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The involvement of protein denaturing activity in the effect of surfactants on skin barrier function

Authors:
  • Sanyo Women's College

Abstract

Abstract Background/purpose: Detailed information on the mechanism by which surfactants affect the skin barrier function is still scarce. We investigated the contribution of protein denaturation to the effect of surfactants on barrier function. Methods: The Transmission Index method, which evaluates the actual effect of sur- factants on barrier function, was combined with a microplate assay measuring pro- tein denaturation activity. The correlation between the TI value and the reciprocal of the median effect concentration (1/EC50) was analyzed for 19 surfactants. The contribution of protein denaturation to the effect of surfactants was discussed based on the 1/EC50 per TI value. Results: A few surfactants showed high TI value. Nonionic surfactants had no effect. The EC50 varied without certain trend. For amino acid-based surfactants, there was a gradual inverse correlation between the TI value and the 1/EC50. Conclusion: The difference in the alkyl structure and the ion source affected the skin barrier function. Protein denaturing activity of the surfactant was not a critical factor. This suggests that the effect on intercellular lipids was the major factor. However, the magnitude of the contribution of protein denaturation activity varied depending on the surfactant, suggesting that each surfactant has a different mechanism of influ- ence on skin barrier function.
Skin Res Technol. 2020;00:1–8.
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  1wileyonlinelibrary.com/journal/srt
1 | INTRODUCTION
Surfactants are used in many daily necessities and pharmaceuti-
cal excipients bec ause of their excellent functions such as strong
washing action and emulsifying action based on amphiphilicity.
Emulsifiers, solubilizers, stabilizers, and other functions of cos-
metics and pharmaceuticals are one of the most important roles
of surfactant s.1,2 They are known to affect skin barrier function3-5
and in recent years have attracted attention for their function as
transdermal absorption enhancers. This function is made possible by
the formation of surfactant-based drug carriers and their effects on
skin barrier function.6-8 Goto's group established a solid-in-oil (S/O)
nanocarrier technology using a surfactant for transdermal deliver-
ing substances with high physiological activity, such as nucleic acids
and hormones, into the body.9 However, surfactants can damage the
skin.10,11 Therefore, many novel "low-irritant" surfac tants have been
proposed by using polymerized or highly biocompatible ion source
components.12-14
Received: 1 June 2020 
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  Accepted: 20 J une 2020
DOI : 10.1111 /sr t.12 939
ORIGINAL ARTICLE
The involvement of protein denaturing activity in the effect of
surfactants on skin barrier function
Koji Kubota1,2 | Mana Okasaka2,3| Asami Kano2| Sadaki Takata2,3
© 2020 John Wiley & Son s A/S. Published by J ohn Wiley & Sons Ltd
1Depar tment of Pharmac y, Faculty of
Pharma cy, Iryo Sosei University, Iwaki-Cit y,
Japan
2Depar tment of Fas hion and B eauty
Science s, Facult y of Liberal Ar ts, Osaka
Shoin Wome n's Univer sity, Higashi-Osaka-
City, Japan
3Divisio n in Fashion and Beaut y Studies,
Gradu ate School of Human Sciences , Osaka
Shoin Wome n's Univer sity, Higashi-Osaka-
City, Japan
Correspondence
Depar tment of Pharmac y, Faculty of
Pharma cy, Iryo Sosei University, 5-5-1,
Chuo-dai Iino, Iwaki-city, Fukushima 970-
8551, Japan.
Email: koji.kubota@isu.ac.jp
Funding information
This work w as suppo rted by JSPS KA KENHI
Grant Number JP19K14014.
Abstract
Background/purpose: Detailed information on the mechanism by which surfactants
affect the skin barrier function is still scarce. We investigated the contribution of
protein denaturation to the effect of sur factants on barrier function.
Methods: The Transmission Index method, which evaluates the actual effect of sur-
factants on barrier function, was combined with a microplate assay measuring pro-
tein denaturation activity. The correlation between the TI value and the reciprocal
of the median effect concentration (1/EC50) was analyzed for 19 surfactants. The
contribution of protein denaturation to the effect of surfactants was discussed based
on the 1/EC50 per TI value.
Results: A few surfactants showed high TI value. Nonionic surfactants had no effect.
The EC50 varied without cer tain trend. For amino acid-based surfactants, there was
a gradual inverse correlation between the TI value and the 1/EC50.
Conclusion: The difference in the alkyl structure and the ion source affected the skin
barrier function. Protein denaturing activity of the surfactant was not a critical factor.
This suggests that the effect on intercellular lipids was the major factor. However, the
magnitude of the contribution of protein denaturation activity varied depending on
the surfactant, suggesting that each surfactant has a different mechanism of influ-
ence on skin barrier function.
KEYWORDS
adsorption property, alkyl structure, amino acid-base surfactant, counter ion, ionic source,
keratinocyte intercellular lipids
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The mechanism of action by which surfac tants affect skin bar-
rier function is not simple and is thought to be a result of two
primar y processes: disruption of the keratinoc ytes and removal of
intercellular lipids from the stratum corneum.10,11 The keratino-
cytes are enucleated at the final stage of skin tissue cell differen-
tiation, leaving the cytoskeletal protein keratin. In the Brick and
Mortar model, the keratinocytes form a tight struc ture with the
intercellular lipids.15-17 The effects of surfactant s on skin barrier
function can be observed using numerous methods, for example,
cell or tissue toxicity test and measuring of transepidermal water
loss (TEWL).15,18 ,19 These factors vary, and it is likely that they do
not function independently.
Previously, we developed the Transmission Index (TI) method,
which evaluates changes in the skin penetration rate of drugs as a
comprehensive measure of the effects of surfactants on the skin.20
The TI value, which comprehensively represents the degree to which
the surfactant effects skin barrier function, is high for surfactants
that are considered to result in "strong skin irritation." Interestingly,
while amino acid surfactants are considered to have low cytotoxicity
and low irritation, we previously repor ted that the TI value of the
glutamate surfactant was relatively high. We have presumed that
the glutamate surfactants have a strong affinity with the stratum
corneum of the skin sur face to reduce the skin barrier function by
adsorbing to the skin.20 In addition, Japan Ministr y of Economy,
Trade and Industry mentioned the anti-virus activity of quaternary
ammonium salt type surfactant at April 202021; however, the effect
on the skin barrier function of them was not considered. Hence, we
investigate the effect of quaternary ammonium salt type surfactant
on skin barrier function.22 In this report, we studied the mecha-
nism in addition to the evaluation of the effects on the skin barrier
function.
In this study, we measured the contribution of protein denatur-
ation to clarify the effects of surfactants on skin barrier function.
The protein denaturing activity was measured by calculating the
50% effective concentration (EC50) by a microplate assay using
a serially diluted surfactant. In this study, we selected hemoglo-
bin, a pigmentary protein, as the target for protein denaturation
activity and measured the change in absorbance upon addition of
various concentrations of surfactant.23 This experiment was per-
formed on several cationic, amphoteric, and nonionic surfactants,
in addition to 12 anionic surfactants whose TI values were pre-
viously reported. The sur factants were also evaluated based on
the TI method. Following calculation of the EC50, the correlation
between the TI value and the ef fect of the surfactant on protein
denaturation was determined. The experiments were conducted
with due consideration of the elimination of pain of laboratory
animals and reduction of use of them. The mice used in the ex-
periments were carefully bred and properly euthanized by animal
husbandry experts. Extraction of skin samples was done carefully
and reliably by experts in animal surgery. Experiment samples
were split to maximize performance from the smallest laboratory
animal samples. Individual and parts differences were statistically
verified to ensure scientific accuracy.
2 | MATERIALS AND METHODS
2.1 | Transmission index method
The TI method, which we reported previously,20 was used to
evaluate the effect of two cationic surfactants, three amphoteric
surfactant s, and two nonionic surfactant s on the skin barrier
function. Dimethyldistearylammonium chloride (Sanyo Chemical
Industries, Ltd.) and trimethylstearylammonium chloride (Toho
Chemical Industry Co., Ltd.) were prepared as cationic sur-
factants. Sodium lauroamphoacetate (Toho), lauryl dimethylami-
noacetic acid betaine (Toho), and cocamidopropyl betaine (BASF
Japan) were prepared as amphoteric surfactants. PEG-60 glyc-
eryl isostearate (Nihon Emulsion Co., Ltd.) and PEG/PPG-25/30
copolymer (ADEKA Corporation) were prepared as nonionic
surfactant s. Each surfac tant was dissolved or diluted in deion-
ized water to a final concentration of 1% to form a homogene-
ous solution. Laboskin® (Hoshino Laboratory Animals, Inc), the
back skin from seven-week-old male Hos:HR-1 hairless mice, was
used as the skin sample. The supplier of the laboratory animals,
Hoshino Laboratory Animals, Inc, is the right supplier, who is
certified by Japanese Society for Laboratory Animal Resources.
Skin permeability testing and concentration measurements of
the permeated drug were evaluated by high-performance liquid
chromatography (HPLC) method according to the Transmission
Index method described previously, and statistical analysis (stand-
ard error, Pearson's square test, Welch's t test) was per formed.
Methylparaben was used as a standard for measuring the time-
dependent rate of change of chemical concentrations. The steady-
state skin permeation rate (Fluxss) was represented by (Formula 1),
where Q is the concentration of methyl paraben that permeated
through the skin and t is the time in hours.
The Transmission Index was determined using (Formula 2) by si-
multaneously comparing the Fluxss of surfactant-treated skin with
the Fluxss of control (water)-treated skin.
2.2 | Evaluation of the effect of surfactants
on proteins
To evaluate the effect of sur factants on proteins, 0.2 g of the
chromoprotein hemoglobin (Sigma-Aldrich Co. LLC.) was dissolved
in pH 7.4 phosph ate -buffered saline (PBS) without Mg2+/C a2+ (Wako
Pure Chemical Industries, Ltd.) for a final weight of 10 g (2% hemo-
globin). This solution was diluted 4 0-fold with cold PBS (4°C) to give
a final concentration of 0.05% hemoglobin. These solutions were
used immediately after preparation.
(1)
Flux
ss =
dQ
dt
(2)
Transmission Index
=
Flux
ss
(surfactant)
Flux
ss
(control)
  
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KUBOTA eT Al.
Nineteen surfactants were prepared and classified according
to ionic proper ties. Sodium laurate (NOF CORPOR ATION), sodium
lauryl sulfate (Kao Corporation), sodium polyox yethylene lauryle-
ther sulfate (Kao), sodium lauroyl glutamate (Ajinomoto Co., Inc),
sodium lauroyl methyl taurate (Nikko Chemicals Co., Ltd.), sodium
cocoyl alaninate (Ajinomoto), sodium cocoyl sarcosinate (Nikko),
sodium cocoyl glutamate (Ajinomoto), potassium cocoyl glutamate
(Ajinomoto), triethanolamine cocoyl glutamate (Ajinomoto), sodium
cocoyl methyltaurate (NOF), and sodium taurine cocoyl methyltau-
rate (NOF) were prepared as anionic surfactants. As cationic surfac-
tants, dimethyldistearylammonium chloride (Sanyo Chemical) and
trimethylstearylammonium chloride (Toho Chemical) were prepared.
As amphoteric surfactants, sodium lauroamphoacetate (Toho), lau-
ryl dimethylaminoacetic acid betaine (Toho), and cocamidopropyl
betaine (BASF) were prepared. PEG-60 glyceryl isostearate (Nihon
Emulsion) and PEG/PPG-25/30 copolymer (ADEKA) were prepared
as nonionic surfactant s. Each was diluted with deionized water to
2.5, 0.5, 0.1, 0.02, 0.004, and 0.0008% (w/w) solutions.
A 100 μL of the 0.05% hemoglobin solution was added to a 96-
well microplate (polystyrene, flat bottom, 400 μL/ well, Fukae Kasei
Co., Ltd.). Then, 100 μL of the serially diluted surfactant sample
solutions was added, diluting the final surfactant concentration 1:2
(1.25, 0.25, 0.04, 0.01, 0.002, 0.0004%). After allowing the reaction
to stand for 10 minutes, the mixture was stirred for 20 seconds using
a BIO RAD iMark microplate reader (Bio-Rad), and the optical den-
sity (OD) at 405 nm was measured. The measured value from which
the absorbance of the blank was subtracted was fitted to a Rodbard
function (Formula 3) using ImageJ software (National Institutes of
Health, Bethesda, MD, USA) to obtain parameters affecting the in-
fluence curve described in (Formula 3).
The median between the upper and lower limits of the plateau
of the measured values was determined as the median effect, and
the concentration at the median effect was determined as the 50%
effect concentration (EC50). The experiment was performed four
times and analyzed using statistical methods.
3 | RESULTS
3.1 | Evaluation of surfactant effects on skin barrier
function by TI method
The effect on skin barrier function was evaluated by the TI method.
The previously reported TI values of the 12 kinds of anionic sur-
factants20 and four kinds of quaternary ammonium salt type sur-
factants22 are shown in Figu re 1, alo ngsid e th e TI valu es of the other
surfactant s measured in this study. The initial 12 surfactants were
arranged on the basis of the alkyl structure: (1) sodium laurate, (2) so-
dium laur yl sulfate, (3) sodium polyoxyethylene laurylether sulfate,
(4) sodium lauroyl glutamate, (5) sodium lauroyl methyl taurate, (6) so-
dium cocoyl alaninate, (7) sodium cocoyl sarcosinate, (8) sodium co-
coyl glutamate, (9) potassium cocoyl glutamate, (9) potassium cocoyl
(3)
y
=d+
a
d
1+
(
x
c
)
b
FIGURE 1 The Fluxss of the surfactant-treated samples (gray bar) and the control samples (white bar). Each value represents the
mean ± SE calculated for at least four experimental replicates. P < .01 is indicated with (*), and P > .1 is indic ated with (†) calculated with
Welch's t test against the value of each control sample
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glutamate, (10) triethanolamine cocoyl glutamate, (11) sodium cocoyl
methyltaurate, and (12) sodium taurine cocoyl methyltaurate. The
subsequent 7 surfactants were arranged by sur fact ant properties:
cationic surfactants, (13) dimethyldistearylammonium chloride, and
(14) trimethylstearylammonium chloride; amphoteric surfactants,
(15) sodium lauroamphoacetate, (16) lauryl dimethylaminoacetic acid
betaine, and (17) cocamidopropyl betaine; and nonionic surfactants,
(18) PEG-60 glyceryl isostearate and (19) PEG/PPG-25/30 copoly-
mer. Skin was washed with either a surfactant or the control (deion-
ized water). Then, methyl paraben was added to the skin, and the
amount of methyl paraben was measured for 1-6 hours. Since the
permeation of methylparaben through the skin was constant (linear)
from 1-6 hours (dat a not shown), the permeation amount per unit
time was determined as Fluxss. Fluxss (control), which was measured
simultaneously with Fluxss (surfactant), was subjected to a signifi-
cant difference test using Welch's t test. Treatment with surfactant
resulted in significant differences (P < .05) in Fluxss for many of the
surfactant s. Treatment with (5) sodium lauroyl methyl taurate or
(15) sodium lauroamphoacetate did not significantly impact Fluxss.
Surfactants marked with (†) had almost no significant difference
with respect to Fluxss (control) (P > .1). Table 1 shows the TI values.
It was shown that (1) sodium laurate and the amphoteric surfactants
(14) trimethylstearylammonium chloride and (16) lauryl dimethylami-
noacetic acid betaine had a par ticularly strong effect on skin barrier
function.
3.2 | Evaluation of protein denaturation activity
The OD 405 nm of the hemoglobin solution treated with (6) sodium
cocoyl alaninate is shown in Figure 2. At the highest concentrations
of sodium cocoyl alaninate (0.00 04% and 0.002%), the OD 405 nm
was measured as the upper limit of the denaturation plateau, the
same level as the surfactant blank; thus, no denaturation in hemo-
globin was observed. At the concentrations of 0.05%, 0.25%, and
1.25%, the OD 405 nm was equivalent to the value of the lower
plateau; thus, hemoglobin denaturation was saturated. Fitting
this measurement to a Rodbard function (Formula 3) using ImageJ
Software provided the parameters; a = 0.824, b = 11.02, c = 0.0022,
and d = 0.530. The correlation coefficient was 0.99. The median ef-
fect was determined as the median between the upper and lower
plateaus. The EC50 was obtained by substituting the intermediate
value of the effect into Y in (Formula 3). Other surfactants were
similarly fitted to the Rodbard function, and all were well-correlated.
TABLE 1 TI value, EC50, and SBF-PDA Index of each surfactant
Surfactant Ionic type
Amino acid
series
Quaternary
ammonium salt TI ± SE
Protein denaturing
EC50 ± SE (%)
SBF-PDA
index
1Sodium laurate Anionic 2. 61 ± 0.19 0.0108 ± 0.0014 35.5
2Sodium lauryl sulfate Anionic 1.41 ± 0.07 0.0022 ± 0.0000 32 7. 3
3Sodium laureth sulfate Anionic 1.48 ± 0.10 0.0051 ± 0.0002 13 0.9
4Sodium lauroyl glutamate Anionic *1.91 ± 0.18 0.0397 ± 0.0074 13.2
5 Sodium lauroyl methyl taurate Anionic * 1.20 ± 0.08 0.0175 ± 0.0005 4 7. 5
6Sodium cocoyl alaninate Anionic *0.99 ± 0.06 0.0108 ± 0.0002 93.5
7Sodium cocoyl sarcosinate Anionic *0 .95 ± 0.07 0.0118 ± 0.0004 89.4
8Sodium cocoyl glutamate Anionic * 1.80 ± 0.25 0.0119 ± 0.0001 46.6
9Potassium cocoyl glutamate Anionic *1.59 ± 0.22 0.0239 ± 0.0011 26.3
10 Triethanolamine cocoyl
glutamate
Anionic *1. 51 ± 0.11 0.0121 ± 0.0001 54.7
11 Sodium cocoyl methyl taurate Anionic *1.17 ± 0 .11 0.0087 ± 0.0003 98.9
12 Sodium t aurine cocoyl methyl
taurate
Anionic *1.17 ± 0.09 0.0154 ± 0.0005 55. 8
13 Dimethyldistearylammonium
chloride
Cationic 1.31 ± 0.23 ND (>0.05) N/A
14 Trimethylstearylammonium
chloride
Cationic 3.12 ± 1.12 0.0 030 ± 0.0001 107.4
15 Sodium lauroamphoacetate Amphoteric 1. 51 ± 0.13 0.0323 ± 0.0014 20.5
16 Lauryl dimethylaminoacetic
acid betaine
Amphoteric 3.18 ± 0.15 0.0238 ± 0.0007 13.2
17 Cocamidopropyl betaine Amphoteric 1.29 ± 0.11 0.0190 ± 0.0003 40.9
18 PEG-60 glyceryl isostearate Nonionic 1.05 ± 0.11 0.0165 ± 0.0008 5 7. 0
19 PEG/PPG-25/30 copolymer Nonionic 0.85 ± 0.07 ND (>1.25) N/A
Note: Values are writ ten as the mean ± the standard error calculated for four experimental replicates.
  
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(19) PEG/PPG-25/30 copolymer did not show hemoglobin dena-
turing ac tivity in the concentration range of this study. Therefore,
we designated the EC50 as >1.25. With (13) dimethyldistearylam-
monium chloride, precipitation occurred at concentrations of 0.25%
or more, so accurate absorbance could not be obtained and the
EC50 could not be calculated. If the absorbance at a concentration
of 0.05% or less was equivalent to the denatured protein, then the
EC50 was designated as at least 0.05% or more. Table 1 shows the
EC50 of each surfactant. Each experiment was performed four times
and analyzed using statistical methods.
The 1/EC50 was determined to ensure proportionality between
the protein denaturing activity and the TI value. Figure 3 shows a
scatter plot of the TI value and 1/EC50. The anionic surfactant so-
dium laur yl sulfate (SDS) was characterized by a relatively small TI
value bu t a lar ge 1/EC50. In contras t, the am photeric surfac ta nt la u-
ryl dimethylaminoacetate betaine had a relatively high TI value and a
low 1/EC50. The TI value and 1/EC50 of anionic surfactant s varied,
and no correlation was found between the values. For the cationic
surfactant, the TI value and the 1/EC50 of dimethyldistearylammo-
nium chloride (for which 1/EC50 could be calculated) both showed
relatively high values. The three amphoteric surfactants had a range
of TI values, but the 1/EC50 values were similar and tended to be
relatively low. PEG-60 glyceryl isostearate, for which 1/EC50 could
be calculated with a nonionic surfactant, exhibited low values for
both TI and 1/EC50.
Figure 4 shows a scatter plot of the TI values and the 1/EC50
of the amino acid-based (including β-amino acid/aminosulfate) an-
ionic surfactants. Among the amino acid surfactants, sodium lauroyl
glutamate had the greatest effect on the skin barrier function, and
sodium cocoyl methyl taurine had the strongest protein denaturing
activity. There was a gradual inverse correlation (R = .557) between
the magnitude of the ef fect on the skin barrier function and the pro-
tein denaturing activity of all nine amino acid surfactants.
4 | DISCUSSION
We have previously shown that the practical effect of surfactants
on skin barrier function can be assessed by the TI method. In this
study, the effect of surfactants other than the previously reported
anionic surfactants on skin barrier func tion was evaluated by the TI
method. It was confirmed that the TI method is scientifically reliable
and technically stable method for assessing the effect of surfactants
other than anionic surfactants on skin penetration. Although the
number of other than anionic surfactants used on the market is not
as large as the number of anionic sur fact ants, the result s are still
meaning ful for a large number of products. The TI method is excel-
lent in that the effect of a surfactant on the skin barrier function is
evaluated as a practical phenomenon by using the skin permeation
rate of a drug as an index. This method is also applicable to artifi-
cially cultured skin tissue samples. The information obtained by this
method can be used as a substitute sample or as a basis for in silico
simulation. Therefore, it can be applied not only to the development
FIGURE 2 A representative graph depicting the change in
absorbance at 405 nm of surfactant (Sodium cocoyl alaninate)
concentration on the horizontal axis. Measurements were
carried out 4 times. Error bars indicate the standard deviation in
absorbance at each concentration
FIGURE 3 Scatter plot with the TI
value on the horizontal axis and 1/EC50
on the vertical axis. The meaning of the
symbols in the figure is shown in the
legend. The names of the surfactants
mapped to characteristic locations are
shown in the figure
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of transdermal preparations in the pharmaceutical field, but also to
the development of prescriptions for cosmetics.
Of the sur factants evaluated for their effects on skin barrier
function, the TI values of the cationic surfactant (14) stear yltrime-
thylammonium chloride and the amphoteric surfactant (16) lauryldi-
methylaminoacetate betaine were the highest. In particular, (16) had
the greatest effect on the skin barrier function among the surfac-
tants investigated so far. However, the number of moles should be
considered when evaluating the TI value. For example, of the cat-
ionic surfactants (13) distearyldimethylammonium chloride and (14)
stearyltrimethylammonium chloride, one of the methyl groups of
trimethylglycine (betaine) chloride was replaced with stearic acid
in (14), and two were replaced in (13). The molecular weight of (13)
is 586.5, and the molecular weight of (14) is 348.1 (PubChem). The
molar ratio is (13):(14) = 1:1.7; therefore, given equivalent weights
of the two compounds, the molar concentrations are different. The
ratio of the TI values of (13):(14) was 1:2.1, which is very close to
the molar ratio. Therefore, it may be appropriate to compare the
effects of surfactants on skin barrier function on a per number of
molecule basis. However, some surfactant s have polymers with
varying degrees of polymerization or hydrophilic polymers added in
an indefinite form, making it difficult to evaluate the exact number of
molecules. Therefore, the evaluation based on weight concentration
is realistic and practical for functional studies.
In this study, we obtained important information by combining
the TI method with a measurement of protein denaturing activity.
Although the protein denaturing activity of surfactants is not a criti-
cal factor for their effec t on skin barrier function, it is not completely
negligible. For cert ain surfac tants, it may even be a major factor. In
the correlation scatter plot of the TI value and 1/EC50 shown in
Figure 3, when both the TI value and the 1/EC50 value are high, it is
possible that the protein denaturing may increase drug permeability.
As a whole, if the scatter plots show a strong direct correlation, the
effect of the surfactant on the skin barrier function is likely to be due
in some part to the protein denaturing activity. However, there was
no clear general correlation between the TI value and the 1/EC50
value for the 19 surfactants in this study. At least in the surfactants
used in this study, there was no clear relationship between protein
denaturing activity and the effect on skin barrier function as a gen-
eral trend.
Both of the two nonionic surfactants (18 and 19) had low TI val-
ues and low 1/EC50 values, suggesting that no factor affects skin
barrier function other than protein denaturation. On the other hand,
for the cationic surfactant (13), both the TI value and the 1/EC50
value were high, suggesting that protein denaturing activity affected
the skin barrier function in some way. Of interest are sur factants
that exhibit (A) relatively high TI and low 1/EC50 values and (B) rel-
atively low TI and high 1/EC50 values. An inverse correlation be-
tween TI value and 1/EC50 value suggests that protein denaturing
activity is not a factor affecting skin barrier func tion. (1) and (14)
show tendency (A), and (2) shows tendency (B). We speculated that
(1) and (14) act on element s other than proteins to affect skin barrier
function. It was shown that protein denaturing activity does not af-
fect the enhancement of skin permeability of the drug for (2). A sim-
ilar tendency was observed in the correlation between the TI value
of the amino acid surfactant and the 1/EC50 value (Figure 4). In
the amino acid surfactant group, the TI value and the 1/EC50 value
showed a relatively strong negative correlation. This indicates that
for amino acid-based surfactants, protein denaturing activity is not a
factor affecting skin barrier function.
Therefore, a measure combining both the 1/EC50 and the TI was
defined as the Skin Barrier Function-Protein Denaturing Activity
Index (SBF-PDA Index). It was calculated as 1/(EC50 * TI value)
(Table 1). The relative magnitude of this value indicates the mag-
nitude that the protein denaturing activity impacts the skin barrier
function. Some surfactants showed extremely high SBF-PDA Index
values, such as (2), while others exhibited very low values, such as
(4) and (16). In the pairs (4) and (8) and the pairs (5) and (11), which
have the same structural and chemical properties other than their
alkyl groups, the laur yl group surfactant had a lower SBF-PDA Index
value. This suggests that differences in the alkyl structure affect the
SBF-PDA Index value and that the effect of the lauryl group on the
protein denaturing activity is relatively weak.
The effect of the ion source on the SBF-PDA Index value was
attributed to a combination of the sodium salt derived from the lau-
ryl group ([1], [2], [3], [4], and [5]) and the sodium salt derived from
cocoyl group ([6], [7], [8], [11], and [12]). When comparing (1), (2),
(3), (4), and (5), the SBF-PDA Index value of (2) is remarkably high. It
was shown that protein denaturation by (2) played an import ant role
in the effect on skin barrier function. Since (2) has a lauryl group,
the sulfate ion was considered to be a major effective factor.24 On
the other hand, the SBF-PDA Index value of (4) was low. As in the
case of (4), since (7) had the lowest SBF-PDA In dex value in the com-
parison between (6), (7) and (8), it is assumed that the influence of
protein denaturing activit y on the skin barrier function of the an-
ionic surfactant using glutamic acid as the ion source is relatively
small. Conversely, SBF-PDA Index values tended to be higher for
FIGURE 4 Plots of amino acid-based surfactants extracted
from Figure 3. The dot ted line indicates the approximate curve
obtained from all plots. R indicates the correlation coefficient of the
approximate curve
  
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alanine, sarcosine, and taurine-based surfactants that had lower TI
values than glutamic acid sur factants. Differences in the ion source
of amino acid-based sur factants can lead to differences in the mech-
anisms of their effec ts on skin barrier function. In a previous report,
we mentioned that glutamate-based surfactants may affect skin bar-
rier function by adsorbing to skin surface proteins. The results of
this study further demonstrate the particularity of glutamate-based
surfactants.
We compared counter ion differences on SBF-PDA Index values
in (5), (6), and (7). In this comparison, the effect of potassium ion
was the smallest, and the effects of sodium ion and triethanolamine
were comparable. The lack of difference between SBF-PDA Index
values for surfactants with counter cations that were either inor-
ganic or organic for anionic surfactants indicates that the identity
of the counter cation may not affect the SBF-PDA Index. SBF-PDA
Index values are also different in comparisons bet ween sur factants
using sodium ions as counter ions; we hypothesize that there is no
relationship between the presence of sodium ions and the relation-
ship between protein denaturation and skin barrier function.
In the case of quaternary ammonium salt type surfactants, there
was no common point among the three parameters: TI value, EC50,
and SBF-PDA. There were groups with low TI values: (13) and (17),
and groups with high TI values (14) and (16). As mentioned above,
these must be considered in terms of molarity. On the other hand,
SBF-PDA Index value of (13) was significantly higher than the other
three. This indicates that the mechanism of action of (13) on the skin
barrier function is likely to be protein denaturing ac tivit y. The strong
protein denaturing activity may effectively function for the outer
coat of the virus, but at the same time, it should be noted that the
strong ef fect on the skin barrier function.
Although the structural and chemical characteristics of the sur-
factant af fec t the SBF-PDA Index value, no decisive fac tor affecting
the SBF-PDA Index value was found. The protein denaturing activity
of the surfactant was not a major factor in the effect of surfactants
on skin barrier function and was not solely determined by struc-
tural and chemical characteristics. It is extremely difficult to infer
the effects of a given surfactant on skin barrier function from the
structural and chemical properties of a surfactant. Therefore, it is
necessary to investigate the factors affecting skin barrier function
for each surfactant.
This study found that the protein denaturing activity of sur fac-
tants was not a critical factor on their effect on skin barrier func-
tion. This suggests that there is a possibility that the influence of
surfactant s on another element constituting skin barrier function,
such as the intercellular lipids, is stronger. However, because the
mechanisms of skin barrier function are complex, other fac tors may
conceivably impact its function. For example, molecular biological
effects on skin tissue differentiation and intercellular lipid secretion
may be relevant. Examination using the TI method will be effective
for clarif ying these mechanisms in future studies. Another limitation
of this study is the use of hemoglobin as the target for protein de-
naturation. While hemoglobin allowed easy detection of denatur-
ation, the main protein constituting skin barrier function is keratin.
Therefore, there is a possibilit y that the protein denaturation effect
on actual skin may not have been accurately evaluated. In addition,
our study was performed on skin samples extracted from mice, and
species differences between humans and mice cannot be ignored.
Also, the possibilit y of residue on the skin surface due to adsorption
remains. In the future, it is necessary to establish a method for more
accurately evaluating the effect of protein denaturation on skin bar-
rier function and to clarify the role of adsorption. Furthermore, the
next challenge is to investigate the effec ts of sur factants on lipids
and skin barrier function.
5 | CONCLUSION
TI values representing the ac tual degree of the effect on the skin
barrier function were determined for a total of 19 types of ani-
onic, cationic, amphoteric, and nonionic surfactants. The EC50
for hemoglobin protein denaturation was determined using a se-
rially diluted surfactant, and the correlation with TI values was
examined. For amino acid surfactants, there was a negative cor-
relation between the effect on skin barrier function and the pro-
tein denaturing activity. For other sur factants, there was no clear
correlation between the TI value and the EC50. This suggests
that the protein denaturing activity of the surfactant was not a
major factor affecting skin barrier function. Among the effects
on skin barrier function, the magnitude of the effect by protein
denaturation was evaluated by defining a Skin Barrier Function-
Protein Denaturing Activity Index (SBF-PDA Index). As a result,
it was suggested that the effects of the alkyl structure and the
ion source may affect protein denaturation. It is presumed that
the effect of surfactants on skin barrier function is greater on
lipids. Therefore, in the future, we plan to investigate the effects
of surfactants on lipids.
ACKNOWLEDGMENT
Miss Asami Kano played a crucial role in this study. Unfortunately,
she passed away suddenly on April 12, 2019. Our research could not
be accomplished without her, so we express our deep appreciation
and respect for her valuable work and contribution to science and
list her as important coauthor. We would like to thank Editage (www.
edita ge.jp) for English language editing.
CONFLICT OF INTEREST
No potential conflict of interest was repor ted by authors.
ORCID
Koji Kubota https://orcid.org/0000-0002-3741-7630
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How to cite this article: Kubota K, Okasaka M, Kano A,
Takata S. The involvement of protein denaturing activity in
the effect of surfactants on skin barrier function. Skin Res
Technol. 2020;00:1–8. htt ps://doi.org/10 .1111/srt .12939
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