日本香粧品学会誌

Vol. 46, No. 4, pp. 335–340（2022）〈Regular Article〉

Applicability of 8-Anilinonaphthalene-1-sulfonic Acid toMeasure the Hydrophobicity of Corneocytes
Hitoshi MASAKI1, 2, *, Yuki SATO1, Taeko MIZUTANI1, Nao KIKUCHI2, Natsuki YOSHIDA2,Nana TAKAYAMA2, Masaki YOSHIDA2, Yuri OKANO1, 2(Accepted: August 10, 2022)

Abstract
This study was conducted to clarify whether 8-anilinonaphthalene-1-sulfonic acid (8,1-ANS), a ﬂuorescent probe thatis used to measure hydrophobic changes of the microenvironment formed by proteins, can be used to measure the degree ofhydrophobicity of corneocytes. Firstly, the ﬂuorescence intensity of 8,1-ANS in keratin ﬁlms in which COOH groups werechemically modiﬁed with hexylamine or with Alexa Fluor 594 cadaverine to increase hydrophobicity was examined. Increase of hydrophobicity in these chemically modiﬁed keratin ﬁlms was indicated by measuring a contact angle of deionized
H2O. The ﬂuorescence intensity of keratin ﬁlms originating from 8,1-ANS showed increases that corresponded to the decreased ﬂuorescence intensity of Alexa Fluor 594 cadaverine, which was used to label COOH groups that remained after thecovalent bonding of hexylamine. Furthermore, keratin ﬁlms with COOH groups that were ﬂuorescence-labelled with Alexa
Fluor 594 cadaverine also showed increased ﬂuorescence intensity of 8,1-ANS. These results showed that 8,1-ANS changed
its ﬂuorescence intensity in response to the hydrophobic modiﬁcation of keratin ﬁlms, which indicated the applicability of8,1-ANS to measure the hydrophobicity of corneocytes. The ﬂuorescence intensity originating from 8,1-ANS labeling of corneocytes, which were chemically modiﬁed to increase their hydrophobicity, was then examined. The ﬂuorescence intensityof corneocytes that originated from 8,1-ANS showed changes corresponding to the hydrophobic modiﬁcations. These results
demonstrated that 8,1-ANS is a useful ﬂuorescent probe to measure the hydrophobicity of corneocytes.
Key words: corneocyte, hydrophilicity, functional group, keratin ﬁlm.

1. Introduction
The stratum corneum is the ﬁrst line of defense against theinvasion of materials from the outer environment and is alsoa ﬁnal barrier to retain endogenous substances in the body.
The stratum corneum is mainly composed of corneocytes,
which are the end-products of the terminal diﬀerentiation ofkeratinocytes, and of lipid lamellae, which are composed ofceramides, cholesterol and fatty acids1) that are located at theinterstices between corneocytes. Considering that lipid lamellae maintain their structure by chemical interactions, it is presumed that corneocytes contribute to the barrier function of
the human body more eﬀectively as robust structures.
Each corneocyte is surrounded by speciﬁc protein complexes that are formed intracellularly inside the plasma membrane, and they are ﬁlled with keratin ﬁbers. Those protein
complexes are called the corniﬁed cell envelope (CE), andthey are formed by the cross-linking of diﬀerentiation marker
proteins, involucrin, loricrin, ﬁlaggrin and small proline rich

CIEL Co. Ltd. (5–4–30 Nishihashimoto, Midori-ku, Sagamihara-shi,Kanagawa 252–0131, Japan)
2
Tokyo University of Technology, School of Bioscience and Biotechnology
1* Corresponding author: CIEL Co., Ltd., 5–4–30 Nishihashimoto,Midori-ku, Sagamihara-shi, Kanagawa 252–0131, Japan (E-mail:h.masaki@ciel-tokyo.com)

protein, by transglutaminases2). Furthermore, each corneocyte
has a lipid envelope in which ω-hydroxy ceramides are covalently bound to carboxyl (COOH) groups of CE proteins suchas involucrin3). Thus, these structures suggest that corneocytes
have a hydrophobic characteristic.
Hydrophobic characteristics are generally reﬂected as awater repellency. On the other hand, water repellency is often
considered as a symbolic feature of young skin. Furthermore,
a phrase of an ancient poem in Chang hen ge written byBai Juyi expresses that water repellency is a symbolic featureof beautiful skin. However, there is no scientiﬁc evidence
indicating whether water repellency reﬂects a condition ofhealthy skin. Although the water repellency of skin is thought
to be achieved due to factors such as sebum and the hydrophobicity of corneocytes, there is no method available as thistime to measure the hydrophobicity of corneocytes. In thecase of hair, water repellency can be evaluated by measuring
the contact angle against water4). A previous study reported
that the ﬂuorescence intensity (FI) originating from 8-anili-