Extraction and chemical characterization of natural dye from western himalayan wild pomegranate peel for textile applications

Sara Iftikhar; Muhammad Qasim Hayat; Atteqa Safdar; Manzar Sohail; Rosario del P Castillo; Vicente Hernández; Aneela Javed

doi:10.1038/s41598-026-57034-y

. 2026 Jul 29;16:27085. doi: 10.1038/s41598-026-57034-y

Extraction and chemical characterization of natural dye from western himalayan wild pomegranate peel for textile applications

Sara Iftikhar ^1,^✉, Muhammad Qasim Hayat ¹, Atteqa Safdar ¹, Manzar Sohail ², Rosario del P Castillo ³, Vicente Hernández ⁴, Aneela Javed ⁵

PMCID: PMC13524945 PMID: 42665590

Abstract

Natural dyes are emerging as sustainable and eco-friendly alternative to synthetic ones in many industries because of health, safety and ecological concerns. The textile sector, being a major producer of dye effluents, is significantly paying attention to eco-friendly organic dyes. This study aimed to extract, apply and characterize a natural dye from the fruit peel of a wild type of Pomegranate (Punica granatum L.) which is widely distributed in several ecological zones of Pakistan. The obtained dye was analyzed for phytochemical screening, UV vis-spectrophotometry, Fourier-transform infrared spectroscopy (FTIR) and Gas chromatography-Mass spectrometry (GCMS). Furthermore, the application of dye on cotton fabric was tested for color efficiencies in terms of fastness properties and CIE L* a* b* values. Three mordants alum, ferrous sulfate and gallnut powder were used for comparison with un-mordanted fabric dyeing. ATR-FTIR analysis was performed to confirm the interaction of dye compounds with cotton fabric with and without mordant. DPPH and MTT assays were employed to check the antioxidant potential and cytotoxicity of the dye. GCMS revealed the presence of 51 compounds with intense color yielding nature. FTIR analysis identifies O–H, C–H and C–O as major functional groups. This study is the first to characterize the Western Himalayan wild pomegranate fruit peel in Pakistan, revealing the high content of biochemical compounds dye potential and antioxidant activity. The overall results suggest that the peel extract of wild pomegranate is suitable for manufacturing ecofriendly dye for various industrial sectors including textiles, pharmaceuticals, food, and cosmetics, as a safer and sustainable alternative to non-biodegradable synthetic dyes.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1038/s41598-026-57034-y.

Keywords: Natural dye, Pomegranate, Color, Fastness, Gas chromatography-mass spectrometry, 4Fourier-transform infrared spectroscopy

Subject terms: Chemistry, Environmental sciences, Plant sciences

Introduction

The pomegranate (Punica granatum L.) is among the world’s oldest cultivated fruits originated in Iran¹ and from there it is diversified to Mediterranean countries, India, China, Pakistan, and Afghanistan, possibly through ancient trade routes. Pomegranate plants have great nutritional and therapeutic importance. The edible portion of the pomegranate peel is 50% and the remaining part consists of non-edible peel. Considering the nutritional and nutraceutical properties of the wild pomegranate, its industrialization in the form of different commercial processed products is very important². Chithra et al.³ explores the pharmacological characteristics and antioxidant potential of pomegranate peel by applying Gas chromatography Mass spectrometry (GCMS), Fourier-transform infrared spectroscopy (FTIR) and 2,2-diphenyl-1picrylhydrazyl (DPPH) assay. Tai et al.⁴ conducted (GCMS) to determine the concentration of bioactive compounds of nutritional and therapeutic importance in peel and seeds of salami variety in Iraq and proposed that this variety can be effectively used for medical treatments. Several biodynamic compounds have been detected in pomegranate peel through GCMS analysis including 4 H-Pyran-4—one, 2,3—dihydro—3,5—di hydroxy—6—methyl which has efficacy as antibacterial, antioxidant, antitussive, antiasthma and anti-inflammatory activities⁵; Oleic acid; as antimicrobial activities⁵, Furan as; antifungal, antibacterial, antiviral and anti-inflammatory activities⁵ and 9,12,15-Octadecatrienoic acid, ethyl ester, (Z, Z,Z); as insectifuge, hepatoprotective, anti-coronary, hypocholesterolemic and cancer protective properties⁶. The wild pomegranate is part of the indigenous flora of Pakistan and is widely distributed in warm temperate Himalayan range². Although pomegranate is native to this region and several local types are widely distributed in different growing regions of the country, the available information is inadequate and limited research had been conducted on pomegranate germplasm collection, characterization, and conservation^7,8. However, the scientific reports are scanty with their dye yielding potential. Therefore, present research is designed to unveil, extract, and chemically characterize the dyes from the fruit peel of this wild pomegranate plant. Highlighting this feature will also cause awareness for biodiversity conservation.

Nature’s expression is full of vibrant colors, providing many dyeing ingredients. Dyeing of diverse objects including textile, leather, carpets, yarns, feathers, hair and even human was a centuries-old craft and its utilization was recorded back to the Bronze age in Europe, when rudimentary dyeing procedures involved rubbing plant extracts and crushed colors on fabrics⁹. This ancient dyeing method was transformed in 1856, when William Henry Perkin developed a synthetic dye, known as Mauvein. As synthetic dyes are less expensive, easier to apply, highly reproduceable and have tremendous fastness properties than natural dyes, they swiftly displaced natural dyes and gained popularity in textile industries, thus sparking a cultural revolution that resulted in the industrialization of synthetic dyes¹⁰. While synthetic dyes were preferred for their easy availability and other advances, a wide range of health and environmental concerns attributed to their non-biodegradable, carcinogenic and recalcitrant nature have increasingly turned the people interest to natural and safe dyes^11–15. The textile processing sector is a major polluter of the environment. It is reported that textile industries involves the production and utilization of nearly 1.3 million tons of pigments, dyes and dye precursors of around $23 billion cost¹⁶ and about 230 to 270 tons of water is required to manufacture a ton of synthetic color, which has been shown to pose critical health dangers. Situation is worse in developing countries like Pakistan^17,18. A substantial portion of synthetic dyes, auxiliaries and dye residues are a serious threat to both humans and the environment due to their unsustainable and carcinogenic nature. One trend since the start of new millennium has been the implementation of limitations on the use of synthetic dyeing chemicals, including toxic finishing agents and azo dyes¹⁶. However, Natural dyes are more environmentally friendly and clinically safer to use than synthetic analogues due to their non-carcinogenic, antiallergic, non-toxic, antibacterial, and biodegradable nature¹⁹. Due to increased acceptance of natural dyes as a significant substitute for synthetic dyes, a considerable increase in their demand in industries such as food processing, medicines, cosmetics, textile and printing has been observed^20,21. Natural dyes have been used for coloring textile materials for ages. In recent years, their applications are extended to antimicrobial finishing textiles, UV protective clothing, food coloring, cosmetics and pharmaceuticals, as they do not require any strong acids and alkalis for their application and production. They can also provide extremely exceptional relaxing mellow shades that match perfectly^22–24.Textile researchers have found considerable potential in natural dyeing as a promising technology to minimize the impact of synthetic dye chemicals, with studies highlighting the advantages of natural dyes such as subtle and elegant colors, biodegradability, non-toxicity and possible medical values^8,25–29.Current research in natural dyeing is focused on developing methods to improve color performance and color fastness properties, such as colorant extraction, laboratory dyeing techniques and physical and chemical techniques including plasma, ozone and radiation treatments^30–33. The growing acceptance of natural colorants as an alternative to synthetic dyes is evident with the advancement of modern techniques such as ultrasonic radiation, plasma, and gamma radiation for natural dyeing^34–37. Pomegranate peel extracts were frequently employed in this concern as a source of yellow dye. Kulkarni et al.³⁸ and Rehman et al.³⁹ explored the dyeing behavior of pomegranate peel extract on cotton and Tencel fiber at varying temperature using mordanting chemicals and presented that good color efficiencies and fastness properties. Bouaziz et al.⁴⁰ extracted dye from pomegranate peel and estimated total phenols and flavonoids but do not pay attention to other phytochemicals which contribute to coloring. Dye was tested on polyamide fabric and antibacterial finishing against S.aureus and E.coli was also considered for analysis. Currently, there is a lack of detailed elemental analysis of wild and cultivated pomegranate peel, as only a few studies have been conducted on its dyeing behaviors and bio-functional activities.

This study involves extraction of dye from the fruit peel of wild pomegranate, phytochemical screening, UV vis-spectroscopy, FTIR and GCMS analysis to understand the chemical nature of compounds and to identify certain functional groups or bonding elements in extracted dye. Dyeing of cotton fabric was carried out with extracted dye and its usage in textile coloration was analyzed by applying standard tests for fastness properties and CIE L* a* b* C* h° values. The effect of three mordants: two conventional metallic mordant chemicals (alum and ferrous sulfate) and one a plant based eco-friendly mordant (gallnut powder) has also been analyzed. Furthermore, antioxidant properties and cytotoxicity of the dye were also considered.

Several studies in different regions of world (e.g., Turkey, China, Brazil, Iran, Iraq) have reported the biochemical richness and dyeing potential of pomegranate peel and other by products, highlighting the color strength and fastness properties under appropriate extraction and mordanting conditions^{3,6,38,40–46}.However, there is a lack of integrated evaluation of chemical profiles alongside functional dyeing properties. In this context, the current research investigates an unexplored wild growing Western Himalayan germplasm, as a sustainable source of natural dye by combining chemical profiling, functional properties assessment and high-altitude ecological context to explain improved dyeing properties and bioactive content. The contribution of study lies in combining the chemical characterization with functional evaluation, thus emphasizing the potential of this untapped natural resource as a sustainable dye source across various industrial sectors including textiles, pharmaceuticals, food and cosmetics. However, utilization of natural dyes derived from wild plant sources at industrial scale requires careful consideration of biodiversity conservation and responsible resource management.

Results

Extraction and optimization of dye

The extraction of natural dye was carried out using wild pomegranate fruit peel powder. The extraction conditions for extracted dye were optimized by evaluating the effect of several parameters including extraction temperature, time, M:L ratio and pH. The efficiency of each parameter was determined based on absorbance measured at 423 nm. Measuring absorbance at this wavelength provides highest sensitivity and reproducibility for evaluating extraction efficiency. Statistical analysis using General Linear Model (GLM) Analysis of Variance (ANOVA) indicated that all of the investigated variables showed a significant effect on dye release. Absorbance increase substantially with temperature upto 70 °C (0.89 ± 0.017), however increasing the temperature to 80 °C (0.91 ± 0.026) resulted in no statistically significant improvement (p > 0.05), with both temperatures grouped in the same statistical category (Group A).Similarly extraction time significantly increased until 150 min (0.97 ± 0.020) approaching a statistical plateau where further extension to 180 min (0.99 ± 0.017) resulted in a slight improvements (p > 0.05).The M: L ratio efficiency enhanced upto 1:20 (0.83 ± 0.017) but no significant increase in dye concentration occurred at 1:25 and 1:30 ratios. However, in comparison to other factors, pH change exhibited a highly significant impact ( F_4,10 = 212.47, P < 0.001) with pH (0.87 ± 0.020) observed as statistically superior condition (Group A) and significantly lower yields at pH 7 and pH 3 (p ≤ 0.05).Consequently, the optimized parameters for extraction were selected as temperature 70 °C, 150 min and 1:20 M: L ratio at pH 5.These conditions represent the statistical optima that balances maximum dye yield considering practical aspects such as moderate temperature, reasonable time, ideal Ph stability and efficient solvent utilization to ensure reproducibility. The overall results are presented in Table 1.

Table 1.

Effect of extraction parameters on extraction of dye.

Parameters	Tested conditions	Absorbance (423 nm)
Temperature (°C)	40	0.53 ± 0.020^D
	50	0.66 ± 0.026^C
	60	0.77 ± 0.026^B
	70	0.89 ± 0.017^A
	80	0.91 ± 0.026^A
Extraction time (minutes)	30	0.44 ± 0.026^D
	60	0.68 ± 0.026^C
	120	0.86 ± 0.026^B
	150	0.97 ± 0.020^A
	180	0.99 ± 0.017^A
M: L ratio	1:10	0.47 ± 0.026^C
	1:15	0.70 ± 0.017^B
	1:20	0.83 ± 0.017^A
	1:25	0.86 ± 0.017^A
	1:30	0.88 ± 0.017^A
pH	3	0.43 ± 0.026^D
	5	0.87 ± 0.020^A
	7	0.80 ± 0.010^B
	9	0.71 ± 0.020^C
	11	0.66 ± 0.020^C

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*Values are expressed as Mean ± SD based on triplicate measurements (n = 3). Values within the same analysis indicated by different letters are significantly different (p ≤ 0.05).

Dye yield

The dye yield was measured through gravimetric method. Based on three experimental replicates, the estimated dye yield was reported as 45.83 ± 0.76% (Table 3) which indicates the recovery of a considerable amount of dyeing matter from fruit peel of wild growing pomegranate variety in Western Himalayan region.

Table 3.

Dye yield, phenolic content, flavonoid content, carbohydrate content, moisture content and ash content.

Parameters	Values
Dye yield %	45.83 ± 0.76%
Total phenolic content (mg/g)	180.33 ± 2.08 mg GAE/g
Total flavonoid content (mg/g)	19.17 ± 2.02 mg CE/g
Carbohydrate content (mg/g)	63.0 ± 1.5 mg/g of extract
Moisture content%	9.61 ± 0.38%
Ash content %	11.68 ± 0.27%

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Phytochemical screening

The phytochemical screening revealed the presence of numerous phytochemicals such as alkaloids, flavonoids, phenols, tannins, glycosides, steroids, sterols, quinones, terpenoids, quinones and saponins (Table 2). These secondary metabolites have enormous significance in pharmaceuticals and other commercial scale products.

Table 2.

Phytochemical screening of extracted dye from pomegranate peel.

Phytochemicals	Results
Alkaloids	+
Flavonoids	+
Phenols	+
Tannins	+
Carbohydrates	+
Glycosides	+
Steroids	+
Sterols	+
Quinones	+
Terpenoids	+
Saponins	+
Anthraquinones	−
Anthocyanins	−
Leuco anthocyanins	−
Phlobatanins	−

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Present (+), Absent (−).

Phenolic and flavonoid content

The total phenolic and flavonoid contents are presented in Table 3. Flavonoids account for approximately 10.6% of total phenolic content which shows that the extract is rich in phenolics. These results are consistent with the previous findings indicating that the polyphenolic composition of pomegranate fruit peel is predominantly made up of tannins⁴⁷.

Carbohydrate content

Carbohydrate content was estimated using phenol sulfuric acid method. The analysis is based on three independent experiments and showed a notable carbohydrates concentration of 63.0 ± 1.5 mg/g of extract (Table 3). The presence of carbohydrates may support solubility and fixation of dye and contribute to the overall functional characteristics as shown in previous reports⁴⁸.

Moisture content

The moisture content was calculated to assess stability and shelf-life characteristics. The estimated moisture content is 9.61 ± 0.38% which indicates less water retention in dye which may help in reduced microbial activity and enhance the stability under storage conditions (Table 3).

Ash content

The ash content was estimated as 11.68 ± 0.27% indicating the proportion of minerals that remained after complete combustion of organic constituents (Table 3). This residual content may contribute to overall functionality and stability of the dye.

UV-visible spectrum analysis

The UV-visible spectrum was recorded by UV-vis spectrophotometric technique. Absorption peaks at variable wavelengths with absorbance values are given in Fig. 1. The spectrum is characterized by two absorption maxima at 423 nm (λ _max) and 336 nm. The recorded absorption values at different wavelengths are characteristics of alkaloids, flavonoids, carotenoids, quinones, and derivatives. The principal absorption maximum at 423 nm (λ _max) corresponds to the main chromophoric transition and supports the use of 423 nm for monitoring extraction efficiency.

Fig. 1 — UV vis spectrum of extracted dye.

FTIR analysis

FTIR spectrum analysis is shown in Fig. 2. Detection was carried out at scan range of 4000 to 400 cm¹. A broad peak conforming the O–H stretch of phenolic compounds is observed at 3317.32 cm⁻¹ and the occurrence of C–H bonds on aromatic ring vibrates at 2922 cm⁻¹, the carbonyl groups of lactones stretch at 1718 cm⁻¹, whereas the aromatic ring skeleton vibrates at 1605 cm⁻¹. Additionally, at 1323 cm⁻¹ there was an OH deformation and C–O stretch (phenols); a C–O–C anti-symmetrical stretch at 1025 cm⁻¹ and at 874 cm⁻¹, 813 cm⁻¹ and 749 cm⁻¹ there was an out of plane C–H enduring twisted vibrations. The predominance of 1025 C–O–C stretching band shows high presence of glycosides or carbohydrates.

ATR-FTIR analysis

ATR-FTIR spectra of both dyed and undyed fabric are presented in Fig. 3.The spectra provides evidence of successful fixation of dye by presenting characteristic shifts in absorption peaks .The undyed cotton (black line) showed a characteristic cellulose peaks at ~ 3330 cm⁻¹(O–H stretching), ~ 2900 cm⁻¹ (C–H stretching), ~ 1640 cm (O–H bending) ~ 1425 cm⁻¹ (CH₂ bending) and ~ 1100–1030 cm⁻¹ (C–O–C stretching of glycosidic linkage). The O–H stretching band (~ 3330 cm⁻¹ became more broader and intense after dyeing indicating the hydrogen bonding between phenolic groups preset in dye and cellulose hydroxyl groups. Dyeing of fabric with pomegranate dye (without mordant) exhibits new absorption bands at ~ 1730 cm⁻¹ (C=O stretching) and ~ 1600 cm⁻¹ (aromatic ring vibration C =C) together with broadening in O–H region confirming the fixation of phenolic constituents of dye on fabric. Samples treated with mordants showed further spectral changes like; gallnut treatment produced stronger peaks at ~ 1730 cm⁻¹ and ~ 1600 cm⁻¹ regions, alum treated sample showed more intense C=O and C–O vibrations which is consistent with metal dye interactions, whereas ferrous sulphate treatment cause broadening of peak and a slight shift at around ~ 1600 to 1700 cm⁻¹ indication interaction between Fe ions and phenolic groups. These spectral changes confirm the interaction of dye compounds with cotton fabric with and without mordant.

GC-MS analysis

Analysis and extraction of plant materials have an important role in the development, transformation, and quality control of herbal preparations. Chemical analysis of extracted dye is performed by applying GCMS technique. The identified chemical constituents with their concentrations (peak area %), retention times (RT) and peak area percentages are provided in supplementary Table S1. Important compounds with plausible natural origin are listed in Table 4. The major identified compound classes include phenolic compounds, furans, hydrocarbons, fatty acids, phenylpropanoids and heterocyclic derivatives. Phenolic compounds and their derivatives such as phenol, 2-methoxy-4-(2-propenyl), phenolic alcohols and phenolic acid were prominent among identified compounds. Furan based compounds including furfural, furyl hydroxymethylketon, 5-hydroxymethylfurfural and 3-furancarboxylic acid methyl ester have also been identified indicating the occurrence of carbohydrate derived components. Additionally, the phenylpropanoid coumaric acid along with typical plant derived constituents such as heptadecanoic acid, oleic acid, linoleic acids and 2,6,10,14,18,22-Tetracosahexaen were also present in extracted dye. According to peak area percentage phenolic compounds, fatty acids and furan derivatives represents a substantial proportion of the identified chemical constituents.

Table 4.

GCMS analysis of various phytochemicals in extracted dye.

Sr#	Compound name	Molecular formula	Mol. weight	R. T	Peak area%	CAS: #	Functional class
1.	4 H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-	C₆H₈O₄	144.13	5.61	1.99	28564-83-2	Pyranone
2.	Furfural	C₅H₄O₂	96.08	9.71	2.64	98-01-1	Furans
3.	5-Hydroxymethylfurfural	C₆H₆O₃	126.11	7.86	3.16	67-47-0	Furans
4.	Furyl hydroxymethylketone	C₆H₆O₃	126.11	12.54	2.27	17678-19-2	Furan derivative
5.	Heptadecanoic acid	C₁₇H₃₄O₂	270.45	15.68	0.33	506-12-7	Fatty acid
6.	Coumaric acid	C₉H₈O₃	164.16	19.43	1.56	501-98-4	Phenylpropanoid
7.	Phenol,2-Methoxy-4-(2-Propenyl)	C₁₀H₁₂O₂	164.20	21.345	9.07	07-53-0	Phenolic compound
8.	3-Furancarboxylic acid, methyl ester	C₆H₆O₃	126.11	21.36	1.68	13129-23-2	Furan derivative/ester
9.	4-Methylbenzyl alcohol (TMS derivative)	C₁₁H₁₈OSi	194.35	25.65	3.84	14642-79-6	Phenolic alcohol
10.	3,4-Bis (trimethylsilyloxy) benzoic acid.	C₁₃H₂₂O₄Si₂	298.48	27.21	2.98	33539-21-4	Phenolic acid
11.	Linoleic acid(9,12-Octadecadienoic Acid)	C₁₈H₃₂O₂	280.45	34.89	6.41	60-33-3	Unsaturated fatty acid
12.	Oleic Acid	C₁₈H₃₄O₂	282.47	35.32	5.10	112-80-1	Monosaturated fatty acid
13.	2,6,10,14,18,22-Tetracosahexaen	C₂₄H₃₈	326.6	36.18	5.65	111-02-4	Polyunsaturated hydrocarbon

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Color fastness properties of dyed cotton fabric

The fastness properties of dyed fabric are given in Table 5. The overall results indicated good to excellent fatness grades across all parameters with washing (4–5), light and dry rubbing (5), wet rubbing (4) and artificial perspiration under both acidic and alkaline conditions (4–5). Gallnut used as tannin-based bio mordant also produced color fastness values comparable to metal mordants indicating its effectiveness as greener alternative. As the presence of mordant chemicals like metal salts makes the textile effluents quite harmful for the environment⁴⁹, these findings suggest that cotton fabric can be dyed without using any chemical mordants in an environmentally friendly, likely due to its polyphenolic constituents that can interact effectively with fiber hydroxyl groups. These interactions are attributed to hydrogen bonding between phenolic groups of dye molecules and hydroxyl groups of cellulose along with other intermolecular forces such as van der Waals interactions. Such molecular adhesion enables strong dye adsorption which explains the comparable fastness values for mordanted and un-mordanted samples.

Table 5.

Color fastness grades of dyed cotton fabric.

Mordant used	Washing fastness	Light fastness	Dry rub fastness	Wet rub fastness	Perspiration (Acidic)	Perspiration (Alkaline)
Without mordant	4	5	5	4	4	4
Ferrous sulfate	4	5	5	4	4–5	4–5
Alum	5	5	5	4	4–5	4–5
Gallnut	4	5	5	4	4–5	4–5

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ISO standard fastness grade; 1-Poor, 2-Moderate, 3-Average, 4-Good, 5-Excellent.

Colorimetric (CIE Lab* C* h°) analysis of dyed fabric

The CIE L* a* b* C* h° values are shown in Table 6. The shades of dyed fabric lies in yellow-red area, which is also confirmed by hue angles. According to b* values, the color of all samples is yellow with slightly reddish shades depending on type of applied chemical mordant. a* values for all the samples are positive representing the red shades. However, fabric samples mordanted with alum are more inclined toward yellow zone showing chroma value of 23.21 making the shade brighter yellow when compared with un-mordanted sample. Ferrous sulfate mordanted samples with a C* value 15.14 and hue angel 81.32 shift towards yellow-red zone thus having a distinctive position of color than other samples. In general, the type of mordant influence the CIE L*, a*, b* and h° values of dyed cotton samples and it is well known through previous studies that various chemical mordants when applied with a same color or dye can brightens, darkens and even completely alters the shades⁵⁰.Notably, the use of gallnut a tannin-based bio mordant produce visually appealing shade comparable to conventional metallic mordants such as alum, thus offering an eco-friendly alternative to support sustainability aspect of textile dyeing process.

Table 6.

Colorimetric values of dyed cotton fabric.

Mordant used	L*	a*	b*	C*	h°	K/S
Without Mordant	73.62	2.93	17.86	18.10	80.65	2.87
Ferrous sulfate	65.66	2.27	14.95	15.14	81.32	3.36
Alum	71.92	2.81	23.06	23.21	83.04	2.68
Gallnut	72.58	3.96	25.63	25.93	81.22	2.71

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L*, lightness; a*, redness (positive) and greenness (negative); b*, yellowness (positive) and blueness (negative); C*, chroma; h°, hue angle.

Color strength (K/S)

Color strength analysis was performed through AATCC procedure (EP-6). The K/S values are presented in Table 6. The sample dyed without mordant showed comparable color strength (K/S = 2.87) to the samples mordanted with ferrous sulphate, alum and gallnut (K/S = 3.36, 2.68 and 2.71) which presents effective interaction between dye constituents and fabric. The slightly lower K/S values of gallnut mordanted samples may be due to tannins polyphenols occupying cellulose binding which limits the dye adsorption. Similar trends in color strength have been reported previously for tannin based bio- mordants which reduce the color strength while improving the fastness properties⁵¹.

Ultraviolet protection factor (UPF)

The ultraviolet protection factor (UPF) is presented in Table 7. The results showed that all the samples dyed with natural dye exhibit excellent UV protection (As per AATCC test method 183 UPF ≥ 50 is reported as 50⁺⁾. This demonstrates that the chemical constituents of the dye are highly effective in absorbing and scattering UV radiation. Control (undyed fabric) is included to show baseline UV protection. UV-protective textiles dyed with natural colorants offers a sustainable approach to address health and ecological concerns^52,53.

Table 7.

Ultravoilet protection factor (UPF) of dyed cotton fabri.

Mordant used	Ultravoilet protection factor (UPF)	UVR protection category
Control (undyed fabric)	08	Low
Without mordant	50⁺	Excellent
Ferrous sulfate	50⁺	Excellent
Alum	50⁺	Excellent
Gallnut	50⁺	Excellent

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According to AATCC test method 183, UPF values above 50 are reported as 50+.

Effect of variable parameters (temperature, time, M:L and pH) on color coordinates of pomegranate peel dye

The color coordinate values at various parameters including temperature, time, M:L and pH are given in Table 7. The color tones of all the samples are yellowish due to positive a* and b* values. However, with the increase in pH a* and b* values also increased thus showing an increase in yellowish shade at pH 9 and 11. All mentioned variables show minor variations in L*, a*, b*, C*, h° values. The primary justification for this finding is that these variables have no significant effect on the structure of coloring components present in dye. Additionally, it is observed that the color coordinate values vary within the tolerance limit and are not visually perceptible.

DPPH assay

The free radical scavenging activities examine by DPPH assay are displayed in Fig. 4. A stable free radical called DPPH interacts with an antioxidant to form R, R-diphenyl-â-picryl hydrazine. The degree of discoloration indicates the potential of radical scavenging activity of the sample. The results obtained from DPPH assay have shown that the extracted dye contains a significant number of free radical inhibitors that can interact with free radicals.

ABTS assay

The antioxidant activity of the dye extract evaluated by ABTS assay is presented in Fig. 5. These results confirm the presence of substantial antioxidant constituents that can effectively quench ABTS ·+radicals. These values are consistent with previously reported high ABTS scavenging activities for pomegranate peel extract^54–56.

Cytotoxic activity

MTT assay was employed to determine the cell viability and the results are presented in Table 8; Fig. 6.The cell viability% remained higher than the control (96.20 ± 0.18)which indicates that the extracted natural dye does not induce cytotoxic effects to HEK-293 cell lines under tested conditions This response can be attributed to the presence of bioactive constituents such as phenolic and flavonoid compounds present in dye which are known to enhance cellular metabolism and provide protection against oxidative stress⁵⁷. Overall, these preliminary in vitro findings present suggest that this natural dye is biocompatible with normal human cells highlighting its potential safety for further application (Table 9).

Table 8.

Effect of extraction temperature on color coordinate values of pomegranate peel dye.

Parameters	Tested values	L*	a*	b*	C*	h°
Temperature (°C)	40	70.49	1.95	16.09	16.21	82.9
	45	71.36	1.93	16.61	16.72	83.4
	50	73.67	2.69	17.41	17.61	81.2
	55	73.56	2.74	18.47	18.79	81.6
	60	74.62	2.82	18.67	18.99	81.4
Time (Min)	120	71.90	1.56	15.91	15.98	84.4
	160	71.47	1.89	15.48	15.60	82.9
	200	71.11	2.16	17.51	17.62	82.6
	220	73.86	2.70	17.19	17.42	81.8
	240	73.19	2.49	17.47	17.61	81.9
M: L ratio	1:10	72.19	1.60	16.88	16.94	84.2
	1:15	72.55	2.04	17.21	17.30	82.5
	1:20	73.70	2.91	17.89	18.14	81.4
	1:25	73.85	2.39	17.58	17.71	82.5
	1:30	73.41	2.77	17.94	18.14	81.8
pH	3	72.34	1.30	15.44	15.50	84.3
	5	72.76	1.73	16.58	16.64	82.5
	7	73.40	2.81	17.98	18.21	81.6
	9	73.93	2.89	18.09	18.34	81.4
	11	73.23	2.91	18.82	19.01	81.8

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Fig. 6 — Cell viability (%) of HEK-293 cells treated with different concentrations of extracted natural dye.

Table 9.

Cytotoxic effect of the dye against HEK-293 cell line by MTT assay. Cell viability (%) is presented as mean ± SD relative HEK-293 cell to control.

Concentrations	%Viability
Control	96.20 ± 0.18
1 ug	159.68 ± 0.04
100 ug	191.26 ± 0.33
5 mg	192.54 ± 0.19

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Discussion

The present study investigated the extraction, chemical characterization and dyeing potential of a truly wild pomegranate population collected from Western Himalayan region which is an underutilized biomass source with promising bioactive properties. These findings reveal a detailed compositional profile expanding the understanding of phytochemical diversity in Punica granatum L. The 45.83 ± 0.76% dye yield is obtained from Himalayan wild pomegranate peel which is notably higher than other varieties reported in previous studies. For example, Kulkarni et al. reported 19.2% dye yield with conventional extraction while Tiwari et al. found 29.2% yield for ultrasound assisted extraction and 26.5% with enzyme assisted extraction^38,58. Similarly, Hosseinnezhad et al. reported extraction yield of 23%, 33% and 36% for madder, Reseda and walnut husk respectively and Mohan et al. documented 20 to 23.3% dye yield for onion skin^59,60.

The secondary metabolites such as alkaloids, flavonoids, phenols, tannins, glycosides, steroids, sterols, quinones, terpenoids and saponins were confirmed in extracted dye. The Phenolic and flavonoid content was found as 180.33 ± 2.08 mg GAE/g and 19.17 ± 2.02 mg CE/g respectively. These phytochemicals have also been identified by Chitra et al.³ and Karthikeyan and vidya, 2019 in in other pomegranate varieties in different regions⁶¹. These values slightly exceed those reported in earlier studies on cultivated pomegranate varieties⁴⁰.The relatively high phenolic content is likely to increase the dye fiber interaction as phenolic hydroxyl groups facilitate hydrogen bonding with cellulose fibers by forming stable coordination complexes, thereby enhancing the dye binding strength and fastness properties^28,51. Additionally, a notable carbohydrate concentration 63.0 ± 1.5 mg/g of extract is also observed. Although no detail experiment was performed to investigate the role of carbohydrates in dye fiber interaction, these compounds may support solubility and fixation of dye supporting the overall functional characteristics as shown in previous reports⁴⁸.FTIR spectrum indicates the presence of hydroxyl, carbonyl, aromatic and ether functional groups confirming the occurrence of phenolic compounds, conjugated structures, aldehydes and carbohydrate related constituents. These absorption bands have also been reported in pomegranate peel by Sharif et al.⁶², Chithra et al.³ and Yang et al.,⁴² indicating that the functional groups identified in the sample in current study are respective to the previous literature findings. The identified functional groups improve the dye-substrate interaction by creating hydrogen bonds or electrostatic interactions with natural fiber such as cotton.

ATR-FTIR analysis of dyed cotton fabric showed a successful interaction of dye with fabric involving hydroxyl, carbonyl, aromatic and C–O functional groups through hydrogen bonding and possible metal coordination in mordanted samples. The undyed cotton showed a characteristic cellulose peaks consistent with the baseline spectra⁶³.Changes in FTIR spectra of dyes fabric indicates the fixation of dye constituents with the fabric is also in agreement with other studies on natural dyes⁶⁴. Treatment of samples with mordants further modify the showing interaction of metal ions with dye phenolic constituents. Similar spectral changes have been reported in study on cotton dyed with natural dyes using mordants and showed enhanced fixation of dye with altered FTIR bands^65,66.

Collectively the FTIR analysis indicated the presence of phenolic –OH, carbonyl (C=O) and aromatic functional groups which constitute polyphenolic compounds commonly present in pomegranate peel and the ATR-FTIR spectra of dyed fabrics reveal an expansion of the O–H band (~ 3330 cm⁻¹) and the appearance of C=O (~ 1730 cm⁻¹) and aromatic C=C (~ 1600 cm⁻¹) bands, suggesting possible interactions between cellulose fiber and dye molecules. In mordant assisted dyeing, fabric treated with alum showed increased C=O and C–O intensities, while ferrous sulphate treatment exhibits broadening of peaks consistent with possible interaction between metal ion and phenolic groups. These molecular interactions explain the observed variations in color strength fastness properties providing a mechanistic understanding of binding of dye molecules with cotton fiber.

The GCMS profiling of pomegranate peel dye indicated the presence of wide array of chemical constituents with prominent phenolic content, furans derivatives fatty acids presenting chemically divers systems responsible for dyeing potential. The results show similarities with previously identified compounds in pomegranate.Previous studies on pomegranate extract reported a variety of compounds like; Furfurals(furfural, 5-Hydroxymethylfurfural), phenolic acids (gallic acid, Protocatechuic acid p-coumric acid), fatty acids (palmitic acid, oleic acid, linoleic acid) and other compounds compounds of medicinal importance^6,67–70.This analysis provides a detailed chemical profile of wild pomegranate cultivar from Western Himalayan region and reveals several promising chromophore candidates (e.g. Conjugated double bonds, aromatic rings), that may contribute to the observed dyeing behavior. However, GCMS based identification is preliminary and cannot directly attribute the dyeing functionality to specific molecules. Therefore, the results presented a supportive chemical profile which aligns with the reported abundance of chemical constituents and their role as natural chromophore in dyeing^6,28,51,71.Further confirmation through compound isolation and spectroscopic characterization is required to determine the structure-function correlation.

The functional groups identified in FTIR analysis are in close alignment with the chemical compounds detected in GCMS. The broad stretch at 3317 cm^− 1 observed in FTIR is associated with phenolic hydroxyl group supporting the presence of Phenol,2-Methoxy-4-(2-Propenyl) and phenolic acid derivative. The C=O stretching at 1718 cm^− 1 corresponds to fatty acids (oleic acid and linoleic acid), furans (furfural and 5-hydroxymethylfurfural) and coumaric acid while aromatic C=C vibrations suggest the occurrence of phenylpropanoid structures. The prominent C–O–C stretching at 1025 cm^− 1 compiles with carbohydrates related compounds and furans. These functional groups particularly phenolics and conjugated carbonyl groups have been shown to be important for color formation and enhanced interaction with fiber supporting the dyeing potential of extract, however their contribution in dye fixation is determined through analytical and dyeing results instead of direct mechanistic confirmation.

Application of dye was carried out on cotton fabric with and without mordant to evaluate textile dyeing potential. Natural dyes are generally reported to show moderate color fastness compared to synthetic dyes which offers more stable color efficiency but raise environmental concerns due to their toxic and non-biodegradable nature^51,72,73. In this context the current analysis presents satisfactory color fastness within the natural dye category. The mordant chemicals did not significantly improve these properties except the light fastness property which is slightly enhanced through pre mordanting. The observed fastness performance is comparable with earlier reports on natural dyeing. Similar studies on dyeing of fabric mordanted with ferrous sulfate and alum have been reported by Das et al.⁷⁴.It is also reported that the use of sustainable natural dyes in textile sector can reduce the environmental challenges and offer a safe alternative to synthetic dyes. ³⁸.

CIE L* a* b* C*h° values of dyed fabric showed the color shades in yellow-red area, with the mordant types influencing the color coordinates as also reported in other studies⁵⁰. Kulkarni et al.³⁸, Rehman et al.³⁹, Bouaziz et al.⁴⁰, and Yang et al.⁴² have perform coloring analysis of pomegranate peel extract on polyamide, cotton, Tencel and silk fiber fabric in terms of its color fastness properties and CIE L* a* b* values, highlighting the potential applications in textile dyeing. The color coordinate values tested at various parameters showed minor variations within the visual tolerance limit. the minor variation observed with broad pH range suggests that the coloring compounds remain largely unaffected under acidic and alkaline conditions supporting fabric durability during laundering and light exposure. The color performance results obtained in current study represent consistent and strong dyeing performance than previously reported cultivated varieties, which can be attributed to unique chemical nature of this freshly explored wild resource. The color strength results also indicate effective uptake of dye consistent with previous reports⁴⁰. The comparable fastness results of bio mordant (gallnut tannins) further supports the potential of replacing harmful metal salts with plant-based alternatives. Moreover, all dyed samples exhibited excellent UV-protective properties(UPF ≥ 50) that aligns with reported UV-protective properties of plant based natural dyes from mangrove bark, pomegranate, madder and weld as described by Santiago et al.⁷⁵ and Maha et al.⁷⁶.

The antioxidant activity of the extracted dye pomegranate peel was observed up to 82.4% and 91.6% through DPPH and ABTS assays respectively, likely due to the presence of flavonoids, tannins and phenols confirming the presence of large amount of free radical inhibitors⁷⁷. Antioxidant potential of pomegranate peel of several cultivars have also been confirmed by Malviya et al.⁷⁸, Mutahar et al.⁷⁹, Jalal et al.⁸⁰and Chithra et al.³ in their studies. In vitro cytotoxicity testing was also performed using MTT assay on human embryonic kidney cell lines (HEK-293) to evaluate the preliminary safety profile of the extracted dye. The findings suggest that the cell viability remains high across the tested concentration. As MTT assay evaluates mitochondrial metabolic activity, the values above 100% may indicate metabolic variations or assay interactions rather than true cellular proliferation. The observed biocompatibility of the dye can be attributed to phenolics, and flavonoid constituents present in dye extract which are known to improve cellular health and protect against oxidative stress⁵⁷. These results are in agreement with previous studies presenting that pomegranate peel shows minimal toxicity towards normal human cells⁸¹. These preliminary findings suggest that the extracted dye exhibits a favorable safety profile, supporting its potential for further investigations in other applications.

This study was performed on laboratory scale, however the scalability of proposed extraction approach shows strong potential for large scale textile dyeing applications as wild pomegranate peel is abundantly available agricultural waste product and conventional solvent extraction method is an already established phenomena in natural product industries and can be easily adopted to pilot and large-scale production. Furthermore, the use of less toxic solvent also allows feasible solvent recovery, minimizing production cost and environmental impacts. In comparison to synthetic dye the extraction process of natural dye offers a substantially lower environmental footprint due to their biodegradable nature and reduced hazardous effluent generation. Although the industrial trials were beyond the scope of this study, the proposed strategy for extraction of natural dye from wild pomegranate peel exhibits strong potential for future integration into large scale textile dyeing applications with economic and environmental advantages. In addition, large scale industrial utilization of Himalayan wild pomegranate peel should be aligned with biodiversity conservation using pomegranate fruit peel as a processing by product rather than fresh harvesting of fruits. Alongside, managed cultivation, controlled harvesting practices and community involvement to minimize pressure on wild population can help to ensure the stable supply of raw material while conserving biodiversity. This approach supports a circular bioeconomy and ensures long term resource sustainability.

Materials and methods

Extraction of dye

The wild pomegranate fruit was collected from Western Himalayan region (Muzaffarabad, Azad Jammu and Kashmir: 34.2718° N, 73.5561° E; Accession No. 47743). The fruits were identified by taxonomist Dr. Muhammad Qasim Hayat, ASAB (Atta-ur-Rahman School of Applied Biosciences), NUST (National University of Sciences and Technology), Islamabad. The plant specimen is present in PMNH (Pakistan Museum of Natural History) herbarium, Islamabad, with voucher specimen no.47743.All fruits were collected during the same harvesting season to minimize the seasonal variability and uniform processing conditions were applied to ensure consistency in phytochemical composition and dye yield. The peels were scraped from the fruit and shade dried for 3–4 days. After drying, the peels were pulverized into powdered form and filtered through 0.074 mm mesh size sieve. The optimized conditions were determined (Section “Dye yield”) and applied for dye extraction. The dye extract was prepared in Soxhlet apparatus using methanol as solvent by applying optimal conditions obtained from performed experiments i.e. material liquor ratio of 1:20 at 70 °C for150 min. The refluxing solvent washed the material repeatedly, extracting the desired components. The solution was then subjected to rotary evaporation to concentrate the solution and finally getting a dry powder. This dry extract (dye) was further subjected to chemical analysis and dyeing applications.

Optimization of extraction conditions

To establish suitable conditions for extraction of dye, systematic optimization experiments were conducted using a single factor design. A series of experiments were designed varying one parameter at a time while keeping others constant. The parameters investigated includes extraction temperature (40 °C ,50 °C, 60 °C ,70 °C and 80 °C ), extraction time (30, 60, 120, 150 and 180 min), M:L ratio (1:10, 1:15, 1:20, 1:30 and 1:40) and Ph (3, 5, 7, 9 and 11). All extractions were performed in a Soxhlet apparatus using 60% methanol as solvent. The absorbance of each extract was measured at 424 nm using UV-Vis spectrophotometer. The wavelength 423 nm was selected based on maximum absorbance observed in UV-Vis spectrum of dye extract (Fig. 1) corresponding to the visible chromophore bands of the dye. Examinations at other spectral regions confirmed no significant peaks indicating consistent chromophore stability across various extraction conditions. Monitoring absorbance at maximum wavelength (λ _max) is a well-established phenomenon in natural dye research where λ _max values provide reflection of visible color intensity and extraction yield^51,82,83. The conditions producing highest absorbance values were selected as optimal for subsequent extraction of dye product. All experiments were performed in triplicate (n = 3) and the results are expressed as mean ± standard deviation (SD). The influence of extraction parameters was analyzed through General Linear Model (GLM) Analysis of Variance (ANOVA) in Minitab. Tukey’s post hoc test was applied to identify significant differences between means and differences were considered statistically significant at p ≤ 0.05. The most appropriate extraction conditions were selected based on statistical significance, and practical considerations such as material economy and reproducibility.

Dye yield determination

Dye yield was estimated gravimetric analysis method. After extraction the dye extract was filtered through Whatman filter paper to remove any residues, and filtrate was transferred to pre weighed dry glass petri plates. The plates containing the dye extract were placed in hot air oven at controlled temperature to evaporate the solvent and achieve solid dye residue. The plates were placed in desiccator for 20 min to avoid absorption of moisture from air and weighed on balance. For reliability the drying, cooling and weighing steps were repeated to obtain a constant weight. The final weight reported the amount of dye extracted from plant raw material²⁰.

The total percentage of dye yield was calculated applying the following formula:

Preliminary phytochemical screening

Preliminary qualitative analysis of was performed to identify various active constituents such as proteins, tannins, alkaloids, flavonoids, glycosides, steroids, sterols, phenols, saponins, quinones, terpenoids, anthraquinones, anthocyanins, leucoanthocyanins, and phlobatanins.

Estimation of phenolic content

Total phenolic content was quantified through Folin-Ciocalteu assay with gallic acid as reference standard. For analysis a mixture of o.4mL dye extract was mixed with 10 mL of diluted Folin-Ciocalteu and left at room temperature for 5 min under continuous stirring. Subsequently 8 mL of 75% sodium carbonate solution was added to the mixture. After incubation for 1 h at room temperature the absorbance was measured at 765 nm. Total Phenolic content was calculated from gallic acid calibration curve and expressed in mg of gallic acid equivalents (GAE)/g of extract.

Estimation of flavonoid content

Total flavonoid content was determined by using aluminum trichloride and sodium hydroxide with a method described by⁸⁴. Catechin is used as reference standard.1Ml volume of extract was mixed with 4 ml distilled water and 5% sodium nitrite. The mixture was kept in dark for 5 min and 0.3 ml of 10% aluminum chloride was added. Following 10 min of incubation in dark 2 ml of 1 M sodium hydroxide was added. After stirring the mixture absorbance was measured at 510 nm and flavonoid content was calculated using a catechin calibration curve and reported as mg catechin equivalent (CE) per gram of extract.

Estimation of carbohydrates

Carbohydrate estimation was carried out applying the method of⁸⁵ with certain modifications.0.1 g of extracted dye was hydrolyzed with 5 ml of 2.5 N HCL for 3 h in a boiling water bath and further neutralized with sodium carbonate and diluted to 25 ml with distilled water and centrifuged at 5000 rpm for 5 min. The supernatant was collected and mixed with 90 µL of 5% phenol and 500 µL of 96% sulfuric acid. The mixture was incubated for 30 min at room temperature and absorbance was recorded at 490 nm in the spectrophotometer. A blank was prepared by replacing the extract with distilled water. Sucrose was employed as standard for quantification of carbohydrates.