Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 247–251 Contents lists available at SciVerse ScienceDirect Journal of Pharmaceutical and Biomedical Analysis jou rn al h om epage: www.elsevier.com/locate/jpba Short communication Simultaneous quantitative determination and validation of quercetin glycosides with peroxynitrite-scavenging effects from Saussurea grandifolia Agung Nugroho a,b , Sang-Cheol Lim a , Chan Mi Lee c , Jae Sue Choi c , Hee-Juhn Park d,∗ a Department of Applied Plant Sciences, Graduate School, Sangji University, Wonju 220-702, Republic of Korea b Department of Agro-industrial Technology, Faculty of Agriculture, Lambung Mangkurat University, Indonesia 70712, Indonesia c Department of Food Science and Nutrition, Pukyong National University, Busan 607-737, Republic of Korea d Department of Pharmaceutical Engineering, Sangji University, Wonju 220-702, Republic of Korea a r t i c l e i n f o Article history: Received 11 October 2011 Received in revised form 10 November 2011 Accepted 14 November 2011 Available online 22 November 2011 Keywords: Saussurea grandifolia Compositae Saxifragin Peroxynitrite HPLC a b s t r a c t The leaves of Saussurea grandifolia (Compositae) are used as chwinamul, a well-known Korean mountainous vegetable, or to treat hepatitis and hematuria. Since the methanolic extract of S. gran- difolia leaves exhibit a potent peroxynitrite-scavenging effect, phytochemical and high-performance liquid chromatographic (HPLC) analyses were employed to identify and simultaneously quantify the active components: chlorogenic acid and 3,5-di-O-caffeoylquinic acid (3,5-DQ) as caffeoylquinic acids, and quercetin, isoquercitrin (quercetin-3-glucoside), saxifragin (quercetin-5-glucoside), and rutin (quercetin-3-rutinoside). Validation of HPLC methods was performed to verify the linearity, LOD, LOQ, intra-day and inter-day variabilities, recovery, and repeatability to ensure that this method is selective, sensitive, precise, accurate and reproducible. In particular, leaves contained saxifragin with potent peroxynitrite-scavenging activity (IC 50 , 0.67 M) as 4.65 mg/g dry weight, which is equivalent to 33.74 mg/g extract. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Mountainous vegetables are consumed to treat or prevent sev- eral diseases in Korea. Numerous mountainous vegetables belong to the family Compositae. Chwinamul is a representative moun- tainous vegetable among this family, but it refers to several species: Aster scaber, Aster yomena, Solidago virga var. gigantea, Solidago virgaaurea var. asiatica, Saussurea grandifolia, Ainsliaea acerifolia, Ligularia fischeri, and Ligularia stenocephala [1,2]. We have studied the polyphenolic substances of chwinamul, since those compounds are commonly beneficial for aging-related diseases. In particular, chwinamul contains high amounts of caffeoylquinic acids [3]. Peroxynitrite is a highly reactive oxidant that is produced from the reaction between superoxide anion radical ( • O 2 - ) and nitric oxide ( • NO) [4]. Overproduction of peroxynitrite results in perox- idation of lipids and proteins, cytotoxicity, and neurotoxicity [5]. Furthermore, it causes hypercholesterolemia, atherosclerosis, obe- sity and diabetes mellitus [6–8]. S. grandifolia is a mountainous vegetable that is used in chwinamul. Since quercetin glycosides were identified from this plant during our study of polyphenolic substances with ∗ Corresponding author. Tel.: +82 33 730 0564; fax: +82 33 730 0564. E-mail address: hjpark@sangji.ac.kr (H.-J. Park). peroxynitrite-scavenging activity, high-performance liquid chro- matographic (HPLC) quantitative analysis and peroxynitrite- scavenging assays of these substances were performed. Validation of the linearity, precision, accuracy and repeatability of our HPLC method was also performed to verify the present method. 2. Experimental 2.1. Plant material S. grandifolia (Compositae) was collected in Gangwon-do province, Korea, and identified by Won-Bae Kim (National Institute of Horticultural & Herbal Science, Rural Development Administra- tion, Korea). This plant was dried and pulverized for extraction. A voucher specimen was deposited in the Laboratory of Natural Prod- uct Chemistry, Department of Pharmaceutical Engineering, Sangji University, Korea. 2.2. Instruments and reagents The Varian HPLC system (Varian, Walnut Creek, CA, USA) used in this study consisted of Prostar 210 solvent delivery module and Prostar 325 UV–Vis detector. A Shiseido (Chuoku, Tokyo, Japan) Capcell Pak C18 column (5 m, 250 mm × 4.6 mm i.d.) was used as the column. Solvents used for the mobile phase were 0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.11.016