Skin Research and Technology 2008; 14: 376380
Printed in Singapore ! All rights reserved
doi: 10.1111/j.1600-0846.2008.00288.x
In vitro antioxidant activity and in vivo efficacy of topical
formulations containing vitamin C and its derivatives
studied by non-invasive methods
Patrcia M. B. G. Maia Campos, Gisele M. S. Goncalves and Lorena R. Gaspar
Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Sao Paulo, Brazil
Background/purpose: Vitamins C and its derivatives,
mainly due to their antioxidant properties, are being used
in cosmetic products to protect and to reduce the signs of
ageing. However, there are no studies comparing the effects
of vitamin C [ascorbic acid (AA)] and its derivatives, mag-
nesium ascorbyl phosphate (MAP) and ascorbyl tetra-
isopalmitate (ATIP), when vehiculated in topical formula-
tions, mainly using objective measurements, which are an
important tool in clinical efficacy studies. Thus, the objective
of this study was to determine the in vitro antioxidant activity
of AA and its derivatives, MAP and ATIP, as well as their
in vivo efficacy on human skin, when vehiculated in topical
formulations.
Methods: The study of antioxidant activity in vitro was
performed with an aqueous and a lipid system. The in vivo
methodology consisted of the application of these formula-
tions on human volunteers forearm skin and the analysis
of the skin conditions after 4-week period daily applications
in terms of transepidermal water loss (TEWL), stratum
corneum moisture content and viscoelasticity using a
s s s
Tewameter , Corneometer and Cutometer , respectively.
Results: In vitro experiments demonstrated that in an aqu-
eous system, AA had the best antioxidant potential, and
MAP was more effective than ATIP, whereas in the lipid
system ATIP was more effective than MAP. In in vivo
studies, all formulations enhanced stratum corneum moist-
T O IMPROVE the aged skin conditions, besides
the use of products aimed at cell renewal
stimulation and skin hydration, the development
of formulations that act on the dermis in the
stimulation of collagen synthesis and in the en-
hancement of skin firmness has also been very
important.
Ascorbic acid (AA) is known for its antioxidant
potential (1), its regulation in collagen production
(2) and for its action on the improvement of UV-
induced modifications in skin relief and ultrastruc-
ture (3); however, it is extremely unstable. To
overcome this problem, AA is chemically modi-
376
r 2008 The Authors
Journal compilation r 2008 Blackwell Munksgaard
Skin Research and Technology
ure content after a 4-week period daily applications when
compared with baseline values; however, only the formula-
tion containing AA caused alterations in TEWL values. The
formulations containing MAP caused alterations in the vis-
coelastic-to-elastic ratio, which suggested its action in the
deeper layers of the skin.
Conclusion: AA and its derivates presented an in vitro
antioxidant activity but AA had the best antioxidant effect.
In in vivo efficacy studies, only the formulation containing
AA caused alterations in TEWL values and the formulation
containing MAP caused alterations in the viscoelastic-
to-elastic ratio. This way, vitamin C derivatives did not
present the same effects of AA on human skin; however,
MAP showed other significant effect-improving skin hydra-
tion, which is very important for the normal cutaneous
metabolism and also to prevent skin alterations and early
ageing.
Key words: antioxidant activity and skin biophysical tech-
niques ascorbyl tetra-isopalmitate magnesium ascorbyl
phosphate vitamin C
& Blackwell Munksgaard, 2008
Accepted for publication 28 December 2007
fied by esterification of the hydroxyl group and its
derivatives, such as ascorbyl palmitate, ascorbyl
tetra-isopalmitate (ATIP) and magnesium ascorbyl
phosphate (MAP), are widely used in topical
formulations to reduce the signs of ageing (4, 5).
Previously, we have reported that AA and MAP
are able to penetrate into the skin in appreciable
amounts (6). The use of MAP in formulations
containing other active substances has been re-
stricted due to the pH necessary for maintenance
of its stability. One way to circumvent this pro-
blem is to use the liposoluble vitamin C derivative
ATIP, which is stable in a wide pH range.
 Antioxidant activity and efficacy of topical formulations
Some in vitro and in vivo studies have been
carried out to evaluate the efficacy of AA and
MAP in the skin; however, most of them used
in vitro methods, such as fibroblast cultures sup-
plemented with AA, which showed enhancement
of collagen synthesis (2), or melanoma cells
culture, showing inhibition of melanogenesis by
MAP (7). On the other hand, in vivo studies
showed that these active substances acted on
the epidermis, causing thickening of the epithe-
lium, with cells of larger volume due to intra- and
extra-cellular hydration (8).
However, the conclusions given by these stu-
dies, mainly those obtained by in vitro studies, are
considered to be a preliminary orientation or a
screening to predict the effects on the human
skin, because many factors such as skin penetra-
tion influence the efficacy of an active substance.
Thus, it is very important to evaluate the formu-
lations containing these active substances in the
human skin to show their benefits under actual
conditions of use.
This way, regarding efficacy testing, several
biophysical methods for objectively characteriz-
ing skin structure and function (without invading
the skin or interfering with the function mea-
sured) have been developed in recent decades.
The skin biophysical methods typically measure
selected properties that depend on the measuring
principle. Hydration, skin elasticity, barrier func-
tion and skin thickness are examples of para-
meters that can be quantified non-invasively
using these techniques (9, 10).
Consequently, this study has an innovative
proposal because there are no studies comparing
the effects of AA, MAP and ATIP when vehicu-
lated in topical formulations, mainly using objec-
tive measurements, which are an important tool
in efficacy studies. In addition, many substances
with a high antioxidant potential may not always
correlate well with the reduction of signs of
ageing such as viscoelasticity, fine lines and
wrinkles under the actual conditions of use of
these products (11).
Thus, the objective of this study was to deter-
mine the in vitro antioxidant activity of vitamin C
(AA) and its derivatives, MAP, ATIP as well as
their in vivo efficacy on the human skin, when
vehiculated in topical formulations. Furthermore,
the results should contribute to a better under-
standing of the topical effects of vitamin C and its
derivatives, MAP and ATIP, evaluated by differ-
ent biophysical techniques, in terms of cell
renewal, skin hydration and skin barrier function
protection, showing that these vitamin C deriva-
tives present efficacy and also that the use of such
active substances is of great interest for topical
formulations, because they are more stable than
free vitamin C and hydration and protection are
considered to be markers of skin health.
Methods
In vitro antioxidant activity
The study of antioxidant activity in vitro was
performed with an aqueous and a lipid system,
the luminol-chemiluminescence and malondial-
dehyde (MDA) assay, respectively.
Chemiluminescence assay
Samples of a solution of the vitamins studied
(final concentration ranging from 0.000125% to
0.002% of AA, from 0.0021% to 0.0337% of MAP
and from 0.0082% to 0.1316% of ATIP) and a
control solution (without any active substance)
were mixed with phosphate buffer, luminol,
H2O2 and horseradish peroxidase (12, 13). Che-
miluminescence in triplicate experiments was
measured in an Autolumat LB953 apparatus
(EG & G Berthold, Gaithersburg, MD, USA) and
the results were expressed as integrated chemi-
luminescence (010 min) in photons counts per
minute (c.p.m.), and the inhibitory effect of each
compound was calculated.
Lipid peroxidation assay
The vitamins studied (with a final concentration
of 0.6% and 1.5% of AA and 0.45% and 0.9% of
MAP and ATIP) were subjected to lipid perox-
idation with MDA generation (14, 15), and the
ability of the vitamins to cause a decrease in
MDA formation was tested, i.e., the lower the
MDA formed the higher the antioxidant activity.
The results obtained were statistically analysed
using the KruskalWallis non-parametric test.
In vivo efficacy on human skin
Formulation studied
The formulation containing 5% (w/w) self-emul-
sifying wax (cetearyl alcohol, ceteareth-12, cetear-
eth-20, glyceryl stearate and cetyl palmitate) and
2% (w/w) isoparafin and polyacrilamide gel was
supplemented or not with 2% of L-AA (pH of the
final formulation adjusted to 3.5) or 2% of MAP
(pH of the final formulation adjusted to 7.0) or 2%
377
Maia Campos et al.

of ATIP (pH of the final formulation adjusted to sion (18). The results obtained were statistically
5.5) because these active substances have shown analysed using the KruskalWallis non-para-
better chemical stability at these pH. metric test.

Study protocol
Approval for the study was obtained from the Results and Discussion
Faculty of Pharmaceutical Sciences Ethics Com- In vitro antioxidant activity
mittee (CEP/FCFRP 06/2001). A total of 40 The findings obtained in the in vitro experiments
healthy volunteers were studied after giving their clearly demonstrate that the vitamins studied
written informed consent. Our research metho- possess good antioxidant free radical-scavenging
dology consisted of application of these formula- properties, even when used at low concentra-
tions on the volunteers forearm skin, followed by tions. On comparing the effect of each vitamin in
analysis of the skin conditions after 2- and 4-week different test systems, we observed that in the
period daily applications. aqueous system, AA had the best antioxidant
potential, and MAP was statistically more effec-
Instrumentation tive than ATIP in free radical-quenching activity
The stratum corneum moisture content, the trans- (aqueous system) (Fig. 1a), whereas in the lipid
epidermal water loss (TEWL) and the skin me- system ATIP was statistically more effective than
chanical properties were determined susing skin MAP (Fig. 1b).
biophysicals techniques, Corneometers CM 825, One of the early cellular events following UV
Tewameter CM 210 and Cutometer SEM 575, light exposure is lipid peroxidation induction (19),
respectively, from Courage1Khazaka (Cologne, and vitamin E (a-tocopherol), which is the major
Germany). s lipophilic antioxidant in membranes and tissues,
The Corneometer , which measures the stra- significantly reduced UV-induced in vivo epider-
tum corneum moisture content, makes use of the mal lipid peroxidation in guinea-pig skin (19, 20).
relatively high dielectric constant of water com- In addition, besides having a ower antioxidant
pared with the ones of the other substances in the activity in lipid systems than vitamin E (data not
skin (16, 17). TEWL, which is related to skin shown), ATIP presents good stability and has a
barrier function,s was expressed as g/m2 ! h (17). good antioxidant potential in lipid systems, and
The Cutometer analyses the mechanical para- consequently it also has a good potential for
meters: Ua/Uf, the ratio of total retraction to total protection of skin barrier function.
distension, called gross elasticity; Ur/Ue, neto-
elasticity of the skin without viscous deforma-
tion; Ur/Uf, the ratio of immediate retraction to In vivo efficacy on human skin
total distension, called biological elasticity; and All the formulations studied enhanced stratum
Uv/Ue, the ratio of viscoelastic to elastic disten- corneum moisture content after 4-week period

(a) (b) C (+)

C ()
AA
140 MAP
Lipid peroxidation assay

120 ATIP
AA d
MAP a
MDA formed (%)

ATIP
100 e
d c
100
80
% inhibition

80
c 60
60
40 a 40
a a
20 a 20
b b b
0 b
0.00000 0.00025 0.00050 0.00075 0.00100 0
Vitamins concentration % (w/w)

Fig. 1. In vitro inhibitory effect of the vitamins studied in reactive oxygen species (a) chemiluminescence assay: inhibition of free radical production
(b) lipid peroxidation assay: free radical production (MDA production). (KruskalWallis test, N 5 3 measurements, mean " SEM, Po0.05).
Different symbols indicate statistically different values (a6b6c6d6e). MDA, malondialdehyde.

378
 Antioxidant activity and efficacy of topical formulations

(a) Vehicle (v) (b) Vehicle(v)

v + AA v + AA
v + MAP v + MAP
v + ATIP 0.4
v + ATIP
7.5
*
* *
0.3

TEWL (g/m2.h)
**
5.0

Uv/Ue
0.2

2.5
0.1

0.0 0.0
before after 2 weeks after 4 weeks before after 2 weeks after 4 weeks

Fig. 2. In vivo effects of the formulations studied (vehicle, vehicle1MAP, vehicle1AA, vehicle1ATIP) before (baseline values) and after a 2- and
4-week period of application, (a) Transepidermal water loss and (b) Skin viscoelastic parameter Uv/Ue (viscoelasticelastic ratio). *Significantly
different from the baseline values (Po0.001); **significantly different from the baseline values and from the vehicle-treated area (Po0.05). AA,
ascorbic acid; ATIP, ascorbyl tetra-isopalmitate; MAP, magnesium ascorbyl phosphate.

daily applications when compared with the base- increase of Uv/Ue, which is a relative parameter,
line values; however, only the formulation con- can indicate a decrease in interstitial fluid visc-
taining AA led to an enhancement in TEWL osity as a result of increased water content (18).
values (Fig. 2a), altering skin barrier function. These results suggest an increase in the skin
During the process of terminal differentiation, hydration that is not limited to the upper cells
several biochemical changes and loss of water layers but is also present in the deeper ones
occur on the skin surface (21) and because AA led (8, 22). According to Maia Campos and Silva
to an enhancement in TEWL, we have an indica- (23), the vehicle influenced AA and MAP pene-
tion that it enhanced the epidermal cell renewal tration into the skin and AA has a higher pene-
process on human skin, which can be measured tration than MAP. However, despite having a
by skin biophysical techniques. Once AA is an a- lower penetration than AA, these authors ob-
hydroxy acid (AHA) in the lactone form, this served in a histopathological study that formula-
effect could be similar to some AHAs effects in tions containing MAP caused hydration (extra-
terms of cell renewal. In addition, Silva and Maia and intra-cellular) in guinea-pig skin after 2- and
Campos (8) observed some alterations caused in 4-week period applications (8).
the epidermis cell nuclei by the application of Finally, in our study we found that MAP
formulations containing AA in guinea-pig skin showed a different in vivo effect from AA, under
and also suggested that they could be due to cell the present experimental conditions, increasing
renewal. the cutaneous hydration in the deeper cell layers.
Once none of the volunteers reported any itch- This way, the different effects observed between
ing or redness (erythema) on the forearms during AA and MAP probably occurred due to the
the experiment, in this study the enhancement of difference in their chemical structures because
TEWL did not occur due to skin irritation. In MAP is a stable L-AA phosphate magnesium
addition, in a previous study, Silva and Maia salt, which could lead to a hydration effect.
Campos (8) did not observe any lymphocytic This observed hydration effect is very important
infiltrate (which may be related to inflammation) for normal cutaneous metabolism and also to
in the guinea-pig dermis after 2- and 4-week prevent skin alterations and early ageing (22).
period application of 2% of L-AA. In addition, although ATIP showed in vitro
The results obtained for the skin mechanical protection of lipid membranes against free radi-
properties showed that the formulations studied cals, no in vivo effect was observed by the
did not alter Ur/Ue, Ua/Uf and Ur/Uf para- application of the formulation containing ATIP.
meters, which suggests that these formulations As this substance is a lipid-soluble vitamin C
did not modify the skin elastic parameters. Only derivative similar to ascorbyl palmitate (24), and
the formulation containing MAP altered the Uv/ consequently has a low penetration into the skin,
Ue parameter (viscoelastic-to-elastic ratio) after its efficacy could reduce when used under actual
2- and 4-week period applications (Fig. 2b). The conditions of use, i.e. on human skin.

379
Maia Campos et al.
Conclusion
AA and its derivates showed an in vitro antiox-
idant activity but AA had the best antioxidant
effect. In in vivo efficacy studies on human skin,
all studied formulations enhanced stratum cor-
neum moisture content when compared with the
baseline values. Only the formulation containing
AA led to provoked alterations in TEWL values
and the formulation containing MAP caused
alterations in the viscoelastic-to-elastic ratio.
This way, vitamin C derivatives did not show
the same effects of AA on human skin; however,
MAP showed other significant effects in improv-
ing skin hydration, which is very important for
normal cutaneous metabolism and also to pre-
vent skin alterations and early ageing.
Acknowledgement
The authors gratefully acknowledge the financial
support of Fundacao de Amparo a Pesquisa do
Estado de Sao Paulo (FAPESP).
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Address:
Patrcia M. B. G. Maia Campos
Faculdade de Ciencias Farmaceuticas de Ribeirao Preto
Universidade de Sao Paulo
Av. do Cafe s/n, Bairro Monte Alegre
Ribeirao Preto
Sao Paulo 14040-903
Brazil
Tel: 155 16 3602 4197
Fax: 155 16 3625 7202
e-mail: pmcampos@usp.br