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Inﬂuence of Aging of Hot Spring Waters on
the Melanin Formation Suppressing E#ect
Shoichi OKOUCHI+, Kouhei NUMATA+, Takao OHAMI+,
Shigeo IKEDA+ and Yuko AGISHI,

Abstract
The melanin formation suppressing e#ect of hot spring water with respect to its
aging was measured using samples of alkaline sulfur spring water collected from Nozawa
spa in Nagano Prefecture and acidic sulfur spring water from Takayu spa in Fukushima
Prefecture, and samples of iron spring water from Tengu spa in Nagano Prefecture. For
aging, the collected spring water samples were left for predetermined periods of time inan open-system environment, air bubbles were forced into them, or combinations of thesemethods were used. After evaluation of the degree of aging based on ORP (oxidationreduction potential)opH relationship, the spring water samples thus prepared were subjected to the measurements of the melanin formation suppressing e#ect. In the measurements, each of the spring water samples was adjusted to pH 1 using a phosphoric acidbu#er, tyrosine and tyrosinase were added to each sample, and then the absorbance (at awavelength of .1/ nm) of dopachrome produced as an intermediate in melanin formation
was measured in terms of variations with aging time. The measurement results showedthat non-aged samples of both sulfur and iron hot spring waters characterized by a low
ORP suppressed melanin formation as expected whereas aged samples of these springwaters having a high ORP did not suppress melanin formation.
Furthermore, since melanin is formed in the skin, we have also carried out experiments to investigate the cutaneous permeability of reductive components of the springwater samples. As a substitute for the skin, a nylon-polyethylene double-layered ﬁlm
having an equivalent-to-skin barrier function was employed. In the experiments, reductive components (H,S) of non-aged sulfur spring water samples permeated through theﬁlm while the ﬁlm permeability of reductive components (Fe (II)) of non-aged iron springwater was not observed. Contrastingly, no ﬁlm permeability was found in the cases of+ +2.ῌ2/2.  -ῌ1ῌ, + Fuculty of Bio Science and Applied
Chemistry, Hosei University, Kajinocho -ῌ1ῌ,, Koganei-shi, Tokyo +2.ῌ2/2., Japan.
,!"#$%&'()  +*2 ῌ **1/ *+*, . ῌ 0 ῌ 2 ῌ ,-*, , ResearchInstitute of HealthResort Medicine, Konan .ῌ0ῌ2ῌ,-*,, Minato-Ku, Tokyo +*2ῌ**1/, Japan.

273

   

aged samples of both sulfur and iron spring waters. Since reductive sulfur and iron hot
spring waters, both of which are capable of suppressing melanin formation, are di#erent
in the degree of cutaneous permeability, it is apparent that the species of reductive components can cause a signiﬁcant di#erence in the e#ect of suppressing melanin formationin the skin. Further, as having been generally said that the beneﬁcial e#ects and e$cacies of hot spring water are lost with its aging, the present work has conﬁrmed that the
aging of hot spring water results in nulliﬁcation of its beneﬁcial e#ect of suppressing
melanin formation in the skin.
Key words : Melanin, dopachrome, aged hot spring water, Nozawa spa, Takayu spa, Tengu
spa, cutaneous permeability.

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et al., ,**, ; Ohnami et al., ,**- ;  ,**2  !

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AB + CD#E6&FG ,-HIJ?*+'()7KLM 7./01)G3"NO 5
ORP & 7PE#'() 01)QRS6R+T5?UV ORP  ! 7Q"  AB4  KLW+8XO8Y43Z7B[# \& ] R'()!^&"#_L+`a bcdef$g>RXH+h %&ij'J7 +56
7() 2 ,**3 ?8"#568 5  HI
XHO1 +87kl"# '()m*+6"3 + .6!
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,**3 ?)o"#pqdr/0 1/s tHu0v2 34s -G -

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+78};<~7 78crp=7>"#?@A7B4 TWA};
C7  7PE3Z"#

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!^)6"3 /0 D!^ : pH2.. E S/+.0 ppm, H,S,., ppm v2 0ῌP!^῍dq`῍!0, !1  : pH,.-1 H,S+.2.2 ppm tHu-)
6"3 5w D-F  : pH /.1* E-/0.1 ppm, CO,32/.2 ppm G&  AB7?!+=> +#E TW%&H??!+=>I8 JF"R qKL?N?KM"# N& TWG ORPopH _7 O.
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

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8 

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r0c®¯°?XY pH 1 " J?*+±²` *., mM tHu01³ ±²`´µ /-1* Uῌcm- 7-G Fig. + S:?*+¶·²¸¹W .1/

nm ;917_`"# Shirota et al., +33. ; aS ,**0 Rt - ῌ 7P«5wtH

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275     

Fig. + Melanin formation mechanism.
 +  

Fig. ,

Scheme of experimental arrangement for ﬁlm permeation of reductive components.
 ,

!"#$% , &' ()*+, /30/ m-ῌm,ῌ-ῌatm ./012 34567%ῌ !"#$%'. Fig. , 89:;<8=>812?8@AB. C?8BDE21 3FG.H
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Figure - 8 YZ [\];^_`B4"Ja%b4cd. ORPopH RSD912 efF276

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ῑn=RSC op6ῑn=TUC K>=

Fig. - Inﬂuence of aging of hot spring waters on ORPo
pH relationships
 : hot spring water. , ,  : aged hot springwater.  : puriﬁed water (pH 1)
 - 
ORPopH 
:    :  :  pH 1

ABC[OPQPqrX Figure - s t
Ruvwx=Ll ORP

y=>?@7119:;<AB ORPKDEz! pH 4{ῌ>?@7u|}~
@F P= 7s\P<6F
RS!iFFh`PX   us=Ll71119:;< ORP y=>?X! pH=I@ ug`7u|}~@F P=7 RS! u῍WXh{῏S!@ pH[IpF4h`PX \F Rus=LX pH S=`PG

6! ORP y=71119:;<>?@F GH  R~ s pH 1  @F[ Fig. - 9s@F! ABC¡Xh!VgX
Figures . g` 0   HLMwx

OPQP¢6£¤@F$%&

Fig. . Absorbance of dopachrome (.1/ nm) as
an intermediate of melanin formation reaction during the aging of Nozawa hot springwaters : Puriﬁed water (pH 1). , , ,  :Hot spring water samples (Nozawa spa) as
shown in Fig. -.
 .     !" #$%&' ()* .1/ nm +, :  pH 1     :  -  '(¥¦§ Fig. + HiX7¨©ª «¬¥­®¯ .1/ nm S[@F  R~ s pH 1  @F° ¥9 s= 6±P4®¯=¨²³56>?@ ª «¬¥­!'(pP $%& '(´¤!µ¶@<6Xh!·¸smX  Fig. . HLM Fig. / 9s HL
MOP`!µ¶@Fs=µ¶¢6 1119 £¤@< ῐ¹®¯=>?@ $%& '(῎)*+!ºjPX»+[@FFigure 0 wx 9 s= ¼=®¯S=G64 +* V³½g`®¯
277 ! "#$% &'( )#*+ ,-῎.

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H7IJKLMNOP
b!c ῐdcῌ`\Fig. 1 Yῒae4N +*2/* ppm fXg

hCijDEFABῑklmX ;<=>?@
no4 .1/ nm pqghNOP `\ ῒ
ab!c ῐdX c ῌ e47 /* ppm Xr9Q
Fig. / Absorbance of dopachrome (.1/ nm) as
an intermediate of melanin formation reaction during the aging of Takayu hot springwaters : Puriﬁed water (pH 1). , , ,  :Hot spring water samples (Takayu spa) as
shown in Fig. -.
 /  

 !"#$%&' .1/ nm () : 
pH 1     :  -  

DEFABNῑkOjsKt^7uQK OQO ce47 /0.1 ppm 

W XY

Fig. 0 RvwxQ9no4756O

DE

FAB7IJOjsK t:Yce4
Yc ῌ ^c ῍ yze4NO {:|}~
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KfX T<UV7IJOc ῌ 7OPQ C9YBu7

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OQO s:Ojc ῌ YDEFῑk
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Figures 2 Q9 +*  Z[ \R]

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- OPT<UV W

b YT<

UV7IJO ORP 7!% ORP 5
Fig. 0 Absorbance of dopachrome (.1/ nm) as
an intermediate of melanin formation reaction during the aging of Tengu hot springwaters : Puriﬁed water (pH 0). , ,  : Hot
spring water samples (Tengu spa) as shownin Fig. -.
 0  *+
 !"#$%&' .1/ nm () : 
pH 0    :  -  

sT<UV W HOP  W N Fig. ,  ¡O 
Y N¢POjlmLMN
Figures 2 \R] 3 Z[ {::  a T<UV W Y £¤ 9 ,**3 ^_῏ῒ¥}~¦§
H,S, 7¨©ª«¬<­® ,- +33. N¯OP°±NO 

W  ORP Nq²p³^`sῌW´µj¶CiP !·b  T<UV W XYΐ ¸W

 ORP q²p

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T<UVRS  x}~¦§7¹ºOjsKt^Q9»¼½LXK!· Fig. +*  XY T<UV W \R]T<UV 278

* /3 + ',*+*( ,( -. /0123456 

 !"#$%&',(  ORP .789:; <=>?@A <'ῌ( BC9DEFG:HI) :J Fig. +*  'a( .KL

MNOF9 /0

12.PQ  pH 9RST.UVW) XY9Z[.\]Y5^_`abcFig. 1 Variations in absorbance (.1/ nm) due to dopachrome
produced as an intermediate in melanin formation reaction for Fe (ῌ) aqueous solution.
 : * ppm.  : +* ppm.  : ,* ppm.  : .* ppm.  :/* ppm 1  ῌ    !"# $% .1/ nm &' : * ppm.  : +* ppm.  : ,* ppm.  : .* ppm.  :/* ppmFig. 2 Inﬂuence of ORPopH variations of Nozawa hot spring waters on ﬁlm permeation.
 : Hot spring water.  : aged hot spring water.  : variations with time in puriﬁed water in ﬁlm enclosure.
 2 () *+()
,-.
 ORPopH &' : 
:
 : 

Fig. 3 Inﬂuence of ORPopH variations of Takayu hot spring waters on ﬁlm permeation.
 : Hot spring water.  : aged hot spring water.  : variations with time in puriﬁed water in ﬁlm enclosure.
 3 () /0()
,-.
 ORPopH &' : 
:
 : 

279

     !"#$%

Fig. +* Inﬂuence of ORPopH variations of Tengu hot spring waters on ﬁlm permeation.
 : Hot spring water.  : aged hot spring water.  : variations with time in puriﬁed water in ﬁlm enclosure.
 +* 
  ORPῌpH  :   :  : 

*+,-./01  pH /23*456789:;<= &> ,**2' ?@A( B C &b' 6DE

F GH?I pH JKLMN OPI62

3Q*+,-.R>ST<=UVW>PA(." # $ %XYZ[\]/^_`Ua6bcd OP> ORP ecf
GHU ORP .gNMh

F

F GH6iEAjkl m\nop q2rOP>

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F GH?I`q2ra~6jkl m\/n.

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4/?.

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q2r

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kl m\n=.PAU.>RUMh( !
!"# &+33.' : Umxy(  +++ +/0+ῌ+/0/.
 #     &,**2' : YZ[62Ajkl
m\n=( 0+ $%  p. +,.
 &,**-' : mdcAUI¡R)¢6£:cm6¤c/¥¦EA)( pp. +2ῌ
/* N§:¨© ª«(  ¬­®¯ °#   !"# &,**3' : YZ[ &`'
62Ajkl m\n=( $% /3 ,ῌ+*.
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/3 ! ,*+*



 

"#$%&' () .2 ,3ῌ-/.
*+,-. */01 23ῒ4 5678 9:;< ,**/a : ORP =>?@ABCDEFGH
IJ# () /. +//ῌ+0,.
*+,-. */01 KLMN *OPQ 9:;< RST1 UVWX ῏Y Z ,**/b :&[$%\]^_`abcd?efg () // //ῌ0-.
*+,-. hOijk l6m4 ῏Ymn oῐpqr st u +333 : abc_`
 ORP "#$%&' v pH wx () .3 /3ῌ0..
*+,-. hOijk ῏Ymn oῐpqr ,*** : y"#zD ORP v pH wx () /* 3.ῌ+*+.
Okouchi, S., Suzuki, M., Sugano, K., Kagamimori, S. and Ikeda, S. (,**,) : Water desirable forthe human body in terms of oxidation-reduction potential (ORP) to pH relationship. J.
Food Sci., 01, +/3.ῌ+/32.

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Ohnami, S., Koumura, K., Ikeda, S., and Okouchi, S. (,**-) : ORP (oxidation-reduction potential)opH
relationship between hot spring waters and human body ﬂuids. Proceedings of the -2th
Conference of Societe Internationale des Techniques Hydrothermales (Beppu, Ohita, Japan), -,*ῌ-,/.

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Shirota, S., Miyazaki, K., Aiyama, R., Ichioka, M., Yokokura, T. (+33.) : Tyrosinase Inhibitorsfrom Crude Drugs. Biol. Pharm. Bull., +1, ,00ῌ,03.

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