2326
Y. WATANABE et al.
1
00
250
200
80
60
40
20
0
f
e
d
1
1
50
00
c
b
a
5
0
0
0
10
20
30
40
50
-2
0
2
4
6
8
10
MMTS (µM)
-
1
Reciprocal of PNPG (mM )
Fig. 2. Inhibition of the ꢀ-Glucuronidase Activity by MMTS.
The results are presented by bars as the mean ꢂ SD of
quadruplicate measurements, unless the error bar is smaller than
the symbol. The means were compared by Tukey’s test (p < 0:05).
Significant differences are indicated by different letters.
Fig. 3. Lineweaver-Burk Plots of ꢀ-Glucuronidase in the Presence of
MMTS.
Control; 1 mM MMTS; 2 mM MMTS. Results are presented
as the mean of quadruplicate measurements.
activity was assayed by monitoring the p-nitrophenol
produced from p-nitrophenyl-ꢀ-D-glucuronide (PNPG;
Sigma-Aldrich Japan, Tokyo, Japan) according to the
unchanged. This pattern indicates that MMTS was an
uncompetitive inhibitor of ꢀ-glucuronidase. This behav-
ior indicates that the inhibitor bound at a site distinct
from the substrate and combined with the enzyme-
substrate complex, but not with the free enzyme.
Thiosulfinates, including MMTS, inhibit platelet aggre-
1
7)
method of Gudiel-Urbano and Go n˜ i with a minor
modification. In brief, 50 mL of each tested compound
in a 20-mM phosphate buffer (pH 7.0), and 50 mL of
1
9)
20)
ꢀ
-glucuronidase from Escherichia coli (12 units/mL;
gation by inhibiting calpain activation. Badol et al.
Sigma-Aldrich Japan, Tokyo, Japan) were added to a
have reported that thiosulfinates rapidly interacted with
sulfhydryls on calpain and inhibited its activity. A
similar mechanism for ꢀ-glucuronidase inhibition by
MMTS could be involved. Further investigations are
required to identify the inhibitory mechanism.
ꢁ
96-well microplate and incubated at 37 C for 10 min. A
50-mL amount of 0.6 mM PNPG was then added to each
well to initiate the reaction. The microplate was read
with a Sunriseꢀ microplate reader (Tecan Japan,
Kanagawa, Japan) at 405 nm and 37 C for 5 min. The
ꢁ
Thiosulfinates are known in Allium species as heat-
labile intermediates generated via the enzymatically
initiated degradation of S-alk(en)yl-L-cysteine sulfox-
enzyme activity is expressed as the change in absorb-
ance at 405 nm per min (slope) calculated by using
Magellanꢀ software (Tecan Japan, Kanagawa, Japan).
We found that DMDS showed no significant inhib-
ition (5 mM; 1:4 ꢂ 1:5%, mean ꢂ SD, n ¼ 4). AMDS and
DADS showed slight but significant inhibition of ꢀ-
glucuronidase activity (5 mM; 8:5 ꢂ 3:3% for AMDS and
1
3)
ides. Garlic (A. sativum L.) has long been used as a
representative vegetable possessing marked pharmaco-
logical potential. This potential has been assumed to be
due to allyl 2-propenylthiosulfinate. However, its insta-
2
1,22)
bility
has been one of the drawbacks for its
8
:5 ꢂ 2:0% for DADS, mean ꢂ SD, n ¼ 4, p < 0:05). In
utilization. Fresh Chinese chive has been shown to
contain up to 9% methyl 2-propene-1-thiosulfinate, 5%
1-propenyl methanethiosulfinate, 13% allyl methylthio-
contrast, MMTS exhibited relatively strong inhibitory
activity at 5 mM (56:4 ꢂ 0:7%, mean ꢂ SD, n ¼ 4,
p < 0:05). There have been few studies in which the
biochemical or physiological functions of thiosulfinates
were compared with those of sulfides. Interestingly,
Merhi et al.1 have reported that MMTS, but not DMDS
or DADS, inhibited cell proliferation, differentiation,
and secretion of leukemic cell lines. Thiosulfinates could
have potential bioactivities which their related sulfides
did not have.
1
4)
23)
sulfinate, and 73% MMTS. However, Seo et al.
have reported that only MMTS and allyl methylthiosul-
finate were isolated from a Chinese chive extract.
MMTS therefore appeared to be relatively stable
8)
2
4)
compared with other thiosulfinates. Shen et al. have
reported that the half-life values of MMTS at an acidic
ꢁ
or neutral pH at 40 C ranged from 1.3 d to 32.9 d. We
therefore consider that MMTS in the processed Chinese
chive could retain its inhibitory activity against ꢀ-
glucuronidase of microbiota in the intestines even after
ingestion, although this needs to be confirmed by further
investigations.
The inhibitory effects of MMTS at different concen-
trations are shown in Fig. 2. MMTS significantly
inhibited the ꢀ-glucuronidase activity, even at 1 mM.
This inhibitory activity increased with increasing con-
centration of MMTS up to 5 mM and gradually leveled
off as the concentration was further increased. Approx-
imately 80% of the enzyme activity was inhibited at
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4)
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Watanabe, Yuko
