Structure-activity relationships of bergenin derivatives effect on α-glucosidase inhibition

Article abstract of DOI:10.3109/14756366.2012.719503

The α-glucosidase inhibitory activities of bergenin derivatives were evaluated. Bergenin derivatives were synthesized from bergenin which is a characteristic compound of B. ligulata. A new bergenin derivative, 11-O-(3',4'-dimethoxybenzoyl)-bergenin showed

Full text of DOI:10.3109/14756366.2012.719503

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; Early Online: 1–9
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.719503
RESEARCH ARTICLE
Structure-activity relationships of bergenin derivatives effect
on α-glucosidase inhibition
Yusei Kashima1, Hidehiko Yamaki2, Takuya Suzuki2, and Mitsuo Miyazawa1
1Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae, Higashiosaka-shi,
Osaka, Japan and 2Koei Kogyo Co., Ltd., Kanda-Awajicho, Chiyoda-ku, Tokyo, Japan
Abstract
The α-glucosidase inhibitory activities of bergenin derivatives were evaluated. Bergenin derivatives were
synthesized from bergenin which is a characteristic compound of B. ligulata. A new bergenin derivative, 11-O-(3′,4′-
dimethoxybenzoyl)-bergenin showed the highest potent inhibitory activity among those of bergenin derivatives.
The presence of substituents at 3′,4′-position in bergenin derivatives altered the α-glucosidase inhibitory activity.
11-O-(3′,4′-dimethoxybenzoyl)-bergenin was noncompetitive inhibitor for α-glucosidase. The present study reveals
that bergenin derivatives could be classified as a new group of α-glucosidase inhibitors.
Keywords: Bergenin, bergenin derivatives, α-glucosidase inhibitor, diabetes, structure-activity relationship
Introduction
have been very interesting biological activities9,10, thus,
there might be value in searching for glucosidase inhibi-
tors derived from food and plant in the view of safety.
In humans, four enzymes are causative for the com-
plete degradation of starch into glucose, which is then
able to enter the bloodstream by a specific transport sys-
tem11,12. Firstly, salivary and pancreatic α-amylases (EC
3.2.1.1) are endohydrolases that cleave the internal α-1, 4
bonds of starch into shorter linear and branched dextrin
chains. e dextrins are then further hydrolyzed at the
nonreducing ends into glucose by two small-intestinal
brush-border exohydrolases: maltase-glucoamylase (EC
3.2.1.20 and 3.2.1.3) and sucraseisomaltase (EC 3.2.148
and 3.2.10)13.
Diabetes mellitus is a worldwide health problem that
affects increasingly people every year. It is also a lifestyle-
related disease known to trigger many complications,
nephropathy, retinopathy, neuropathy, cardiovascular
disease and so on. Diabetes mellitus type 2, which is
caused by a secretory decrease in insulin from pancre-
atic Langerhans β cells or a lowering of the insulin resis-
tance1,2, is a serious problem in developed countries.
One of the most beneficial therapies for diabetes melli-
tus type 2 is said to be one that achieves optimal blood
glucose control after a meal, and α-glucosidase inhibi-
tors perform a very useful activity by delaying glucose
absorption3–5. Glucosidase inhibition is applicable to the
treatment of numerous diseases including diabetes mel-
litus type 26, cancer7 and HIV8. is wide rage of uses for
glucosidase inhibitors exemplifies the pervasive impor-
tance of glycosylation/deconjugation of sugars in such
varied processes as cell signalling/recognition, cell cycle
regulation and metabolism.
In previous studies, human pancreatic α-amylase
has been widely known as family 13 of the glycoside
hydrolases (GH13), which gathers a large variety of
enzymes with varying substrate and product specificities
as reflected by the 26 different EC numbers found in this
family of enzymes: glycoside hydrolases (EC 3.2.1.X, the
most abundant), glycoside transferases (EC 2.4.1.X) and
Interest in glucosidase inhibitors is growing because
the number of diabetes mellitus cases has been increas-
ing worldwide in recent years. Glucosidase inhibitors
even isomerases (EC 5.4.99.15 and EC 5.4.99.16)14–16
Regardless of the low amino acid sequence similarity
.
Address for Correspondence: Mitsuo Miyazawa, Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University,
Kowakae, Higashiosaka-shi, Osaka, Japan. Tel: +81-6-6721-2332. Fax: +81-6-6727-2024. E-mail:miyazawa@apch.kindai.ac.jp
(Received 28 October 2011; revised 09 June 2012; accepted 05 August 2012)
1

2
Y. Kashima et al.
between the evolutionarily distant members of this
family, some highly conserved residues are still present.
Consequently, it has been established by structural
studies that all GH13 members share similar folding17.
e α-glucosidase (EC 3.2.1.20) from Saccharomyces
cerevisiae (baker’s yeast), coded by the MAL12 gene, is
also one of the GH13 family. It can catalyze the hydrolysis
of terminal, nonreducing 1,4-linked α-d-glucose resi-
dues with release of α-d-glucose molecule and retention
of configuration at the anomeric carbon, characteristic
of GH13 enzymes15,18. Because of its ready availability
and ease of handling, baker’s yeast α-glucosidase (EC
3.2.1.20; GH13 family) has been widely used for enzy-
matic assays to find novel α-glucosidase inhibitors with
different biological activities19–24
.
Bergenia ligulata (Wall.) Engl. (syn. Saxifraga) is a
perennial herb that grows wild in North India and partic-
ularly between the stones and rocks. is plant has been
used in the Ayurvedic formulations for various ailments.
B. ligulata contained β-sitosterol25, a number of phenolic
compounds like (+)-afzelechin26 and catechin and other
compounds27–29. Among these compounds, bergenin
were identified as the major unique phenolic compound
of B. ligulata. It has been reported that bergenin has such
as anti-inflammatory30, antioxidant31, hepatoprotective32,
neuroprotective33, anti-HIV34, antiarrhythmic35, anti-
fungal36 and gastroprotective37 activities. Furthermore,
bergenin and its esterified derivatives occur widely in
a number of plants and have been found as ingredients
in plant extracts38–42. Bergenin is modified by coupling
with a variety of fatty acids on hydroxyl groups through
chemical catalysis, Takahashi et al. synthesized a series of
esterified derivatives of bergenin with greatly enhanced
biological activity33. Some other chemical modifications
of bergenin derivatives were also reported43.
In present study, bergenin derivatives were prepared
from the naturally occurring bergenin and evaluated
to the inhibitory effects of the bergenin derivatives on
α-glucosidase from baker’s yeast (E.C. 3.2.1.20), to study
the structure-activity relationships and kinetics of inhibi-
tory activity (Figure 1).
Figure 1. Chemical structures of bergenin (1) and its derivatives
(2–9).
4000–450. Nuclear magnetic resonance (NMR) spectra
were recorded at 400 MHz for 1H and 100 MHz for 13C on a
JEOL ECA 400 spectrometer in DMSO or CDCl3 with TMS
as internal standard (chemical shift in d, ppm). J values
are reported in Hertz. EI-MS spectra were obtained on
a JEOL the Tandem MS station JMS-700 (Japan Electron
Optics Laboratory Co., Ltd., Tokyo, Japan). e absor-
bance was measured with a MTP-800Lab microplate
reader. α-glucosidase from baker’s yeast (EC 3.2.1.20;
GH13 family) was purchased from Oriental Yeast Co., Ltd
(Tokyo, Japan). p-nitrophenyl α-d-glucopyranoside was
purchased from Wako Pure Chemistry (Osaka, Japan).
Materials and methods
Chemistry
All reagents were purchased from Wako Pure Chemistry
(Osaka, Japan). All solvents were purchased from Kanto
Chemical (Tokyo, Japan). Roots of Bergenia ligulata
(Wall.) Engl. were provided by Koei Kogyo Co., Ltd.
(Tokyo, Japan). in layer chromatography (TLC) was
performed on precoated plates (silica gel 60 F254, 0.25 mm,
Merk, Darmstadt, Germany). Column chromatography
was carried out using 70–230 mesh silica gel (Kieselgel
60, Merk, Germany). Optical rotations were measured
on a Japan Spectroscopic Co. LTDDIP-1000. Infrared
(IR) spectra were recorded with a JASCO FT/IR-470
plus Fourier transform infrared spectrometer (JASCO
Co., Ltd, Japan) with an ordinate scale in the region
Extraction and isolation
e barks (5.0 kg) of B. ligilata were extracted once with
MeOH. e solution was evaporated to dryness in vac-
cuo to give a methanol extract (480 g). e residue was
re-extracted successively with hexane, dichloromethane,
Journal of Enzyme Inhibition and Medicinal Chemistry

Bergenin derivatives as α-glucosidase inhibitor
3
ethyl acetate, butanol and water. Each fraction was con-
centrated to dryness in vaccuo to give hexane, dichlo-
romethane, ethyl acetate, butanol and water fractions
respectively.
3.83–4.01 (m, 1H), 3.91 (s, 3H), 3.98 (d, J = 9.2 Hz, 1H),
4.41–4.55 (m, 4H), 4.72 (d, J = 10.4 Hz, 1H), 5.26 (dd, J1 =
1.8 Hz, J2 = 10.8 Hz, 2H), 5.40 (dd, J1 = 1.8 Hz, J2 = 17.2 Hz,
2H), 5.97–6.12 (m, 2H), 7.34 (s, 1H).
e butanol extract was fractionated to fraction 1–3 by
silica gel column chromatography with dichloromethane
and methanol as eluants. Fraction 2 was recrystallized,
thereby bergenin (1) (3.7 g) was isolated. e structure
of 1 was identified by the comparison of its physical and
11-O-benzoylbergenin (2)
Yield 25%, amorphous powder; [α]25 +66.8° (c = 0.15,
D
MeOH); IR (KBr) νmax cm−1: 3474 (OH), 1722 (ester),
1610 (arom. C=C); 1H NMR (DMSO-d6, 400 MHz) δ 3.41
(dd, J1 = 9.2 Hz, J2 = 8.8 Hz, 1H), 3.69 (m, 1H), 3.74 (s,
3H), 3.93 (ddd, J1 = 9.2 Hz, J2 = 7.2 Hz, J3 = 2.0 Hz, 1H),
4.03 (t, J = 10.0 Hz, 1H), 4.34 (dd, J1 = 12.4 Hz, J2 = 7.2
Hz, 1H), 4.78 (dd, J1 = 12.4 Hz, J2 = 2.0 Hz, 1H), 5.03 (d, J
= 10.8 Hz, 1H), 7.00 (s, 1H), 7.54 (m, 2H), 7.67 (m, 1H),
8.01 (m, 2H); 13C NMR (DMSO-d6, 100 MHz): Table 1;
EI-MS, m/z (rel. intensity) 432 [M]+ (2), 370 (4), 256 (6),
122 (53), 105 (100), 77 (56).
spectral data with those described in the literature39,44
.
Synthesis
General procedure for the synthesis of 8,10-diallyloxy-
bergenin (1a)
First, the phenolic hydroxy groups in bergenin (1)
(700 mg, 2.13 mmol) were selectively allylated. Allyl
bromide and NaI were added to a solution bergenin
and K2CO3 in anhydr. DMF (5 mL). After the mixture was
stirred 2 h at 55°C under nitrogen, it was concentrated
in vacuo. e residue was treated with water (15 mL)
and extracted with ethyl acetate (3 × 10 mL). e organic
extract was washed successively with brine (3 × 15 mL).
e extract was dried over Na2SO4 and concentrated in
vacuo. e residue was chromatographed on silica gel to
afford 1a (554 mg, 64%) as a white powder.
11-O-p-hydroxybenzoylbergenin (3)
Yield 38%, amorphous powder; [α]25 +18.3° (c = 0.15,
MeOH); IR (KBr) νmax cm−1: 3380 (OH)D, 1712 (ester), 1608
1
(arom. C=C), 1238 (C-O); H NMR (DMSO-d6, 400 MHz)
δ 3.40 (m, 1H), 3.69 (t, J = 9.2 Hz, 1H), 3.73 (s, 3H), 3.87
(ddd, J1 = 9.2 Hz, J2 = 6.4 Hz, J3 = 1.6 Hz, 1H), 4.03 (t, J =
9.8 Hz, 1H), 4.26 (dd, J1 = 12.0 Hz, J2 = 6.4 Hz, 1H), 4.71
(dd, J1 = 12.0 Hz, J2 = 1.6 Hz, 1H), 5.02 (d, J = 10.0 Hz, 1H),
6.86 (d, J = 8.8 Hz, 2H), 7.00 (s, 1H), 7.85 (d, J = 8.8 Hz,
2H); 13C NMR (DMSO-d6, 100 MHz): Table 1; EI-MS, m/z
(rel. intensity) 448 [M]+ (2), 328 (1), 208 (24), 121 (100), 81
(13), 65 (28).
General procedure for the synthesis of bergenin derivatives
(2–4, 6–9)
Firstly, to a solution of 1a in anhydr. THF (2 mL/mmol),
various benzoic acids and Ph3P (2 equiv) were dissolved.
DIAD (1.7 equiv) was added dropwise under 0°C. e
mixture was stirred for 2 h under nitrogen. It was concen-
trated in vacuo. e residue was chromatographed on
silica gel to afford benzoic acid ester of 1a (yield 79–92%)
as a white powder.
Secondly, the allyl protected esters and Pd(PPh3)4
(1 mol%, freshly prepared) were dissolved in degassed
anhydrous. THF (2mL/mmol) and morpholine (10 equiv
per allylgroup to be cleaved) was added dropwise. e
mixture was stirred at r.t and concentrated in vacuo. e
residue was taken up in EtOAc. e organic layer was
washed several times with small amounts of 1 N HCl,
dried and concentrated. e crude material was purified
by silica gel column chromatography. e purified mate-
rial was bergenin derivatives (2–4, 6–9) in yield 28–59%
(Scheme 1).
11-O-p-methoxybenzoylbergenin (4)
Yield 24%, amorphous powder; [α]25 +44.4° (c = 0.15,
MeOH); IR (KBr) νmax cm−1: 3373 (DOH), 2959 (C-H),
1720 (ester), 1609 (arom. C=C), 1237 (C-O); 1H NMR
(DMSO-d6, 400 MHz) δ 3.40 (t, J = 8.8 Hz, 1H), 3.69 (t, J
= 8.8 Hz, 1H), 3.74 (s, 3H), 3.89 (m, 1H), 3.83 (s, 3H), 4.04
(t, J = 10.0 Hz, 1H), 4.29 (dd, J1 = 12.0 Hz, J2 = 6.8 Hz, 1H),
4.74 (dd, J1 = 12.0 Hz, J2 =2.1Hz, 1H), 5.03 (d, J = 10.0 Hz,
1H), 7.00 (s, 1H), 7.05 (d, J = 8.8 Hz, 2H), 7.95 (d, J = 8.8 Hz,
2H); 13C NMR (DMSO-d6, 100 MHz): Table 1; EI-MS, m/z
(rel. intensity) 462 [M]+ (17), 250 (11), 208 (50), 152 (93),
135 (100), 92 (34), 77 (17).
11-O-protocatechuoylbergenin (5)
Yield 16%, amorphous powder; [α]25 +25.2° (c = 0.25,
D
MeOH); IR (KBr) νmax cm−1: 3364 (OH), 1709 (ester),
Synthesis of 11-O-protocatechuoylbergenin (5)
1
Firstly, the phenolic hydroxy groups in protocatechual-
dehyde were selectively allylated. Secondly, 3,4-dially-
loxy-protocatechuic acid was accomplished according
to previous study45. en, the same procedure as for 2–4,
6–9 was used by starting from 1a (Scheme 2).
1610 (arom. C=C), 1227 (C-O); H NMR (DMSO-d6, 400
MHz) δ 3.39 (t, J = 9.2 Hz, 1H), 3.69 (t, J = 9.2 Hz, 1H),
3.74 (s, 3H), 3.86 (m, 1H), 4.01 (t, J = 10.0 Hz, 1H), 4.26
(dd, J1 = 11.6 Hz, J2 = 6.0 Hz, 1H), 4.67 (brd, J = 11.6 Hz,
1H), 5.05 (d, J = 10.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 7.00
(s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 1.6 Hz, 1H);
13C NMR (DMSO-d6, 100 MHz): Table 1; EI-MS, m/z (rel.
intensity) 464 [M]+ (11), 328 (23), 262 (15), 137 (100), 77
(54), 55 (32).
8,10-diallyloxy-bergenin (1a)
1
Yield 64%, white powder. H NMR (CDCl3, 400 MHz) δ
3.54–3.59 (m, 1H), 3.54–3.59 (m, 1H), 3.68–3.73 (m, 1H),
© 2012 Informa UK, Ltd.

4
Y. Kashima et al.
Scheme 1. Reagents and conditions: (a) allylbromide, K2CO3, NaI, DMF; (b) DIAD, P(Ph)3, RCOOH, THF; (c) 10% Pd(PPh3)4, 20 equiv.,
morpholine, THF.
11-O-(3′,5′-dimethoxybenzoyl)-bergenin (8)
Yield 26%, amorphous powder; [α]25 +157.9° (c = 0.025,
D
MeOH); IR (KBr) νmax cm−1: 3355 (OH), 1717 (ester), 1602
1
(arom. C=C), 1237 (C-O); H NMR (DMSO-d6, 400 MHz)
δ 3.38 (m, 1H), 3.69 (m, 1H), 3.73 (s, 3H), 3.81 (s, 6H), 3.91
(ddd, J1 = 9.2 Hz, J2 = 7.6 Hz, J3 = 1.6 Hz, 1H), 4.04 (dd, J1 =
10.4 Hz, J2 = 9.6 Hz, 1H), 4.23 (dd, J1 = 12.0 Hz, J2 = 7.6 Hz,
1H), 4.90 (dd, J1 = 12.0 Hz, J2 = 1.6 Hz, 1H), 5.05 (d, J = 10.4
Hz, 1H), 6.79 (t, J = 2.4 Hz, 1H), 7.00 (s, 1H), 7.19 (d, J = 2.4
Hz, 2H); 13C NMR (DMSO-d6, 100 MHz): Table 1; EI-MS,
m/z (rel. intensity) 492 [M]+ (12), 328 (5), 208 (31), 182
Scheme 2. Reagents and conditions: (a) DIAD, P(Ph)3,
3,4-diallyloxy-protocatechuic acid, THF; (b) 10% Pd(PPh3)4, 20
equiv., morpholine, THF.
(78), 165 (100), 137 (18), 122 (17), 55 (15).
11-O-syringylbergenin (9)
11-O-(3′,4′-dimethoxybenzoyl)-bergenin (6)
Yield 20%, amorphous powder; [α]25 +24.5° (c = 0.1,
MeOH); IR (KBr) νmax cm−1: 3371 (OH),D1718 (ester), 1610
(arom. C=C), 1235 (C-O); H NMR (DMSO-d6, 400 MHz)
Yield 28%, amorphous powder; [α]25 +32.6° (c = 0.025,
D
MeOH); IR (KBr) νmax cm−1: 3395 (OH), 1716 (ester), 1598
1
1
(arom. C=C), 1225 (C-O); H NMR (DMSO-d6, 400 MHz)
δ 3.38 (m, 1H), 3.69 (m, 1H), 3.72 (s, 3H), 3.85 (s, 6H), 3.92
(ddd, J1 = 10.0 Hz, J2 = 8.1 Hz, J2 = 2.4 Hz, 1H), 4.06 (t, J =
10.0 Hz, 1H), 4.12 (dd, J1 = 11.6 Hz, J2 = 8.8 Hz, 1H), 4.89
(dd, J1 = 11.6 Hz, J2 = 2.4 Hz, 1H), 5.07 (d, J = 10.8 Hz, 1H),
7.00 (s, 1H), 7.26 (s, 2H); 13C NMR (DMSO-d6, 100 MHz):
Table 1; EI-MS, m/z (rel. intensity) 508 [M]+ (2), 328 (8),
237 (16), 208 (100), 182 (50), 165 (83), 152 (75).
δ 3.38 (m, 1H), 3.70 (t, J = 9.6 Hz, 1H), 3.73 (s, 3H), 3.83
(s, 3H), 3.84 (s, 3H), 3.91 (m, 1H), 4.04 (dd, J1 = 10.0 Hz, J2
= 9.6 Hz, 1H), 4.21 (dd, J1 = 12.0 Hz, J2 = 7.2 Hz, 1H), 4.83
(brd, J = 12.0 Hz, 1H), 5.05 (d, J = 10.0 Hz, 1H), 7.00 (s, 1H),
7.09 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 1.6 Hz, 1H), 7.63 (dd, J1
= 8.4 Hz, J2 = 1.6Hz, 1H); 13C NMR (DMSO-d6, 100 MHz):
Table 1; EI-MS, m/z (rel. intensity) 492 [M]+ (9), 208 (20),
182 (50), 165 (100), 152 (9), 79 (9).
α-glucosidase inhibitory activity
11-O-vanilloylbergenin (7)
α-glucosidase inhibitory activity was determined using
the modified version of the method according to Li
et al.46 α-glucosidase (25 µL, 0.2 U/mL), 25 µL of various
concentrations of samples, and 175 µL of 50 mM sodium
phosphate buffer (pH 7.0) were mixed at room tempera-
ture for 10 min. e reaction was started by the addition
of 25 µL of 2.5 mM p-nitrophenyl-α-d-glucopyranoside.
e activities of glucosidase were detected in 96-well
plate, and the absorbance was determined at 405 nm (for
p-nitrophenol) in a microplate reader (Corona Electric
Co., Ltd). Control sample contained 25 µL DMSO in place
of test samples. Percentage of enzyme inhibition was cal-
culated as (B/A) × 100, where A represents absorbance
Yield 30%, amorphous powder; [α]25 +56.4° (c = 0.1,
MeOH); IR (KBr) νmax cm−1: 3365 (OH),D1717 (ester), 1599
(arom. C=C), 1222 (C-O); H NMR (DMSO-d6, 400 MHz)
1
δ 3.39 (m, 1H), 3.69 (dd, J1 = 9.6 Hz, J2 = 8.8 Hz, 1H), 3.73
(s, 3H), 3.85 (s, 3H), 3.89 (ddd, J1 = 8.0 Hz, J2 = 7.6 Hz, J3
= 2.0 Hz, 1H), 4.04 (dd, J1 = 10.4 Hz, J2 = 9.6 Hz, 1H), 4.19
(dd, J1 = 11.6 Hz, J2 = 8.0Hz, 1H), 4.81 (dd, J1 = 11.6 Hz, J2
= 2.0 Hz, 1H), 5.05 (d, J = 10.8 Hz, 1H), 6.88 (d, J = 8.4 Hz,
1H), 7.00 (s, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.51 (dd, J1 = 8.4
Hz, J2 = 2.0 Hz, 1H); 13C NMR (DMSO-d6, 100 MHz): Table
1; EI-MS, m/z (rel. intensity) 478 [M]+ (7), 328 (16), 208
(53), 180 (17), 151 (100), 97 (13), 43 (18).
Journal of Enzyme Inhibition and Medicinal Chemistry

Bergenin derivatives as α-glucosidase inhibitor
5
Table 1. 13C NMR spectral data for compounds 2–9 (δ in ppm).
Position
2
78.5
3
78.6
4
78.5
5
78.6
6
78.5
7
78.5
8
78.3
9
78.5
2
3
70.2
70.2
70.2
70.1
70.5
70.5
70.5
70.8
4
73.5
73.5
73.5
73.5
73.5
73.5
73.5
73.5
4a
79.6
79.6
79.6
79.6
79.5
79.6
79.5
79.5
6
163.4
118.1
109.5
150.9
140.6
148.0
115.8
72.2
163.4
118.1
109.5
150.9
140.6
148.0
115.8
72.2
163.4
118.1
109.5
150.9
140.6
148.0
115.8
72.2
163.4
118.1
109.5
150.9
140.6
148.0
115.8
72.2
163.3
118.1
109.6
151.0
140.6
148.0
115.7
72.1
163.3
118.1
109.6
151.0
140.6
148.0
115.8
72.1
163.3
118.1
109.6
151.0
140.6
148.0
115.7
72.1
163.2
118.1
109.6
151.1
140.6
148.0
115.7
72.1
6a
7
8
9
10
10a
10b
11
64.0
63.5
63.7
63.4
64.0
63.8
64.1
64.0
9-OCH3
1'
59.7
59.7
59.7
59.8
59.7
59.7
59.7
59.7
129.5
129.2
128.8
133.5
128.8
129.2
165.6
120
121.7
131.4
114.1
163.3
114.1
131.4
165.4
120.2
116.4
145.1
150.7
115.3
122.0
165.6
123.4
111.2
148.4
153.1
111.7
121.6
165.3
55.5
120.2
112.6
147.4
151.8
115.2
123.7
165.4
55.6
131.5
106.9
160.5
105.5
160.5
106.9
165.2
55.5
119.0
107.0
147.6
141.1
147.7
107.0
165.3
56.1
2'
131.6
115.4
162.2
115.4
131.6
165.5
3'
4'
5'
6'
COO
3′-OCH3
4′-OCH3
5′-OCH3
55.6
55.7
55.5
56.1
13C NMR spectral data were recorded at 100 MHz in DMSO-d6 using tetramethylsilane (TMS) as internal standard.
of control without test samples, and B represents absor-
bance in presence of test samples. All the tests were run
in triplicate. Acarbose and 1-deoxynojirimycin were
used as the positive control in this study. e fifty percent
inhibitory concentration (IC50) values were expressed as
mean SD (n = 3).
excellent inhibitions on yeast α-glucosidase a with
extremely high potency, and its 50% inhibition of enzyme
activity (IC50) values were calculated as 24.6 µM which
were much lower than that of reference drugs, acarbose
(907.5 µM) and 1-deoxynojirimycin (278.0 µM). Other
active compounds, compound 2–4 and 7 also exhibited a
potent inhibition with IC50 values of 118.3 µM, 143.2 µM,
143.8 µM and 186.9 µM, which were much more potent
to that of 1-deoxynojirimycin, respectively, on the other
hand, compound 5 (IC50: 289.6 µM) showed much the
same effect as a potent inhibition of 1-deoxynojirimycin.
Furthermore, we evaluated the inhibitory activities
of benzoic acid derivatives and bergenin (1) to clarify
whether inhibitory activities of compound 2–7 are due
to benzoic acid moiety or bergenin moiety. Benzoic
acid (inhibition: 7.4 0.9% at 300 µM), p-hydroxyben-
zoic acid (inhibition: 3.1 1.3% at 300 µM), anisic acid
(inhibition: 1.6 1.4% at 300 µM), protocatechuic acid
(inhibition: 10.7 2.1% at 300 µM), 3,4-dimethoxyben-
zoic acid (inhibition: 17.7 3.0% at 300 µM), vanilic acid
(inhibition: 22.3 1.0% at 300 µM) and bergenin (1)
(inhibition: 22.7 0.6% at 300 µM) exhibited low inhibi-
tory activity. Each benzoic acid derivatives exhibited
a relatively poor inhibitory activity as compared with
these bergenin derivatives (Table 2). erefore, we con-
sidered the linkage of benzoic acid and bergenin as well
as benzoic acid moiety and bergenin moiety are critical
factor of α-glucosidase inhibitory activity. ese results
accorded with previous reports that α-glucosidase
Results and discussion
e synthesis of ester analogues 2–4, 6–9 of bergenin at
C-11 position were accomplished by coupling various
benzoic acids with bergenin (Scheme 1) by employ-
ing Mitsunobu protocol in dry THF. For the synthesis of
analogues 5, the phenolic hydroxy groups in protocat-
echualdehyde were selectively allylated. en, the same
procedure as for 2–4, 6–9 was used by starting from 1a
(Scheme 2). e pure compound 2, 3, 5, 7, 9 were identi-
fied through analysis of spectroscopic data and compari-
son with previous studies39,40. e structures of the new
bergenin derivatives (4, 6 and 8) have been assigned
from 1H and 13C NMR spectra (Table 1), IR and EI-MS.
e α-glucosidase inhibitory activities of bergenin
derivatives (2–9) were investigated and compared with
that of acarbose and 1-deoxynojirimycin, a clinically
used α-glucosidase inhibitors, were employed as a
reference drugs. ese results were shown in Figure 2.
Compounds 2–7 demonstrated a significant inhibition
on α-glucosidase in a dose dependent manner. In par-
ticular, compound 6, new bergenin derivative, showed
© 2012 Informa UK, Ltd.

6
Y. Kashima et al.
activity. e evidence supported is the previous study48.
On the other hand, compound 9 had no effect on
α-glucosidase inhibitory activity. e inhibitory effect
of each compound on α-glucosidase activity is shown in
Table 2.
Inhibitory mechanisms of compounds 2, 3, 4, 6, acar-
bose and 1-deoxynojirimycin on α-glucosidase were
analysed by a Lineweaver–Burk plot (Figures 3–6). is
analysis (Figures 3 and 4) showed that Vmax decreased
without changing Km in the presence of increasing con-
centration of inhibitors: as can be seen directly from
the graph, −1/Km (the x-intercept) was unaffected by
inhibitor concentration, whereas 1/Vmax became more
positive. is behaviour indicates that compounds 2
and 6 exhibit noncompetitive inhibition characteristics
for α-glucosidase. e inhibitory constants (Ki) were
determined to be 104.9 µM for compound 2, 19.5 µM for
compound 6, by using Dixon plot (Figures 3B and 4B). In
contrast, as shown representatively in Figure 5, the kinetic
plot shows that acarbose and 1-deoxynojirimycin were
noncompetitive inhibitors with Ki value of 943.15 µM and
353.6 µM, respectively (Figure 5B and 5D). On the other
hand, Figure 6 shows that the plot of 1/V versus 1/[S] give
a family of parallel straight lines with the same slopes.
Accompanying the increase of the inhibitor concentra-
tion the values of both Km and Vm increase, but the ratio
of Km/Vm remained unchanged. e slopes were inde-
pendent of the concentration of compound 3 or 4 which
indicated that compounds 3 and 4 were uncompetitive
inhibitors of the enzyme. e Ki values of compound 3
and 4 were 217.5 µM and 235.8 µM, respectively.
Figure 2. Dose-dependent inhibitory effects of bergenin (1)
and its derivatives (2–9). Acarbose and 1-deoxynojirimycin
were used a positive control. e result represents the means of
triplicate experiments. (See colour version of this figure online at
www.informahealthcare.com/enz)
Table 2. Inhibition effects of bergenin (1) and bergenin
derivatives (2–10) against α-glucosidase.
Compound
IC50 (µM)a Compound
IC50 (µM)a
1
2
3
4
5
>300 Benzoic acid
>300
118.3 0.7 p-Hydroxybenzoic acid
143.2 2.0 Anisic acid
>300
>300
143.8 2.3 Protocatechuic acid
>300
289.6 1.4 3,4-Dimethoxybenzoic
acid
>300
To date, the microbial α-glucosidase is known to be
structurally different to those of mammalial origins. e
microbial α-glucosidase inhibitors are not necessarily
the mammalial α-glucosidase inhibitors. For example,
(+)-catechin, a natural inhibitor of yeast α-glucosidase
does not show any inhibitory activity on mammalial
α-glucosidase49. On the other hand, voglibose shows
very high inhibitory activity on porcine small intestine
α-glucosidase, but both of them show very low inhibi-
tory activity on microbial α-glucosidase, suggesting that
ongoing experiments should be focused on the inhibi-
tory activity of these compounds against mammalian
intestinal α-glucosidases. Nevertheless, the inhibition of
yeast α-glucosidase by bergenin derivatives served as an
interesting structural activity relationship of this group of
inhibitors.
In conclusion, we have undertaken synthesis of ber-
genin derivatives which abounds in many plants and a
thorough investigation into the α-glucosidase inhibition
of these compounds. In this study, we reported the cor-
relation between α-glucosidase inhibition and structures
of bergenin derivatives for the first time. Compound 6
(IC50 = 24.6 µM) showed a most potent α-glucosidase
inhibition. Furthermore, we have uncovered some of
more potent inhibitors of α-glucosidase than positive
control. From the structural-activity point of view, linkage
of benzoic acid and bergenin as well as benzoic moiety
6
7
8
9
24.6 0.3 Vanilic acid
>300
186.9 2.3
>300
>300
Acarboseb
1-Deoxynojirimycinb
907.5 2.2
278.0 1.7
aData represents means SD of triplicate samples.
bis compound was used as a positive control.
was inhibited to a great extent when the compound
contained a fused-aromatic ring and a heteroaromatic
component47.
From the structural-activity point of view, the presence
of methoxy group at 3′-position on bergenin derivative
moiety was potent factor of the α-glucosidase inhibitory
activity. In the case of compound 3, 4′ position replace-
ment with OMe group (compound 4) is similar to that
of compound 3, however, the introduction of another
methoxy group in an ortho-position to a methoxy group
(compound 7) led to dramatically increase inhibitory
activity relative to compound 4. When the α-glucosidase
inhibitory activities of compound 5 and 7 were compared,
it was found that the potency increased in the order of
7 > 5. e observation revealed that replacement of the
3′-hydroxy substituted in the compound 5 by OMe group
increased α-glucosidase inhibitory activity. In addition
to these reports, the observation of the inhibitory activi-
ties of compound 5 and 7 indicated that 4′ position OMe
group is also potent factor of the α-glucosidase inhibitory
Journal of Enzyme Inhibition and Medicinal Chemistry

Bergenin derivatives as α-glucosidase inhibitor
7
Figure 3. Lineweaver–Burk plot (panel A) and Dixon plot (panel B) for inhibition of compound 2 (0, 75, 150 and 300 µM) on α-glucosidase
activity.
Figure 4. Lineweaver–Burk plot (panel A) and Dixon plot (panel B) for inhibition of compound 6 (0, 7.5, 15 and 30 µM) on α-glucosidase
activity.
Figure 5. Lineweaver–Burk plot (panels A and C) and Dixon plot (panels B and D) for inhibition of acarbose (0, 0.5, 1.0 and 2.0 mM) and
1-deoxynojirimycin (0, 200, 400 and 800 μM) on α-glucosidase activity.
and bergenin moiety are critical factor of α-glucosidase
Acknowledgments
inhibitory activity. We hope that these compounds could
I am grateful to Professor I. Horibe for number of valuable
suggestions and discussions on this study. e author is
be employed as a lead for the design of new potential
α-glucosidase inhibitors.
© 2012 Informa UK, Ltd.

8
Y. Kashima et al.
Figure 6. Line weaver–Burk plots for the inhibition of the hydrolysis activity of α-glucosidase by compounds 3 (0, 75 and 150 mM) (a) and 4
(0, 75, 150 and 300 mM) (b).
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Declaration of interest
Dividing the large glycoside hydrolase family 13 into subfamilies:
towards improved functional annotations of α-amylase-related
e authors report no conflicts of interest.
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