Phenolic Compounds from the Branches of Malus sieboldii (Regel) Rehder and Their Antioxidative Activities

Malus is a genus of about 35-50 species of deciduous woody plants in the family Roseaceae that includes apple (M. pumila), a common orchard fruit. The species M. sieboldii (Regel) Rehder is a shrub 2-6 m tall native to Japan and Korea. 1 This plant has been used as an ornamental tree for its showy flowers in the spring. The extracts from the leaves of M. sieboldii have been reported to exhibit scavenging activities on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals 2 and inhibition activities on nitric oxide production. 3 However, as far as we know, no phytochemical study on M. sieboldii has yet been published. Antioxidants exert protective effects in human health against oxidative damage associated with reactive oxygen species (ROS). ROS are chemically reactive substances that can attack lipids, proteins and nucleic acids within living organisms. ROS include free radicals (O2 ·� , HO·, RO· and ROO·) and neutral molecules ( 1 O2 and H2O2). Even though ROS generation is a normal metabolic process, 4 its excessive formation can cause peroxidation in human tissues, ultimately leading to various diseases related with aging. 5 When the skin is exposed to ultraviolet (UV) rays, ROS are generated in excess to induce aged-skin symptom such as wrinkles. In order to reduce the cumulative effects of ROS-related damage, the addition of antioxidants to food or cosmetic formulations has been considered. 6 The development of novel antioxidative agents, especially from natural sources, has attracted attention in terms of their ecologically friendly properties. 7 We are continuously conducting studies to find biologically active natural products, especially for use in cosmetic formulations from plants in Jeju Island, a place of great biodiversity in Korea. 8 Preliminary study indicated that the ethanol extract prepared from the branches of Malus sieboldii exhibited significant DPPH radical scavenging activities. Therefore, we decided to carry out a full phytochemical investigation of the M. sieboldii extract to determine the bioactive metabolites. Described herein are the isolation and structural determination of the chemical constituents in the plant extract. In addition, the antioxidative properties of the isolates were examined using the DPPH and ABTS + radical scavenging activity tests. The ethanol extract (147.4 g) was prepared from the branches of M. sieboldii (1.0 kg) with a 14.7% yield. The extract suspended in water was partitioned successively to provide n-hexane, ethyl acetate (EtOAc), and n-butanol fractions. The extract and fractions were then tested for their anti-oxidative capacities using DPPH radical scavenging assay first reported by Blois. 9 Ascorbic acid showing an SC50 (50% radical scavenging concentration) value of 7.1 μg/mL was used as a positive control in this exper