3-2014
English Edition
International Journal for Applied Science
• Personal Care • Detergents • Specialties
T. Rudolph, S. Eisenberg, J. Grumelard, B. Herzog
State-of-the-art Light Protection
against Reactive Oxygen Species
Cosmet ic s
T. Rudolph, S. Eisenberg*, J. Grumelard, B. Herzog**
State-of-the-art Light Protection against
Reactive Oxygen Species
■■ Introduction
Light, in particular ultraviolet irradiation
(UV), but also visible and near infrared
irradiation (IR) are the major sources to
generate reactive oxygen species (ROS)
such as singlet oxygen, radicals or peroxides.
Today´s »sun protection systems« are
more and more developing into fu-
Abstract
T
his paper aims at a scientific comparison of two substantially different anti-ROS test methodologies: the beta-carotene test (BCT test)
and the radical status factor test method (RSF method). The photometer-based BCT test uses the light-sensitive and lipophilic probe of betacarotene to be inserted into the formulation. This probe degrades under
sun-like irradiation through its ability to quench lipophilic reactive oxygen
species (ROS) like singlet oxygen. The RSF method indirectly measures the
UV light-triggered formation of hydroxyl radicals that are trapped by a watersoluble spintrap (electron spin resonance technique).
Two state-of-the-art light protection actives, Bis-ethylhexyl hydroxydimethoxy benzylmalonate (HDBM), a liquid, stable, non-yellowing, and non-absorbing skin care antioxidant, plus Bis-ethylhexyloxyphenol methoxyphenyl
triazine (BEMT), a broadband and photostable UV filter were combined at
various ratios in a light protection cream to be tested in both assays.
Under solar simulated light in the BCT test, HDBM performed optimal due
to its lipophilic nature and its ability to quench lipophilic ROS in the direct
neighbourhood of beta-carotene. By contrast BEMT predominates in the
RSF test. Here the product performance mainly originates from BEMT´s
broadband UV absorbance power that covers both, UVB and UVA.
In the discussion about relevant antioxidant testing it appears essential to
combine diverse protocols into the evaluation strategy. Here two complementary methods are proposed as well as a combination of modern actives that can pass those assays with distinction.
ture »light protection systems«. Modern products may provide photostable
broadband UVB/A protection against
sunburn and photoaging but may also
need to counteract all sources for ROS
generation while maintaining an optimal skin feel as well as appealing galenic
properties such as being white, stable
and non-yellowing.
Anti-ROS efficacy test methods are
countless. Some include irradiation steps
while others do not. Some might only
measure a specific group of ROS, like
radicals, but do not monitor non-radical
ROS like singlet oxygen or peroxides.
Some involve hydrophilic measurement
techniques, others concentrate on the
lipophilic environment.
(Photo)oxidative stress is a complex
process. To a large extent it is linked to
the presence of air oxygen, its transformation into reactive species and their
chemical reaction with oxidation-sensitive compounds. Light in the presence
of a sensitizer often initiates oxidative
stress or autoxidation. Air oxygen is remarkably lipophilic and concentrated
Fig. 1 Spectrum of the Atlas CPS+
irradiance
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SOFW-Journal | 140 | 3-2014
Co sm etics
on the skin surface (5), so that lipophilic environments such as sebum are
in particular prone to autoxidation (6).
Regarding this lipophilic aspect we herein describe the BCT assay (beta carotene
test) to capture lipophilic ROS like singlet oxygen, lipoperoxides and lipophilic radicals. In comparison the RSF test
(radical status factor) is more directed
towards hydrophilic ROS with a focus on
the hydroxyl radical.
Five cosmetic emulsions were tested for
their overall antioxidant performances
in both assays. Varying levels of »primary« and »secondary« actives were chosen
and their impact on the overall antioxidant protection level investigated.
Bis-ethylhexyloxyphenol
methoxyphenyl triazine (BEMT (1)), a broadband and photostable UV filter served
as »primary« performance active. It
was combined with its »secondary«
active counterpart Bis-ethylhexyl hydroxydimethoxy benzylmalonate (HDBM (2, 3)). HDBM is a liquid, stable,
non-yellowing, and non-absorbing
skin care antioxidant.
■■ Materials and methods
a) BCT test (beta-carotene test)
A solution of 0.5 % (w/w) beta-carotene
(Fluka, Art. Nr. 22040) in o-xylene was
freshly prepared and 2µLcm-2 of the solution homogeneously applied over the
roughened side of a PMMA plate (PMMA
plates from Schönberg, sandblasted, 2µm
roughness). The plate was allowed to dry
(RT, 10 min) before the formulation was
applied onto the plate with a positive displacement pipette (2µLcm-2) and homogeneously distributed with a finger. In total
8 replicates per product were prepared.
4 replicates were stored in the dark and 4
replicates were subjected to the irradiation step. As irradiation source we used an
Atlas CPS+ solar simulator equipped with
a water cooled sample tray. As an irradiation dose we applied 150kJm-2 (total UVB
plus UVA) corresponding to an erythemal
effective dose of about 3 MED. The total
irradiance was set to 500Wm-2 of which
58.9 Wm-2 was UV, the remaining visible
and IR. From Fig. 1, which shows the irradiance spectrum of the Atlas CPS+, it
becomes clear, that the samples in this test
SOFW-Journal | 140 | 3-2014
Fig. 2 Absorbance in terms of specific extinction of BEMT and HDBM in the
spectral range between 290 and 450 nm
were exposed to significant amounts of
visible radiation.
After irradiation the PMMA plates
were extracted with isopropanol and
diluted to a volume of exactly 50mL.
Beta-carotene was photometrically
quantified at 452nm. The final recovery of beta-carotene was calculated in
comparison to the equally treated dark
references.
b) Radical Status Factor (RSF) Test
This method is based on EPR spectroscopy using a spin probe for the detection of radicals. A spin probe is a stable
radical, for instance a nitroxide, which
reacts with the radicals induced during UV irradiation, such that the EPR
signal of the probe decreases with UV
dose (4, 6). Skin biopsies of porcine origin
(taken from pig ears) are incubated with
a 50/50 water/ethanol 1 mM solution
of 3-carboxy-2,2,5,5-tetramethylpyrrolidone-1-oxyl (PCA) for five minutes.
Radicals are induced in the skin samples
by irradiation with simulated solar UV
light (Newport, 300 W; irradiance in the
UVB range = 23.5 W/m2). EPR signals are
measured after UV doses of 7, 14, 28,
42, and 70 kJ/m2), and a rate constant is
determined from the decrease of the signal. It is important to note, that the light
source used here was comparable in the
UV range to the output of the lamp used
for the BCT test, but radiation above 450
nm was filtered off with the RSF tests.
In order to reduce experimental scatter due to the variability of the biological substrates, a calibration curve is
measured for each set of skin samples
originating from the same animal, using neutral density filters with known
transmission, and thus known protection
factor (RSF). The rate constant of the
degradation process of the spin probe is
determined from the experimental data
obtained after the different UV doses,
and is normalized to its value without
protection (7). The calibration curve is
constructed by plotting the normalized
rate constants against the RSF values
of the neutral density filters. Test formulations are assessed by distributing
them at a rate of 2mg/cm2 on pig skin
substrates. After 15 minutes normalized
rate constants are determined from the
decrease of the EPR signal as function
of UV dose. The value of the RSF can
then be read from the calibration curve.
Each formulation was measured on four
skin biopsies. The RSF tests were carried
out at Gematria Test Lab GmbH, Berlin,
Germany, using an X-band EPR spectrometer Miniscope MS300 (Magenttech GmbH, Berlin, Germany).
c) Test formulations
Oil-in-water cream containing various
amounts of Bis-ethylhexyl hydroxydimethoxy benzylmalonate (HDBM, RonaCare
AP®, Merck KGaA) and Bis-ethylhexyloxyphenol methoxyphenyl triazine (BE-
11
Cosmet ic s
MT, Tinosorb® S, BASF SE) were prepared.
The compositions are listed in Table 1.
Fig. 2 shows results of UV spectroscopic
measurements of BEMT and HDBM in
terms of specific extinction (E11, absorbance at a concentration of 1 % (w/v)
and an optical path length of 1 cm) in
the spectral range between 290 and 450
nm. While BEMT is efficient in absorbing
UV over a broad spectral range, there is
no UV attenuation by HDBM.
■■ Results and discussion
In this study two types of tests were used,
the BCT test (beta carotene test) and the
RSF test (radical status factor test). From
the differences in testing conditions,
complementary results can be expected.
The main differences in the test conditions are summarized in Table 2.
a) BCT test (Beta carotene test)
Carotenoids, mainly beta-carotene (Provitamin A) and lycopene are part of the
natural skin´s defense system against
light-induced oxidative stress (8). Ca-
demonstrated in the BCT test (Fig. 3).
Here we used the extreme light-sensitivity of beta-carotene as a suitable
criterion to measure the product´s ability to prevent beta-carotene from being photooxidized. The photooxidation
level may then be transferred to other
light-sensitive skin lipids like squalene,
unsaturated fatty acids or retinoids (10).
The test results indicate (Fig. 3) that in
the placebo formulation A) almost 80 %
of beta-carotene were photodegraded
under sun-simulated light containing a
total UV dose of 150kJm-2 (equivalent to
3 MED). The addition of 1 % of the antioxidant HDBM in formulations B), D) and
F) drastically reduced the beta-carotene
degradation from 80 % back to 15 % in
BCT test
RSF test
Substrate
PMMA
Pig ear skin
Irradiation
UV + visible
UV
Nature of probe
hydrophobic
hydrophilic
Table 2 Comparison of BCT- and RSF testing conditions.
INCI Name
A
B
C
D
E
F
Emulgin Prisma (BASF)
Disodium Cetearyl Sulfosuccinate
0.50
0.50
0.50
0.50
0.50
0.50
Lanette O (BASF)
Cetearyl Alcohol
1.00
1.00
1.00
1.00
1.00
1.00
Cetiol B (BASF)
Dibutyl Adipate
5.00
4.00
5.00
4.00
5.00
4.00
X-Tend 226 (ISP)
Phenethyl Benzoate
12.00
12.00
12.00
12.00
12.00
12.00
RonaCare® AP (MERCK)
Bis-Ethylhexyl Hydroxydimethoxy
Benzylmalonate
Tinosorb® S (BASF)
Bis-Ethylhexyloxyphenol
Methoxyphenyl Triazine
Water
Aqua
83.88
Rhodicare S (Rhodia)
Xanthan Gum
Edeta®BD (BASF)
1.00
1.00
3.00
3.00
83.88
82.78
82.78
80.58
80.58
0.50
0.50
0.50
0.50
0.50
0.50
Disodium EDTA
0.20
0.20
0.20
0.20
0.20
0.20
DC 246 Fluid
(Dow Corning)
Cyclohexasiloxane (and)
Cyclopentasiloxane
4.00
4.00
4.00
4.00
4.00
4.00
Tinovis® ADE (BASF)
Sodium Acylates Copolymer (and)
Hydrogenated Polydecene (and) PPG-1
Trideceth-6
0.40
0.40
0.40
0.40
0.40
0.40
Germall Plus (ISP)
Diazolidinyl Urea (and) Iodopropynyl
Butylcarbamate
0.15
0.15
0.15
0.15
0.15
0.15
Part B
1.00
Part C
1.00
Part D
1.00
Part E
Part A
Trade Name
rotenoids are ingested through the diet
and can reach the outermost skin layers were they are exposed to solar light
and can be degraded by light-induced
singlet oxygen, lipophilic radicals and/
or lipoperoxides.
It is well known that under light exposure
beta-carotene can form lipophilic ROS
like peroxyl radicals and lipoperoxides
that further degrade beta carotene itself.
In addition beta-carotene is a highly efficient singlet oxygen quencher. In plants it
neutralizes singlet oxygen that is produced
in a sensitized reaction by the green leaf
pigment chlorophyll. Beta-carotene thus
helps to maintain the photosynthesis (9).
The quenching of lipophilic ROS leads
to beta-carotene´s photodegradation as
Table 1 Compositions of formulations
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SOFW-Journal | 140 | 3-2014
Co sm etics
The high RSF of 6.7 of emulsion C containing 1 % of BEMT can be explained by
the broadband absorption of UV-radiation of this molecule, protecting the skin
sample from radical formation. Effects
of visible light are not to be expected
here, as the Newport solar simulator
shows a steep decrease of irradiance at
wavelengths above 400nm. Again, the
presence of 1 % HDBM in emulsion D)
leads only to a small increase of the RSF
to 7.7.
■■ Conclusion
Fig. 3 BCT test (beta-carotene test); six emulsions A) to F) tested for their antioxidative potential; emulsion A) is the control without HDBM or BEMT (UV dose
150kJm-2); error bars represent ± standard deviation of four replicates.
formulation F). In this assay the influence of the UV absorber BEMT on the
photostabilization of beta-carotene was
less obvious: In formulation E), 3 % BEMT
reduced the beta-carotene photodegradation only from 80 % to about 50 %.
One possible mechanistic explanation for
the outstanding performance of HDBM in
this assay might be the impact of visible
light present in the irradiation spectrum
(11). The UV absorber BEMT does efficiently absorb broadband UV, but does not absorb visible light. The lipophilic antioxidant
HBDM however neutralizes singlet oxygen
and other lipophilic ROS irrespectively of
the spectral light situation. It hence may
extend the broadband UV protection by
BEMT into the visible region.
b) RSF test results
Fig. 4 shows the results of the RSF tests,
which have been performed with emulsions A, B, C, and D. The RSF of the placebo
formulation (control) without HDBM or
BEMT shows as expected a value of 1.0.
The RSF of emulsion B) which contains
1 % HDBM is only slightly higher, at 1.4.
This can be explained by the fact that the
radical probe mainly goes into the water
phase of the system (the n-octanol/water partition coefficient of the PCA spin
probe is 0.0047 (12)), whereas HDBM is
located in the lipophilic phase. Another
SOFW-Journal | 140 | 3-2014
reason for the small effect of HDBM
could be that it does not penetrate very
deep into the skin, where still radicals
may have formed. As HDBM does not
absorb in the UV/Vis region, the slight
increase of the RSF could be explained by
small amounts of the spin probe (about
0.5 % of its total concentration) partitioning into the oil phase.
Together, both tests, RSF and BCT, cover
a broad range of most important parameters in the discussion about relevant anti-ROS protection. Firstly they
combine lipophilic as well as hydrophilic
environments, and thus for instance can
take the lipophilic nature of air oxygen
into account. Both tests cover radical
and non-radical reactive oxygen species
of which non-radical singlet oxygen
plays an important role at the origin of
the radical chain reaction. Finally they
include sun simulating UV light conditions comprising not only UV, but also
visible and IR as additional source for
ROS generation (as lately discovered)
(11).
Fig. 4 RSF results with emulsions A) to D) tested for their antioxidative potential;
emulsion A) is the control without BEMT or HDBM (UV dose 70 kJm-2); error bars
represent ± standard deviation of four replicates.
13
Cosmet ic s
In conclusion it appears essential to
combine diverse protocols into the
evaluation of the antioxidative potential
of cosmetic skin and sun care products.
Here two complementary methods, the
lipophilic BCT and the hydrophilic RSF
test, were proposed as well as the actives
combination of HDBM and BEMT that
have been shown to pass those assays
with distinction.
References
(1)
B. Herzog, D. Hüglin, E. Borsos, A. Stehlin,
H. Luther; New UV Absorbers for Cosmetic
Sunscreens – A Breakthrough for the Photoprotection of Human Skin, Chimia 58, 554
– 559 (2004)
(2) R
. Graf, J. Beck, T. Rudolph, K. Jung, T. Herrling, F. Pflücker; Antioxidative Power of
Formulations Over Life Time: Unique Active
Superior than Vitamins; SOFW Journal,
134(9) (2008) 52, 54-56, 58, 60.
(3)
T. Rudolph, J, Pan, R. Scheurich, F. Pfluecker, R.
Graf, H. Epstein; Superior two step approach
to completely photoprotect avobenzone with
a designed organic redox pair; SOFW Journal
(2009), 135(9), 14, 16-18.
(4)
T. Herrling, L. Zastrow, J. Fuchs, N. Groth.
Electron spin resonance detection of UVA-induced free radicals; Skin Pharmacol. Physiol.
17 (2002) 381 – 383
(5)
M. Stücker, C. Moll, P. Altmeyer; Sauerstoffversorgung der Haut – Unter besonderer
14
Berücksichtigung der kutanen Sauerstoffaufnahme aus der Atmosphäre; Hautarzt 55
(2004) 273-279.
(6) T. Herrling, J. Fuchs, J. Rehberg, N. Groth;
UV-induced free radicals in the skin detected
by ESR spectroscopy ans imaging using nitroxides. Free Radical Biology & Medicine 35
(2003) 59 – 67
(7)
K. Jung, M. Seifert, T. Herrling, J. Fuchs; UVgenerated free radicals in skin: Their prevention by sunscreens and their induction by
self-tanning agents. Spectrochimica Acta
Part A 69 (2008) 1423 – 1428
(8) I. Ermakov, J. Lademann, M Ermakova, W.
Gellermann; Noninvasive selective detection
of lycopene and β-carotene in human skin
using Raman spectroscopy; J Biomed Opt,
9(2) (2004) 332-338.
(9) F. Ramel et al.; Chemical quenching of singlet
oxygen by carotenoids in plants; Plant Physiology, 158(3) (2012) 1267-1278.
(10) B. Auffray; Protection against singlet oxygen,
the main actor of sebum squalene peroxidation during sun exposure, using Commiphora
myrrha essential oil; Int J Cosmet Sci, 29(1)
(2007) 23-29.
(11) L . Zastrow, N. Groth, F. Klein, D. Kockott,
J. Lademann, R. Renneberg, L. Ferrero; The
Missing Link – Light-Induced (280-1600
nm) Free Radical Formation in Human
Skin; Skin Pharmacol Physiol, 22 (2009)
31-44.
(12) F. Hyodo. K. Yasukawa, K. Yamada, H. Utsumi; Spatially resolved time-course studies
of free radical reactions with an EPRI/MRI
fusion technique; Magnetic Reson Med, 56
(2006) 938 – 943
Acknowledgement
The authors thank Dr. Katinka Jung
(Gematria Test Lab GmbH) for measurements of the radical status factor
(RSF).
Authors´ addresses:
*Thomas Rudolph
Sylvia Eisenberg
Merck KGaA
Frankfurter Str. 250
64293 Darmstadt
Germany
Email: [email protected]
**Julie Grumelard
Bernd Herzog
BASF Grenzach GmbH
Köchlinstr. 1
79639 Grenzach-Wyhlen
Germany
Email: [email protected]
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