- tive damage to a critical set of biological macromolecules, such as mutations in mitochondrial DNA. 5 The nematode (Caenorhabditis elegans), a multicellular organism with a completely sequenced genome, is widely used as an animal model in aging research because of its relatively short lifespan, morphological simplicity, ease of genetic manipulation, and capacity for self-fertilization, which facil- itates the generation of genetically homogeneous populations. 68 On the basis of the identi fication and unraveling of multiple mechan- isms and corresponding genes limiting the lifespan of nematodes, it has been clarified that the insulin signaling pathway has a close relationship with lifetime determination. It has also been re- ported that the homologues of these genes are preserved in Drosophila and mice, indicating that the aging process of organ- isms is controlled by a universal molecular mechanism from lower to higher animals. 911 Moreover, some of the identified genes have been reported to affect the insulin signaling pathway and mitochondrial function, which are of particular interest with respect to the free radical theory of aging. 12,13 The present work focuses on the isolation and characterization of the main flavonoids from a variety of onions using HPLC and NMR. We also analyzed the antioxidative and antiaging activities of the flavonoids and discuss the correlativity among these activities. ’ MATERIALS AND METHODS Materials and Chemicals. Onions of different colors [a yellow cultivar, Super Kita-momiji (SK); a red cultivar, Syonan-red (SR); and a white cultivar, Aichi-shiro (AS)] were purchased from the Japan Agricultural Cooperatives. After washing with water, the onions were divided into three parts: the peel (dried protective outer scales), flesh (fleshy scales), and core. Each part was cut into small pieces, frozen in liquid nitrogen, and stored at 80 °C. All chemicals and solvents unless otherwise stated were purchased from Wako (Osaka, Japan) or Sigma- Aldrich (St. Louis, MO). Sample Preparation. The onion pieces were chopped into smaller ones and then incubated overnight with 5 times the volume of 80% methanol at 4 °C. After homogenization, the mixture was stirred for 1 h Received: December 14, 2010 Revised: May 3, 2011 Accepted: May 4, 2011