Effect of Rubia tinctorum Root Age on Coloration and Antibacterial Properties of Dyed Silk

Natural dyes are increasingly valued as eco‐friendly alternatives to synthetic colourants, with intrinsic functional properties such as antimicrobial activity [1, 2]. In this study we investigate how the age of Rubia tinctorum L. (madder) plants affects the dyeing of silk. Silk fabric was mordanted with potash alum and dyed with extracts from one-, two- and three-year-old madder roots harvested in the Edirne region of Turkey, and compared with a commercial madder root. Dyeing was carried out at a fixed dyestuff concentration (specified as % weight of fabric) and temperature; the baths were acidified (to pH≈4–5) to optimise anthraquinone uptake. Colour strength and CIElab coordinates were measured with a DataColor spectrophotometer, and colourfastness to washing, light and perspiration was assessed by ISO methods. The extracts and dyed fabrics were analysed by HPLC (Thermo Ultimate 3000) and FTIR (PerkinElmer Spectrum Two), and their surface morphology by SEM-EDX (Zeiss EVO LS10). Antibacterial activity of the dyed fabrics was tested against Staphylococcus aureus (ATCC 6538) by ASTM E2149. We performed one-way ANOVA on the colour and antibacterial results to test the effect of plant age. Results: Older plants yielded significantly lighter, stronger red shades: for example, L* (lightness) increased from 35.7 (±1.0) for 1-year roots to 39.6 (±1.2) for 3-year roots (p<0.05), while K/S (colour strength) likewise rose (3-year > 2-year > 1-year). All dyed samples showed excellent wash fastness (4–5) and good light and perspiration fastness (3–5), with a positive trend in ratings as plant age increased. In antibacterial tests, the 3-year madder gave the highest S. aureus reduction (76.4%), significantly greater than the 1-year sample (68.4%) (ANOVA, p<0.05). In summary, two- and three-year-old plants produced deeper red hues, stronger colour yield and higher antibacterial efficacy on silk. HPLC confirmed that older roots contained more anthraquinones (alizarin, purpurin, rubiadin) [2]. Novelty: Although Rubia (madder) has long been used as a red dye [2], the effect of harvest age on silk dyeing performance has not been systematically reported. This work uniquely correlates plant maturity with silk dyeing outcomes, justifying optimisation of harvest time. We further integrate recent literature on protein fibres: for example, Kovačević et al. (2021) showed that flavonoid-rich Spartium junceum extracts dye wool (a protein fibre) effectively with alum mordant [3], and Muruganandham et al. (2025) demonstrated sustainable silk dyeing with Bixa orellana seed extracts, achieving strong antibacterial action [4]. Reviews emphasise the rise in natural dye research and functional textiles [5]. Our study advances this by quantifying the influence of madder root age.