Journal of Pharmaceutical and Biomedical Analysis 48 (2008) 579–586 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Determination of phenolic compounds and their antioxidant activity in Erigeron acris L. extracts and pharmaceutical formulation by flow injection analysis with inhibited chemiluminescent detection Edyta Nalewajko-Sieliwoniuk a , Jolanta Nazaruk b , Ewelina Antypiuk a , Anatol Kojło a,∗ a Institute of Chemistry, University of Bialystok, ul. Hurtowa 1, 15-399 Bialystok, Poland b Department of Pharmacognosy, Medical University of Bialystok, ul. Mickiewicza 2a, 15-089 Bialystok, Poland article info Article history: Received 28 December 2007 Received in revised form 21 May 2008 Accepted 21 May 2008 Available online 28 May 2008 Keywords: Erigeron acris L. Antioxidant activity Polyphenols Luminol chemiluminescence Flow injection analysis (FIA) abstract It was found that the chemiluminescence (CL) produced from the reaction of luminol with iodine in the alkaline medium was strongly inhibited by plant phenolic compounds. Based on this find- ing, a new flow injection CL method was developed for the determination of caffeic acid and 6 ′ -caffeoylerigeroside. The latter compound was isolated for the first time from Erigeron acris L. herb. The method was simple, rapid and sensitive with a detection limit of 4 × 10 -3 ng mL -1 (caffeic acid) and 0.18 ng mL -1 (6 ′ -caffeoylerigeroside), linear range of 0.1–1.5 ng mL -1 (caffeic acid) and 1–200 ng mL -1 (6 ′ - caffeoylerigeroside), relative standard deviation of 3.3% for 10 measurements of 0.45 ng mL -1 caffeic acid and 2.9% for 40 ng mL -1 6 ′ -caffeoylerigeroside. This method was successfully applied to determine the content of phenolic compounds/antioxidant activity of E. acris L. extracts and phenolic acids content in pharmaceutical formulation. A possible mechanism of the inhibition of the proposed CL system was discussed. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Erigeron acris L., blue fleabane, from the Asteraceae family, is a biennial or perennial plant occurring in dry grasslands and fields [1]. In folk medicine it was applied to relieve toothache and arthritic pains [2]. Phytochemical studies showed the pres- ence of polyphenolic compounds in this plant. Flavonoid aglycones (quercetin, luteolin, apigenin and kaempferol), luteolin 7-O- glucoside, scutellarin and erigeroside were isolated from the herb [3–5]. Polyphenols in general play a key role in the antioxidant activity of plant sources. Many publications concern searching for methods to measure that activity, most of which are spec- trophotometric methods based on the capacity of the sample to inhibit the production of free radicals generated in the system [6]. Over the past few years, there have been increasing atten- tion to the chemiluminescence (CL) as a sensitive and fast assay for the screening of antioxidant activity. Among differ- ent chemiluminogenic species exploited for the evaluation of antioxidant activity, luminol seems to be the most commonly used CL reagent. The assays employing a luminol CL detection are based on the scavenging of free radicals (including reactive ∗ Corresponding author. Tel.: +48 85 7457804; fax: +48 85 7470113. E-mail address: kojlo@uwb.edu.pl (A. Kojło). oxygen species) involved in the sequence leading to an elec- tronically excited 3-aminophtalate dianion (3-APA*), which emit the light on return to its ground state [7]. The determination of antioxidants is mainly based on the inhibiting of CL derived from few enzymatic reactions: luminol–H 2 O 2 –horseradish per- oxidase [8,9], luminol–xanthine–xanthine oxidase [8,10,11], luminol–hypoxanthine–xanthine oxidase [12], but also luminol–H 2 O 2 –Co(II)/EDTA [13], luminol–H 2 O 2 –Fe(II) [14], luminol–ClO - [15] and luminol-2,2 ′ -azo-bis-(2-amidinopropane) [16] CL system. Majority of those methods are manual, only three employs flow injection methodology [10,13,15]. CL detection combined with flow injection analysis considerably reduces the time of analysis and is very useful for studying the inhibition effect of antioxidants on unstable oxygen species with a short lifetime (e.g. O 2 •- ) [10]. To the best of our knowledge, there are no reports using the CL of luminol–I 2 system for the determination of antioxidant activity. As far in the literature there are only few studies which exploited luminol–I 2 detection system for the CL determination of different analytes [17–20]. Babko et al. [17] as first found that luminol–I 2 detection system could be used to measure iodine concentra- tion. Min et al. [18] have reported use of a sequential injection analysis (SIA) system for the analysis of penicillin, based on the formation of penicilloic acid by penicillinase, which was quanti- fied by its quenching effect on the CL originating from luminol oxidized by I 2 . Ratanawimarnwong et al. [19] used gas-diffusion 0731-7085/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2008.05.026