Schepky, AG, Holtzmann, U., Bohnsack, K., Dörschner, A., Skibbe, V

Improved skin compatibility of mild surfactant
systems results in protection of enzyme activity
in human skin
Schepky, A.G., Holtzmann, U., Bohnsack, K., Dörschner, A., Skibbe, V., Wenck, H., Rippke, F.
Beiersdorf AG, Hamburg, Germany
Abstract
The trypsin assay, a new ex vivo method, was employed to investigate the effects of various surfactants on skin enzymes. After
repeated washing of human forearm skin with either water or different shower or shampoo formulations the uppermost layers of the
stratum corneum were extracted and the specific activity of stratum corneum tryptic enzyme (SCTE) was determined in the extracts.
The remaining trypsin activity was significantly higher after use of
anionic surfactant / cocoamphoacetate system than after use of a
commonly used standard anionic surfactant / betaine system.
Therefore, by determining the inactivation of this physiologically important skin enzyme during the cleansing procedure, a clear differentiation of mild and less mild shower and shampoo formulations
could be achieved. The results prove the mild surfactant system, as
applied in Eucerin® pH5 products, to be highly skin compatible
and thus suitable for the cleansing of sensitive, irritation-prone
skin.
Introduction
Sensitive skin is a common skin condition of subjective cutaneous
hyperreactivity to environmental factors (5). For these patients, a
careful selection of mildest skin cleansing and care preparations is
highly warranted in order to stabilize their delicate skin status. It
has been known for a long time that anionic surfactants (e.g. sodium lauryl sulfate, SLS) can act as strong irritants, binding to skin
proteins and leading to dose-dependent skin damage and exsiccation (figure 1).
Therefore other classes of surfactants (e.g. cationic and nonionic) are
considered preferable in sensitive skin conditions. In addition, the
skin compatibility of a skin cleanser depends on its pH in aqueous
solution with slightly acid formulations (approx. pH 5) being superior to neutral or alkaline ones (1, 2). Only recently, we found significant correlations between the mildness of applied surfactant systems and the activity of several stratum corneum enzymes responsible for barrier formation and desquamation. Application of SLS
resulted in a significant inhibition of stratum corneum tryptic enzyme
(SCTE), stratum corneum chymotryptic enzyme, acid phosphatase
and β-glucocerebrosidase (6). In our new study, we set out to investigate the effect of a range of acidic medical skin cleansers on
SCTE activity, employing a novel trypsin assay, in comparison to a
market standard cleanser and tap water.
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Material and Methods
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Study design: The study was conducted in a single-center, randomized design, double-blind for the test products and the reference
(standard shower gel) in 16 healthy female volunteers aged 18 to
55 years.
Test products:
1. Market standard shower gel
(based on sodium laureth sulfate / sodium sodium
cocoamidopropyl betaine surfactant system)
2. Eucerin® pH5 Shampoo
(based on sodium myreth sulfate/ sodium laureth sulfate /
sodium amphoacetate surfactant system)
3. Eucerin® pH5 Shower Oil
(based on MIPA-laureth sulfate)
Ex vivo determination of surfactant effect
on trypsin activity in human epidermis
Treatment: For standardisation of the skin condition, the volunteers
were asked to use only a reduced irritancy shower formulation
(based on sodium myreth sulfate and an amphoteric surfactant)
for washing over a period of two weeks. Following this preconditioning, the volar forearms were divided into two test areas each.
The test areas were treated on three consecutive days with 1 mL
cleansing product or tap water control, respectively, for 45 s. After
treatment, the test area was rinsed with tap water for 30 s and
dabbed with a one-way paper towel. On day 1 and 2, the test areas
were treated three times (morning, noon, late afternoon), on day 3
they were treated twice (morning, noon).
Fig. 2a: Stratum corneum stripping
with saccharide-coated glass slide
Fig. 1b: Exsiccated stratum corneum
100
20
4. Eucerin® pH5 Soft Shower
(based on sodium myreth sulfate/ sodium amphoacetate
surfactant system)
Fig. 1a: Healthy stratum corneum
100
Trypsin Enzyme Activity (%)
Cleansing of the skin with surfactant containing formulations
should effectively remove surface lipids and dirt but should affect
the skin’s enzyme function as little as possible in order to maintain
the protective skin barrier functions and prevent cumulative irritation.
Fig. 2b: Stratum corneum biopsy
Skin biopsy extraction and measurement of SCTE activity:
On day 4, stratum corneum samples were stripped of the test areas,
using a saccharide-coated glass slide (figure 2). Subsequently, the
corneocytes attached to the glass slide were rinsed off with PBS
buffer solution, and the specific SCTE activity was determined.
The SCTE activity in the tap-water-treated area was set to 100 %,
serving as a reference for the test areas treated with cleansing
products.
Market
standard
pH5
Shampoo
pH5
Shower Oil
pH5
Soft Shower
Tab
Water
Figure 3: SCTE activity in human forearm skin after treatment with tap water, market standard shower gel, and 3 acidic skin cleansing formulations with improved skin compatibility
Stratum corneum tryptic enzyme (SCTE) activity assay:
100 µl porcine epidermis extract were incubated with 150 µL N-tBoc-Phe-Ser-Arg-7-amido-4-methylcoumarin (33 µM in PBS; Sigma,
St. Louis, USA) at 37 °C for 24 h. The SCTE-specific release of fluorescent 7-amino-4-methylcoumarin was measured using a fluorescence well plate reader (filters ex = 360 nm ± 40, em = 460 nm
± 40 nm, CytoFluor 4000, PerSeptive Biosystems, Framingham,
USA).
Protein concentration measurement: For calculating the specific enzyme activity of the sugar-strip biopsies, their protein content
was determined using the ninhydrin method after alkaline hydrolysis. In brief, the corneocyte solutions were dried, and the proteins
were hydrolysed with 2 mL sodium hydroxide solution (6 M) for
5 h at 150 °C. The solution was neutralized with 2 mL hydrochloric acid (6 M) and 1 mL sodium propionate buffer (3.35 M, pH 5.5)
was added. After that, 50 µL of the lysate were diluted in 450 µL
aqua bidest. and incubated with 25 µL formic acid (0.4 % (v/v)) and
500 µL ninhydrin solution (2 % (w/ v) ninhydrin in 3.35 M
sodium propionate buffer with 50 % (v/ v) ethylene glycol monomethylene ether; Sigma, St. Louis, USA) at 70 °C for 20 min. After
cooling, 5 mL ethanol (50 % (v/v) in aqua bidest.) were added. The
absorption was measured with a spectral photometer at a wavelength of 570 nm (UVICON 942, Kontron, Milano, Italy), and the
corresponding protein concentration was calculated.
Discussion and Conclusion
Employing our novel trypsin assay, we were able to demonstrate
clear and significant differences in the ability of various surfactant
systems to inhibit stratum corneum enzyme functions. Whereas
SCTE activity was markedly suppressed by a standardized washing
procedure with a SLS-based standard surfactant system, much
higher enzyme activities remained after the application of advanced surfactant systems at acidic pH values. These findings are significant for patients with sensitive skin conditions as, in addition
to an enhanced immune responsiveness and altered neurosensory input, a diminished barrier function is regarded as one hallmark
of sensitive skin pathogenesis (3). Stratum corneum barrier function, from its formation through desquamation, is driven by a
plethora of enzymatic processes (4). Inhibition of certain enzymatic steps, e.g. by harsh surfactants, can therefore impair skin
resilience and / or desquamation, leading to increased skin irritability and roughness. Based on the data presented here, the tested
Eucerin® pH5 cleansing products proved to be much better compatible with the skin’s own enzymatic processes, approaching the
level of mildness of tap water. They can therefore be considered
suitable for sensitive and irritable skin conditions.
References
Results
Figure 3 shows the highest specific SCTE activity in the tap water
treated area. The area treated with the SLES / betaine based product (i.e. market standard shower gel) shows a 45 % inhibition of
specific SCTE activity compared to the tap water control. The areas
treated with the sodium myreth sulfate / sodium laureth sulfate /
sodium amphoacetate based shampoo (product 2), the MIPAlaureth sulfate based shower oil (product 3), and the sodium
myreth sulfate / sodium amphoacetate based shower gel (product
4) showed a remaining SCTE acitivity of 73%, 79%, and 82%
compared to tap water control. The SCTE inactivation by the improved formulas are all significantly lower than the SCTE inactivation by the market standard formula, or, in other words, the skin
enzymes are significantly less influenced and better protected by the
new formulas compared to the market standard formula.
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