A non-invasive method for the in vivo determination

of skin antioxidant capacity (IAC-S

s
)
Paola Ziosi
1
, Elena Besco
2
, Silvia Vertuani
2
, Nicola Solaroli
1,3
and Stefano Manfredini
1
1
Dipartimento di Scienze Farmaceutiche, Universita` degli studi di Ferrara, Ferrara, Italy,
2
AmbrosiaLab s.r.l., Via Fossato di Mortara 17/19, Ferrara,
Italy and
3
Division of Metabolic Diseases, Karolinska University Hospital, Stockholm, Sweden
Background/purposes: Skin antioxidant network protects
cells against oxidative injury and prevents the production of
oxidation products. When oxidative stress overwhelms the
skin antioxidant capacity, the subsequent modication of the
cellular redox apparatus leads to an alteration of cell home-
ostasis leading to degenerative processes. In the dermo-
cosmetic eld, the topical application of antioxidants is often
suggested as a possible strategy to prevent and modulate
oxidative skin damages. Continuing our studies addressed
to set-up new bio-engeneering protocols for the claim sub-
stantiation of antioxidant cosmetic products, we have devel-
oped a new non-invasive methodology for the evaluation of
antioxidants cosmetics ingredients and nished products.
Methods: The effects of a pre-treatment on forearm skin
with an antioxidant ingredient were investigated on 15
volunteers, in a double-blind randomised fashion. A non-
invasive method was devized that comprises the collection
of forehead SC layers of the pre-treated area and control
and the next evaluation of skin antioxidant capacity (IAC-
S
s
) by a luminescence-based method.
Results: The results showed that the antioxidant prepara-
tion was able to increase, to a statistically signicant extent
(Po0.01), the IAC-S
s
in comparison with the control
area. The data were conrmed (Po0.05) by comparison
with a method, previously developed by us, based on
DermAnalyzer
s
.
Conclusions: In view of the simplicity and reliability of the
method here presented, this new technique is proposed as a
possible tool for the routine evaluation of in vivo efcacy of
antioxidant functional ingredients and nished products.
Key words: photochemiluminescence skin antioxidant
capacity Dermanalyzer
s
tocopherol
& Blackwell Munksgaard, 2006
Accepted for publication 3 January 2006
S
KIN IS a highly metabolic tissue that possesses
the largest surface area in the body, and is
also a major candidate and target of damaging
free radicals. It is well-known that free radicals
and reactive oxygen species (ROS) are involved
in the mechanism leading to cutaneous damages,
such as early ageing, inammatory disorders and
skin cancers. The skin can be protected from
oxidative damage with elaborate and diversied
antioxidant mechanisms, of enzymatic and non-
enzymatic nature. Antioxidants, such as a-toco-
pherol, ascorbic acid, and other active substances
with high radical scavenging activity, modulate
this damage by scavenging free radicals and lipid
peroxyl radicals. An important goal of modern
cosmetic science should be the development of
appropriate skin care products based on topical
application of radical scavengers to prevent the
onset and propagation of the free radical cascade.
Although many studies strongly suggest that
some cosmetic ingredients, such as vitamins,
minerals and botanicals, have the potential to
improve skin ageing, rigorous studies, that can
withstand critical review, are still lacking in this
eld, as most of the studies conducted are either
small in scale, in vitro or utilise non-human
models.
After its description by Pinkus (1), tape strip-
ping has become a standard method in dermato-
logical research (2). Tape stripping is a relatively
non-invasive technique, which permits samples
of the stratum corneum to be collected from the
skin. Taking this into account and continuing our
studies (3) addressed to set up new bio-engeneer-
ing protocols for the claim substantiation of anti-
oxidant cosmetic products, we have developed a
non-invasive and quantitative method that can
reliably measure the integral skin antioxidant
capacity (IAC-S
s
, AmbrosiaLab sri, Ferrara,
Italy), by means of the tape stripping technique
303
Skin Research and Technology 2006; 12: 303308 & 2006 The Authors
Journal compilation & 2006 Blackwell Munksgaard 2006
Printed in Singapore All rights reserved
Skin Research and Technology
coupled to an efcient method for antioxidant
activity determination (4). In particular, in this
study we have validated our approach using a
simple vitamin E-based formula to modify the
IAC-S
s
of the stratum corneum. To this end, the
antioxidant capacity of the stratum corneum was
determined by a photo-chemiluminescence
(PCL)-based method and compared with that
obtained after a pre-treatment with the study
antioxidant formula.
Materials and Methods
Antioxidant formula
In order to reduce the number of parameters, the
formula was limited to the simplest possible and
thus to the lowest number of ingredients and
excipients. The antioxidant ingredient was cho-
sen from the panel of ingredients available at our
laboratory on the basis of the highest antioxidant
activity and lowest skin irritation. The chosen
formula contained (according to INCI terminol-
ogy): cyclometicone (Polichimica, Bologna, Italy)
and tocopherol (Copherol F1300, Cognis, Care
Chemicals, Fino Mornasco, Como, Italy). Ratio
of the assay mixture was 1:1.
A standardized patch test of the mixture was
carried out. The test involves the application of
the examined substance to the skin under adhe-
sive tape, which is then left in place for 48 h. The
skin is then examined a further 48 h later for any
response. The results demonstrated that topical
application of the antioxidant mixture is not
associated with acute skin irritation or with
allergic sensitization.
Subject and experimental design: in vivo study
Fifteen Caucasian healthy volunteers between 23
and 30 years participated in the study after giving
written consent. The study was approved by the
Ethics Committee. The study was carried out in a
partly air-conditioned room at a temperature of
23 2 1C and with an average relative humidity
of 33%. The study was performed in a double-
blind and randomized manner. The anatomical
sites chosen were four areas (10 cm
2
) of the
internal forearm. Two areas (A and B) were
treated with the product, whereas C and D
were control areas. The study was performed in
two steps. The rst was the pre-treatment phase
consisting of the application of 20 mL of the
antioxidant mixture onto skin areas of the human
volunteers twice a day for 15 days. The second
phase, starting from day 16, involved the evalua-
tion of the effects of the pre-treatment on the
forearm skin of the 15 volunteers, using two non-
invasive techniques: tape stripping/PCL assay in
comparison with Dermanalyzer
s
(AmbrosiaLab
sri, Ferrara, Italy).
Tape stripping
The areas of the forearm were marked and
rectangles of exactly tting pieces of adhesive
tape (cut to a size of 2 cm 5 cm) were consecu-
tively applied to the skin.
Before sample acquisition, the skin was
cleaned with a piece of gauze soaked in 50 mL of
ethanol. The stratum corneum of the treated area
was removed by 11 successive tape strippings
using 3M (3M, Minneapolis, MN, USA) invisible
adhesive tape. The tape was applied to the test
site with forceps, attened equally twice, and
removed gently using moderate and even trac-
tion. Owing to the possibility of surface contam-
ination and to remove surface lipids, the rst
(uppermost) tape stripping was discarded. Sub-
sequent tapes were then placed, adhesive side
downwards, into a glass box with 15 mL of
MeOH (HPLC grade) and then treated for
30 min in an ultrasonic bath. Solutions were
puried by centrifugation (10 min with 4000 cy-
cles/s) to separate small horny layer particles,
and the uppermost part was analysed.
Antioxidant capacity
Measurements of antioxidant capacity of the
tocopherol formula and stratum corneum sam-
ples were performed by photochemilumines-
cence assay (PCL).
Skin antioxidant capacity of volunteers was
measured at baseline and after 15 days (at the
end of the treatment period). The method princi-
ple (PCL) is briey described: dened free radi-
cals (superoxide anion radicals) are generated in
the measuring system by the exposure of a
photosensitizer to a UV-light source. The free
radicals are detected by their reaction with a
chemiluminogenic substance and the measure-
ment of the emitted light. The light ashes are
detected by a photomultiplier. These generated
radicals are partially scavenged by reaction with
the sample antioxidants and remaining radicals
are quantied by the above-described detec-
tion principle. The results are presented in
304
Ziosi et al.
equivalent concentration units of Trolox
s
(Sigma-
Aldrich, St. Louis, MI, USA) (synthetic vitamin E)
for lipid-soluble substances or ascorbic acid for
water-soluble substances. Different concentra-
tions of these standard compounds are used to
establish a calibration curve, and the detector
signal of each run is monitored for 180 s. The
PCL assay is suitable to measure the radical
scavenging properties of single antioxidants as
well as more complex systems in the nanomolare
range (5). The antioxidant potential is measured
by means of the lag phase at different concentra-
tions, calculated by a Trolox
s
calibration curve
and expressed as mmol equivalents in antioxi-
dant activity of a reference compound (i.e.
Trolox
s
). The PCL method was carried out as
described by Popov and Lewin (6, 7) and, as
decribed above, can be conducted by two differ-
ent protocols, ACW and ACL, that consent to
measure the antioxidant capacity of the water-
and lipid-soluble components, respectively. In
the water-soluble fraction antioxidants such are
avonoids, ascorbic acid, aminoacids, etc. are
detected, while in the lipid-soluble fraction, toco-
pherols, tocotrienols, carotenoids, etc. are mea-
sured.
ACW and ACL sample preparation general
procedure
An exact quantity of the product was poured in
1 mL methanol HPLC grade, for the measure
with the ACL kit, or 1 mL water, HPLC grade,
for the measure with the ACW kit, and they were
mixed by vortex for 1 min at room temperature.
The obtained solution was then ltered
through HPLC lter (Chemtek Analitica, Bo-
logna, Italy) by a syringe and diluted with Re-
agent 1 of ACL or ACW kit (AnalytikJena, Jena,
Germany). Different dilutions of the various
samples were evaluated to obtain a calibration
curve.
Results are expressed as mmol equivalents, in
antioxidant activity, of Trolox
s
for each litre of
product under examination.
DermAnalyzer
s
The data obtained with the protocol described
above, were compared with those collected by a
method, previously developed by us, based on
DermAnalyzer
s
. In this method, the intensity and
duration of skin redness generated by methyl
nicotinate (MN) is used to assess the efcacy of
functional ingredients, included in cosmetic for-
mulations to prevent and modulate skin oxida-
tive damages (3). The reliability of the method in
the measurement of skin redness has been pre-
viously assessed by us in comparison with other
well-recognized instruments. In particular, the
correlation with Minolta Chromameter CR300
(Minolta, Tokyo, Japan) was very high both in
vitro and in vivo (R50.99 and 0.86, respectively)
(3). We used the standardized protocol as de-
scribed before (3). Briey, after the pre-tratment
phase, consisting of the application of the pro-
duct studied twice a day for 15 days, the selected
areas were re-treated with the products, 30
0
before the application of the irritant agent. At
this time the baseline value of redness of the
treated and control areas (T0) was determined.
The irritation was induced in both areas by the
application of paper disks (5 mm of diameter)
soaked with 15 mL of 0.5% aqueous solution of
methyl nicotinate (99% purity, Fluka Chemie
GmbH, Sigma-Aldrich, St. Louis, MI, USA) for
3
0
. After removal of the lter disk, excess solution
was gently removed using a paper tissue. A
circular erythema spot was induced by this treat-
ment. Measurements were performed before MN
exposure (T0) and 15 min after MN removal.
Image recording
In order to develop a simple and cost-effective
method, a simple digitalizer was chosen and
coupled with specically developed software
described below. True colour images of the ob-
served skin area were taken under the same
constant lighting conditions, using a digital
photocamera (Nikon coolpix4300, Nikon, Tokyo,
Japan) placed over a specically designed dark
box. The position of the camera was adjusted so
that the examined areas were in the middle of the
images. In particular, great attention has been
given to the type and modality of illumination
and in consequence to white calibration. The
camera was used in manual mode and each
parameter was accurately dened. In particular,
for illumination, a diffuse light (D-65, 6500 Kel-
vin, Osram GmbH, Berlin, Germany), which is
considered a standard for average daylight, with
a minor yellow component, was used to reduce
shadows on the border of the arm and to avoid
reections on the skin. The size of the skin area
under study was 10 cm
2
, and it was located on
the volar forearm. The arm was placed inside the
305
In vivo determination of skin antioxidant capacity (IAC-S
s
)
box, volar forearm upwards, and xed to avoid
large movements at suitable distances and angle
from the camera and light source. To analyse the
degree of the erythema response, true colour
images were taken at baseline and 15 min after
MN removal (T0), which represents the highest
peak of redness. The reliability of such a device
has been already demonstrated by us during a
previous comparative study (3).
Software
The images were downloaded from the digital
camera and archived on the computer hard disk
and afterwards analysed singularly with the
DermAnalyzer
s
. The program was selected each
area to be treated simply by drawing a line
around the zone to be analysed. After this step,
the program algorithm, developed by us, auto-
matically pulls apart the treated skin from the
untreated skin, and measures the CIE a* compo-
nents of the red area, excluding the other colour
components such as b* (greenyellow). The nal
result consists in the mean a* value of the whole
area selected. The reliability of such a program
has been already demonstrated by us during a
previous comparative study (3).
Erythemapruritusredness
s
index
The self-pruritus sensation and the degree of
oedema after MN removal was also determined.
In this test the volunteers were asked to score the
self-pruritus sensation induced by topical MN
application at the same time skin erythema was
determined by DermAnalyzer
s
. The simple scor-
ing system is as follows: 0, no pruritus; 0.5, weak
pruritus sensation; 1, moderate pruritus sensa-
tion; 2, high pruritus sensation; 3, intolerable.
Moreover, erythema and oedema were scored
visually using the following scale: 0, no reaction
(negative); 0.5, very weak erythema or minute
scaling (doubtful); 1, weak erythema, slight oe-
dema, slight scaling and/or slight roughness
(weak); 2, moderate degree of erythema, oedema,
scaling and/or roughness, or minor degree of
erosion, vesicles, bullae, crusting and/or ssur-
ing (moderate); 3, marked degree of erythema,
oedema, scaling, roughness, vesicles, bullae,
crusting and/or ssuring (strong); 4, as 3, with
necrotic area (very strong/caustic).
According to that we have introduced a new
parameter termed EPR
s
index which represents the
sum of the three parameters: redness, measured by
DermAnalyzer
s
, degree of erythema/oedema
scored visually and self-pruritus sensation.
Statistical analysis
For both test mean values and standard devia-
tions were calculated using the software program
Graph Pad prism. Paired one-tailed Students
t-test was used in comparison with signicance
difference between the treated and control area.
A signicance of Po0.05 was used as the value
for signicant differences.
Results
The antioxidant capacity of the 1:1 tocopherol
formula was 1540 mmol Trolox
s
equivalents/L.
This result is quite consistent and may be as-
cribed to the natural vitamin E preparation used
for the formula.
Measurements of IAC-S
s
Results are summarized in Table 1 and represents
the sum of ACW and ACL mean values both
expressed in Trolox
s
equivalents per litre of
solution. In order to evaluate skin antioxidant
capacity, 10 sequentially tape-stripped layers was
obtained from each volunteer. Measurements
were taken in triplicate and overall mean values
for the 15 volunteers were calculated. Data
showed that IAC-S
s
was signicantly higher in
pre-treated areas with respect to the control
(Po0.01). In fact, whereas the value of area A
increase from 24.76 mmol Trolox
s
/L (T0) to
36.86 mmol Trolox
s
/L (T15), the control area
shows no difference between T0 and T15 (the
end of the treatment period) (Fig. 1).
Measurements of EPR
s
index
Mean values among the 15 volunteers and the
standard error of measurements (SEM) for EPR
s
index are shown in Table 2. As it can be observed
(Fig. 2) the antioxidants formulation tested was
able to reduce, in statistically signicant extent,
the intensity of skin redness induced by MN
TABLE1. Mean of values of IAC-S
s
expressed as mmol Trolox/L and
standard deviation (n 515) at baseline (T0) and after the pre-treatment
with antioxidant formulation (T15)
T0 T15 % variation
Area A 24.76 (1.3) 36.86 (1.6) 48.86
Control 25.19 (1.2) 25.21 (1.4) 0.08
306
Ziosi et al.
(Po0.05), with respect to the control area. In fact,
after 15 min from MN removal, the increase of
EPR
s
index of the control area, compared with B
(pre-treated area) was 17%.
Discussion and Conclusions
The outermost layer of the skin is directly ex-
posed to chemical oxidants, air pollutants and
ultraviolet (UV) solar light, all of which are
potent inducers of ROS. Growing experimental
evidences sustain the development of powerful
cosmeceutical strategies involving antioxidant
formulations to prevent UV-induced carcinogen-
esis and photoageing, as well as to modulate skin
disorders sustained by inammatory process,
mediated by oxidative species. During our stu-
dies aimed to highlight antioxidant properties of
functional ingredients, we faced with the pro-
blem of the determination of the real efcacy of
the nished formulae. Thus, in a previous report
(3), we demonstrated that pre-treatment with an
antioxidant formulation counteracts the skin oxi-
dative damages induced by MN, reasonably in
view of the integration of skin physiological
defences. This protocol allows to determine
the EPR
s
index which represents the sum of
three parameters: redness, measured by Derm
Analyzer
s
, degree of erythema/oedema scored
visually and self-pruritus sensation.
In the present study, we hypothesised that the
previously observed efcacy, in the MN-based
model, of an antioxidant formulation could be
quantitatively correlated, by means of the bio-
engineering method, with the real antioxidant
skin capacity, by measuring the stratum corneum
antioxidant status using the photochemilumines-
cence (PCL), uorescence (FL) or other spectro-
scopic methods (4). In order to investigate this
issue, we have thus developed a new protocol,
IAC-S
s
, based on a combination of tape stripping
and PCL, suitable for the direct determination of
the real contribution of an ingredient to antiox-
idant capacity of the stratum corneum, and the
investigation has been conducted in parallel with
the previously developed MN-based protocol in
order to give further support to our observations.
Thus, the in vivo antioxidant efcacy of a topical
application of a simple tocopherol-based for-
mula, taken as the simplest example, was eval-
uated on 15 volunteers by (i) measuring the IAC-
S
s
basal value of the stratum corneum by PCL,
(ii) measuring the IAC-S
s
value after pre-treat-
ment with the given formula and (iii) evaluating
on the volunteers the protective role of the
formula against oxidative stress by means of the
ROS driven MN-microinammatory model pre-
viously developed by us (3).
To the best of our knowledge, this is the rst
time that skin antioxidant capacity has been
evaluated by means of a bio-ongenering method
able to describe the whole antioxidant capacity
independently from the effective concentration of
antioxidants present. Indeed, nothwistanding
their presence, the different antioxidants may
interact in a sinergistic or detrimental manner
with other bio-molecules, thus rising up to anti-
oxidant activities that may not be related to their
effective concentration (8). Indeed, the proposed
evaluation of the protective mechanism of topical
skin application of antioxidants has made use of
measurements of antioxidant compounds con-
tent, in the dermis and epidermis, by means of
analytical techniques such as HPLC and UV
spectroscopy (9, 10). These techniques account
for the content of antioxidants but not for their
Fig. 1. Mean values (n 515) are given for skin antioxidant capacity
(IAC-S
s
) at baseline (T0) and after the pre-treatment with the
antioxidant formulation (A area).
TABLE2. Mean of values of EPR
s
index and standard deviation (n515)
at baseline (T0) and after 15 min from the exposure to MN
T0 T15 % variation
Area B 7.53 (0.59) 10.57 (0.71) 39.84
Control 7.74 (0.49) 12.12 (0.75) 56.5
Fig. 2. Mean values (n 515) are given for the EPR index after 15 min
from the exposure to MN for formulation (B area) and the control.
Higher values reect increase of redness.
307
In vivo determination of skin antioxidant capacity (IAC-S
s
)
real efcacy into the skin. In contrast, the IAC-S
s
is a value that considers the antioxidant capacity
of the whole stratum corneum, considering the
possible interactions with other biomolecules and
baseline antioxidants content. Moreover, when
combined with MN-based assay, it attributes
functional signicance to the observed increase
in the antioxidant skin capacity, consenting a full
correlation between the antioxidant content of a
given formula and its antioxidant-based activity.
Finally, although preliminary, the data reported
here indicate that the proposed method is suita-
ble for the quantication of the real capacity of a
given formula to increase antioxidant skin de-
fence. Indeed the antioxidant capacity of the
stratum corneum of volunteers resulted, measur-
able to a statistically signicant extent (Po0.01)
and average values for treated and untreated
subjects were achieved. In particular, a 15 days
pre-treatment with a 1:1 tocopherol topical for-
mulation having an antioxidant potency of
1540 mmol Trolox
s
equivalents/L induced a
48.86% increase in the overall skin antioxidant
capacity of the stratum corneum.
We can thus conclude that this approach repre-
sents a suitable method to determine the antiox-
idant status of the skin and the real efcacy of the
topical application of antioxidant-based cosmetic
formulations. Taken together, the presented data
suggest that a pre-treatment with the tested anti-
oxidant preparation containing tocopherol was able
(i) to increase the IAC-S
s
in comparison with the
control area and (ii) to reduce and modulate the
effects deriving from oxidative stress owing to MN
stimulation, with a signicant reduction of EPR
s
index of the treated area in comparison with the
control area. A few preliminary results coming in
from another study currently ongoing at our la-
boratory also indicate the possibility to correlate an
increase in IAC-S
s
deriving not only from topical
application but also from oral intake of foods or
supplements rich in antioxidants and in particular
for those with high skin afnity (8).
The goal of these studies is to correlate the
observed increase in the antioxidant skin activity
with the capability of a given antioxidant formula
to increase skin defence against other oxidative
stressors such is UVradiation. The results will be
of oustanding importance in the comprehension
on how UV lters at low concentrations can be
combined with other functional ingredients in
order to get better protection from solar exposure.
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Address:
Stefano Manfredini
Department of Pharmaceutical Sciences
University of Ferrara
Via Fossato di Mortara 17-19 I-44100 Ferrara
Italy
Tel: 139 0532 291292
Fax: 139 0532 291296
e-mail: stefano.manfredini@unife.it
308
Ziosi et al.