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Cosmetic effectiveness of topically applied hydrolysed
keratin peptides and lipids derived from wool
C. Barba
1
,S.Me
´ndez
1
, A. Roddick-Lanzilotta
2
, R. Kelly
3
, J. L. Parra
1
and L. Coderch
1
1
Department of Surfactant Technology, Chemical and Environmental Research Institute of Barcelona, Barcelona, Spain,
2
Canesis Network Limited,
Christchurch, New Zealand and
3
Keratec Limited, Canterbury, New Zealand
Background/purpose: Skin moisturisation, elasticity, feel
and appearance can all be improved through the topical
application of protein hydrolysates. Recent studies suggest
that supplementing intercellular lipids of the stratum cor-
neum can enhance the functioning of the skin.
Methods: In this study, a hydrolysed keratin peptide (mo-
lecular weight o1000 Da) was prepared from wool and
tested on skin in two different formulations: an aqueous
solution and an internal wool lipids (IWL) liposome suspen-
sion. In vivo long-term studies were performed to evaluate
the water barrier function of the skin after topical application
of different formulations. During the treatment period, hy-
dration and elasticity were determined. A sorption –deso-
rption test was also performed to assess the hygroscopic
properties and water-holding capacity of the different trea-
ted skin sites.
Results: Significant differences were found between the
control and treated sites, with the treated areas showing
an increase in hydration and elasticity as a result of keratin
peptide application. Measurements also indicated that the
keratin formulations reinforce the skin barrier integrity,
improving its water-holding capacity.
Conclusion: A combination of the keratin peptide with the
IWL showed beneficial effects, indicating that this combina-
tion is suitable for designing new cosmetics products.
Key words: keratin peptides – internal wool lipids – lipo-
somes – hydration – elasticity – sorption –desorption test
&Blackwell Munksgaard, 2007
Accepted for publication 24 May 2007
WOOL IS primarily (85–95%) composed of
keratin proteins that combine to give it
desirable properties such as strength, insolubility
and moisture regain. Different classes of keratin
proteins are represented in the complex macro-
molecular structure, each of which has specific
functions and characteristics. Protein hydroly-
sates from various sources have long been used
in skin and hair personal care products and are
known to confer improved compatibility, feel,
moisturisation and help maintain the natural
structure (1, 2) by interacting favourably with
the keratin and other components of skin and
hair (3).
Wool also contains a minor (1.5%) internal
lipid component that is also known to impact on
the physical, chemical and mechanical properties
of the fibre (4). Internal wool lipids (IWL) are rich
in cholesterol, free fatty acids, cholesterol sul-
phate and ceramides and are very similar in both
structure and composition to those membranes of
other keratinic tissues such as human hair and
skin stratum corneum (5).
Recent studies have shown that formulations
containing lipids that are similar to the natural
components of the skin and, in particular some
ceramide supplementation, can improve disturbed
skin conditions. IWL extracts have been shown to
form liposomes with a stable bilayer structure (6, 7)
and topical application of IWL liposomes on intact
and disturbed skin has been demonstrated to
improve the skin barrier properties (8, 9).
In this work, the effect on the skin of a novel
wool keratin peptide, obtained from an enzy-
matic hydrolysis of the intermediate filament
protein, is investigated. Depending on the mole-
cular weight and chemical composition, the abil-
ity of peptides to penetrate into the skin and
provide nutrition, moisturisation and skin pro-
tection, is well established (2, 10).
The aim of this work is to determine the effect
of the wool keratin peptide when applied topi-
cally on the water-holding capacity, hydration
and elasticity of undisturbed skin. Two different
formulations, an aqueous solution and IWL lipo-
some solution, have been evaluated.
243
Skin Research and Technology 2008; 14: 243–248
Printed in Singapore All rights reserved
doi: 10.1111/j.1600-0846.2007.00280.x
r2007 The Authors
Journal compilation r2007 Blackwell Munksgaard
Skin Research and Technology
Materials and Methods
Chemicals
Chloroform (Merck, Darmstadt, Germany), kera-
tin peptide (Keratec Limited, Christchurch, New
Zealand), methanol (Merck) and sodium chloride
(Panreac, Barcelona, Spain) were used.
Sample preparation
The IWL were soxhlet extracted with a chloro-
form/methanol azeotrope (11). IWL liposomes
were prepared by dissolving the IWL in chloro-
form/methanol 2:1 (v/v) and evaporating to
dryness under a stream of dry nitrogen to form
a thin film on the flask. The film was hydrated
with 0.9% NaCl solution to give a final suspen-
sion that contained 2% liposomes. Multilamellar
vesicle liposomes were formed by sonication of
the suspension in a sonicator, Labsonic 1510 (B.
Braun, Melsungen, Germany), at 100 W for about
15 min, maintaining the temperature at 65 1C.
Two different keratin formulations were pre-
pared for application on the skin: keratin sample 1,
an aqueous keratin formulation, where a 15%
solution of the keratin peptide was diluted to a
final concentration of 0.5%, and keratin sample 2,a
liposome formulation prepared by mixing the
IWL liposomes (2%) with the aqueous keratin
solution to obtain a final concentration of 0.5% of
the keratin peptide and 2% of IWL.
Subjects
Six healthy Caucasian volunteers (all females)
phototype III–IV, mean age 28 6 years (range
24–36 years), participated in both studies (Table
1). The subjects were advised to avoid topical
drugs or moisturisers on the tested zones for a
week before the experiments. To obtain reliable
measurements, the volunteers were acclimatised
for 15 min in a conditioned room (20 1C, 60% RH)
before the experiments.
Biophysical measurements
Skin hydration was determined using a Corne-
ometer CM 85 (Courage & Khazaka, Colonge,
Germany), which measures skin capacitance in
arbitrary units (AU). Elasticity was determined
by a Cutometer SEM 575 (Courage & Khazaka)
using Mode 1 where the measurements are per-
formed with a constant negative pressure. Results
are visualised in a curve that points out the
viscoelastic qualities of the skin. The parameters
that were considered in these studies are: R5, the
net elasticity, and R7, portion of elasticity com-
pared with the complete curve (the closer these
parameters to 1, the more elastic the skin). All
parameters were recorded in accordance with
established guidelines (12–14).
Efficacy of the keratin peptides on healthy human skin
A long-term study was performed to test the
effect of the keratin formulations when applied
repeatedly to undisturbed skin (15). Baseline
measurements of skin capacitance and skin elas-
ticity were taken on six marked zones of the volar
forearm of the volunteers before topical applica-
tion: three zones for topical treatment of the
different samples [keratin peptide aqueous solu-
tion (keratin sample 1), keratin peptide liposome
solution (keratin sample 2), and IWL liposomes]
and two zones for the placebo solutions [water
(placebo 1) and 0.9% NaCl solution (placebo 2)].
Placebos and solutions (50 mL) were randomly
applied onto marked areas of 9 cm
2
using an
Exmire microsyringe (ITO Corp., Fuji, Japan).
After 24 h all biophysical parameters, skin capa-
citance and skin elasticity, were evaluated and
then 50 mL of the solutions were applied again.
The application of the solutions was repeated for
three more days and the parameters were mea-
sured on days 2, 3, 4 and 7 (3 days after the last
application).
Sorption–desorption test
Following the long-term study, a sorption–deso-
rption test (16, 17) was performed on the sites
treated with the keratin peptides. This test is
based on a kinetic method to measure stratum
corneum water uptake and water-holding capacity
following the application of the different samples.
Baseline measurement of skin capacitance was
takenoneachofthethreetestareas.Then,
250 mL of distilled water was applied onto each
test area for 90 s using an Exmire microsyringe
TABLE1. Age and skin phototype of the volunteers who participated in the
study
Volunteer Age Phototype
1 24 III
230IV
3 24 III
423IV
536IV
635IV
244
Barba et al.
(ITO Corp.). Afterwards, the water droplet was
removed with a soft paper towel and the water
desorption kinetics was recorded every 90 s (t
0
,
t
90
,t
180
,t
270
,t
360
,t
450
,t
540
and t
630
) using a
Corneometer.
Data treatment
The mean values and standard deviations (SD)
were calculated. Dixon’s test was used for detect-
ing outliers, which were excluded from the data.
One-way analysis of variance, with the Kruskal–
Wallis test, was used to determine significant
differences between the values obtained from
the different treatments (significance level ac-
cepted
*
Po0.05).
Results and Discussion
Efficacy of the keratin peptides on undisturbed skin
A long-term study was performed on undisturbed
skin to determine the efficacy of the keratin pep-
tide samples (18, 19). Evaluation of skin capaci-
tance and skin elasticity was performed 24h after
a daily application (days 1, 2, 3 and 4) and 3 days
after the last application (day 7).
The mean values of skin capacitance and elas-
ticity parameters for the different treatments were
calculated and are listed in Table 2 and Table 3.
Significant differences were obtained for skin
capacitance and elasticity parameters with appli-
cation of the keratin samples.
Values for skin capacitance (Table 2) show a
greater increase on the skin capacitance in the
sites to which keratin samples were applied
during the treatment period. Figure 1 displays
the variation of the skin capacitance after sample
application during the treatment period. Changes
were evaluated vs. both the basal and the place-
bos values. It was observed that the zones treated
with the keratin peptide samples had a higher
skin capacitance and this was maintained 3 days
after the last application. Keratin peptide samples
1 and 2 showed very similar abilities to increase
the hydration of the skin, indicating that the
peptides are effective when applied from an
aqueous solution or in liposome form.
Evaluation of the parameters R5 and R7 for the
skin elasticity (Table 3) showed a trend of
increasing elasticity for the zones to which the
keratin peptides were applied. The keratin solu-
tion 1 yielded an increase in skin elasticity that
was maintained over the duration of the treat-
TABLE2. Skin capacitance values (mean values SD) obtained before (basal) and during the treatment period
Zones
Time (days)
Basal 1 2 3 4 7
Placebo 1 37.78 4.37 39.00 5.93 39.61 4.78 41.28 6.15 38.78 5.21 38.28 5.11
Placebo 2 39.67 8.58 41.28 6.93 38.56 5.63 39.00 8.81 40.78 5.73 35.78 6.70
IWL liposomes 38.95 8.54 41.28 6.59 39.50 4.64 40.94 5.68 38.89 7.69 35.50 6.46
Keratin sample 1 35.56 5.30 39.00 5.48 38.61 5.83 41.29 4.99 41.06 5.83 41.33 5.89
Keratin sample 2 37.61 6.64 40.94 6.36 40.72 5.55 40.61 8.25 41.28 3.63 40.11 3.97
IWL, internal wool lipids; SD, standard deviation.
TABLE3. Skin elasticity parameter values (mean values SD) obtained before (basal) and during the treatment period
Parameter Zones
Time (days)
Basal 1 2 3 4 7
R5 Placebo 1 0.811 0.10 0.826 0.08 0.814 0.08 0.812 0.11 0.826 0.08 0.818 0.10
Placebo 2 0.781 0.06 0.704 0.11 0.740 0.14 0.730 0.14 0.730 0.18 0.764 0.12
IWL liposomes 0.728 0.15 0.693 0.13 0.744 0.16 0.631 0.13 0.726 0.15 0.774 0.13
Keratin sample 1 0.777 0.13 0.834 0.09 0.825 0.06 0.789 0.09 0.824 0.07 0.825 0.10
Keratin sample 2 0.763 0.11 0.729 0.12 0.838 0.16 0.731 0.12 0.837 0.12 0.805 0.09
R7 Placebo 1 0.670 0.07 0.673 0.07 0.668 0.07 0.671 0.08 0.686 0.05 0.667 0.07
Placebo 2 0.640 0.04 0.589 0.08 0.629 0.10 0.605 0.10 0.613 0.12 0.643 0.10
IWL liposomes 0.520 0.10 0.622 0.07 0.565 0.10 0.569 0.11 0.605 0.12 0.629 0.07
Keratin sample 1 0.616 0.09 0.680 0.08 0.653 0.08 0.638 0.07 0.679 0.07 0.658 0.12
Keratin sample 2 0.641 0.09 0.625 0.09 0.688 0.12 0.626 0.09 0.691 0.09 0.683 0.07
IWL, internal wool lipids; SD, standard deviation.
245
Cosmetic effectiveness of topically applied keratin peptides and lipids
ment period. The combination of the keratin
peptide with the IWL liposomes showed a sig-
nificant beneficial effect, resulting in a 17% in-
crease in skin elasticity during the treatment
period. An increase in skin elasticity was also
obtained for the zones treated with the IWL
liposomes only, supporting the beneficial effects
of skin lipid supplementation (Fig. 2 and Fig. 3).
The results obtained in this first study show
that application of the keratin peptides conferred
beneficial effects to the skin. These effects were
demonstrated by an increase in the skin hydra-
tion and elasticity. Wool keratin peptides, with a
low molecular weight range (o1000 Da), are able
to penetrate into the skin and improve the water-
holding capacity of the skin, as demonstrated by
an increase in its hydration and elasticity. A
combination of the keratin peptides with the
IWL extract in the form of liposomes confers
similar beneficial effects on hydration and im-
proves elasticity. Therefore, this combination may
be a good approach when formulating new
cosmetic products for skin care, as it combines
the ability to increase skin hydration and elasti-
city due to the presence of keratin peptides and
also offers the potential to increase the skin
barrier and decrease transepidermal water loss
attributed to the presence of IWL (8, 9).
Sorption–desorption test
This method is a dynamic measurement of skin
capacitance using a non-invasive test (16, 17, 20).
Following the application of water to the skin, the
capacitance value increased sharply, and then
declined slowly with an almost complete recovery
to the baseline value reached after 10min (Table 4).
90.00
95.00
100.00
105.00
110.00
115.00
120.00
12 347
Time
(
Da
y
s
)
Skin Capacitance Change (%)
Keratin sample 1
Keratin sample 2
IWL Liposomes
*
*
**
*
Fig. 1. Variation of skin capacitance after sample application during
the treatment period. Changes were evaluated vs. both basal and
placebo values (
*
Po0.05, calculated between samples and placebos).
90.00
95.00
100.00
105.00
110.00
115.00
120.00
12345
Time (Da
y
s)
Elasticity R5 Change (%)
Keratin sample 1
Keratin sample 2
IWL Liposomes
*
Fig. 2. Variation of the elasticity parameter R5 after sample applica-
tion on volunteers during the treatment period. Changes were
evaluated vs. basal and placebos values. (
*
Po0.05, calculated between
samples and placebos).
90.00
95.00
100.00
105.00
110.00
115.00
12345
Time (Days)
Elasticity R7 Change (%)
Keratin sample 1
Keratin sample 2
IWL Liposomes
*
*
*
Fig. 3. Variation of the elasticity parameter R7 after sample applica-
tion on volunteers during the treatment period. Changes were
evaluated vs. both basal and placebo values. (
*
Po0.05, calculated
between samples and placebos).
TABLE4. Skin capacitance values (mean values SD) obtained before
(BV) and during the treatment period
Time (s)
Samples
Water Keratin sample 1 Keratin sample 2
BV 42.81 5.27 42.06 7.80 39.89 1.75
0 85.56 5.91 96.72 11.28 100.31 5.22
90 61.61 6.47 59.50 7.01 57.17 2.84
180 54.06 4.30 55.06 7.61 51.00 3.84
270 51.00 4.75 53.11 7.52 48.64 4.63
360 49.67 4.73 50.89 7.18 47.83 3.44
450 48.33 4.95 50.44 7.27 47.11 3.48
540 47.84 3.73 50.45 7.37 46.33 3.72
630 47.83 4.24 50.45 7.13 46.22 2.20
SD, standard deviation.
246
Barba et al.
To assess the final results, the baseline values
were subtracted to obtain the increase in capaci-
tance (in arbitrary units) at each time point
(added capacitance, AC); then, the ln(AC) was
calculated and plotted vs. time (Fig. 4). Figure 4
shows that the two zones treated with the keratin
peptides yielded a higher absorption (value at
time 0).
The sorption–desorption test demonstrated the
moisturisation properties of the keratin samples.
Higher values of initial absorption of water and a
greater capacity for retention were observed for
the areas treated with the keratin samples. There
was a slightly more pronounced effect for the
aqueous keratin peptide solution 1.
Conclusions
In summary, in the long-term study, the beneficial
effect of application of the wool keratin peptide
samples on healthy skin has been demonstrated.
Improved hydration and elasticity were observed
following treatment with the keratin peptide
samples. Furthermore, the moisture sorption–
desorption profile obtained showed higher va-
lues of initial absorption of water for the skin
zones treated with the two keratin formulations.
Higher skin elasticity values were observed fol-
lowing treatment with the IWL liposomes; these
results support the beneficial effects of skin lipid
supplementation with IWL, which have been
shown, in previous work, to strongly resemble
that of the stratum corneum.
Therefore, hydrolysed keratin peptides derived
from wool may be applied alone or combined
with wool internal lipids structured as liposomes,
improving in both cases the hydration, elasticity
and moisture sorption–desorption profile. This
new combination of derivatives from wool fibre
can be suitable for designing new pharmaceutical
or cosmetic products for skin care.
Acknowledgements
We thank all the volunteers who participated in
these trials. Thanks are also due to EVIC Hispa-
nia for the scholarship awarded to C. Barba.
References
1. Fo
¨ster TH, Waldmann-Laue M, Both W, Jassoy C. Lipo-
proteins creams: utilization of multifunctional ingredi-
ents for the preparation of cosmetic emulsions with
excellent skin compatibility. Int J Cosmet Sci 1999; 21:
253–264.
2. Teglia A, Secchi G. New protein ingredients for skin
detergency: native wheat protein–surfactant complexes.
Int J Cosmet Sci 1994; 16: 235–246.
3. Hart J, Polla C, Hull JC. Oat fractions. Cosmet Toiletries
1998; 113: 45–52.
4. Leeder JD. The cell membrane complex and its influence
on its properties of the wool fibre. Wool Sci Rev 1986; 63:
3–35.
5. Coderch L, de la Maza A, Soriano C, Erra P, Parra JL.
Chromatographic characterization of internal polar li-
pids from wool. J Am Oil Chem Soc 1995; 72: 1715–1720.
6. Coderch L, de la Maza A, Pinazo A, Parra JL. Physico-
chemical characteristics of liposomes formed with inter-
nal wool lipids. J Am Oil Chem Soc 1996; 73: 1713–1718.
7. Ko
¨rner A, Petrovic S, Ho
¨cker H. Cell membrane lipids of
wool and human hair form liposomes. Text Res J 1995;
65: 56–58.
8. De Pera M, Coderch L, Fonollosa J, de la Maza A, Parra
JL. Effect of internal wool lipid liposomes on skin repair.
Skin Pharmacol Appl Skin Physiol 2000; 13: 188–195.
9. Coderch L, de Pera M, Fonollosa J, de la Maza A, Parra
JL. Efficacy of stratum corneum lipid supplementation
on human skin. Contact Dermatitis 2002; 47: 139–146.
10. Teglia A, Mazzola G, Secchi G. Chemical characteristics
and cosmetic properties of protein hydrolysates. Cosmet
Toiletries 1993; 108: 56–65.
11. Coderch L, Fonollosa J, Martı
´M, Garde F, de la Maza A,
Parra JL. Extraction and analysis of ceramides from
internal wool lipids. J Am Oil Chem Soc 2002; 79: 1–6.
12. Berardesca E, Maibach HI. Transepidermal water loss
and skin surface hydration in the non invasive assess-
ment of stratum corneum function. Derm Beruf Umwelt
1990; 38: 50–53.
13. Berardesca E, the EEMCO Group. EEMCO Guidance for
the assessment of stratum corneum hydration: electrical
methods. Skin Res Technol 1997; 3: 126–132.
14. Rodrigues L, the EEMCO Group. EEMCO Guidance
to the in vivo assessment of tensile functional properties
of the skin. Skin Pharmacol Appl Skin Physiol 2001; 14:
52–67.
15. Fisher TW, Wigger-Alberti W, Elsner P. Assessment of
dry skin: current bioengineering methods and testing
designs. Skin Pharmacol Appl Skin Physiol 2001; 14:
183–195.
16. Agache P, Mary S, Muret P, Matta AM, Humbert P.
Assessment of the water content of the stratum corneum
using a sorption–desorption test. Dermatology 2001;
202: 308–313.
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
0 120 240 360 480 600 720 840
Time (s)
ln (AC)
Water
Keratin sample 1
Kerat in sample 2
Fig. 4. Plot of ln(AC) vs. time according to the sorption–desorption
test. AC, added capacitance.
247
Cosmetic effectiveness of topically applied keratin peptides and lipids
17. Hachiro T, Yuko K, Kunio I, Shoko S, Fumio I, Keiji I,
Kohdo Y, Mizuho Y. Water sorption–desorption test of
the skin in vivo for functional assessment of the stratum
corneum. J Invest Dermatol 1982; 78: 425–428.
18. Serup J, Winther A, Blichmann CW. Effects of repeated
application of a moisturizer. Acta Derm Venerol (Stock-
holm) 1989; 69: 457–459.
19. Gregor BE, Renhua NA. Hydration and plasticity fol-
lowing long-term use of a moisturizer: a single blind
study. Acta Derm Venerol 2002; 82: 322–324.
20. Fluhr JW, Lazzerini S, Distance F, Gloor M, Berardesca E.
Effects of prolonged occlusion on stratum corneum
barrier function and water holding capacity. Skin Phar-
macol Appl Skin Physiol 1999; 12: 193–198.
Address:
Clara Barba
Department of Surfactant Technology
Chemical and Environmental Research Institute of Barcelona
Jordi Girona 18-26
Barcelona 08034
Spain
Tel: 134 93 400 6179
Fax: 134 93 204 5904
e-mail: cbaesl@iiqab.csic.es
248
Barba et al.
