**1. Introduction**

Brazilwood dyestuffs and pigments are derived from a number of closely related and often historically confused species of trees of the Leguminosae family. Heartwood from the so-called 'soluble redwoods', sappanwood (*Caesalpinia sappan* L.) from Southeast Asia and several South American species commonly called brazilwoods (including *Caesalpinia brasiliensis* L. and *Paubrasilia echinata* (Lam.)), contain brazilin, the main colorant in brazilwood dyes. Brazilin (Figure 1a) is a tetracyclic homoisoflavonoid which converts to a more deeply red colored molecule, brazilein, through autoxidation of a hydroxyl group into a carbonyl group (Figure 1b). Other phenolic components isolated from brazilwood include xanthone, coumarin and various chalcones, flavones and other homoisoflavonoids [1].

The fugitive nature of brazilwood has been documented since the Middle Ages, and its use as both a dyestuff and a pigment has been regulated or proscribed as a result, particularly for use as a sole colorant [2]. It was, however, less expensive than other

**Citation:** Doherty, B.; Degano, I.; Romani, A.; Higgitt, C.; Peggie, D.; Colombini, M.P.; Miliani, C. Identifying Brazilwood's Marker Component, Urolithin C, in Historical Textiles by Surface-Enhanced Raman Spectroscopy. *Heritage* **2021**, *4*, 1415–1428. https://doi.org/ 10.3390/heritage4030078

Academic Editor: Lucia Burgio

Received: 24 June 2021 Accepted: 22 July 2021 Published: 25 July 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

natural red dyestuffs and gave an attractive color when fresh, so found great use as a textile dye, an organic lake pigment in manuscripts (where light exposure was less of an issue) and in paintings. Evidence of the use of brazilwood-derived lake pigments has been found in paintings by a diverse range of artists, from Raphael to Rembrandt and Van Gogh [3–5]. For textiles, the dyestuff was generally extracted in neutral or basic aqueous solutions to which the mordanted textile was added. To prepare brazilwood lake pigments, various recipes are known. In some, the dyestuff is extracted from the raw material under alkaline conditions, then added with alum to precipitate the pigment from the solution. Alternatively, the dyestuff could be extracted in a slightly acidic solution (e.g., alum solution) and the pigment precipitated on addition of an alkaline solution or of a source of calcium carbonate or sulphate [1,6,7].

The analytical examination of brazilwood's main colorants is generally straightforward for the characterization of unaltered homoisoflavonoid constituents by nondestructive FT-Raman and Infrared spectroscopies as well as micro-destructive chromatographic methods alike [8–10]. However, accurately identifying aged and faded brazilwoodbased dyes and pigments in historic artworks is challenging. Brazilwood's renowned lack of permanence has limited the identification of severely faded brazilwood in artefacts, in the heritage science domain, to only the most sensitive and specific micro-destructive techniques, such as HPLC with spectrophotometric or mass spectrometric detection [11]. Such studies have indicated the presence of a minor non-dyestuff component that appears to be a brazilwood marker, highlighted by Nowik [10]. This marker component has been recently identified as 3,8,9-trihydroxy-6H-benzo[c]chromen-6-one, urolithin C (Figure 1c), and unequivocally linked to brazilwood by Peggie et al. [12].

**Figure 1.** Molecular structures of (**a**) brazilin, (**b**) brazilein and (**c**) marker component urolithin C.

Urolithin C had initially been thought to be a degradation product of brazilein, yet its natural and unquantified presence in the heartwood has been highlighted, suggesting its unrelated presence [13]. Furthermore, observations by Peggie et al. have established a link between an alkaline extraction method and the increased presence of urolithin C in brazilwood dyes and lake pigments. Tamburini has subsequently noted that a sample dyed with sappanwood at alkaline pH showed a higher relative abundance of urolithin C than in a sample dyed at neutral pH, but also highlighted therein a much lower abundance of protosappanins [14]. Such deductions may provide information as to the origins of urolithin C within these dyes and pigments. In nature, tannin constituents play a major role in the formation of the urolithin metabolite. Studies have observed that in animal guts microbia can metabolize ellagitannin and ellagic acid into dibenzopyran-6-one derivatives with different hydroxyl substitutions, through the loss of one of the two lactones present in ellagic acid (lactonase/decarboxylase) followed by subsequent removal of hydroxyls through dehydroxylase activity [15,16]. In the context of the study of historical textiles and pigments, what is of particular interest is that urolithin C appears to be relatively stable, and still persists in degraded samples of brazilwood dyes or pigments even when other colorant components have been lost [13]. This provides an ideal opportunity for further

analytical methods to be examined to assess their suitability in determining the presence of urolithin C.

Surface-enhanced Raman spectroscopy (SERS) is an ideal candidate for such studies, and is being increasingly adopted for the investigation of natural and synthetic organic dyes (and pigments derived from such colorants) associated with historical artefacts by combining Raman spectroscopy's fingerprinting ability with plasmonic-enabled enhanced sensitivity. The SERS effect is due to a combination of an electromagnetic (EM) enhancement associated with plasmon excitation in metal particles and a chemical (CHEM) enhancement associated with the transfer of electrons from the analyte molecule to/from the metal particles in both ground and excited states, often forming a metal−molecule bond. The application of SERS techniques has expanded in key application areas of heritage science in the last twenty years [17] and is nowadays competitive in terms of sample requirements to HPLC for ultra-sensitive and selective constituent identifications with current in situ potential. The construction of in-house databases and optimized analytical protocols help to improve the reproducibility of measurements for the characterization of the low quantities and/or unknown components that are typically encountered in the analysis of historic samples. Although still a micro-destructive and qualitative technique when used for the analysis of real, historical samples, the versatility of SERS methodologies can allow a single sample to be used in a series of sequential analyses. First, a single micro-sample can be mixed with an activated colloidal solution and investigated as such. Following drying of this same sample, it can undergo suitable acid-based pre-treatments such as treatment with nitric acid or controlled exposure to hydrofluoric acid vapors before examination [18]. SERS screening methods are therefore potentially of great value for the investigation of naturally aged artefacts suspected of containing low concentrations of brazilwood dyestuffs and pigments which are of historical relevance and which necessitate less destructive investigations or even in situ analyses when sampling is prohibited, or where chromatographic methods are unavailable. The use of conventional SERS methodologies is most likely to be successful in cases where the brazilwood colorant is potentially present in the most superficial layer. The use of this technique when analyzing paint samples is, however, much more complicated, as high degrees of heterogeneity can be expected, given that the brazilwood is present as a lake pigment that may have been mixed with other pigments. The embedding of such samples in resins or other materials as cross-sections further adds to the complexity. The use of SERS methodologies thus seems most viable, at this stage, as a first approach, to use during the examination of textiles dyed with brazilwood, even in a faded condition.

In this work a reference sample of urolithin C was examined by classical Raman and colloidal SERS to understand whether characteristic features of the relatively persistent urolithin C marker component can be successfully distinguished. Based on the results of this study and additional work on reference samples, colloidal SERS was used to examine four historical textiles (with pre-treatments as necessary). Samples of the four textiles had been previously analyzed by HPLC-DAD during restoration campaigns (data included in the restoration documentation stored by the corresponding institutions) and were all known to contain urolithin C together with varying proportions of surviving brazilwood colorant components, other dyestuffs and a range of other molecules typical of historical dyed textile samples.

#### **2. Materials and Methods**

Urolithin C was purchased from Dalton Research Molecules, 349 Wildcat Road, Toronto, Ontario, M3J 2S#11, Canada, purity greater than 95% by HPLC, and used as such. Brazilin was purchased from ICN Biomedical Inc. Cat #205613. The powdered brazilwood lake reference was prepared at the National Gallery in 2003 from Sappan Lignum (*Caesalpinia sappan*), no further details of the recipe are available. Details of the four historical textile samples, two from tonacelles (ecclesiastical garments) and two from tapestries examined, are given in Table 1.

Samples S#11 and S#5 belong to the series of the Valois Tapestries, woven in Brussels in the sixteenth century and belonging to the Gallerie degli Uffizi, Florence, directed by Eike Schmidt. The series consists of eight sixteenth-century tapestries, representing Catherine de Medici and her family observing courtly festivities, collectively known as The Valois Tapestries [19]. The samples were collected during the restoration performed by Restauro Tessile di Bayer e Perrone da Zara, Florence, Italy, directed by Alessandra Griffo (Gallerie degli Uffizi) and funded by the Friends of the Uffizi, chaired by Maria Vittoria Colonna Rimbotti, with the contribution of Mrs. Veronica Atkins. Samples S#27 and S#9 are part of a collection of eighteenth-century Italian ecclesiastical garments belonging to a public authority, which was subjected to a restoration campaign in 2011–2012 during which the private company 'Giordano Passarella' was appointed, who performed the sampling.

**Table 1.** Outline of samples investigated in this work.

