Structural elucidation of the main coloured compounds of Justicia spicigera based on experimental and computational electronic features Lucie Arberet a,b,* , Lea-Maria Ibele a , Alain Blond c , Anne Michelin b , Witold Nowik b,d , Alain Tchapla a , Federica Agostini a , Christine Andraud b , Sylvie H´eron a a Universit´e Paris-Saclay, CNRS, Institut de Chimie Physique, Orsay 91405, France b Centre de Recherche sur la Conservation (CRC), Mus´eum national d’Histoire naturelle, CNRS, Minist`ere de la Culture, 36 rue Geoffroy-Saint-Hilaire, Paris 75005, France c Mol´ecules de Communication et Adaptation des Micro-Organismes (MCAM), Mus´eum national d’Histoire naturelle, CNRS, 57 rue Cuvier, Paris 75005, France d Laboratoire de recherche des monuments historiques, 29 Rue de Paris, 77420 Champs-sur-Marne, France A R T I C L E INFO Keywords: Natural dye Structural elucidation Justicia spicigera Computational chemistry Electronic spectroscopies Phenoxazones ABSTRACT In continued efforts to characterise the dye extracted from Justicia spicigera leaves, the two main coloured compounds were isolated. In this study, a multi-analytical techniques characterisation of their structures was carried out by HRMS, NMR, vibrational (IR and Raman) and electronic (UV–Vis absorption and fluorescence) spectroscopies, completed by computational studies. Two unreported compounds from the phenoxazone (aka phenoxazine-3-one) family were identified, the 2-amino-7‑hydroxy-8‑methoxy-3H-phenoxazin-3-one, a posi- tional isomer of the already described perisbivalvin B and the 2-N-(4‑hydroxy-γ-glutamyl)-7‑hydroxy- 8‑methoxy-3H-phenoxazin-3-one. The simulated UV–Vis absorption and fluorescence spectra of the suggested structures showed good agreement with the experimental ones, supporting the structural elucidation and giving more insights into the chemistry of these compounds. 1. Introduction Justicia spicigera Schltdl. (Acanthaceae) – also known as Jacobinia spicigera (Schtdl.) L.H. Bailey, Mexican honeysuckle, muicle, sacatinta or many other vernacular names – has been used for its therapeutic and dyeing properties in Central America since pre-Hispanic times [1–4]. For instance, its use as paint in the Codex Borbonicus, a 16th century manuscript, has been revealed by Raman and UV–Vis fluorescence spectroscopies [5,6]. Most of research studies regarding J. spicigera focused on its pharmacological properties: anti-oxidant [4,7–9], anti-diabetic [2], anti-hypertensive [10], anti-depressant [11,12], anti-cancer [13], anti-inflammatory [14] and other characteristics. On the otherhand, phytochemical studies of the plant extract are less frequent and the bioactive compounds are still not always well identi- fied. Various flavonoids were identified: kaempferitrin and tri-rhamnosides of kaempferol in ethanolic extracts [2,15,16], kaemp- ferol 7-O-rhamnoside and kaempferol 3-O-acetyl-rhamnoside 7-O-rhamnoside in ethyl acetate extracts [17], hesperidin, naringenin and kaempferol in chloroform-based extracts [10]. As for other com- pounds, the presence of β-sitosterol and 3-O-glucoside of β-sitosterol, allantoin and cryptoxanthin I in hexane-isopropyl-ether-methanol ex- tracts was also shown [16]. In ethanol-water extracts analysed by GC–MS, eucalyptol, phytol and azulene were identified as the main compounds in fresh leaves while phytol, linoleic acid and oleic acid were found in dried leaves [4]. Procumbenoside B was identified in meth- anolic extracts [14]. Regarding the compounds responsible for the purple colour of the aqueous extract used for basketry or textile dyeing, they were little studied. Several papers dismissed the presence of indi- gotin [18–20]. Anthocyanins were identified in ethyl acetate extracts [9] while they appeared to be absent from aqueous or ethanol extracts [4]. Before the start of our project, it was not yet possible to link the violet dye extracted from the leaves of J. spicigera to any precise chemical structure. In our preliminary study, two coloured compounds, namely X1 and X2, were detected by HPLC-diode array detector (DAD) in the extract of Justicia spicigera leaves, along with non-coloured compounds [6]. To perform extensive structural analyses of these compounds, a method for their purification was optimised and the two compounds were success- fully isolated [21]. Based on high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy, one of them, X2, * Corresponding author at: Sorbonne Universit´e, CNRS, Laboratoire d’Arch´eologie Mol´eculaire et Structurale, LAMS, Paris 75005, France. E-mail address: lucie.arberet@sorbonne-universite.fr (L. Arberet). Contents lists available at ScienceDirect Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstr https://doi.org/10.1016/j.molstruc.2025.142281 Received 14 January 2025; Received in revised form 28 March 2025; Accepted 2 April 2025 Journal of Molecular Structure 1338 (2025) 142281 0022-2860/© 2025 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.