Synthesis of Plant Phenol-derived Polymeric Dyes for Direct or Mordant-based Hair Dyeing

Podsumowanie

Here, we present a protocol to use pre-synthesized polymeric products derived from fungal laccase-catalyzed polymerization of plant phenols, either with or without mordant agents (e.g., FeSO₄), to induce detergent-resistant keratin hair dyeing within 2.5 hours.

Streszczenie

Effective hair dyeing through in situ incubation of keratin hair with the products of fungal laccase-catalyzed polymerization of plant phenols has been previously demonstrated. However, the dyeing process takes a long time to complete compared to commercial hair-dyeing products. To overcome this bottleneck, pre-synthesized polymeric products of the oxidative reaction of Trametes versicolor laccase on catechin and catechol, either with or without mordant agents (e.g., FeSO₄), were here employed to achieve permanent keratin hair dyeing in various colors and shades. The laccase action in acidic sodium acetate buffer led to a deep black coloration after coupling reactions between the plant phenols. The colored dye products were then desalted and concentrated with ultrafiltration. The dyes, with or without mordant agents, caused a significant increase in ΔE values (i.e., color difference value) in gray human hair within 2.5 hours. In addition, different keratin colors and shades were induced depending upon the mordanting and pH changes. The dyed hair also exhibited a strong resistance to detergent treatments, indicating that our methods can give rise to permanent hair dyeing. Overall, our work has provided novel insight into developing eco-friendly hair-dyeing methods as alternatives to commercial toxic diamine-based dyes.

Wprowadzenie

Laccases are oxidases that are active towards phenolic and polyphenolic compounds. They have been identified in various living organisms, including plants, fungi, insects, and bacteria. Their enzymatic actions contribute to several morphogenetic phenomena¹. The enzymes catalyze single-electron oxidation of the substrates, resulting in the formation of radicals that are further coupled to small organics and to solid surfaces. Such coupling processes lead to syntheses of oligomers and polymers and to surface functionalizations^{2, 3}. When laccase substrates are from natural sources, such as plant phenolics, the enzymatic reactions are of great interest with respect to green chemistry. Here, both reactants and catalysts are from natural sources. In addition, the resulting products are similar to the natural products, since the overall reactions mimic the in vivo syntheses of natural phenolic polymers-including plant lignin, poly(flavonoid), and humus-wherein small plant phenolic compounds are highly cross-linked by oxidase-induced radical coupling⁴.

Products derived from laccase-catalyzed coupling reactions of plant-derived phenols can be used to dye gray hair through in situ incubation and can be developed as alternatives to commercially available dyes¹. Such alternatives are important, since commercial hair-dyeing agents are based on p-phenylenediamine (PPD), PPD-related diamine compounds, and hydrogen peroxide, which have been shown to be toxic, carcinogenic, and allergenic to humans^{5, 6}. In the laccase-catalyzed coupling reactions, the laccases and plant phenols functionally replace hydrogen peroxide and p-phenylenediamine, respectively⁷. However, the dyeing speed of the laccase-based systems is much slower than that of the commercial one. In general, the PPD-based dyeing agents require less than one hour to achieve effective color change in keratin hair, while the laccase-based reactions require an overnight incubation⁷. The slow dyeing kinetics could be explained by two possible phenomena. First, the use of a low-pH buffer (e.g., pH 5) to maximize laccase activity has been observed to decrease the degree of swelling in the keratin matrices, thus inhibiting deep penetration of dyes into the matrices. Indeed, agents allowing the dyeing reactions to proceed in high-pH conditions have been shown to be integral to commercial hair-dyeing products⁸. Second, the number of possible chromophore molecules exhibiting strong adsorption to keratinous surfaces during the polymerization reaction has been shown to be proportional to the incubation time (i.e., the extent of polymerization). For example, the transformation of dopamine to polydopamine was shown to induce a strong adhesion to many surfaces that was concomitant with the formation of a black color⁹.

In the current work, pre-synthesized polymeric products obtained from T. versicolor laccase-catalyzed oxidation of catechin and catechol were used to treat keratin hair for dyeing. We hypothesized that the adsorption ability of the polymers would be much stronger than that of the monomeric plant phenols and that they would initially form low-molecular-weight oligomers. Results demonstrated that, when using the pre-synthesized polymers, the enzymatic oxidation power was no longer necessary. This indicates that the pH can be controlled and that metal ions can be used in hair-dyeing treatments, regardless of enzyme activity. This protocol provides a simple and fast method to dye keratin hair in various shades of color while using eco-friendly and renewable plant-derived phenolics (Figure 1).

Protokół

1. Preparation of Plant Phenol-derived Polymeric Dyes

1. Dissolve catechol (0.1 g) and (+)-catechin hydrate (0.1 g) in 32 ml of 100 mM sodium acetate buffer (pH 5.0) and 8 ml of absolute ethanol.
2. Add 10 mg of T. versicolor laccase to the catechol- and catechin-containing buffer. Mix vigorously and pour the solution into a square Petri dish. Incubate the dish at room temperature in a shaking incubator (25 rpm) for 24 hr. Dramatic color change of the solution from transparent to dark black can be observed with the naked eye after laccase-induced coupling reactions.
3. Centrifuge the solution for 10 min at 20,000 x g in order to spin down insoluble polymeric particles. Use the deep black supernatant for further desalting.
4. Desalt the reaction solution with a 5 kDa ultrafiltration disc. After concentrating the reaction volume to 20 ml through ultrafiltration, exchange the reaction buffer by adding 300 ml of distilled water. Finally, using filtration, concentrate the volume of the solution to 25 ml.

2. Dyeing Solutions for Gray Keratin Hair

1. Prepare the following six polymeric solutions: polymeric dyes, polymeric dyes/FeSO₄, polymeric dyes/FeSO₄ in pH 3 water, polymeric dyes/FeSO₄ in pH 11 water, polymeric dyes/FeSO₄ with acetic acid, and polymeric dyes/FeSO₄ with ammonia.
   1. For the polymeric dye, mix 5 ml of distilled water with 1 ml of the desalted polymeric dyes (1.4).
   2. For the mordant solutions, add 0.33 g of FeSO₄ to the mixture from step 2.1.1. Vigorously vortex to dissolve the FeSO₄ completely.
   3. For the pH 3 or 11 water solution, adjust the pH of 5 ml distilled water using 1 N HCl or 1 N NaOH. Then add 1 ml of the desalted polymeric dyes (1.4) and 0.33 g of FeSO₄.
   4. For acetic acid- or ammonia-treated solution, mix 1.0 ml of glacial acetic acid or 1.0 ml of ammonia water with 5 ml of distilled water. Then add 1 ml of the desalted polymeric dyes (1.4) and 0.33 g of FeSO₄.
2. For the plant monomers, mix catechol (0.1 g) and (+)-catechin hydrate (0.1 g) in 6 ml of distilled water, with or without 0.33 g of FeSO₄, to soak the hair in.
3. As soon as the dyeing solutions from step 2.1 and 2.2 are prepared, completely soak 5-cm long gray human hair tresses (0.2 g) in the solutions. Incubate the hair tresses at 32 °C in a shaking incubator (160 rpm) for 2.5 hr.
4. Afterwards, take out the hair tresses and rinse them with running water. Use an electronic hair drier to remove the moisture. Color change caused by the polymeric dyes can be visualized with the naked eye.
   1. To obtain the color parameters (i.e., L*, a*, and b*), employ a conventional colorimeter according to the manufacturer's protocol. Crumple the hair tresses into a ball, thus allowing them to be measured with the lens of a colorimeter. Repeat this process on a different region of the hair with the colorimeter lens.
   2. Measure the color parameters of each dyed tress seven times. Calculate averages and standard deviations of the parameters. Calculate ΔE using the formula: [(100 - L*)² + (a*)² + (b*)²]^1/2.

3. Color Durability Tests

1. Dissolve 200 mg of sodium dodecyl sulfate (SDS) in 40 ml of distilled water. Soak the dyed hair completely in the SDS-containing water for 5 min at room temperature. Take out the hair tresses and then rinse them with enough running water to remove the detergents. Use an electronic hair drier to remove the moisture.
2. To obtain the color parameters (i.e., L*, a*, and b*), employ a conventional colorimeter according to the manufacturer's protocol. Repeat the soaking described in step 3.1 once more with the same dyed hair and then measure the parameters again.
3. Dissolve 800 mg of SDS in 40 ml of distilled water. As described in step 3.1, repeat the soaking twice more with the same dyed hair. Overall, treat each dyed hair tress with the SDS solution a total of four times.

Wyniki

First, the dyeing ability of polymeric dyes was compared to that of plant-derived monomers (i.e., catechin and catechol). The polymeric dyes induced a significant change in the color of gray keratin hair (Figure 2A and Figure 3), while the innate gray color of the hair remained very stable with plant monomers (data not shown). The effects of mordanting agents on the dyeing abilities of polymeric products were then evaluated. As s...

Dyskusje

Interestingly, our method reduced the time it took to dye keratin hair with oxidant-induced polymerizations of natural phenolics. It also induced diverse colors in the hair through simple manipulations of the polymeric dyes, such as changing the pH and applying mordant.

In situ incubation of keratin hair with laccase-catalyzed oxidation of plant phenols requires overly long incubation times to attain effective dyeing⁷. Such slow dyeing kinetics may be due to the poor bindin...

Materiały

Name	Company	Catalog Number	Comments
Sodium dodecyl Sulfate	Promega	H5114
Laccase from Trametes versicolor	Sigma	38429-1G	Enzyme activity is denoted as 0.53 U/mg
(+)-catechin hydrate	Sigma	C1251-5G
1,2-dihydroxybenzene (catechol)	Sigma	135011-5G
Ammonia water	Duksan	701	Ammonia contents is denoted as 25 ~ 30%
Acetic acid, glacial	Duksan	448
Iron(II) sulfate heptahydrate	JUNSEI	83380-1250
Ultracell 5 kDa	Amicon	PLCC06210
Stirred ultrafiltration cells	Millipore	Model 8200
Human gray hair	PheonixKorea	Not available
Colorimeter	SPEC	JCS-10
Square dish	SPL	10125	125 * 125 * 20 (mm)