Journal of Pharmaceutical and Biomedical Analysis 76 (2013) 139–144 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 quantification and validation of caffeoylquinic acids and flavonoids in Hemistepta lyrata and peroxynitrite-scavenging activity Agung Nugroho a,b , Sang-Cheol Lim a , Jeong Su Byeon 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, 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 8 October 2012 Received in revised form 19 December 2012 Accepted 19 December 2012 Available online 27 December 2012 Keywords: Hemistepta lyrata Compositae Peroxynitrite Phenolic substances HPLC a b s t r a c t Traditionally, Hemistepta lyrata is consumed as a mountainous vegetable or a medicinal herb to treat inflammation, fever, hemorrhage, and hemorrhoids. In order to provide the scientific evidence of tradi- tional uses of this plant, we identified and quantified thirteen active substances (caffeic acid, chlorogenic acid, and 3,5-di-O-caffeoylquinic acid as caffeoylquinic acids; apigenin, isorhoifolin, acacetin, linarin, diosmetin, diosmin, pectolinarigenin, and pectolinarin as flavones or their glycosides; kaempferol 3-O-rutinoside and rutin as flavonol glycosides) from H. lyrata and evaluated their peroxynitrite- scavenging activity. The chromatographic separation was performed on a Capcell Pak C18 column (5 m, 250 mm × 4.6 mm i.d.) with a gradient elution of 0.05% TFA (trifluoroacetic acid) and 0.05% TFA in MeOH–CH 3 CN (60:40). Validation of HPLC methods on the linearity, LOD, LOQ, intra-day and inter-day variabilities, recovery, and repeatability proved that this method is selective, sensitive, precise, accurate, and reproducible. In peroxynitrite-scavenging assay, caffeic acid derivatives (chlorogenic acid, caffeic acid, and 3,5-di-O-caffeoylquinic acid) exhibited relatively lower IC 50 values than other substances tested. And HPLC simultaneous quantification showed that the 70% MeOH extract and the BuOH fraction contain a higher quantity of caffeic acid derivatives (17.82 and 30.09 mg/g, consecutively). Therefore, caffeic acid derivatives could be the main contributors to the peroxynitrite-scavenging activity of H. lyrata than other phenolic substances. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Peroxynitrite (ONO 2 - ) formed from a combination of the super- oxide anion radical ( • O 2 - ) and nitric oxide ( • NO) is necessary in the living cell. However, its excess production causes a vari- ety of diseases due to lipid peroxidation, cytotoxicity, and rapid neurotoxicity [1]. Such diseases include hypercholesterolemia, atherosclerosis, obesity, diabetes mellitus, and Alzheimer dis- ease [2–4]. Therefore, peroxynitrite-scavengers contribute to the prevention of these diseases. Peroxynitrite-scavengers, such as polyphenols which include caffeoylquinic acids and flavonoids, are being identified from a variety of fruits and vegetables [5,6]. A great number of polyphenols with peroxynitrite-scavenging activ- ity have been reported, and we can consider that a higher content of such polyphenols should contribute more to that scavenging activity. Therefore, a quantitative evaluation of such polyphenols ∗ Corresponding author. Tel.: +82 33 730 0564; fax: +82 33 730 0564. E-mail address: hjpark@sangji.ac.kr (H.-J. Park). is required for utilization of those vegetables as peroxynitrite- scavengers. Hemistepta lyrata, belonging to the family Compositae, is a biennial herbaceous plant used as a mountainous vegetable or a medicinal herb. The aerial part of H. lyrata has been used to treat cancer, inflammation, fever, hemorrhage, and hemorrhoids in the folkloric medicinal society of Korea [7]. Many constituents of H. lyrata have been known: two guaianolides, isoamber- boin and 8-hydroxyzaluzanin [8], acetylenes [9], acylated flavone O-glycosides, acylated flavone C-glycosides [10], caffeic acid, tracheloside, uracil, 8-carboxymethyl-p-hydroxycinnamic acid, 3- O-coumaroylquinic acid [11], apigenin, apigenin 7-O-rutinoside, acacetin 7-O-rutinoside, astragalin as flavonoids [12], hemice- ramide, hemisterpene ether [13], and triterpenes [14]. However, a quantitative evaluation of the constituents has not been deter- mined from this plant. In this research, qualitative and quantitative analyses on the phenolic substances of H. lyrata were undertaken includ- ing the optimization of extraction method and an experiment for validation. Quantitative evaluation was also performed in the CHCl 3 fraction with less polar substances, and the BuOH fraction 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.12.021