Plants to dye for…

We are told that the future’s bright, the future’s orange. If true, that’s rather monochrome, and drab, and sad. So, what better time to celebrate a more colourful – and botanical! – past (and one which might also help to make the future a little more polychrome)? Take, for instance, illuminated manuscripts, such as the 9^th Century Book of Kells, the 13^th Century Roman de la Rose, and the 15^th Century Trivulzio Book of Hours. Although such artworks are hundreds of years old, they continue to display vivid and vibrant colours today. How have those pigments survived, and in such intensity, for so long? What is their ‘secret’?

Key to answering those questions is knowing the source of the pigments, and their methods of preparation. Unfortunately, we don’t always know both – or sometimes neither – of those for all colours found in the artworks. That may be because ancient recipes and methodologies – where they are written down – may appear vague to modern eyes or hard to decipher because of the language they are recorded in, or both. It’s therefore great news that the chemical identity and method of production of one such plant-derived dye has now been elucidated by Paula Nabais et al. (Science Advances 17 Apr 2020: Vol. 6, no. 16, eaaz7772; doi: 10.1126/sciadv.aaz7772).

The pigment of concern was used to add blue or purplish colours (depending on pH, as for litmus) to ‘illuminate’ ancient manuscripts and other artworks, and is known as folium or turnsole in ancient texts. The source of folium has variously been stated as: Crozophora tinctoria, Solsequium, Morella, Heliotropium tricoccum and Croton tinctorium, which range of possibilities somewhat hinders accurate identification of its source. Under the name of turnsole, the pigment’s true source was further obscured because that name is also used for blue/purple colours obtained from lichens such as Roccella tinctoria and Lasallia pustulata (Maria Melo et al., Phil. Trans. R. Soc. A 374: 20160050; http://dx.doi.org/10.1098/rsta.2016.0050).

With those identity issues as a background, a considerable amount of ‘detective work’ – including the deciphering of an ancient recipe written in Judaeo-Portuguese (“the extinct language used by the Jews of medieval Portugal”) in a 15^th century book whose title translates to “The book on how to make all the colour paints for illuminating books” (Maria Melo et al. Herit Sci (2018) 6:44; https://doi.org/10.1186/s40494-018-0208-z) – was needed to unravel the mystery of folium.

The challenge was taken up, and Nabais et al. achieved three outcomes – confirming the identity of the botanical source of the pigment as Chrozophora tinctoria (a member of the Euphorbiaceae, the spurge family), successfully demonstrating a method to extract the pigment from its plant source, and identifying the pigment molecule.

The details of the steps involved in unravelling this mystery are detailed in the scientific paper (as you’d expect!), and make for a satisfying read. It is here sufficient to say that using an impressive range of sophisticated 21^st century methodology and analytical and computational techniques – which included: high-performance liquid chromatography; high-resolution mass spectrometry; diode array detector (HPLC-HRMS-DAD); gas chromatography–mass spectrometry (GC-MS) ; nuclear magnetic resonance (NMR); heteronuclear single-quantum coherence (HSQC); and electron paramagnetic resonance (EPR) – they isolated and identified the blue pigment molecule as 6′-hydroxy-4,4′-dimethoxy-1,1′-dimethyl-5′-{[3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]oxy}-[3,3′-bipyridine]-2,2′,5,6(1H,1′H)-tetraone. Rather pleasingly they’ve given this chemical the common name of chrozophoridin, and note that it is distinct from other plant-derived blue dyes such as anthocyanins and indigo.

So, that 1000-year-old mystery is now solved: The ancient illuminated manuscript blue pigment known as folium comes from Chrozophora tinctorial and is now called chrozophoridin. Well, almost. As the authors recognise in summarizing their study, “this molecular structure is key to identifying folium in works of art…” This will be a crucial next part of the story: Sampling folium from ancient manuscripts* and comparing its chemical identity with chrozophoridin should confirm that ‘folium’ in such artworks is highly likely to have been sourced from Chrozophora tinctoria.

But, isn’t this phytopigmentoforensics story just ‘blue sky’ research? Or does it have a more tangible benefit? No, and yes – respectively.

To elaborate. It is said that a thing of beauty is a joy forever. Sadly, that’s not always the case. Take for instance highly-coloured works of art from hundreds and even thousands of years ago. Over time some of their originally vibrant colours may have faded, or parts of the artwork been damaged or lost. To appreciate those items at their original best – as their creators presumably intended – it may be necessary to restore some of the now-faded colours. Unless you have the exact replacements you may not be able to faithfully ‘rejuvenate’ the original. However, for those decorated with ‘folium’ that should now be possible – thanks to the work of Nabais et al. Arguably, having been able to confirm the source of this pigment, and a reproducible method for its manufacture, the knowledge will help in attempts to conserve/preserve/restore ancient artworks where that pigment was originally used.

This botanical sleuthing tale has underlined the value of bringing together appropriate experts from different disciplines – in this case a “team comprised of chemists, conservation scientists and a biologist specializing in botany” – to address a specific problem. It also emphasises that there is much plant knowledge in the works of the ancients, you just have to work at it to extract and interpret it for a 21^st century audience**.

From that vivid pigment of the illumination tale, we go now to a much less colourful – albeit more black-and-white – plant pigment story about oak gall ink by Derek Niemann. Oak galls, also known as oak apples, are outgrowths that develop on an oak tree in response to eggs laid in its tissues by the oak gall wasp. Oak apples can be objects of great beauty, but, when crushed, mixed with water, iron sulphate, and gum arabic***, produce one of the most famous of black inks [“the ink of choice in the Western world for well over a millennium”, “one of the most important inks in the history of western civilisation” (Jana Kolar et al., Analytica Chimica Acta 555: 167–174, 2006; https://doi.org/10.1016/j.aca.2005.08.073%5D, iron gall ink.

Iron gall ink has been around for centuries and used in the drawings of artists such as Leonardo da Vinci, Rembrandt, Delacroix, and Vincent van Gogh, musical scores by JS Bach, Grieg, Mahler, Beethoven, and Mozart, and to produce such important documents as the Magna Carta and the American Declaration of Independence. Not a bad legacy for something that began life as a plant’s response to an insect-irritation****.

And, to end this item in a pleasingly circular way (which is reminiscent of the outline of an oak apple), Paula Nabais (and her Sci. Adv. co-authors Maria Melo, Natércia Teixeira, Victor Freitas, and Fernando Pina) contributed to a study that provided new insights into … iron-gall inks (Rafael Javier Díaz Hidalgo et al., Herit Sci 6, 63 (2018); https://doi.org/10.1186/s40494-018-0228-8). Plants, helping to record – and beautify – human endeavours for centuries.

* Whether this will be permitted is another matter. Since such artworks are usually so precious that obtaining permission to sample parts of the item – however small such samples may be – could be difficult.

** In that regard, this news item bears some resemblance to a previous Cutting that considered medical research work that examined an ancient remedy for treating an eye infection contained in the 9^th century ancient text known as Bald’s Leechbook.

*** For the chemistry that makes it all happen, see here. For more on ink from oak galls, try the web site dedicated to this remarkable material. And, if you’d like to have a go at creating your own oak gall ink, detailed, illustrated recipes can be found at The Schoenberg Institute for Manuscript Studies, and the Axbridge Museum Artist’s blog.