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Changing the shape of hair with keratin peptides†

Cite this: RSC Adv., 2017, 7, 51581 C. F. Cruz,‡a M. Martins,‡a J. Egipto,a H. Osório,b A. Ribeiroa and A. Cavaco-Paulo *a

Chemical straightening of curly human hair fibres involves the use of strong reducing agents at alkaline pH.
Human hair is made of keratin, and the fixation of fibre shape involves the reduction and reformation of new
disulphide bonds between keratin molecules. Here, we propose an alternative and green methodology
using keratin peptide sequences (10–13 residues) derived from the human genome. In a previous study,
we analysed 1235 cysteine-containing peptides encoded by all human genes of hair keratin and keratin-
associated proteins. These peptide fragments have been designed by nature to interact with keratin.
Here we tested eight peptides, which were select based on their affinity for human hair keratin solution
as shown by Matrix-Assisted Laser-Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-
TOF/TOF) and by molecular dynamics simulation. The peptides were characterized in detail regarding
their ability to act as hair straightening modulators and to improve the tensile strength and elasticity of
hair. Of the eight tested peptides, PepE, PepG and KP showed the highest ability to interact with
a keratin peptide model, and to improve hair mechanical properties and straightening efficiency. The
proposed solutions presented here will replace harsh reducing agents at alkaline pH by peptide
Received 20th September 2017
Accepted 26th October 2017
formulations acting at neutral pH to change hair shape through the re-conformation of disulphide
bonds. Here, we provide experimental evidence which explains at a molecular level how keratin
DOI: 10.1039/c7ra10461h
decapeptides can interact with large keratin molecules in human hair, opening an innovative green
rsc.li/rsc-advances approach to changing the shape of hair fibre.

repeated hair chemical treatments produce irreversible changes

Introduction in hair texture, and can result in a fracture of the hair bre once
The haircare industry has developed a plethora of products to the cuticle is removed, and cortex exposure leads to
modify hair characteristics, with hair straightening being one of breakage.4,8,10,12–18 The use of chemicals also represents an
the most popular processes. Many of these products contain important concern for the health of the consumer and stylist, as
toxic or environmentally hazardous chemicals such as strong well as for the environment.
alkaline agents, thioglycolates, sodium or lithium hydroxide, Biologically, the human hair is a complex bre with various
guanidine or even formaldehyde. These chemicals have high morphological components. Hair essentially consists of keratin,
toxicity and the potential to generate poisonous gases, and they a brous structural protein, which is a member of the super-
have a negative impact on human health.1–7 Table 1 presents the family of intermediate lament proteins, containing 18%
common methods for hair straightening. These treatments cysteine as calculated from the overall amino acid composi-
damage the hair bre, reduce the cross-linking density and tion.19,20 The hair bre is divided into three main regions:
decrease the hair's physicomechanical properties.8,9 Extremely cuticle, cortex, and medulla.21,22 The medulla is located in the
damaged hair suffers several negative impacts such as the core of the hair bre, and may be partially or entirely absent in
appearance of cracks and liing of the cuticle, the removal of ne hair bres. The cortex is the major bulk of the hair bre and
the hydrophobic top-layer of the hair cuticle and a consequent is constituted of macrobrils of intermediate laments, which
reduction of surface hydrophobicity.10–12 A further consequence are in turn composed of a-keratins, found in a-helical form,
is the alteration of the diffusion rate into keratin bres, which is acting as mechanical support.23 The individual laments of
dependent on the cross-linking density of the hair. Excessive or cortical cells are separated by a cell membrane complex of
keratin associated proteins (KAPs), with a high cysteine content
and without a well-dened spatial organization.24 Keratin a-
a
CEB – Centre of Biological Engineering, University of Minho, 4710-057 Braga, helices are coiled by ionic forces, hydrogen bonds, van der
Portugal. E-mail: artur@deb.uminho.pt; Tel: +351 253604407 Waals forces and disulphide bonds. The cuticle, enclosing the
b
IPATIMUP – Institute of Molecular Pathology and Immunology, University of Porto,
hair cortex, is composed of superimposed layers of cells in
Porto, Portugal
a scaled structure. Each cuticle cell contains various sub-
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra10461h lamellar layers (epicuticle, A-layer, exocuticle, and endocuticle)
‡ Both authors contributed equally to this work. with proteins cross-linked mainly by cysteines. The outer

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RSC Advances Paper

Table 1 Common methods and agents used in hair straightening

Reducing methods Agents Methodologies References

Method with reductive agents (thio-relaxers) Ammonium thioglycolate, Disruption of disulphide 9 and 69–72
thioglycolic acid, cysteamine bonds and possible
hydrochloride, rearrangement through
glycerylmonothioglycolate, disulphide interchange
ammonium sulphite, processes
ammonium bisulphite
Method with Lye relaxer Potassium hydroxide, sodium Disruption of disulphide 9, 69 and 72
alkaline agents hydroxide bonds and possible
(hydroxides) No-lye mix relaxer Calcium hydroxide and an rearrangement to disulphide
activator of the relaxation bonds or lanthionine bonds
reaction containing guanidine (lye relaxer with pH $ 12)
carbonate
No-lye no-mix Lithium hydroxide
relaxer
Brazilian keratin method Formaldehyde Cross-links hair keratin with 9, 72 and 73
the keratin introduced in the
formulation

epicuticle layer contributes to the lubricity of the hair and acts peptides containing two to ve cysteines based on genes for
as a barrier to the penetration of compounds into the hair.9,21 human hair keratin and KAPs.34 It is known that cysteine can be
The free lipids on the surface and the involvement of proteins applied as a reducing agent for the substitution of environ-
are critical factors in the overall structure of the hair bre. mentally harmful chemicals.35 The origin of the peptides, their
Hair proteins are able to create stronger or weaker interac- high cysteine content, and their hydrophobicity are intended to
tions.25,26 For example, the presence of disulphide bonds in improve hair properties, enabling the restructuring and rein-
cysteine residues of the hair keratin protein determine the forcement of hair bres. A detailed characterization of the
maintenance of the shape of the hair, and can be altered by peptides was conducted regarding their ability to act as hair
perming or relaxing. The disulphide bonds are much stronger straightening agents and their ability to recover and/or increase
when thiol groups are closer together, making these bonds hair tensile strength and elasticity. Herein, we explore the
easier to form, and in consequence the hair is much curlier application of peptides as innovative ecological and healthy
once a curve in the linear protein chain is induced. Hair bre is alternatives for hair straightening, without compromising hair
sensitive to changes in the pH. Alkaline reducing agents, when integrity, so opening an innovative way to change the shape of
in contact with the cortex, break and rearrange the disulphide the hair bre.
bonds, stretching the spiral coil of keratin molecules.10 This
makes the hair more susceptible to friction, leading to lower
hair strength and resistance. However, hair damage caused by
Experimental
the use of chemicals can be minimized, avoided or repaired by Materials
the controlled use of proteins or peptides.27 The use of peptides Human virgin hair samples were provided by International Hair
for hair treatments including hair gloss, hair soness and Importers & Products Inc. (New York, USA). All chemicals were
manageability and hair damage repair is well described. purchased from Sigma-Aldrich (Madrid, Spain), except when
Peptides and amino acids have high ability to diffuse into the stated otherwise. A total of eight peptides were designed to
hair bre cortex, high substantivity in a practical pH range, and interact with hair keratin. These peptides are based on frag-
the ability to aid recovery from hair cuticle damage.10,13,16,27–32 ments of human hair keratin and KAPs and were selected on the
Numerous bonds such as ionic, hydrophobic and hydrogen basis of a previous study which used a microarray of peptides.34
bonds, can be established between the exogenous proteins and Specically, the peptide sequences are based on keratin asso-
the hair keratin. Covalent binding, such as that in disulphide ciated protein 5–3 (PepA), keratin associated protein 4–8 (PepB),
bonds, may also occur.22,27,32,33 Hair products based on synthetic keratin associated protein 9–7 (PepC), keratin type I cuticular
peptides are a reality today, with applied dosages of 0.01%. Ha4 (PepD), keratin type I cuticular Ha5 (PepE), keratin asso-
Disulphide bond disruption and formation are known to be ciated protein 16–1 (PepF), keratin associated protein 5–9
associated with changes of hair shape.9 Here, we explore the use (PepG) and keratin type II cuticular Hb5 (KP). The KP peptide
of peptides as substitutes for alkaline agents and thio-relaxers has already been analysed in previous studies.16,28 The peptides
which are commonly used for hair straightening. Our goal is were synthesised by JPT Peptide Technologies GmbH (Berlin,
to present a novel green approach to validate the use of Germany). PepA, PepB, PepC, PepD, PepE, PepF and PepG were
decapeptide sequences as eco-friendly alternatives for the covalently linked via their N-terminal amines to the uorescent
control of hair shape changes. These peptides were selected dye 5(6)-carboxyuorescein (lex ¼ 492 nm; lem ¼ 517 nm), while
from a previous broad study which analyzed over 1000 different KP was covalently linked via its N-terminal amines to the

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Paper RSC Advances

uorescent dye 5(6)-carboxytetramethyl- Qualitative and quantitative analysis of peptide penetration

rhodaminesuccinimidyl ester, 5(6)-TAMRA (lex ¼ 544 nm and into the hair bre. The penetration of the peptides into African
lem ¼ 572 nm), to facilitate the analysis of peptide penetration. hair was assessed qualitatively by uorescence microscopy. The
The peptides were supplied as lyophilized materials. They were hair bres treated with the peptides were embedded in an epoxy
analysed by high performance liquid chromatography (HPLC) resin. Transversal cuts of 15 mm width were prepared using
and mass spectrometry (MS), and their purities were over 70% a microtome (Microtome Leitz) and the hair cross sections were
(HPLC, 220 nm, C18, linear gradient). The peptides' character- analysed by uorescence microscopy (Olympus BX51) using
istics are shown in Fig. 2A. 40 amplication. The images were collected with the same
The hair protein-like peptide, KeraPep, (peptide sequence conditions of brightness, time of exposure and gain, and were
from N-terminal to C-terminal: DDDDDKPCCCSSGCGSS- examined at the spectral setting of 40 ,6-diamidino-2-
CCQSSCCKPCCSQSSCCVPVCCQCKIDDDDD), has a sequence phenylindole (DAPI – lex ¼ 358 nm; lem ¼ 461 nm) for the
based on the gene for human hair keratin-associated protein peptides PepA, PepB, PepC, PepD, PepE, PepF and PepG, and at
5–1, and was synthesised by GenScript Inc. (Piscataway, USA). the spectral setting of tetramethylrhodamine (TRITC – lex ¼
The selected sequence contains a high content of cysteine to 557 nm; lem ¼ 576 nm) for the peptide KP. As auto-uorescence
model the hair cysteine content and it can be considered to be was unavoidable for these samples, the auto-uorescence of the
a good model for the study of the redox behaviour of disulphide control sample was measured and subtracted from the uo-
bonding of hair proteins and smaller peptides. The penta- rescence of the other samples. For homogenization purposes,
aspartate sequences at each extremity were added to induce the KP, labelled with TAMRA and observed with TRITC spectral
peptide solubility. settings, was depicted as blue. The most representative images
were chosen.
The quantication of peptide uptake by hair bres was
Methods assessed from the variation in concentration of each peptide
Matrix-assisted laser-desorption ionization time-of-ight aer the peptide hair treatments (procedure adapted from
mass spectrometry (MALDI-TOF/TOF) analysis of peptides. Fernandes and Cavaco-Paulo16). Briey, the hair-peptide solu-
Each peptide was processed for MALDI-TOF/TOF analysis. The tion was monitored by the measurements of the absorbance at
peptides were desalted, concentrated and spotted onto the 497 nm for peptides PepA, PepB, PepC, PepD, PepE, PepF, and
MALDI plate using reversed-phase C18 Zip Tips (Millipore, MA, PepG, and at 555 nm for KP, before and aer the hair treatment.
USA), according to the manufacturer's instructions. The matrix Calibration curves for each peptide were obtained. Three
used was a solution of 7–8 mg mL1 a-cyano-4-hydroxycinnamic independent experiments were performed for each peptide
acid, prepared in 50% (v/v) acetonitrile and 0.1% (v/v) tri- condition. The concentration variation (%) or peptide uptake
uoroacetic acid. Samples were analysed using a 4800 Proteo- was calculated using the equation:
mics Analyzer MALDI-TOF/TOF (AB SCIEX, Framingham, MA, concentrationinitial  concentrationfinal
USA). The peptide mass ngerprint data was collected in posi- Peptide uptake ð%Þ ¼
concentrationinitial
tive MS reector mode for the mass-to-charge ratio (m/z) 700–  100
4000 using trypsin autolysis peaks for internal calibration.
Interaction analysis of peptides with a hair protein peptide
model by MALDI-TOF/TOF. The peptides were analysed by Molecular dynamics simulations of the interactions between
MALDI-TOF/TOF regarding their binding ability to a peptide the peptides and the hair protein peptide model. All molecular
hair protein model, a 45-mer peptide based on human hair KAP dynamics simulations were performed with the GROMACS 4.0.7
(abbreviated as KeraPep), to make the identication of peptide package using the GROMOS 54A7 united-atom force-eld,36–40
binding easier, and to allow for a closer comparison to the with periodic boundary conditions, applying the LINCS41 and
simulations. Each peptide was incubated with the KeraPep the SETTLE42 algorithms to constrain the peptide and water
(1 mg mL1 in phosphate buffer pH 7), in a ratio of 2 : 1 (w/w) bonds respectively. The simulations ran with a time step of
for 1 hour. This incubation was performed at two tempera- integration of 2 fs, and the single point charge (SPC) model43
tures: 37  C and 120  C. For analysis, a solution of 8–10 mg was used to represent the water. The temperature was main-
mL1 sinapic acid, prepared in 50% (v/v) acetonitrile and 0.1% tained at 300 K with the V-rescale thermostat44,45 and the Par-
(v/v) triuoroacetic acid was used as the matrix. The samples rinello–Rahman barostat46,47 (with isotropic coupling) was used
were spotted onto the MALDI plate and analysed using a 4800 to maintain the pressure at 1 atm. van der Waals and electro-
Proteomics Analyzer MALDI-TOF/TOF (AB SCIEX, Framingham, static interactions were treated by a twin-range method, with
MA, USA). The peptide mass ngerprint data was collected in short and long range cutoffs (0.8 and 1.4 nm, respectively), and
linear mode for the mass-to-charge ratio (m/z) 4000–30 000 with a reaction eld correction for electrostatic interactions
using trypsin autolysis peaks for internal calibration. The ratio using a dielectric constant of 54,48 and the neighbour list was
of the different peaks was determined using the equation: updated every 10 steps. The molecular model of the KeraPep
areapeak was built through PYMOL soware using its amino acid
Ratio ð%Þ ¼
 100 sequence and assigning the KeraPep secondary structure as a-
sum areaall peaks
helix (the majority of this specic sequence of keratin-

This journal is © The Royal Society of Chemistry 2017 RSC Adv., 2017, 7, 51581–51592 | 51583
RSC Advances Paper

associated protein 5–1 was predicted to be a-helical49,50). The Statistical signicances were determined using SPSS, using
same procedure was used to build the remaining PepC, PepD, the one-way ANOVA test, followed by the Tukey's post-hoc test.
PepG and KP computational models, but in these models no p-Values # 0.05 were considered statistically signicant
secondary structure was dened, due to their small size. The (expressed in the gures with an asterisk, *) and p-values # 0.01
soware VMD (Visual Molecular Dynamics)51 was used to create were considered very signicant (**).
all simulation pictures presented. Straightening evaluation of single curly hair treated with
All simulated systems were initially energy-minimised for peptides. The hair straightening was performed using curly
around 2000 steps with the steepest descent method. The African hair (curliness type VII). Hair tresses were washed with
systems initiated a series of three short equilibration simula- a solution of 0.5% SDS, with constant agitation for 1 hour, and
tions (500 ps in each simulation), with position restraints dried naturally. The procedure to straighten the curly hair was
allowing the slow relaxation of the structures for the nal adapted from the Kirby method .53,54 Single hair bres of curly
production run (the atoms were restrained with a harmonic hair were forcefully straightened by gluing the root ends on the
force of 103 kJ mol1 nm2). The rst simulation was performed frame to ensure a straight shape. Hairs were kept evenly straight
in the NVT ensemble (number of particles, volume and without any twisting. While straightened, the hair was incu-
temperature constant), applying position restraints to all heavy bated at 37  C for 1 hour in a formulation of phosphate buffer
atoms. The second simulation was performed in the NPT pH 7 with 0.01% (w/v) of each peptide (PepA, PepB, PepC, PepD,
ensemble (number of particles, pressure and temperature PepE, PepF, PepG or KP). Aer treatment, hair was washed with
constant), with position restraints for the same atoms. Lastly, distilled water and allowed to dry naturally, still straightened. In
the third simulation was also run in the NPT ensemble, but with order to evaluate the effect of the peptides, three sequential
position restraints only for the a-carbon atoms of all the amino treatments with the same concentration (0.01% (w/v)) and
acids. Aer the equilibration of the systems by the preparatory a separate treatment with a higher peptide concentration (0.1%
simulations, 100 ns of NPT simulations in aqueous systems (w/v)) were performed. Three independent experiments were
were performed. performed for each peptide treatment. Hair straightened by
Evaluation of mechanical properties of over-bleached a chemical relaxing treatment was used as a positive control.
straight hair treated with peptides. The recovery of the Briey, in the chemical treatment, hair tresses were washed
mechanical properties of severely chemically damaged straight with 0.75 M sodium hydroxide pH 12–14 for 30 minutes, and
hair was performed using straight Caucasian hair (curliness then combed and washed with water. Then, the hair was
type II52). Before the bleaching treatment, hair tresses were washed with 0.1 M acetic acid, as a neutralising agent, and
washed with a solution of 0.5% sodium dodecyl sulphate (SDS) nally the hair was washed with a commercial shampoo.
with constant agitation for 1 hour and dried naturally. Chemi- Straightening efficiency was calculated based on the change of
cally damaged hair underwent eight cycles of bleaching. The the hair twists and length aer the treatments, as depicted in
bleaching process consisted of the application of 12% (v/v) H2O2 Fig. 1, using the equation:
in the presence of 0.1 mol L1 Na2CO3/NaHCO3 buffer, pH 9.0 at
Straightness efficiency ð%Þ ¼
50  C for 1 hour, in a bath ratio of 1 : 10. The hair tresses were
number of twistsafter treatment
washed with distilled water aer each cycle of bleaching. Aer
hair lengthafter treatment
that, hair tresses were treated with 0.01% (w/v) of each peptide 100   100
number of twistsbefore treatment
(PepA, PepB, PepC, PepD, PepE, PepF, PepG, KP) in a formula-
hair lengthbefore treament
tion of phosphate buffer pH 7, for 1 hour at 37  C with agitation.
Aer hair treatment, the roughly bound peptides were washed
off in distilled water. Straightening evaluation of curly hair tresses treated with
The mechanical properties of the chemically damaged hair peptides in serum formulation. The efficiency of the peptides at
samples were evaluated following guidelines outlined in ASTM straightening curly hair was evaluated in an alcoholic-based
D1445-95 for bre tensile testing. The measurements were formulation. The effect of the formulation was computation-
performed in an Instron 4505 tensile tester, with a 2.5 N load ally reproduced in a previous study regarding strong
cell. For each type of hair treatment, 15 single hair bres with
low variability in diameter were randomly taken from the hair
tress and individually mounted in the tensile jig, using a paper
device with a xed gauge length of 20 mm. All the hair samples
were kept under the same conditions before the measurements.
The measurements were performed with a constant rate of 1.5
mm min1 until breakage. The measurements were performed
assuming an average mean bre diameter of 70 mm (the value
that was obtained through previous measurements with light
microscopy). The data recorded in the equipment (applied load
against extension) was converted to stress (load/unit area)
against strain (% extension).
Fig. 1 Scheme of hair measurement and twist counting.

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Paper RSC Advances

interactions between the hair and alcoholic components.55 The with a thiol group which has a strong tendency to reduce
peptides (PepE, PepG and KP) were incorporated into alcoholic disulphide bonds in hair keratin.59 Disulphide bonds can be
formulations at pH 7.5 (1.5% propylene glycol, 10% ethanol, formed with the cysteine residues when there is an available
and 0.5% benzyl alcohol). Each serum formulation was applied electron acceptor, such as molecular oxygen in the solution, to
onto the hair tresses and le in for 15 minutes. The hair oxidise the sulydryl groups of the cysteine residues into
straightening process was induced using a at iron at 200  C. disulphide bonds. The peptides analysed contain two to ve
The method was repeated until each hair tress was dried and cysteines, Fig. 2A. The relative ratios of species were analysed by
straight. Then, the hair tresses were washed with distilled water MALDI-TOF/TOF (Fig. 2B), revealing that PepA, PepB, PepD,
and dried at 50  C. The straightening efficiency was calculated PepF, PepG and KP formed as dimers; PepA, PepB, PepD and KP
using the above-mentioned equation. The hair tresses were then showed a ratio of approximately 50% dimer conguration; PepG
washed and shampooed 20 times. was mainly found as a dimer (around 94%); PepC and PepE
were only found in monomeric congurations; while PepF was
Results and discussion found as a monomer with a ratio of around 87%.
To further analyse the peptide conformations and their
Characteristics of chosen peptides predisposition for the formation of internal disulphide bonds,
The modication of hair shape was the driving force for the molecular dynamics simulations were performed for PepC,
selection of appropriately engineered peptides based on the PepD, PepG and KP. The selection of these peptides was based
sequences of keratin and KAPs. These peptides were selected on the desirability of including a monomeric conguration
based on a previous study of 1235 cysteine-containing peptides (PepC), a dimeric conguration (PepG) and two peptides with
of all genes of human hair keratin and KAPs.34 This study the ability to form both congurations (PepD and KP). At the
analysed the interaction of the peptides with keratin extracted end of the simulations with these peptides in water, the GRO-
from human hair, and the peptides with the best characteristics MACS tool “g_cluster” was used to determine the most frequent
were selected for further analysis. Due to their high cysteine spatial conformation of these peptides that occurred in the
content and hydrophobic properties, these peptides are ex- analysed simulation time. The tool was applied to 100 ns of
pected to present higher affinities towards the hair proteins. simulation for each peptide, using a 0.1 nm root mean square
Furthermore, decapeptides are small enough to be able to cutoff for the calculations. The distances between the b-carbons
penetrate into the hair cortex, but 10 to 13 amino acids residues of the cysteine residues in the peptides were measured in the
make these peptides big enough to provide enhancement of most common spatial conformation according to the
strength and restructuring of the hair protein linkage “g_cluster” results. In addition, the same distance was
network.10,16,30,32 measured along the simulation time using the “g_dist” GRO-
Firstly, we made a comparative analysis to determine the MACS tool and the average distance for the last 10 ns of simu-
best peptides, based on their amino acid sequences, isoelectric lation was calculated. This could provide some insights into the
points, cysteine contents, and hydrophobic and polar amino possible propensity of these peptides to form intramolecular
acids34 (Fig. 2A). The affinity towards the hair bre is deter- disulphide bonds.
mined by the specic characteristics of each peptide.56,57 The Several properties such as the variation of temperature,
isoelectric point of the peptides inuences the interaction pressure and potential energy of the systems were calculated
prole with the hair keratin. The isoelectric point of human hair using GROMACS tools at the end of the interaction simulations.
has been reported to be around 3.7,58 indicating that hair The parameters evaluated indicated that the systems were well
presents a negative net surface charge under treatments per- equilibrated during the simulation time.
formed at pH 7. At this pH, the peptides PepD, PepE, PepF, The average distance between the b-carbons of the cysteine
PepG and KP also present a negative net charge, which will not residues of each peptide was measured for the last 50 ns
favour the adsorption mechanism onto the hair bre by these (Fig. 2C); this time allowed the simulation to reach equilib-
peptides. The hydrophobic character of the peptides is another rium. The distances obtained are much longer than the
important parameter for their cosmetic effect.10 Hydrophobic distances occurring in nature; disulphide bond lengthening at
and hydrogen interactions contribute to the overall binding a transition state has been described from 0.24 Å to 0.78 Å.60
force of peptide–hair interactions. In terms of hydrophobic These distances may indicate peptide conformations with
amino acid content, PepE is the most hydrophobic (60%), PepD, a predisposition for internal bond formation. PepD may have
PepF and KP present medium hydrophobicity (around 40%), a predisposition for the formation of intramolecular disul-
while PepA and PepG are the least hydrophobic (less than 10%). phide bonds. PepG has ve close cysteines in a total of 11
Due to the presence of some reactive groups in the amino acid amino acids which may form several different bonds, such as
sequences, other interactions such as polar interactions may be Cys1–Cys3, Cys1–Cys10, Cys3–Cys5, Cys3–Cys6, Cys3–Cys10,
formed, enhancing the capability of some peptides for specic and Cys5–Cys6. The distances between the cysteines of PepC
and directional binding.22 The KP peptide presents a high and KP did not indicate a propensity for the formation of
content of polar amino acids (46%). Peptides containing bonds between the cysteines. Most peptides alone showed
cysteine may also interact covalently with hair keratin where a predisposition to form internal disulphide bonds between
partial recombination or disruption of the disulphide bonds of the cysteines.
the hair keratin is involved.59 Cysteine is a standard amino acid

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RSC Advances Paper

Fig. 2 (A) Peptide nomenclature and characteristics: name, number of amino acids (N.a.a), molecular weight (MW), isoelectric point (pI),
percentage of cysteines (Cys), percentage of hydrophobic (Hydr.) and polar amino acids in the peptide sequence, and peptide chemical
structures; (B) relative ratio of species determined by MALDI-TOF/TOF; (C) distance between b-carbons of the cysteines of the peptides PepC,
PepD, PepG and KP in the last 50 ns of simulation.

Interaction of peptides with hair bre transversal cuts of hair treated with the peptides (Fig. 3A) and by
the peptide uptake as determined by the variation in absor-
From the previous study with the microarray, the peptides were
bance of the peptide solutions before and aer hair treatments
already known to interact with keratin extracted from human
(Fig. 3B).16 The peptide uptake is dependent on both the hair
hair.34 Here, the peptides were analysed regarding their ability
to interact with the entire hair bre. Fig. 3 reveals the efficient bre peptide binding ability and the extent of hair damage.
interaction of the peptides with the human hair bre, as well as The obtained results were as expected: the damaged hair
resulting from the over-bleaching treatment allowed a higher
their capability to recover the strength/elasticity of damaged
peptide uptake since the access to the hair cortex was easier in
hair and to aid in curl straightening.
a hydrophilic bre.
The affinity towards human hair bre was analysed for all
The hair's physical structure and the interactions between
peptides using uorescence microscopy analysis and peptide
hair components endow the hair with remarkable mechanical
uptake by the hair bre. These approaches were performed to
properties. Chemical over-bleaching affects both the hair's
conrm the degree of penetration in the binding of peptides
along the structure of African hair (Fig. 3A and B). For a broader structure and its interactions, leading to a signicant loss of
study, the ability of the peptides to penetrate and bind into strength and elasticity.11,61,62 The treatment of the hair bres
with the peptides may re-establish some of the hydrogen and
straight over-bleached Caucasian hair was also veried (ESI,
disulphide bonds lost during chemical treatment, improving
Fig. S3 and S4†). The peptides had the ability to penetrate into
the hair's mechanical properties. The effect of the peptides on
the hair cuticle and cortex for both types of hair as shown by the

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Paper RSC Advances

Fig. 3 (A) Fluorescence microscopy of hair treated with peptides at 0.01% (w/v). (B) Peptide uptake by curly African hair measured in terms of
absorbance at 497 nm for peptides PepA, PepB, PepC, PepD, PepE, PepF and PepG, and at 555 nm for KP. The hair samples were treated with
0.01% (w/v) of each peptide. (C) Young's modulus (part A) and stress at peak (part B) of Caucasian over-bleached hair (from left to right): virgin
hair; over-bleached hair; over-bleached hair treated with 0.01% (w/v): PepA; PepB; PepC; PepD; PepE; PepF; PepG; and KP. p-Values # 0.05
were considered statistically significant (*) and p-values # 0.01, very significant (**), when compared with over-bleached hair. (D) Straightening
efficiency of hair treatments for single curly African hair containing 0.01% (w/v) and 0.1% (w/v) of each peptide: control (phosphate buffer); PepA;
PepB; PepC; PepD; PepE; PepF; PepG; KP; and with chemical relaxer (from left to the right). Data represent the mean  standard deviation of
independent experiments. (E) Peptide nomenclature and characteristics: name, number of amino acids (N.a.a), molecular weight (MW),
isoelectric point (pI), percentage of cysteines (Cys), percentage of hydrophobic (Hydr.) and polar amino acids in the peptide sequence, and
peptide chemical structures.

the hair's mechanical properties was assessed through the chains unfold without a high level of resistance. The measure-
evaluation of hair elasticity (Young's modulus) and stress at ment of the stress at peak corresponds to the resistance of b-
peak (Fig. 3C). The Young's modulus results from a-keratin sheets to stretch until reaching the rupture point.63–65
stretching, which is resisted by disulphide bonds and by The treatments with the peptides increased the mechanical
hydrogen bonds between turns that stabilize the helical struc- properties of the hair, as measured by both the Young's
ture of the keratin. Aer a-keratin stretching, a transition of the modulus and stress at peak. PepA, PepB, PepC, PepD, PepE,
keratin from a-helix to b-sheet occurs, in which the keratin PepF, PepG and KP improved the Young's modulus resistance

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RSC Advances Paper

by up to 40% when compared with the over-bleached hair. PepC revealed that KeraPep exists as a monomer (98.33% as mono-
has high attachment with a positive net charge during the hair mer and 1.67% as dimer). The binding of the peptides to this
treatments, which increases its resistance to washing and its model was analysed using MALDI-TOF/TOF at temperatures of
attraction to the hair surface, especially in damaged hair due to 37  C and 120  C. All peptides formed aggregates with the hair
the more negative net charge of the hair cuticle. In addition, protein model by the formation of new disulphide bonds,
this peptide did not show a propensity to form intramolecular conrming their ability to interact with hair proteins (ESI,
disulphide bonds but was able to interact with hair proteins. Fig. S2†), as already proven by the microarray research study34
Both properties contribute to the high efficiency of this peptide and the previous tests with the human hair bre. At 37  C, some
to improve the mechanical properties of damaged hair. PepD aggregates of KeraPep and three units of PepB and PepG were
has a close conformation which promotes the formation of observed (Fig. 4A). An increase of temperature from 37  C to
intramolecular disulphide bonds, decreasing its propensity to 120  C promoted an increase in the size of aggregates between
interact with the hair surface, resulting in low bre uptake and the hair protein model and PepE, PepG and KP. The higher
low improvement of hair mechanical properties. temperature increased the reformation of new disulphide
The modication of African hair shape was demonstrated bonds. These results show the ability of these peptides to bind
through the ability of the peptides to restructure the disulphide to the hair proteins at higher temperatures, as is required when
bonds between the peptide and hair bre. The straightening of the hair is subjected to a hair dryer or at iron, and conrm that
African hair was ensured with the binding of the peptides to the a higher temperature favours the formation of the disulphide
hair keratin, which created new bonds, xed the protein bonds.66 Hence, the temperature of the reaction is a crucial
conformation and consequently straightened the hair. The parameter for an enhanced interaction between the peptides
straightening effect of all peptides was clearly demonstrated by and hair proteins. The re-modulation of the disulphide bonds
quantitative (Fig. 3D) and by visual analysis (for low peptide occurred throughout the conjugation between the small
concentration, 0.01% (w/v), see ESI, Fig. S5†). peptides and the KeraPep.
The straightening efficiency of the peptides for curly African The KeraPep was further simulated using GROMACS. The
hair ranged between 17% and 52%, with the best result being peptides used for this simulation were the same as used for the
obtained for the KP peptide. In contrast, the hair treated with simulations of the distances between b-carbons of the cysteines
phosphate buffer in the absence of peptides presented of each small peptide (PepC, PepD, PepG and KP). For this
a straightening efficiency of around 5%. A typical chemical simulation, several properties were calculated to evaluate the
relaxing treatment using sodium hydroxide as the relaxing interaction between these peptides and the KeraPep, including
agent led to a straightening efficiency of approximately 65%. the distance, the number of hydrogen bonds, and the radial
For peptides PepC, PepD, PepF, PepG and KP, the straightening distribution function (RDF) between the KeraPep and the
effect was dependent on concentration; a higher straightening peptides. It was assumed that a smaller distance between
efficiency was obtained for the highest peptide concentration. a peptide and the KeraPep predicts a higher affinity between
For 0.1% (w/v) peptide concentrations, the straightening effi- them (Fig. 4B). PepD gave a greater distance to the KeraPep. KP,
ciency of these peptides was around 60%, close to the result and in particular PepG, maintained a closer distance to the
obtained with the chemical relaxing treatment. The sequential KeraPep, suggesting their higher affinity for it and consequently
treatment which consisted of a series of three treatments using for the hair proteins.
the same initial concentration (0.01% (w/v)) induced The capability to straighten African curly hair was also
a straightening effect close to that of the chemical treatment for assessed by the incorporation of peptides into a serum formu-
PepE, PepG and KP. The results indicate that the straightening lation. The formulation containing the peptides was applied to
efficiency of the peptides for the African hair bre was deeply hair tresses. The hair tresses were combed straight and
inuenced by the peptide interactions, suggesting that the mechanically straightened with a at iron. The shape of the hair
peptides were able to create stable disulphide bonds with the tresses was analysed for each peptide aer treatment. The
hair keratin. penetration of the peptides into the hair bre was facilitated
through their incorporation into the serum formulation con-
taining benzyl alcohol, propylene glycol and ethanol. The hair
Peptides can reform disulphide bonds with a large-chain tresses were treated with two concentrations (0.01% and 0.1%
model of hair protein and induce hair shape changes (w/v)) of the selected peptides: PepE, PepG and KP. The selec-
The binding interaction between the peptides and hair proteins tion of these peptides was based on their straightening effi-
was demonstrated using a 45-mer peptide with high cysteine ciencies in the three conditions used previously in the
content, KeraPep, as a hair protein model. This peptide was straightening hair treatments (Fig. 3D). Fig. 4 shows the quali-
based on the KAP 5–1 protein of the human hair. This peptide tative and quantitative analysis of these treatments. The inclu-
has the sequence: DDDDDKPCCCSSGCGSSCCQSSCCKP- sion of the peptides into a serum formulation was done in order
CCSQSSCCVPVCCQCKIDDDDD. It has ve aspartates to provide to simulate the effect of the peptides as straightening agents in
improved solubility in aqueous solutions and 15 cysteine resi- a commercial formulation. The development of a cosmetic
dues. It can be considered as a good model of hair protein to product as a green alternative to the chemical relaxing treat-
study the redox behaviour of disulphide bonding between hair ment was the motivation behind the work here presented. Thus,
proteins and smaller peptides. MALDI-TOF/TOF analysis

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Paper RSC Advances

Fig. 4 (A) Relative ratios for the reaction of the peptides and the KeraPep at 37  C and at 120  C obtained by MALDI-TOF/TOF. (B) Molecular
dynamics simulations of the average distances in the last 10 ns of simulation between dimers of KeraPep and the peptides PepC, PepD, PepG and
KP. The images reflect the final state of the simulations. (C) Curly hair tresses before and after peptide treatments; after treatments, the hair
tresses were washed and dried at 50  C. The bottom images present the samples treated with the serum formulations containing peptides (PepE,
PepG, KP) at 0.1% (w/v); the images in the middle present the samples treated with the serum formulations containing peptides (PepE, PepG, KP)
at 0.01% (w/v); the images at the top show the samples treated without peptides (buffer, chemical and base serum formulation treatments). (D)
Straightening efficiency of serum treatments with peptides for curly hair tresses. (E) Peptide nomenclature and characteristics: name, number of
amino acids (N.a.a), molecular weight (MW), isoelectric point (pI), percentage of cysteines (Cys), percentage of hydrophobic (Hydr.) and polar
amino acids in the peptide sequence, and peptide chemical structures.

it was essential to evaluate the straightening effect of the concentrations. These effects were of the same magnitude as for
peptides when included in a formulation. the chemical treatment. For peptides PepE and PepG, the
The straightening effects of PepE, PepG and KP when straightening efficiency was improved by increasing the
applied in a serum formulation were not affected when concentration of peptide in the serum formulation. The base
compared to their effects in phosphate buffer (Fig. 4C and D), serum formulation itself (without peptide) showed a slight
with efficiencies of up to 60% for all peptides and for both improvement of the straightening efficiency, at around 30%,

This journal is © The Royal Society of Chemistry 2017 RSC Adv., 2017, 7, 51581–51592 | 51589
RSC Advances Paper

when compared with the phosphate buffer treatment. This These results open an innovative way to repair damaged hair
could be attributed to the formation of bonds with the hair bre from severe and repetitive treatments (e.g. coloration, and
keratin. The serum formulation facilitates the penetration of perming) and to straighten curly hair, while avoiding conven-
peptides into the hair bre, which promotes the re-shaping of tional chemicals and preserving the integrity of the hair
disulphide bonds, changing the shape of the hair. The re- structure.
shaping of the hair was demonstrated qualitatively and quan-
titatively (Fig. 4C and D, respectively), where PepE, PepG and KP
were able to promote a signicant change in the hair shape. The Conclusions
effect of these peptide formulations on the African hair lasted
for at least 20 washes with shampoo, with the exception of PepE Although conventional chemical treatments are the most
at the concentration of 0.1% (w/v). popular methods used to change the style of the hair, they
Comparing the straightening efficiency of the peptides represent a threat to the hair, the user and the environment.
without the serum formulation (single curly hair) and with the The repetitive use of conventional cosmetic chemical treat-
serum formulation (curly hair tresses) it was evident that there ments can severely affect hair physicochemical properties. Eight
was an improvement in the effects of the peptides when they engineered small peptides based on fragments of human hair
were included in the serum formulation. The serum formula- keratin and KAPs were designed to restore hair properties and
tion has previously been used to promote the penetration of to straighten curly hair. These peptides, when applied to
peptides into the hair cortex.55 Besides, it may also promote chemically over-bleached hair and curly African hair, were
a stabilization and change of peptide conformation aiding the shown to penetrate the hair bre into the cortex and to bind to
formation of bonds between the peptide and the hair. Peptide the hair proteins. Due to this binding, they induced a signicant
interactions are affected by their functional groups, as well as by recovery in the tensile strength and elasticity of severely
their involvement in building new bonds or interactions with damaged hair. Of the eight tested peptides, PepE, PepG and KP
hair proteins. Peptides have the ability to bind to hair proteins, showed the highest ability to interact with a keratin peptide
be absorbed by the hair bre, improve damaged hair mechan- model. Based on this result, the three peptides were explored as
ical properties and aid the straightening of curly hair. Each modulators of hair shape. When applied to curly hair, these
peptide has a specic amino acidic sequence which is crucial peptides were shown to enable a high straightening efficiency
for its behaviour towards hair proteins. Due to the conforma- without the use of harsh chemicals. These results were sup-
tion of each peptide, the nal results are highly dependent on ported by molecular dynamics simulations and by MALDI-TOF/
the reactivity of each cysteine and its accessibility; on the resi- TOF, which demonstrated effective conjugation between the
dues in the vicinity of the cysteine; on the charge–charge small peptides and the peptide hair protein model, based on the
interactions and on the location of the cysteine.67 Basically, the re-conformation of disulphide bonds. Peptide treatments at
peptide mechanism involves the breakage of the disulphide neutral pH involve the formation of intra and intermolecular
bonds between hair keratin laments and then the re- disulphide bonds between cysteine-based peptides and hair
modulation of the hair occurs by the disulphide bond rear- proteins and show great potential for cosmetic use as modula-
rangements between the hair proteins and peptides. The forma- tors of hair shape. The new method here proposed, based on
tion of the disulphide bonds between the cysteine thiols peptide formulations, is environmentally friendly and consti-
presented in the peptides with the hair keratin or KAPs is due to tutes a real alternative to the conventional chemical treatments,
the reduction of the hair protein disulphide bonds and the opening a new chapter for a green haircare cosmetic industry.
reformation of these disulphide bonds with the thiol of the
peptides and even with themselves. Sulphur atoms of cysteines
may also be linked by hydrogen bonds or electrostatic interac- Conflicts of interest
tions.67 PepE, despite presenting a negative charge during the There are no conicts to declare.
treatment, improved the hair's mechanical properties. However,
its efficiency as a straightening agent was not as evident as that of
peptides PepG and KP. This is mainly due to the eventual Acknowledgements
formation of assemblies based on hydrophobic and polar inter-
actions in PepE. This type of binding and penetration prole is This work was supported by the Portuguese Foundation for
possible in peptides with net negative charge.28,68 PepG and KP Science and Technology (FCT) under the scope of the strategic
present net negative charges; however, their abilities to form funding of the UID/BIO/04469/2013 unit and COMPETE 2020
disulphide bonds with the hair bre were more effective. Their (POCI-01-0145-FEDER-006684) and under the Project RECI/
conformation and the accessibility of their cysteine residues were BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). This
imperative for the higher conjugation displayed with hair keratin study was also supported by BioTecNorte operation (NORTE-01-
which led to the effective recovery of chemically damaged hair 0145-FEDER-000004) funded by the European Regional Devel-
and efficient straightening of African hair. The effect of the opment Fund under the scope of Norte2020 – Programa Oper-
treatments with these peptides was sustained for at least 20 acional Regional do Norte. Célia F. Cruz and Artur Ribeiro
washes with shampoo, conrming the long durability of the thank FCT for SFRH/BD/100927/2014 and SFRH/BPD/98388/
treatments. 2013 grants, respectively.

51590 | RSC Adv., 2017, 7, 51581–51592 This journal is © The Royal Society of Chemistry 2017
Paper RSC Advances

27 Principles of Polymer Science and Technology in Cosmetics and

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51592 | RSC Adv., 2017, 7, 51581–51592 This journal is © The Royal Society of Chemistry 2017