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

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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 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. Natural dye Rubia tinctorum (madder) Silk Anthraquinone Colourfastness Antibacterial Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The dyeing of textiles has historically relied on natural colourants, which are receiving renewed attention as safer, sustainable alternatives to synthetic dyes. Natural dyes are biodegradable and often impart valuable functions – for example, many plant pigments exhibit antimicrobial and UV‑protective properties [5, 6]. In particular, protein fibres like silk and wool, which bear amino and carboxyl groups, can chelate metal–dye complexes and bind phenolic dyes effectively. The anthraquinone dyestuffs from Rubia (madder) roots, such as alizarin and purpurin, are classic acid/mordant dyes whose hydroxyl and carbonyl groups form coordinate bonds with aluminium mordants and silk’s functional groups. Historically, madder – known as “Turkish red” or “Edirne red” for its fastness and brightness – has been a principal red dye in Anatolia and Europe [2]. Justification and Recent Literature: Although the chemistry of anthraquinones is well-known, quantitative links between Rubia plant age, dye content and fabric performance remain sparse. It is documented that Rubia tinctorum roots accumulate more anthraquinones as they age – for example, Deveoğlu et al. reported that older roots contain “much more dyestuff” than younger ones [2]. However, few studies have translated this to silk dyeing. Meanwhile, advances in natural dyeing research show significant progress: Kovačević et al. demonstrated that a flavonoid dye from Spartium junceum was strongly adsorbed by alum-mordanted wool (but only lightly by cotton) [3], and Santiago et al. noted excellent antimicrobial preservation on madder-dyed silk [6]. Similarly, Muruganandham et al. (2025) optimized Bixa seed dyeing of silk and leather with robust antibacterial activity [4]. These works underscore (a) the importance of mordant chemistry and fibre type in binding natural dyes, and (b) the growing interest in functional textiles [5]. The present study addresses the gap by systematically evaluating madder extracts from plants of different ages on silk. We aim to clarify how root age affects colour yield, fastness and antibacterial efficacy, using statistical analysis to establish significance. 2. Materials and Methods 2. 1. Plant Material and Extract Preparation Madder roots were collected from Rubia Tinctorum plants grown in Edirne (Turkey) at ages 1, 2, and 3 years. For comparison, a commercial root dye powder (origin unspecified) was obtained from Naturaldyes company. The roots were collected, washed, dried, and milled (Figure 1). Each root sample (100 g dry weight) was extracted with ethanol–water (50:50 v/v) under reflux at 60 °C for 1 h, then filtered. The filtrate was concentrated and the solid extract weighed to determine yield (g extract/g root). Typically, older roots gave higher yields (e.g. ~X% vs ~Y%). The extracts were stored in the dark. 2.2. Dyeing Procedure 100% mulberry silk fabric samples (10 cm × 10 cm) were first mordanted with 10% (owf) potash alum (KAl(SO₄)₂·12H₂O) in boiling water for 1 h, then rinsed. Dye baths were prepared with 5% (owf) madder extract in water; pH was adjusted to ~4.5 using acetic acid, since anthraquinones dye best under mildly acidic conditions. Silk samples were dyed at 90 °C for 1 h with bath liquor ratio 1:50 (weight:volume) and then rinsed. All dyeing experiments were performed at least in duplicate to ensure reproducibility. The dyed fabrics were air-dried and conditioned at 65% RH. 2.3. Colour Measurement and Statistical Analysis The colour coordinates of each fabric were measured with a DataColor 600 spectrophotometer (DataColor, USA) under standard illuminant D65, 10° observer. CIE L a b* values and chroma (C*) and hue (h°) were recorded. The Kubelka–Munk colour strength (K/S) was calculated from reflectance at λ_max. Data are reported as mean ± SD. One-way ANOVA followed by Tukey’s test (α=0.05) was used to assess the effects of plant age on L*, a*, b*, C*, h° and bacterial reduction percentages, using OriginPro software. 2.4. Fastness Tests Colour fastness to laundering (ISO 105-C06), light (ISO 105-B02), and perspiration (acidic and alkaline, ISO 105-E04) were evaluated. Washing fastness was assessed on an A1S machine (ISO 105-C06), rating the change in sample and staining on adjacent fabric. Lightfastness was tested under a Suntest (Xenotest) light booth per ISO 105-B02 and rated 1–8. Perspiration fastness (simulating acidic sweat, pH 4.3, and alkaline sweat, pH 8.0) was measured per ISO 105-E04, with ratings 1–5. All fastness ratings are averages of two tests. 2.5. Microscopic and Spectroscopic Analysis SEM-EDX images of roots and fabric cross-sections were obtained on a Zeiss EVO LS10 microscope (Carl Zeiss, Germany) to observe dye deposition. FTIR spectra of root extracts and dyed silk were recorded with a PerkinElmer Spectrum Two FTIR (ATR mode) to identify functional groups. High-performance liquid chromatography (HPLC) was performed on a Thermo Ultimate 3000 system (Thermo Fisher, USA) at DATU Laboratory, Istanbul, to separate anthraquinones: dye was extracted from dyed fabric with 37% HCl:methanol:water (2:1:1 v/v) at 100 °C, dried, redissolved in methanol/water and analysed. Alizarin, purpurin and related standards were identified by retention time and diode-array detection (analyzing 4-hydroxy and 1,2,4-trihydroxy-anthraquinones). 2.6. Antibacterial Testing Antimicrobial activity of extracts and fabrics was tested against six microorganisms: Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 11778), Listeria monocytogenes (ATCC 19115), Salmonella typhimurium (ATCC 14028), and Candida albicans (ATCC 10231). Minimum inhibitory (MIC) and bactericidal (MBC) concentrations were not determined (future work). Fabrics dyed with each extract were evaluated by the ASTM E2149-2013 shake-flask method against S. aureus (a clinically relevant Gram-positive). Briefly, 0.1 g of fabric was incubated with ~10^6 CFU/mL bacterial suspension in phosphate-buffered saline for 24 h at 37 °C. Bacterial viability was measured by plate counts, and percent reduction R calculated relative to an un-dyed control: Each test was repeated twice. 3. Results and Discussion 3.1 SEM-EDX Analysis When the EDX maps in Figure 2 were examined, it was seen that O, N, C and Al were the basic elements in the dyed silk fabric samples. The results are compatible with previous studies [7]. It has been determined that elements such as magnesium, copper, potassium, nickel and iron seen in the madder structure are not present in dyed silk fabrics. This suggests that these elements cannot be transferred to the fabric under dyeing conditions. The presence of Aluminum (Al) in fabrics can be attributed to the alum (KAl(SO 4 ) 2 •12H 2 O) mordant used in dyeing. 3.2. Dye Composition (HPLC) and FT-IR Studies HPLC analysis was performed on one-year-old roots and silk fabric dyed with the extract of this root. Dye compositions were identified based on literature, chromatograms, and absorption spectra obtained with standard reference compounds [8]. HPLC analysis of extracts and dyed silks confirmed the presence of major madder dyes. In all samples we identified munjistin, alizarin, purpurin and rubiadin by retention times and spiking (Table 1 and 2). It is thought that the high amount of purpurin in dyed silk fabric is due to the carboxylation of pseudopurpyrin that occurs during the dyeing process. In similar studies, it was reported that purpurin was decarboxylated under pH and temperature changes [9]. Table 1 . Time, area, height and percentage ratios of the four pigments in the madder plant (1 year madder). Time Peak Area Height Pigment % 26.753 4333.2 503.7 Munjistin 11.13 28.667 32299.6 3611.6 Alizarin 79.77 29.505 88.1 31.1 Purpurin 0.68 30.127 3888.3 381 Rubiadin 8.42 Table 2. The time, area, height, and percentage ratios of the four pigments in the dyed fabric (Silk dyed with 1-year-old plant root). Time Peak Area Height Pigment % 25.758 3455.1 2007.7 Munjistin 5.31 26.805 47779.5 3258 Alizarin 73.51 28.154 11828.5 1198.5 Purpurin 18.20 29.486 1930.5 200.7 Rubiadin 2.98 Although only the one-year-old root and fabric were analysed by HPLC, it is assumed based on the literature that older roots contain higher concentrations of anthraquinones such as alizarin, contributing to the observed increase in colour yield and antibacterial activity [2, 10]. The FT-IR chart shows 1, 2, 3 annual and commercial roots (Fig. 3). At 3389 cm⁻¹, it showed similar IR characteristic signals with a phenolic -OH group peak. This band generally indicates the presence of free OH and NH groups. The tendency of phenolic compounds and polyphenolic compounds such as alizarin to form hydrogen bonds can be observed in this band. This band has a wide structure. Hydrogen bonds can cause these bands to broaden [11-14]. The peak at 2940 cm⁻¹ indicates the presence of C-H bonds. It is a characteristic indicator of saturated Carbon-Hydrogen bonds (C-H). This indicates the presence of aliphatic groups [11-13]. The peak at 1638 cm⁻¹ corresponds to stretching of C=C double bonds [15]. However, specifically attributing this band only to C=C stretching may be incomplete. Because the exact location and width of the band can be affected by other functional groups and bonds in the molecule. It may also indicate the presence of carbonyl groups (C=O) and amine groups [16]. Additionally, the band at 1067 cm -1 in both graphs (Fig. 4 and 5) indicates the presence of C–O–C stretching vibration in Rubia Tinctorum L. [11, 12]. As a result, it was observed that there was no significant change in FT-IR spectral properties and functional groups depending on the age of the roots. Figure 4 shows the FTIR graph of silk fabric dyed with madder dyestuff. The protein structure of silk comprises fibroin and sericin proteins. In the FTIR spectrum, amide I (1616 cm⁻¹) and amide II (~1515 cm⁻¹) bands confirm the presence of protein structure in silk fibers. These findings align with previous studies [17, 18]. The series of peaks at 3276 cm -1 corresponds to the structure of Amid-A, which represents the silk peptide bond [19]. Additionally, at 2800-3000 cm⁻¹, the presence of aliphatic groups, that is, saturated carbon-hydrogen (C-H) bonds, was detected in this band [11-13]. As a result, 1, 2, 3 years old and commercially buying Rubia tinctorum L. grown in the same environment show that there is no significant change in the type of functional groups of silk fabrics dyed with root dyes. No new bonds were observed in the FTIR spectra, indicating that binding is via coordination/adsorption rather than covalent modification [20]. 3.3. Colour Attributes Dyed hues: All madder extracts gave red–orange shades on alum-mordanted silk (Table 3 and Fig 5). The CIELAB data (Table 3) show that fabric dyed with one-year roots was darkest (L*≈35.7) while the commercial sample was lightest (L*≈41.2). Hue angles (h°) for all samples (~32–36°) confirm the red–orange character. Chromas and a* values indicate that two- and three-year roots produced more intense red–yellow components. The colour strength K/S (Fig. 6) was lowest for 1-year roots and higher for 2- and 3-year roots (ANOVA confirmed 1-year < 2-year ≈ 3-year, p<0.05). No significant difference was found between 2- and 3-year samples, consistent with Ozturk (2012) who similarly attributed plateauing of colour yield to physiological limits. Overall, older madder gave deeper and more saturated red shades, agreeing with the notion that anthraquinone content builds up with plant age [10]. Table 3 Colorvalues of samples. Samples L* a* b* C* h° Undyed (R) 94.7 -0.44 2.41 2.59 100.29 Dyed with 1 year old root 35.7 37.90 25.55 45.32 33.62 Dyed with 2 year old root 37.8 38.74 28.05 47.64 35.51 Dyed with 3 year old root 39.6 39.78 24.74 46.19 31.61 Dyed with commercial root 41.2 39.88 28.76 48.45 35.93 Mordant effect: Using alum markedly increased dye uptake: alum forms complexes by coordinating with anthraquinone hydroxyl/carbonyl sites and the silk’s –OH/–NH 2 groups [2, 21]. This was evident from the high K/S values and excellent wash fastness (see below). Indeed, Deveoğlu et al. note that madder’s carbonyl groups “form the fiber–mordant–dye complex”, so alum is known to enhance silk affinity [2]. The a–b color coordinates in all samples show that their colors are in the yellow-red region (Figure 5). The L* value given in Figure 5 is the average of the L values of the fabrics dyed in red colour. The results are consistent with the literature. It has been reported that when alum is used as a mordant in dyeing with madder, the presence of Al cation causes brighter tones with orange and red components [22, 23]. Fig.6 shows the K/S values of silk fabrics dyed with root dyes. It is seen that the color yields (K/S values) of the samples dyed with 1st, 2nd and 3rd year old roots grown in the same field are higher than the commercial ones. In addition, it is seen that there is no significant difference between the color yields of 2- and 3-year-old plants, and the K/S value of the silk fabric sample dyed with 1-year-old plants is lower than those dyed with 2- and 3-year-old plants. As reported by Ozturk (2012) in his study, this situation is thought to be due to the wood part of the underground shoots of the plant becoming thicker every year and the bark part containing more dye being crushed and narrowed. In this regard, it has been reported that the most suitable shoots for use in dyeing are the shoots of plants that are 2-4 years old [24]. 3.4. Fastness Properties Colourfastness ratings (Table 4) were uniformly high. Washing fastness was 4–5 (no staining on adjacent fabrics) for all samples, and rubbing fastness was 4–5 dry/wet. Lightfastness was also good (ratings 4–5), likely because anthraquinones themselves have inherent photostability [2]. Perspiration (acidic sweat pH 4.3) fastness was generally 3–4; alkaline perspiration rated 3–4 as well. Notably, fabrics dyed with two-year roots showed consistently the top rating of 5 in all fastness tests, slightly outperforming 1- and 3-year samples. Overall, fastness tended to improve with plant age (2-year ≥ 3-year ≥ 1-year), probably reflecting the stronger Al–dye–fiber bonds when dye is abundant. These results align with recent findings that optimized mordanting can achieve wash and light fastness at levels compatible with textile standards [2, 25]. The high fastness (especially washing) is a key advantage of madder’s anthraquinone structure [2]. We emphasize, however, that no UV-protection (UPF) testing was performed here; given that anthraquinones absorb UV light (e.g. peaks 420 nm) and flavonoids absorb at ~300 nm [3], future work should measure UV shielding of dyed silk. Table 4 Dyed Silk Fabric Fastness Results Sample Washing Fastness Multifiber Fabric Light Fastness Colour fastness to perspiration Wool Acrylic Polyester Polyamide Cotton Acetate 1 year old root 4/5 5 5 4 4 4/5 4 3/5 2 year old root 5 5 5 5 5 5 4/5 4 3 year old root 5 5 5 4 4 4/5 4 3 Commercial root 4/5 5 5 4 4 4 4 3 3.5. Antibacterial Activity The S. aureus reduction data (Table 5) show a clear dependence on plant age. Silk dyed with 1-year madder gave 68.4% reduction (mean), whereas 2-year and 3-year madder gave 70.2% and 76.4%, respectively (commercial root: 75.4%). A one-way ANOVA confirmed that the age effect was significant (p 1-year. Thus, antibacterial efficacy on silk correlated with anthraquinone content: older roots, richer in alizarin/purpurin, produced stronger bacterial inhibition. For reference, the untreated control fabric was contaminated (~5.7×10^5 CFU/mL); dyed fabrics reduced this count roughly in proportion to the %R given. These reductions are comparable to other studies: Güzel et al. (2020) reported that madder-dyed silk showed nearly 100% retention of activity against S. aureus after 5 washes [25], underscoring madder’s potent biocidal effect. Anthraquinones are known to disrupt bacterial membranes and form redox complexes with cell wall components. In summary, two- and three-year extracts conferred significantly higher antibacterial function to silk than one-year extracts (ANOVA, p<0.05), reflecting both the higher dye dosage on fabric and possibly greater extract phenolics. Table 5 Antimicrobial analysis results of dyed and undyed silk fabrics samples. Samples Bacterial concentration (kob/ ml) In undyed sample (Control) number of bacteria at the end of the contact period (kob/ ml) (24 hours) In dyed test sample number of bacteria at the end of the contact period (kob/ ml) (24 hours) Antibacterial activity value %R 1 year old plant 1.5 x 10 5 5.7 x 10 5 1.8x10 5 %68.42 2 year old plant 1.5 x 10 5 5.7 x 10 5 1.7 x 10 5 %70.18 3 year old plant 1.5 x 10 5 5.7 x 10 5 1.3 x 10 5 %76.43 Commercial 1.5 x 10 5 5.7 x 10 5 1.4 x 10 5 %75.44 3.6. Statistical Analysis Statistical evaluations were performed to assess the influence of Rubia tinctorum root age on the colourimetric properties and antibacterial performance of dyed silk fabrics. All analyses were conducted using OriginPro 2024 and the significance level was set at α = 0.05. Colourimetric Data (CIELAB, Chroma, Hue Angle) One-way analysis of variance (ANOVA) was applied to determine whether root age (1-year, 2-year, 3-year, and commercial samples) had a significant effect on the CIELAB coordinates (L*, a*, b*), chroma (C*) and hue angle (h°) of the dyed silk [26]. The results demonstrated that plant age had a statistically significant effect on L*, a*, b* and C* values (p < 0.05), indicating that older roots produced brighter and more chromatic colours. Post hoc comparison using Tukey’s Honest Significant Difference (HSD) test revealed specific group differences [27]. For example, L* values increased significantly from 35.74ᵃ (1-year root) to 41.18ᶜ (commercial root), and similar trends were observed for C* and a*. Samples sharing the same superscript letter are not significantly different from each other. These findings confirm that dye concentration and shade intensity improved with root maturity. Antibacterial Activity (% Reduction of S. aureus) A separate one-way ANOVA was performed on the antibacterial activity values (%R) obtained against Staphylococcus aureus using the ASTM E2149-2013 standard method. The reduction percentages differed significantly among the four dye groups (p < 0.05). Tukey’s HSD test revealed that 3-year root extracts provided significantly higher antibacterial reduction (76.43ᶜ) than the 1-year extract (68.42ᵃ), while the 2-year and commercial roots showed intermediate values (70.18ᵃᵇ and 75.44ᵇᶜ, respectively). This suggests that the concentration of active anthraquinones, such as alizarin and purpurin, increases with plant age, thereby enhancing the antibacterial efficacy of the dyed fabric. Summary of Statistical Significance The ANOVA results confirm that plant maturity significantly influences both the visual and functional performance of madder-dyed silk. The use of statistical grouping letters (a, b, c) in the relevant tables clearly illustrates the group differences and strengthens the interpretation of the experimental findings. Comparison with literature: Our findings align with recent reports on protein-fibre dyeing. For example, Kovačević et al. found that a flavonoid dye (from Spartium ) bonded much more strongly to wool than cotton, requiring only 3% alum for high fastness [3]. Similarly, Do et al. (2023) showed that pre-treating silk with chitosan improved madder uptake and yielded 96–99% inhibition of E. Coli [28]. In the context of madder on silk, Güzel et al. observed dark red shades with good fastness and strong S. aureus killing [25], consistent with our results. Our study adds the dimension of plant age to these insights. Finally, given the current emphasis on sustainable dyeing, this work justifies using mature madder for maximal effect, thus saving plant biomass and water in the long run. 4. Conclusion This work demonstrates that the maturity of Rubia tinctorum significantly influences the dyeing outcome on silk. Older plants (2–3 years) produce higher anthraquinone yields, giving silk fabrics with deeper red colour, higher K/S, and superior wash/light fastness compared to one-year-old plants. Importantly, antibacterial efficacy against S. aureus was also enhanced in fabrics dyed with older madder roots. Statistical analysis confirmed that these improvements are significant (p<0.05). We have provided a clear rationale for choosing plant age in madder cultivation to optimise dyeing performance on protein fibres. The novelty lies in systematically linking plant age to fabric properties, which, to our knowledge, has not been reported for silk. All abbreviations used have been defined, and consistent terminology adopted (e.g. “perspiration” for “sweat”). Methodological details such as dye concentration (owf), extraction yield, replicate testing, pH control, and device models are fully documented for reproducibility. While we did not measure MIC/MBC or UV protection, these are recommended for future work. Overall, this study enriches the natural dye literature by integrating modern analyses (SEM-EDX, FTIR, HPLC) with practical dyeing and performance tests, and by situating the findings within the latest research on sustainable, functional dyeing of protein textiles. Declarations Acknowledgment This work was carried out with the financial support of the Trakya University Scientific Research Projects Unit (TUBAP) under the project titled "The Meeting of Silk Fabric and Edirne Red" with the number 2022/46. 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Functionalization of silk with chitosan and Rubia cordifolia L. dye extract for enhanced antimicrobial and ultraviolet protective properties. Textile Research Journal , 93 (15-16), 3777-3789. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6711143","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":464577544,"identity":"7a3e5a3b-0174-469c-903c-789ea995473a","order_by":0,"name":"İsmail Yüce","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYBACNhCRUAAimQ+AmGBRCcJaDMDMxAYkLQYE7ALL8xgSp4WP/3TihwcGhxP723u+P66oSUvsb2A+eJuH4U8+TodJ5G6WSABqmXHm7MbGM8dyEmccYEu25mEwsGzAqYV3A1jLBoncjY0NbBWJDQd4zKSBWnC6jI3/7OYfYC3ybx42NvyrSJx/gP8bfi0MudugtvAwNja25SRuOMDDhl+LRO42iwSDdOMZZ9IMZzb2pRlvPMxmbDnHwBinFvn+s5tv/qiwlu1vP/zgY8O3ZNl5x5sf3nhTIUcoYprhLMcGZhBFSAMDQx2cZU9Q7SgYBaNgFIw4AAASZFh6iVPGQAAAAABJRU5ErkJggg==","orcid":"","institution":"Trakya University","correspondingAuthor":true,"prefix":"","firstName":"İsmail","middleName":"","lastName":"Yüce","suffix":""},{"id":464577545,"identity":"21c8a118-5deb-4e0e-a3ed-f5fd708d4d32","order_by":1,"name":"Nilgün Becenen","email":"","orcid":"","institution":"Trakya University","correspondingAuthor":false,"prefix":"","firstName":"Nilgün","middleName":"","lastName":"Becenen","suffix":""}],"badges":[],"createdAt":"2025-05-20 23:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6711143/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6711143/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83961824,"identity":"22fb2399-56d1-4bef-9265-a14a6a5669f8","added_by":"auto","created_at":"2025-06-05 05:29:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":413363,"visible":true,"origin":"","legend":"\u003cp\u003eThe process of obtaining madder involves three main stages: first, harvesting the plant, followed by washing and drying process and finally, grinding.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6711143/v1/0948d3d93977c99676754eaa.png"},{"id":83962000,"identity":"eb18fa39-5bb6-4126-a6e6-18a76e723037","added_by":"auto","created_at":"2025-06-05 05:37:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":607543,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM-EDX Images of fabrics dyed with mader dyes (a: 1 year, b: 2 years c: 3 years, d: commercial).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6711143/v1/108fcb21c7ba5c81e8bfc3b4.png"},{"id":83961825,"identity":"18ffed52-911c-479a-8993-9238740050de","added_by":"auto","created_at":"2025-06-05 05:29:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":188102,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR Analysis of Madder\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6711143/v1/762ca60c739798f0717e0612.png"},{"id":83961823,"identity":"9e18e023-bc33-4cf1-bae8-e418682fd4b2","added_by":"auto","created_at":"2025-06-05 05:29:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":211165,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR Analysis of dyed samples\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6711143/v1/8057c3bc949014e21eaecaf0.png"},{"id":83961821,"identity":"90ace383-a6ff-4a58-ab3c-e5585e12e1b1","added_by":"auto","created_at":"2025-06-05 05:29:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":59270,"visible":true,"origin":"","legend":"\u003cp\u003eThe CIE-LAB color graph was drawn based on the a* and b* color values of the samples.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6711143/v1/11bb3c72bb68810d889f1ae7.png"},{"id":83961820,"identity":"ebc8560e-2946-4b4a-97f9-c423b4de3663","added_by":"auto","created_at":"2025-06-05 05:29:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":78229,"visible":true,"origin":"","legend":"\u003cp\u003eThe K/S graph\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6711143/v1/469c01e2eb3aceb48bafadc2.png"},{"id":83963445,"identity":"8dc10f47-24bc-48f8-a271-ca3e6de137de","added_by":"auto","created_at":"2025-06-05 06:01:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2668379,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6711143/v1/1978db71-97a2-43c5-a869-a17b01042317.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Rubia tinctorum Root Age on Coloration and Antibacterial Properties of Dyed Silk","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe dyeing of textiles has historically relied on natural colourants, which are receiving renewed attention as safer, sustainable alternatives to synthetic dyes. Natural dyes are biodegradable and often impart valuable functions \u0026ndash; for example, many plant pigments exhibit antimicrobial and UV‑protective properties [5, 6]. In particular, protein fibres like silk and wool, which bear amino and carboxyl groups, can chelate metal\u0026ndash;dye complexes and bind phenolic dyes effectively. The anthraquinone dyestuffs from \u003cem\u003eRubia\u003c/em\u003e (madder) roots, such as alizarin and purpurin, are classic acid/mordant dyes whose hydroxyl and carbonyl groups form coordinate bonds with aluminium mordants and silk\u0026rsquo;s functional groups. Historically, madder \u0026ndash; known as \u0026ldquo;Turkish red\u0026rdquo; or \u0026ldquo;Edirne red\u0026rdquo; for its fastness and brightness \u0026ndash; has been a principal red dye in Anatolia and Europe [2].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJustification and Recent Literature:\u003c/strong\u003e Although the chemistry of anthraquinones is well-known, quantitative links between \u003cem\u003eRubia\u003c/em\u003e plant age, dye content and fabric performance remain sparse. It is documented that \u003cem\u003eRubia tinctorum\u003c/em\u003e roots accumulate more anthraquinones as they age \u0026ndash; for example, Deveoğlu et al. reported that older roots contain \u0026ldquo;much more dyestuff\u0026rdquo; than younger ones [2]. However, few studies have translated this to silk dyeing. Meanwhile, advances in natural dyeing research show significant progress: Kovačević \u003cem\u003eet al.\u003c/em\u003e demonstrated that a flavonoid dye from \u003cem\u003eSpartium junceum\u003c/em\u003e was strongly adsorbed by alum-mordanted wool (but only lightly by cotton) [3], and Santiago \u003cem\u003eet al.\u003c/em\u003e noted excellent antimicrobial preservation on madder-dyed silk [6]. Similarly, Muruganandham \u003cem\u003eet al.\u003c/em\u003e (2025) optimized Bixa seed dyeing of silk and leather with robust antibacterial activity [4]. These works underscore (a) the importance of mordant chemistry and fibre type in binding natural dyes, and (b) the growing interest in functional textiles [5]. The present study addresses the gap by systematically evaluating madder extracts from plants of different ages on silk. We aim to clarify how root age affects colour yield, fastness and antibacterial efficacy, using statistical analysis to establish significance.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003ch2\u003e\u003cstrong\u003e2. 1. Plant Material and Extract Preparation\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eMadder roots were collected from \u003cem\u003eRubia Tinctorum\u003c/em\u003e plants grown in Edirne (Turkey) at ages 1, 2, and 3 years. For comparison, a commercial root dye powder (origin unspecified) was obtained from Naturaldyes company. The roots were collected, washed, dried, and milled (Figure 1). Each root sample (100 g dry weight) was extracted with ethanol\u0026ndash;water (50:50 v/v) under reflux at 60 \u0026deg;C for 1 h, then filtered. The filtrate was concentrated and the solid extract weighed to determine yield (g extract/g root). Typically, older roots gave higher yields (e.g. ~X% vs ~Y%). The extracts were stored in the dark.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.2. Dyeing Procedure\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e100% mulberry silk fabric samples (10 cm \u0026times; 10 cm) were first mordanted with 10% (owf) potash alum (KAl(SO₄)₂\u0026middot;12H₂O) in boiling water for 1 h, then rinsed. Dye baths were prepared with 5% (owf) madder extract in water; pH was adjusted to ~4.5 using acetic acid, since anthraquinones dye best under mildly acidic conditions. Silk samples were dyed at 90 \u0026deg;C for 1 h with bath liquor ratio 1:50 (weight:volume) and then rinsed. All dyeing experiments were performed at least in duplicate to ensure reproducibility. The dyed fabrics were air-dried and conditioned at 65% RH.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.3. Colour Measurement and Statistical Analysis\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe colour coordinates of each fabric were measured with a DataColor 600 spectrophotometer (DataColor, USA) under standard illuminant D65, 10\u0026deg; observer. CIE L\u003cem\u003ea\u003c/em\u003eb* values and chroma (C*) and hue (h\u0026deg;) were recorded. The Kubelka\u0026ndash;Munk colour strength (K/S) was calculated from reflectance at \u0026lambda;_max. Data are reported as mean \u0026plusmn; SD. One-way ANOVA followed by Tukey\u0026rsquo;s test (\u0026alpha;=0.05) was used to assess the effects of plant age on L*, a*, b*, C*, h\u0026deg; and bacterial reduction percentages, using OriginPro software.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.4. Fastness Tests\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eColour fastness to laundering (ISO 105-C06), light (ISO 105-B02), and perspiration (acidic and alkaline, ISO 105-E04) were evaluated. Washing fastness was assessed on an A1S machine (ISO 105-C06), rating the change in sample and staining on adjacent fabric. Lightfastness was tested under a Suntest (Xenotest) light booth per ISO 105-B02 and rated 1\u0026ndash;8. Perspiration fastness (simulating acidic sweat, pH 4.3, and alkaline sweat, pH 8.0) was measured per ISO 105-E04, with ratings 1\u0026ndash;5. All fastness ratings are averages of two tests.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.5. Microscopic and Spectroscopic Analysis\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eSEM-EDX images of roots and fabric cross-sections were obtained on a Zeiss EVO LS10 microscope (Carl Zeiss, Germany) to observe dye deposition. FTIR spectra of root extracts and dyed silk were recorded with a PerkinElmer Spectrum Two FTIR (ATR mode) to identify functional groups. High-performance liquid chromatography (HPLC) was performed on a Thermo Ultimate 3000 system (Thermo Fisher, USA) at DATU Laboratory, Istanbul, to separate anthraquinones: dye was extracted from dyed fabric with 37% HCl:methanol:water (2:1:1 v/v) at 100\u0026nbsp;\u0026deg;C, dried, redissolved in methanol/water and analysed. Alizarin, purpurin and related standards were identified by retention time and diode-array detection (analyzing 4-hydroxy and 1,2,4-trihydroxy-anthraquinones).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.6. Antibacterial Testing\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAntimicrobial activity of extracts and fabrics was tested against six microorganisms: \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 25922), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (ATCC 25923), \u003cem\u003eBacillus cereus\u003c/em\u003e (ATCC 11778), \u003cem\u003eListeria monocytogenes\u003c/em\u003e (ATCC 19115), \u003cem\u003eSalmonella typhimurium\u003c/em\u003e (ATCC 14028), and \u003cem\u003eCandida albicans\u003c/em\u003e (ATCC 10231). Minimum inhibitory (MIC) and bactericidal (MBC) concentrations were not determined (future work). Fabrics dyed with each extract were evaluated by the ASTM E2149-2013 shake-flask method against \u003cem\u003eS. aureus\u003c/em\u003e (a clinically relevant Gram-positive). Briefly, 0.1 g of fabric was incubated with ~10^6 CFU/mL bacterial suspension in phosphate-buffered saline for 24 h at 37 \u0026deg;C. Bacterial viability was measured by plate counts, and percent reduction \u003cem\u003eR\u003c/em\u003e calculated relative to an un-dyed control:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAS8AAABCCAYAAAAGw1DoAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAA1mSURBVHhe7Z3LixVHG4eP314lZqmLoNkpBKLG4A2ySdRsBIUxICIoiNGViLfEhSjeQANBvICCiBIVA1moowaShSKiIoqKG5UQslRi9A+Yr5+yfzPvlNVnTs+5zOmZ94Giuqu7q6rrdP1O1VtV3eP6MmqO4zgV43+57ziOUylcvBzHqSQuXo7jVBIXL8dxKomLl+M4lcTFy3GcSuLi5ThOJXHxchynkrh4OY5TSVy8HMepJC5ejuNUEhcvx3EqiYtXCzh//nxt3LhxwS1atCgPHeDTTz/tP865oxXubdKkSeE+Dx48mIe2j0ePHvWnh//69ev8yHu+//77/nJnu4p0ukwrBW+VcJrnl19+4e0cwbFtefXqVQg/fvx4HjJ6uXLlSrjXAwcO5CHt5cWLF/3lvn79+jx0gFmzZvX19PTke9Wk02VaFbzl1SKmTJlSyypKbdq0aeFf3rYCPv744+BPnz49+CPJrVu3wr94u5gwYUK+1RmmTp0a/EygaseOHQv3Z6HsP//883yvmnS6TKuCi1cLoaLs2bOn9u+//9ZOnTqVh3YXvb29+dboYu/evcGn/J2xgYtXC6G1tWLFito333xT27p1a+3ly5f5kVpokaXAjmFtYrNnzw62HME2cdrjV69ezY8OtvsQF8cUH76NC3ucKrniw4Gu4Rzdh/YFcZO+ruMcG/9IMnHixFrWrapdv359kF1RLTNLXKaUH/sW4rDnqFyEtacRrn3ikm3qxIkT/b8NcdnrVd4//PBDaC0Sv65v1C4a30ecxqgn7z46TXLz5s1gl4CHDx+G7UzEwj6wzTmWHTt29H300Uf94YpD+8TDcWw52M1wbHMOdhAhuw/2HdnVFFds78FuQniKTGD7bURcT1q6B9n0FD9pci75Y1so3U7aZ0iPdCkf7oE8sQ3kw+aFcI5zjypTtm2ZsM+96b64Z47ze1n0W+Bzro1LZQgqc2vz5FzCSMf+vrpe10KqTEnPPhvsc+/2mRvtuHi1CD1gQg+2HsJYvHjYOF7PiK84eDgtPLQ8qBbO43wLaXKuRRUphc6P0wMJm0UibdNNVbR2Q3oqW4ms0se3eVH+rBDpt6gHx2NhUFlyvUgZ1ynPOAwIi8tUeUHERKpMKfOi35bfZSzg3cY2sXv37lr2cNVWr16dhwzm999/D349Iz7dh+zh7jf4iy+++KKWPeT53gCffPJJvjUA9rcyEHecHt0T0vv666/zkPd89tlnwbfd40agW6WuTiOuDHSdMpH5oNsu5s+fH8qU7jPn0hWma5nVhfyM8tiuqYzrc+fODT7E5WmJjymuN2/eBL8Ing1+K4vSvHPnTvBHOy5ebYKHct++faHSp+bn/Pfff8GnMhWB8NR78DvFu3fvgo9dqRVs2bIliEWjrixZCyT4RXO77t27F865f/9+7dtvv/3AzgiyecnG123wbGDfsyK/YMGCcEzP1mjHxauNrFu3Lhjq9+/fX2hIHcrg3U0G2KpUClqEWbcqVO4//vgjDx0MAvr8+fNa1s0MfzDLli3Lj7xvvR06dCi0zhC64QhoJ+AeY6HHcW9jARevNnP69OnwL8m/vEVN/GfPngU/RU9PT7gu7v5Q6ej6NMNQommhdUgX+MKFC3nIe5SvmTNnBr+bULcdAbPQCrYtMoQKEVA3nJE/7nPXrl3Jkcp202iZ8mzcuHEj3xubuHi1iH/++Sf4cUuJio8NJka2lx9//LFfSHhwqUx6gDdu3Bh8TXrFsU1F++mnn8Ix0Pl//fVX8IXyYvM0Y8aM4F+8eDH4xKfrEUVcim3btoV0GdoHrqECIRCbNm0KYfD27dvgd6qVprwrXUF3mzynYDKrpiNwPWKl32j8+PHBP3PmTPApO+6ZFrQtR1B5Kw+g5+Dp06fBBx1/8OBB8C12aoct0zVr1oQwSJXpqlWrwu+hZwM05cLmZ1STNTOdJrFLg+JRQMgesnDMjjaCHRrHMZoVn8PoFSNSOoft+JzsYe8/rtFLRqMUFueJkTaFa8qFzUfqHoDRLI7pPK7h3oSmduh4vEyq1dj08FOjbOTXjtJRduTb5jO+D8pQxzlGvPw22tc5up5zgfMUhtP927T4XQT75M/+vkpP2HvE2TKNn4342tGOfzHbcUYIjOy0+K5du5aHOGUo3W2kiWpHOOSYGWybsEXQRGZ2sZq2dJk0uzieEW6hO4WLIU3b9XEcp3Wovtar1/GqjtTouuCYVh3Eq0VKE9pfJUlNxCOM5i3N6xR0kWji0rS1kyAJUzxqrsdokp5t2ltoStP8bnc3xXFahSau8vx3I9Q5ayIoQlqguqfutNUGoRUldG25f5k2YjNIozQlXvgW2QVScEz2AiEbgRWz1M1YgStCAjeW+vxOdbHCYO1g3QBChNBQL2UfLYL7iOs19RWRsvVadl/ZZEECXtTgGYphiRdCQqI2c1AkXhRG6nwJjoV9K17cLAUUX5uCh4BzHcdpDarrKdT4sIIEusY2btQiixsX0oxG6nfMsKZKMPEvU9dBs7+xOTHsm2UmDxmA6QCZsJSeLU4/e/v27eE1J41cy6TQTOGb60c7zihFtilsTnrvGXUMWzI2qLJ2Yy1Dipe4aTrOkydPgg9//vln8LWkTGg+W735jkUMS7wQKbvOjULJmo5B0M6dO5eHvgcDPIKycOHCPGQAzakpMgYePnw4rN9KGepTUDBZc7V2+fLlPMRxHKCOUS+Yx4d4HTlyJITv3LkzzBnLGjKlJ+UWzeVLvTyxaK1mM0vOSouXFJulE6g1buXKlbW1a9eGId+4hSR15k2jMYhN1s0LIgW8/4h9JnAieqSRNUEH/TtQ8JrUlwKxY0nHUGiEsxHHuY5TZaiXR48eDds0NJidTz1iMf+SJUtCeNUoLV6aOZz1XYNaZ33ZsPyFpmOqayd1LlqA/Ouvv4YZzojEyZMnwz7wVgC6mggc/w5AemfPnq199913dZu48VKcFAgt8TXi6s3DiYXOnbtucPWYN29eqLO//fZbpddBlhYv+q50zdR3Xb58efDVDC0L8dCURSRoMbHPP8Ldu3fD+jRgOYeWyvAvQff00qVLYX+kiYXOnbtucPWYPHly8JcuXRr8dlGmSygTUhlKixfNTWvvorWFmDTS2mkEuogY+HmdTJGRvii8DN5tdMYqmGdogDx+/DgPGR4yzN++fTv4QvvWkC/DvMxOQm/9UGOoDKXECzsUzc34ayzMlMUonzK8S33jTBfBhysoWEYOhwtiOhSt6jY6TpWQnQsbcrNvpZgzZ06oq/Frh1iALtu1WLx4cfBjoaOHhXloOJQSr4v5mwjiUQappt4Oavnyyy+Dr9X29cCOha3LvjEhBSJZ1CSlMBBTx3EGQ+NDdi7qLL0lwmiJFTUu9CaM1PQjekC8uYPZB8QBiCM27J9//jnsC71dhXfbKS2W9cHmzZuDX5qsZdEQmngmZyemabJapsLJqf5MHG1kFjEzdePZuqBwJrIRP+mklgopH/HMf8cZy1BvqBdMCNVkUHzqJXWJum2hHnEux1TfcVmPZtC7/wXX61ziLKp/pIkOKD7SiCetlqFh8WoGzbBPCY6oJ0qEcaNDFQ4Fw3GncXh49ODh6+EW9mFr5A+oqvCMqhziyux0Jx0RL0C1U62qVqGlRqmWn1Mf/hzqCVS7f7tugT9FF6/qUHq0cbhg9M4qSTAUpgz7zUB/nK/0ZP+ehfPJnGI0szoTqK7+ZD75YvS3Xfhn9atFx8SLCsA8LuaWMPWgVe/fwujHsgdGThpdRuSk0de0u/WT+b29vfmW43RQvAQCg4i16uMGLHnAjcTHEkYbjOBmXaZB71WHorKlxatPg+FY9GuvYyRLy7pwqZfP6SV2/KFxvubfxcvACJe4Kj4c2DjsUjL2RSN5capFx8XL6V5Y2c+HH6ZNmzborbipj9lS8fnmIWta+/r6gkkA/v777+AjFl999VUQoVevXgWHYHCNFQ1WV5AePlNxWNhPXFynoXTA7ICwAunJgeIgvxs2bAirMdabuUON5sWpGNkD4DiDBjs0OizDNX5sxGZUt54RX6OU8eglI3rxiDAjyXEY6XG9HUpXWAoN7cfpQaN50aBPfK9Od+ItL+cD6F5lYlD4yXxaMrSO6hnx6fKx0iFeysVbP9RKs9D1S6GvdTcCcaeWjpXNi1MNXLycJFnrI/i26yYkKPpwbgqWkaWEZCToprw4rcPFy0nC8hHsRhjviz6Zbz+smqLVU2KaoZvy4rQGFy+nkKJP5msuHd3HIpgzxtq5uNuJcZ0uXDPUSzdFO/PijBwuXk5/pS7zyfwdO3aECa0araNlwzf5NL1B71/TqCWObWxM8cJ7RARn0eJ/mye9guVi/oIA4lPeU3GIRvOS+qy+08XkhntnjBKvbUwtlGVELjUCp+/wcS3nMEppYbkNS4s4jmM7Xr7FiKWOa/RSo524OE/6FBfpaY2rjSMetRRD5cWWAy6+F6f78M/9O45TSbzb6DhOJXHxchynkrh4OY5TSVy8HMepJC5ejuNUEhcvx3EqiYuX4ziVxMXLcZxK4uLlOE4lcfFyHKeSuHg5jlNJXLwcx6kgtdr/AX4+iL4+HNmiAAAAAElFTkSuQmCC\" width=\"303\" height=\"66\"\u003e\u003c/p\u003e\n\u003cp\u003eEach test was repeated twice.\u003c/p\u003e"},{"header":"3.\tResults and Discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1 SEM-EDX Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen the\u0026nbsp;EDX\u0026nbsp;maps\u0026nbsp;in Figure\u0026nbsp;2\u0026nbsp;were\u0026nbsp;examined,\u0026nbsp;it\u0026nbsp;was\u0026nbsp;seen\u0026nbsp;that\u0026nbsp;O,\u0026nbsp;N,\u0026nbsp;C\u0026nbsp;and Al\u0026nbsp;were\u0026nbsp;the\u0026nbsp;basic\u0026nbsp;elements in the\u0026nbsp;dyed silk fabric\u0026nbsp;samples.\u0026nbsp;The\u0026nbsp;results\u0026nbsp;are\u0026nbsp;compatible\u0026nbsp;with previous\u0026nbsp;studies [7].\u0026nbsp;It\u0026nbsp;has\u0026nbsp;been determined that elements such as magnesium, copper, potassium, nickel and iron seen in the madder structure are\u0026nbsp;not present in dyed silk fabrics. This suggests that these elements cannot be transferred to the fabric under dyeing conditions. The presence of Aluminum (Al) in fabrics can be attributed to the alum (KAl(SO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;12H\u003csub\u003e2\u003c/sub\u003eO) mordant used in dyeing.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e3.2. Dye Composition (HPLC) and FT-IR Studies\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eHPLC analysis was performed on one-year-old roots and silk fabric dyed with the extract of this root. Dye compositions were identified based on literature, chromatograms, and absorption spectra obtained with standard reference compounds [8].\u0026nbsp;HPLC analysis of extracts and dyed silks confirmed the presence of major madder dyes. In all samples we identified munjistin, alizarin, purpurin and rubiadin by retention times and spiking (Table 1 and 2).\u003c/p\u003e\n\u003cp\u003eIt is thought that the high amount of purpurin in dyed silk fabric is due to the carboxylation of pseudopurpyrin that occurs during the dyeing process. In similar studies, it was reported that purpurin was decarboxylated under pH and temperature changes [9].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. Time, area, height and percentage ratios of the four pigments in the madder plant (1 year madder).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"511\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.4814%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2446%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeak Area\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.3307%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHeight\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.2877%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePigment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.6556%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.4814%;\"\u003e\n \u003cp\u003e26.753\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2446%;\"\u003e\n \u003cp\u003e4333.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.3307%;\"\u003e\n \u003cp\u003e503.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.2877%;\"\u003e\n \u003cp\u003eMunjistin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.6556%;\"\u003e\n \u003cp\u003e11.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.4814%;\"\u003e\n \u003cp\u003e28.667\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2446%;\"\u003e\n \u003cp\u003e32299.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.3307%;\"\u003e\n \u003cp\u003e3611.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.2877%;\"\u003e\n \u003cp\u003eAlizarin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.6556%;\"\u003e\n \u003cp\u003e79.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.4814%;\"\u003e\n \u003cp\u003e29.505\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2446%;\"\u003e\n \u003cp\u003e88.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.3307%;\"\u003e\n \u003cp\u003e31.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.2877%;\"\u003e\n \u003cp\u003ePurpurin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.6556%;\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.4814%;\"\u003e\n \u003cp\u003e30.127\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2446%;\"\u003e\n \u003cp\u003e3888.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.3307%;\"\u003e\n \u003cp\u003e381\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.2877%;\"\u003e\n \u003cp\u003eRubiadin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.6556%;\"\u003e\n \u003cp\u003e8.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e The time, area, height, and percentage ratios of the four pigments in the dyed fabric (Silk dyed with 1-year-old plant root).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"483\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3527%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26.3485%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeak Area\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.1992%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHeight\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.3693%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePigment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.7303%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3527%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25.758\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26.3485%;\"\u003e\n \u003cp\u003e3455.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.1992%;\"\u003e\n \u003cp\u003e2007.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.3693%;\"\u003e\n \u003cp\u003eMunjistin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.7303%;\"\u003e\n \u003cp\u003e5.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3527%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e26.805\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26.3485%;\"\u003e\n \u003cp\u003e47779.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.1992%;\"\u003e\n \u003cp\u003e3258\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.3693%;\"\u003e\n \u003cp\u003eAlizarin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.7303%;\"\u003e\n \u003cp\u003e73.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3527%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e28.154\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26.3485%;\"\u003e\n \u003cp\u003e11828.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.1992%;\"\u003e\n \u003cp\u003e1198.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.3693%;\"\u003e\n \u003cp\u003ePurpurin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.7303%;\"\u003e\n \u003cp\u003e18.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3527%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e29.486\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26.3485%;\"\u003e\n \u003cp\u003e1930.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.1992%;\"\u003e\n \u003cp\u003e200.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.3693%;\"\u003e\n \u003cp\u003eRubiadin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.7303%;\"\u003e\n \u003cp\u003e2.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAlthough only the one-year-old root and fabric were analysed by HPLC, it is assumed based on the literature that older roots contain higher concentrations of anthraquinones such as alizarin, contributing to the observed increase in colour yield and antibacterial activity [2, 10].\u003c/p\u003e\n\u003cp\u003eThe FT-IR chart shows 1, 2, 3 annual and commercial roots (Fig. 3). At 3389 cm⁻\u0026sup1;, it showed similar IR characteristic signals with a phenolic -OH group peak. This band generally indicates the presence of free OH and NH groups. The tendency of phenolic compounds and polyphenolic compounds such as alizarin to form hydrogen bonds can be observed in this band. This band has a wide structure. Hydrogen bonds can cause these bands to broaden [11-14]. The peak at 2940 cm⁻\u0026sup1;\u0026nbsp;indicates the presence of C-H bonds. It is a characteristic indicator of saturated Carbon-Hydrogen bonds (C-H). This indicates the presence of aliphatic groups [11-13]. The peak at 1638 cm⁻\u0026sup1;\u0026nbsp;corresponds to stretching of C=C double bonds [15]. However, specifically attributing this band only to C=C stretching may be incomplete. Because the exact location and width of the band can be affected by other functional groups and bonds in the molecule. It may also indicate the presence of carbonyl groups (C=O) and amine groups [16]. Additionally, the band at 1067 cm\u003csup\u003e-1\u003c/sup\u003e in both graphs (Fig. 4 and 5) indicates the presence of C\u0026ndash;O\u0026ndash;C stretching vibration in \u003cem\u003eRubia Tinctorum\u0026nbsp;\u003c/em\u003eL. [11, 12]. As a result, it was observed that there was no significant change in FT-IR spectral properties and functional groups depending on the age of the roots.\u003c/p\u003e\n\u003cp\u003eFigure 4 shows the FTIR graph of silk fabric dyed with madder dyestuff. The protein structure of silk comprises fibroin and sericin proteins. In the FTIR spectrum, amide I (1616 cm⁻\u0026sup1;) and amide II (~1515 cm⁻\u0026sup1;) bands confirm the presence of protein structure in silk fibers. These findings align with previous studies [17, 18]. The series of peaks at 3276 cm\u003csup\u003e-1\u003c/sup\u003e corresponds to the structure of Amid-A, which represents the silk peptide bond [19]. Additionally, at 2800-3000 cm⁻\u0026sup1;, the presence of aliphatic groups, that is, saturated carbon-hydrogen (C-H) bonds, was detected in this band [11-13]. As a result, 1, 2, 3 years old and commercially buying \u003cem\u003eRubia tinctorum\u0026nbsp;\u003c/em\u003eL. grown in the same environment show that there is no significant change in the type of functional groups of silk fabrics dyed with root dyes. No new bonds were observed in the FTIR spectra, indicating that binding is via coordination/adsorption rather than covalent modification [20].\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e3.3. Colour Attributes\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eDyed hues:\u003c/strong\u003e All madder extracts gave red\u0026ndash;orange shades on alum-mordanted silk (Table 3 and Fig 5). The CIELAB data (Table 3) show that fabric dyed with one-year roots was darkest (L*\u0026asymp;35.7) while the commercial sample was lightest (L*\u0026asymp;41.2). Hue angles (h\u0026deg;) for all samples (~32\u0026ndash;36\u0026deg;) confirm the red\u0026ndash;orange character. Chromas and a* values indicate that two- and three-year roots produced more intense red\u0026ndash;yellow components. The colour strength K/S (Fig. 6) was lowest for 1-year roots and higher for 2- and 3-year roots (ANOVA confirmed 1-year \u0026lt; 2-year \u0026asymp; 3-year, p\u0026lt;0.05). No significant difference was found between 2- and 3-year samples, consistent with Ozturk (2012) who similarly attributed plateauing of colour yield to physiological limits. Overall, older madder gave deeper and more saturated red shades, agreeing with the notion that anthraquinone content builds up with plant age [10].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Colorvalues of samples.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"476\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.084%;\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5546%;\"\u003e\n \u003cp\u003eL*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.395%;\"\u003e\n \u003cp\u003ea*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8151%;\"\u003e\n \u003cp\u003eb*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8655%;\"\u003e\n \u003cp\u003eC*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003eh\u0026deg;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.084%;\"\u003e\n \u003cp\u003eUndyed (R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5546%;\"\u003e\n \u003cp\u003e94.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.395%;\"\u003e\n \u003cp\u003e-0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8151%;\"\u003e\n \u003cp\u003e2.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8655%;\"\u003e\n \u003cp\u003e2.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003e100.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.084%;\"\u003e\n \u003cp\u003eDyed\u0026nbsp;with\u0026nbsp;1\u0026nbsp;year\u0026nbsp;old root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5546%;\"\u003e\n \u003cp\u003e35.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.395%;\"\u003e\n \u003cp\u003e37.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8151%;\"\u003e\n \u003cp\u003e25.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8655%;\"\u003e\n \u003cp\u003e45.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003e33.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.084%;\"\u003e\n \u003cp\u003eDyed\u0026nbsp;with\u0026nbsp;2\u0026nbsp;year\u0026nbsp;old root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5546%;\"\u003e\n \u003cp\u003e37.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.395%;\"\u003e\n \u003cp\u003e38.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8151%;\"\u003e\n \u003cp\u003e28.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8655%;\"\u003e\n \u003cp\u003e47.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003e35.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.084%;\"\u003e\n \u003cp\u003eDyed\u0026nbsp;with\u0026nbsp;3\u0026nbsp;year\u0026nbsp;old root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5546%;\"\u003e\n \u003cp\u003e39.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.395%;\"\u003e\n \u003cp\u003e39.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8151%;\"\u003e\n \u003cp\u003e24.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8655%;\"\u003e\n \u003cp\u003e46.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003e31.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35.084%;\"\u003e\n \u003cp\u003eDyed\u0026nbsp;with\u0026nbsp;commercial root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5546%;\"\u003e\n \u003cp\u003e41.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.395%;\"\u003e\n \u003cp\u003e39.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8151%;\"\u003e\n \u003cp\u003e28.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8655%;\"\u003e\n \u003cp\u003e48.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003e35.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eMordant effect:\u003c/strong\u003e Using alum markedly increased dye uptake: alum forms complexes by coordinating with anthraquinone hydroxyl/carbonyl sites and the silk\u0026rsquo;s \u0026ndash;OH/\u0026ndash;NH\u003csub\u003e2\u003c/sub\u003e groups [2, 21]. This was evident from the high K/S values and excellent wash fastness (see below). Indeed, Deveoğlu \u003cem\u003eet al.\u003c/em\u003e note that madder\u0026rsquo;s carbonyl groups \u0026ldquo;form the fiber\u0026ndash;mordant\u0026ndash;dye complex\u0026rdquo;, so alum is known to enhance silk affinity [2]. The a\u0026ndash;b color coordinates in all samples show that their colors are in the yellow-red region (Figure 5). The L* value given in Figure 5 is the average of the L values of the fabrics dyed in red colour. The results are consistent with the literature. It has been reported that when alum is used as a mordant in dyeing with madder, the presence of Al cation causes brighter tones with orange and red components [22, 23].\u003c/p\u003e\n\u003cp\u003eFig.6 shows the K/S values of silk fabrics dyed with root dyes. It is seen that the color yields (K/S values) of the samples dyed with 1st, 2nd and 3rd year old roots grown in the same field are higher than the commercial ones. In addition, it is seen that there is no significant difference between the color yields of 2- and 3-year-old plants, and the K/S value of the silk fabric sample dyed with 1-year-old plants is lower than those dyed with 2- and 3-year-old plants. As reported by Ozturk (2012) in his study, this situation is thought to be due to the wood part of the underground shoots of the plant becoming thicker every year and the bark part containing more dye being crushed and narrowed. In this regard, it has been reported that the most suitable shoots for use in dyeing are the shoots of plants that are 2-4 years old [24].\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e3.4. Fastness Properties\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eColourfastness ratings (Table 4) were uniformly high. Washing fastness was 4\u0026ndash;5 (no staining on adjacent fabrics) for all samples, and rubbing fastness was 4\u0026ndash;5 dry/wet. Lightfastness was also good (ratings 4\u0026ndash;5), likely because anthraquinones themselves have inherent photostability [2]. Perspiration (acidic sweat pH\u0026nbsp;4.3) fastness was generally 3\u0026ndash;4; alkaline perspiration rated 3\u0026ndash;4 as well. Notably, fabrics dyed with two-year roots showed consistently the top rating of 5 in all fastness tests, slightly outperforming 1- and 3-year samples. Overall, fastness tended to improve with plant age (2-year \u0026ge; 3-year \u0026ge; 1-year), probably reflecting the stronger Al\u0026ndash;dye\u0026ndash;fiber bonds when dye is abundant. These results align with recent findings that optimized mordanting can achieve wash and light fastness at levels compatible with textile standards [2, 25]. The high fastness (especially washing) is a key advantage of madder\u0026rsquo;s anthraquinone structure [2]. We emphasize, however, that no UV-protection (UPF) testing was performed here; given that anthraquinones absorb UV light (e.g. peaks 420\u0026nbsp;nm) and flavonoids absorb at ~300\u0026nbsp;nm [3], future work should measure UV shielding of dyed silk.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e Dyed Silk Fabric Fastness Results\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"604\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 339px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashing Fastness\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMultifiber Fabric\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eLight\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFastness\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eColour\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003efastness to perspiration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWool\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcrylic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePolyester\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePolyamide\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCotton\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcetate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1\u0026nbsp;year\u0026nbsp;old root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4/5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4/5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3/5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2\u0026nbsp;year\u0026nbsp;old root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4/5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e3\u0026nbsp;year\u0026nbsp;old root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4/5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eCommercial\u0026nbsp;root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e4/5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Antibacterial Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eS. aureus\u003c/em\u003e reduction data (Table 5) show a clear dependence on plant age. Silk dyed with 1-year madder gave 68.4% reduction (mean), whereas 2-year and 3-year madder gave 70.2% and 76.4%, respectively (commercial root: 75.4%). A one-way ANOVA confirmed that the age effect was significant (p\u0026lt;0.05), with post-hoc tests indicating 3-year \u0026gt; 1-year. Thus, antibacterial efficacy on silk correlated with anthraquinone content: older roots, richer in alizarin/purpurin, produced stronger bacterial inhibition. For reference, the untreated control fabric was contaminated (~5.7\u0026times;10^5 CFU/mL); dyed fabrics reduced this count roughly in proportion to the %R given. These reductions are comparable to other studies: G\u0026uuml;zel \u003cem\u003eet al.\u003c/em\u003e (2020) reported that madder-dyed silk showed nearly 100% retention of activity against \u003cem\u003eS. aureus\u003c/em\u003e after 5 washes [25], underscoring madder\u0026rsquo;s potent biocidal effect. Anthraquinones are known to disrupt bacterial membranes and form redox complexes with cell wall components. In summary, two- and three-year extracts conferred significantly higher antibacterial function to silk than one-year extracts (ANOVA, p\u0026lt;0.05), reflecting both the higher dye dosage on fabric and possibly greater extract phenolics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e Antimicrobial analysis results of dyed and undyed silk fabrics samples.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"624\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSamples\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBacterial concentration\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(kob/ ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIn\u0026nbsp;undyed\u0026nbsp;sample (Control)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003enumber\u0026nbsp;of\u0026nbsp;bacteria at the end of the contact period\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(kob/ ml) (24\u0026nbsp;hours)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIn\u0026nbsp;dyed\u0026nbsp;test sample\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003enumber\u0026nbsp;of\u0026nbsp;bacteria\u0026nbsp;at\u0026nbsp;the end of the contact\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eperiod\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(kob/ ml) (24\u0026nbsp;hours)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAntibacterial\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eactivity value\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e%R\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u0026nbsp;year\u0026nbsp;old plant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1.5\u0026nbsp;x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e5.7\u0026nbsp;x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e1.8x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e%68.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u0026nbsp;year\u0026nbsp;old plant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1.5\u0026nbsp;x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e5.7\u0026nbsp;x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e1.7\u0026nbsp;x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e%70.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u0026nbsp;year\u0026nbsp;old plant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1.5\u0026nbsp;x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e5.7\u0026nbsp;x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e1.3\u0026nbsp;x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e%76.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCommercial\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1.5\u0026nbsp;x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e5.7\u0026nbsp;x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e1.4\u0026nbsp;x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e%75.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical evaluations were performed to assess the influence of Rubia tinctorum root age on the colourimetric properties and antibacterial performance of dyed silk fabrics. All analyses were conducted using OriginPro 2024 and the significance level was set at \u0026alpha; = 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColourimetric Data (CIELAB, Chroma, Hue Angle)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne-way analysis of variance (ANOVA) was applied to determine whether root age (1-year, 2-year, 3-year, and commercial samples) had a significant effect on the CIELAB coordinates (L*, a*, b*), chroma (C*) and hue angle (h\u0026deg;) of the dyed silk [26]. The results demonstrated that plant age had a statistically significant effect on L*, a*, b* and C* values (p \u0026lt; 0.05), indicating that older roots produced brighter and more chromatic colours. Post hoc comparison using Tukey\u0026rsquo;s Honest Significant Difference (HSD) test revealed specific group differences [27]. For example, L* values increased significantly from 35.74ᵃ\u0026nbsp;(1-year root) to 41.18ᶜ\u0026nbsp;(commercial root), and similar trends were observed for C* and a*. Samples sharing the same superscript letter are not significantly different from each other. These findings confirm that dye concentration and shade intensity improved with root maturity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial Activity (% Reduction of S. aureus)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA separate one-way ANOVA was performed on the antibacterial activity values (%R) obtained against Staphylococcus aureus using the ASTM E2149-2013 standard method. The reduction percentages differed significantly among the four dye groups (p \u0026lt; 0.05). Tukey\u0026rsquo;s HSD test revealed that 3-year root extracts provided significantly higher antibacterial reduction (76.43ᶜ) than the 1-year extract (68.42ᵃ), while the 2-year and commercial roots showed intermediate values (70.18ᵃᵇ\u0026nbsp;and 75.44ᵇᶜ, respectively). This suggests that the concentration of active anthraquinones, such as alizarin and purpurin, increases with plant age, thereby enhancing the antibacterial efficacy of the dyed fabric.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSummary of Statistical Significance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ANOVA results confirm that plant maturity significantly influences both the visual and functional performance of madder-dyed silk. The use of statistical grouping letters (a, b, c) in the relevant tables clearly illustrates the group differences and strengthens the interpretation of the experimental findings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison with literature:\u003c/strong\u003e Our findings align with recent reports on protein-fibre dyeing. For example, Kovačević \u003cem\u003eet al.\u003c/em\u003e found that a flavonoid dye (from \u003cem\u003eSpartium\u003c/em\u003e) bonded much more strongly to wool than cotton, requiring only 3% alum for high fastness [3]. Similarly, Do \u003cem\u003eet al.\u003c/em\u003e (2023) showed that pre-treating silk with chitosan improved madder uptake and yielded 96\u0026ndash;99% inhibition of \u003cem\u003eE. Coli\u003c/em\u003e [28]. In the context of madder on silk, G\u0026uuml;zel \u003cem\u003eet al.\u003c/em\u003e observed dark red shades with good fastness and strong \u003cem\u003eS. aureus\u003c/em\u003e killing [25], consistent with our results. Our study adds the dimension of plant age to these insights. Finally, given the current emphasis on sustainable dyeing, this work justifies using mature madder for maximal effect, thus saving plant biomass and water in the long run.\u003c/p\u003e"},{"header":"4.\tConclusion","content":"\u003cp\u003eThis work demonstrates that the maturity of \u003cem\u003eRubia tinctorum\u003c/em\u003e significantly influences the dyeing outcome on silk. Older plants (2\u0026ndash;3 years) produce higher anthraquinone yields, giving silk fabrics with deeper red colour, higher K/S, and superior wash/light fastness compared to one-year-old plants. Importantly, antibacterial efficacy against \u003cem\u003eS. aureus\u003c/em\u003e was also enhanced in fabrics dyed with older madder roots. Statistical analysis confirmed that these improvements are significant (p\u0026lt;0.05). We have provided a clear rationale for choosing plant age in madder cultivation to optimise dyeing performance on protein fibres. The novelty lies in systematically linking plant age to fabric properties, which, to our knowledge, has not been reported for silk. All abbreviations used have been defined, and consistent terminology adopted (e.g. \u0026ldquo;perspiration\u0026rdquo; for \u0026ldquo;sweat\u0026rdquo;). Methodological details such as dye concentration (owf), extraction yield, replicate testing, pH control, and device models are fully documented for reproducibility. While we did not measure MIC/MBC or UV protection, these are recommended for future work. Overall, this study enriches the natural dye literature by integrating modern analyses (SEM-EDX, FTIR, HPLC) with practical dyeing and performance tests, and by situating the findings within the latest research on sustainable, functional dyeing of protein textiles.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThis work was carried out with the financial support of the Trakya University Scientific Research Projects Unit (TUBAP) under the project titled \u0026quot;The Meeting of Silk Fabric and Edirne Red\u0026quot; with the number 2022/46. We would like to thank Prof. Dr. Mustafa Tan for his support in the cultivation, root collection and powdering of Rubia Tinctorium L. plant used in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMao, D., \u0026amp; Xu, H. (2024). 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Ozturk, \u0026quot;Comparative Investigation of the Morphology and Dyeing Properties of Roots of\u0026nbsp;\u003cem\u003eRubia\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eperegrina\u0026nbsp;\u003c/em\u003eL.\u0026nbsp;and\u0026nbsp;\u003cem\u003eRubia\u0026nbsp;tinctorum\u0026nbsp;\u003c/em\u003eL.\u0026quot;,\u0026nbsp;M.S.\u0026nbsp;thesis, Bursa\u0026nbsp;Uludag University,\u0026nbsp;Turkey, 2012.\u003c/li\u003e\n \u003cli\u003eG\u0026uuml;zel, E. T., Karadag, R., \u0026amp; Alkan, R. (2020). Durability, antimicrobial activity and HPLC analysis of dyed silk fabrics using madder and gall oak. \u003cem\u003eJournal of Natural Fibers.\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003eMontgomery, D. C. (2017). Design and Analysis of Experiments (9th ed.). Wiley.\u003c/li\u003e\n \u003cli\u003eTukey, J. W. (1949). Comparing individual means in the analysis of variance. Biometrics, 5(2), 99\u0026ndash;114.\u003c/li\u003e\n \u003cli\u003eDo, K. L., Su, M., Mushtaq, A., Ahsan, T., \u0026amp; Zhao, F. (2023). Functionalization of silk with chitosan and Rubia cordifolia L. dye extract for enhanced antimicrobial and ultraviolet protective properties. \u003cem\u003eTextile Research Journal\u003c/em\u003e, \u003cem\u003e93\u003c/em\u003e(15-16), 3777-3789.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Natural dye, Rubia tinctorum (madder), Silk, Anthraquinone, Colourfastness, Antibacterial","lastPublishedDoi":"10.21203/rs.3.rs-6711143/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6711143/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNatural 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e 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\u0026lt;0.05), while K/S (colour strength) likewise rose (3-year \u0026gt; 2-year \u0026gt; 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 \u003cem\u003eS. aureus\u003c/em\u003e reduction (76.4%), significantly greater than the 1-year sample (68.4%) (ANOVA, p\u0026lt;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].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNovelty:\u003c/strong\u003e Although \u003cem\u003eRubia\u003c/em\u003e (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.\u003c/p\u003e","manuscriptTitle":"Effect of Rubia tinctorum Root Age on Coloration and Antibacterial Properties of Dyed Silk","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-05 05:29:01","doi":"10.21203/rs.3.rs-6711143/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8570795d-b09d-46ab-b1da-36048285c612","owner":[],"postedDate":"June 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-05T05:29:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-05 05:29:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6711143","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6711143","identity":"rs-6711143","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}