Chrozophora tinctoria in medieval and 19th century written sources

The use of the plant Chrozophora tinctoria (L.) A.Juss. to produce colors for illuminated manuscripts is extensively described in medieval written sources (1–4). What distinguishes C. tinctoria from other medieval natural sources to dye or produce paints is that, until now, the blue color structure remained elusive (5–7), despite the efforts by many groups in the last decades of the 20th century and into the 21st century (8–11). To tackle this mystery, our interdisciplinary group assembled a team of chemists who have expertise in natural products identification; conservation scientists, working in the reproduction of medieval colors; and a biologist with a great deal of botanical and field knowledge of the Portuguese flora, who oversees plant sourcing. This interdisciplinary approach proved essential to solving the complex structure of the blue dye.

On the basis of the detailed descriptions that were selected from three medieval treatises, we planned the field expeditions and the sampling methods for collecting plant materials. Fruits were collected during July, August, and September 2017 and 2018 (unripe and ripe) in southern Portugal (Granja/Mourão). C. tinctoria extracts were prepared following the treatises’ instructions, and the main colorant was isolated, purified, and characterized through a multi-analytical approach: high-performance liquid chromatography–high-resolution mass spectrometry–diode array detector (HPLC-HRMS-DAD), gas chromatography–mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) [1H, 13C, correlation spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), heteronuclear multiple-bond correlation (HMBC), INADEQUATE], and electron paramagnetic resonance (EPR) (12, 13). On the basis of the experimental results, the molecular structure for the blue color compound has finally been solved and will be discussed in this paper. Theoretical calculations supported this assignment, and the predicted ultraviolet-visible (UV-VIS) absorption spectra overlapped well the experimental spectra. Other families of chromophores, present in minor amounts, were also detected and characterized by HPLC-HRMS.

C. tinctoria and its uses were well known in antiquity and medieval times. However, the practice of creating the blue color fell out of use and it was lost in the 19th century. Its medicinal properties were first described by Dioscorides (De Materia Medica, 1st century) and were also mentioned in medieval pharmacopoeia texts, and studies focusing on its anti-inflammatory properties have been published recently (14–16). The dyeing properties of this species and their applications are a fascinating subject that will be revisited, as it is relevant to this research.

In medieval times, the blue and purple solutions extracted from C. tinctoria were stored, after adsorption onto cloth and drying, as watercolors (clothlets), and were applied as paint by cutting a piece of cloth and extracting its color with the appropriate binding medium. Complete descriptions of the plant, when to collect it, and how it was processed are found in important medieval treatises such as The book on how to make all the colour paints for illuminating books (15th century), Montpellier liber diversarum arcium (14th century), and Theophilus on divers arts (12th century), hereafter referred to as Book of all color paints (1), Montpellier (2), and Theophilus (3). In these treatises, only the fruits were collected (Fig. 1). The paint thus obtained was named folium or tornasol (turnsole); this latter designation is common to the blue/purple watercolors obtained from lichens (e.g., Roccella tinctoria and Lasallia pustulata), and consequently, C. tinctoria dyes remained forever interlinked to these lichens. Clarke, in his critical edition of texts written in the 14th and 15th centuries, goes further and suggests that the term tornasol was used generically to designate any water-based color stored in cloth (17). This had been already posited by Wallert (9) in his proposal to identify folium in manuscript illuminations based on molecular fluorescence. Fortunately, we were able to select some medieval recipes to produce C. tinctoria blues, because they are so detailed and offer accurate descriptions of the fruit and instructions to not break the fruits and free the seeds, thus pointing to the use of C. tinctoria (and not of lichens). We also have 19th century documental evidence that this medieval knowledge was preserved, until nearly the turn of that century, in the region of Grand-Gallargues (now, Gallargues-le-Montueux), in France. In 1842, Joly (18) published a clear, concise, and complete text on this subject, including references to earlier works such as Nissolle’s (19) publication that offers a precise description of the plant, accompanied with an equally accurate illustration. Joly’s text is remarkable in several aspects; first, he visits Grand-Gallargues to interview a priest by the name of Hugues and to gather information directly from the makers; precisely during the period when the plant should be collected (August/September), “vers la fin de septembre 1838, et dans les derniers jours du mois d’août 1839.” According to Joly, in this region, this activity has been documented since 1600 (when records are available), but even so, according to Father Hugues, it is one of the most mysterious of crafts (18, 20):

Fig. 1 The molecule of this study, chrozophoridin. Left: Close-up of C. tinctoria fruits (collected in Alentejo, Portugal) and clothlets prepared with the juice of the fruits following the instructions in the Book of all color paints. Light green fruits were used in this study shortly after collection. Right: Molecular structures of the blue colorants, hermidin (from M. perennis), and chrozophoridin (from C. tinctoria). Photo credit: Paula Nabais, Universidade NOVA de Lisboa.

“peu d’industries sont aussi mystérieuses: ceux qui l’exploitent n’en connaissent point la destination; ceux qui en profitent n’en connaissent point la préparation, et ceux qui l’ont décrite n’ont débité que des mensonges, parce qu’ils ne transcrivaient que de fausses indications” [few industries are so mysterious: those who exploit it do not know its final applications; those who profit by it do not know how to prepare it, and those who have described it have only told lies, because they have only transcribed false directions] (18).

Joly describes the process using the entire plant to produce the dark blue color and the innovations that were introduced in the medieval process, and concludes that, at that time, the clothlets were mainly sold to dye cheese rinds red in the Netherlands (21): “mais il paraît que l’usage en est borné à donner aux croûtes du fromage de Hollande cette teinte rouge qui les distingue, (...) Il suffit de tremper les formages dans un baquet d’eau bleuie par les chiffons, et de les en retirer presque aussitôt pour les faire sécher” [but it seems that the use is limited to giving the rind of Dutch cheese that red hue that distinguishes it, (...) All you have to do is soak the cheese in a bucket of water dyed blue by the clothlets (rags), and to remove them almost immediately to left them dry] (18). Joly proposes that not only other species could be tested for dyeing more efficiently, such as other Chrozophora species, but also Mercurialis perennis (dog’s mercury) and Mercurialis tomentosa. This is extraordinary, as now from this current project, we know that these dyes share a common molecular structure (Fig. 1). Joly also tested and concluded that the precursors for the blue may be found in all the parts of the plant and are more abundant in fruits. He concluded, “Sous l’influence de la vie, il existe dans ces organes à l’état incolore; après la mort du végétal, et sous l’influence de l’oxygène atmosphérique et d’une prompte dessiccation, il peut devenir bleu” [Under the influence of life, it exists in these organisms in a colorless state; after the death of the plant, and under the influence of atmospheric oxygen and a rapid desiccation, it becomes blue] (18), which is precisely the way the appearance of the blue color in M. perennis is described in more recent studies performed by Swan and Lorentz et al. (22–25). These authors have characterized the molecular structure for the blue chromophore as will be described below.