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j. Cosmet. Sci., 50, 147-157 (May/June 1999)
Skin penetration properties of cosmetic formulations using
a perfused bovine udder model
TH. F(SRSTER, W. PITTERMANN, M. SCHMITT, and
M. KIETZMANN, Henkel KGaA, D-40191 Diisseldorf and Institute
of Pharmacology, Toxicology and Pharmac•y, School of Veterinary
Medicine, D-30559 Hannover, Germany
Accepted for publication April 30, 1999. Presented at the XXth
Congress of the International Federation of the Societies of Cosmetic
Chemists, Cannes, France, September 14-18, 1998. Presented in part at
Penetration, Leiden, The Netherlands, September 22-26, 1998.
Synopsis
Bioactives are a major focus of the recent developments in cosmetics. Most of these ingredients will work
only if they penetrate the stratum corneum and reach the living epidermis. The extent and rate of pen-
etration of bioactive ingredients from cosmetic products is of paramount interest. A systematic in vitro study
of penetration into stratum corneum and viable skin was performed using the isolated perfused bovine udder
skin (BUS) model.
As bioactive ingredients, oil-soluble vitamin E, vitamin E acetate, and water-soluble D-panthenol were
incorporated into different cream formulations and a shower gel. Under leave-on conditions (exposure
periods of one hour and five hours), the changes in phase behavior and viscosity have a strong impact on the
penetration pattern. Generally, the water-soluble D-panthenol penetrates faster and in a higher amount into
the stratum corneum than the oil-soluble vitamin E. Regarding the choice of the galenic vehicles, the
penetration of both the oil-soluble and water-soluble vitamin is faster and much higher from w/o-cream than
from lameIlar o/w-cream due to changes in phase behavior. In contrast to the lamellar o/w-emulsion, for the
w/o-emulsion there is no difference in the penetration of vitamin E between one hour and five hours (e.g.,
a steady state is already reached after an exposure period of 1 h). The top ten skin layers (200 pm depth)
again reflect the influence of the emulsion formulation on the vitamin E absorption. From the w/o-cream
the penetration is faster and more efficient than from the lameliar o/w-cream.
Under rinse-off conditions (3*2 minutes), the water-soluble D-panthenol penetrates in a higher amount
than the oil-soluble vitamin. However, the penetration effect is detectable even after the extremely short
period of exposure. Due to the fact that the shower gel will not change the phase behavior, different routes
of penetration may be considered for water- and oil-soluble vitamins.
INTRODUCTION
Today's cosmetic emulsions are not simply mixtures of oil, water, and emulsifier, but
they also contain a lot of active ingredients whose function is to ameliorate the condition
147
148 JOURNAL OF COSMETIC SCIENCE
of the skin, e.g., use in skin hydration or on the barrier properties of the stratum
corneum. Most of these ingredients will work only if they penetrate into the stratum
corneum or the deeper layers of the epidermis. The extent and rate of penetration of an
active ingredient from a topical formulation depends on its passive diffusion into the
skin, which in principle obeys Fick's law of diffusion. This takes account of the con-
centration of the active ingredient in the galenic vehicle, the mobility (diffusion con-
stant) of the active ingredient molecule, and--in the form of the distribution coeffi-
cient-the interaction of the active ingredient with the vehicle and the skin (overview
in references 1 and 2). In practice, the application of this simple law is frequently
difficult, because the composition of a topical formulation only rarely remains constant
during the course of application; volatile components vaporize, water evaporates (3-5),
and the mobility and the distribution coefficient of the active ingredient change as a
function of its concentration and of the viscosity and phase behavior of the galenic
vehicle.
Moreover, the penetration properties of a formulation are also dependent on the inter-
action of the vehicle with the skin (2,6). The application of the emulsion can cause an
occlusive effect, under which the hydration of the horny layer increases, resulting in
increased penetration. Certain components of the formulation may accumulate in the
outermost skin layer, the actual diffusion barrier, thus accelerating penetration (2,7).
Simple penetration models cannot take these different, mutually interacting effects into
account, so that an optimal galenical can only be developed on the basis of experimental
penetration studies.
On ethical grounds, in vivo studies on humans and animals are only carried out on a
limited scale, mainly in connection with dermatics (8). The isolated perfused bovine
udder skin (BUS) model is a good substitute for in vivo tests on humans (9). As a living
skin model it takes also into account metabolic processes in the skin, so that it can be
used to study both skin penetration and skin irritation by cosmetic formulations (10,11).
Due to the different rheology of the emulsions under living conditions (physiological
surface temperature, physiological TEWL) and the expected identical substantivity
(same components as far as possible), it was intended to compare the emulsions under the
conditions of infinite dose, i.e., more than 10 or 20 mg/cm 2. The high dosage prevents
any depletion of vitamin concentration in the vehicle during the penetration experiment.
Furthermore, the study design allows a comparison with the results of the testing of
certain types of ointments and dermatics using this in vitro model (9).
Based on an extensive investigation of the influence of emulsion type and structure on
vitamin penetration into the skin (10), the objective of this work was to study the
kinetics of the penetration of oil- and water-soluble vitamins such as vitamin E, vitamin
E acetate, and D-panthenol. An emulsion of the water-in-oil (w/o) type and a lamellar
oil-in-water (o/w) emulsion were chosen as leave-on products since they showed big
differences in phase behavior and penetration properties (10). At an extreme, the short-
term penetration from a surfactant-based shower gel is investigated under rinse-off
conditions.
MATERIALS AND METHODS
CHEMICALS
The galenic formulations selected for the penetration studies (Table I) exhibit basic
SKIN PENETRATION PROPERTIES 149
physicochemical differences. Two creams (w/o and lameliar o/w emulsions) were studied
as typical leave-on products. As a rinse-off product, a shower gel formulation based on
an aqueous surfactant solution was chosen.
All surfactants, emulsifiers and, consistency-imparting factors were Henkel products.
The surfactants used were sodium lauryl sulfate (Texapon © N70), lauryl glucoside
(Plantacare © 1200), potassium cocoyl hydrolyzed collagen (Lamepon © S), PEG-7 glyc-
eryl cocoate (Cetiol © HE), and cocoamidopropyl betaine (Dehyton © K). The emulsifiers
used were PEG-7 hydrogenated castor oil (Dehymuls © HRE 7) and sodium cetearyl
sulfate (Lanette © E); the consistency-imparting products and iipids were glyceryl oleate
(Monomuls © 90-O-18), cetearyl alcohol (Lanette © O), and behenyl alcohol (Lanette ©
22).
The oils used were dicaprylyl ether (Cetiol © OE, Henkel KGaA), decyl oleate (Cetiol ©
V, Henkel KGaA), and dimethicone (Bayslion © 350, Bayer AG). The vitamins were
natural vitamin E (Copherol © F1300, Henkel KGaA), vitamin E acetate (Hoffmann-La
Roche), and provitamin B5 (D-panthenol, Hoffmann-La Roche). Glycerol and
MgSO4*7H20 (p.a. quality, J.T. Baker).
METHODS
Sample preparation. Lameliar oil-in-water (o/w) emulsions were prepared by mixing the
Table I
Galenic Formulations (in wt.% active substance)
O/W cream
W/O cream (lameliar) Shower gel
Ingredients
Dicaprylyl ether 7.0 7.5
Decyl oleate 7.0 7.5
Behenyl alcohol 7.0
Glyceryl oleate 1.2
Sodium lauryl sulfate 9.5
Lauryl glucoside 5.4
Cocoamidopropyl betaine 1.0
Potassium cocoyl hydrolyzed collagen 1.0
PEG-7 glyceryl cocoate 1.0
Laureth-2 0.5
Sodium cetearyl sulfate 0.2
PEG-7 hydrogenated castor oil 3.5
Dimethicone 0.5
Vitamin E 0.7 0.7
Vitamin E acetate 0.1
D-Panthenol 0.4 0.4 0.2
Glycerol 5.0 5.0
MgSO4*H20 0.7
NaC1 0.5
Preservative 0.3 0.3 1.4
Water 75.4 71.9 78.2
Physical parameters
Droplet size/pm 0.7 _+ 0.2 2.4 _+ 2.2
Viscosity/mPas at 30/s 1000 1000 1160
150 JOURNAL OF COSMETIC SCIENCE
components with an Ikavisc MR-D1 mixer (Janke & Kunkel) for 30 minutes during a
heating and cooling cycle between 25 øC and 95 øC. The emulsions were homogenized for
five minutes at 40øC.
The w/o cream was made by heating the oil/emulsifier mixture to 80øC and stirring in
the hot water phase. The mixture was stirred for five minutes at 80øC and then cooled
to room temperature while being stirred.
The shower gel was prepared by mixing the components for 30 minutes at room
temperature.
Phase behavior of the emu/siom. Liquid crystalline phases were identified by polarization
microscopy (Zeiss, K61n, Germany). The emulsion type (o/w or w/o) was determined by
conductivity measurements (Radiometer, Copenhagen, Denmark).
Viscosity measureme, t. Flow and viscosity curves in the shear rate range from 0 to 100/s
were studied with a thermosrated, shear-rate-controlled rotation rheometer RFS 2
(Rheometrics, Piscataway, NJ) with a plate-plate measuring system (2 mm gap) at 25 øC.
Partide sizes. The particle size distribution in the undiluted emulsions was determined
with an optical microscope with the help of the Optimetrix digital image analyser
(Stemmer, Meerbusch, Germany).
Bovine udder skin (B US) model. The in vitro "isolated perfused bovine udder skin" model
makes use of material from slaughterhouses. Immediately after an animal has been
slaughtered, the udder skin is perfused with heated and oxygen-enriched Tyrode's
solution (80-100 mm Hg pressure; approx. 120 ml/minute) under laboratory condi-
tions. The viability of the skin is monitored biochemically in the perfusate by deter-
mining the pH, the lactate-dehydrogenase activity, and the lactate and glucose concen-
trations. The skin surface temperature (approx. 32øC) and skin fold thickness 3mm are
measured physically (9). The skin of the udder is thin and exhibits all the morphological
characteristics of mammalian skin, including the cutaneous appendages such as seba-
ceous glands and hair follicles. The skin of the udder functionally resembles human skin
(9). The experimental procedures such as preparation, perfusion, viability checks, the
topical application of the coded test substances, and sampling were performed at
SIMRED GmbH (Gro[3burgwedel, Germany).
Skin penetration. The penetration of oil- and water-soluble vitamins from the different
formulations into perfused bovine udder skin was studied in two ways.
Leave-on application. Fifteen minutes after perfusion started, 3 g of each different vita-
min-containing cream formulation were applied topically to skin areas measuring 75
cm 2. This high dosage was intended to prevent any depletion of the vitamin concen-
tration in the vehicle during the penetration experiment (infinite dose). After one hour
and five hours, respectively, the residual cream was carefully removed with a paper towel,
and then adhesive tape strips and samples for preparing dermatome sections were taken.
The adhesive strips and skin samples were kept deep-frozen at -20øC.
Rinse-off application. Fifteen minutes after perfusion started, 3 g of each different vi-
tamin-containing shower gel formulation were applied topically to skin areas measuring
75 cm 2. After two minutes the shower gel was rinsed off with a surplus of warm water
(40øC). The skin was carefully dried with a paper towel. This washing-rinsing procedure
was repeated three times, and then adhesive tape strips were taken.
SKIN PENETRATION PROPERTIES 151
Adhesive tape stripping. Stripping (Tesa © 4204, BDF, Hamburg) was used to remove the
outermost layers of the stratum corneum in sequence; about ten layers of horny-layer
cells (roughly 10 l•m) were removed. The mass of the removed horny-layer cells was
constant for all layers, being approx. 1.10 mg per adhesive strip (1.9'10 cm), with a
standard deviation of +0.36 mg (n = 20). The dermatome sections parallel to the skin
surface were cut into 20-pm-thin sections and went down 200 pm through the epider-
mis into the upper layer of the dermis.
Detection of the vitamins. The detection of vitamin E, vitamin E acetate, and D-panthenol
in the adhesive strips from the outermost skin layer and in the dermatome sections of the
dermis and epidermis was based on extraction of the analytes and subsequent liquid
chromatographic analysis of the obtained extracts. The selective determination of the
vitamins in the complex matrix required substance-specific detection to be carried out
after the chromatographic separation. The quantification was based on external standard
calibrations and comparison of the treated and untreated skin.
Detection of vitamin E and vitamin E acetate. Vitamin E/vitamin E acetate were extracted
from the relevant skin sections with ethanol, and then the solvent was quantitatively
removed and the residue was taken up in hexane. Extraction from adhesive strips was
carried out directly with hexane. Vitamin E was separated from unwanted accompanying
substances in a diol phase (Lichrospher 100 Diol) by means of liquid chromatography.
The chromatography was carried out under isocratic conditions with n-hexane/te•-t.
butyl methyl ether as eluent. Selective detection was performed with a fluorescence
detector. The chromophoric system in the vitamin E molecule enabled the work to be
carried out at an excitation wavelength of 295 nm.
Detection ofpanthenol. In contrast to vitamin E and vitamin E acetate, panthenol has no
molecular properties that would facilitate sensitive and selective detection in difficult
matrices. After extraction from the skin or adhesive strips, therefore, the obtained extract
was first hydrolyzed alcoholically and the panthenol quantitatively converted to ami-
nopropanol. This hydrolysis product was separated from unwanted accompanying sub-
stances in an ion-exchange column with diluted sodium hydroxide solution as eluent,
and was converted to a strongly fiuorescing isoindole derivative in a postcolumn reaction
with orthophthaldialdehyde. The fluorescence emission was measured with an HPLC
fluorescence detector at a wavelength of 455 nm.
RESULTS AND DISCUSSION
PHASE BEHAVIOR OF THE COSMETIC FORMULATIONS
For the leave-on products, consideration also has to be given to their behavior after open
topical application. When they are applied, the emulsions are spread over the skin,
forming a film (approx. 400 l•m). As the temperature of the emulsions increases to skin
temperature, components with a high vapor pressure, in our case water, start to evaporate
(3,4). Drying experiments on thin emulsion films show that•clepending on atmo-
spheric humidity and the thickness of the layer--almost all the water escapes from the
emulsions within five to ten minutes. During this period the viscosity and sometimes
also the structure of the emulsion change (10).
152 JOURNAL OF COSMETIC SCIENCE
Directly after application, the lameliar cream contains 70% water and has a moderate
viscosity of about 1000 mPas (Table I). When water evaporates, the lameliar layers are
packed together more densely, leading to an appreciable viscosity increase (about 7,000
mPas). When the water content becomes less than about 15%, the amount of water is
insufficient to allow o/w-emulsions to be formed any longer, and w/o-emulsions are
formed (10).
The situation is different with the w/o-cream. Here the oil is the outer phase and the
viscosity rises exponentially by increasing the proportion of the inner water phase (10).
During drying, therefore, the viscosity of the w/o-cream decreases below 10 mPas, in
contrast to the o/w-emulsion.
For the shower gel, the phase behavior will not change during the short application
period. The surfactant product is washed away with a surplus of water after only two
minutes. Therefore, phase changes caused by water loss will not take place under
rinse-off conditions.
KINETICS OF VITAMIN PENETRATION INTO THE SKIN UNDER LEAVE-ON CONDITIONS
After application on the skin, an active ingredient begins to diffuse out of the galenic
vehicle into the top layers of the skin. If, for purposes of simplification, it is assumed that
the horny layer is a homogeneous diffusion barrier, then, according to Fick's law (Equa-
tion 1), the penetration rate after a steady state has been reached depends only on the
mobility of the active-ingredient molecule, given by the diffusion constant D, the
coefficient of distribution K of the active ingredient between the vehicle and the stratum
corneum layer, and the concentration c of the active ingredient (1,2).
Penetration rate J = K/d ß D * c (Eq. 1)
where d is the thickness of the horny layer, D is the diffusion constant, c is the
concentration, and K is the distribution coefficient.
Figures 1 to 4 show the penetration profiles of vitamin E and D-panthenol into the
stratum corneum from w/o and o/w creams. The amount of naturally occurring vitamin
E in the skin is several orders of magnitude smaller than the amount absorbed after
topical application of the creams. For the oil-soluble vitamin E, a steep concentration
gradient over the stratum corneum is observed (Figures 1 and 2), which indicates that
the stratum corneum is the main penetration barrier. In contrast to the lameliar o/w
emulsion, for the w/o emulsion there is no difference in penetration between 1 hour and
5 hours. This means that a steady state is already reached for the vitamin E penetration
from the w/o emulsion after 1 hour. In general, the penetration is faster and much higher
from the w/o cream than from the lameliar o/w cream.
The water soluble D-panthenol penetrates faster and in a higher amount into the stratum
corneum compared to the oil-soluble vitamin E (Figure 3, 4). The concentration gradient
is smaller than with vitamin E, indicating a low skin barrier capability against pen-
etration. Again, absorbed amounts are much higher for the w/o than for the o/w emul-
sion.
The reason for the poor vitamin penetration from the o/w emulsion relative to the w/o
cream is the lameliar gel network that is responsible for the viscosity buildup in this o/w
SKIN PENETRATION PROPERTIES ! 5 3
1,6
1,2
0,4
0,0
1st 2rid 3rd 4th 5th 6th 7th 8th 9th loth
Tape strip
-•- untreated --•-w/o cream (1 h) --•-w/o cream ($ h)
Figure I. Kinetics of vitamin E penetration (pg/cm 2 tape strip) from a w/o cream into the stratum corneum
after exposure periods of one hour and five hours (SEM for n = 4).
cream (12--14). These lameliar layers impede the free diffusion of vitamin E from the oil
phase into the skin (10,15). There are no differences in the oil components and vitamin
contents between both emulsion types and only small differences in emulsifiers. There-
fore, the differences observed in the penetration properties are attributed mainly to the
1,2
0,8
0,4
0,0
1st 2nd 3rd 4th 5th 6th 7th 8th 9th loth
Tape strip
-e-untreated -x- o/w cream (1 h) -•- o/w cream ($ h)l
Figure 2. Kinetics of vitamin E penetration (pg/cm 2 tape strip) from a lameilar o/w cream into the stratum
corneum after exposure periods of one hour and five hours (SEM for n = 4).
154 JOURNAL OF COSMETIC SCIENCE
5,0
•4,0
3,0
1,0
0,0 1st 2nd 3rd 4th 5th 6th 7th 8th 9th loth
Tape strip
--•-w/o cream (1 h) --*-w/o cream (5 h)]
Figure 3. Kinetics of D-panthenol penetration (tig/cm 2 tape strip) from a w/o cream into the stratum
corneum after exposure periods of one hour and five hours (SEM for n = 4).
different phase and viscosity behavior of the emulsions in the course of drying on the
skin.
After passing the stratum corneum, which represents the actual penetration barrier, the
vitamins enter into deeper skin layers. Therefore, for vitamin E not only adhesive tape
1,0 .......... ,
0,0 1st 2nd 3rd 4th 5th 6th 7th 8th 9th loth
Tape strip
-•-o/w cream (1 h) -*-o/w cream (5 h)l
Figure 4. Kinetics of D-panthenol penetration (pg/cm 2 tape strip) from a lameliar o/w cream into the
stratum corneurn after exposure periods of one hour and five hours (SEM for n = 4).
SKIN PENETRATION PROPERTIES 155
Table II
Kinetics of Vitamin E Penetration From W/O and Lameliar O/W Emulsions
O/W cream
W/O cream (lameliar)
Amount of vitamin E in 200 lain skin in pg/cm 2
% Absorbed vitamin E in 200 pm skin
After 1 h 24.9 + 4.6 14.5 + 2.7
After 5 h 30.0 + 5.1 21.8 + 2.7
After 1 h 8.9 5.1
After 5 h 10.7 7.8
Total applied amount of vitamin E = 280 pg/cm 2 skin; SEM for n = 4.
Table III
Penetration Properties of Oil- and Water-Soluble Vitamins From O/W Emulsions and a Shower Gel
Leave-on Rinse-off
(5 h) (3* 2 min)
O/W cream
W/O cream (lameliar) Shower gel
Applied total Vitamin E 280 280
amount of vitamins Vitamin E acetate 3'40
in pg/cm 2 D-panthenol 160 160 3*80
Amount of vitamins Vitamin E 4.3 + 1.4 2.9 + 1.0
in 10 stratum Vitamin E acetate 0.1 + 0.02
corneum strips in ].lg/cm 2 D-panthenol 25.3 + 8.5 6.6 _+ 1.2 1.2 + 0.09
% Absorbed vitamin Vitamin E 1.5 1.0
in 10 stratum Vitamin E acetate 0.1
corneum strips D-panthenol 15.8 4.1 0.5
SEMforn = 4.
strips of the outermost stratum corneum layers but also skin sections were examined to
get information about vitamin absorption in the living epidermis and dermis.
In general, the cumulative absorption of vitamin E in the top ten skin layers (200 pm
skin depth) is determined by the penetration of vitamin E into the stratum corneum. As
a consequence, the influence of the emulsion formulation on vitamin E absorption is the
same for the stratum corneum as for the epidermis. From the w/o cream the penetration
is faster and higher than from the lameliar o/w cream (Table II).
The naturally occuring vitamin E content in the skin (200 pm) is about 0.312 (1 hour)
and 0.191 (5 hours) i•g/mg. Thus the results in Table II show that, by typical leave-on
applications, high amounts of vitamin E are transported from the cream vehicle into the
outer skin.
VITAMIN PENETRATION FROM SHOWER GEL UNDER RINSE-OFF CONDITIONS
The rinse-off conditions chosen in the experimental setup should resemble the short
exposure periods found for body cleansing products on one hand and the frequent
156 JOURNAL OF COSMETIC SCIENCE
Vitamin E acetate and D-panthenol (rinse-off)
BUS-model (rinse off): Penetration of Vitamin E Acetate and
D-panthenol into the horny layer (n=4)
0,16
0,14
0,10
0,08
0,06 ........................................................................................
0,04 I ......................
0,02
0,001 ,
1st 2rid 3rd 4th 5th 6th 7th 8th 9th 10th
tape strip
•Vitamin E acetate •D-panthenol 1
Figure 5. Kinetics of D-panthenol and vitamin E penetration (tlg/cm 2 tape strip) from a surfactant-based
shower gel (rinse-off condition) into the stratum corneum after an exposure period of 3 * 2 minutes (SEM
for n = 4).
applications on the other hand. Considering the very short exposure periods (two min-
utes compared to one or five hours for topical leave-on application), the low vitamin
concentrations in the surfactant formulation, and the missing of any vitamin enrichment
caused by water evaporation, it is astonishing that penetration of vitamins into the skin
is achievable at all (Table III, Figure 5).
The vitamin penetration from a surfactant-based shower gel formulation after three
rinse-off applications is much lower than from the cream formulations under leave-on
conditions, but still detectable. Again, as found for the leave-on products, the water-
soluble D-panthenol penetrates to a higher extent into the stratum corneum than the
oil-soluble vitamin E acetate. It can be assumed that the difference is also influenced by
different routes of penetration into the stratum corneum, e.g., the lipid-soluble vitamin
is following the intercellular spaces whereas the water-soluble vitamin penetrates more
through the corneocytes.
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