Journal of Pharmaceutical and Biomedical Analysis 52 (2010) 701–706 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Liquid chromatography/tandem mass spectrometry study of anti-inflammatory activity of Plantain (Plantago L.) species Ivana N. Beara a,∗ , Dejan Z. Orˇ ci´ c a , Marija M. Lesjak a , Neda M. Mimica-Duki ´ c a , Biljana A. Pekovi ´ c b , Mira R. Popovi ´ c a a Department of Chemistry, Biochemistry and Environmental Protection, University of Novi Sad, Faculty of Sciences, Trg Dositeja Obradovi´ ca 3, Novi Sad, Serbia b The Institute for Blood Transfusion of Vojvodina, Hajduk Veljkova 9a, Novi Sad, Serbia article info Article history: Received 13 October 2009 Received in revised form 9 February 2010 Accepted 10 February 2010 Available online 18 February 2010 Keywords: Cyclooxygenase Lipoxygenase Anti-inflammatory activity Plantago LC–MS/MS Platelets abstract To evaluate anti-inflammatory activity of selected Plantago species (P. lanceolata L. and P. major L.) an optimized in vitro test for determination of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibition potency was undertaken. By using intact cell system (platelets) as a source of COX-1 and 12- LOX enzymes and highly sensitive and specific LC–MS/MS technique for detection of main arachidonic acid metabolites formed by COX-1 and 12-LOX, this test provides efficient method for evaluation of anti-inflammatory potential of plant extracts and isolated compounds. Our results validated the well- known COX-1 inhibitory activity of P. lanceolata and P. major methanol extracts (concentration required for 50% inhibition (IC 50 ) was 2.00 and 0.65 mg/ml, respectively). Furthermore, 12-LOX inhibitory activity of examined extracts was reported for the first time (IC 50 = 0.75 and 1.73 mg/ml for P. lanceolata and P. major, respectively). Although renowned inhibitors, such as acetylsalicylic acid and quercetin showed higher activity, this study verifies P. lanceolata and P. major as considerable anti-inflammatory agents. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Arachidonic acid, usually derived from the second position of phospholipids in the plasma membrane by the action of phos- pholipase A 2 , is precursor to the eicosanoids, physiologically and pharmacologically active compounds, which biochemical effect is expressed as local hormones act, functioning through G-protein- linked receptors. Arachidonic acid can be converted to these products by three different pathways: cyclooxygenase, leading to the formation of prostanoids (prostaglandins and thrombox- anes), lipooxygenase, where leukotrienes and certain mono-, di- and tri-hydroxy acids are synthesized, and epoxygenase pathway, which includes cytochrome P-450 and epoxides as final products. Accordingly, cyclooxygenases, lipooxygenases and epoxygenases are enzymes involved in these pathways [1]. Cyclooxygenase (COX), implicated in cyclooxygenase pathway, exists in two forms, named COX-1 and COX-2. COX-1 is expressed constitutively in different tissues, blood monocytes and platelets, and transforms arachidonic acid to prostanoids, which are involved Abbreviations: 12-HETE, 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid; 12-HHT, 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid; 12-LOX, 12- lipoxygenase; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; MRM, multiple reaction monitoring; MS2SIM, single-stage mass spectrometry. ∗ Corresponding author. Tel.: +381 21 4852755; fax: +381 21 454065. E-mail address: ivanak@eunet.rs (I.N. Beara). in normal cellular homeostasis. In contrast, COX-2 may be induced by a series of pro-inflammatory stimuli and its role in the progress of inflammation, fever and pain has been known [2]. Furthermore, three types of lipoxygenases, termed 5-, 12- and 15-lipoxygenase are engaged in lipoxygenase pathway. Some compounds, like 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE), a product of 12-lipoxygenase (12-LOX), has influence on the regula- tion of platelet aggregation, but is also found to be involved in the progression of several human diseases like various cancers [3], pso- riasis [4] and rheumatoid arthritis [5]. Aforementioned enzymes can be found in different cell types. Thus, in human platelets, COX-1 and 12-LOX are the initial enzymes responsible for arachi- donic acid metabolism leading to the formation of thromboxane B 2 , 12-HHT (12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid) and 12-HETE. Other minor metabolites such as prostaglandins F 2 ,E 2 and D 2 are also formed [1]. Therefore, the relationship between these enzymes and their potential inhibitors can be established by quantifying COX-1 and 12-LOX metabolites 12-HHT and 12- HETE, respectively. Several assays [6–13] have been founded on this principle, but they differ significantly in source of platelets (intact human, rat or rabbit platelets), addition of exogenous arachi- donic acid or inflammation induction agent (calcium ionophore A23187) and technique used for quantification of metabolites (high performance liquid chromatography (HPLC) with UV or radio- chemical detection, preparative thin layer chromatography (TLC) with subsequent radioactive counting or enzyme immunoassay (EIA)). 0731-7085/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2010.02.014