Inhibitive effects of alkyl gallates on Hyaluronidase and collagenase

Article abstract of DOI:10.1271/bbb.90365

A series of the gallate esters of n-alkanols (C₁-C₁₂) was examined to determine their inhibitory activities against hyaluronidase and collagenase. Hexyl, heptyl, octyl, nonyl, and decyl gallates inhibited both hyaluronidase and collagenase, and the most potent inhibitor was octyl gallate against both enzymes. Octyl 3, 5- dihydroxybenzoate showed inhibitory effects on hyaluronidase, whereas collagenase was inhibited by octyl 3, 4-dihydroxybenzoate.

Full text of DOI:10.1271/bbb.90365

Biosci. Biotechnol. Biochem., 73 (10), 2335–2337, 2009
Note
Inhibitive Effects of Alkyl Gallates on Hyaluronidase and Collagenase
Florin BARLA,1 Hayato HIGASHIJIMA,1 Shingo FUNAI,1 Keiichiro SUGIMOTO,1 Naoki HARADA,1
y
1;
Ryoichi YAMAJI,1 Tomoyuki FUJITA,2 Yoshihisa NAKANO,1;3 and Hiroshi INUI
1Department of Applied Biological Chemistry, Graduate School of Life and Environmental Sciences,
Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
2Department of Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University,
Minami-minowa, Nagano 399-4598, Japan
3Osaka Women’s Junior College, Fujiidera, Osaka 583-8558, Japan
Received May 21, 2009; Accepted July 20, 2009; Online Publication, October 7, 2009
[doi:10.1271/bbb.90365]
A series of the gallate esters of n-alkanols (C1–C12)
was examined to determine their inhibitory activities
against hyaluronidase and collagenase. Hexyl, heptyl,
octyl, nonyl, and decyl gallates inhibited both hyalur-
onidase and collagenase, and the most potent inhibitor
was octyl gallate against both enzymes. Octyl 3,5-
dihydroxybenzoate showed inhibitory effects on hyalur-
onidase, whereas collagenase was inhibited by octyl
3,4-dihydroxybenzoate.
Gallic acid (3,4,5-trihydroxybenzoic acid) is a phe-
nolic compound that is found as a constituent of
hydrolyzable tannins in many plants. Gallic acid and
hydrolyzable tannins have many biological activities,
including antioxidation and hyaluronidase inhibition.9,10)
The gallate esters of n-alkanols also act as potent
antioxidants, and propyl, octyl, and dodecyl (lauryl)
gallates are permitted additives for antioxidation in
foods in the United States11) although they are not
natural compounds. Furthermore, some of these alkyl
gallates, including octyl gallate, have been reported to
be useful as multifunctional food additives, as they have
antibrowning,12) antifungal,13) and antibacterial14) ef-
fects. To determine whether alkyl gallates are also useful
as cosmeceutical ingredients, in the present study, we
have identified their inhibitory activities against hyalur-
onidase and collagenase.
Methyl gallate, butyl gallate, octyl gallate, and octyl
4-hydroxybenzoate were purchased from Wako Pure
Chemical Industries (Osaka, Japan); gallic acid, propyl
gallate, and hyaluronidase (from bovine testes) were
from Nacalai Tesque (Kyoto, Japan); ethyl gallate,
dodecyl gallate, 3,4-dihydroxybenzoic acid, 3,5-dihy-
droxybenzoic acid, and 3-hydroxybenzoic acid were
from Tokyo Chemical Industry (Tokyo); hyaluronic acid
sodium salt (from rooster comb) and collagenase (from
Clostridium histolyticum; type IV) were from Sigma;
Pz-peptide (Pz-Pro-Leu-Gly-Pro-D-Arg-OH) was from
Bachem (Bubendorf, Switzerland); and recombinant
human matrix-metalloproteinase-1 (MMP-1) and Fluo-
rogenic Peptide Substrate IX (Mca-Lys-Pro-Leu-Gly-
Leu-Dpa-Ala-Arg-NH2) were from R&D systems (Min-
neapolis, MN).
Key words: alkyl gallates; hyaluronidase inhibitory ac-
tivity; collagenase inhibitory activity; anti-
oxidant activity
Hyaluronic acid is found in the extracellular matrix of
soft connective tissues, such as the umbilical cord, skin,
synovial fluid, and vitreous humor. This polysaccharide
is involved in cell adhesion and protection, the forma-
tion of skin tissue, water retention in tissues, and the
maintenance of flexibility in the skin due to its unique
hydroscopic, rheologic, and viscoelastic properties.1)
Hyaluronidase is a mucopolysaccharase that hydrolyzes
glycosaminoglycans, including hyaluronic acid, in the
extracellular matrix during tissue remodeling.2) When
the level of hyaluronic acid decreases under conditions
in which hyaluronidase activity increases, the moisture
and tension of the skin are reduced. Thus hyaluronidase
inhibitors are useful cosmeceutical ingredients as they
have antiwrinkle and antiaging effects on the skin. In
addition, hyaluronidase induces histamine release from
mast cells during inflammatory reactions, and some hyalu-
ronidase inhibitors, including disodium cromoglycate,
have been used to suppress allergies and inflammation.3,4)
Collagen is the major fibrous component of the
extracellular matrix in the skin, and the collagen content
in the skin decreases greatly during the aging process
and due to long-term exposure to ultraviolet radiation.5,6)
Collagenase (matrix-metalloproteinase, MMP) plays an
important role in unbalanced turnover and the rapid
breakdown of collagen in human inflamed/ultraviolet-
irradiated skin.7,8) Hence, collagenase inhibitors, in
addition to hyaluronidase inhibitors, have useful func-
tions as cosmetic materials.
Hexyl gallate, heptyl gallate, nonyl gallate, decyl
gallate, octyl 3,4-dihydroxybenzoate, octyl 3,5-dihy-
droxybenzoate, and octyl 3-hydroxybenzoate were syn-
thesized as follows: The corresponding phenolic acid
(5 g) and n-alkanol (50 g) were mixed with H2SO4
(approximately 2 ml) and kept at 110 ꢀC for 5 h. After
cooling, tert-methyl butyl ether (approximately 250 ml)
was added to the mixture, and successive washing with
saturated aqueous solutions of NaCl and NaHCO3 (5
times each) was done. After removal of the solvent by
y
To whom correspondence should be addressed. Tel: +81-72-254-9453; Fax: +81-72-254-9454; E-mail: inui@biochem.osakafu-u.ac.jp
Abbreviations: DPPH, 1,1-diphenyl-2-picrylhydrazyl; DMSO, dimethyl sulfoxide; EC50, 50% effective concentration; IC50, 50% inhibitory
concentration; MMP, matrix-metalloproteinase

2336
F. BARLA et al.
Table 1. Hyaluronidase and Collagenase Inhibitory Activities of
n-Alkyl Gallates and Related Compounds
evaporation, the crude product dissolved in methanol
was purified by silica gel column chromatography with a
solvent system of chloroform/methanol (98:2).15) To
confirm the structure of the synthesized products, the
1H-NMR spectrum was recorded on a JEOL JNM-
AL400 spectrometer (400 MHz) in deuterium methanol
and compared with previously reported data.12,13,15–17)
To evaluate radical scavenging activity against the
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, a sample
(alkyl gallate) was dissolved in 2 ml of methanol and
mixed with 0.5 ml of a DPPH solution (0.4 mg/ml) in
methanol. After incubation at room temperature for
30 min in air, the remaining DPPH was determined by
measuring the absorbance at 520 nm, and the radical-
scavenging activity was estimated as described by
Yoshida et al.9)
Hyaluronidase inhibitory activity was examined fol-
lowing Ono et al.,18) with some modifications. Briefly,
hyaluronidase (160 units) was incubated with an
inhibitor (alkyl gallate) dissolved in 10 ml of 40%
dimethyl sulfoxide (DMSO) and 70 mg of compound
48/80 in 0.1 M sodium acetate buffer, pH 4.0, in a total
volume of 0.25 ml at 37 ꢀC for 20 min. Then 0.275 mg of
hyaluronic acid sodium salt dissolved in 0.25 ml of 0.1 M
sodium acetate buffer, pH 4.0, was added to the enzyme
solution to start the hyaluronidase reaction, and the
mixture was incubated for an additional 40 min.
DPPH
Hyaluronidase Collagenase
radical
inhibitory
inhibitory
scavenging
activity EC50
(mM)
activity IC50 activity IC50
(mM)
(mM)
Gallic acid
Methyl gallate
>1,000
>1,000
>1,000
>1,000
>1,000
253
2.07
>10
>10
>10
>10
3.43
1.19
1.08
1.25
2.64
>10
>10
>10
3.16
>10
nd
18.5
18.6
21.0
24.1
22.7
19.8
21.7
20.5
22.8
16.6
19.6
>100
>100
15.4
>100
nd
Ethyl gallate
Propyl gallate
Butyl gallate
Hexyl gallate
Heptyl gallate
Octyl gallate
112
106
Nonyl gallate
Decyl gallate
167
580
Dodecyl gallate
Octyl 3-hydroxybenzoate
Octyl 4-hydroxybenzoate
Octyl 3,4-dihydroxybenzoate
Octyl 3,5-dihydroxybenzoate
Disodium cromoglycate
EDTA
>1,000
>1,000
>1,000
902
113
450
nd
0.58
nd
nd, not determined
onidase (their IC50 values were higher than 1 mM). In
contrast, hexyl, heptyl, octyl, nonyl, and decyl gallates
exhibited potent hyaluronidase inhibitory activities. It is
hence thought that the hyaluronidase inhibitory activity
(but not the antioxidant activity) associated with the
length of the hydrophobic alkyl chain to a large extent.
Octyl gallate was the most potent inhibitor among the
alkyl gallates examined, and its IC50 value (106 mM) was
substantially lower than that of disodium cromoglycate
(450 mM). Octyl 3,5-dihydroxybenzoate was a potent
inhibitor of hyaluronidase, with an IC50 value of 113 mM,
comparable to that of octyl gallate; however, other
derivatives of octyl benzoate (octyl 3,4-dihydroxyben-
zozate, octyl 3-hydroxybenzoate, and octyl 4-hydrox-
ybenzoate) exhibited virtually no inhibitory activity.
These results suggest that the two hydroxy groups at the
3 and 5 positions in the benzoate moiety are essential for
hyaluronidase inhibition. This is in contrast with the
catechol structure necessary for antioxidative effects.
The kinetics of the hyaluronidase reaction were
examined to the presence of octyl gallate. As shown in
Fig. 1, the inhibition kinetics, analyzed by Lineweaver-
Burk plots, indicated that octyl gallate exhibited un-
competitive inhibition. Thus it is less likely that octyl
gallate binds directly to the catalytic domain of
hyaluronidase. The inhibition constant, the Ki value,
was estimated to be 45 mM.
Collagenase (from C. histolyticum) was inhibited by
gallic acid, with an IC50 value of 2.07 mM, but gallic
acid esters of n-alkanols with short chain lengths
(C1–C4) did not inhibit the enzyme (Table 1). These
results suggest that the carboxyl group in gallic acid is
essential for collagenase inhibition. However, similarly
to the case of hyaluronidase inhibition, the collagenase
reaction was strongly inhibited by gallate esters of
n-alkanols when the alkyl chain length ranged between
C6 and C10, and the most potent inhibitor was octyl
gallate. The IC50 value of octyl gallate (1.08 mM) was
distinctly lower than that of gallic acid although it was
Collagenase inhibitory activity was determined using
´
the Pz-peptide as a substrate by the method of Hernandez
et al.,19) with some modifications. The reaction mixture
contained 2.5 mg of collagenase from C. hystolyticum, an
inhibitor (alkyl gallate) dissolved in 25 ml of 10%
DMSO, 0.5 mg of Pz-peptide, 20 mM CaCl2, and 0.1 M
Tris–HCl buffer, pH 7.1, in a total volume of 0.5 ml.
Collagenase inhibitory activity was also evaluated using
MMP-1 (mammalian collagenase). The reaction mixture
(0.5 ml) contained 10 ng of MMP-1, which had been
activated by preincubation with p-aminophenylmercuric
acetate, an inhibitor (octyl gallate) dissolved in 25 ml of
DMSO, 10 mM Fluorogenic Peptide Substrate IX, 10 mM
CaCl2, 150 mM NaCl, 0.05% Briji-35, and 50 mM Tris–
HCl buffer, pH 7.5, and the enzyme activity was
determined by measuring the increase in fluorescence
intensity (Ex: 320 nm; Em: 405 nm) at 25 ꢀC.
To determine the antioxidative activity of gallic acid
and gallic acid esters of n-alkanols with different chain
lengths (C1–C12), the radical scavenging activity, which
can be measured on the basis of decolorization follow-
ing the trapping of unpaired electrons from DPPH, was
examined. As shown in Table 1, all of the alkyl gallates
examined as well as gallic acid were found to have
strong radical scavenging activities against DPPH, and
their 50% effective concentration (EC50) values were
almost constant (approximately 20 mM) regardless of the
alkyl chain length. These results are similar to those of a
previous report.12)
The gallate esters of n-alkanols were examined as to
their hyaluronidase inhibitory activity, and their 50%
inhibitory concentration (IC50) values were compared
with that of disodium cromoglycate, a well-known
hyaluronidase inhibitor, which is used as an antiinflam-
mation and antiallergic agent4) (Table 1). Methyl, ethyl,
propyl, butyl, and dodecyl gallates as well as gallic acid
exhibited virtually no inhibitory activity against hyalur-

Alkyl Gallates
2337
Acknowledgment
We are grateful to Dr. Kiyoshi Furukawa (Nagaoka
Perfumery Co., Ltd., Osaka, Japan) for his help in the
synthesis of alkyl gallates.
References
1) Sujith S and Ivo D, Acta Medica, 50, 225–228 (2007).
2) Sugimoto K, Iizawa T, Harada H, Yamada K, and Katsumata M,
Cartilage, 12, 1006–1014 (2004).
3) Sakamoto K, Nagai H, and Koda A, Immunopharmacology, 2,
139–146 (1980).
4) Kakegawa H, Matsumoto H, and Satoh T, Chem. Pharm. Bull.,
33, 642–646 (1985).
5) Uitto J, Dermatol. Clin., 4, 433–446 (1986).
6) Plastow SR, Lovell CR, and Young AR, J. Invest. Dermatol.,
88, 145–148 (1987).
7) Lim H and Kim HP, Planta Med., 73, 1267–1274 (2007).
8) Herrmann G, Wlaschek M, Lange TS, Prenzel K, Goerz G, and
Scharffetter-Kochanek K, Exp. Dermatol., 2, 92–97 (1993).
9) Yoshida T, Mori K, Hatano T, Okumura T, Uehara I, Komagoe
K, Fujita Y, and Okuda T, Chem. Pharm. Bull., 37, 1919–1921
(1989).
Fig. 1. Lineweaver-Burk Plots of the Hyaluronidase Reaction in the
Presence of Octyl Gallate.
The hyaluronidase reaction was done in the presence of octyl
gallate at the concentrations cited.
10) Sugimoto K, Nakagawa K, Hayashi S, Amakura Y, Yoshimura
M, Yoshida T, Yamaji R, Nakano Y, and Inui H, Food Sci.
Technol. Res., 15, 331–336 (2009).
approximately 2-fold higher than that of EDTA
(0.58 mM), the most often used collagenase inhibitor.20)
Octyl 3,4-dihydroxybenzoate as well as octyl gallate
inhibited collagenase, whereas octyl 3,5-dihydroxyben-
zoate did not. This result is in contrast with that in the
case of hyaluronidase inhibition. In addition to the
bacterial collagenase, MMP-1 (a mammalian collage-
nase) was also inhibited by octyl gallate. The IC50 value
of octyl gallate for MMP-1 was estimated to be 0.35 mM,
lower than that for the bacterial enzyme (1.08 mM).
The data obtained in the present study show that the
gallate esters of n-alkanols with alkyl chain lengths
between C6 and C10 exhibit hyaluronidase and collage-
nase inhibitory activities, in addition to their antioxida-
tive,11,12) antibrowning (tyrosinase inhibitive),12) anti-
fungal,13) and antibacterial14) effects. Thus, these alkyl
gallates, in particular octyl gallate, which is a permitted
food additive for antioxidation in the United States,11)
might be useful cosmeceutical ingredients to improve
inflammation and allergic reactions, and to prevent
wrinkles and skin aging.
11) Aruoma OI, Murcia A, Butler J, and Halliwell B, J. Agric. Food
Chem., 41, 1880–1885 (1993).
12) Kubo I, Kinst-Hori I, Kubo Y, Yamagiwa Y, Kamikawa T, and
Haraguchi H, J. Agric. Food Chem., 48, 1393–1399 (2000).
13) Kubo I, Xiao P, Nihei K, Fujita K, Yamagiwa Y, and Kamikawa
T, J. Agric. Food Chem., 50, 3992–3998 (2002).
14) Kubo I, Fujita K, Nihei K, and Nihei A, J. Agric. Food Chem.,
52, 1072–1076 (2004).
15) Shibata H, Kondo K, Katsuyama R, Kawazoe K, Sato Y,
Murakami K, Takaishi Y, Arakaki N, and Higuti T, Agents
Chemother., 49, 549–555 (2005).
16) Nihei K, Nihei A, and Kubo I, J. Agric. Food Chem., 52, 5011–
5020 (2004).
17) Ha TJ, Nihei K, and Kubo I, J. Agric. Food Chem., 52, 3177–
3181 (2004).
18) Ono M, Masuoka C, Tanaka T, Ito Y, and Nohara T, Food Sci.
Technol. Res., 7, 307–310 (2001).
´
19) Hernandez M, Duflos G, Malle P, and Bouquelet S, Food Res.
Int., 36, 141–147 (2003).
20) Woolley D, Roberts D, and Evanson J, Biochem. Biophys. Res.
Commun., 66, 747–754 (1975).