Industrial Crops and Products 49 (2013) 697–704 Contents lists available at SciVerse ScienceDirect Industrial Crops and Products journal h om epage: www.elsevier.com/locate/indcrop Characterization of Pinus brutia bark tannin by MALDI-TOF MS and 13 C NMR Mualla Balaban Ucar a,∗ , Gunes Ucar a , Antonio Pizzi b , Oktay Gonultas a a Department of Wood Chemistry and Technology, Faculty of Forestry, Istanbul University, 34473 Istanbul, Turkey b ENSTIB-LERMAB, University of Lorraine, Epinal, France a r t i c l e i n f o Article history: Received 10 January 2013 Received in revised form 10 June 2013 Accepted 11 June 2013 Keywords: MALDI-TOF 13 C NMR GC–MS Pinus brutia bark extract tannins Structural composition a b s t r a c t The structures of condensed tannin from Pinus brutia bark were characterized by means of GC–MS, solid and liquid state 13 C NMR and MALDI-TOF MS analyses. The results indicated that the monomeric units building up the condensed tannin of brutia pine bark are mainly composed of catechin/epicatechin, gallo- catechin/epigallocatechin units accompanied by possibly tri- or tetra hydroxyl-flavanols. Some oligomers appear to present p-hydroxy- or 3,4-dihydroxy benzoic acid residues or glucose at their terminal unit. Apart from oligomers, monomeric flavanoids were also present in the brutia bark extract. Taxifolin was especially found in appreciable amounts. Additionally, in this tannin procyanidin oligomers predom- inated while prodelphinidin units were present to a lesser extent. The NMR results showed that the oligomers in brutia pine bark tannin Exhibit 2,3-cis stereochemistry and C4–C6 bonds with a notice- able portion of C4–C8 interflavonoid links being present. The MALDI-TOF MS showed a series of peaks corresponding to flavonoid hexamers of the condensed tannin (1750 Da). © 2013 Elsevier B.V. All rights reserved. 1. Introduction Tannins are polyphenols. They can be classified as hydrolysable tannins and condensed or flavanoid tannins (e.g., oligomeric to polymeric proanthocyanidins (PAs)). In procyanidins or phlobaphenes, the flavan-3-ol sub-units such as cate- chin/epicatechin, gallocatechin/epigallocatechin are joined by C4–C8 and/or C4–C6 linkages. The analysis of PAs is difficult because of the heterogeneous structure of these compounds. Currently, solid state CPMAS 13 C NMR is used to identify and differentiate the chemical structure of proanthocyanidins. Another powerful technique, MALDI-TOF MS has also been used to determine the kind and the number of flavan-3-ol sub-units in tannin. Pasch et al. (2001) have introduced MALDI-TOF as a technique to analyze the structure of mimosa-, quebracho- and pecan nut tannins. Many articles have appeared thereafter, dealing with the analysis of different bark tannins by using jointly 13 C NMR- and MALDI-TOF MS. The characterization of many tannins have been investigated using these techniques. Thus, were investigated the tannins isolated from the barks of species such as Pinus radiata (Ku and Mun, 2007), Pinus pinaster and P. radiata (Jerez et al., 2009), P. pinaster and Pinus massoniana (Weber et al., 2007), maritime pine ∗ Corresponding author. Tel.: +90 212 226 11 00; fax: +90 212 226 11 13. E-mail address: mbalaban@istanbul.edu.tr (M.B. Ucar). (Navarrete et al., 2010), Acacia mangium (Hoong et al., 2010), man- grove (Oo et al., 2008), Acacia confuse (Wei et al., 2011); from the wood of Acacia auriculiformis (Ishida et al., 2005); from the leaves of Kandelia candel and Rhizophora mangle (Zhang et al., 2010), and from grape pomace (Ping et al., 2012). However, to our knowledge, there are no MALDI-data available to-date for Pinus brutia bark. P. brutia (Turkish red pine) is grown in the Mediterranean- and Black Sea regions of Turkey covering 25% of the total Turkish forest area. Therefore its bark as a waste material has a big potential in the wood working industry. Ayla (1980) examined the bark tannin of P. brutia by performing some chemical analyses. Recently, by means of LC and LC–MS analyses, the polyphenolic constituents and biologi- cal activity of P. brutia bark tannin were subject to the comparison of pycnogenol content (Yesil-Celiktas et al., 2009). Tannin produc- tion from P. brutia bark was studied by Ozdemir (2010). The authors reported that ethyl acetate extracts from P. brutia bark, similar to other pine bark extracts, contained monomeric taxifolin in large amounts, together with some catechin and catechin ferulate. The aim of this study was to analyze the bark tannin of P. brutia by using solid state-, liquid 13 C NMR and MALDI-TOF MS techniques in order to estimate its composition and molecular characteristics. 2. Materials and methods The bark material of Turkish red pine was obtained from Mediterranean region (province Mersin, south part of Turkey). Bark samples were air dried, ground in a Wiley mill and screened. The 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.06.010