Efﬁcacy of antioxidants in human hair
, Blanca Martínez-Teipel
, Ricard Armengol
, Clara Barba
, Luisa Coderch
Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain
Provital, Barberá del Vallés, Barcelona, Spain
Received 11 June 2012
Received in revised form 14 September 2012
Accepted 25 September 2012
Available online 13 October 2012
Hair is exposed every day to a range of harmful effects such as sunlight, pollution, cosmetic treatments,
grooming practices and cleansing. The UV components of sunlight damage human hair, causing ﬁbre deg-
radation. UV-B attacks the melanin pigments and the protein fractions (keratin) of hair and UV-A produces
free radical/reactive oxygen species (ROS) through the interaction of endogenous photosensitizers. Hair
was dyed and the efﬁcacy of two antioxidant formulations was demonstrated after UV exposure by eval-
uating, surface morphology, protein and amino acid degradation, lipidic peroxidation, colour and shine
changes and strength/relaxation properties. UV treatment resulted in an increase in protein and lipid deg-
radation, changes in colour and shine and in adverse consequences for the mechanical properties. Natural
antioxidants obtained from artichoke and rice applied to pretreated hair improved mechanical properties
and preserved colour and shine of ﬁbres, coating them and protecting them against UV. Furthermore, the
lipidic peroxidation of the protein degradation caused by UV was reduced for some treated ﬁbres, suggest-
ing an improvement in ﬁbre integrity. This was more marked in the case of the ﬁbres treated using the
artichoke extract, whereas the rice extract was better preserving shine and colour of hair ﬁbres.
Ó2012 Elsevier B.V. All rights reserved.
It is well known that the UV components of sunlight damage
human hair. This observation has been reported using a laboratory
simulation of sunlight . UV irradiation (mainly UV-B) attacks
both the melanin pigments and the protein fractions (keratin) of
hair . The effects of UV-B irradiation can be severe, resulting
in the breakdown of disulﬁde bonds inside the hair ﬁbre and on
the surface of the cuticle. However, UV-A irradiation mainly pro-
duces free radical/reactive oxygen species (ROS) through interac-
tion with endogenous photosensitizers. Studies have shown that
photo-oxidation of hair ﬁbre involves the fracture of C–S linkages
from proteins , oxidation of internal lipids,  and melanin
granules  and tryptophan degradation of keratin . Moreover,
exposure to sunlight leads to hair decoloration due to melanin oxi-
dation via free radicals, [7,8]. Melanin is a homogeneous biological
polymer containing a population of intrinsic, semiquinone-like
radicals. There are two types of melanin, the brown–black pig-
ments (eumelanins) and the less prevalent red pigments (pheo-
melanins) . Melanin granules selectively absorb radiation and
offer photoprotection but become degraded or bleached in the pro-
The lipid and protein fractions play a major role in the structure
and integrity of the hair ﬁbre, protecting it against external agents.
Therefore, aggressions to the proteic and lipidic fraction [3,4] and
the degradation of amino acids susceptible to photodegradation
such as tryptophan, [6,9,11] can help us to evaluate the decompo-
sition of hair ﬁbre .
The most harmful effect of sunlight on hair is cystine oxidation
to cisteic acid, which alters its mechanical properties (long UV
exposure) . Shorter irradiations cause unwanted effects such
as a decrease in hydration, increased permeability, leading to a loss
of colour and shine and to an increase in combing resistance
The rapid increase in hair treatments (bleaching, dyeing, etc.)
has led to a proliferation of hair cosmetics that facilitate repair
and prevent adverse effects on the capillary structure. Vitamins
and antioxidants have been included in cosmetic formulations spe-
cially designed to reduce the adverse effects on hair ﬁbre. The most
effective ingredients are antioxidants that can interrupt radical-
chain processes, help to repair skin/hair systems, protect against
oxidative damage and are frequently used in food and cosmetics
. For instance, vitamin B5 has been used for many years in hair
care products because it functions as humectant, increases the
water content and improves the elasticity of hair . Incorpora-
tion of antioxidants, at low concentrations, in cosmetics can better
protect and possibly correct the damage by neutralising free radi-
cals and retard lipid oxidation.
The aim of this work is to study the efﬁcacy of different antiox-
idant formulations after application on hair subjected to UV radia-
tion and to the most common capillary treatments (dyeing). Two
1011-1344/$ - see front matter Ó2012 Elsevier B.V. All rights reserved.
Corresponding author. Tel.: +34 934 006 100; fax: +34 932 045 904.
E-mail address: email@example.com (E. Fernández).
Journal of Photochemistry and Photobiology B: Biology 117 (2012) 146–156
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journal homepage: www.elsevier.com/locate/jphotobiol
natural antioxidant active ingredients were selected to evaluate
their effect on hair ﬁbres; a Cynara scolymus L. extract obtained
from the leaves of artichoke (KERACYN tm) and an Oryza sativa L.
extract obtained from rice grain (KERARICE tm).
The leaves of artichoke (C. scolymus L.) contain the greatest con-
centration of active principles. Hydroxycinnamic acids (Fig. 1)in
artichoke have the general C6–C3 structure with the same aro-
matic ring and hydroxyl group as phenolic compounds, and a car-
boxylic function. These hydroxycinnamic acids are rarely free. They
are usually found in the form of derivatives, e.g., caffeic acid is
esteriﬁed with quinic acid, resulting in chlorogenic acid, isochloro-
genic acid and neochlorogenic acid. Shahidi and Chandrasekara
conducted an extensive review of the antioxidant activity ,
both in vitro and in vivo, of hydroxycinnamic acids (ferulic, caffeic,
coumaric, sinapic and derivatives). Flavonoids, which are other
molecules present in Artichoke leaves, act as antioxidant agents
by inactivating free radicals generated by irradiation.
Rice can grow in diverse media. It will grow faster and more vig-
orously in hot and humid environments. The chemical composition
of the active ingredient is characterised by the presence of biofunc-
tional peptides and amino acids, polysaccharides and phytic acid.
The amino acid composition of the peptides present in the ac-
tive ingredient is characterised by a high concentration of glutamic
acid (up to 17%) and large amounts of aspartic acid, arginine, leu-
cine, phenylalanine, serine, valine and tyrosine (up to 10%).
Approximately 80% of the protein hydrolyzate has a molecular
weight between 500 and 3000 Da. The molecular weight of the
peptides present is directly related to its function in the hair.
Low molecular weight peptides (<1000 Da) can penetrate and re-
pair hair ﬁbres from the inside. Medium molecular weight peptides
(1000–3000 Da) can repair the cuticle [17,18], and account for 42%
of the extract.
The main polysaccharide present in the rice active ingredient is
amylopectin (Fig. 2a).This is a water-soluble branched polymer
composed of glucose units connected linearly by bonds
Each molecule of amylopectin can contain between 100.000 and
200.000 glucose units, and each branch is formed by 20–30 glucose
units in length. This type of three-dimensional structure prevents
an excessive accumulation of the active ingredient and hair mat-
ting, compared with normally used linear polymers.
Phytic acid (myo-inositol hexaphosphoric acid, abundant in edi-
ble legumes, cereals and seeds) (Fig. 2b) is another key component
of rice and acts as an antioxidant agent by inactivating free radicals
generated by irradiation. Phytic acid is capable of chelating metals,
especially divalent metals. It forms an iron chelate which greatly
mediated oxygen reduction yet blocks iron-driven
hydroxyl radical generation and suppresses lipid peroxidation .
Protein and amino acid degradation, lipid peroxidation,
strength, shine and colour measurements were used to evaluate
proteic and lipidic photodecomposition in hair ﬁbres subjected to
A Scanning Electron Microscopy (SEM) study was also per-
formed with all hair samples to evaluate the possible changes in
the surface morphology of the hair ﬁbres due to the different treat-
All these methodologies were applied to virgin or dyed hair
tresses subjected to UV radiation and pre-treated with the two
antioxidant formulations in order to achieve a possible protection
of hair properties.
2. Materials and methods
Revlon 7.45 dye was provided by The Colomer Group (Barce-
lona, Spain). Natural dark brown hair tresses (20 cm in length)
were purchased from De Meo Brothers Inc (New York). Hair tresses
were treated with a shampoo base formulation (Table 1), placebo
and a protective serum which contained C. scolymus antioxidant
active (Cyn) and O. sativa antioxidant active (Rice) (Table 2).
2.2. Hair Treatments
Hair was chemically treated by dyeing and also subjected to
2.2.1. Dyeing procedures
Untreated hair (32 g) was dyed with 70 ml of dye solution con-
taining 50 ml of Revlon 7.45 dye and 20 ml of H
(20 vol.%). The
hair was covered and maintained for 30 minutes at 25–29 °C. Final-
ly, the hair was washed with neutral shampoo and was dried at
2.2.2. Antioxidant treatments
Four dyed (D) hair tresses of 8 g each were prepared. For com-
parison, four tresses of 10 g untreated (UT) hair (virgin hair) were
Fig. 1. Hydroxycinnamic acid.
Fig. 2a. Amilopectin.
Fig. 2b. Phytic acid.
E. Fernández et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 146–156 147
also prepared as control. All tresses were washed daily with a neu-
tral shampoo for two minutes and then rinsed with water. Then,
three tresses of each kind of hair (UT, D) were treated with the pro-
tective serums: placebo (P) and Cyn and Rice antioxidant agents,
respectively. The treatment of approximately 0.5 g of each formu-
lation for 8 g of dyed tresses, and 0.6 g for 10 g of untreated hair
was repeated everyday for 10 days. The formulations were applied
to the hair tresses for 24 h. Next, all the tresses were washed and
the formulations were applied again. The other UT, D tresses were
only washed but not post treated.
All hair tresses are described below:
- Untreated hair (UT).
- Untreated hair post treated with placebo (UTP).
- Untreated hair post treated with Cyn (UTC).
- Untreated hair post treated with Rice (UTR).
- Dyed hair (D).
- Dyed hair post treated with placebo (DP).
- Dyed hair post treated with Cyn (DC).
- Dyed hair post treated with Rice (DR).
2.3. Hair UV exposure
Human hair was irradiated using a light source simulating UV
solar irradiation (500 W/m
equivalent to 3.0 J/min cm
CPS, Atlas, USA). The irradiation of 500 W/m
is equivalent to
2 days in June in Catalonia (1.50–1.84 J/min cm
per day ). Moreover, the UV radiation intensity
was modiﬁed by different exposure times depending on the test
applied: 500 W/m
during 18, 24, 36 and 48 h.
2.4. Tensile properties of the hair ﬁbres
A stress–strain test was performed with 25 irradiated (at
during 36 h), and non-irradiated UT, UTP, UTC and
UTR ﬁbres, which were randomly taken from the hair samples pre-
viously conditioned for 48 h in a standard atmosphere (20 °C and
65% relative humidity) and centrally attached to a pair of card-
board frames with an internal rectangular cut frame of
50 25 mm
following the longest length.
Samples on the cardboard were attached to an Instron 5500R
dynamometer with a gauge length of 50 mm. The two sides of
the cardboard were cut before the beginning of the stress–strain
test to enable just the ﬁbre under testing to be stressed. The test
was performed according to the ASTM Standard D 3822 methodol-
ogy. A gauge length of 50 mm, a rate of strain of 30 mm/min, and
the breaking stress (MPa) and strain (%) were recorded. The multi-
plication of breaking stress and percentage strain gave rise to
breakage work, which evaluated ﬁbre conditions .
2.5. Protein degradation
A total of 100 mg of UT, UTP, UTC and UTR hair was exposed to
UV radiation (500 W/m
) at different times (18 and 36 h). Proteins
and peptide solubilisation was carried out with 100 mg of hair sub-
jected to UV radiation, diluted in 1 ml of 2% SDS aqueous solution
and sonicated using a Labsonic 1510 device (B. Braun, Melsungen,
Germany) for 5 h at 45 °C. Hair extracts were then diluted to 0.01%
SDS concentration and the Bradford colorimetric assay was used to
quantify the proteins and peptides solubilised . This assay is
based on the formation of a complex between the dye, Brilliant
Blue G, and the proteins in solution, which leads to an increase
in absorption at 595 nm and is proportional to the amount of pro-
tein in the solution . Bovine Serum Albumin (BSA) was used as
a standard to calculate the amount of protein. This procedure was
also performed for non irradiated hair as a control.
2.6. Tryptophan decomposition
Tryptophan measurements were carried out with UT, UTP, UTC
and UTR hair unexposed and exposed to UV radiation (500 W/m
at different times (24 and 48 h).
A protocol to hydrolyse the ﬁbre was followed to measure the
tryptophan in the alkaline solution with a spectroﬂuorimeter .
A total of 50 mg of irradiated and non-irradiated hair was chopped
from hair tresses and then dissolved in 50 ml of 2 M NaOH solution
for 24 h. This led to a release of tryptophan from hair and enabled
us to quantify tryptophan in order to correlate the variations in
hair at different UV exposure times . The measurements of tryp-
tophan intensity were obtained at 345 nm in the ﬂuorescence
spectrum, which is the emission wavelength of tryptophan in solu-
Shampoo base formulation.
Ingredient % (w/
Aqua (water) 73.16
Acrylates/C10-30 alkyl acrylate crosspolymer 1.00
Sodium laureth sulfate, aqua (Water), sodium chloride 12.00
Cocamidopropyl betaine 6.00
Sodium hydroxide, aqua (Water) 2.14
Phenoxyethanol, propylparaben, ethylparaben, methylparaben,
Maris sal (Sea Salt) 0.10
Protective serum formulations.
Ingredients Cyn Rice Placebo
Aqua (Water) 76.90 76.90 81.90
Glycerin 2.00 2.00 2.00
Tetrasodium glutamate diacetate, sodium hydroxide, aqua (Water) 0.20 0.20 0.20
Hydroxypropyl starch phosphate 5.50 5.50 5.50
Sucrose laurate, aqua (water), alcohol denat. 5.00 5.00 5.00
Prunus amygdalus dulcis (Sweet Almond) oil 4.00 4.00 4.00
Phenoxyethanol, methylparaben, ethylparaben, butylparaben, propylparaben, isobutylparaben 0.60 0.60 0.60
Parfum (Fragrance) 0.30 0.30 0.30
C. scolymus active (Cyn) 5.00 – –
O. sativa active (Rice) – 5.00 –
Aqua (Water), lactic acid 0.50 0.50 0.50
148 E. Fernández et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 146–156
2.7. Lipid peroxidation measurements
Lipids from UT, UTP, UTC and UTR human hair unexposed and
exposed to UV radiation (500 W/m
) at different times (18 and
36 h) were extracted with methanol (500 mg hair/10 ml methanol)
in a sonicating device Labsonic 1510 (B. Braun, Melsungen, Ger-
many) for 15 min. Next, the hair extracts were dried under a N
ﬂow and diluted again in 1.5 ml of methanol. Lipid peroxides
(LPO species) were measured by thiobarbituric acid (TBA) assay
[24,25]. The thiobarbituric acid-reactive species (TBARS) were
quantiﬁed by spectrophotometry at 534 nm (Cary 300 Bio UV–Vis-
ible Spectrophotometer, Varian, USA). At low pH and at a high tem-
perature, MDA readily participates in nucleophilic addition
reaction with TBA, generating a red, ﬂuorescent 1:2 MDA: TBA ad-
duct according to the reaction shown in Fig. 3.
The results were expressed as malonaldehyde bis(dimethylace-
tal) equivalents (
M MDA) using a standard curve for the pure
MDA–TBA complex. The calibration curve was obtained by using
MDA at different concentrations (0.5–30
M). Negative and posi-
tive controls were also quantiﬁed using 0 and 100
M of MDA.
The calibration curve was prepared on each day of the study.
Brieﬂy, the LPO species present in the samples (0.5 mL) were added
to aliquots (1 mL) of a solution made up of 0.4% of TBA and 15% of
trichloroacetic acid (TCA) in 100 mL of the HCl solution (0.25 M).
The mixture was incubated in a bath of boiling water for 1 h. Fresh
TBA/TCA stock solution was prepared on each day of the analysis.
This procedure was also performed for non irradiated hair as a
2.8. Scanning electron microscopy (SEM)
A SEM study was performed with non-irradiated and irradiated
(at 500 W/m
during 36 h) UT, UTP, UTC and UTR hair samples to
evaluate the possible changes in the surface morphology of the hair
ﬁbres due to the different treatments. To this end, the samples
were studied with a TM-1000 Tabletop scanning electron micro-
scope (Hiatchi). The microscope was operated at 15.0 kV. Several
ﬁbres from each hair sample were viewed, and representative
images were taken.
2.9. Shine measurements
Shine measurements of irradiated (at 500 W/m
during 36 h),
and non-irradiated UT, UTP, UTC and UTR; D, DP, DC and DR hair
tresses were obtained using a micro-TRI-gloss BYK-Gardner GmbH
(Geretsried, Germany) with standard geometries of 20°,60°or 85°.
Hair tresses were aligned by combing and the measurement at the
three angles was performed through a plate glass (slide) pressed
over the tresses. The alignment of ﬁbres showed to be more impor-
tant than roughening or hair surface on brightness . The mea-
surements were performed under controlled conditions (23 °C and
50% relative humidity). Mean values were obtained for the 15 val-
ues of 85°incidence angle of the 24 strands .
2.10. Colour measurements
Changes in hair colour of irradiated (at 500 W/m
during 36 h),
and non-irradiated UT, UTP, UTC and UTR; D, DP, DC and DR hair
tresses were measured using a spectrophotometer (Macbeth Col-
or-eye 3000, Neurtek Instruments, Spain). The colour measure-
ments were obtained using The CIE L
bmodel, developed by
the Commission Internationale de l’Eclairage. Colour on three axes
corresponds to trichromatic human perception and reﬂects the de-
gree of change in colour that humans can perceive . This model
has the advantage of corresponding to the human perception of
colour and has the additional beneﬁt of giving a grid point for each
The lightness or intensity of a colour is measured on the ‘L
on a scale from 0 (black) to 100 (white). The colour is also measured
on the ‘a
’ axis that gives a value from 100 (green) to +100 (red).
’ axis measures colour from 100 (blue) to +100 (yellow).
One unit on the L
axes is considered to be the smallest dif-
ference that the human eye can perceive . This grid point allows
the mathematical comparison of colour and also enables colours to
be corrected for different conditions. It should be noted that, theo-
retically, the a
axes have no maximum or minimum values.
Our research has used the cut off point of +/100 because this is the
practical limit of the instrument used to measure colour .
On the order hand, the total colour loss (
E) was calculated by
assessing the changes in L
readings on the treated tresses
using a spectrocolorimeter.
The equation used to calculate the total colour loss was :
Standard deviations were calculated for all mean values. Analy-
sis of variance (ANOVA) with a one-way layout was applied to
group comparisons. The software used was the STATGRAPHICS
plus 5. Signiﬁcant differences in the mean values were evaluated
by the F-test. A pvalue below 0.05 was considered signiﬁcant.
3. Results and discussion
3.1. Tensile properties of the hair ﬁbres
There is a long-standing hypothesis, that the cortex is primarily
responsible for the tensile properties of human hair  and that
the cuticle has little involvement. Then, the tensile properties are
primarily an index of cortical damage . Moreover, the mechan-
ical properties of hair depend on the condition of the hair ﬁbre, pri-
marily of keratin and its three-dimensional arrangement .In
this study a stress–strain test was performed on untreated (UT)
and treated ﬁbres with placebo (UTP) and the two antioxidant for-
mulations (UTC and UTR) with and without UV radiation. Mean
values of breaking stress and deformation at break for the different
hair samples are given in Table 3.
Fig. 3. Reaction proposed for the detection of MDA after lipid peroxidation.
E. Fernández et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 146–156 149
Breaking stress evaluates ﬁbre integrity. Therefore, higher val-
ues of this parameter indicate a larger amount of bonds present
in the ﬁbre structure. First results showed that the UV irradiation
leads to a modiﬁcation of the hair ﬁbre integrity with a decrease
in the values of stress at break from 239.62 to 230.32 Mpa for UT
ﬁbres. When evaluating the different hair treatments (placebo
and the two antioxidant formulations), an increase in the breaking
stress after 36 h of UV irradiation was observed, suggesting an
improvement in ﬁbre integrity. This increase was statistically sig-
niﬁcant for the ﬁbres treated with the Rice formulation.
Moreover, results of deformation at break indicated a signiﬁcant
increase in irradiated ﬁbres treated with the Rice formulation
(Fig. 4). An increase in the deformation at break indicates an in-
crease in ﬁbre plasticity. This increase may be attributed to (1)
an increase in the water content of the ﬁbres due to the antioxidant
formulations; the higher the moisture content, the greater the abil-
ity of the ﬁbre to be deformed; and to (2) a binding of some func-
tional groups in the formulations to cystine in the hair ﬁbre, which
restores some of the disulﬁde bonds broken upon UV radiation.
This could also confer greater elasticity onto the ﬁbre. When defor-
mability is increased, the resulting hair ﬁbres are softer and more
resistant to breakage.
As stated in the experimental part, considering stress and defor-
mation at break values, breakage work can be calculated. The re-
sults of the different hair ﬁbres are detailed in irradiated samples
showed a signiﬁcant increase in breakage work for the samples
treated with the placebo and the two antioxidants. Again, this
improvement was more marked for the hair ﬁbres treated with
the Rice antioxidant formulation, reaching values higher than those
obtained for untreated ﬁbres. The mechanism for protection of the
ﬁbre strength could arise from the low molecular weight compo-
nents of the active. The Rice extract has biofunctional peptides with
high substantively for keratin. More than 45% of the peptides in
this extract have molecular weights lower than 1000 Da. According
to Stern and Johnson, peptides with a molecular weight lower than
1000 Da, can penetrate and perform a repairing action of the hair
ﬁbres from the inside . The effects of these peptides on hair
have been described in numerous studies which demonstrate pro-
tection of hair against alkali and oxidizers  or improvement of
esthetic characteristics of hair (elasticity, body) [34,35]. Further-
more, these peptides provide protection from radical mediated
damaged due to UV insult through the radical scavenging ability
associated, as in other works [17,18,36], with the cystine residues
present within the peptides.
3.2. Protein degradation
Hair is composed primarily of proteins (88%). These proteins are
of a hard ﬁbrous type known as keratin. Loss of protein due to UV
radiation will lead to damaged hair structure. The decrease of cys-
tine due to photodamage of hair does not necessarily imply large
protein solubilisation. Breaking disulphide bridges with formation
of cysteic acid and by main chain scission causes a formation of
secondary crosslinks between protein residues such as lanthionine
and dityrosine which limit the extractability of the protein .
However the amount of protein solubilisation due to UV radiation
could be enough to demonstrate antioxidant protection.
Protein degradation from hair was measured using the Bradford
assay. This assay is based on the formation of a complex between
the Brilliant Blue G dye and the proteins in solution, which leads
to an increase in absorption at 595 nm and is proportional to the
amount of protein in the solution . Bovine Serum Albumin
(BSA) was used as a standard to calculate the amount of protein.
The Bradford assay was performed on UT and on treated ﬁbres
with placebo (UTP) and the two antioxidant formulations (UTC
and UTR) with and without UV radiation. The results for the differ-
ent hair ﬁbres are detailed in Table 4 and Fig. 5.
Non-irradiated samples presented protein solubilisation ranging
between 8.42 and 13.29
g protein/mg hair. However, it should be
pointed out that hair treated with the Cyn and Rice extracts showed
signiﬁcantly lower values of protein solubilisation with respect to
untreated hair, suggesting protection of the ﬁbres due to these
Breaking Stress, deformation at break and breakage work of untreated (UT) and treated ﬁbres (UTP, UTC and UTR) with and without
irradiation at 500 W/m
for 36 h.(Mean ± SD) (
Breaking stress (Mpa) Deformation at break (%) Breakage work
UT 239.63 ± 72.10 44.12 ± 4.28 10525.78 ± 2999.71
UTP 226.23 ± 52.51 44.19 ± 4.81 10001.14 ± 2948.61
UTC (Cyn) 227.74 ± 74.27 44.11 ± 4.18 10129.30 ± 3442.29
UTR (Rice) 217.61 ± 60.93 45.90 ± 3.21 10076.21 ± 3165.84
UT 36 h UV 230.32 ± 52.26 45.11 ± 4.51 10315.38 ± 2476.73
UTP 36 h UV 235.58 ± 59.71 45.75 ± 2.95 10844.38 ± 2721.67
UTC (Cyn) 36 h UV 237.57 ± 75.70 45.77 ± 3.44 11084.64 ± 3187.65
UTR (Rice) 36 h UV 303.88
± 101.67 46.80
± 2.75 13761.58
36h UV0h UV
Fig. 4. Breakage work of UT, UTP, UTC and UTR samples with and without UV irradiation (
150 E. Fernández et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 146–156
formulations. Irradiated and non treated samples showed an in-
crease in protein solubilisation ranging between 21.78 and
g protein/mg hair for 18 h and 36 h UV, respectively. When
evaluating the protective capacity of the different hair treatments, a
signiﬁcant decrease in protein degradation for the hair ﬁbres trea-
ted with the Cyn and Rice extracts after 18 h of UV radiation was
observed. Furthermore, after 36 h of irradiation, ﬁbre improvement
was also signiﬁcant for the ﬁbres treated with the placebo and the
two antioxidant treatments, suggesting a conservation of the ﬁbres.
The protection of hair with the Cyn and Rice extracts was clearly
demonstrated even with the non-irradiated ﬁbres. Moreover, after
18 h irradiation and at 36 h irradiation, protection of protein deg-
radation is signiﬁcant in the two treatments (Fig. 5). The percent-
age of increase in protein degradation with respect to the UT
sample is shown in Table 4. UT and UTP irradiated samples showed
an increase of 160 and 200% in protein degradation, whereas sam-
ples treated with the Rice and Cyn range between 110 and 135%
after irradiation. Other strong antioxidants based on procyanidins
together with tocopherols also preserve protein degradation but
to a lower extent . Comparison of the two antioxidants showed
a higher protection of protein degradation of the Cyn extract. This
is probably due to the strong antioxidants such as hydroxycin-
namic derivatives in the extract. These derivates could promote
the protection of the ﬁbre.
3.3. Tryptophan degradation
The aromatic amino acids, tyrosine phenylalanine and mainly
tryptophan, which are associated with photo-yellowing of hairs,
are easily degraded by light . Of all the amino acids present
on the ﬁbre surface, tryptophan is the most sensitive to UVB radi-
ation. Therefore, it is commonly used to evaluate the protective ef-
fect of cosmetic active ingredients for hair. Tryptophan from hair is
usually measured as a solid using a spectroﬂuorimeter [6,11].A
protocol to hydrolyse the ﬁbre was followed to measure the tryp-
tophan in the alkaline solution . A total of 50 mg of hair was
chopped from hair tresses and then dissolved in 50 ml of 2 M
NaOH solution for 24 h. This led to a release of tryptophan from
hair and enabled us to quantify it in order to correlate the varia-
tions in hair at different UV exposure times. The measures of ﬂuo-
rescence intensity at different UV exposure times were obtained at
355 nm in the ﬂuorescence spectrum, which is the emission wave-
length of tryptophan in solution. Tryptophan ﬂuorescence mea-
surements were performed on UT and treated ﬁbres with placebo
(UTP) and the two antioxidant formulations (UTC and UTR) with
and without UV radiation. The decrease in tryptophan degradation
for irradiated hair ﬁbres treated with the antioxidant formulations
is shown in Table 5 and Fig. 6.
Non-irradiated samples presented a ﬂuorescence intensity of
about 66–67 (a.u), whereas irradiated samples (at 500 W/m
24, and 48 h) showed a decrease in ﬂuorescence intensity between
47 and 60 (a.u). When evaluating tryptophan on hair subjected to
the extracts, higher values in ﬂuorescence intensity were observed
for the hair ﬁbres treated with the Cyn and Rice extracts after 24
and 48 h of UV radiation. However, comparison of the behaviour
of the two formulations after 48 h of radiation showed that the
improvement was more marked in the ﬁbres treated with the Rice
formulation (Fig. 6).
Protein hair solubilisation with 2% SDS of untreated (UT) and treated ﬁbres (UTP, UTC
and UTR) with and without irradiation at 500 W/m
for 36 h (Mean ± SD). Percentage
of increase in protein degradation with respect to UT sample.
g Protein/mg hair Increase in protein
UT 12.38 ± 1.35
UTP 13.29 ± 1.63 107.35
UTC (Cyn) 9.23 ± 0.78 74.58
UTR (Rice) 8.42 ± 0.45 68.01
UT 18 h UV 21.78 ± 1.72 175.93
UTP 18 h UV 19.58 ± 1.21 158.16
UTC (Cyn) 18 h UV 14.04 ± 0.45 113.41
UTR (Rice) 18 h UV 14.55 ± 0.30 117.53
UT 36 h UV 25.38 ± 0.63 205.00
UTP 36 h UV 20.18 ± 0.27 163.00
UTC (Cyn) 36 h UV 13.62 ± 0.16 110.02
UTR (Rice) 36 h UV 16.77 ± 0.30 135.46
36h UV18h UV0h UV
µg prot/mg hair
Fig. 5. Degradation of hair proteins of UT, UTP, UTC and UTR samples with and without UV irradiation (
Fluorescence intensity values obtained at 355 nm of untreated (UT) and treated ﬁbres
(UTP, UTC and UTR) with and without irradiation at 500 W/m
for 36 h (Mean ± SD).
Percentage of conservation of trp with respect to UT sample.
Fluorescence intensity at 355 nm
(50 mg hair)
UT 66.45 ± 4.40
UTP 66.75 ± 4.11 100.45
UTC (Cyn) 67.77 ± 4.39 101.98
UTR (Rice) 67.23 ± 2.64 101.18
UT 24 h UV 47.13 ± 1.61 70.93
UTP 24 h UV 49.82 ± 1.07 74.93
UTC (Cyn) 24 h UV 56.80 ± 0.63 83.82
UTR (Rice) 24 h UV 60.80 ± 0.56 90.43
UT 48 h UV 48.78 ± 3.48 73.41
UTP 48 h UV 48.57 ± 4.93 72.76
UTC (Cyn) 48 h UV 50.12 ± 2.64 73.95
UTR (Rice)48 h UV 57.07 ± 3.88 84.88
E. Fernández et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 146–156 151
The percentages of tryptophan conservation with respect to the
UT sample are shown in Table 5. UT and UTP irradiated samples
presented a trp conservation of about 70% and 73% after 24 and
48 h of irradiation, respectively. By contrast, samples treated with
the Rice extract reached 90% and 85% after 24 and 48 h UV. The re-
sults obtained using this formulation could indicate a higher ﬁbre
UV protection due to the composition of the Rice extract. The pres-
ence of phytic acid antioxidant and moreover the medium molec-
ular weight peptides (1000–3000 Da) may form a protective layer
on the ﬁbre surface. Similarly to other protein and peptides
[17,18,36] the UV damage should occur preferentially to the active
rather than the ﬁbre beneath.
3.4. Lipid peroxidation
Human hair contains 1.9-5% of internal lipids  but despite
this low amount, the cell membrane lipids are very important as
they make possible a continuous pathway of diffusion into the ﬁ-
bre . Furthermore, according to recent studies on human hair,
a correlation has been found between the amount of internal lipids
and the moisture content in the hair ﬁbre . Integral lipids of
hair ﬁbre are degraded by UV light as well as visible light, cause
weakening of the cell membrane complex exposed to light radia-
Lipid peroxides (LPO species) were measured by thiobarbituric
acid (TBA) assay [27,28]. The thiobarbituric acid-reactive species
(TBARS) were quantiﬁed by spectrophotometry at 534 nm (Cary
300 Bio UV–Visible Spectrophotometer, Varian, USA). Lipid perox-
idation measurements were performed on UT and treated ﬁbres
with placebo (UTP) and the two antioxidant formulations (UTC
and UTR) with and without UV irradiation.
Non-irradiated samples presented lipidic peroxidation ranging
between 0.0019 and 0.0024
M MDA /mg hair (Table 6 and
Fig. 7). Irradiated samples (at 500 W/m
, during 18 and 36 h)
showed an increase in lipidic peroxidation ranging between
0.0021 and 0.0060
M MDA/mg hair. Evaluation of the antioxidant
ability of the different hair treatments showed a signiﬁcant de-
crease in lipidic peroxidation for the hair ﬁbres treated with the
placebo and the Cyn and Rice extracts after 18 h of UV radiation
(Fig. 7). However, after 36 h of irradiation, the improvement was
only signiﬁcant for the ﬁbres treated with the two antioxidant
0h UV 24h UV 48h UV
Fluorescence Intensitya (a.u)
Fig. 6. Fluorescence intensity of UT, UTP, UTC and UTR samples with and without UV irradiation (Mean ± SD) (
Lipid peroxides of untreated (UT) and treated ﬁbres (UTP, UTC and UTR) with and
without irradiation at 500 W/m
for 36 h (Mean ± SD). Percentage of increase in lipid
peroxidation with respect to UT sample.
M MDA/mg hair Increase in lipidic
UT 0.0019 ± 0.0000
UTP 0.0022 ± 0.0002 115.79
UTC (Cyn) 0.0022 ± 0.0003 115.79
UTR (Rice) 0.0024 ± 0.0005 126.31
UT 18 h UV 0.0039 ± 0.0003 205.26
UTP 18 h UV 0.0026 ± 0.0001 136.84
UTC (Cyn) 18 h UV 0.0021 ± 0.0002 110.52
UTR (Rice) 18 h UV 0.0023 ± 0.0003 121.05
UT 36 h UV 0.0061 ± 0.0000 321.05
UTP 36 h UV 0.0064 ± 0.0001 336.84
UTC (Cyn) 36 h UV 0.0035 ± 0.0000 184.21
UTR (Rice) 36 h UV 0.0043 ± 0.0005 226.31
0h UV 18h UV 36h UV
µM MDA/mg hair
Fig. 7. Lipid peroxidation of UT, UTP, UTC and UTR samples with and without UV irradiation (Mean ± SD) (
152 E. Fernández et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 146–156
The percentages of lipidic peroxidation with respect to the UT
sample are shown in Table 6. UT samples presented an increase
in lipidic peroxidation of about 205–321% after 18 and 36 h,
respectively. In contrast, the samples treated with Rice attained
only 121 and 226% after 18 and 36 h UV, respectively. Moreover,
samples treated with Cyn only increased lipid peroxidation to
110% and 184% after 18 and 36 h UV, respectively. The similarity
between Fig. 5 (protein degradation) and Fig. 7 (LPO formation)
indicates that proteins and lipids from the hair ﬁbre are affected
similarly when subjected to UV irradiation. This similarity could
be used to determine the efﬁcacy of antioxidant treatments on hu-
man hair. As in the case of protein degradation, the antioxidant
UT 36 h UV UTP 36 h UV
UTC (Cyn) 36 h UV UTR (Rice) 36 h UV
Fig. 8. Representative SEM micrographs of UT, UTP, UTC and UTR samples with and without UV irradiation.
E. Fernández et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 146–156 153
behaviour of the Cyn extract is more marked than the behaviour of
the Rice extract. Phytic acid in Rice was demonstrated to inactivate
free radicals generated by radiation and be capable of chelating
metals and inhibit lipid peroxidation  However, the presence
of strong antioxidants such as hydroxycinnamic derivatives and
ﬂavonoids in Cyn, provides better protection in this extract, pre-
serving the inner substance of the hair (proteins and lipids).
3.5. Surface changes
SEM images revealed some differences in the conditions of the
surface morphology of the hair samples due to the treatments.
Some hair ﬁbres were evaluated and images of UT ﬁbres and those
treated with the placebo and the two antioxidant formulations
UTP, UTC and UTR were obtained (Fig. 8). Images of irradiated ﬁ-
bres (at 500 W/m
during 36 h) were also obtained and the restor-
ative capacities were compared. Irradiation of UT hair (UT 36 h)
showed the largest differences as clearly lifted cuticles when com-
pared with the non-irradiated hair sample (UT). The application of
the placebo and the Cyn and Rice formulations led to a slight
improvement in the hair surface without open cuticles when sub-
jected to UV radiation.
The improvement in hair surface of the two formulations could
be attributed to the antioxidant role of hydroxycinnamic derivates
of the Cyn and phytic acid in the Rice extracts. These molecules
help to prevent the oxidation of hair ﬁbres subjected to UV, repair-
ing the cuticle and protecting the hair.
3.6. Shine measurements
Shine is one of the most valued hair properties, as it implies hair
health and beauty. This property is closely linked to the structure
of the hair ﬁbre and, in particular, it depends on the condition of
the cuticle. Antioxidants are usually applied to protect shine and
colour not only in natural hair but especially in dyed hair . Un-
treated and dyed ﬁbres (D, DP, DC and DR) were also evaluated.
Shine measurements were performed on UT and D ﬁbres treated
with placebo (UTP and DP) and the two antioxidant formulations
(UTC, UTR, DC and DR) with and without UV radiation. As de-
scribed in the experimental part, shine measurements were per-
formed using a micro-TRI-gloss BYK-Gardner GmbH with
standard geometries of 20°,60°or 85°. The measurements were
carried out under controlled conditions (23 °C and 50% relative
humidity) and mean values were obtained for the 15 evaluations
for each incidence angle. The results for shine for the three differ-
ent incident angles are shown in Table 7.
Non-irradiated (D) ﬁbres showed a small but signiﬁcant in-
crease in shine values when treated with the two antioxidant for-
mulations. As expected, the UV irradiation leads to a reduction in
shine. This is evidenced by comparing UT and D ﬁbres with irradi-
ated samples (UT 36 h UV and D 36 h UV).
Fig. 9 shows the values of shine for the 60°incidence angle for
UT and D ﬁbres and those treated with the placebo and the two
antioxidant formulations before and after 36 h of UV radiation
(UTP, UTC, UTR, DP, DPC and DPR). The graph shows a signiﬁcant
increase in shine values in the antioxidant treated ﬁbres UTC,
UTR, DPC, and DPR after irradiation. This improvement in shine is
signiﬁcant for both ﬁbres treated with Cyn and Rice formulations.
This suggests that the presence of actives in the formulations pre-
serves the shine of the ﬁbres. The irradiated samples (DR 36 h UV)
attained the same values as the non-irradiated ones. Moreover, a
higher increase is observed in both irradiated ﬁbres using the Rice
extract. The Rice formulation probably contributes to the higher
protective effect due to the presence of phytic acid and moreover
the peptides with restorative properties preserving hair brightness
of natural and dyed hair.
3.7. Colour measurements
Hair melanins provide some photoprotection to hair proteins by
absorbing and ﬁltering the impinging radiation and subsequently
dissipating this energy as heat. However, red and dark-brown hairs
photo-yellow when exposed to near ultraviolet plus visible radia-
tion . Hair colour resulting from a cosmetic treatment (artiﬁcial
colour) is very sensitive to sunlight. UVA radiation resulted in a sig-
niﬁcant and perceivable change in the dye red hair colour as the
one assayed in this work. The red pigment introduced into the hair
ﬁbre by the dyeing process acts as a photoreceptor, absorbing pho-
tons and photochemically degrading after UVA and visible light
It is therefore essential to evaluate colour changes in dyed ﬁbres
subjected to UV radiation [10,45]. Colour measurements were per-
formed on UT and D ﬁbres treated with placebo (UTP and DP) and
the two antioxidant formulations (UTC, UTR, DC and DR) with and
without UV irradiation. As described in the experimental part, col-
our measurements were performed using a spectrophotometer
(Macbeth Color-eye 3000). The colour measurements were ob-
tained using The CIE L
bmodel, in which colour on the three axes
correspond to trichromatic human perception. This model has the
advantage of corresponding to the human perception of colour and
has the additional beneﬁt of giving a grid point for each speciﬁc
colour. Then the total colour loss (
E) parameter was calculated
using the following equation:
The results of total the colour loss parameter for hair ﬁbres be-
fore and after 36 h UV irradiation are shown in Table 8 and Fig. 10.
The results showed a signiﬁcant decrease in the colour loss
parameter in the UT and D ﬁbres when placebo and the two anti-
oxidants were applied, indicating a good colour conservation of the
ﬁbres. As in the case of shine measurements, this effect is more
marked in the case of the Rice extract, which probably protects
the colour of ﬁbres better than the Cyn extract.
Melanins, mainly located in the cortex, provide some photo-
chemical protection to hair proteins, however in the process of
protecting the hair proteins from light; the pigments are degraded
or bleached. The main result of this process is hair colour changes
[9,10]. Besides, tryptophan associated with photo-yellowing, of
hair is one of the most degraded amino acid after exposure to UV
[9,10]. This amino acid of the cuticle is altered to a greater extent
than those of the cortex. Therefore the results of color loss preven-
tion could be related to the ones obtained for thryptophan evalua-
tion. The higher oxidation prevention of Rice extract could be
Shine values of untreated and dyed (UT, D) and treated ﬁbres (UTP, UTC, UTR, DP,
DC,DR) with and without irradiation at 500 W/m
for 36 h (Mean ± SD).
UT 128.67 ± 1.75 142.00 ± 0.00 118.07 ± 1.69
UTP 127.33 ± 2.44 141.00 ± 0.55 115.27 ± 3.33
UTC (Cyn) 117.87 ± 7.71 141.69 ± 0.48 115.87 ± 4.02
UTR (Rice) 126.93 ± 2.37 140.80 ± 1.01 115.00 ± 3.23
UT 36 h UV 117.73 ± 3.24 133.47 ± 2.47 113.40 ± 2.16
UTP 36 h UV 119.80 ± 2.54 133.93 ± 1.75 112.00 ± 2.54
UTC (Cyn) 36 h UV 121.60 ± 6.00 135.27 ± 4.44 107.93 ± 4.04
UTR (Rice) 36 h UV 120.60 ± 1.01 136.67 ± 0.90 114.23 ± 2.13
D 122.52 ± 4.05 137.24 ± 2.20 113.52 ± 2.16
DP 122.96 ± 3.60 136.86 ± 1.40 113.03 ± 3.60
DC (Cyn) 123.77 ± 2.80 137.90 ± 1.74 113.83 ± 1.92
DR (Rice) 124.36 ± 2.16 138.11 ± 0.99 113.90 ± 1.95
D 36 h UV 124,14 ± 2.50 135.20 ± 1.71 108.37 ± 2.87
DP 36 h UV 120.37 ± 2.59 134.46 ± 1.64 109.30 ± 4.43
DC (Cyn) 36 h UV 124.57 ± 3.01 136.57 ± 1.38 113.67 ± 2.37
DR (Rice) 36 h UV 124.90 ± 1.90 137.60 ± 1.30 114.60 ± 1.22
154 E. Fernández et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 146–156
ascribed to the phytic acid antioxidant and moreover to the pep-
tides that as in the case of triptophan evaluation were supposed
to be the reason of a stronger protection effect at the ﬁbre surface.
Irradiated hair samples were observed to be damaged chemi-
cally and mechanically with a decrease in breakage work and
adverse consequences for the mechanical properties. Furthermore,
the physical appearance of hair showed a decrease in shine and
loss of colour. Treatments with antioxidants improved the
mechanical properties of irradiated hair especially in the case of
the Rice extract. Low molecular weight peptides of the rice formu-
lation probably penetrate hair and improve the mechanical proper-
ties at the cortex level and higher molecular weight peptides may
form a protective layer preventing tryptophan degradation and
Moreover, lipidic peroxidation and protein degradation of hair
are signiﬁcantly reduced in the antioxidant treated samples, sug-
gesting an improved integrity of the ﬁbre. This is more marked
in the case of the Artichoke extract, indicating higher antioxidant
properties for hydroxycinnamic derivates contained in this formu-
lation. SEM images showed that the application of antioxidants to
hair ﬁbres leads to an improvement in the cuticle scale with a
smoothing of the scale edge. These images coincide with the con-
servation of colour and shine in the dyed tresses.
The two formulations provided protection against UV irradia-
tion and corroborated a signiﬁcant protection capacity; the Cyn
formulation proved to be more effective in preventing protein deg-
radation and lipid peroxidation. The Rice extract led to higher
breakage work, and a lower degradation of colour and shine. The
different methodologies assayed help to evaluate the degree of
damage in the ﬁbre subjected to UV radiation. Moreover they
determine the effectiveness and the action mechanism of the dif-
ferent antioxidant formulations applied to human hair.
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