2
32
A. Kato et al. / Phytochemistry Letters 3 (2010) 230–233
Table 1
3.3. Extraction and isolation
Concentration of iminosugars giving 50% inhibition against glycosidases.
Enzyme
IC50
(
mM)
The roots (5 kg) of A. triphylla were extracted three times with
hot water for 1 h. The filtrate was applied to a column of Amberlite
a
4
DIL
+
4
IR-120B (1000 mL, H form). The 0.5 M NH OH eluate was
a-Glucosidase
Rice
b
concentrated to give a brown syrup (30.8 g). This syrup was
NI
845
NI
ꢀ
applied to Dowex 1-X2 (OH form) short column to remove amino
Yeast
NI
b
-Glucosidase
Almond
acids and pigments, and eluted with H
2
O. This pool treated with
NI
329
NI
Dowex 1-X2 was further chromatographed with Amberlite CG-50
Bovine liver
-Galactosidase
Coffee bean
-Galactosidase
Bovine liver
-Mannosidase
Jack bean
222
+
(
2.2 cm ꢁ 56 cm, NH
4
form) with H
2
O as eluant and/or Dowex 1-
a
b
a
b
a
ꢀ
X2 (OH form) with H
2
O as eluant to give 1-deoxynojirimycin
8
0.5
NI
(20 mg), 1-deoxymannojirimycin (11 mg), DMDP (366 mg), and
the DIL glucoside (4) (241 mg).
NI
NI
NI
NI
39
NI
0
0
0
0
0
0
3
.4. (1S,2S,Z)-1-((2 R,3 R,4 S,5 R)-3 -O-
a
-
D
-glucopyranosyl-4 -
-Mannosidase
Snail
0
hydroxy-5 -(hydroxymethyl)pyrrolidin-2-yl)undec-4-ene-1,2,11-triol
-L-Fucosidase
1
Bovine epididymis
98
Colorless syrup; [
a
]
D
+ 11.8 (c 1.89, H
2
O); H NMR (500 MHz,
a
Ref.: Mercer et al. (2009).
D
2
O)
d
/ppm 5.55–5.60 (1H, m, H-5), 5.39–5.44 (1H, m, H-4), 4.53
b
00
0
NI: no inhibition (less than 50% inhibition at 1000
mM).
(1H, d, J = 7.9 Hz, H-1 ), 4.29 (1H, t, J = 4.1 Hz, H-4 ), 4.23 (1H, dd,
J = 4.1, 7.3 Hz, H-3 ), 3.85 (1H, d, J = 12.6 Hz, H-6 a), 3.75 (1H, dd,
J = 6.3, 11.4 Hz, H-6 a), 3.75 (1H, m, H-2), 3.71 (1H, dd, J = 4.4,
12.6 Hz, H-6 b), 3.62 (1H, dd, J = 6.6, 11.4 Hz, H-6 b), 3.55 (2H, t,
J = 6.3 Hz, H-11), 3.51 (1H, dd, J = 3.8, 6.3 Hz, H-1), 3.38–3.48 (3H,
0
0
0
Previously, a variety of 1,4-dideoxy-1,4-imino-
D
-lyxitol (DIL)
00
0
derivatives with long-side chains have been isolated from B.
kajinoki (Moraceae) and were designated as broussonetines A, B, Q,
and V (Shibano et al., 1997, 2000; Tsukamoto et al., 2001).
Interestingly, all these broussonetines were characterized with C13
00
00
00
0
m, H-3 , 4 , 5 ), 3.34 (1H, dd, J = 6.3, 7.4 Hz, H-2 ), 3.29 (1H, dd,
J = 7.9, 9.1 Hz, H-2 ), 3.23 (1H, ddd, J = 4.1, 6.3, 6.6 Hz, H-5 ), 2.21–
00
0
side chains at C-1
has a C11 side chain. B. kajinoki (Moraceae) and A. triphylla
Campanulaceae) belong to quite unrelated families. Thus, we need
more investigation of the biosynthetic pathways of these plant
families.
a
position of DIL, while our isolated new alkaloid
2.37 (2H, m, H-3), 1.95–2.08 (2H, m, H-6), 1.47–1.55 (2H, m, H-10),
1
3
4
(
1.23–1.38 (6H, m, 3x CH
2 2
-7, 8, 9); C NMR (126 MHz, D O) d/ppm
00
0
00
133.5 (C-5), 124.9 (C-4), 102.8 (C-1 ), 83.4 (C-3 ), 75.7 (C-3 ), 75.7
00 00 0 00
(C-5 ), 73.5 (C-1), 72.9 (C-2 ), 72.1 (C-4 ), 71.0 (C-2), 69.3 (C-4 ),
00
0
0
0
61.8 (C-11), 60.5 (C-6 ), 60.4 (C-2 ), 60.2 (C-6 ), 60.1 (C-5 ), 31.2 (C-
The IC50 values of compound 4 and DIL toward various
glycosidases are shown in Table 1. We have previously reported
3), 30.8 (C-10), 28.8 (C-8), 28.6 (C-7), 26.7 (C-6), 24.0 (C-9).
+
HRFABMS m/z 496.2763 [M+H] (C22
H
42NO11 requires 496.2759).
that DIL was a potent inhibitor of coffee bean
moderate inhibitor of jack bean -mannosidase, with IC50 values of
.5 and 39 M, respectively (Mercer et al., 2009). Compound 4
gave no inhibition toward rice -glucosidase, almond -glucosi-
dase, and jack bean -mannosidase, while it still kept strong
galactosidase inhibition, with IC50 value of 8 M.
a-galactosidase and a
a
3
.5. Enzyme assays
0
m
a
b
The enzymes
yeast, assayed at pH 6.8),
pH 5.0), -galactosidase (from coffee bean, pH 6.5),
dase (from bovine liver, assayed at pH 6.8), -mannosidase (from
Jack bean, pH 4.5), -mannosidase (from snail), -fucosidase
from bovine kidney), p-nitrophenyl glycosides, and disaccharides
a
-glucosidase (from rice, assayed at pH 5.0; from
-glucosidase (from almond, assayed at
-galactosi-
a
a-
b
m
a
b
a
b
a-L
3
. Experimental
(
were purchased from Sigma–Aldrich Chemical Co. (St. Louis, Mo.
USA). Glycosidase activities were determined using an appropriate
p-nitrophenyl glycoside as substrate. The reaction was stopped by
3
.1. General
The purity of samples was checked by HPTLC on silica gel 60F254
E. Merck) using the solvent system PrOH–AcOH–H O (4:1:1), and
2 3
adding 400 mM Na CO . The released p-nitrophenol was measured
(
2
spectrometrically at 400 nm.
a chlorine-o-tolidine reagent or iodine vapor was used for
detection. Optical rotations were measured with a Jasco DIP-370
digital polarimeter (Tokyo, Japan). Structure elucidation was
References
1
carried out using NMR (Bruker DRX500, 500 MHz) for full
H
13
and C assignments. Chemical shifts were expressed in ppm
downfield from tetramethylsilane in D O as an internal standard.
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medicine shop (Uchida Wakanyaku Co., Tokyo, Japan) in July 2008.
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herbarium of the Institute of Biological, Environmental and Rural
Sciences, Aberystwyth, UK.