α-amylase and lipase inhibitory activity and structural characterization of acacia bark proanthocyanidins

Article abstract of DOI:10.1021/np100372t

The bark extract of Acacia mearnsii showed strong lipase and α-amylase inhibition activities. Fractionation of the extract by column chromatography and subsequent ¹³C NMR and MALDI-TOF-MS analysis revealed that the active substances are proanthocyanidin oligomers mainly composed of 5-deoxyflavan-3-ol units. In addition, 4′-O- methylrobinetinidol 3′-O-β-d-glucopyranoside, fisetinidol-(4α, 6)-gallocatechin, and epirobinetinidol-(4β,8)-catechin were isolated as new compounds, and their structures were determined from spectroscopic data. Furthermore, a modified thiol degradation method using strongly acidic conditions was applied to the extract to yield three thiol degradation products derived from robinetinidol units. This method is useful for characterizing acacia proanthocyanidins (wattle tannins).

Full text of DOI:10.1021/np100372t

ARTICLE
pubs.acs.org/jnp
R-Amylase and Lipase Inhibitory Activity and Structural
Characterization of Acacia Bark Proanthocyanidins
Rie Kusano,† Sosuke Ogawa,‡ Yosuke Matsuo,† Takashi Tanaka,*,† Yoshikazu Yazaki,§ and Isao Kouno†
†Laboratory of Natural Product Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi,
Nagasaki 852-8521, Japan
‡Mimozax Co. Ltd., 4291-1 Miyauchi, Hatsukaichi, Hiroshima 738-0034, Japan
§Department of Chemical Engineering, Monash University, Wellington Road, Clayton, Victoria 3800, Australia
S Supporting Information
b
ABSTRACT: The bark extract of Acacia mearnsii showed strong
lipase and R-amylase inhibition activities. Fractionation of
the extract by column chromatography and subsequent 13C NMR
and MALDI-TOF-MS analysis revealed that the active substances
are proanthocyanidin oligomers mainly composed of 5-deoxyflavan-
3-ol units. In addition, 40-O-methylrobinetinidol 30-O-β-D-glucopyr-
anoside, fisetinidol-(4R,6)-gallocatechin, and epirobinetinidol-
(4β,8)-catechin were isolated as new compounds, and their structures were determined from spectroscopic data. Furthermore, a
modified thiol degradation method using strongly acidic conditions was applied to the extract to yield three thiol degradation products
derived from robinetinidol units. This method is useful for characterizing acacia proanthocyanidins (wattle tannins).
cacia mearnsii of the family Fabaceae, commonly called
“black wattle”, is a tree native to Australia. The bark is rich
In contrast, the acacia proanthocyanidins are resistant to thio-
lysis, making the chemical characterization of these oligomers
difficult. This paper presents a modified thiolysis method that
can discriminate acacia proanthocyanidin oligomers from other
proanthocyanidins.
A
in condensed tannins (syn. proanthocyanidins) known as “wattle
tannins”,1-4 which are industrially used for particleboard adhe-
sives and leather tanning. Proanthocyanidins are known to have a
variety of health benefits, including antitumor effects,5 hairgrowth
promotion,6 antihypertension,7 and antiallergic properties.8 Antio-
xidative activity9 and tyrosinase inhibitory activity10 have been
reported for acacia proanthocyanidins. Recently, antidiabetes
and antiobesity activities of acacia bark extract have been demon-
strated in mouse models.11 Since proanthocyanidins constitute
68 wt % of bark extract, acacia proanthocyanidins are a readily
available and promising food additive with health benefits.
Gastrointestinal absorption of monomeric flavan-3-ols and
proanthocyanidin dimers, such as catechin and procyanidin B-2,
has also been reported;12 however, the absorption of proantho-
cyanidin oligomers is known to be very low.13 Therefore, the in
vivo biological activities of proanthocyanidin oligomers, such as
suppression of sugar and lipid uptake, are believed to be mainly
attributable to the inhibition of digestive enzymes in the gastro-
intestinal tract.14-16 In this study, we evaluated the effect of
acacia proanthocyanidins on two digestive enzymes, R-amylase
and lipase, and then characterized the active fractions of the bark
extract using spectroscopic and chemical methods. It is known
that the acacia proanthocyanidin oligomers are mainly composed
of 5-deoxyflavan-3-ol units, such as robinetinidol (1) and fiseti-
nidol (2) (Figure 1),17 and their chemical properties are different
from those of many other proanthocyanidins. The interflavan
linkages of proanthocyanidins composed of 5-hydroxyflavan-3-ol
units are readily cleaved by acid-catalyzed thiolysis.18,19 This
reaction is commonly used for their structural characterization.
’ RESULTS AND DISCUSSION
Measurement of r-Amylase and Lipase Inhibitory Effects.
Acacia bark extract showed strong R-amylase inhibition activity
(73.7 ( 3.8% inhibition at 250 μg/mL), which is comparable to
that of black tea (68.5 ( 4.6%) and much stronger than that of
green tea (21.0 ( 3.7%), oolong tea (10.9 ( 2.7%), or guava leaf
(32.4 ( 9.5%) extracts. To identify the active substance, the
acacia bark extract was separated into three major fractions by
column chromatography over Diaion HP20SS resin, and inhibi-
tion activities (IC50 values) of the fractions were compared
(Table 1). Adjusting for yields, the second fraction (Fr. 2) had
the highest activity (86.0% at 250 μg/mL). Fraction 2 was further
separated into four fractions by size-exclusion column chroma-
tography using Sephadex LH-20 eluted with aqueous acetone
containing a high concentration of urea.20 Although three frac-
tions (212, 221, and 222) showed strong activities (92.8, 89.2,
and 65.8% inhibition, respectively, at 250 μg/mL), fraction 221
was obtained with the highest yield and was therefore further
fractionated by Sephadex LH-20 column chromatography to
afford the most active fraction, 2217. The R-amylase inhibition
activity of fraction 2217 was stronger than that of robinetinidol
Received: June 3, 2010
Published: December 30, 2010
r
Copyright
2010 American Chemical Society and
119
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American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
Figure 1. Structures of compounds 1-17.
Table 1. R-Amylase Inhibitory Activity and Yield of Fractions
Table 2. Lipase Inhibitory Activity
Fr. no.
IC50 (μg/mL)
yield (%)a
Fr. no.
inhibition (%)a
1
>500
68.4
20.6
70.4
1.8
1
0.3 ( 0.7
79.6 ( 11.5
69.3 ( 8.7
22.7 ( 7.3
71.3 ( 8.9
87.6 ( 7.9
86.4 ( 9.1
28.4 ( 0.8
64.1 ( 4.0
58.7 ( 8.2
69.1 ( 6.4
75.1 ( 6.0
87.9 ( 6.4
82.8 ( 1.8
2
2
3
80.1
3
211
212
221
222
2211
2212
2213
2214
2215
2216
2217
>500
60.9
0.6
211
16.1
44.4
7.5
212
60.8
221
83.7
222
>500
>500
>500
335.0
58.8
0.6
2211
0.9
2212
1.8
2213
4.1
2214
2215
7.1
52.6
8.8
2216
38.0
15.9
2217
a At 80 μg/mL.
robinetinidol (1)
137.5
368.5
robinetinidol-(4R,8)-catechin (10)
a Yield calculated from the original extract.
at 80 μg/mL), and subfractions 2215, 2216, and 2217 obtained
from fraction 221 exhibited strong inhibitory effects (75.1, 87.9,
and 82.8%, respectively, at 80 μg/mL). Adjusting for yield,
fraction 2217 exhibited the highest lipase inhibition activity. Frac-
tion 2217 was qualitatively characterized as containing proantho-
cyanidins by its characteristic coloration following the addition of
FeCl3 (dark blue) and vanillin-HCl (red). HPLC analysis of the
fraction provided two broad humps; there were no sharp peaks
arising from flavan-3-ols or proanthocyanidin dimers (see Sup-
porting Information), suggesting that this fraction is a complex
mixture of proanthocyanidin oligomers with relatively high mole-
cular weights.
and robinetinidol-(4β,8)-catechin,1-4 which are known acacia
polyphenols (Table 1). The activity was also compared to the
activities of other R-amylase inhibitors. The activity of fraction
2217 (92.4 ( 1.9% inhibition at a concentration of 250 μg/mL)
was comparable to that of epigallocatechin 3-O-gallate
(77.7 ( 6.5%) from green tea15 and theaflavin 3,30-di-O-gallate
(88.7 ( 2.5%) from black tea15 and stronger than the activities of
sanguiin H-6 (65.3 ( 5.4%) from Chinese sweet tea21 and rasp-
berry.14 R-Amylase inhibition activities of the acacia proantho-
cyanidin fractions roughly paralleled their inhibition effects on
lipase (Table 2). Fractions 212, 221, and 223 showed high lipase
inhibition activity (71.3, 87.6, and 86.4% inhibition, respectively,
Identification of Low Molecular Weight Constituents.
The extract was chromatographically separated on several different
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Journal of Natural Products
ARTICLE
two rotamers exist in equilibrium, as evidenced by strong
NOESY exchange peaks between equivalent protons of each
rotameric pair.36 The large coupling constants (J2C,3C = J3C,4C
=
9.5 Hz, J2F,3F = 6.8 Hz) of the C and F rings indicated 2,3- and
3,4-trans configuration of the C-ring and a 2,3-trans configuration
of the F-ring; this was supported by the 13C NMR chemical
shifts.27 The HMBC correlations of H-2 (C) with C-6 (B) and
H-2 (F) with C-2 (E) and C-6 (E) indicated that this compound
is composed of fisetinidol and gallocatechin units (see Support-
ing Information). In the HMBC spectrum, the carbon at δ 155.7
[C-8a (D)] was correlated with aromatic singlet signals of the
D-ring (δ 5.92 and 6.08) and H-2 (F); therefore, the carbon
signal was assigned to C-8a (D). This observation indicated that
the aromatic proton is located at C-8 (D), and thus the gallo-
catechin unit is attached at C-6 of the fisetinidol unit (Figure 2).
In addition, compound 12 showed a negative CE at 236 nm,
indicating an R-orientation of the interflavan linkage.37 There-
fore, 12 was determined to be fisetinidol-(4R,6)-gallocatechin.
HRFABMS analysis showed the molecular formula of 13 to be
C30H26O12, indicating that 13 is an isomer of 12. The 1H NMR
spectrum measured at room temperature exhibited broad peaks
due to rotational hindrance, as observed for 12. However, the
spectrum measured at -20 °C showed sharp peaks arising from
two flavan-3-ol units (see Supporting Information). The terminal
unit was shown to be catechin, based on the C-ring coupling
constant of H-2 (F) (5.1 Hz) and the appearance of a long-range
1H-1H correlation between H-2 (F) and H-2 (E) in the 1H-1H
Figure 2. Selected HMBC correlations for compound 12.
columns to identify the components. Sixteen compounds, including
a new flavan-3-ol glycoside (3) (Figure 2) and two new pro-
anthocyanidin dimers (12 and 13), were isolated. The known com-
pounds were identified as robinetinidol (1),22 syringic acid (4),23
gallocatechin (5),24 catechin (6),24 taxifolin (7),25 butin (8),26
robinetinidol-(4R,8)-gallocatechin (9),27 robinetinidol-(4R,8)-
catechin (10),27 fisetinidol-(4R,8)-catechin (11),27 1,6-di-O-
galloyl-β-D-glucose (14),28 4-hydroxy-2-methoxyphenyl 1-O-β-
D-glucopyranoside (15),29 3,5-dimethoxy-4-hydroxybenzyl alco-
hol 4-O-β-D-glucopyranoside (16),30 and multifidol glucoside
(17)31,32 (Figure 1). The identifications were made by compar-
ing the 1H and 13C NMR spectra of the isolated compounds with
those of authentic samples or with literature data. Fisetinidol (2),
a constituent unit of profisetinidins, was not isolated in this
experiment.
1
COSY spectrum. Long-range H-1H coupling was also ob-
Compound 3 showed an [M]þ peak at m/z 466.1471 in
HRFABMS analysis, confirming its molecular formula to be
served between H-5 (A) and H-4 (C) protons. The HMBC
spectrum showed correlations of C-8a (D) (δ 155.4) with H-4
(C) of the extension unit, as well as correlations with H-2 (F) and
H-4 (F). These observations revealed the C-4-C-8 linkage
between two flavan units in 13. The configuration of the C-ring
was deduced to be 2,3-cis and 3,4-trans on the basis of compar-
1
C22H26O11. The H NMR spectrum was related to that of
robinetinidol (1) and showed ABX-type signals at δ 6.29 (1H,
d, J = 2.4 Hz), 6.35 (1H, dd, J = 2.4, 8.3 Hz), and 6.86 (1H, d, J =
8.3 Hz), attributable to the aromatic protons of a resorcinol-type
A-ring. In addition, signals of methylene protons at δ 2.67 (1H, d,
J = 7.8, 15.6 Hz) and 2.84 (1H, d, J = 4.9, 15.6 Hz) and two
oxygenated methine protons at δ 4.03 (1H, m) and 4.69 (1H, d,
J = 6.6 Hz) indicated that 3 is a 5-deoxyflavan-3-ol with a 2,3-trans
configuration. The appearance of mutually m-coupled aromatic
protons at δ 6.59 and 6.68 (each d, J = 1.8 Hz) suggested that the
B-ring is an asymmetric pyrogallol ring. In addition to these
signals, one methoxy proton signal at δ 3.83 (3H, s) and a sugar
anomeric proton signal at δ 4.91 (1H, d, J = 7.5 Hz), accom-
panied by oxygenated methine proton signals in the range
δ 3.35-3.75, indicated the presence of a methoxy group and a
sugar moiety. The sugar was determined to be β-D-glucose on the
basis of its 13C NMR chemical shifts33 and the results of acid
hydrolysis.34 The glucose and methoxy groups were located at
C-30 and C-40 of the B-ring, respectively, on the basis of the
HMBC correlations of the methoxy protons with C-40 (δ 137.3),
and the anomeric proton with C-30 (δ 151.4) (see Supporting
Information). As for absolute configuration, the circular dichro-
ism (CD) spectrum of compound 3 showed a negative Cotton
effect (CE) at 284 nm, indicating a 2R configuration.35 Thus,
compound 3 was established to be 40-O-methylrobinetinidol 30-
O-β-D-glucopyranoside (Figure 1).
ison of the coupling constants of the C-ring protons (J2,3 = J3,4
2.5 Hz) with those of epifisetinidol-(4β,8)-catechin (J2,3
=
=
2.5 Hz, J3,4 = 3.5 Hz).37 This was confirmed by the NOESY
correlations between H-2 (C) and H-3 (C) and between H-4 (C)
and B-ring H-2, H-6. In addition, the CD spectrum of compound
13 showed a positive CE at 239 nm, being consistent with the
β-orientation of the interflavan linkage.37 Therefore, 13 was
concluded to be epirobinetinidol-(4β,8)-catechin (13).
Spectroscopic Analysis of the Active Fraction. Fraction
2217, which exhibited strong enzyme inhibition activities, was
examined by 13C NMR spectroscopy and MALDI-TOF-MS.
The 13C NMR spectrum of the fraction was compared with that
of 10 (Figure 3).27,38 The appearance of signals attributable to
resorcinol-type A-ring, pyrogallol-type B-ring, catechol-type
E-ring, and hydroxypyran C- and F- rings indicated that fraction
2217 contains a mixture of oligomers composed of robinetinidol
(major) and fisetinidol (minor) units. The chemical shifts of C-2
(C), C-2 (F), C-3 (C), C-3 (F), and C-4 (C) were similar to
those of 10 and 12, indicating that the configuration of the C-ring
is mainly 2,3-trans and 3,4-trans. No signals corresponding to D-6
and F-4a of the phloroglucinol-type aromatic ring of 10 were
detected, suggesting that the terminal units of the oligomers are
5-deoxyflavan-3-ols.
HRFABMS analysis of compound 12 provided an [M þ Na]þ
The MALDI-TOF-MS spectrum of fraction 2217 showed
a series of peaks arising from proanthocyanidin oligomers at
m/z 1177, 1465, 1753, 2041, and 2329 (tetramer to octamer)
(Figure 4). The distance between the major peaks was 288 Da,
which coincides with the mass of a robinetinidol unit. The major
peak at m/z 601, consistent with the molecular formula
1
C30H26O12 and indicating that 12 is an isomer of 10. The H
and 13C NMR spectra of 12 showed duplicated signals arising
from two conformational isomers of a proanthocyanidin dimer
created by rotational hindrance at the interflavan linkage. The
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Figure 3. 13C NMR spectra of the active fraction Fr. 2217 (A) and robinetinidol-(4R,8)-catechin (10) (B) measured at 100 MHz in methanol-d4.
Figure 4. MALDI-TOF-MS spectrum of Fr. 2217.
peaks were accompanied by peaks 16 Da smaller or larger arising
from molecules with fisetinidol or gallocatechin units. These
results are similar to the mass spectra of the original extracts,
except the peaks arising from trimers (m/z 905 corresponding to
the [M þ K]þ of trimers) were much larger in the original
Figure 5. HPLC of the active fraction 2217 (A), the thiol degradation
products of the active fraction under common reaction conditions (B),
and the thiol degradation products under modified reaction conditions
(C) (18-23: see Figure 6, ꢀ indicates peaks also observed in the blank).
extracts.
Size-Exclusion High-Performance Chromatography of the
Active Fraction. Size-exclusion high-performance chromatog-
raphy of fraction 2217 suggested that the number average molec-
ular weight (Mn) and weight average molecular weight (Mw) are
1185 and 1556, respectively. The Mn value corresponded with
the molecular weight of proanthocyanidin tetramer composed of
robinetinidol and is consistent with the MALDI-TOF-MS data.
Thiolysis of the Extract. Thiol degradation is commonly
used for characterization of proanthocyanidin oligomers com-
posed of 5-oxygenated flavan-3-ols units.19 Substitution reaction
with thiol groups at the C-4 position under moderately acidic
conditions cleaves interflavan linkages to produce catechins from
the terminal units and 4-thioethers from the extension units.
However, the method is not effective for proanthocyanidins with
5-deoxyflavan-3-ol units because the 4-8 (or 6) interflavan
linkages are stable and resist the substitution reaction.18 Accord-
ingly, we attempted thiolysis reactions of fraction 2217 under
strongly acidic conditions (HSCH2CH2OH in EtOH containing
0.75 mol/L of HCl, 80 °C for 7 h) (Figure 5). Although not
quantitative, the degradation reaction proceeded gradually, and
three characteristic products, 18-20, derived from a 5-deoxy-
flavan-3-ol were obtained, as well as three from catechin and
epigallocatechin (Figure 6). Acid thiolysis of the active fraction
and the original extract provided similar results; therefore, due to
the limited quantity of the active fraction, characterization of the
products was conducted using the extract.
Product 18 exhibited an [M þ Na]þ peak at m/z 389,
indicating its molecular weight is 366. On the basis of HRFABMS
data, the molecular formula was determined to be C17H18O7S,
which is consistent with a structure composed of the -SCH2-
CH2OH group and robinetinidol. The 1H NMR spectrum
(Table 3) showed signals of the SCH2CH2OH moiety and a
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Figure 6. Structures of the thiol degradation products.
1
Table 3. H (500 MHz) and 13C (125 MHz) NMR Data of 18-20 in Methanol-d4
18
19
20
position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
4.39, brs
δC
1
4.24, dd (1.1, 4.6)
4.30, dd (1.6, 4.6)
4.58, d (1.6)
56.1
81.2
3.96, d (3.4)
4.33, t (3.4)
4.42, d (3.4)
57.8
88.5
54.7
82.6
2
4.43, d (4.7)
3
50.0
49.5
4.84, brd (4.7)
49.5b
136.7
104.8
147.7
133.0
144.2
120.0
116.9
158.1
103.7
158.3
107.4
133.6
35.2
3a
121.7
143.8
133.9a
146.7
104.7
134.0a
119.5
157.1
103.4
158.0
107.2
128.9
35.1
121.9
147.1
134.2
143.3
104.8
133.4
121.1
156.7
103.2
157.8
107.5
130.6
34.7
4
5.98, d (1.1)
5
6
7
6.49, d (1.1)
6.33, d (2.5)
6.46, s
7a
10
20
30
40
50
60
6.32, d (2.5)
6.32, d (2.5)
6.10, dd (2.5, 8.6)
6.27, d (8.6)
6.16, dd (2.5, 8.2)
6.66, d (8.2)
6.26, dd (2.5, 8.2)
6.92, d (8.2)
SCH2
CH2OH
2.66-2.76, m
3.67, t (6.6)
2.65-2.78, m
3.67, t (7.0)
2.73-2.84, m
3.72-3.81, m
62.8
62.7
62.9
a Assignments may be interchanged in the column. b Overlapped with solvent signal.
of a -CH-CH-CH- partial structure. In addition, allylic
1H-1H couplings of the pyrogallol-ring proton H-7 with the
methine proton H-1, and the resorcinol H-60 with the methine
proton H-3, were observed. The HSQC spectrum revealed that
H-2 was attached to an oxygenated carbon (δ 81.2). In the
HMBC spectrum, H-1 was correlated to C-7, C-7a, and C-3a,
confirming that C-1 was attached to the pyrogallol C-7a.
Correlation of H-1 with a methylene carbon of the mercap-
toethanol moiety was also observed. Furthermore, the HMBC
correlations of H-3 with C-7a, C-3a, C-4, C-20, and C-60 indicated
that the methine carbon C-3 was connected to the pyrogallol
C-3a and to the resorcinol C-10 (Figure 7). Accordingly, the
structure of 18 was determined to be as shown in Figure 6. The
relative configuration was determined by a NOESY experiment
(Figure 7). The NOE correlations of H-1 with H-7 and H-60,
and H-2 with the methylene protons of the mercaptoethanol
moiety, revealed 1,2-trans and 2,3-cis configurations. The abso-
lute configuration was established by CD spectroscopic
analysis,39 which showed a positive CE at 278 nm and a negative
CE at 244 nm. Positive CE at lower energy (longer wavelength)
indicated two aromatic rings oriented with positive helicity,
Figure 7. Selected HMBC and NOESY correlations for 18 and 20.
set of ABX-type aromatic signals [δ 6.33 (d, J = 2.5 Hz, H-30),
6.10 (dd, J = 2.5, 8.6 Hz, H-50), and 6.27 (d, J = 8.6 Hz, H-60)]
attributable to a 1,2,4-trisubstituted benzene ring (A-ring). In
addition, a singlet signal at δ 6.49 was assigned to a proton (H-7)
attached to a pyrogallol ring (B-ring) based on the HMBC
1
correlations with aromatic carbons (Figure 7). The H-1H
COSY correlations of the remaining aliphatic methine protons
resonated at δ 4.24 (dd, J = 1.1, 4.6 Hz, H-1), 4.30 (dd, J = 1.6,
4.6 Hz, H-2), and 4.58 (d, J = 1.6 Hz, H-3), showing the presence
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Figure 8. Possible mechanism for the production of 18-20.
corresponding to the R configuration at C-3 (see Supporting
Information). Therefore, the structure of product 18 was deter-
mined to be as shown in Figure 6.
zenetriol, obtained as a byproduct of thiol degradation with
R-toluenethiol.40 On the basis of the HMBC, HSQC, and 1H-1H
COSY spectra, the structures of these two products were deter-
mined to be 2-hydroxyethylthio analogues, as shown in Figure 6.
The configuration of the benzylic methine carbon of these prod-
ucts was not determined. The production of these compounds
indicated the presence of catechin at the terminal units. Product
23 was identified as 4β-(2-hydroxyethylsulfanyl)epigallocatechin
by comparing the sample's spectroscopic data with those of an
authentic sample.41
Thiol degradation of 10 under similar conditions also yielded
18, 19, and 20. On the basis of the results, a mechanism for the
generation of 18 and 19 from the 5-deoxyflavan-3-ol extension
units of proanthocyanidins is proposed as shown in route a of
Figure 8. In contrast, the mechanism for the production of 20 is
not clear. In a possible mechanism (route b in Figure 8), an
intermediate with two thioether groups is produced, and sub-
sequent elimination of one of the ethylthio groups accompanied
by migration of a resorcinol moiety produce the product 20.
McGraw et al. reported that products with an indane skeleton
related to 18-20 were produced from typical proanthocyanidins
composed of 5-hydroxyflavan-3-ols upon extended thiol degra-
dation (for 24 to 72 h).42,43 They proposed a mechanism that
begins with cleavage of the carbon-carbon bond between C-4
and C-4a, which is different from the mechanism for proantho-
cyanidins with 5-deoxyflavan-3-ol units.
HRFABMS indicated that the molecular formula of product
19 was the same as that of 18, and the 1H and 13C NMR spectra
were also closely related to those of 18 (Table 3). The 1H-1H
COSY, HSQC, and HMBC spectra indicated that the planar
structure of 19 was identical to that of 18. However, differences
were observed in the NOESY spectrum, which showed cross-
peaks between the resorcinol H-60 and methylene protons of the
mercaptoethanol moiety, indicating that these two groups are
located on the same side of the molecule. Furthermore, H-60 also
showed an NOE correlation with H-2, confirming the 1,2-trans
and 2,3-trans configuration. The CD spectrum resembled that of
18 and showed a positive CE at 269 nm and a negative CE at 243 nm.
Therefore, the configuration of C-3 was concluded to be R,
and the structure of 19 was established to be as illustrated in
Figure 6.
Product 20 was shown to be an isomer of 18 and 19 by
1
its HRFABMS and H and 13C NMR spectra (Table 3). An
1
important difference in the H-1H COSY spectrum of 20
compared to that of 19 was the appearance of an allylic coupling
(J = 1.1 Hz) between the benzylic H-3 and a pyrogallol aromatic
proton (δ 5.98, H-4). In addition, the HMBC spectrum showed a
long-range coupling between H-4 and C-3 (Figure 7). Moreover,
the NOESY correlations of the pyrogallol methine proton H-4
with H-3 and the resorcinol H-60 were observed. These observa-
tions indicated that C-4 of the pyrogallol ring is a methine car-
bon. Consistent with this, the HMBC spectrum showed another
benzylic methine H-1 (δ 4.39) correlated both with the C-7 (δ
144.2) bearing a hydroxy group and the methylene carbon of the
mercaptoethanol moiety. NOESY correlations of H-1 and the
resorcinol ring proton, and H-2 with H-3 and the mercaptoetha-
nol methylene protons, indicated that the relative configuration
of the five-membered ring of 20 is the same as that of 18. A
positive CE at 288 nm and a negative CE at 246 nm in the CD
spectrum indicated that the absolute configuration of 20 is the
same as that of 18 and 19. Therefore, the structure of compound
20 was concluded to be as shown in Figure 6.
In conclusion, proanthocyanidins from the bark of A. mearnsii
exhibited strong inhibitory activities toward R-amylase and
lipase. The most active fraction was characterized by spectro-
scopic and chemical methods and shown to contain tetrameric to
octameric compounds mainly composed of robinetinidol units.
The R-amylase and lipase inhibitory activities of these oligomeric
proanthocyanidins suggest that they may be a promising func-
tional food material for suppressing sugar and lipid uptake. In this
study, we also developed a chemical method for characterization
of proanthocyanidins with robinetinidol extension units.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Ultraviolet (UV) spectra
were obtained with a Jasco V-560 UV/vis spectrophotometer. The CD
spectra were measured with a Jasco J-725N spectrophotometer. 1H and
13C NMR spectra were recorded in methanol-d4 or acetone-d6 with a
Varian Unity Plus 500 spectrometer operating at 500 MHz for 1H and
125 MHz for 13C and with a JEOL JNM-AL 400 spectrometer operating
Compounds 21 and 22 showed signals arising from phlor-
oglucinol, catechol, mercaptoethanol, one methylene, and two
methine groups in the 1H and 13C NMR spectra. These spectro-
scopic features are closely related to those of 2-[3-(3,4-dihydro-
xyphenyl)-2-hydroxy-3-[(phenylmethyl)thio]propyl]-1,3,5-ben-
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1
at 400 MHz for H and 100 MHz for 13C. MS were recorded on a
Fractionation of the Acacia mearnsii Extract. Dried extract
of A. mearnsii bark (50 g) was first fractionated by Diaion HP20SS
column chromatography (5.5 cm i.d. ꢀ 25 cm) with H2O, and the eluate
was monitored by TLC. Elution of the column with H2O gave Fr. 1,
which contained sugars (10.3 g). Further elution of the column with 10-
100% aqueous MeOH yielded Fr. 2, containing polyphenols (35.3 g),
and Fr. 3, containing nonpolar compounds (0.9 g). Fr.2 was dissolved in
a small amount of 6 M urea-acetone mixture (adjusted to pH 2 with
HCl) (2:3, v/v), applied to a Sephadex LH-20 column (8 ꢀ 60 cm), and
eluted with 6 M urea-acetone pH 2 solution to give two fractions: Fr.
21, which contained relatively high molecular weight polyphenols, and
Fr. 22, which contained lower molecular weight polyphenols. After
evaporation of the acetone, the fractions were separately applied to an
MCI-gel CHP20P column (4.5 cm i.d. ꢀ 25 cm). The HCl and urea
were eluted with H2O, and subsequent elution with H2O containing an
increasing concentration of MeOH (0-100% MeOH, 10% stepwise
elution) furnished Fr. 211 (0.3 g) and Fr. 212 (8.1 g) from Fr. 21, and Fr.
221 (22.2 g) and Fr. 222 (3.7 g) from Fr. 22. Fr. 221 was further
fractionated by Sephadex LH-20 column chromatography (5 cm i.d. ꢀ
25 cm) with EtOH containing increasing proportions of water (0-40%
H2O, 20% stepwise elution), and then elution with 60% aqueous
acetone, to give seven fractions: Fr. 2211 (0.31 g), Fr. 2212 (0.48 g),
Fr. 2213 (0.95 g), Fr. 2214 (2.19 g), Fr. 2215 (3.83 g), Fr. 2216 (4.73 g),
and Fr. 2217 (8.61 g) (the separation scheme is shown in the Supporting
Information).
Isolation of Compounds. Dried extract of A. mearnsii bark (100 g)
was dissolved in 500 mL of H2O and successively partitioned with
Et2O and EtOAc. The Et2O layer (1.13 g) was subjected to Sephadex
LH-20 (3 cm i.d. ꢀ 28 cm) column chromatography with 60-100%
MeOH in H2O (10% stepwise elution, each 200 mL) and then with 60%
aqueous acetone to give seven fractions: E-1-E-7. Fr. E-1 was succes-
sively subjected to Sephadex LH-20 and Cosmosil 75C18-OPN (2 cm
i.d. ꢀ 20 cm) column chromatography with 0-40% aqueous MeOH
(5% stepwise elution, each 100 mL) to afford syringic acid (4) (3.2
mg)23 and multifidol glucoside (17) (18.5 mg).31,32 Crystallization of Fr.
E-3 and E-5 from H2O yielded robinetinidol (1) (87.3 mg)22 and
catechin (6) (29.0 mg),24 respectively. Fr. E-6 was subjected to Cosmosil
75C18-OPN (2 cm i.d. ꢀ 20 cm) column chromatography with 0-40%
aqueous MeOH (5% stepwise elution, each 100 mL) to afford butin
(8)26 (9.5 mg, eluted with 15% aqueous MeOH) and taxifolin (7)25
(8.6 mg, eluted with 20% aqueous MeOH). The EtOAc layer (31.1 g)
was separated by Sephadex LH-20 (5 cm i.d. ꢀ 35 cm) column
chromatography (0-20% H2O in EtOH, 10% stepwise elution, each
500 mL) into five fractions: EA-1-EA-5. Fr. EA-3, containing flavan-3-
ols and proanthocyanidin dimers, was separated by Sephadex LH-20
(2 cm i.d. ꢀ 20 cm) column chromatography with 60-100% aqueous
MeOH (10% stepwise elution, each 200 mL) to give eight fractions, EA-
31-EA-38. Crystallization of EA-34 from H2O afforded gallocatechin
(5)24 (53.1 mg). EA-36 was subjected to MCI-gel CHP20P (3 cm i.d. ꢀ
30 cm) column chromatography with 0-50% aqueous MeOH (10%
stepwise elution, each 200 mL) to give a mixture of proanthocyanidin
dimers. Separation of the mixture by Chromatorex ODS (3 cm i.d. ꢀ 25
cm) column chromatography with 0-30% aqueous MeOH (5% step-
wise elution, each 200 mL) afforded fisetinidol-(4R,8)-catechin (11)27
(54.6 mg) and a mixture of 12 and 13. This mixture was separated by
preparative HPLC (elution program A) to yield fisetinidol-(4R,6)-
gallocatechin (12) (2.3 mg) and epirobinetinidol-(4β,8)-catechin
(13) (10.4 mg). The aqueous layer was fractionated into five fractions,
AQ-1-AQ-5, by Sephadex LH-20 (5 cm i.d. ꢀ 35 cm) column
chromatography with H2O containing increasing proportions of MeOH
(0-100%, 20% stepwise, each 300 mL), and then the column was
washed with 50% aqueous acetone. Fr. AQ-1 was subjected to MCI-gel
CHP 20P (5 cm i.d. ꢀ 35 cm, 0-80% aqueous MeOH, 10% stepwise)
and Chromatorex ODS column chromatography (2.5 cm i.d. ꢀ 20 cm,
Voyager-DE Pro MALDI-TOF spectrometer in positive linear ion
mode, with 2,5-dihydroxybenzoic acid (10 mg/mL in 50% aqueous
MeOH) used as the matrix. HRFABMS were recorded on a JMS 700N
spectrometer (JEOL Ltd., Tokyo, Japan), with m-nitrobenzyl alcohol or
glycerol used as the matrix. Column chromatography was performed
using Sephadex LH-20 (25-100 mm, GE Healthcare UK Ltd., Little
Chalfont, UK), MCI-gel CHP 20P (75-150 mm, Mitsubishi Chemical
Co., Tokyo, Japan), Diaion HP20SS (Mitsubishi Chemical Co.),
Bondapak C18 125A (Waters Co., Ltd., Milford, U.S.A.), Cosmosil
75C18-OPN (Nacalai Tesque, Inc., Kyoto, Japan), and Chromatorex
ODS (Fuji Silysia Chemical Ltd., Kasugai, Japan). The flow rate for
column chromatography was about 3 mL/min. TLC was performed on
precoated Kieselgel 60 F254 plates (0.2 mm thick, Merck) with toluene-
ethyl formate-formic acid (1:7:1, v/v) and CHCl3-MeOH-H2O
(7:3:0.5, v/v). Spots were detected using UV illumination and by
spraying with 2% ethanolic FeCl3 or 5% H2SO4 reagent followed by
heating. Analytical HPLC was performed on a Cosmosil 5C18-AR II
(Nacalai Tesque Inc.) column (4.6 ꢀ 250 mm i.d.) with gradient elution
from 4 to 30% (39 min) and 30 to 75% (15 min) of CH3CN in 50 mM
H3PO4 at 35 °C (flow rate, 0.8 mL/min; detection, Jasco photodiode
array detector MD-910). Preparative HPLC was performed on a
Cosmosil 5C18-PAQ (Nacalai Tesque, Inc.) column (20 ꢀ 250 mm)
using gradient program A: elution with 4-10% (60 min) and 10-30%
(240 min) of CH3CN (flow rate, 2 mL/min), and with gradient program
B: elution with 4-10% (60 min) and 10-20% (120 min) of CH3CN
(flow rate, 2 mL/min). Size-exclusion HPLC was performed on a TSK
gel R-3000 (TOSOH) column (7.8 ꢀ 300 mm) with N,N-dimethylfor-
mamide containing 10 mM LiCl as the mobile phase. Absorbance
measurements for the R-amylase and lipase inhibitory assays were
performed using an Emax microplate reader (Molecular Devices Inc.,
Sunnyvale, CA, U.S.A.).
Dried Extract of the Bark of Acacia mearnsii. Spray-dried
aqueous extract of A. mearnsii bark was prepared in 2007 according to
the method reported by Cutting1 and was provided by mimozax Co.,
Ltd. (Hiroshima, Japan). Briefly, the bark was chipped and extracted
with hot H2O (100 °C) for 30 min. After filtration, the filtrate was spray-
dried.
Preparation of Extracts. Green tea, black tea, oolong tea, and
guava leaf (each 2.0 g) were separately extracted with 100 mL of boiling
H2O for 5 min, and the filtrates were lyophilized. The polyphenol
fraction of guava leaf was prepared as follows. The dried leaf (2.0 g) was
extracted with 200 mL of boiling H2O for 5 min, and the filtrate was
subjected to Diaion HP20SS chromatography. After washing the
column with H2O, the polyphenols were eluted with 50% aqueous
acetone.
Chemicals. Iodine solution (0.5 mol/L) and L-cysteine methyl
ester hydrochloride were purchased from Nacalai Tesque, Inc. Pancrea-
tic R-amylase, pancreatic lipase (type II), orlistat, glyceryl trioleate, and
2,5-dihydroxybenzoic acid were purchased from Sigma (St. Louis, MO,
U.S.A.). NEFAC-test Wako, acarbose, N,N-dimethylformamide, sodium
cholate, lecithin, and soluble starch were purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). LiCl and TES [N-tris-
(hydroxymethyl)methyl-2-aminoethanesulfonic acid] were purchased
from Kishida Chemical Co., Ltd. (Osaka, Japan). Isothiocyanic acid
o-tolyl ester was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo,
Japan). (þ)-Catechin was isolated from Uncaria gambir extract. San-
guiins H-6 and H-11 were isolated from Sanguisorba officinalis and Rubus
suavissimus,21,44 respectively. Epicatechin-(4β,8,2β,7)-epicatechin-(4R,8)-
catechin and procyanidin B-1 were isolated from Vaccinium ashei.45
Epigallocatechin-3-O-gallate was isolated from commercial green tea
and recrystallized from H2O. Theaflavin-3,30-di-O-gallate was synthe-
sized by enzymatic oxidative coupling of epigallocatechin-3-O-gallate
and epicatechin-3-O-gallate.46
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0-60% aqueous MeOH, 10% stepwise) to give 4-hydroxy-2-methox-
yphenyl 1-O-β-D-glucopyranoside (15)29 (11.2 mg) and 3,5-dimethoxy-
4-hydroxybenzyl alcohol 4-O-β-D-glucopyranoside (16)30 (16.3 mg).
Fr. AQ-3 was applied to Chromatorex ODS (3 cm i.d. ꢀ 22 cm)
and Cosmosil 75C18-OPN (3 cm i.d. ꢀ 23 cm) columns and eluted
with 0-50% aqueous MeOH (5% stepwise, each 100 mL) to afford
40-O-methylrobinetinidol 30-O-β-D-glucopyranoside (3) (165.8 mg).
Successive column chromatography of Fr. AQ-5 using Diaion HP20SS
(5 cm i.d. ꢀ 22 cm), Chromatorex ODS (5 cm i.d. ꢀ 25 cm), Sephadex
LH-20 (3 cm i.d. ꢀ 25 cm), and Chromatorex ODS (3 cm i.d. ꢀ 22 cm)
furnished 1,6-di-O-galloyl-β-D-glucose (14)28 (40.6 mg), robinetinidol-
(4R,8)-gallocatechin (9)27 (22.3 mg), and robinetinidol-(4R,8)-
catechin (10)27 (312.7 mg) (the separation scheme is shown in the
Supporting Information).
(C-2F), 82.6 (C-2C), 97.4 (C-6D), 100.3 (C-4aD), 102.9 (C-8A), 104.6
(C-2, 6B), 105.8 (C-8D), 108.6 (C-6A), 113.8 (C-2E), 114.2 (C-4aA),
115.9 (C-5E), 118.2 (C-6E), 129.8 (C-5A), 131.8, 132.0 (C-1B, 1E),
132.9 (C-4B), 145.2, 145.6 (C-3E, 4E), 146.5 (C-3B, 5B), 154.5, 156.2,
156.4 (C-7A, 7D, 8aA), 155.4 (C-8aD), 157.5 (C-5D); HRFABMS m/z
601.1338 [M þ Na]þ (calcd. for C30H26O12Na, 601.1322).
Hydrolysis of 3. Compound 3 (4 mg) was dissolved in 1 M H2SO4
(0.5 mL) and heated at 100 °C for 5 h. After neutralization with
Amberlite IRA400 (OH form), the resin was removed by filtration and
the filtrate was dried in vacuo. The residue was dissolved in pyridine
(1 mL) containing L-cysteine methyl ester (10 mg) and heated at 60 °C
for 1 h. The mixture was mixed with a solution (0.5 mL) of pyridine
o-tolylisothiocyanate (10 mg) in pyridine and heated at 60 °C for 1 h.
The final mixture was directly analyzed by HPLC [Cosmosil 5C18 AR II
(250 ꢀ 4.6 mm i.d., Nacalai Tesque Inc.) with isocratic elution at 25%
(40 min) and 25-90% gradient elution (5 min) with CH3CN in 50 mM
H3PO4]. The tR of the peak at 16.9 min coincided with that of
the thiocarbamoyl thiazolidine derivative of D-glucose (the tR of the
L-diastereomer was 15.4 min).
40-O-Methylrobinetinidol 30-O-β-D-glucopyranoside (3): pale
brown, amorphous powder; [R]18 -74.0 (c 0.09, MeOH); UV
D
(MeOH) λmax (log ε) 280 (3.62) nm; CD (MeOH) Δε242 þ8.1,
Δε255 -2.6, Δε259 þ2.7, Δε284 -15.3; IR νmax 3399, 2931, 1599, 1511,
1
1453, 1348, 1158, 1079 cm-1; H NMR (methanol-d4, 400 MHz);
δ 2.67 (1H, d, J = 7.8, 15.6 Hz, H-4), 2.84 (1H, d, J = 4.9, 15.6 Hz, H-4),
3.35-3.50 (4H, m, H-200, 300, 400, 500), 3.64 (1H, dd, J = 5.1, 12.2 Hz,
H-600), 3.73 (1H, dd, J = 2.1, 12.2 Hz, H-600), 3.83 (3H, s, OMe), 4.03
(1H, m, H-3), 4.69 (1H, d, J = 6.6 Hz, H-2), 4.91 (1H, d, J = 7.5 Hz,
H-100), 6.29 (1H, d, J = 2.4 Hz, H-8), 6.35 (1H, dd, J = 2.4, 8.3 Hz, H-6),
6.59 (1H, d, J = 1.8 Hz, H-60), 6.68 (1H, d, J = 1.8 Hz, H-20), 6.86 (1H, d,
J = 8.3 Hz, H-5); 13C NMR (acetone-d6þD2O, 100 MHz) δ 33.1 (C-4),
61.1 (C-OMe), 62.1 (C-600), 67.8 (C-3), 70.8 (C-400), 74.3 (C-200), 77.4,
77.5 (C-300, 500), 82.5 (C-2), 101.7 (C-100), 103.2 (C-8), 107.3 (C-20),
109.1 (C-60), 109.7 (C-6), 112.1 (C-4a), 130.9 (C-5), 136.1 (C-10),
137.3 (C-40), 151.1 (C-50), 151.4 (C-30), 155.6 (C-7), 157.6 (C-8a);
HRFABMS m/z 466.1471 [M]þ (calcd for C22H26O11, 466.1475).
Fisetinidol-(4R,6)-gallocatechin (12): pale brown, amorphous
powder; [R]D -87.7 (c 0.06, MeOH); UV (MeOH) λmax (log ε)
281 (3.92) nm; CD (MeOH) Δε216 -267.9, Δε236 -134.9, Δε274
Measurement of r-Amylase Inhibitory Activity. The activ-
ity was measured using the method reported by Xiao et al.47 and
Yoshikawa et al.48 with slight modifications. Acarbose was used as the
positive control. Substrate solution was prepared as follows: soluble
starch (500 mg) was dissolved in 25 mL of 0.4 M NaOH and heated for 5
min at 100 °C. After cooling in ice H2O, the solution was adjusted to pH
7 with 2 M HCl, and H2O was added to adjust the volume to 100 mL.
Sample solutions were prepared by dissolving each sample in acetate
buffer (pH 6.5) to make 2, 0.2, and 0.02 mg/mL solutions. The substrate
(40 μL) and sample (20 μL) solutions were mixed in a microplate well,
and the mixtures were preincubated at 37 °C for 3 min. Then 20 μL of R-
amylase solution (50 μg/mL) was added to each well, and the plate was
incubated for 15 min. The reaction was terminated by addition of 80 μL
of 0.1 M HCl; then 200 μL of 1 mM iodine solution was added, and the
absorbances were measured at 650 nm. Inhibitory activity (%) was
calculated as follows:
1
þ15.2, Δε287 -17.6; H NMR (methanol-d4, 500 MHz) δ of two
rotational isomers 2.47, 2.61 (1H, dd, J = 7.5, 16.4 Hz, H-4F), 2.74, 2.77
(dd, J = 5.5, 16.4 Hz, H-4F), 3.73, 4.05 (1H, m, H-3F), 4.42, 4.76 (1H, d,
J = 6.8 Hz, H-2F), 4.45 (2H, m, H-2C, H-3C), 4.53 (1H, d, J = 9.5 Hz,
H-2C), 4.54, 4.62 (1H, br d, J = 9.5 Hz, H-4C), 4.65 (1H, t, J = 9.5 Hz,
H-3C), 5.92, 6.08 (1H, s, H-8D), 6.03, 6.46 (2H, s, H-2E, 6E), 6.15, 6.18
(1H, d, J = 2 Hz, H-8A), 6.20, 6.27 (1H, dd, J = 2.0, 8.7 Hz, H-6A), 6.54,
6.83 (1H, dd, J = 2.0, 8.0 Hz, H-6B), 6.61, 6.64 (1H, dd, J = 1.5, 8.7 Hz,
H-5A), 6.70, 6.76 (1H, d, J = 8 Hz, H-5B), 6.75, 6.96 (1H, d, J = 2.0 Hz,
H-2B); 13C NMR (methanol-d4, 100 MHz) δ of two rotational isomers
27.5, 28.7 (C-4F), 41.8, 42.0 (C-4C), 68.6, 68.8 (C-3F), 71.1, 71.2
(C-3C), 82.5, 82.7 (C-2F), 84.3 (C-2C), 96.0, 97.1 (C-8D), 99.9, 101.7
(C-4aD), 103.2, 103.6 (C-8A), 106.8, 107.6 (C-20E, 60E), 108.1, 108.3
(C-6D), 109.4 (C-6A), 115.9, 116.1 (C-5B), 116.2, 116.5 (C-2B), 119.6
(C-4aA), 120.8, 121.1 (C-6B), 130.0, 130.1 (C-5A), 131.3, 131.1 (C-1E),
132.6, 132.8 (C-1B), 133.5 (C-4E), 145.6, 146.1 (C-3B, 4B), 146.4, 146.8
(C-3E, 5E), 155.1 (C-5D), 155.7, 157.2 (C-8aD, 7D), 156.4 (C-7A);
HRFABMS m/z 601.1331 [M þ Na]þ (calcd for C30H26O12Na,
601.1322).
Epirobinetinidol-(4β,8)-catechin (13): pale brown, amorphous
powder; [R]D -45.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 280
(3.85) nm; CD (MeOH) Δε212 -280.8, Δε239 þ5.2, Δε278 -65.7; 1H
NMR (methanol-d4, 400 MHz at -20 °C) δ 2.56 (2H, m, H-4F), 3.98
(1H, m, H-3F), 4.20 (1H, t, J = 2.5 Hz, H-3C), 4.68 (1H, d, J = 5.1 Hz,
H-2F), 4.81 (1H, d, J = 2.5 Hz, H-4C), 5.15 (1H, d, J = 2.5 Hz, H-2C),
5.92 (1H, s, H-6D), 6.20 (2H, s, H-2B, 6B), 6.23 (1H, dd, J = 2.4, 8.5 Hz,
H-6A), 6.38 (1H, d, J = 2.4 Hz, H-8A), 6.39 (1H, dd, J = 1.8, 8.3 Hz,
H-6E), 6.53 (1H, d, J = 8.5 Hz, H-5A), 6.58 (1H, d, J = 1.8 Hz, H-2E),
6.60 (1H, d, J = 8.3 Hz, H-5E); 13C NMR (methanol-d4, 100 MHz at
-20 °C) δ 26.6 (C-4F), 31.7 (C-4C), 67.8 (C-3F), 73.2 (C-3C), 81.1
Inhibition ð%Þ ¼ f1 - ðAbs 2 - Abs 1Þ=ðAbs 4 - Abs 3Þ ꢀ 100g
where Abs 1 is the absorbance of incubated solution containing sample,
starch, and amylase, Abs 2 is the absorbance of incubated solution
containing sample and starch, Abs 3 is the absorbance of incubated
solution containing starch and amylase, and Abs 4 is the absorbance of
incubated solution containing starch.
IC50 value was determined by curve-fitting using the graphing soft-
ware DeltaGraph 5 for Windows (RockWare Inc., Golden, CO, U.S.A.).
Measurement of Pancreatic Lipase Inhibitory Activity. Li-
pase inhibitory activity was measured according to the method of
Han et al.49 with slight modifications. Orlistat was used as the positive
control. Substrate solution was prepared by sonication (10 min in an ice
bath) of a mixture of glyceryl trioleate (80 mg), lecithin (10 mg), and
sodium cholate (5 mg) suspended in 9 mL of 0.1 M TES buffer (pH 7.0).
Samples were separately dissolved in 0.1 M TES buffer to make
0.2 mg/mL solutions. The substrate (20 μL) and sample solutions
(20 μL) in microplate wells were preincubated for 3 min; then 10 μL of
lipase solution (20 μg/mL) was added to each reaction mixture and
incubated for 30 min at 37 °C. The amount of released fatty acid was
measured by a NEFAC-test Wako at 550 nm using a microplate reader.
Inhibitory activity (%) was calculated as follows:
Inhibition ð%Þ ¼ f1 - ðAbs 6 - Abs 5Þ=ðAbs 8 - Abs 7Þ ꢀ 100g
where Abs 5 is the absorbance of incubated solution containing sample,
substrate, and lipase, Abs 6 is the absorbance of incubated solution
containing sample and substrate, Abs 7 is the absorbance of incubated
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Journal of Natural Products
ARTICLE
solution containing substrate and lipase, and Abs 8 is the absorbance of
incubated solution containing substrate.
(C-60), 132.3 (C-10), 145.6 (C-30, 40), 157.5 (C-400), 158.0 (C-200, 600);
HRFABMS m/z 391.0849 [M þ Na]þ (calcd for C17H20O7SNa,
391.0828).
MALDI-TOF-MS Spectrum of Active Fraction. Fr. 2217 was
dissolved in 50% MeOH (1 mg/mL) and mixed 1:1 (v/v) with a 50%
aqueous MeOH solution of 2,5-dihydroxybenzoic acid (10 mg/mL).
The mixture (0.5 μL) was placed on a MALDI-TOF-MS target plate.
Size-Exclusion Chromatography of Active Fraction. High-
performance size-exclusion chromatography was performed on a TSK
gel R-3000 column (7.8 ꢀ 300 mm) with DMF containing 10 mM LiCl
as the mobile phase at 40 °C.50 Samples were dissolved in the elution
solvent, and 5 μL was individually applied to the column and eluted
using a flow rate of 0.5 mL/min; the eluate was monitored at 275 nm.
Catechin (MW 290, tR 15.9 min), procyanidin B-1 (MW 578, tR
14.9 min), A-type trimer [epicatechin-(4β,8,2β,7)-epicatechin-(4R,8)-
catechin, MW 864, tR 14.3 min], and sanguiin H-11 (MW 3738, tR 12.9
min) were used as standards for molecular weight estimation. The
number average molecular weight (Mn) and weight average molecular
weight (Mw) of Fr. 2217 were calculated from the SEC profile using a
807-IT integrator (Jasco).
Compound 22: pale brown, amorphous powder; [R]18 -22.6
D
(c 0.05, MeOH); UV (MeOH) λmax (log ε) 281 (3.48) nm; IR νmax
1
3309, 1614, 1518, 1455, 1358, 1287 cm-1; H NMR (methanol-d4,
500 MHz) δ 2.50 (1H, dd, J = 9.5, 14.0 Hz,, H-3a), 2.48 (2H, m, SCH2),
2.87 (1H, dd, J = 3.5, 14.0 Hz, H-3b), 3.53 (2H, t, J = 7.0 Hz, CH2OH),
3.81 (1H, d, J = 7.0 Hz, H-1), 3.97 (1H, m, H-2), 5.83 (2H, s, H-300, 600),
6.69 (1H, d, J = 7.8 Hz, H-50), 6.67 (1H, dd, J = 1.7, 7.8 Hz, H-60), 6.87
(1H, d, J = 1.7 Hz, H-20); 13C NMR (methanol-d4, 125 MHz) δ 30.0
(C-3), 34.3 (SCH2), 57.9 (C-1), 62.3 (CH2OH), 77.5 (C-2), 95.8
(C-300, 500), 105.6 (C-100), 115.9 (C-50), 116.9 (C-20), 121.5 (C-60),
133.7 (C-10), 146.1 (C-30), 145.5 (C-40), 157.7 (C-400), 158.2 (C-200,
600); HRFABMS m/z 369.0999 [M þ H]þ (calcd for C17H21O7S,
369.1008).
Thiolysis of 10. Compound 10 (1 mg) was dissolved in 62%
aqueous EtOH (2.6 mL) and mixed with concentrated HCl (0.12 mL)
and 2-mercaptoethanol (0.2 mL). After heating at 80 °C for 7 h, the
reaction mixture was analyzed by HPLC. Production of 18 (tR 17.5 min),
19 (tR 14.6 min), and 20 (tR 19.5 min) was confirmed by comparisons of
the retention times and UV absorptions.
Thiolysis of Dried Extract of Acacia mearnsii Bark. Bark
extract (5.0 g) was dissolved in 50 mL of H2O and mixed with 12 mL of
concentrated HCl, 110 mL of EtOH, and 20 mL of 2-mercaptoethanol.
After heating at 80 °C for 7 h, the solution was concentrated by rotary
evaporation to remove the EtOH, the resulting aqueous solution was
applied to a Sephadex LH-20 column (4 ꢀ 27 cm), and the HCl and
mercaptoethanol were eluted with H2O (Fr. 1). Subsequent elution
of the column with 0, 50, and 100% aqueous MeOH afforded Fr. 2
(404 mg) and Fr. 3 (2.5 g). Fr. 2 was fractionated by Sephadex LH-20
column chromatography (2 ꢀ 30 cm) with 0-50% aqueous MeOH into
four fractions. Fr. 23 thus obtained was further subjected to Bondapak
C18 125A column chromatography (2 ꢀ 16 cm) with 0-15% aqueous
MeOH to afford compounds 18 (24.2 mg) and 19 (22.0 mg). Fr. 3 was
separated into five fractions by Sephadex LH-20 column chromatogra-
phy (4 ꢀ 27 cm) with 100-80% aqueous EtOH and 50% aqueous
acetone. Then Fr. 33 was further fractionated on a Diaion HP20SS
column (3.5 ꢀ 22 cm) with 0-50% aqueous MeOH into five fractions.
Fr. 334 was subjected to Cosmosil 5C18-PAQ preparative HPLC
(gradient elution program B) to afford 20 (4.1 mg), 21 (4.5 mg), 22
(3.3 mg), and 23 (8.0 mg).
’ ASSOCIATED CONTENT
S
Supporting Information. Separation schemes, HPLC
b
chromatograms of the active fractions and the thiol degradation
products, and selected NMR and CD spectra of the new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 81-95-819-2433. Fax: 81-95-819-2477. E-mail: t-tanaka@
nagasaki-u.ac.jp.
’ ACKNOWLEDGMENT
Compound 18: pale brown, amorphous powder; [R]28 þ76.1
D
The authors are grateful to Professor K. Fujita and Professor
M. Tanaka (Nagasaki University) for use of the CD spectrometer
and also thank Mr. K. Inada, Mr. N. Yamaguchi, and Mr. Y. Ohama
(Nagasaki University) for NMR and MS measurements.
(c 0.03, MeOH); UV (MeOH) λmax (log ε) 282 (3.61) nm; CD
(MeOH) Δε244 -34.2, Δε278 þ30.5; IR νmax 3351, 1605, 1510, 1463,
1304, 1210 cm-1; HRFABMS m/z 389.0648 [M þ Na]þ (calcd for
C17H18O7SNa, 389.0671); 1H and 13C NMR data, see Table 3.
Compound 19: pale brown, amorphous powder; [R]18 -38.7
D
’ REFERENCES
(c 0.17, MeOH); UV (MeOH) λmax (log ε) 282 (3.60) nm; CD
(MeOH) Δε243 -28.6, Δε269 þ0.7; IR νmax 3297, 1605, 1514, 1459,
1304, 1206 cm-1; HRFABMS m/z 389.0705 [M þ Na]þ (calcd for
C17H18O7SNa, 389.0671); 1H and 13C NMR data, see Table 3.
(1) Cutting, N. J. J. Soc. Leather Technol. Chem. 1995, 81, 89–93.
(2) Yazaki, Y.; Gu, R.; Lin, Y.; Chen, W.; Nguyen, N. K. Holz-
forschung 1993, 47, 57–61.
(3) Hoong, Y. B.; Pizzi, A.; Tahir, P, M.; Pasch, H. Eur. Polym. J.
2010, 46, 1268–1277.
(4) Pasch, H.; Pizzi, A.; Rode, K. Polymer 2001, 42, 7531–7539.
(5) Singh, A. P.; Singh, R. K.; Kim, K. K.; Satyan, K. S.; Nussbaum,
R.; Torres, M.; Brard, L.; Vorsa, N. Phytother. Res. 2009, 23, 1066–1074.
(6) Takahashi, T.; Kamiya, T.; Hasegawa, A.; Yokoo, Y. J. Invest.
Dermatol. 1999, 112, 310–316.
(7) Kwak, C.-J.; Kubo, E.; Fujii, K.; Nishimura, Y.; Kobuchi, S.;
Ohkita, M.; Yoshimura, M.; Kiso, Y.; Matsumura, Y. J. Hypertens. 2009,
27, 92–101.
Compound 20: pale brown, amorphous powder; [R]18 þ67.7
D
(c 0.2, MeOH); UV (MeOH) λmax (log ε) 282 (3.73) nm; CD (MeOH)
Δε246 -28.6, Δε288 þ23.4; IR νmax 3358, 1604, 1505, 1457, 1300, 1210
cm-1; HRFABMS m/z 389.0655 [M þ Na]þ (calcd for C17H18O7SNa,
389.0671); 1H and 13C NMR data, see Table 3.
Compound 21: pale brown, amorphous powder; [R]18 þ61.0
D
(c 0.09, MeOH); UV (MeOH) λmax (log ε) 282 (3.57) nm; IR νmax
1
3334, 1615, 1519, 1446, 1367, 1284 cm-1; H NMR (methanol-d4,
500 MHz) δ 2.36 (1H, dd, J = 9.6, 14.4 Hz,, H-3a), 2.47 (2H, t, J =
7.0 Hz, SCH2), 3.02 (1H, dd, J = 2.4, 14.4 Hz, H-3b), 3.55 (2H, t, J =
7.0 Hz, CH2OH), 3.84 (1H, d, J = 5.3 Hz, H-1), 4.00 (1H, m, H-2), 5.85
(2H, s, H-300, 600), 6.70 (1H, d, J = 8.0 Hz, H-50), 6.75 (1H, dd, J = 2.1, 8.0
Hz, H-60), 6.98 (1H, d, J = 2.1 Hz, H-20); 13C NMR (methanol-d4, 125
MHz) δ 30.1 (C-3), 34.3 (SCH2), 56.9 (C-1), 62.3 (CH2OH), 76.7
(C-2), 95.8 (C-300, 500), 105.8 (C-100), 115.7 (C-50), 117.6 (C-20), 122.4
(8) Kanda, T.; Akiyama, H.; Yanagida, A.; Tanabe, M.; Goda, Y.;
Toyoda, M.; Teshima, R.; Saito, Y. Biosci., Biotechnol., Biochem. 1998, 62,
1284–1289.
(9) Noferi, M.; Masson, E.; Merlin, A.; Pizzi, A.; Deglise, X. J. Appl.
Polym. Sci. 1997, 63, 475–482.
(10) Takagi, K.; Mitsunaga, T. J. Wood Sci. 2003, 49, 461–465.
127
dx.doi.org/10.1021/np100372t |J. Nat. Prod. 2011, 74, 119–128

Journal of Natural Products
ARTICLE
(11) Ikarashi, N.; Toda, T.; Okaniwa, T.; Ito, K.; Ochiai, W.;
Sugiyama, K. Evid.-based Complementary Altern. Med. DOI: 10.1093/
ecam/neq043.
(12) Holt, R. R.; Lazarus, S. A.; Sullards, M. C.; Zhu, Q. Y.; Schramm,
D. D.; Hammerstone, J. F.; Fraga, C. G.; Schmitz, H. H.; Keen, C. L. Am.
J. Clin. Nutr. 2002, 76, 798–804.
(13) Prior, R. L.; Gu, L. Phytochemistry 2005, 66, 2264–2280.
(14) McDougall, G. J.; Shpiro, F.; Dobson, P.; Smith, P.; Blake, A.;
Stewart, D. J. Agric. Food Chem. 2005, 53, 2760–2766.
(15) Hara, Y.; Honda, M. Agric. Biol. Chem. 1990, 54, 1939–1945.
(16) Deguchi, Y.; Osada, K.; Uchida, K.; Kimura, H.; Yoshikawa, M.;
Kudo, T.; Yasui, H.; Watanuki, M. Nippon Nogeikagaku Kaishi. 1998, 72,
923–931.
(44) Tanaka, T.; Nonaka, G.; Nishioka, I. J. Chem. Res., Synop. 1985,
176–177.
(45) Matsuo, Y.; Fujita, Y.; Ohnishi, S.; Tanaka, T.; Hirabaru, H.;
Kai, T.; Sakaida, H.; Nishizono, S.; Kouno, I. Food Chem. 2010, 121,
1073–1079.
(46) Tanaka, T.; Mine, C.; Inoue, K.; Matsuda, M.; Kouno, I. J. Agric.
Food Chem. 2002, 50, 2142–2148.
(47) Xiao, Z.; Storms, R.; Tsang, A. Anal. Biochem. 2006, 351, 146–
148.
(48) Yoshikawa, M.; Nishida, N.; Shimoda, H.; Takada, M.; Kawa-
hara, Y.; Matsuda, H. Yakugaku Zasshi 2001, 121, 371–378.
(49) Han, L.-K.; Kimura, Y.; Okuda, H. Int. J. Obes. 1999, 23,
174–179.
(17) Drewes, S. E.; Roux, D. G.; Saayman, H. M.; Eggers, S. H.;
Feeney, J. J. Chem. Soc. (C) 1967, 14, 1302–1308.
(18) Young, D. A.; Cronje, A.; Botes, A. L.; Ferreira, D.; Roux, D. G.
J. Chem. Soc., Perkin Trans. 1 1985, 2521–2527.
(50) Fujihara, T.; Nakagawa-Izumi, A.; Ozawa, T.; Numata, O.
Biosci. Biotechnol. Biochem. 2007, 71, 711–719.
(19) Thompson, R. S.; Jacques, D.; Haslam, E.; Tanner, R. J. N.
J. Chem. Soc., Perkin Trans. 1 1972, 1387–1399.
(20) Yanagida, A.; Shoji, T.; Shibusawa, Y. J. Biochem. Biophys.
Methods 2003, 56, 311–322.
(21) Li, H.; Tanaka, T.; Zhang, Y.-J.; Yang, C.-R.; Kouno, I.;
Rubusuaviins, A. F. Chem. Pharm. Bull. 2007, 55, 1325–1331.
(22) Gao, S.; Fu, G.-M.; Fan, L.-H.; Yu, S.-S.; Yu, D.-Q. J. Integr. Plant
Biol. 2005, 47, 759–753.
(23) Inoshiri, S.; Sasaki, M.; Kohda, H.; Otsuka, H.; Yamasaki, K.
Phytochemistry 1987, 26, 2811–2814.
(24) Seto, R.; Nakamura, H.; Nanjo, F.; Hara, Y. Biosci. Biotechnol.
Biochem. 1997, 61, 1434–1439.
(25) Han, X.-H.; Hong, S.-S.; Hwang, J.-S.; Lee, M.-K.; Hwang,
B.-Y.; Ro, J.-S. Arch. Pharm. Res. 2007, 30, 13–17.
(26) Lee, M.-H.; Lin, Y.-P.; Hsu, F.-L.; Zhan, G.-R.; Yen, K.-Y.
Phytochemistry 2006, 67, 1262–1270.
(27) Duan, W.; Ohara, S.; Hashida, K.; Makino, R. Holzforschung
2005, 59, 289–294.
(28) Kashiwada, Y.; Nonaka, G.; Nishioka, I.; Yamagishi, T. Phyto-
chemistry 1988, 27, 1473–1477.
(29) Zhong, X.-N.; Otsuka, H.; Ide, T.; Hirata, E.; Takeda, Y.
Phytochemistry 1999, 52, 923–927.
(30) Kitajima, J.; Ishikawa, T.; Tanaka, Y.; Ono, M.; Ito, Y.; Nohara,
T. Chem. Pharm. Bull. 1998, 46, 1587–1590.
(31) Zhang, Y.-J.; Abe, T.; Tanaka, T.; Yang, C.-R.; Kouno, I. Chem.
Pharm. Bull. 2002, 50, 841–843.
(32) Kosasi, S.; Van der Sluis, W. G.; Labadie, R. P. Phytochemistry
1989, 28, 2439–2441.
(33) Lokvam, J.; Coley, P. D.; Kursar, T. A. Phytochemistry 2002, 65,
351–358.
(34) Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I.
Chem. Pharm. Bull. 2007, 55, 899–901.
(35) Slade, D.; Ferreira, D.; Marais, J. P. J. Phytochemistry 2005, 66,
2177–2215.
(36) Rayyan, S.; Fossen, T.; Solheim Nateland, H.; Andersen, Ø. M.
Phytochem. Anal. 2005, 16, 334–341.
(37) Steynberg, P. J.; Steynberg, J. P.; Brandt, E. V.; Ferreira, D.;
Hemingway, R. W. J. Chem. Soc., Perkin Trans. 1 1997, 1943–1950.
(38) Steynberg, J. P.; Brandt, E. V.; Hoffman, M. J. H.; Hemingway,
R. W.; Ferreira, D. Conformations of proanthocyanidins. In Plant
Polyphenols, Synthesis, Properties, Significance, 1; Hemingway, R. W., Laks,
P. E., Eds.; Plenum Press: New York, 1992; pp 501-520.
(39) Zhang, M.; Kazlauskas, R. J. Org. Chem. 1999, 64, 7498–7503.
(40) Laks, P. E.; Hemingway, R. W. J. Chem. Soc., Perkin Trans. 1
1987, 465–470.
(41) Tanaka, T.; Takahashi, R.; Kouno, I.; Nonaka, G. J. Chem. Soc.,
Perkin Trans. 1 1994, 3013–3022.
(42) McGraw, G. W.; Steynberg, J. P.; Hemingway, R. W. Tetra-
hedron Lett. 1993, 34, 987–990.
(43) Steynberg, P. J.; Steynberg, J. P.; Hemingway, R. W.; Ferreira,
D.; Mcgraw, G. W. J. Chem. Soc., Perkin Trans. 1 1997, 16, 2395–2403.
128
dx.doi.org/10.1021/np100372t |J. Nat. Prod. 2011, 74, 119–128