Helene Hine, Neil James, Kathryn McNeil – Croda, UK
HAIR CARE
Proteomics: next generation
biomimetic hair care actives
Biomimetics is a cutting edge technology
platform that involves the concept of taking
inspiration from nature; its models, systems
and processes and exploiting them to meet
consumer demands. In personal care,
the biomimetic approach refers to using
proteins naturally found in skin and hair.
Keratin proteins have commonly been
used in hair care products as a biomimetic
solution to replenish and restore the hair
and recent innovations in biopolymer
synthesis have taken biomimetic keratin
one step further.
Laurdimonium Hydroxypropyl Hydrolyzed
Keratin is a keratin mimetic that replaces
‘like with like’ by mimicking the amino acid
sequences found in the hair’s natural
composition.
Product synthesis
Maintaining hair architecture at the
molecular level is important to achieve
healthy hair structure. Ultimately, proper
hair protein structure has an important
impact on the cosmetic value of the
hair shaft. About 95% of human hair’s
constitution is based on proteins, the
majority of which are keratin proteins.
Keratins belong to a multigene family
that comprises more than 30 members.
They are grouped into type I acidic
proteins and type II basic to neutral
proteins which form the 10 nm
intermediate filament network in epithelial
cells. Depending on their function and
expression site, keratins can be divided
into either the large group of epithelial
cytokeratins (i.e. soft a-keratins) expressed
in the various types of epithelia or the
hair keratins (i.e. hard a-keratins), which
are involved in the formation of hard
keratinised structures such as hairs, nails,
hoofs, and claws.
The human hair proteome has been
investigated using two dimensional Liquid
Chromatography and Tandem Mass
Spectrometry (LCMS/MS). Several hundred
proteins have been identified and fully
sequenced, many of which are keratin
proteins. These human hair keratin protein
sequences are available on public
databases such as UniProt (Universal
Rod
Head
IA
IB
2
L1
Tail
L2
Figure 1: Keratin proteins with a central rod domain dominated by a-helical subsegments (1A,
1B and 2) and separated by short linker regions (L1 and L2). The rod is flanked by non-helical
head and tail domains at the amino and carboxyl termini, respectively.
Protein Resource). UniProt is a
comprehensive, high quality and freely
accessible database of protein sequence
and functional information. The database is
a central access point for extensive curated
protein information, including function,
classification, and crossreference and is
available at www.uniprot.org. Keratins are
highly conserved proteins across species,
meaning there is high sequence homology
within the keratin protein family.
The term keratin protein covers a class
of proteins which includes intermediate
filament proteins (IFPs) and intermediate
filament associated proteins (IFAPs) that
are known to occur in nature in a variety
of different cell types. The IFPs are so
named because they have been found to
be associated in intermediate filaments
(IFs), a class of intracellular filamentous
structures that are intermediate in size
between microtubules and microfilaments.
Keratin intermediate filaments consist of
a globular head region, a highly conserved
helical rod domain and a globular tail
domain. The rod domain itself is composed
of a-helical segments, named segment
Coil 1A, 1B and Coil 2A and 2B, which
are separated from each other by globular
linkers. It is this complex matrix of helical
protein bundles and amorphous protein
junctions that are the basis for the special
mechanical properties of the composite
human hair.
In the past, manufacturers have
hydrolysed keratin protein sources using
conventional methods to develop products
that meet the ‘like with like’ trend. Croda
has taken the next step forward to further
build on this platform and to deliver
advanced claims for the next generation
of hair care products. Croda has targeted
peptides in these a-helical rod domains,
which are responsible for the structural
integrity of hair, as the foundation for its
new keratin mimetic. Building on Croda’s
Primary ions
Mass spectrometer
Hair fibre
Figure 2: ToFSIMS uses primary ions to create an image of the hair’s surface.
September 2015 P E R S O N A L C A R E
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HAIR CARE
Root
Middle
Tip
Figure 3: ToFSIMS images compiled using the mass spectrometer data, each pixel represents a mass spec peak.
The green highlighted areas represent deposition of active onto different parts of the hair fibre.
extensive knowledge of biopolymer and
keratin products, a new synthesis
methodology has been developed in order
to create defined molecular mass species
from a keratin source.
The first hurdle to overcome involves
breaking the disulfide bridges present in
keratin. These disulfide bridges create a
helix shape that is extremely robust, as
sulfur atoms from cysteine residues bond
to each other from across the helix,
creating a fibrous matrix which is not
readily soluble. Once the disulphide bridges
have been cleaved, a dual system of
endoprotease and exopeptidase enzymes
are used to hydrolyse keratin to yield the
peptides of interest. The use of this novel
synthesis process ensures the keratin
mimetic ingredient contains biofunctional
peptides that replicate amino acid
sequences naturally found in hair keratin
proteins. Croda has proven this using the
science of proteomics. The peptide
sequences of the keratin components of
this ingredient were determined by Tandem
Mass Spectrometry technique (MSMS),
equipped with electrospray ionisation (ESI),
time of flight (ToF)–MS in coupling with
quadruple MS (ESIQUADTOFMSMS).
A Basic Local Alignment Search Tool
(BLAST) was used to screen each peptide
to see which human hair keratin proteins
contained the peptide in question.
These biofunctional peptides are
ideal chemical precursors for cationic
conditioning agents as they contain amino
functional groups as part of their structure.
The final step in the process involves
Table 1: Control shampoo formulation.
Product
Water
INCI Name
Aqua
% w/w
qs
Sodium Lauryl Ether Sulphate [23% Active SLES solution]
20.00
Crodateric CAB 301
Aqua (and) Cocamidopropyl Betaine
15.00
Euxyl K1002
Benzyl Alcohol (and) Methylchloroisothiazolinone
(and) Methylisothiazolinone
Sodium Chloride
0.15
qs
Suppliers: 1 Croda 2 Schülke & Mayr
Table 2: Shampoo formulation containing 0.2% active of the keratin mimetic.
Product
Water
INCI Name
Aqua
Sodium Lauryl Ether Sulphate [23% Active SLES solution]
% w/w
qs
20.00
Crodateric CAB 301
Aqua (and) Cocamidopropyl Betaine
5.00
Versathix1
PEG150 Pentaerythrityl Tetrastearate (and) PPG2 Hydroxethyl
Cocamide (and) Aqua
3.00
Kerestore 2.01
Aqua (and) Laurdimonium Hydroxypropyl Hydrolyzed Keratin
1.00
Crovol A701
PEG60 Almond Glycerides
0.50
Euxyl K1002
Benzyl Alcohol (and) Methylchloroisothiazolinone (and)
Methylisothiazolinone)
0.15
Suppliers: 1 Croda 2 Schülke & Mayr
42
P E R S O N A L C A R E September 2015
grafting quaternary ammonium groups onto
the peptide backbone to become covalently
bound. The resulting product is a mixture of
biofunctional peptides with a chemically
bound quaternary ammonium group, which
imparts a cationic charge. These cationic
groups enhance the product’s substantivity
to anionic substrates, such as hair. As a
result the product enhances consumer
sensory and conditioning properties.
Claim substantiation
of targeted repair
Constantly under attack from everyday
styling, chemical processing and UV
exposure, the hair cuticle has a major
impact on the appearance and
manageability of the hair. Damage to the
cuticle directly affects the look, feel and
condition of the hair.
Analysis of a hair fibre from root to tip
demonstrates that the degree of damage
to the cuticle increases towards the tip as
exposure to chemical treatments and
mechanical manipulation takes its toll.
At the root which is the closest
resemblance to virgin hair the least
amount of damage has occurred of any
section of the hair while repetitive combing,
drying, straightening and curling all start to
impact upon the health of the midsection
of the hair. After months of exposure to
colourants, chemical treatments and
various other styling treatments, the hair
tip has the most cuticular damage.
Time of Flight Secondary Ion Mass
Spectroscopy (ToFSIMS) is a novel method
of acquiring detailed information about
the surface chemistry of hair. ToFSIMS
is a technique which provides detailed
information about the surface chemistry of
human hair by visually mapping the hair
fibre and the deposition of actives on the
hair fibre.
The ToFSIMS method shown
schematically in Figure 2 involves the firing
of primary ions directed to the surface of a
HAIR CARE
Hair evaluation
after shampooing
In order to assess the conditioning benefits
delivered by the keratin mimetic from a
shampoo formulation, a comprehensive
build up and wash off hair sensory
study was conducted. Four successive
applications of a basic shampoo (Table 1)
or a shampoo containing the keratin
mimetic (Table 2) were performed in order
to assess build up. This was followed by
four consecutive washes in order to evaluate
the lasting benefits of the keratin active.
8
*
Quantitative sensory scale
Statistically significant compared to control shampoo where p=<0.05
*
7
6
5
*
*
3
2
1
0
Wet tangle removal
Wet ease of combing
Wet smoothness
n Treatment 1 (with Kerestore 2.0) n Treatment 4 (with Kerestore 2.0) n Wash n Control shampoo
Figure 4: Wet sensory properties of bleached European hair are greatly improved following
treatment with a shampoo containing the keratin mimetic.
The performance was assessed by
Croda’s specialist in-house method of
evaluating the sensorial impact of actives
on the hair. Objective studies were carried
out by a trained panel of experts profiling
hair attributes against industry benchmarks.
The panel follows a strict testing routine,
which includes calibration sessions and
statistical reproducibility. The sensory
analysis of three wet attributes, was
performed after each treatment, the results
of which can be seen in the Figure 4.
The sensory performance of the control
shampoo, which is represented by the
yellow line, does not change over time.
This provides a baseline for comparison
while also validating the results obtained
by Croda’s trained panellists, demonstrating
their reproducibility.
The red bars represent the sensory
performance of the active shampoo after
one treatment, while the purple bars
illustrate the sensory performance of the
active shampoo after four treatments.
Finally, the green bars show the sensory
profile of the hair after four washes with
the active shampoo followed by four
washes with the control shampoo.
When compared to a control shampoo,
the keratin mimetic can greatly improve the
sensory properties of the hair while wet.
These benefits are noticeable from the first
application and continue to improve with
INCI Name
% w/w
Aqua
Incroquat™ CTC301
(Aqua (and) Cetrimonium Chloride
3.20
Crodacol™ CS901
Cetearyl Alcohol
5.00
Preservative
To 100.00
qs
Lactic Acid [To pH 55.5]
Supplier: 1 Croda
*
4
Water
Kerestore 2.01
*
*
*
Table 3: Base conditioner formulation containing 1% active of the keratin mimetic.
Product
*
*
Improved wet sensory properties
hair fibre which dislodge species from the
cuticle. Although most of the dislodged
species are neutral, between 1% and 10%
are charged these are known as secondary
ions. The dislodged species are identified
by a mass spectrometer then used to
map areas of damage to the hair surface
and regions where active ingredients
have been deposited on treated samples.
The secondary ions are focused by charged
detector plates and analysed by a mass
spectrometer, allowing an image to be
created, with each pixel representing a
peak in the mass spectrum.
This high resolution, mass spectroscopy
method has been used to identify and
map the deposition of the keratin mimetic
ingredient along the hair fibre (Fig. 3),
demonstrating that it deposits on the
most damaged areas of the hair cuticle,
intelligently repairing the most damaged
areas of the hair’s surface. In this particular
study, European hair tresses were bleached
and then treated with an aqueous solution
of the keratin mimetic ingredient.
The image map produced for the tresses
treated with the active ingredient, Figure 3,
shows the deposition of the active at the
most damaged areas of the hair’s surface.
The hair becomes progressively more
anionic from root to tip as the degree of
damage increases. There is less deposition
of keratin mimetic at the root of the hair
fibre, where there is less damage and
extensive coverage at the severely damaged
tip. The images prove that this keratin
mimetic ingredient provides intelligent
repair by selectively targeting the areas of
the hair surface with the most damage.
Laurdimonium Hydroxypropyl Hydrolyzed Keratin
5.00
successive treatments. Furthermore, this
improved sensory performance remains
after four wash cycles, demonstrating that
this keratin active can impart enhanced
long-lasting sensory performance from
shampoo formulations.
Hair evaluation after
conditioning
The look and feel of the hair is most
obviously related to the hair cuticle.
Everyday grooming methods such as
combing and styling practices, as well as
chemical treatments and UV exposure,
cause damage to the hair cuticle. This
leads to increased friction between strands,
cuticle decementation and force required
to pull a comb through the hair. Care for
the cuticle requires good conditioning
which improves the appearance of hair and
makes combing easier. Measuring the force
required to comb a hair tress pre- and
post-treatment allows the conditioning
effect of an active to be calculated.
Conditioner evaluation
on European hair tresses
One of the most damaging cosmetic hair
treatments commonly used in Europe is
bleaching. In order to measure the ability of
this keratin mimetic to treat damaged
European hair, tresses were bleached and
treated with either a basic conditioner or a
basic conditioner containing 1% active of
the ingredient, as shown in Table 3. The
basic conditioner used is exactly the same
but without the keratin active.
The tresses were tested using the Mini
Tensile Tester to assess their effectiveness
at reducing combing forces. The change in
force required to pull a comb through the
hair tress before and after treatment
provides an indication of the conditioning
properties of the active.
The change in total work required to
comb the hair tress after treatment with
September 2015 P E R S O N A L C A R E
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HAIR CARE
the keratin mimetic ingredient is
significantly reduced by 21.5% compared
to a basic conditioner (Fig. 5). This clearly
demonstrates the excellent conditioning
properties of this ingredient.
0
0
–10
–10
–20
–20
–30
–40
–50
–60
–70
–80
–90
–100
Statistically significant
compared to basic
conditioner where
p=<0.05
*
*
Statistically significant
compared to basic
conditioner where
p=<0.05
basic conditioner containing the keratin
mimetic ingredient (see Table 3 for the
formulation). The change in total work
required to comb the hair tress after
treatment with the active ingredient is
significantly reduced by 9% compared to
a basic conditioner (Fig. 6).
composition to move biomimetics to the
next generation. Using the powerful
technology of proteomics, we have proved
that this active ingredient mimics the
proteins found in the hair’s natural
composition.The cuticle has a major impact
on the appearance and manageability of
hair and is therefore the target of most hair
care products. This keratin mimetic is a
mixture of biofunctional peptides with a
chemically bound quaternary ammonium
group, which imparts a cationic charge.
Quaternisation of this highly specialised
keratin protein enhances the substantivity
to the cuticular surface of the hair. This
ensures the active ingredient deposits at
the most damaged sites of the cuticle
where it is needed most. Images produced
by the advanced ToFSIMS technique prove
that this keratin mimetic provides intelligent
repair by selectively targeting the most
damaged areas of the hair’s surface.
The visual and sensorial health status of
hair is directly related to the condition of
the hair cuticle. Hair with damaged cuticles
feels rough and creates a greater force
required to pull a comb through the hair.
In a sensory study by a trained panel the
keratin mimetic was shown to improve
wet tangle removal, wet combing and
wet smoothness, with an effect that
remains even after four washes. In addition
using instrumental combing studies the
excellent conditioning properties of this
keratin mimetic ingredient have been
demonstrated.
Substantiated by the latest techniques
in protein sequencing, advanced imaging
techniques and consumer relevant combing
studies this product helps formulators meet
the demands of today’s science savvy
consumer who is also looking for real
PC
results.
Conclusion
Laurdimonium Hydroxypropyl Hydrolyzed
Keratin is a keratin quat that mimics
the proteins found in the hair’s natural
Cetearyl Alcohol
11.16
Crodacol CS90EP1
Cetearyl Alcohol
1.33
Tween 60
Polysorbate 60
2.67
Solan E
PEG75 Lanolin
1.33
Polychol15
Laneth15
1.33
White Petroleum Jelly
Petrolatum
11.00
Paraffinum Liquidum
10.00
Aqueous Phase
Propylene Glycol
4.00
Sodium Hydroxide
2.40
Aqua
To 100.00
Supplier: 1: Croda
Table 5: Neutralising shampoo used on relaxed Brazilian hair.
Product
INCI Name
Crodateric CAB 301
Aqua (and) Cocoamidopropyl Betaine
5.0
Incromine Oxide1
Aqua (and) Cocoamidopropylamine Oxide
3.0
Deionised Water
Aqua
Ammonium Lauryl Sulphate [30%]
P E R S O N A L C A R E September 2015
–70
Figure 6: Average percentage change in
total work required to comb relaxed Brazilian
tress after treatment with either the control
or the keratin mimetic.
Crodacol CS501
44
–60
Figure 5: Average percentage change in total
work required to comb bleached European
tresses after treatment with either the control
or the keratin mimetic ingredient.
Oil Phase
Supplier: 1: Croda
–50
–100
% w/w
Citric Acid
–40
–90
*
INCI Name
Deionised Water
–30
–80
*
Table 4: Hair relaxer system used in the combing study on Brazilian hair.
Product
n Control conditioner
n Conditioner with Kerestore (1% active)
Change in total work (%)
Human hair differs vastly around the world
and within Latin America there is a wide
range of hair types, many of which
are excessively curly and difficult to style.
Chemical straightening is a common
cosmetic treatment in Latin America to
tame curly and unruly tresses, it works by
breaking disulphide bonds, reshaping the
hair into a straight style and then reforming
the bonds. Although considered permanent,
this method of straightening is generally
repeated every 6 weeks to 3 months and
the caustic ingredients in these relaxer
systems are known to cause damage to
the hair fibres, resulting in raised cuticles
and thus making combing more difficult.
A lye relaxer system (Table 4) was applied
to virgin Brazilian hair with mechanical
straightening and left on the hair for 20
minutes. The relaxer cream was then rinsed
off with water before the tress was washed
with a neutralising shampoo (Table 5).
The hair was then washed a further two
times with the neutralising shampoo before
allowing the hair to dry naturally.
The relaxed tresses were then treated
with either a basic control conditioner or a
Change in total work (%)
Conditioner study on relaxed
Brazilian hair tresses
n Control conditioner
n Conditioner with Kerestore (1% active)
% w/w
20.0
1.0
To 100