Skin Research and Technology 2012; 18: 421430 2011 John Wiley & Sons A/S

Printed in Singapore
All rights reserved Skin Research and Technology

doi: 10.1111/j.1600-0846.2011.00588.x

Methods for evaluation of cosmetic antioxidant capacity

Anna Ratz-Lyko, Jacek Arct and Katarzyna Pytkowska
Academy of Cosmetics and Health Care, Department of Cosmetics Chemistry, Warsaw, Poland

The skin as the largest part of human body is one of the main but a large diversity of methods for determination of cosmetics
targets for ultraviolet radiation, environmental pollution, toxic antioxidant activity is also accessible. These methods can be
chemicals and some metal ions, which share responsibility for divided into three main groups: in vitro, in vivo, and ex vivo as
the formation of free radicals. The resulting free radicals, both reported herein. Due to lack of standardization and validation it
oxygen and nitrogen species are one of the main causes of is necessary to use a variety of methods as well as conditions
aging due to impaired regulation of cell respiratory metabolism for those purposes, which are presented to the context.
involving incomplete oxygen reduction in mitochondria and
production a superoxide anion, hydroxyl radicals et al. In Key words: free radicals antioxidants cosmetics antioxi-
modern cosmetology to minimize the adverse effects of free dant capacity
radicals, antioxidants, which inhibit free radical reactions,
mainly autoxidation processes are used. Currently, not only 2011 John Wiley & Sons A/S
many cosmetic products containing antioxidants are available, Accepted for publication 14 November 2011

as the main organ of the human In healthy organisms the right balance
T HE SKIN
body is constantly exposed to external
factors such as ultraviolet radiation, pollution,
between formation and neutralization of free
radicals is maintained. This balance disorder
cigarette smoke, toxic chemicals, and ions of associated with excessive production of free
certain metals, pathogenic bacteria and viruses radicals and reactive oxygen and nitrogen
that are to some extent responsible for the for- species, leads to oxidative and nitrosative stress,
mation of free radicals (1). which is an etiopathological factor of degenera-
In addition to exogenous factors, internal tive diseases and cancer, as well as the aging
factors also contribute to the formation of free process (3). Oxidative and nitrosative stress
radicals. Metabolic reactions in all living cells overwhelms the natural skins antioxidant
are accompanied with the oxidation of nutrients defense system leading to changes in redox
while reducing oxygen to the water molecules. apparatus and subsequently to alternation of
Incomplete reduction of triplet oxygen mole- cell homeostasis and a generation of degenera-
cule, results in the formation of reactive oxygen tive processes (4).
species (ROS), most of which are classified as Free radicals, reactive oxygen and nitrogen
free radicals. These include the superoxide species contribute to the changes both at the
anion O2 , hydroperoxyl radical HOO, hydro- stratum corneum and the living layers of the
xyl radical OH, NO nitric oxide, and com- epidermis and dermis (4).
pounds that are not free radicals, but because
of their greater reactivity than that of oxygen in One of the negative aspects of free radical
a triplet position, they are among the group of activity is the peroxidation of intercellular
reactive oxygen, such as singlet oxygen 1O2, cement lipids and cell membrane phospholip-
H2O2 hydrogen peroxide. ids (5). Both generated lipid peroxides and
It is also worth mentioning about organic free their break down products, such as malondial-
radicals resulting from the reaction of reactive dehyde and 4-hydroxy-2-nonenal, can directly
oxygen and nitrogen species with nucleic acids, or indirectly affect cellular and organ homeo-
proteins, lipids. These free radicals contribute to stasis, by damage to proteins due to carbonyla-
lipid peroxidation in intercellular cement, and tion and release of pro-inflammatory mediators
oxidative damage to skin structures (2,3). (6).

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Ratz-Lyko et al.

Moreover, the peroxidation leads to changes reduce the rate of free radical reactions occur-
in the organization of liquid-crystalline lipid ring in both the cosmetics and skin structures
structures, which in turn causes disturbances in are used in modern cosmetology. In the last
the functioning of epidermal barrier and the decade, antioxidants have been proposed as one
consequent increase in transepidermal water of the most effective functional ingredients for
loss (TEWL). Dehydration of the stratum corneum anti-aging cosmetics, which counteract the
may leads to weakening of the epidermal bar- effects of oxidative damage to the skin (6).
rier, and further to dehydration and increase in The antioxidant compounds are also used to
susceptibility to irritants, allergens, and the protect the raw materials and the final product.
aging processes (3,5). In cosmetic formulations oily components are
The ROS can act as second messengers in the widely used. They are very easy to oxidize,
induction of several biological responses, such which leads to changes in sensory parameters
as the activation of transcription factors NF-jB and physicochemical forms of cosmetics. Addi-
and AP-1, which generate pro-inflammatory tion of antioxidant, synthetic or natural,
cytokines (TNF-a, IL-1, IL-6, IL-8) and thus prevents cosmetic adverse oxidation.
propagate skin inflammation (6,7). Formation of Currently, the market offers a variety of cos-
hydrogen peroxide and peroxide radical anion metic products containing antioxidants. The
in skin cells may lead to oxidative stress. As a active substances in these preparations are both
result, the release of keratinocyte interleukin 1 isolated compounds such as vitamins and mix-
(IL-1) and tumor necrosis factor alpha (TNFa) tures such as plant extracts rich in polyphenols,
comes into effect, leading to the activation of triterpenes etc. (8). Determination of specific
mitogen-activated protein (MAP) kinases, which antioxidant activity is difficult and in most
in turn stimulate the synthesis of the compo- cases not possible, mainly due to the presence
nents of the transcription factor AP-1. The effect of synergistic effect. Therefore, in plant mate-
of activation of AP-1 is a distortion of cyto- rial, food and cosmetic formulations, the total
plasm signal pathways responsible for the regu- antioxidant activity (TAA) is determined, which
lation of gene expression and collagen synthesis includes content of all compounds showing
in fibroblasts. This leads to damage to the skin antioxidant properties. Currently, there are
by fibrous protein degradation of collagen type many analytical methods that allow determina-
II and III and elastin production and hypertro- tion of the level of antioxidants in plant materi-
phy (6,8). als and foods. Not all of them are appropriate
to analyze cosmetic products, which are mix-
To counteract adverse changes caused by free tures of compounds with different physico-
radicals and reactive oxygen and nitrogen spe- chemical properties (913).
cies, the skin is equipped with effective defense To determine the total cosmetics antioxidant
mechanism, which aims to increase the physical activity, special testing methods, which take
stability and counteract the effects of oxidative into account the presence of hydrophilic and
stress. The antioxidant defense system of the hydrophobic compounds, should be applied.
skin consists of protective enzymes and low It is also worth mentioning Porters polar para-
molecular weight antioxidants (endogenous and dox. According to this paradox, nonpolar
exogenous). Protective enzymes include: super- antioxidants function best in polar lipid emul-
oxide dismutase, catalase, peroxidase, and some sion and membranes, while polar antioxidants
supporting dehydrogenase enzymes glucose- are relatively more effective in nonpolar lipids
6-phosphate dehydrogenase and glutathione due to their interfacial properties (14).
reductase, whereas low molecular weight an-
tioxidants include ascorbic acid, vitamin E, glu- The methods for determination of total anti-
tathione, carnosine, uric acid, lipoic acid, and oxidant capacity can be divided into in vitro, in
coenzymes Q (3,8). vivo, and ex vivo (15).
The knowledge of the skins natural defense In vitro studies rely on the ability of anti-
mechanisms significantly influenced the develop- oxidants to neutralize free radicals. These reac-
ment of the cosmetic industry. Antioxidant tions may proceed by hydrogen atom transfer
substances including low molecular weight (HAT) and electron transfer (ET)-based assays.
antioxidants of high capacity to inhibit or They may occur in parallel and the type of anti-

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 Evaluation of cosmetic antioxidant capacity

oxidant, solubility, the solvent used, and most disadvantage of DPPH is steric inaccessibility
of all, the bond dissociation energy and ioniza- and narrow linear range of absorbance vs. con-
tion potential decide the superiority of one over centration.
the other (1620). Some authors (16,17,21) differ Similar restrictions create the FRAP (ferric
HAT assays for two reactions: hydrogen atom reducing antioxidant power) method, which is
transfer (HAT) processes, which take place in based on the reduction ability of Fe(III) complex
nonpolar solvents and non-HAT processes spe- with 2,4,6-tris (2-pyridyl)-s-triazine, to Fe(II).
cific for polar solvents. The non-HAT reactions The method proposed by Benzie and Strain (25)
are classified as a proton-coupled electron trans- was originally designed for the determination
fer (PCET), sequential proton-loss electron of TAA in blood plasma, and later was
transfer (SPLET), and electron transfer-proton extended to test food ingredients, and actively
transfer (ET-PT). used in pharmaceuticals. This method allows
evaluation of antioxidant activity of the hydro-
philic substance thus, it is not useful for deter-
Electron transfer (ET) reactions
mination of antioxidant properties in the
One of the most commonly used techniques finished cosmetic product.
based on a single electron transfer mechanism Some hope for the possibility of using this
is a method using stable synthetic radical 2,2- method in the cosmetic industry creates a modi-
diphenyl-1-picrylhydrazyl DPPH. This com- fication of this technique using the complex Fe
pound in methanol or ethanol solution exhibits (III) with Ferroin, which was used for determin-
a maximum absorbance in the visible range at a ing the antioxidant activity of edible oils (26).
wavelength of 515520 nm. The radical solution A method for determining the ability of
has purple color, which changes as a result of reducing copper (II) (Copper Reduction Assay,
electron transfer reactions after the addition of CRA) also creates the possibility of potential
an antioxidant, containing CH, NH, OH, SH use in the cosmetic industry. A variation of this
groups in its molecule. It is accompanied by a method is CUPRAC (Cupric Reducting Antioxi-
change in absorbance being a function of anti- dant Capacity) which is based on the ability to
oxidant concentration in the sample. Antioxi- reduce Cu (II) to Cu (I). The Cu (II) ion occurs
dant activity of samples using the DPPH as a complex with neocurpine, allowing the use
radical is measured by suitable colorimetric/ of the hydrophilic or hydrophobic solvents.
spectrophotometric techniques or by HPLC or Thus, it can be applied in research of both
EPR techniques (20,22). lipophilic and hydrophilic antioxidants (27,28).
The method was proposed in the late 50s of Further CUPRAC advantages over other elec-
XX century by Blois (23), and its subsequent tron-based assays are superior with regard to
modifications were designed to determine the its realistic pH close to the physiological pH,
antioxidant activity of food and medicines. In favorable redox potential, accessibility, and
cosmetic industry it is the most common stability of reagents (24,26,27).
method used to determine the antioxidant activ- A method using 2,2-azino-di-(3-etylobenzo-
ity of cosmetic products and their active ingre- tizoline-6-sulfonate), ABTS, has found practical
dients such as vitamins and polyphenols. application in the cosmetics industry as well.
Difficulties associated in measuring overall This compound is converted to ABTS + cation
activity of a cosmetic product are related with radical by enzymatic reaction with methmioglo-
the presence of both fractions hydrophilic as bin, hemoglobin, and horseradish peroxidase
well as hydrophobic ones. According to litera- and hydrogen peroxide, or by chemical reaction
ture data, DPPH method is particularly useful with potassium persulfate (K2S2O6) or manga-
for determination of antioxidants that are solu- nese dioxide (MnO2) (or with 2,2-azo-bis(2-am-
ble in organic solvents, especially alcohols (20). inopropane) ABAP radicals) (24). ABTS + cation
Furthermore, the evaluation of antioxidant radical is blue-green and has a maximum
activity by DPPH method should be carefully absorbance at several wavelengths: 417, 645, 734,
interpreted because the absorbance of DPPH 815 nm. In the presence of antioxidants in the
radical after reaction with an antioxidant is solution, it is reduced and accompanied by the
decreased by light, oxygen, pH, and the type of disappearance of the color of the solution. Fall
solvent. According to Apak et al. (24), a further color intensity is proportional to the antioxidant

423
Ratz-Lyko et al.

content in the sample. In this method, the oxidation in foods, cosmetics. and in the biolog-
antioxidant activity is expressed in Trolox ical systems (24,31,34).
equivalents as TEAC (Trolox Equivalent Antioxi- The HAT mechanism is also used to deter-
dant Capacity). Trolox (6-hydroxy-2,5,7, mine the absorption capacity of oxygen radicals
8-tetramethylochroman-2-carboxylic acid) is a (Oxygen Radical Absorbance Capacity, ORAC)
synthetic, water-soluble derivative of vitamin E, (35,36). This method involves measuring the
which was introduced by Hoffman-LaRoche. decrease in fluorescence of molecular probe by
Due to the high antioxidant activity this com- the reaction of thermally generated free hydro-
pound is used as a reference substance (2931). peroxyl radicals. In the presence of antioxidant
The advantage of using ABTS + cation is substances the drop of fluorescein distribution,
that it permits determination of antioxidants, extending the reaction time defined as the time
both hydrophilic and lipophilic, which have lag, and the decrease or increase in the constant
been used in the cosmetics industry. In a study rate is observed. Kinetics of this reaction is
conducted by Estee Lauder researchers, the compared with the reference substance, for
antioxidant activity of cosmetic emulsion example Trolox. As the fluorescent substance in
containing a mixture of antioxidants: 2% Toco- the measurements of antioxidant activity of cos-
pheryl Acetate, 0.5% N-acetyl-L-cysteine, 0.1% metics, dichlorofluoroscein H2DCF-dA (2, 7-
Tocopherol Cysteamine, 0.1% BHT, 1.0% Ascorbyl dichlorodihydrofluorescein diacetate) is applied.
Magnesium Phosphate, 0.1% Ubiquinone 50, DCF tests allow in vitro assessment of antioxi-
0,1% Rosmarinus Officinalis (Rosemary) Leaf dant capacity of cosmetics carried out in the
Extract, was evaluated . Because of the abun- living cell cultures (37).
dance of active ingredients in studied formula- Kinetics of chemiluminescence reactions
tion authors indicated the need for proper occurring between the detector and generated
preparation of the test sample by diluting it in free radicals can also be applicable in assessing
isopropyl alcohol. The results of this study of total antioxidant capacity of cosmetics. In this
show that the total antioxidant activity of the method, as the detectors luminol or lucygenine
test preparation compared with the sample whether bioluminescence protein (like Pholasin
without antioxidant increased by 86.8%, which isolated from shellfish Pholas dactylus) can be
corresponds to 52.7 Trolox nmol/mg (10). This used. Compounds with antioxidant activity
method has also been used in patents and their have the ability to respond to the hydrogen per-
authors showed the advantages of this analyti- oxide produced under the action of horseradish
cal technique (32,33). peroxidase or hemin, oxygen free radicals and
peroxyl. As a result, the inhibition of radiation
is observed. Activity of the antioxidant in the
Hydrogen atom transfer reactions testing sample is determined by comparing the
The HAT mechanism-based methods are recom- kinetics reaction of the control sample without
mended to measure the ability of antioxidants antioxidants to the sample containing the active
to neutralize free radicals by the reaction of ingredients (34,38).
hydrogen atom transfer. The principle of these The method proposed by Knight Scientific
methods is to monitor the kinetics of competing uses Pholasin as a bioluminescence protein for
reactions occurring between the antioxidant measuring the relative activity of both water
contained in the sample and the molecular and fats soluble antioxidants, as well as ready-
probe in the presence of peroxide radical. Rela- made emulsion. This measurement is helpful in
tive antioxidant activity is determined by the assessing the quality of finished products and
bond dissociation energy of hydrogen donor monitoring different stages of manufacturing
group (24). The measurement is based on moni- process of cosmetics (39,40).
toring the kinetics of quenching free radicals The Photochem measurement system, pro-
through the dedication of the hydrogen atom. posed by the Analytik Jena company, with the
The antioxidant capacity of a quantitative detector in the form of luminol allows to
sample analysis is calculated from the curves. evaluate water (ACW) or fat (ACL) soluble
The described method is preferable to measure antioxidants and also the finished cosmetic
the lipophilic antioxidant due to peroxide radi- product. The measurement is based on the prin-
cals that are involved in the process of lipid ciple of photo-luminescence. As a result of

424
 Evaluation of cosmetic antioxidant capacity

optical excitation of a photosensitizer, the free directly into the test preparations without
radicals are created and then monitored. Photo- pretreatment of the sample. A comparison of
chemical excitation of samples leads to oxida- the anode potential for 11 test preparations, the
tion and the process goes a thousand times active substances (vitamins E, C and BHT,
faster than normal. The presence of the antioxi- BHA), and the base of the sample (without the
dant results in reduction of reaction rate, which antioxidant compounds) allow the estimation of
is then compared with the Trolox standard (for the total antioxidant activity of cosmetics. It
fat-soluble substances) or ascorbic acid (for was observed that the greatest possible accu-
water-soluble substances) (41,42). racy of measurements was characterized by a
The advantages of the fluorescent and chemi- platinum electrode. The voltammetry method is
luminescent methods are a low threshold of one of the faster, easy to use, and effective
detection of antioxidants, either hydrophilic or methods for measuring the antioxidant activity
hydrophobic, and ability to determine the of cosmetics. The lack of biological reference of
content of reactive oxygen species in different obtained results is a disadvantage of this
biological systems. These methods can be used method (43).
for indirect analysis of the antioxidant activity
of cosmetics.
Determination of the radical protection
factor (RPF) for cosmetic and radical
Electrochemical methods for skin/status factor (RSF)
assessment of antioxidant activity of Nowadays, the methods of indirect determina-
cosmetics tion of antioxidant activity of cosmetics based on
Increasingly, the voltammetry method is used the radical protection factor (RPF) have become
to determine the antioxidant activity of the cos- more popular (44). Measurement of the RPF
metics (9). This method belongs to the electro- defines the scavenging activity of an antioxidant
chemical measure technique in which the against a tested substance. In this method,
intensity of current flowing through the mea- the nitroxides are used as reactive radicals,
suring cell, depending on the voltage applied to for example: Proxo (2,2,5,5-tetramethyle-1-dihy-
the electrode is determined The analysis is pre- dropyrrolinoxy-nitroxide), Tempol (2,2,6,6-te-
sented on the voltammetric curves graph. tramethyl-1-piperidinoxy-4-ol-nitroxide), DTBN
Among the reported methods, different mea- (di-tert-butyl-nitroxide) or preferably DPPH (1,1-
surement techniques can be distinguished. Vol- diphenyl-2-picryl-hydrazyl) (45). The RPF mea-
tammetry with the linear sweep of the working surement is carried out using the electron spin
electrode (LSV) and cyclic voltammetry (CV) resonance spectroscopy (ESR) or equivalently
are most commonly practiced in cosmetic electron paramagnetic resonance (EPR). It is a
industry. During measurements namely, mea- magnetic resonance-based technique that allows
suring, reference, and auxiliary, three elec- detection of species with unpaired electrons and
trodes, which are placed in a flask with the test thus indirect assessment of the antioxidant
electroactive substance are involved. The cur- capacity of cosmetic (44).
rent flowing through the working electrode is The RPF method allows simple classification
recorded, and a voltage is varied at a constant of cosmetic products, which are designed for
rate in a cyclical manner, accompanied by the neutralizing free radicals. This method is also
potential change in the value of E1E2 and then useful to monitor changes in the antioxidant
to E1. This process is presented graphically on properties over a long period of time, thus to
the voltammetry curve, which has in this case control the quality of cosmetic formulations.
two peaks corresponding to the cathode current According to the studies conducted by Herling
(potential from E1 to E2) and anode current et al. (44), the antioxidant properties of a cos-
(potential from E2 to E1) (43). metic product can be reduced by up to 60%
In a study conducted by Guitton et al. (9) to after 3 months of storage compared to baseline.
measure overall antioxidant activity of cosmet- The effect of external treatments of the skin
ics the cyclic voltammetry was used. In the by cosmetic antioxidants and UV filter influence
quoted experiment working electrodes (plati- on the free radical status of the skin (46). This
num, gold, and glassy carbon) were introduced influence can be characterized by the Radical

425
Ratz-Lyko et al.

Skin/Status Factor (RSF) (47) that describes One of the most interesting studies on the
relation between the radical status (RS) of the in vivo methods for assessing the antioxidant
untreated (RSuntr) and treated skin (RStr). The effectiveness of a cosmetic product was pre-
measurement is based on electron spin reso- sented by Vertuani et al. (50). In these studies
nance spectroscopy (ESR). By this method it is proposed by Manfredini et al. (59), DermAna-
possible to characterize the whole radical status lyzer was used as the measurement device. This
as a function of various exogenous and endoge- allows one to evaluate the skin color by micro-
nous influences and also to distinguish between inflammatory model. In the experiment, the
substances acting as a radical protector or as skin of healthy volunteers was exposed to irri-
radical promoter. Furthermore, RSF values tating factor-methyl nicotinate, and then it was
measured as a function of time give information deposited on preparation containing antioxi-
about the penetration behavior of reactive dants (vitamin C and E). Protective effects of
ingredients (48). the antioxidant substance were determined
using the DermAnalyzer system, and the results
of measurements have been validated using
In vivo and ex vivo methods
standard measuring instruments: chromameter
In vivo and ex vivo assays of cosmetics antioxi- (assessing the color of the skin), laser Doppler
dant activity are usually performed by noninva- (for measuring perfusion) and tewameter
sive instrumental methods, which also (assessing transepidermal water loss). Signifi-
indirectly determine anti-aging properties (49 cant differences between the control not treated
51). area, the area treated with cosmetic base, and
A valuable and complementary way for a area treated with cosmetic formulation contain-
direct and noninvasive measurement of free ing antioxidants were observed. The methyl nico-
radicals is EPR technique and its modifications, tinate microinflammatory model confirmed the
i.e. nitroxide-based EPR spectroscopy and mitigating effects of antioxidant preparations.
imaging (EPRI) of spin labels (52). These tech- The results of the study suggest the usefulness
niques allow one to measure the kinetics and of instrumental techniques for noninvasive,
mapping the spatial distribution of free radi- indirect assessment of the antioxidant activity
cals in biological systems, thus they have of cosmetics. This method can complement the
become a useful tool in experimental oncology, research carried out by in vitro method.
cardiology, neurology, toxicology, and cosme- Stripping of stratum corneum, which can be
tology researches (53). EPRI technique uses the classified as an ex vivo method, can also be use-
resonators like nitroxides as a redox indicators ful in the evaluation of antioxidant capacity of
and spin labels (52,54). When topically applied, cosmetics. This is a commonly used method for
these compounds penetrate from the skin evaluation of kinetic and penetration depth of
surface into the dermis and react with skin topically applied drugs and cosmetics (60, 61).
antioxidants, i.e. ascorbate, ubiquinone, gluta- It is also helpful in evaluation of inhibition of
thione, alpha-tocopherol to form stable adducts the lipid peroxidation by natural skin antioxi-
(51,55). EPR-based techniques allow monitoring dants and cosmetic ingredients (61). For exam-
of the complex enzymatic and nonenzymatic ple, Alonso et al. applied this approach for
redox processes in human tissues and the dis- ex vivo characteristic of antioxidant properties of
tribution and metabolism of nitroxides in the emulsion containing vitamin E and C (60). The
body. On the other hand, it also allows indi- presence of MDA, one of the lipid oxidation
rect evaluation of cosmetics antioxidant capac- end products, was detected by TBA test. Each
ity (51). layer of skin remaining on the tape was
In noninvasive in vivo measurement of lipid dissolved in a mixture of chloroform and thio-
peroxidation, the proton-transfer reaction mass barbituric acid, and then the concentration of
spectrometry (PTR-MS) can also be useful (56 MDA-TBA adducts was fluorimetrically deter-
58). With this technique it is possible to mined. This method allowed one to determine
measure the markers of lipid peroxidation such the degree of inhibition of the s.c. lipid peroxi-
as aldehydes and hydrocarbons in multi- dation and thus indirectly allowed one to assess
component mixtures of various volatile organic the antioxidant properties of the applied
compounds (VOCs). cosmetics (62).

426
 Evaluation of cosmetic antioxidant capacity

The 2, 7-dichlorodihydrofluorescein diacetate

In vitro antioxidant activity in skin
is further converted to DCFH dichlorohydroflu-
cultured cells
orescin by cellular esterases. The presence of
A large amount of literature concerning the free radicals in a cell, especially superoxide
in vitro effects of antioxidant and the cosmetic anion, causes the transformation of this com-
antioxidant activity is commonly assessed in pound into dichlorofluorescein DCF, which
cell-free systems. This approach however, does emits a characteristic fluorescence spectrum
not consider the cellular mechanisms, and showing maxima at 520 nm wavelength (68).
therefore is incomplete. Thus, to assess the This method allows one to monitor specific cel-
antioxidative capacity of a certain cosmetic lular mechanisms in response to oxidative
compound in a more physiological way, experi- stress-inducing factors (37, 67, 69).
ments should be performed in a cellular setting. Cultured cells are also a valuable model for
Human keratinocyte cell lines HaCaT and in evaluation of the antioxidant activity of cos-
vitro homogeneous fibroblasts and keratinocytes metic ingredients and for control the lipid per-
more accurately reflect the biological reality, oxidation (70). During the oxidation of
thus allowing one to evaluate the properties of polyunsaturated fatty acids incorporated in cell
the antioxidants in cosmetics and their effect on membrane phospholipids malondialdehyde is
the skin (37). They were used as an appropriate formed. This compound often serves as a
experimental in vitro model to evaluate the marker of lipid peroxidation processes, because
effects of antioxidant on cell viability, prolifera- in the presence of malondialdehyde and tio-
tion, free radicals generation, lipid peroxidation, barbituic acid (TBA) colored adducts with chro-
and intercellular enzymes level measurement. mophores group are formed, which can be
For this reason, the cultured cells, especially determined spectrophotometrically of fluoro-
skin cells (for example keratinocytes and fibro- metrically (71). Svobodova et al. used human
blasts) are a valuable model for indirect evalua- keratinocytes as an appropriate experimental
tion of antioxidant activity of cosmetic model. The cells were exposed to UV radiation
ingredients and control the lipid peroxidation and then the lipid peroxidation level was mea-
process. The results, however, should be inter- sured by TBA test (70).
preted cautiously as the cell lines are only a
part of the phenotype of normal skin cells
(6365).
Summary
Most of the presented skin-cultured cells tests Although many methods are described in the
based on determination of antioxidant enzymes literature for assessing the antioxidant activity,
(i.e. catalase, glutathione peroxidase and super- not all can be used in the cosmetics industry.
oxide dismutase activity assay), total antioxi- These restrictions are mainly due to the pres-
dant capacity (TAC) of active ingredients, and ence of both hydrophilic and hydrophobic com-
lipid peroxidation are assayed by commercially pounds in cosmetics. Therefore, it seems
available assay kits. For example Grazul-Bilska appropriate to choose the method to a type of
et al. (66) determined the effects of skin mois- antioxidant present in the sample. It is worth
turizers on total antioxidant capacity of human mentioning that the mechanism of the reaction
skin using EpiDerm (MatTek Corporation, between a compound and various antioxidants
Ashland, MA, USA) model and glutathione per- depends on the type of free radicals. This
oxidase activity as a marker of antioxidant causes considerable inconvenience in analysis
capacity. and points to the fact that we are not really
In a study conducted by Jentzsch et al. (37), certain, whether the used method allows us to
HaCaT cell line derived from human skin kerat- measure the overall activity of the antioxidants.
inocytes has been used to assess the antioxidant Moreover, most in vitro methods do not reflect
activity of vitamin C and E in cosmetics. In this the actual biological processes in skin cells,
method, the 2, 7-dichlorodihydrofluorescein di- and the results obtained by using cell lines,
acetate was used as detector. This substance can although most similar to natural conditions
diffuse through the cell membrane to the intra- should be interpreted with caution. To obtain
cellular space and is incorporated into hydro- reliable results, it seems appropriate to use
phobic lipid regions of the cell (67).

427
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several analytical techniques both in vivo, ex vivo ent methods of assessing the antioxidant activity
and in vitro. of cosmetics. Certainly, this would allow a better
In literature there is no objective evidence in understanding of the mechanisms of oxidation,
the form of in vitro and in vivo research, which countermeasures, and thus would provide a
allows one to compare the effectiveness of differ- more effective tool for cosmetics quality control.

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