Journal of Chromatography A, 1315 (2013) 15–20 Contents lists available at ScienceDirect Journal of Chromatography A jou rn al hom epage: www.elsevier.com/locate/chroma Molecularly imprinted polymers for the isolation of bioactive naphthoquinones from plant extracts Stella K. Tsermentseli a , Panagiotis Manesiotis b,∗ , Andreana N. Assimopoulou a,∗∗ , Vassilios P. Papageorgiou a a Organic Chemistry Laboratory, Chemical Engineering Department, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece b School of Chemistry and Chemical Engineering, Queen’s University Belfast, David Keir Building, Stranmillis Road, BT9 5AG Belfast, Northern Ireland, United Kingdom a r t i c l e i n f o Article history: Received 15 June 2013 Received in revised form 7 August 2013 Accepted 11 September 2013 Available online 17 September 2013 Keywords: Shikonin Alkannin Naphthazarin Plant extracts Molecularly Imprinted Solid Phase Extraction (MI-SPE) Wound healing agents a b s t r a c t Molecularly Imprinted Polymers (MIPs) targeting shikonin, a potent antioxidant and wound heal- ing agent, have been prepared using methacrylic acid (MAA) and 2-diethylaminoethyl methacrylate (DEAEMA) as functional monomers. An investigation of solution association between shikonin and both acidic and basic functional monomers by UV–vis titrations, suggested stronger affinity towards the basic functionality. Strong inhibition of the co-polymerisation reaction of such basic monomers was observed, but was overcome by reduction of the amount of template used during polymer synthesis. Polymer morphology was severely impacted by the template’s radical scavenging behaviour as demonstrated by solid state NMR spectroscopy measurements. HPLC evaluation of the final materials in polar conditions revealed limited imprinting effects and selectivity, with the MAA polymers exhibiting marginally better performance. During application of the polymers as MI-SPE sorbents in non-polar solvents it was found that the DEAEMA based polymer was more selective towards shikonin compared to the MAA counterpart, while shikonin recoveries of up to 72% were achieved from hexane solutions of a commercial sample of shikonin, hexane extract of Alkanna tinctoria roots and a commercial pharmaceutical ointment. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Alkannin and its enantiomer shikonin, are naturally occurring isohexenylnaphthazarins, found in the external layer of the roots of numerous plant species that belong mainly to the genera Alkanna, Lithospermum, Echium, Onosma and Anchusa of the Boraginaceae family [1,2]. They are potent pharmaceutical substances with a wide spectrum of antimicrobial [3], anti-inflammatory [4], antiox- idant [5] and antitumor activity [6]. Shikonin, alkannin and related naphthazarin derivatives (Fig. 1) are also established as strong wound healing agents and are nowadays commercially available in the form of a wound healing pharmaceutical ointment under the trademark HELIXDERM ® . Extraction of bioactive constituents, such as alkannin, shikonin and their esters, from medicinal plants is typically performed by Soxhlet extraction, ultrasound-assisted extraction, maceration extraction using organic solvents and accelerated solvent extrac- tion either at ambient temperature or at reflux conditions [7]. ∗ Corresponding author. Tel.: +44 28 9097 4515; fax: +44 28 9097 6524. ∗∗ Corresponding author. Tel.: +30 2310 994242. E-mail addresses: p.manesiotis@qub.ac.uk (P. Manesiotis), adreana@eng.auth.gr (A.N. Assimopoulou). Techniques that enable faster extraction, higher sample through- put and require less organic solvent such as microwave-assisted extraction and rapid solid-liquid dynamic extraction have recently been proposed [8,9]. Nonetheless, these techniques are energy intensive and contribute to sound pollution, especially in industrial scale. Furthermore, active ingredients of plant origin are in many cases thermally labile. All these techniques require multiple fur- ther steps and time for isolating and purifying alkannin, shikonin and their derivatives, resulting in their extraordinarily high price. In order to address the above limitations we employed molec- ular imprinting as an alternative extraction technique, aiming to isolate shikonin from natural extracts and to purify commer- cial samples. Molecular imprinting is a technique that introduces specific binding sites in the matrix of a synthetic polymer, by co-polymerisation of appropriate functional and cross-linking monomers in the presence of a target substance, the so-called template [10,11]. These binding sites are complementary to the template in terms of size, shape and functional group orientation and are able to rebind and extract it from complicated sam- ples. Thus, such materials have been extensively used as sorbents in solid-phase extractions (MI-SPE) [12], chiral separations [13], catalysis [14] and sensing [15]. In contrast to natural receptors, imprinted polymers are stable in a wide range of temperatures, 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.09.044