Microencapsulated Myrciaria cauliflora Berg. fruit peel extract as a natural pigment for neutralizing shampoo preparation

Hair is a complex natural fiber consisting mainly of keratin that can be divided into four main structural units, namely: cuticle, cortex, medulla, and membrane complex. The use of pigments in hair care preparations, mainly derivatives of aromatic amines, has been a topic of discussion due to toxicity and ecotoxicity concerns. In this background, Myrciaria cauliflora Berg is a native fruit occurring from northeastern to southern Brazil. Among the diversity of compounds detected in this fruit species are bioactive flavonoids and anthocyanins, which are responsible for the purple pigmentation of the fruit peels. This study reports on the use of a microencapsulated Myrciaria cauliflora Berg. peel extract as a temporary pigment to reduce the yellow tone of the hair. The extract was obtained by maceration with 70% ethanol (v/v) in an acidic milieu (6.0% citric acid). The extract was submitted to phytochemical profiling and a shampoo formulation was developed and applied onto a chemically treated hair. The preliminary characterization of the extract confirmed the presence of phenolic compounds and anthocyanins, in addition to the acid/base character of the extract. When tested on discolored hair samples with a yellow tone, the Myrciaria cauliflora Berg. shampoo reduced the yellow color, which was visually perceived by the enhancement of the hair brightness. These results demonstrate the potential of M. cauliflora extract – obtained from a fruit widely found in Brazil, as a temporary pigment to be used in cosmetic hair care.


Introduction
Hair is a complex natural fiber consisting mainly of keratin that can be divided into four main structural units, namely: cuticle, cortex, medulla, and membrane complex. The cuticle, the outermost portion of a hair strand, is composed of amorphous protein material responsible for protecting the cortex from external aggression. The cortex is the middle part of the hair fiber, which constitutes about 70% of the hair mass and is the largest constituent of the fiber. The cortex is formed by aligned keratin microfibers organized in the direction of the strand, where melanin granules can be found. Interestingly, the type, size and quantity of melanin granules are key factors determining the hair color. The medulla is a structure forming the fiber core, consisting of specialized cells rich in glycogen and citronella. This structure corresponds to a small percentage of the capillary mass. Lastly, the membrane complex is a secondary component of the capillary fiber, composed mainly of lipids and proteins, which is responsible for joining adjacent cortical and cuticular cells (Peters et al. 2011;Hanumanthayya et al., 2018).
Mechanical damages and chemical treatments, such as discoloration of the hair strands, can cause changes in the chemical composition of the cuticle. Discoloration alters the natural melanin content in the cortex of the hair fiber, causes degradation of essential amino acids, and a number of other damages, which include increased porosity of the hair fibers and loss of brightness; in addition, the hair can be naturally damaged by solar radiation. Both prolonged exposure to radiation and the process of hair discoloration cause capillary oxidation, alter the melanin content and cause degradation of keratin, ultimately resulting in a yellowing hair (Peters et al. 2011;Nogueira et al., 2004).
The use of pigments in hair care preparations, mainly derivatives of aromatic amines, has been a topic of discussion due to toxicity and ecotoxicity concerns (Oliveira et al., 2014;Guerra-Tapia et al., 2014). Furthermore, common synthetic pigments such as anilines and gentian violet may dry the hair strands, which reinforces the need for novel alternatives to these products. As naturally occurring products are readily available and widely used in cosmetology (Scotti et al., 2007), the exploration of their potential, in particular within the Brazilian biodiversity, has proven a promising possibility.
In this background, Myrciaria cauliflora Berg is a native fruit occurring from northeastern to southern Brazil (Sasso et al., 2010). Among the diversity of compounds detected in this fruit species are bioactive flavonoids and anthocyanins, which are responsible for the purple pigmentation of the fruit peels (Volp et al., 2008;Alves et al., 2013). In addition, the presence of free electrons in the nucleus and of free anthocyanins can be related to the antioxidant activity reported for these compounds (Baldin et al., 2016).
Both in discolored and natural hair, the fiber yellowing process takes place due to oxidative reactions. Hence, the antioxidant potential of anthocyanins and their effects in reducing the yellow hair tone warrant further investigation. As the stability and bioactivity of anthocyanins can be further increased with the use of microencapsulation technology (Baldin et al., 2016), we proposed in this pioneer study the development of a microencapsulated M. cauliflora peel extract formulation to neutralize the capillary yellow tone in discolored (with reflections, wicks and/or lights) hair.

Extract preparation
M. cauliflora fruits were collected in the countryside of Sinop,Mato Grosso,Brazil (11.80'.43'S and 55.57'43.2'W) between August and September 2016. Initially, the fruits were peeled manually to obtain the extract. Then, M. cauliflora peels were crushed in a knife mill and submitted to extraction with 70% ethanol (v/v) in acidic milieu (6.0% citric acid). This mixture was kept under refrigeration (4ºC) in the absence of light for 24 hours. The filtered extracts were concentrated to eliminate the solvent under reduced pressure at 40°C (rotary evaporator Buchi ® R-3), remaining 30% of the initial volume. A portion of the extract material was concentrated and used for the characterization analysis while the remaining portion was used for microencapsulation.

Microencapsulation of M. cauliflora extract
The microencapsulation of M. cauliflora extract was performed using the lyophilization technique, as described elsewhere (Laine et al., 2008;Kechinski et al., 2010;Nogueira et al., 2017). To prepare the microencapsulated extract formulation, the wall material chosen (10% maltodextrin) was dissolved first, then the extract was added. The mixture was stirred for 3 hours and then submitted to freezing at -5ºC. The frozen material was lyophilized on a JJ Científica ® equipment (Model LJJ02).

Total Phenol Content of M. cauliflora extract
The content of total phenolic compounds was established by the Folin-Ciocalteau method, modified by Shahidi & Naczk (1995). Briefly, samples were prepared with a concentration of 1 mg/mL in ultrapure water. The procedure was performed by adding 100 μL of the diluted extract into water, 500 μL of Folin-Ciocalteau (Merk ® ) reagent, and 6.0 mL of ultrapure water, in an amber glass. After 1 minute, 6.0 mL of a sodium carbonate solution (7.5 %) were added, stirred for a few seconds, and allowed to stand for 1 hour at room temperature. Lastly, the absorbance of the samples was read at 750 nm (Perkin Elmer ® , UV / VIS Lambda 20). Gallic acid (Vetec ® ) was used as a reference standard and the curve was plotted with six concentrations ranging from 20 to 600 μg/mL (R 2 = 0.9987). The results were expressed as gallic acid equivalent per gram of extract (GAE/g).

Total monomeric anthocyanins content
The analysis of total monomeric anthocyanins content was performed according to the pH differential method described by Terci & Rossi (2002). An aliquot of 3.0 mL of the lyophilized material at 1 mg/mL was diluted into 10 mL of an acidified ethanol solution (pH 1.0 and 4.5). Absorbance readings were performed at wavelengths ranging from 517 nm to 700 nm (Perkin Elmer®, UV / VIS Lambda 20). The same acidified ethanol solution was used as a blank solution. The concentration of anthocyanins was calculated as cyanidin-3-glycoside (M = 449.2 g.mol-¹) from the equations I (A = (A517-A700) pH 1.0 -(A517-A700) pH 4.5) and II (C = A.Mm / Ɛ.b, where Ɛ = 26,900 L / mol.cm and b = 1.0).

Characterization reactions
In aqueous solutions, anthocyanins can present different structures depending on the pH. Due to their acid/base character, the color of these compounds may change and therefore can be used as a pH indicator (Terci & Rossi, 2002). pH indicators are weakly acid (acid indicators) or weakly basic (basic indicators) organic substances that present different colors for their protonated and deprotonated forms. The color variation occurs when the hydrogen ion (acid) is added to, or removed from, the molecule. Anthocyanins may produce blue, violet, red and pink coloration of flowers and fruits (Kong et al., 2003). To verify the acid-base character of the M. cauliflora peel extract, the concentrated extract was added to citratephosphate buffer solutions at pH 2.0, 5.0, 7.0, and 9.0.

Ferric Chloride Reaction and Cyanidin Reaction or Shinoda Assay
To determine the presence of polyphenols in the M. cauliflora extract, two drops of 2.5% (p/v) ferric chloride were added to 1.0 mL of M. cauliflora peel extract. In the presence of ferric chloride, the extract may develop coloration varying between green, yellow-brown and blue-violet depending on the type of compound present in the sample (Li et al., 2018).
For the cyanidin reaction, 3.0 mL of M. cauliflora peel extract were used and oven-heated at 80°C until complete drying. Thereafter, 1.5 ml of methanol, magnesium (1.0 g) and 0.5 mL of concentrated HCl, were added.

Antioxidant activity by DPPH assay
The quantitative evaluation of the antioxidant activity of M. cauliflora extract against the stable DPPH radical was performed by adding 0.0237 mg of DPPH into 1.0 ml of the methanolic solution of M. cauliflora extract at different concentrations (25, 50 and 100 μg/mL). The decrease in absorbance was evaluated after 30 minutes of reaction at λ = 517 nm (Perkin Elmer®, UV / VIS Lambda 20) (Kordali et al., 2005;Sharma et al., 2009;Ratz-Lyko et al., 2012). Methanol was used as a blank solution and a 25% (p/v) DPPH solution was used as a control. Hydroxytoluene-butylated (BHT) was used as a reference antioxidant compound. A sample of the shampoo containing 10% (w/w) of the microencapsulated M. cauliflora peel extract (see topic 2.8 for further details) was also evaluated at the concentrations mentioned above. In such a case, the base shampoo was used as a control. The experiments were performed in triplicate and the antioxidant activity was calculated according to the equation: % of DPPH radical scavenging = [(control absorbance -sample absorbance) / (control absorbance)] × 100.

Shampoo development
A shampoo containing the microencapsulated M. cauliflora peel extract was prepared. For the development of the shampoo base (SH0) for normal hair, an aqueous solution containing 25% sodium lauryl ether sulphate was initially prepared. Then 5% triethanolamine lauryl sulphate, 3% fatty acid diethanolamine and 1% methyl paraben were added to the solution and homogenized slowly. The pH was adjusted to 5.5 using a 20% citric acid solution and the viscosity was obtained with a solution of 20% sodium chloride. The shampoo samples containing the microencapsulated M. cauliflora peel extract were prepared at the concentrations of 1.0, 5.0 and 10.0% (SH1.0, SH5.0, SH10.0). Additionally, a shampoo sample containing 0.1 % BHT, a synthetic antioxidant agent, was prepared.

Macroscopic analysis, Thermal stress, Freeze-thaw cycle and Centrifuge test
The macroscopic characteristics of the formulations were determined based on the following parameters: appearance, color, odor and homogeneity.
For the thermal stress testing, the samples were submitted to gradual heating in a water bath (Quimis ® -Model Q334M-28) until reaching a temperature of 80ºC, which was maintained for 30 minutes (22).
For the freeze-thaw cycle, the samples were submitted to freezing conditions at -5 ± 2ºC for 24 hours and then incubated at 40 ± 2ºC for 24 hours, thus completing one cycle. Twelve freeze-thaw cycles were performed, totaling twelve days of testing (Brazil, 2004).
For the centrifuge test, 5.0 g of each formulation (kept at room temperature after thermal stress and the freeze-thaw cycle) were added to a conical test tube and subjected to centrifugation (Fanem-Excelsa II -Model 206 BL) with cycles of 1000, 2500 and 3500 rpm for 15 minutes at room temperature (23).

Determination of pH and Viscosity
The pH of the M. cauliflora extract and shampoo samples was determined by dilution in distilled water (1:20 w/v) at 25 °C. The pHmeter (Tecnopon-Model mPA210) was calibrated with known pH standard solutions (4.0, 7.0 and 10.0), and pH readings were performed in triplicate (Brazil, 2004).
Ford cup viscosity analysis were performed at 25 °C, with the hole number 4 in the Ford cup closed with the finger and filled with the samples. The orifice was released, and the chronometer was started simultaneously. When the first flow interruption occurred, the chronometer was stopped, and the time spent was then recorded. The calculation was performed according to the hole used in the measurement to obtain the viscosity in the centipoise unit (cP).

Accelerated Stability
A sample of the shampoo containing the microencapsulated M. cauliflora extract at the highest concentration (SH10) was tested for the accelerated stability analysis. Test (SH10) and control formulations (SH0 and SHBHT) were submitted to three different temperature conditions, as follows: 4 ± 2 o C (Compact Consul Refrigerator 120 liters), 25 ± 2 o C (controlled room temperature) and 45 ± 2 o C (New Ethics Climate 420 -CLD 300) for a period of 90 days (22). Macroscopic, pH and viscosity analyses were performed at days 0, 30, 60 and 90.

Hair sample analysis
Samples of discolored hair with reflections, wicks and/or lights, with yellow color tone, were kindly donated by beauty salons. The different types of hair were treated with the shampoo (1.5 g) containing the microencapsulated M. cauliflora peel extract [10% (w/w)] (SH10.0). In each sample, a manual process was performed simulating a capillary wash. After 3 minutes, the samples were washed twice with water to remove the product. The hair samples were dried with a hairdryer and analyzed by visual comparison to an untreated hair.

Extract preparation and total phenol content of M. cauliflora extract
To evaluate the chemical composition on M. cauliflora, the peels were extracted by the maceration method using 70% ethanol (v/v) and the yield of 45.8% was obtained, resulting in an appropriate extraction. The total phenolic content of M. cauliflora peel extract was 580.41 mg GAE/g of extract.

Total monomeric anthocyanins content
In the analysis of total anthocyanins, the M. cauliflora extract had a cyanidin concentration of 11.5 mg/ml, which was the major anthocyanin detected in the extract.

Characterization reactions
The ethanolic extract of M. cauliflora fruit peels presented blue color when in contact with 10% sodium hydroxide, therefore indicating the presence of anthocyanins therein. As anthocyanins present different structures based on the pH of the solution, the extract was submitted to differential pH analysis. The M. cauliflora extract presented different staining according to the pH tested, as shown in Figure 1. In acidic milieu (pH 2.0), the anthocyanins presented red color, since there is a predominance of the flavilic cation form. When the pH was adjusted to 5.0 the staining reduced considerably, with a predominance of the pseudocarbinol form. At pH higher than 6.0, there was a disruption of the heterocyclic ring. Therefore, the staining promoted by the different types of anthocyanins varied based on the pH of the solution (Marco et al., 2008).

Ferric chloride reaction and cyanidin reaction or Shinoda assay
When ferric chloride was added to the diluted sample, a visible violet coloration was observed, thereby indicating the presence of polyphenols in the extract, especially flavonoids and hydrolysable tannins.
The reaction of cyanidin was developed by the action of magnesium in the presence of concentrated HCl and M. cauliflora extract. A blood red staining (Figure 2) was observed, indicating the presence of flavonols or dihydroflavonol in the extract. The presence of different compounds leads to different colors.

Antioxidant activity by DPPH assay
The M. cauliflora extract showed a doseresponse curve showing an increase in potential antioxidant activity as the extract concentration increased, a characteristic observed even after incorporation of the extract into the shampoo base (Table 1). The M. cauliflora extract and BHT at the concentration of 100 μg/mL produced a percentage of inhibition of DPPH around 90%, indicating that the natural product presented antioxidant activity equivalent to that of the synthetic compound.

Shampoo development and preliminary stability
The macroscopic analysis demonstrated that the experimental shampoos (SH0, SH1.0, SH5.0, SH10.0 and SHBHT) were transparent, homogeneous and viscous at room temperature. The bases containing the extract of M. cauliflora showed characteristic color, which increased as a function of the extract concentration. The formulations demonstrated stability even under thermal stress since these characteristics were maintained throughout the experimental period.
None of the experimental shampoo formulations showed any physical instability, such as phase separation (coalescence, cremation or flocculation) after centrifugation, as well as thermal stress or freeze-thaw cycles followed by centrifugation throughout the duration of the study.
A pH analysis of the ethanolic extract and shampoo samples was also carried out. The findings at day 0 and after the freeze-thaw cycle showed no significant difference in pH values at day zero even after thermal stress (Table 2). This result implies in the stability of the formulation containing or not M. cauliflora extract at different concentrations. It is also noted that increasing the concentration of extract in the shampoos did not modify the pH of the formulations. However, there was a significant increase in the pH of the shampoo without M. cauliflora extract after 12 days of the feeze-thaw cycle. The initial pH of the formulation was 5.88 and after the stability study, it increased to 7.16. This phenomenon could be attributed to the loss of citric acid activity or interaction between the components of the formulation. It is worth noting, therefore, that the presence of M. cauliflora extract provided physical-chemical stability to the system, keeping the pH close to baseline levels.

Accelerated Stability
The SH0 and SHBHT formulations did not show macroscopic changes in color and odor as a function of time, while shampoos containing the extract (SH10.0) presented constant purple coloration. This is a desired situation for a formulation that exhibits antiyellowing activity for the hair. Figure 3 shows the pH variation of the samples as a function of time and environmental conditions. The SH0 and SHBHT formulations maintained at 4 ± 2 o C had an increased pH after 30 days of study, remaining unchanged up to 90 days. The SH10.0 formulation presented a slight decrease in this parameter; however, with no significant difference between the days analyzed. The formulations maintained at 45 ± 2 o C, SHBHT and SH10.0, presented a decrease in pH after 30 days of study, which was reverted 60 days following the initial tests.
The viscosity analysis showed that all samples kept in refrigerator remained unaltered regarding this parameter (Figure 4). On the other hand, the base shampoo maintained at 25 ± 2 o C and 45 ± 2 o C showed significant reduction in viscosity after 30 days of analysis. At room temperature, this change was reversible over time. However, this characteristic was irreversible when the sample was stored at a higher temperature.

Hair sample analysis
Discolored hair samples were treated with the base shampoo containing 10% microencapsulated M. cauliflora extract, and then dried with a hairdryer. The hair strands samples with lights/wicks showed excellent results in color neutralization. We observed that the yellowish tone was neutralized and showed a clear natural color ( Figure 5).
The chemical composition and content of metabolites in plant extracts is variable and depends on the fruit species and extraction method. The extraction of M. cauliflora peels by the maceration method using 70% ethanol (v/v) was shown to be more effective than those reported by Santos, Veggi, Meireles (2012) (Santos et al., 2012). These authors used ethanol as extracting agent under ultrasound, continuous agitation and Soxhlet, resulting in yields ranging from 9 to 12 % (p/v). On the other hand, the extract obtained in our study resulted in a low viscosity liquid, showing a dark red to purple color, with characteristic odor and pH, consistent with that obtained by Nazaré, et al. (2002).
The development of a more stable extract through microencapsulation allows for its use in the food, pharmaceutical, and cosmetic industry. Kechinski (2010) concluded that, as compared to carboxymethylcellulose and hydroxymethylpropylcellulose, maltodextrin at 10% is the best encapsulating polymer used for microencapsulation of anthocyanins. Maltodextrin is a low-cost polysaccharide obtained by the acid hydrolysis of starches, for instance, potatoes and others, and it does not influence the flavor and final aroma of the formulations, which makes it an interesting encapsulation polymer. Therefore, 10% maltodextrin was chosen as the encapsulation agent for the microencapsulation of M. cauliflora peel extract.
Importantly, the microencapsulation technique preserves the color of the pigments, reducing oxidation and maintaining the quality of the formulation for long periods of time.
The total phenol amount observed herein was higher than that found by Hamm (2009) (499.10 mg GAE/g of extract) and Silva (2011) (435.38 mg GAE/g of extract). The extraction process along with lyophilization and the use of maltodextrin as the encapsulation agent may have possibly contributed to retaining the phenolic compounds more efficiently.
Characterization reactions were developed for the phtyochemical screening, and M. cauliflora peels were found to have two major anthocyanins, delphinidin (3-O-glucoside) and cyanidin (cyanidin-3glucoside), in which cyanidin concentration is four times higher than that of delphinidin (Wu et al., 2012). The cyanidin amount observed in our study was higher than that reported by Santos et al. (2010) for all extracts obtained (˂ 7 mg cyanidin-3-glycoside/g dry material). Possibly, the slightly acidified extractive solvent (70% ethanol, acidified with 6.0% citric acid) may have facilitated the release and solubilization of anthocyanins from the fruit peels (Baldin et al., 2010). Thus, the extraction method used herein proved to effectively extract anthocyanins from M. cauliflora fruit peels.
For instance, when flavones are present in the sample, the yellow coloration turns to red, flavonols lead to a red coloration, while flavonones turn the solution from red to purple; anthocyanin derivatives turn to red, becoming pink; and chalcones, aurones, dihydrochalcones, isoflavones and isoflavanones do not react, presenting no coloration (Pedroso et al., 2009).
For the DPPH assay, the result found in this study was superior to that found in a study performed by Hacke et al. (2016), who observed 82.79% of the antioxidant potential. There was no significant difference between the results obtained with the extract before and after incorporation into the shampoo. The shampoo base and BHT did not influence the test, demonstrating that the extract presents an excellent antioxidant potential and can therefore be incorporated into shampoos for this purpose. Therefore, the M. cauliflora extract constitutes a great alternative for use as an antioxidant agent in cosmetic formulations.
The evaluation of formulation stability is commonly accomplished through methods that accelerate instability, in which the formulation is subjected to stressing conditions such as thermal variation, exposure to light, among others (Montagner et al., 2004). Based on this, the thermal stress test was developed in order to accelerate the physicalchemical interactions between the raw materials by increasing the temperature in a short period of time (Rieger, 1996). In our study, we observed that as the temperature increased, the viscosity of the formulation decreased, although this expected characteristic was found to be reversible when samples returned to room temperature. No signs of phase separation, release of gases and/or color changes were observed. Another effective method for determining the physical stability of cosmetic products is the centrifuge test. In this test, a change in the gravity force occurs, thereby accelerating the physical processes of sedimentation, cremation or coalescence of the system through phase separation. The pH analysis also showed results within the requirements of legislation. The results found in this study suggest these shampoo formulations are physically stable, since there were no macroscopic changes in color and odor after all the stability assays.
The hair sample assay showed that the yellowish tone was neutralized. The main capillary structure related to the response to such a procedure is the cuticle. Agents acting on this structure are responsible for malleability, softness and combability, and mainly for hair shine (Souza & Antunes-Jr, 2009). Based on the findings of this study, we can infer that the daily use of capillary preparations containing anthocyanins as a natural pigment in the formulation, in discolored hairs with lights/wicks, positively affect the process of removing the yellow color of the hair, which is mainly observed by the increase in capillary brightness. The ethanolic extract of M. cauliflora peel was found to possess a high content of phenolic compounds and anthocyanins. The latter present different structures as a function of different pH values, which makes them potentially useful natural pH indicators. In addition, the DPPH assay showed that the antioxidant potential of the extract was close to the synthetic antioxidant and that the shampoo base did not influence the antioxidant capacity of these compounds upon incorporation.
The shampoo samples containing M. cauliflora extract were stable even under adverse conditions. In the treatment of hair with yellowish coloration, the formulation containing 10% microencapsulated ethanolic extract of M. cauliflora presented a positive result, neutralizing the yellowing process. Taken altogether, these findings indicate the potential use of anthocyanins in shampoo formulations aimed at treating hair yellowing. Thus, M. cauliflora, an abundant and low-cost Brazilian fruit mainly composed of anthocyanins in its peels, demonstrated promising antioxidant and hair-toner characteristics. The potential of this fruit for cosmetic purposes, especially cosmetic hair care, warrants further investigation.

Conclusion
Taken altogether, these findings indicate the potential use of anthocyanins in shampoo formulations aimed at treating hair yellowing. Thus, M. cauliflora, an abundant and low-cost Brazilian fruit mainly composed of anthocyanins in its peels, demonstrated promising antioxidant and hair-toner characteristics. The potential of this fruit for cosmetic purposes, especially cosmetic hair care, warrants further investigation.