1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 DOI: 10.1002/elan.201600753 Electrochemical Determination of the Antioxidant Activity in Echinacea Purpurea Roots Using Square Wave Voltammetry Emad F. Newair,* [a] Refat Abdel-Hamid, [a] and Paul A. Kilmartin [b] Abstract: A simple and rapid electrochemical method was established to quantify the total polyphenol (TP) content and assess their antioxidant activity (AA) in roots of three Echinacea purpurea (E. purpurea) species using square wave voltammetry (SWV). Individual polyphenol compo- nents were identified, and then quantified by ultra-high performance liquid chromatography coupled with mass spectrometery (UPLC-MS). Two major polyphenols, chicoric (ChA) and caftaric (CFT) acids, were identified by mass spectroscopy in the extract of E. purpurea samples. The Accuracy of the proposed SWV electro- chemical method for TP content and AA analysis was validated by the highly sensitive UPLC-MS technique and standard ABTS method, respectively. A high correlation was noticed between the results, indicating the high sensitivity and reliability of the proposed SWV method for polyphenols analysis and AA evaluation in natural herbal samples. Keywords: Voltammetry · Caffeic acid derivatives · Antioxidant activity · Liquid chromatography · Echinacea purpurea herbs 1. Introduction Echinacea purpurea (E. purpurea) roots have become extremely popular as medicinal herbs in the United States as well as in Europe [1, 2]. E. purpurea is one of three species of Echinacea that are generally used: Echinacea purpurea (L.) Moench, Echinacea angustifolia (DC.) Hell and Echinacea pallid (Nutt.) Nutt. [3]. E. purpurea has four important active compounds which are polysacchar- ides, caffeic acid derivatives (CAFDs) especially chicoric (ChA) and caftaric (CFT) acids, alkamides, and glycopro- teins [4]. CAFDs possess many bioactive roles, including antiviral activity [5], antihyaluronidase activity [6], inhib- ition of immunodeficiency virus type 1 [7], collagen protection against radical-induced degradation [8], phag- ocyte activity [9], and high free-radical scavenging activity [10]. Also, CAFDs are commonly used as important markers of the medicinal quality of Echinacea herbal extracts [11]. Moreover, CAFDs and alkamides are considered to be the main important constituents of Echinacea which are responsible for its AA [5, 12]. It is worth mentioning that the biological properties of CAFDs depend on their absorption and their metabolism. The absorption and bioavailability processes have been well characterized in animals and in humans [13–17]. The extracts of E. purpurea have complex chemical compositions. Stanisavljevic et al. reported that the chem- ical composition of root extracts is different from the chemical composition of the extracts from other parts of the plant [18]. Chicoric acid (ChA, Figure 1) is the main phenolic in E. purpurea roots [19]. ChA is generally used as a marker to evaluate the quality of herbal products [20]. In addition to ChA, there are important constituents of E. purpurea roots such as caftaric acid (CFT), chlorogenic acid (CGA), ferulic acid (FA), coumaric acid (CA), caffeic acid (CAF) and cynarin (CYN) as shown in Figure 1. All of these compounds inhibit the production of free-radicals and lipid peroxidation involved in the development of inflammation [18]. Bauer et al. [21] made use of reversed-phase HPLC for the analysis of individual phenolic compounds in Echinacea. A proposed standard extraction and HPLC analysis method have been used to measure typical levels of various phenolic compounds in medicinal Echinacea species. CFT was the other main phenolic compound in E. purpurea roots [22]. ChA and CFT were reported to be highly susceptible to enzymatic degradation during the preparation of E. purpurea [23]. Several reports have previously studied the total polyphenol content as well as antioxidant activity of Echinacea roots using chemical 2,2’-Azino-bis(3-ethyl- benzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl- 1-picrylhydrazyl (DPPH * ) radical scavenging methods [10, 24, 25]. Generally, chemical methods require several preparation steps, and a long time to complete the measurement. Consequently, the need for fast and simple [a] E. F. Newair, R. Abdel-Hamid Unit of Electrochemistry Applications (UEA), Department of Chemistry, Faculty of Science, University of Sohag, Sohag 82524, Egypt E-mail: emad.newair@science.sohag.edu.eg newair.emad@gmail.com [b] P. A. Kilmartin School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/elan.201600753 Full Paper www.electroanalysis.wiley-vch.de  2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2017, 29, 1 – 11 1 These are not the final page numbers! ÞÞ