Freeze-Drying as a Stabilization Strategy for Natural Dyes Derived from Lawsonia inermis L. and Indigofera suffruticosa

Abstract

This study focuses on the stabilization of a natural hair dye derived from Lawsonia inermis L. (henna) and Indigofera suffruticosa (indigo). Although various formulations already exist, they are designed for immediate use and cannot be stored. Lawsonia, a primary component of the dye, tends to degrade after release. To ensure its stability, freeze-drying was implemented as a protective measure. Colorimetric analysis confirmed the dye’s ability to maintain an intense, uniform coloration even after multiple washing cycles. Stability tests demonstrate that freeze-drying effectively enhances the dye’s stability and capacity to retain its physical properties and color under various environmental conditions, demonstrating its potential for long-term use. The dye’s pH (5.05) aligns with the natural pH of hair, promoting cuticle sealing and improving hair health. Cytotoxicity tests confirmed the dye’s safety, showing no harmful effects. Gray hair exhibited a total color difference (ΔE) of 64.06 after the initial application, using natural gray hair as a reference. By the third application, ΔE increased to 69.86 and gradually decreased to 68.20 after 15 washing cycles, highlighting its long-term durability. Gray hair exposed to 720 h of UV radiation showed a ΔE of 17.34, whereas dyed gray hair exhibited a ΔE of 2.96 compared to non-UV-exposed samples. This indicates superior resistance to color degradation in dyed hair. Also, SEM imaging revealed the dye’s restorative effects, progressively improving hair cuticle structure with each application.

1. Introduction

Dyeing hair has become a common beauty practice across various age groups, serving both as a means of personal expression and to conceal gray hair. The most commonly used hair dyes today are oxidative, permanent types, known for their excellent color durability. However, the ingredients in these oxidative hair dyes have the potential to trigger allergic reactions and may cause damage to the hair [1]. Para-phenylenediamine, a key ingredient in oxidative hair dyes, can irritate the eyes, trigger asthma when inhaled, and may cause skin irritation, contact dermatitis, or even kidney damage through skin exposure [2]. The hair dye industry is actively working to develop products that are safe for the body, easy to use, and free from para-phenylenediamine. As part of this initiative, there is growing interest in temporary or semi-permanent hair dyes, which, although offering less color durability than permanent dyes, are considered safer and more user-friendly [1]. One type of semi-permanent hair dye is basic dye, which consists of cationic pigments [3]. Basic hair dye colors the hair without the need for para-phenylenediamine, hydrogen peroxide, or alkaline substances [4]. While permanent hair dye creates color within the hair’s cortex through an oxidation reaction, basic hair dye deposits the basic (cationic) pigment onto the hair in its pre-colored form.

Synthetic dyes are popular as colorants due to their availability, cost-effectiveness, vibrant hues, and excellent color fastness properties [5]. However, some synthetic dyes have been linked to allergic reactions, carcinogenicity, and pose risks to human health and the environment [6]. This has spurred renewed interest in natural dyes, which are non-allergenic, non-toxic, environmentally friendly, and readily available in nature [5]. Natural dyes are non-oxidative colorants that can penetrate the hair cuticle and, to some extent, the cortex, resulting in color deposition [7]. Lawsonia inermis L., commonly known as henna, has been used for nearly 5000 years, dating back to ancient Egypt, where it was applied to color hair and nails [8]. Henna is widely used as a dye for leather, silk, wool, and for staining the skin and hands [9]. The active dyeing agent in henna, lawsone (2-hydroxy-1,4-naphthoquinone), is found in the henna plant (Lawsonia alba and Lawsonia inermis). On gray hair, it imparts an orange-red color, while on brown hair, it produces shades ranging from copper to red. Lawsonia inermis L. dye has been extensively studied for its application on vegetable, protein, and synthetic fibers [9]. The dyeing property of henna is attributed to lawsone, identified by Color Index Number 75480, also known as Natural Orange 6. In 2013, the European Union confirmed that henna, when applied using traditional methods, is safe for health, with experts specifically recommending pure, natural henna powder for hair dyeing, except for individuals with glucose-6-phosphate dehydrogenase deficiency [10]. Additionally, the antibacterial properties of henna-dyed wool against E. coli and S. aureus have been reported [11]. Lawsonia inermis L. is known in traditional medicine for its antifungal properties [12], which can be used to manage dandruff and hair loss. It also exhibits a range of beneficial properties, including antitumor, antiangiogenic, antileishmanial, anti-inflammatory, antiproliferative, antimalarial, analgesic, antipyretic, enzyme-inhibiting, larvicidal, hepatoprotective, wound-healing, memory-enhancing, and antioxidant effects [12].

Another natural dye is indigo (Indigofera suffruticosa). It is used to dye hair, nails, fabrics (such as jeans), leather, silk, wool, and more. Indigo can be obtained from various species, including Indigofera suffruticosa, Indigofera tinctoria, and Isatis tinctoria. The chopped plants are soaked in hot water, with heat maintained to facilitate fermentation. The violet-blue color of indigo is produced through the oxidation of indican, a glycosylated precursor, which occurs upon exposure to air [13]. Indigo is insoluble in water, and its use as a dye is challenging due to poor absorption by keratin fibers. This dye imparts a blue color to gray hair and an ash to violet hue to brown hair. The indigo plant has various applications in folk medicine, including as an antiepileptic, antispasmodic, purgative [14], antioxidant [15], diuretic [16], chemopreventive [17], and as an aid during labor [18]. It is also used to treat snake bites [19]; assist in cases of malnutrition, flu, and cough; function as an antipyretic, analgesic [20], and gastroprotective [21]; and alleviate colic and kidney diseases. Additionally, it exhibits anti-inflammatory, pain-relieving, and antimicrobial properties [22]. GC-MS analysis has identified 25 chemical compounds in Indigofera suffruticosa [23], which show anti-seborrheic properties, stimulate hair growth, assist in treating alopecia, and possess antiviral and antiparasitic effects [23,24].

The aim of this study was to produce an organic dye for repairing and coloring damaged hair that had undergone a discoloration process and dyeing gray hair. The organic dye was formulated using Lawsonia inermis L. and Indigofera suffruticosa, with additional components for specific purposes: Chamaemelum nobile infusion (to prolong pigment retention), citrus lemon (to facilitate the release of hennotannic acid from Lawsonia inermis leaves), and tea tree essential oil (to enhance pigment release). A key focus of the study was the stability of the organic dye, which was achieved through a freeze-drying process to extend its shelf life. Additionally, the color performance of the organic dye on gray and discolored hair was evaluated through three dyeing processes, each followed by 15 wash cycles. In this study, discolored hair refers to hair that has undergone bleaching as a result of exposure to chemical agents, particularly hydrogen peroxide. The dye’s ability to protect colored hair exposed to UV radiation was also examined, along with the effect of storage time on dye performance.

2. Materials and Methods

2.1. Chemical and Biological Material

Lauryl sulfate sodium (CH₃(CH₂)₁₁OSO₃Na, ≥99.0%) and phosphate-buffered saline tablets (PBS) were bought from Sigma-Aldrich. Metallic bags were bought from Sellatodomx. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was purchased from Merck/Sigma-Aldrich (México City, México). The HaCaT human epidermal keratinocytes (PCS-200-011) were acquired from the ATCC and cultured in Dulbecco’s Modified Eagle’s Medium purchased from Sigma-Aldrich, supplemented with 10% Fetal Bovine Serum (FBS, Gibco, México City, México) and 1% penicillin-streptomycin (10,000 μ/mL, Sigma-Aldrich, México City, México), at 37 °C and 5% CO₂. Lawsonia inermis L. was obtained from Sharmili Spices PVT, a company located at coordinates 20.8334° N, 73.6759° E. Indigofera suffruticosa was obtained from a Mexican supplier in Santiago de Niltepec, Oaxaca, located at coordinates 16.5593° N, 94.6130° W. Chamaemelum nobile (commonly known as chamomile) and lemon were cultivated in Mexico. Essential oil of the melaleuca tree was bought from Bonita Organics. Dr. Teresa del Carmen Flores Flores performed the botanical identification on 19 October 2023, using dichotomous keys and specialized literature to analyze the plant’s vegetative and reproductive characteristics. Discolored hair and gray hair were donated by two women, aged 25 and 55, respectively. Deionized water with a resistance of 18 MΩcm was used.

2.2. Organic Dye Formulation

An infusion was prepared using 4 g of chamomile in 100 mL of water, maintaining the temperature at 45 °C. A volume of 250 mL of this infusion was then mixed with 100 g of Lawsonia inermis L. Subsequently, 17 mL of lemon juice and 100 µL of tea tree oil (Melaleuca alternifolia) were added and thoroughly mixed. The mixture was allowed to stand for 2 h. At room temperature, the aglycone of Lawsonia inermis L. remains stable in the mixture for a maximum of 12 to 24 h. To ensure the long-term preservation of the aglycone, 100 g of Indigofera suffruticosa was added and mixed after a 2 h rest period. The mixture was then frozen at −15 °C for 48 h. Subsequently, the samples were freeze-dried for 24 h, with the condenser maintained at −45.5 °C and a vacuum pressure of 0.065 mbar. This process preserved the properties of the organic dye by removing water content and producing a solid, which was then pulverized using a mortar.

To discolor the hair, a mixture was prepared by combining 30 g of bleaching powder with 60 mL of 9% hydrogen peroxide (30 vol., suitable for moderate to severe discoloration), yielding a 1:2 ratio. This formulation was applied to dry hair and allowed to process for 30 to 45 min. Subsequently, the hair was rinsed, a violet toner was applied to neutralize yellow hues, and a repairing mask was used to help maintain the integrity of the hair fiber.

To apply the dye to hair (discolored hair or gray hair), the hair samples were washed and dried. For each gram of organic dye, 5.5 mL of distilled water at 25 °C was added and manually stirred with a glass rod until a homogeneous mixture was obtained. This mixture was allowed to rest for 10 min. The organic dye was then applied to the hair using a brush. The samples were covered with parafilm to retain heat, facilitating the interaction of the active components of lawsonia and indigo with keratin. The dye was left to set for two hours before being rinsed thoroughly with water at 25 °C.

2.3. Characterization

To evaluate the interaction between the components of the organic dye and hair (gray and discolored hair), Fourier-transform infrared spectroscopy (FTIR) analysis was performed. The measurements were conducted using an Agilent Cary 600 spectrometer equipped with an attenuated total reflectance (ATR) accessory. The data were collected over a range of 4000 to 400 cm⁻¹. For each sample type, spectra were recorded in triplicate, and the raw data remained unaltered.

The surface area of both discolored and gray hair, before and after treatment with the organic dye, was measured using nitrogen adsorption at −195.8 °C with a surface area analyzer (Quantachrome AS1Win™, Quantachrome Instruments, Boynton Beach, NY, USA). Prior to the measurements, the samples were degassed at 50 °C for 5 h. BET measurements were performed in triplicate for each sample. The Brunauer–Emmett–Teller (BET) equation was applied to the adsorption data to calculate the BET surface area of the samples. The BET equation in its linear form is expressed as follows:

$${\displaystyle \frac{\raisebox{1ex}{$P$}\!\left/ \!\raisebox{-1ex}{$P^o$}\right.}{V(1-\raisebox{1ex}{$P$}\!\left/ \!\raisebox{-1ex}{$P^o$}\right.)}}={\displaystyle \frac{1}{V_{m}C}}+{\displaystyle \frac{C-1}{V_{m}C}}{\displaystyle \frac{P}{P^o}}$$

(1)

where $V_m$ represents the monolayer adsorption capacity; V is the volume of gas adsorbed at the equilibrium pressure; $P^o$ corresponds to the saturation pressure of nitrogen at the measurement temperature; and C is a constant related to the energy of interaction between the adsorbate and the surface, which must take a positive value. The BET surface area is calculated using the following equation:

$$Sg=\left({\displaystyle \frac{V_{m}N}{V}}\right)\alpha$$

(2)

where N is Avogadro’s number (6.023 × 10²³ molecules/mol); V is the molar volume of nitrogen at standard conditions (22,400 cm³/mol); and α represents the cross-sectional area of a nitrogen molecule (1.372 × 10⁻¹⁵ cm²/molecule).

The cytocompatibility of Lawsonia inermis L., Indigofera suffruticosa, and the organic dye was evaluated using MTT metabolic assays. HaCaT cells (8 × 10⁴) were seeded in each well of 96-well culture plates and incubated overnight to allow adhesion (at 37 °C, 5% CO₂, and 95% humidity). Once the cells formed monolayers, they were synchronized for 24 h in the absence of FBS. Media containing different concentrations of Lawsonia inermis L., Indigofera suffruticosa, or the organic dye (1, 10, 100, and 1000 μg/mL) were then added and incubated for 4 h under the same environmental conditions. The cells were gently rinsed with 0.01 M PBS (pH 7.4), followed by incubation with MTT salt for 3 h. The resulting formazan crystals were dissolved in Dimethyl sulfoxide (DMSO), and absorbance was measured at 560 nm with a reference wavelength of 670 nm. Cell viability was calculated using the following Equation (3):

$$\% \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}={\displaystyle \frac{{\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{{\mathrm{A}}_{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}}\times 100$$

(3)

In this equation, ${\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}$ represents the absorbance of cells treated with the sample, while ${\mathrm{A}}_{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}$ refers to the absorbance of untreated cells. The percentage of cell viability is reported as the mean ± standard deviation from triplicate experiments. Triton X-100 was used as a positive control for cytotoxicity due to its ability to induce cell damage. HaCaT cells served as a negative control to demonstrate the absence of cytotoxicity when treatment does not affect cell viability. A two-way ANOVA (Analysis of Variance) was conducted, followed by Tukey’s multiple comparison tests, to determine statistically significant differences between groups. The data were analyzed using GraphPad Prism 10 software.

To determine the chromaticity parameters of discolored and gray hair before and after treatment with an organic dye, a colorimetric analysis was performed using a Konica Minolta CM-5 spectrophotometer. Measurements were taken using a white ceramic tile as a reference, and the instrument was calibrated with a standard D65 illuminant. Each colorimetric measurement was carried out in triplicate to ensure reproducibility. The hair dyeing procedure was repeated three times, with 15 washing cycles performed between each application using a 10% sodium lauryl sulfate solution. After each wash, the hair was towel-dried. Colorimetric analysis was carried out using the CIELAB color space (L*, a*, b*), where L* represents lightness (L = 0 corresponds to black, and L = 100 corresponds to white), a * represents the green (−) to red (+) axis, and b* represents the blue (−) to yellow (+) axis. The total color change (∆E) was calculated using the following equation [25]:

$$∆E={[{∆L}^2+{∆a}^2+{∆b}^2]}^{1/2}$$

(4)

where ΔL = L_standard − L_sample, Δa = a_standard − a_sample, and Δb = b_standard − b_sample. Discolored and gray hair samples without prior application of the organic dye were used as reference standards. These samples allowed for the quantification and comparison of the total color variation in hair treated with the formulated organic dye.

A wooden box was constructed for the UV resistance tests, equipped with a Schalter S-BAR185UV UV radiation lamp. The lamp operated at 110 V with a power output of 90 W, measuring 100 cm in length and 7 cm in width, and providing a light output range of 25 m to ensure uniform radiation across all samples. Hair samples weighing 100 mg each (4 cm in length) were arranged in a single layer and exposed to UV radiation for durations of 200, 400, and 720 h. The color of the samples was measured after each exposure period and compared to their initial color recorded at zero hours of UV exposure.

Scanning Electron Microscopy (SEM) was used to examine the surface of the hair. The dried samples were mounted on aluminum stubs and coated with gold for 40 s using an SPI-MODULE™ Sputter Coater. Images were captured using a JEOL JSM-6510 (Tokyo, Japan) scanning electron microscope at an accelerating voltage of 5 kV. The following parameters were assessed: overall appearance, presence of the cuticle, uniformity of layering, lifting or separation, and the presence of holes or cracks.

Optical images of the samples were acquired using a KEYENCE VHX-7000 digital microscope. This high-resolution system allows for detailed surface characterization under variable magnifications and lighting conditions, providing sharp and accurate visualization of sample morphology.

For the stability test, to investigate variations in weight, 0.5 g of dye sample was placed in one-gram metallic bags (4.5 cm × 5.0 cm) made of triple-layer polyethylene. The samples were weighed before and after the process. Ten milligrams of the organic dye were then placed in 20 mL of water, homogenized using a vortex, and subsequently measured. Particle size was determined using dynamic light scattering (DLS) with the Beckman Coulter Delsa Nano C Particle Analyzer, equipped with software version 3.73/2.30. The accelerated stability test involved storing the samples at either 5 °C or 40 °C. Samples were placed in a refrigerator or laboratory oven, both equipped with thermometer controls. After 24 h, an initial check was performed, followed by daily checks for 10 days [26]. Further readings were taken on the fifteenth and thirtieth days. For the long-term shelf stability test, samples were maintained at 25 °C, with monitoring on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 30, 60, and 90 days after the organic dye preparation [27]. Chromaticity parameters were measured on each check-up day. Hair samples (4 cm long, weighing 50 mg) were dyed, and the presence of color in the hair was assessed throughout the stability test period.

3. Results and Discussion

3.1. Fourier Transform Infrared (FTIR) Spectrometry Analysis

Figure 1 presents the FTIR (Fourier Transform Infrared) spectra of the Lawsonia inermis L. (henna), Indigofera suffruticosa (indigo), and the organic dye formed by the interaction of Lawsonia inermis L., Indigofera suffruticosa. The most relevant chemical components of Lawsonia inermis L. include compounds such as lawsone (2-hydroxy-1,4-naphthoquinone), apigenin, luteolin, gallic acid, ellagic acid, limonene, geraniol, tannins, carotenoids, glucose, and mucilages. The main chemical components of Indigofera suffruticosa include indican (indoxyl glucoside), a water-soluble compound that serves as the precursor to indigo. Upon hydrolysis and oxidation, indican is converted into the characteristic blue pigment. Indigotin (natural indigo) is the primary compound responsible for the plant’s intense blue color, formed through the oxidation of indoxyl. Other components include kaempferol, quercetin, isorhamnetin, saponins, tannins, gallic acid, caffeic acid, and mucilages. The spectra display characteristic vibrational bands corresponding to various functional groups in three distinct regions: 3600–2700 cm⁻¹, 1800–1200 cm⁻¹, and 1200–500 cm⁻¹. A broad absorption band is observed around 3288 cm⁻¹ in all three samples, attributed to the O–H stretching vibration, indicating the presence of hydroxyl groups. In the region between 3300 and 3500 cm⁻¹, the stretching of primary amines is observed, due to the presence of the N-H bond. Peaks in the range of 2970–2800 cm⁻¹ correspond to C–H stretching vibrations (asymmetric and symmetric), representing aliphatic or aromatic hydrocarbon structures. Characteristic bands between 1650 and 1600 cm⁻¹ are indicative of C=O stretching vibrations. The 1542 cm⁻¹ and 1555 cm⁻¹ bands are associated with the deformation or bending vibration of the N-H bond in secondary (or primary) amines. It can also be related to the stretching vibrations of the C-N bond in amide II. Peaks around 1710 cm⁻¹ and 1450–1400 cm⁻¹ correspond to C=O, C–N, and O–H deformations, showing the presence of carbonyl and hydroxyl groups. Below 1200 cm⁻¹, the spectra show a variety of C–H bending vibrations and out-of-plane bending modes typical of aromatic structures. The bands in the range of 1100–1000 cm⁻¹ suggest C–C vibrations linked to polysaccharides or related compounds. Peaks around 1072 cm⁻¹, 1020 cm⁻¹, and 895 cm⁻¹ indicate the presence of aromatic or olefinic compounds. In the wavelength range of 886–1030 nm, characteristic vibrations of gallic acid are observed, similar to those reported by Delgado et al. [28]. Gallic acid, a tannin found in the leaves of Lawsonia inermis L. and Indigofera suffruticosa, likely acts as a mordant, aiding in the fixation of the extracted dye. It is important to note that, although some minor peaks are small and not explicitly labeled in Figure 1, they correspond to meaningful chemical interactions. Peak assignments presented in this study are based on well-established literature references and are consistent with common FTIR characterization practices.

Henna’s coloring ability comes from a chemical component in its structure, 2-hydroxy-1,4-naphthoquinone, known as lawsone. Lawsone is not a free molecule in henna leaves; rather, it is derived from its precursors, the hennosides, during the preparation of henna. These hennosides consist of three isomers that arise from the tautomeric forms of naphthoquinone (1,4-naphthoquinone, 1,2-naphthoquinone, and 1,2,4-naphthotrione) [29], formed through the keto-enol tautomerism of the naphthoquinone structure. In this process, the second ring undergoes triple oxidation, allowing for the formation of three potential hydroxyl groups, which can result in a diketone form. Each of these hydroxyl groups may undergo glycosylation, leading to the three isomers. The aglycone, released through hydrolysis of these compounds, is further oxidized into lawsone, an active but unstable compound that tends to degrade over time in aqueous solutions. This reaction is typically facilitated by hot and acidified infusions that promote the hydrolysis of the precursors. Glycosylation plays a key role in maintaining the stability of active components, as glycosides are more soluble, transported more efficiently within the cells, or stored in vacuoles, unlike the aglycone, which, despite its activity, is unstable [30]. When brown hair is dyed with henna (lawso), it produces a copper to red hue; however, on gray hair, it results in an orange color, which may not be visually appealing; see Figure 2. The leaves of Indigofera suffruticosa contain a β-glucoside (indican) that acts as a precursor to indigo. Indigo is obtained by hydrolyzing the leaves, forming two types of indoxyls depending on the species: 2-hydroxyindole and 3-hydroxyindole. These compounds exhibit both enolic and ketonic properties, with position two being the most favorable for aldol-type condensation reactions. Indoxyl’s significance lies in its ability to oxidize easily in alkaline conditions, resulting in a stable, insoluble dark blue dye known as indigo [31]. When brown hair is dyed with indigo, it produces an ash to violet hue, while gray hair dyed with indigo results in a blue color; see Figure 2. In the FTIR spectrum (Figure 1), the C-N and N-H groups in henna and indigo shift from 1555 cm⁻¹ to 1563 cm⁻¹, 1542 cm⁻¹ to 1549 cm⁻¹, 1520 cm⁻¹ to 1530 cm⁻¹, and 1337 cm⁻¹ to 1346 cm⁻¹ in the organic dye. The OH group in henna and indigo shifts from 1337 cm⁻¹ (and 1340 cm⁻¹) to 1346 cm⁻¹ in the organic dye. Based on these FTIR shifts, it can be inferred that hydroxyl groups in henna may form hydrogen bonds with NH groups in indigo; see Figure 2.

Figure 3 shows the FTIR spectra of gray hair, organic dye, and dyed gray hair. A band at 3272 cm⁻¹ in an FTIR spectrum of hair is assigned to the N-H stretching vibrations of amine or amide groups. This band is typical of hydrogen bonds in proteins, especially keratin, which is the main protein in hair. This band can also be influenced by the presence of water (O-H) in the hair, as water can form hydrogen bonds with amine groups. A band at 3068 cm⁻¹ is associated with the C-H stretching vibrations in aromatic compounds. This signal is usually related to the presence of benzene rings, which may be found in certain proteins or compounds present in hair, such as aromatic side-chain amino acids (e.g., tyrosine or phenylalanine). Peaks at 2919 and 2850 cm⁻¹ correspond to C-H symmetric stretching of CH₃ and CH₂ groups (from lipids), respectively. A band at 1576 cm⁻¹ can be assigned to the C=C stretching vibrations in aromatic rings, such as those present in aromatic amino acids of hair proteins, specifically tyrosine and phenylalanine. It may also be related to N-H bending vibrations, which are part of the protein structure of keratin, the main protein in hair. Bands in this range (1500–1600 cm⁻¹) are characteristic of protein structure and are associated with the secondary structure of proteins, such as alpha-helices and beta-sheets. The bands located at 1555 cm⁻¹, 1539 cm⁻¹, and 1521 cm⁻¹ are assigned to the stretching vibrations of the combined N-H bending and C-N stretching vibrations in proteins, specifically keratin. The exact position of the band may be influenced by the molecular environment and the specific conformation of the proteins (e.g., alpha-helices or beta-sheets). These variations can be affected by factors such as hydration level, chemical treatments, or interactions with other compounds in the hair. The bands at 1472 cm⁻¹, 1456 cm⁻¹, and 1456 cm⁻¹ are typically assigned to the C-H bending vibrations in methyl (-CH₃) and methylene (-CH₂) groups. These groups are present in the side chains of amino acids that form hair proteins, such as keratin. They may also be associated with the bending vibrations of aliphatic groups in proteins or other lipids present in hair. The band at 1472 cm⁻¹ is related to the C-H bending vibrations in aliphatic compounds, which are part of the amino acid side chains in hair proteins and the lipids found in hair structure. The band at 1339 cm⁻¹ is associated with C-H bending vibrations in methyl (-CH₃) and methylene (-CH₂) groups. This signal may also be related to C-N stretching vibrations in hair proteins such as keratin and other compounds present in the hair fiber. The band at 1234 cm⁻¹ can be assigned to C-N stretching vibrations in proteins, particularly keratin. This band is also related to N-H bending vibrations and contributes to the identification of the protein structure in the hair fiber. The band at 1171 cm⁻¹ is attributed to the stretching vibration of the C-O bond in esters or carbohydrates. It may also be associated with stretching vibrations of the C-N bond, indicating the presence of protein structures in the hair. The band at 1147 cm⁻¹ of Indigofera suffruticosa typically corresponds to the stretching vibration of the C-O bond in esters or carbohydrates. This band may also be associated with the vibration of the C-N bond in polyphenolic structures, such as flavonoids. A band at 1074 cm⁻¹ is assigned to C-O stretching vibrations in ester and ether groups, and vibrations related to methylene (-CH₂) and methyl (-CH₃) groups. This band is characteristic of compounds found in the lipids that may be present in the hair cuticle or inner layers, as well as in protein structures. The band assigned to the S=O (sulfoxide) group typically appears in the range of 1030 to 1160 cm⁻¹. Around 1030–1080 cm⁻¹, the band may correspond to asymmetric S=O stretching, while around 1130–1160 cm⁻¹, it may relate to symmetric S=O stretching. These peaks may indicate the presence of sulfur-containing amino acids, such as cysteine [32]. A band at 1019 cm⁻¹ corresponds to the stretching vibration of the C-O bond in alcohol or ester functional groups. It may also be associated with C-O-C bond vibrations in ethers. This type of band is related to the protein and lipid components present in the hair structure. A band at 930 cm⁻¹ is generally associated with C-H stretching vibrations in unsaturated bonds or vibrations of phosphate groups. A band at 547 cm⁻¹ is associated with C-H bending vibrations in more complex structures, such as hydrocarbon groups present in keratin and other compounds that may be found in hair. Although some minor peaks are low in intensity and not labeled, they still represent relevant chemical interactions. Their assignments are supported by the established literature and align with standard FTIR practices.

Figure 4 shows the FTIR spectra of discolored hair, organic dye, and dyed discolored hair. In discolored hair compared to hair, changes in the position of spectral bands can occur due to chemical alterations induced by the bleaching process, such as the breakdown of disulfide bonds and the formation of cysteic acid. A band at 1633 cm⁻¹ is attributed to the C=O stretching vibrations. The bands at 1574, 1556, 1533, and 1530 cm⁻¹ are associated with the N-H bending and C-N stretching vibrations in proteins like keratin. A band at 1515 cm⁻¹ is linked to the C=C stretching vibrations of aromatic compounds, such as tyrosine and phenylalanine, found within the keratin structure. The bands at 1471 and 1454 cm⁻¹ correspond to C-H bending vibrations in methyl (-CH₃) and methylene (-CH₂) groups, relating to the vibrations of aliphatic chains present in the hair’s protein structure, primarily keratin. A band at 1341 cm⁻¹ is assigned to the C-H bending vibrations of methyl and methylene groups and can also be associated with C-N stretching vibrations in protein structures, particularly keratin. Similarly, a band at 1311 cm⁻¹ is associated with the C-H bending vibrations of methyl (-CH₃) groups, indicative of the aliphatic regions in the protein structure. A band at 1217 cm⁻¹ is attributed to C-N stretching vibrations and may also be related to the bending vibrations of N-H groups. The band at 1174 cm⁻¹ corresponds to C-O stretching vibrations in ether and ester functional groups and may additionally be associated with C-N stretching vibrations. The bands at 1124, 1079, and 1040 cm⁻¹ are all assigned to C-O stretching vibrations in ether and ester functional groups, with potential associations to C-N bond vibrations in protein structures. A band at 934 cm⁻¹ is attributed to P=O stretching vibrations in phosphate groups and may also relate to C-H bending vibrations in aliphatic compounds. The band at 577 cm⁻¹ is associated with C-S stretching vibrations, highlighting the presence of sulfur-containing compounds relevant to the keratin structure. Although some minor low-intensity peaks are unlabeled, they still indicate relevant chemical interactions. Peak assignments follow recognized literature and standard FTIR practices.

The cuticle, the outermost layer of the hair, is composed of flattened cells that shield the inner cortex and medulla from external influences and predominantly adopts a β-sheet conformation [33]. Approximately 80% of hair weight is due to proteins (polymers of amino acids), with keratin being the primary component, formed by 18 amino acids. The major amino acids that comprise keratin include cystine (17.5%), serine (11.7%), glutamic acid (11.1%), threonine (6.9%), glycine (6.5%), and arginine (5.6%). The cuticle consists mainly of keratin (65–95%), lipids (1–9%), primarily 18-methyleicosanoic acid or 18-MEA [34], and pigments (0.3–0.9%). This layer has the highest concentration of cystine and thus contains numerous disulfide bonds [33]; free cysteines form a thioester bond with the protective 18-MEA-rich layer [35]. Based on the infrared (FTIR) spectrum of gray and discolored hair, organic dye (composed of Lawsonia inermis and Indigofera suffruticosa), and dyed hair, significant changes in the positions of certain bands are observed. These shifts suggest that the functional groups of the dye are binding to the functional groups of the hair. The following potential interactions between the dye and hair’s functional groups were identified: (1) In the 1634–1611 cm⁻¹ region, corresponding to C=O stretching, a shift in the band is observed in dyed hair. This could indicate that the carbonyl groups from the dye are forming bonds with the hair proteins, or carbonyl groups from the hair are forming bonds with the dye. (2) Bands in the 1546–1234 cm⁻¹ region, corresponding to the C-N and N-H vibrations, exhibit significant differences. This suggests that the dye may be forming bonds with the amine groups of the hair, or the hair may be forming bonds with the amine groups of the dye [36,37]. Figure 5 illustrates the structural differences between natural hair and dyed hair at the molecular level, focusing on the interactions between dye molecules and hair keratin. The molecular structure of natural hair (left side) is primarily composed of keratin, which consists of amino acids with amine (-NH) and carbonyl (C=O) groups. Cysteine residues within keratin form disulfide bonds (S-S bonds), which contribute to the strength and structure of the hair. The zoomed-in SEM (scanning electron microscope) image at the bottom (left side) of Figure 5 shows the natural cuticle structure of hair. The surface is relatively smooth, with visible scales that form the outer protective layer of hair. The structure of dyed hair (right side) is shown with added dye molecules interacting with the keratin structure. The dye molecules Lawsonia inermis L. and Indigofera suffruticosa are represented by structures in green and blue, symbolizing the chemical structure of the dye components. These dye molecules can interact with keratin through hydrogen bonds (represented by red dotted lines), which connect the dye to amino and carbonyl groups in the keratin. This bonding is critical for the attachment of the dye to the hair. Figure 5 shows that dyeing hair introduces new hydrogen bonds between the dye molecules and keratin, altering the hair’s structure at the molecular level. This interaction explains the durability of hair color after dyeing, as the dye molecules become integrated into the keratin structure. The SEM image highlights the external impact on the hair cuticle, which could change with dyeing and treatments. The application of the organic dye on gray or discolored hair seals the cuticle layer. This effect is attributable, among other factors, to the dye’s pH of 5.05, which aligns well with the pH range of hair, typically between 5 and 5.5. Consequently, a compatible dye layer is achieved, enhancing hair health. Synthetic commercial dyes typically possess a pH of 9, causing damage to and opening of the cuticle, thereby allowing external agents to penetrate the hair fiber [38]. In contrast, the dye formed from Lawsonia inermis L. and Indigofera suffruticosa presented here maintains a pH of 5.05, aiding in the closure of damaged cuticles.

3.2. Brunauer–Emmett–Teller (BET) Surface Area Analysis

Table 1. Brunauer–Emmett–Teller (BET) surface area.

Sample	Surface Area (m2/g)
Gray hair	0.4550
Dyed gray hair	0.5981
Discolored hair	0.4182
Dyed discolored hair	0.2276

presents the results of Brunauer–Emmett–Teller (BET) surface area analysis. Gray hair exhibits a surface area of 0.455 m²/g, while dyed gray hair demonstrates a significantly larger surface area of 0.598 m²/g. This increase suggests that the dyeing process enhances the porosity of gray hair, facilitating greater dye absorption and potentially improving color adherence. The increased surface area may also indicate a rougher or more irregular hair structure, which could aid in color retention. In contrast, discolored hair shows a surface area of 0.418 m²/g. This indicates that the discoloration process alters the hair’s structure, but does not increase its porosity. The reduced surface area may result from the loss of essential structural components during discoloration, such as melanin, keratin, lipids, water, and disulfide bonds. Keratin, the primary structural protein in hair, can be weakened, rendering it more prone to breakage and damage. The removal of natural lipids, which help maintain hydration and softness, can lead to dryness and roughness. Furthermore, the water content of the hair may decrease, reducing flexibility. Disulfide bond disruption further compromises hair strength and integrity [39]. These cumulative changes make discolored hair more fragile and vulnerable to further damage without appropriate post-discoloration care. Interestingly, dyed discolored hair exhibits an even lower surface area of 0.228 m²/g. This significant reduction suggests that the discoloration process adversely affects the hair structure, resulting in decreased porosity and surface area. Such changes could impair the hair’s ability to retain dye, potentially reducing color durability and intensity. The dyeing process appears to have a positive effect on gray hair, increasing its surface area and improving dye adherence. However, for discolored hair, dyeing results in a decreased surface area, potentially compromising color retention. Discoloration involves the breakdown of chemical bonds and the removal of natural pigments, damaging the cuticle and cortex and resulting in a less porous, more fragile structure. Severe cuticle damage may create a smoother, less exposed surface, reducing the overall surface area. The interaction of dyed discolored hair with dye is likely influenced by these structural and chemical changes. The modification of keratin during discoloration and dyeing could alter the surface properties of the hair. Additionally, the cumulative effects of discoloration and dyeing chemicals might lead to reduced porosity. For instance, the saturation of discolored hair with dye could result in a layering effect that diminishes porosity and surface area. Although the reduced surface area of dyed discolored hair might seem counterintuitive, it is reasonable to attribute this outcome to the complex structural and chemical changes induced by both discoloration and dyeing. These processes significantly alter the physical properties of the hair, impacting its ability to retain dye effectively.

3.3. Cell Viability Evaluation

MTT assays were performed to evaluate the impact of the organic dye on the metabolic activity and viability of human keratinocytes (HaCaT). Figure 6 presents the cell viability (%) of samples treated with various concentrations (1, 10, 100, and 1000 μg/mL) of Lawsonia inermis L., Indigofera suffruticosa, and the organic dye. Cytotoxicity levels are categorized as non-cytotoxic, slightly cytotoxic, or cytotoxic, with thresholds indicated by dotted lines. According to Rodríguez et al. [40], materials are classified as cytotoxic if cell viability falls between 0% and 50%, slightly cytotoxic if viability ranges from 50% to 70%, and non-cytotoxic if viability exceeds 70%. The positive control (+) exhibited nearly 100% cell viability, indicating optimal cell health, while the negative control (−) showed almost 0% viability, reflecting high cytotoxicity. For Lawsonia inermis L., cell viability exceeded 100% at all tested concentrations (1–1000 μg/mL), demonstrating no cytotoxicity and even suggesting a potential for promoting cell proliferation. For Indigofera suffruticosa, cell viability was above 100% at lower concentrations (1 and 10 μg/mL), indicating no cytotoxic effects. However, at 100 μg/mL, viability dropped close to 50%, approaching the threshold of cytotoxicity. At 1000 μg/mL, viability increased significantly, suggesting slight cytotoxicity. Notably, the standard deviation at 1000 μg/mL was higher than at 100 μg/mL, indicating variability in the response and reduced apparent toxicity at lower concentrations. The IC50 for Indigofera suffruticosa was calculated as 513.6 μg/mL, representing the concentration at which 50% cell viability was observed. For the organic dye, cell viability exceeded 100% at lower concentrations (1 and 10 μg/mL), indicating no cytotoxicity. At higher concentrations (100 and 1000 μg/mL), viability decreased but remained above 80%, confirming the absence of cytotoxicity across all tested concentrations. These findings suggest that the organic dye is potentially safer for applications requiring minimal cellular damage. In comparison, the literature reports that the commercial dye Arianor Ebony, at concentrations ranging from 100 to 800 μg/mL, induced cytotoxic effects in HepG2 cells [41]. The primary toxic component in industrial hair dyes is para-phenylenediamine. Additional ingredients in commercial dyes, such as resorcinol, propylene glycol, liquid paraffin, cetostearyl alcohol, sodium lauryl sulfate, herbal extracts, preservatives, and fragrances, may exhibit additive toxic effects, contributing to significant morbidity and mortality [42,43]. The cytotoxicity results of the organic dye proposed in this study, when compared with industrial hair dyes, highlight its negligible health risks. This underscores its potential as a safer alternative for applications requiring reduced cellular damage.

As shown in Figure 7, the control HaCaT cells exhibit healthy morphology and strong adherence, consistent with normal epithelial growth. In the image corresponding to HaCaT cells treated with the organic dye (1000 μg/mL), the cell morphology appears slightly altered; however, the cells are still visible and attached. This observation suggests that the dye may partially obscure the optical contrast due to the presence of aggregates or precipitates, but does not necessarily indicate cytotoxicity or cell detachment. The metabolic activity observed in the MTT assay supports this hypothesis. In the image of HaCaT cells treated with the dye and incubated with MTT, numerous dark formazan crystals are visible, indicating active mitochondrial function in many cells. This suggests that, although some cells may have been affected, a substantial proportion remained viable and metabolically active.

3.4. Colorimetric Analysis

The colorimetric parameters of Lawsonia inermis L. in an aqueous medium are L = 18.1, a = 2.6, and b = 4.1. For Indigofera suffruticosa in an aqueous medium, the parameters are L = 17.8, a = −6.6, and b = 4.7. For the organic dye, the values are L = 16, a = −0.6, and b = 2.6.

Table 2. The colorimetric parameters for the three dye applications on discolored and gray hair after various wash cycles.

Dye	Wash	L	a	b	∆E	Photographs	Optical Micrographs (Magnification: 100×)
Discolored hair		46.01	12.75	29.43	---
First application	1	20.6	0.99	1.61	39.47
	5	21	0.95	1.55	39.26
	10	21.20	1.20	1.84	38.86
	15	21.54	1.31	2.07	38.45
Second application	1	18.45	0.11	0.83	41.68
	5	18.68	−0.25	0.97	41.54
	10	19.73	−0.51	1.20	40.78
	15	20.43	−0.82	1.03	40.56
Third application	1	17.72	−0.76	0.50	42.66
	5	18.16	−0.68	0.46	42.37
	10	18.54	−0.66	0.52	42.07
	15	18.77	−0.64	0.57	41.88
Gray hair		85.54	1.01	14.33	---
First application	1	22.72	1.35	1.99	64.06
	5	22.9	1.15	2.05	63.86
	10	23.15	0.9	2.34	63.56
	15	23.22	0.06	2.57	63.42
Second application	1	20.15	1.12	0.13	66.95
	5	20.85	0.99	0.38	66.20
	10	20.98	0.42	0.47	66.04
	15	21.23	0.35	0.54	65.79
Third application	1	17.51	1.78	−1.16	69.83
	5	17.79	1.66	−0.69	69.44
	10	18.15	1.25	−0.53	69.04
	15	18.97	0.94	−0.38	68.20

summarizes these parameters for gray hair (used as one standard) and discolored hair (used as another standard). These values served as references for the calculation of ∆L, ∆a, and ∆b parameters. The hair samples were dyed three times. After each dyeing process, the hair was washed 15 times before proceeding with the next application. The first wash removed excess organic dye. Table 2 also includes photographs of discolored and gray hair samples alongside their corresponding optical micrographs. The optical micrographs reveal the shine and color of the hair samples post-dyeing, showing a noticeable increase in color intensity with successive dye applications.

Figure 8 underscores the influence of dye layering on color retention across different hair types, offering valuable insights into the durability of hair dye formulations under repeated washing conditions. Figure 8a presents the total color difference (ΔE) in gray hair following dye application over multiple washing cycles (0 to 15 cycles). The first dye application consistently demonstrates the lowest ΔE values, beginning at 64.06 and gradually decreasing to 63.43 after 15 cycles. The second dye application starts at a higher ΔE of 66.95, dropping to 65.79 by the 15th cycle. The third dye application exhibits the highest initial ΔE (69.83), with a gradual decline to 68.20 after 15 washing cycles. Figure 8b illustrates the total color difference (ΔE) in discolored hair subjected to dye application across multiple washing cycles (0 to 15 cycles). The first dye application results in the lowest ΔE values, ranging from 39.47 initially to 38.45 by the end of the washing cycles. The second dye application follows a similar pattern, starting at 41.68 and decreasing to 40.56. The third dye application yields the highest ΔE values, beginning at 42.66 and gradually reducing to 41.88 after 15 cycles. For both gray and discolored hair, repeated dye applications lead to higher initial ΔE values, indicating enhanced color differences. Although color retention decreases with increased washing cycles, the rate of decline is slower for samples that have undergone multiple dye applications. Gray hair consistently exhibits higher ΔE values compared to discolored hair, suggesting greater color uptake or retention. It is important to consider that the higher ΔE values observed in gray hair may be partly due to its lighter base color, which makes color changes more noticeable, while the altered porosity and chemical structure of discolored (chemically treated) hair can affect its color uptake and retention differently.

3.5. Resistance to Ultraviolet Radiation

Table 3. Color degradation of hair and dyed hair subjected to UV radiation.

Sample	UV Radiation Time (h)	L	a	b	∆E	Photographs	Optical Micrographs (Magnification: 100×)
Discolored hair	0	46.01	12.75	29.43	---
	200	46.52	14.82	22.46	7.29
	400	58.32	7.51	24.62	14.22
	720	64.84	6.77	26.59	19.96
Dyed discolored hair	0	18.77	−0.64	0.57	---
	200	19.04	0.33	0.21	1.07
	400	19.74	−0.51	−0.37	1.36
	720	20.15	−0.24	0.46	1.44
Gray hair	0	85.54	−1.01	14.33	---
	200	86.94	−2.31	4.58	9.93
	400	88.73	2.39	3.19	12.08
	720	84.80	5.30	−1.80	17.34
Dyed gray hair	0	18.97	0.94	−0.38	---
	200	19.87	0.36	0.29	1.07
	400	19.58	−0.22	0.49	1.57
	720	20.80	−1.00	0.9	2.96

presents experimental data regarding the total color change (ΔE) of different hair samples, both discolored and gray, exposed to UV radiation over time (0, 200, 400, and 720 h). After 720 h of UV exposure, discolored hair shows a substantial change in ΔE, reaching 19.96. Gray hair also exhibits a significant color change, with ΔE increasing to 17.34. In contrast, dyed discolored hair shows a much smaller overall color difference, with ΔE values increasing only slightly to 1.44, and dyed gray hair also shows smaller ΔE values, with a maximum ΔE of 2.96. This suggests that dyed hair, whether discolored or gray, resists color change from UV exposure more effectively than untreated hair. The quality of color, as determined by the total difference between two colors, follows these guidelines: a ΔE between 0 and 1 indicates excellent quality, 1–2 denotes good quality, 2–4 is considered normal, and a difference exceeding 5 suggests poor quality [44]. Based on the total color difference (ΔE), discolored hair shows a ΔE of 7.29 after 200 h of UV exposure, indicating poor color quality. After 400 h, ΔE rises to 14.22, further reinforcing the poor quality. At 720 h, ΔE reaches 19.96, reflecting significant color degradation and very poor quality. In contrast, dyed discolored hair has a ΔE of 1.07 after 200 h, which falls within the “good quality” range. After 400 h, ΔE increases slightly to 1.36, still indicating good quality, and after 720 h, ΔE reaches 1.44, remaining in the good quality range. For gray hair, after 200 h of UV exposure, ΔE is 9.93, indicating poor color quality. This increases to 12.08 after 400 h, showing significant color change and maintaining poor quality. By 720 h, ΔE reaches 17.34, indicating very poor color quality. However, dyed gray hair shows a ΔE of 1.07 after 200 h, indicating good color quality. After 400 h, ΔE rises to 1.57, still reflecting good quality, and at 720 h, ΔE reaches 2.96, falling within the “normal” range but showing a noticeable color difference. Dyed hair, whether discolored or gray, maintains better color quality under UV exposure compared to untreated discolored or gray hair. Untreated hair shows substantial color degradation, with ΔE values exceeding 5, indicating poor quality, while dyed hair mostly remains in the “good” or “normal” range. To provide a visual representation of the colors, Table 3 includes photographs of hair samples and optical images taken at 100X magnification.

Figure 9 shows the comparison of total color difference (ΔE) in hair samples following UV radiation exposure. To establish this difference, standard references included untreated gray or discolored hair samples and samples treated with three applications of organic dye without UV exposure. These were compared to samples subjected to UV radiation for 200, 400, and 720 h, both in untreated hair and hair treated with organic dye. The graph in Figure 9a shows a consistent increase in color difference for gray hair with prolonged UV exposure, indicating significant visual changes, reaching a total color difference of approximately 17.34 ΔE after 720 h. In contrast, dyed gray hair exhibits a slower and more gradual increase, with a maximum color difference of around 2.96 ΔE after 720 h. This suggests that dyed hair is more resistant to color changes compared to untreated gray hair. Discolored hair, as shown in Figure 9b, exhibits a significant increase in color difference with longer UV exposure, indicating substantial changes in appearance. After 720 h of UV radiation, the total color difference reaches 19.96 ΔE. Dyed discolored hair shows minimal color change over time, with ΔE remaining relatively constant and low at 1.44 ΔE, suggesting that the dye provides strong resistance to UV-induced color changes. The results indicate that color degradation intensifies with increased UV exposure. Although hair possesses a protective protein layer, it is insufficient to shield the hair follicle from UV radiation [44]. After prolonged exposure, this natural barrier degrades easily. The micrographs on the right clearly show that dyed hair exhibits less color degradation compared to untreated hair, whether it is gray or discolored. For discolored hair, visible surface degradation is observed, corresponding to the large increase in ΔE seen in the graph. Meanwhile, the surface of dyed discolored hair remains relatively intact, consistent with the minimal color change shown in the graph. Overall, discolored hair undergoes significant color change and structural damage when exposed to prolonged UV radiation. In contrast, dyed discolored hair demonstrates much greater resistance to both color change and structural degradation, as evidenced by the small ΔE increase and less noticeable surface damage in the micrographs.

3.6. Scanning Electron Microscopy (SEM) Analysis

The sequence of SEM (scanning electron microscope) images illustrates the effects of an organic dye derived from Lawsonia inermis L. (henna) and Indigofera suffruticosa (indigo) on discolored hair, as shown in Figure 10. Fifteen washing cycles were performed between each dye application. The four micrographs offer a comparative view of the structural changes in the hair cuticle with successive dye applications. Figure 10a presents a micrograph of discolored hair (without dye). The hair cuticle exhibits significant damage and degradation. The scales are rough, lifted, and uneven, likely due to the discoloration process. This degraded appearance suggests that the hair is fragile, lacking protection, and therefore more susceptible to environmental damage. Figure 10b displays discolored hair after the first application of the organic dye. There is noticeable improvement in the hair’s surface structure. The cuticle appears smoother, with fewer lifted scales. This indicates that the dye is beginning to coat and repair the hair, likely due to the conditioning properties of the natural ingredients in Lawsonia inermis and Indigofera suffruticosa. Figure 10c shows the discolored hair after the second application of the organic dye. Further improvement is observed, as the cuticle looks more compact, and the surface appears more uniform. The dye seems to have continued to repair the hair, filling in gaps and smoothing rough edges. This reinforces the protective barrier, enhancing the hair’s strength and resilience. Figure 10d shows the discolored hair after the third application of the organic dye. The cuticle now looks almost fully restored. The scales are tightly packed and lie flat, indicating that the organic dye has effectively repaired the surface damage. The smooth appearance of the cuticle suggests that the organic compounds from Lawsonia inermis and Indigofera suffruticosa have successfully nourished and sealed the hair, providing long-lasting protection against further damage. The organic dye demonstrates restorative properties for discolored hair. After each application, the hair cuticle shows visible signs of repair, becoming progressively smoother and more uniform. This suggests that the dye not only imparts color but also improves hair health by protecting and reinforcing the hair fiber, particularly in damaged or discolored hair.

Figure 11 shows the SEM images illustrating the effects of Lawsonia inermis L. and Indigofera suffruticosa dye on gray hair, along with the impact of UV radiation exposure. Fifteen washing cycles were performed between each dye application. Figure 11a shows gray hair, where the cuticle appears rough, with visibly lifted scales, suggesting natural wear or environmental damage [45]. Figure 11b displays gray hair after 720 h of UV radiation exposure. The cuticle shows increased roughness and surface degradation, with more lifted and irregular scales, indicating that prolonged UV exposure exacerbates damage, likely weakening the hair structure and affecting its appearance. The colorimetry results confirm that with prolonged exposure to UV, there is greater damage to the lipid layer and subsequent color loss in the hair samples [46,47]. Despite the hair’s protective protein layer, it is insufficient to shield the hair follicle from radiation [44]. After prolonged exposure to UV radiation, the natural barrier provided by proteins easily degrades. The UV radiation induces photo-oxidation of the proteins within keratin, leading to the rupture of disulfide bonds formed between the organic dye and keratin, consequently breaking thioester bonds [48]. These reactions result in deteriorated hair properties, noticeable to consumers as poor manageability, dryness, fragility, loss of shine, and, in severe cases, reduced strength. Figure 11c presents gray hair after three applications of the organic dye. The cuticle appears significantly smoother compared to the untreated sample, with more compact and flatter scales, suggesting the dye has repaired the hair surface by creating a protective layer and potentially sealing the cuticle. Figure 11d shows gray hair (dyed three times) after 720 h of UV radiation. Despite prolonged exposure, the cuticle remains relatively intact compared to the untreated hair in Figure 11b. While minor surface degradation is visible, the overall hair structure remains mostly smooth, suggesting the organic dye provided a protective barrier that helped resist UV-induced damage. This can be attributed to the presence of fraxetin and esculetin in Lawsonia inermis L., compounds known for their photoprotective properties, which help preserve the color of dyed hair even under UV radiation exposure.

3.7. Stability

Accelerated stability (40 °C and 5 °C) simulates extreme conditions to predict how the dye may degrade or change over time. At 40 °C, the stability of the dye is assessed under high temperatures, representing storage conditions in warm climates or during transportation. At 5 °C, the resistance of the dye to low temperatures is evaluated, reflecting storage in cold climates or refrigeration. Changes in color, phase separation, precipitation, or loss of efficacy indicate stability issues. Dyes that remain stable under these conditions are expected to have a longer shelf life across various climates. In contrast, long-term stability (25 °C) evaluates the performance of the dye under normal storage conditions. The behavior of the dye is monitored over extended periods to ensure it retains its color, texture, and properties. The positive results indicate that the dye maintains its characteristics without significant alterations throughout its shelf life. Products demonstrating good stability preserve their color and effectiveness, minimizing returns or customer complaints. Conversely, unstable products may degrade rapidly, leading to inconsistent results, color loss, or texture changes.

Table 4. Accelerated stability test (at 40 °C and 5 °C) and long-term stability test (at 25 °C) of the dye.

Temperature (°C)	Day	Final Weight (g)	Colorimetric Parameters
			ΔE (1)	ΔE (2)	ΔE (3)	Average ΔE
40	0	0.5
40	1	0.5	1.37	1.36	1.41	1.38 ± 0.02
40	2	0.48	1.52	1.36	1.36	1.42 ± 0.09
40	3	0.49	1.36	1.45	1.36	1.39 ± 0.05
40	4	0.49	1.48	1.45	1.38	1.44 ± 0.04
40	5	0.49	1.45	1.52	1.52	1.50 ± 0.03
40	6	0.49	1.42	1.42	1.38	1.41 ± 0.02
40	7	0.49	1.42	1.49	1.42	1.44 ± 0.04
40	8	0.49	1.42	1.49	2.01	1.64 ± 0.32
40	9	0.49	1.52	1.36	1.36	1.42 ± 0.09
40	10	0.49	1.36	1.45	1.36	1.39 ± 0.05
40	15	0.49	1.42	1.42	1.38	1.41 ± 0.02
40	30	0.49	1.42	1.49	1.42	1.44 ± 0.03
5	0	0.5
5	1	0.49	1.60	1.66	1.63	1.63 ± 0.03
5	2	0.49	1.54	1.65	1.65	1.61 ± 0.06
5	3	0.49	1.60	1.59	1.59	1.59 ± 0.01
5	4	0.49	1.53	1.59	1.57	1.56 ± 0.02
5	5	0.49	1.63	2.18	1.57	1.79 ± 0.33
5	6	0.49	2.26	1.56	1.50	1.78 ± 0.42
5	7	0.49	1.55	1.49	1.55	1.53 ± 0.03
5	8	0.49	1.45	1.55	1.55	1.52 ± 0.05
5	9	0.49	1.55	1.51	1.51	1.52 ± 0.02
5	10	0.49	1.55	1.44	1.55	1.51 ± 0.06
5	15	0.48	1.49	1.49	1.55	1.51 ± 0.03
5	30	0.49	1.42	1.49	1.42	1.44 ± 0.03
25	0	0.5
25	1	0.498	1.605	1.665	1.635	1.63 ± 0.03
25	2	0.499	1.547	1.654	1.654	1.61 ± 0.06
25	4	0.495	1.607	1.594	1.594	1.59 ± 0.01
25	6	0.499	1.538	1.594	1.573	1.56 ± 0.02
25	8	0.491	1.634	2.186	1.579	1.79 ± 0.33
25	10	0.494	2.266	1.56	1.508	1.77 ± 0.42
25	15	0.499	1.557	1.497	1.557	1.53 ± 0.03
25	30	0.498	1.458	1.557	1.557	1.52 ± 0.05
25	60	0.498	1.494	1.494	1.555	1.51 ± 0.03
25	90	0.498	1.555	1.443	1.555	1.51 ± 0.06

illustrates how temperature affects the stability and color retention of the dye, with higher ΔE values indicating greater color changes over time. The particle size of all samples remained constant at 61.58 μm throughout the study. The aroma of the samples did not change over the storage period, and the appearance consistently resembled a fine, homogeneous dry powder. These results suggest that the dye samples did not absorb moisture. The prepared organic dye, in powder form, was moistened prior to use, resulting in a pH of 5.05 in all cases. The pH was monitored with a potentiometer during the stability test by placing 0.25 g of the dye powder in 0.625 mL of water at 25 °C. Both accelerated and long-term stability testing showed minimal changes in the product. No water absorption was observed, as the sample weight even slightly decreased. If water molecules were to adsorb onto the surface, they could potentially cause dye degradation. Table 4 summarizes the results of an accelerated stability test conducted at 40 °C and 5 °C, and a long-term stability test at 25 °C for a hair dye. For each temperature (40 °C, 5 °C, and 25 °C), Table 4 presents the final weight of the dye over several days, along with colorimetric parameters represented as ΔE values (1, 2, and 3), followed by the average ΔE. At 40 °C, starting from day 0 with a weight of 0.5 g, there is a slight reduction in weight after the first few days. The ΔE values fluctuate slightly but remain relatively consistent, with a small increase in average ΔE from day 1 (1.385) to day 30 (1.446). At 5 °C, the dye also starts with 0.5 g. There is a more significant fluctuation in ΔE, especially between days 1 and 6, where the average ΔE increases from 1.446 to 1.799, indicating a change in color stability at low temperatures. For the 25 °C long-term stability test, the values show a consistent trend similar to those at 40 °C, though ΔE values exhibit a smoother transition over 90 days, from 1.635 to 1.517, with an average ΔE of 1.517 on day 90. Unlike other drying methods, solvent removal through freeze-drying does not alter the basic structure or composition of the treated material, allowing its properties to remain intact [49]. The stability tests demonstrate that the dye is reliable and safe for end users, preserving its quality across a range of storage and usage conditions.

4. Conclusions

In this study, an organic dye was obtained from Lawsonia inermis L and Indigofera suffruticosa, resulting in an organic powder dye. The interaction between henna and indigo is evident in the FTIR spectral shifts, which suggest hydrogen bonding between the hydroxyl groups of henna and the NH groups of indigo. These interactions may play a role in the stability and color outcomes of the dyeing process. Also, the dye binds to hair through hydrogen bonding with amino and carbonyl groups in keratin, as evidenced by FTIR spectral shifts, forming stable attachments that enhance the durability of the color. The pH of the organic dye (5.05) aligns with the natural pH of hair (5–5.5), facilitating the sealing of the cuticle layer and contributing to improved hair health.

The increased surface area of dyed gray hair (0.598 m²/g) suggests enhanced porosity and roughness, which likely contribute to improved dye adherence and color retention. In contrast, discolored hair exhibits a reduced surface area (0.418 m²/g), indicating structural damage from the discoloration process, including the loss of melanin, keratin, lipids, water, and disulfide bonds. Notably, dyed discolored hair demonstrates a further reduction in surface area (0.228 m²/g), which could reflect a smoother, less exposed structure resulting from cumulative chemical treatments.

Lawsonia inermis L. showed no cytotoxic effects on human keratinocytes and even promoted cell proliferation at all concentrations, while Indigofera suffruticosa displayed slight cytotoxicity only at higher concentrations, with an IC50 value of 513.6 μg/mL. The organic dye exhibits no cytotoxic effects across all tested concentrations (1–1000 μg/mL).

The colorimetric analysis of the organic dye demonstrates the ability of the organic dye to achieve balanced coloration. Repeated dyeing and washing cycles revealed that the organic dye maintains its color intensity and uniformity. The increased color intensity with successive dye applications underscores the dye’s cumulative effect and adherence to the hair fibers in both gray and discolored hair. The enhanced color retention, even after multiple washes, demonstrates its potential for long-term use while minimizing the environmental and health impacts typically associated with synthetic hair dyes.

The analysis of total color difference (ΔE) under prolonged UV exposure demonstrates that dyed hair, whether discolored or gray, exhibits significantly better resistance to color degradation compared to untreated hair. Untreated discolored and gray hair show substantial increases in ΔE values over time, reaching levels that indicate poor to very poor color quality, with ΔE exceeding 5 after 200 h and continuing to deteriorate with extended exposure. In contrast, dyed discolored hair consistently maintains ΔE values within the “good quality” range (1–2), even after 720 h of UV exposure, while dyed gray hair shows slightly higher ΔE values that remain within the “good” to “normal” range.

The SEM images highlight the restorative effects of the organic dye on discolored hair. The progressive improvement in the hair cuticle with each dye application demonstrates the dual functionality of the organic dye: not only does it impart color, but it also repairs and strengthens damaged hair. After the first application, the dye begins to smooth the cuticle, reducing visible damage and lifting scales. With subsequent applications, the cuticle becomes more compact and uniform, ultimately achieving a near-restored state by the third application. The tight, flat alignment of the cuticle scales after the final application suggests enhanced protection and resilience against external aggressors.

Untreated gray hair exhibits a rough cuticle with lifted scales, which becomes more pronounced after 720 h of UV exposure, indicating substantial structural degradation. The effects of UV radiation, including the photo-oxidation of keratin proteins and the rupture of disulfide and thioester bonds, lead to weakened hair properties, manifesting as dryness, fragility, and loss of shine. In contrast, gray hair treated with three applications of the organic dye shows a significantly smoother and more compact cuticle, suggesting the dye’s ability to repair damage and form a protective barrier. After prolonged UV exposure, the dyed hair retains much of its structural integrity, with only minor surface degradation observed. This resilience is likely due to the photoprotective properties of fraxetin and esculetin present in Lawsonia inermis L., which help preserve the cuticle and maintain color stability under UV radiation.

The stability tests of the prepared organic dye, including both accelerated and long-term tests, demonstrate that the dye maintains consistent physical properties and color stability over time. The particle size remained constant at 61.58 μm, and the appearance of the dye retained its fine, homogeneous powder form throughout the study. The absence of moisture absorption suggests the dye is resistant to environmental factors that could lead to degradation. The pH of the dye remained stable at 5.05, indicating that no significant chemical changes occurred during the stability tests. The accelerated stability test at 40 °C showed a slight reduction in weight and a minor increase in the ΔE values over 30 days, suggesting minimal color change. At 5 °C, more fluctuation in color stability was observed, with a more noticeable increase in ΔE, reflecting a change in color retention at lower temperatures. However, the long-term stability test at 25 °C exhibited a consistent trend, with ΔE values gradually transitioning over 90 days, indicating that the dye maintains a stable color profile under standard storage conditions.