Dyes and Pigments 104 (2014) 194e196 Contents lists available at ScienceDirect Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig Short communication Identification of indirubin and monobromoindirubins in Murex brandaris Dimitrios Mantzouris a, Ioannis Karapanagiotis a,b,* a Ormylia Foundation, Art Diagnosis Center, Ormylia 63071, Greece b University Ecclesiastical Academy of Thessaloniki, Department of Management and Conservation of Ecclesiastical Cultural Heritage Objects, Thessaloniki 54250, Greece article info abstract Article history: For the first time, indirubin, 6-bromoindirubin and 60-bromoindirubin are detected in hypobranchial Received 10 December 2013 glands, removed from Murex brandaris molluscs. Liquid chromatography coupled to a diode array de- Received in revised form tector is used to analyse dimethyl sulfoxide extracts of brandaris molluscs, which were collected from the 4 January 2014 coastline of Thera island, Greece. The three aforementioned compounds are detected in small amounts Accepted 6 January 2014 compared to 6,60-dibromoindigotin and 6,60-dibromoindirubin, which correspond to very high chro- Available online 18 January 2014 matographic peaks. Finally, small amounts of indigotin and 6-bromoindigotin are detected in the molluscan extracts. Keywords: Ó Tyrian purple 2014 Elsevier Ltd. All rights reserved. Murex brandaris Bolinus brandaris HPLC Indirubin Monobromoindirubin 1. Introduction dibromoindigotin [1], its composition was investigated with HPLC in only four studies [3,5,6,8]. Since 1909, when 6,60-dibromoindigotin was identified in Tyrian HPLC is used herein to analyse extracts of M. brandaris. The re- purple [1], the analysis of the chemical composition of the ported results are important for three reasons: (i) for the first time, molluscan dye has attracted considerable attention, not only indirubin and monobromoindirubins are detected in brandaris ex- because Royal purple is a material of extreme importance for ob- tracts. (ii) Integrated HPLC peak areas, measured at 288 nm, are jects of the cultural heritage, but also because its chemical carried out to enrich the available data regarding the relative composition can be probably used to identify the biological source composition of M. brandaris. (iii) The investigated brandaris mol- (molluscan species) of a purple pigment/dye detected in a historical luscs were collected from an area of the Aegean Sea, nearby the or archaeological object [2e8]. For this reason, high performance island of Thera, where the earliest direct chemical evidence for the liquid chromatography (HPLC) has been employed to investigate use of true purple in painted objects were reported [8,9]. the chemical compositions of the three Mediterranean molluscan species: Hexaplex trunculus L. (Murex trunculus), Bolinus brandaris L. (Murex brandaris) and Stramonita haemastoma (Thais haemastoma) 2. Experimental e [2 8]. However, the majority of the previously published reports 0 focused on M. trunculus, while the compositions of the other two Indigotin (IND) was purchased from Fluka. Indirubin (INR), 6 - bromoindirubin (60MBIR), 6-bromoindirubin (6MBIR), 6- species have been scarcely investigated. Although M. brandaris 0 0 was used in the 1909 article for the identification of 6,60- bromoindigotin (MBI), 6,6 -dibromoindigotin (DBI) and 6,6 - dibromoindirubin (DBIR), were synthesized in pure forms accord- ing to procedures published elsewhere [8] and were used as stan- dards for identification purposes. The structures of these * Corresponding author. University Ecclesiastical Academy of Thessaloniki, compounds are shown in Fig. 1. Department of Management and Conservation of Ecclesiastical Cultural Heritage The hypobranchial glands were removed from the shells of fresh Objects, N. Plastira 65, Thessaloniki 54250, Greece. Tel.: þ30 2310 397730; fax: þ30 2310 300360. M. brandaris molluscs, collected in the coastline of Thera island E-mail address: [email protected] (I. Karapanagiotis). (Greece), and exposed to direct sunlight for 3 h. The glands became 0143-7208/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dyepig.2014.01.011 D. Mantzouris, I. Karapanagiotis / Dyes and Pigments 104 (2014) 194e196 195 Fig. 1. Structures of indigotin (IND), indirubin (INR), 6-bromoindigotin (MBI), 60-bromoindirubin (60MBIR), 6-bromoindirubin (6MBIR), 6,60-dibromoindigotin (DBI) and 6,60- dibromoindirubin (DBIR). Except for IND, the other compounds were synthesized and produced in pure forms according to previously published procedures [8]. coloured and were dissolved in dimethyl sulfoxide (DMSO) which The HPLC system (Thermoquest) was coupled to a Diode Array was heated at 80 C for 30 min. DMSO was selected for sample Detector (UV 6000LP) and was equipped with a 20 mL sample loop treatment as it results in better extraction yields than N,N- and an Alltima C18 (Alltech) column (5 mm particle size, dimethylformamide (DMF) or pyridine [8], which were used in 250 mm  3.0 mm) thermostated at 35 C. The gradient elution other studies [3e5]. The mixture was subjected to centrifugation program was consisted of H2O þ 0.1% TFA and CH3CN þ 0.1% TFA, as and the upper clear phase/solution was analysed by HPLC. described in detail elsewhere [8]. Fig. 2. Chromatogram collected for Murex brandaris at 540 nm. Photographs of the molluscs and the extracted hypobranchial glands which gave the analysed purple are shown. Absorbance maxima of the detected compounds are included in the table. Enlargement of the HPLC graph for 6e15 min is provided to clearly demonstrate the detections of IND, INR, MBI, 60MBIR and 6MBIR, which correspond to low HPLC peaks, compared to DBI and DBIR (high HPLC peaks). 196 D. Mantzouris, I. Karapanagiotis / Dyes and Pigments 104 (2014) 194e196 3. Results and discussion indigotin, 6-bromoindigotin, 6,60-dibromoindigotin, 6,60-dibro- moindirubin (as shown previously [3,6,8]), indirubin, 6-bro- Fig. 2 shows the identifications of IND, INR and their brominated moindirubin and 60-bromoindirubin (reported herein for the first derivatives (Fig. 1) in the DMSO extract of the hypobranchial glands time). of several M. brandaris molluscs. The HPLC graph was collected at 540 nm. This wavelength helps to visualize the detected com- 0 pounds, especially INR, 6 MBIR and 6MBIR (see absorbance maxima Appendix A. Supplementary data in Fig. 1) which were previously detected in M. trunculus [3,5e8] but not in M. brandaris [3,5,6,8]. Consequently, Fig. 2 is the first Supplementary data related to this article can be found at http:// 0 HPLC analysis that reveals the presence of INR, 6 MBIR and 6MBIR dx.doi.org/10.1016/j.dyepig.2014.01.011. in M. brandaris. The three compounds are detected in the graph of Fig. 2 in small amounts. Similarly, small amounts of INR, 60MBIR and 6MBIR are usually reported in HPLC results of M. trunculus [8]. References According to the literature, DBI is the major colouring component of M. brandaris, followed by DBIR which is detected in lower, but [1] Friedländer P. Über den Farbstoff des antiken Purpurs aus murex brandaris. Ber usually considerably high amounts [3,6]. These previously reported Dtsch Chem Ges 1909;42:765e70. fi [2] Wouters J, Verhecken A. High-performance liquid chromatography of blue and results are in agreement with the HPLC pro le of Fig. 2. Finally, IND purple indigoid natural dyes. J Soc Dye Colour 1991;107:266e9. and MBI are detected in M. brandaris in small amounts, according to [3] Wouters J. A new method for the analysis of blue and purple dyes in textiles. Fig. 2 and previously published studies [3,5,6,8]. Dye Hist Archaeol 1992;10:17e21. [4] Koren ZC. High-performance liquid chromatographic analysis of an ancient Relative (%) integrated HPLC peak areas of the detected M. Tyrian purple dyeing vat from Israel. Isr J Chem 1995;35:117e24. brandaris components are provided in the Supplementary file. [5] Karapanagiotis I, de Villemereuil V, Magiatis P, Polychronopoulos P, Measurements are reported at 288 nm (monitoring wavelength), Vougogiannopoulou K, Skaltsounis A-L. Identification of the coloring constit- e which is commonly used to report HPLC semi-quantitative data of uents of four natural indigoid dyes. J Liq Chrom Rel Tech 2006;29:1491 502. [6] Koren ZC. Archaeo-chemical analysis of Royal purple on a Darius I stone jar. Tyrian purple. The results in the Supplementary information offer Microchim Acta 2008;162:381e92. support to the above discussion related to the relative composition [7] Surowiec I, Nowik W, Moritz T. Mass spectrometric identification of new minor fi of M. brandaris. indigoids in shell sh purple dye from Hexaplex trunculus. Dye Pigment 2012;94:363e9. [8] Karapanagiotis I, Mantzouris D, Cooksey C, Mubarak MS, Tsiamyrtzis P. An 4. Conclusions improved HPLC method coupled to PCA for the identification of Tyrian purple in archaeological and historical samples. Microchem J 2013;110:70e80. fi [9] Karapanagiotis I, Sotiropoulou S, Chryssikopoulou E, Magiatis P, Using HPLC, the following compounds were identi ed in M. Andrikopoulos KS, Chryssoulakis Y. Investigation of Tyrian purple occurring in brandaris molluscs, collected from the sea of Thera (Greece): prehistoric wall paintings of Thera. Dye Hist Archaeol 2013;23 [in press]..