Potential α-glucosidase inhibitors from thermal transformation of (+)-catechin

Article abstract of DOI:10.1016/j.bmcl.2014.01.027

Thermal transformation of the (+)-catechin (1) with heating processing afforded a new oxidation product, gambiriin D (2), along with catechin [6′-8]-catechin (3), and (+)-epicatechin (4). The structure of a new catechin dimer with C-C linkage was determined on the basis of spectroscopic data interpretation. The catechin dimers 2 and 3 exhibited significantly improved inhibitory activities against α-glucosidase, with IC₅₀ values of 0.16 ± 0.2 and 0.14 ± 0.2 μM, respectively, when compared to parent (+)-catechin. Kinetic analysis showed that the two effective compounds 2 and 3 have noncompetitive modes of action.

Full text of DOI:10.1016/j.bmcl.2014.01.027

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1621–1624
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potential
a-glucosidase inhibitors from thermal transformation
of (+)-catechin
Taewan Kim a, Hyo Jin Choi b, Sung-Hwan Eom c, Jaemin Lee d, Tae Hoon Kim d,e,
⇑
a Department of Food Science and Biotechnology, Andong National University, Andong 760-749, Republic of Korea
b College of Pharmacy, Kyungpook National University, Daegu 702-701, Republic of Korea
c Department of Food Science and Technology, Pukyong National University, Busan 608-737, Republic of Korea
d Department of Herbal Medicinal Pharmacology, Daegu Haany University, Gyeongsan 712-715, Republic of Korea
e Blue-Bio Industry RIC, Dongeui University, Busan 614-714, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Thermal transformation of the (+)-catechin (1) with heating processing afforded a new oxidation product,
gambiriin D (2), along with catechin [60–8]-catechin (3), and (+)-epicatechin (4). The structure of a new
catechin dimer with C–C linkage was determined on the basis of spectroscopic data interpretation. The
Received 13 December 2013
Revised 8 January 2014
Accepted 10 January 2014
Available online 20 January 2014
catechin dimers 2 and 3 exhibited significantly improved inhibitory activities against
a-glucosidase, with
IC50 values of 0.16 0.2 and 0.14 0.2 M, respectively, when compared to parent (+)-catechin. Kinetic
l
analysis showed that the two effective compounds 2 and 3 have noncompetitive modes of action.
Keywords:
Thermal transformation
(+)-Catechin
Ó 2014 Published by Elsevier Ltd.
a-Glucosidase
(+)-Catechin dimers
Kinetic analysis
Diabetes mellitus is one of the most common chronic diseases
in populations worldwide and belongs to the group of metabolic
disorders which are characterized by high blood glucose levels.
Diabetes mellitus is closely associated with cardiovascular disease,
as the major cause of morbidity and mortality.1 Also, the serious
complications associated with diabetes mellitus, such as peripheral
vascular disease, diabetic neuropathy, amputations, renal failure,
stroke, and blindness result in increasing disability, reduced life
expectancy, and enormous health costs.2 It is an important and
growing public health problem, due largely to the substantial
financial loss incurred for appropriate disease control and manage-
ment of chronic complications. The strategy of the most effective
treatment for type 2 diabetes mellitus is to achieve optimal blood
glucose levels after a meal. Recently, a more effective strategy for
the treatment of type 2 diabetes mellitus has involved the distur-
sugar mimics with tedious multi-steps during preparation and
problems of gastrointestinal side-effects. In recent years substan-
tial efforts to discover effective non-sugar inhibitors of
a-glucosi-
dase from natural sources have been conducted and have
received great attention because of the high levels of natural abun-
dance and biological efficacies.5,6
Catechins are among the most well-known flavonoids and are
potentially beneficial to human health. The naturally occurring
compounds are widely distributed in various fruits, green tea, red
wine, juices, and in chocolate.7 Catechin derivatives have been
shown to possess a wide spectrum of significant biological effica-
cies, such as antioxidant, anticancer, and anti-inflammatory prop-
erties. In addition, recent studies have demonstrated that naturally
occurring catechin polymers, procyanidins, are also known to have
various physiological functions, including antioxidant, anti-inflam-
mation, antitumor, and vascular protective effects.8–10 Recently,
several researchers have attempted to create new useful procyani-
din derivatives by structural modifications11,12 and have verified
that thermal processing of inactive compounds is a useful method
for bringing about structural modifications and improving the bio-
activity of natural products.13,14 In addition, the presence of cate-
chin derivatives by thermal processing in various primary foods
is closely related to human health and it is necessary to evaluate
their levels under thermal conditions because of possible biological
effects and safety. As part of our continuing investigation into
creating bioactive compounds of inactive ubiquitous natural
bance of dietary monosaccharide absorption by inhibition of
cosidase.3 Thus,
-glucosidase inhibitors are considered to be a
valuable therapeutic reagent for treating type 2 diabetes mellitus
in humans. Several -glucosidase, including acarbose and vogli-
a-glu-
a
a
bose from natural sources are representative anti-diabetic agents
that affect blood glucose levels after food intake, are clinically used
in the effective treatment of type 2 diabetes mellitus.4 Only a few
a-glucosidase inhibitors are clinically available and they are all
⇑
Corresponding author. Tel.: +82 53 819 1371; fax: +82 53 819 1339.
E-mail address: skyey7@dhu.ac.kr (T.H. Kim).
0960-894X/$ - see front matter Ó 2014 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2014.01.027

1622
T. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1621–1624
OH
OH
Upper unit
OH
O
HO
OH
4a
O
HO
OH
8a
2
OH
OH
HO
HO
O
OH
O
9
OH
OH
O
HO
OH
HO
9
HO
OH
8a
4a
2
OH
Lower unit
OH
OH
OH
OH
OH
1 : 2R
4 : 2S
2
3
Figure 1. Structures of thermal transformation products 2–4 of (+)-catechin.
Table 1
1H and 13C NMR chemical shifts of compound 2a
Position
Upper unit
Lower unit
b
b
dH (J in Hz)
dC, mult.
dH (J in Hz)
dC, mult.
2
3
4
4.61 (d, 8.4)
4.02 (ddd, 8.4, 7.4, 5.4)
2.90 (dd, 16.2, 5.4)
83.4
67.5
28.9
4.61 (d, 8.4)
4.02 (ddd, 8.4, 7.4, 5.4)
2.90 (dd, 16.2, 5.4)
83.4
67.5
28.9
4
2.51 (dd, 16.2, 8.4)
2.51 (dd, 16.2, 8.4)
4a
5
6
—
—
100.9
155.2
96.8
—
—
100.9
155.2
96.8
6.00 (s)
6.00 (s)
7
8
8a
9
10
11
12
13
14
—
—
—
—
155.0
105.1
152.4
130.5
115.4
145.9
145.6
115.8
120.0
—
—
—
—
155.0
105.1
152.4
130.5
115.4
145.9
145.6
115.8
120.0
6.93 (d, 1.8)
—
—
6.80 (d, 7.8)
6.75 (dd, 7.8, 1.8)
6.93 (d, 1.8)
—
—
6.80 (d, 7.8)
6.75 (dd, 7.8, 1.8)
a
1H NMR measured at 600 MHz, 13C NMR measured at 150 MHz; obtained in acetone-d6 + D2O with TMS as internal standard. Assignments based on HSQC and HMBC NMR
spectra.
b
J values (Hz) are given in parentheses.
constituents using
herein report the biotransformation of (+)-catechin using a heating
c
-irradiation and polyphenol oxidase,15,16 we
OH
HO
OH
processing, forming a new catechin dimer that exhibited signifi-
HO
HO
O
OH
O
OH
cantly improved inhibitory effects against
parent (+)-catechin.
a-glucosidase than their
HO
A sample solution containing (+)-catechin in H2O was directly
heated for 3 h17 and the converted products were monitored by
HPLC analysis. The dried resultant exhibited significant inhibitory
OH
OH
COSY
HMBC
2
activity, with an IC50 value of 11.3 1.3 lg/mL in a a-glucosidase
Figure 2. Selected COSY and HMBC correlations of 2.
inhibition assay (Table 3). In the present investigation, column
chromatographic purification of the 3 h reactant led to the isola-
tion of the new catechin derivative, gambiriin D (2), along with cat-
echin [60–8]-catechin (3),18 and (+)-epicatechin (4).19 The known
compounds were identified by comparison of their spectroscopic
data with literature values (Fig. 1).
8.4 Hz, H-4). Consistent with these 1H NMR observations, the 13C
NMR and HSQC experiments of 2 further displayed the presence
of signals for catechin skeleton at dC 83.4 (C-2), 67.5 (C-3), 28.9
(C-4).22 These data closely resemble those for the parent com-
pound, (+)-catechin (1),18 except for the absence of an aromatic
proton at the C-8 position in 1. Peak at m/z 601 in the positive HRE-
SIMS of the sodiated molecule was consistent with 2, being a dimer
of (+)-catechin (1). The absence of a H-8 proton signal in the 1H
NMR spectrum of 2 suggested that the intermolecular linkage
was at C-8 in both halves of the dimer. HMBC experiments allowed
for the complete assignment of the 1H and 13C NMR spectra of 2,
and a summary of key correlations observed for 2 is shown in Fig-
ure 2. The absolute configurations at C-2 and C-3 were determined
by circular dichroism (CD) spectroscopy. The CD spectrum
displayed a negative Cotton effect around 280 nm, indicating
2R,3S-configuration of the upper and lower units.22,23 The R-config-
uration of the biphenyl moiety in 2 was supported by a negative
Cotton effect at 212 nm and a positive Cotton effect at 241 nm by
comparison of the CD spectrum of theasinensin C having an R
Compound 2 was obtained as a yellow amorphous powder,
½
a 2D5
ꢀ
ꢁ 134:3ꢂ (MeOH). Its molecular formula was determined to
be C30H26O12 using positive HRESIMS, which showed a pseudomo-
lecular ion peak at m/z 601.1317 [M+Na]+. The absorption maxima
at 280 nm in the UV spectrum were attributed to a flavan-3-ol
skeleton.20 In the 1H NMR spectrum of 2 (Table 1), the four protons
in the aromatic region exhibited characteristic signals for one ABX-
type aromatic signals at dH 6.93 (1H, d, J = 1.8 Hz, H-10), 6.80 (1H,
d, J = 7.8 Hz, H-13), and 6.75 (1H, dd, J = 7.8, 1.8 Hz, H-14), one iso-
lated aromatic proton at dH 6.00 (1H, s, H-6), indicating the pres-
ence of two aromatic ring systems with different substitution
patterns. Also observed were two oxygenated methine protons at
dH 4.61 (1H, d, J = 8.4 Hz, H-2) and 4.02 (1H, ddd, J = 8.4, 7.4,
5.4 Hz, H-3), which showed a characteristic large coupling constant
associated with the 2,3-trans configuration,21 and one methylene
at dH 2.90 (1H, dd, J = 16.2, 5.4 Hz, H-4) and 2.51 (1H, dd, J = 16.2,

T. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1621–1624
1623
Table 2
Relative amounts of individual constituents in the reacted mixtures
Compound
Time (min)
1 h
2 h
3 h
6 h
12 h
Content (%)
Content (%)
Content (%)
Content (%)
Content (%)
1
2
3
4
8.25
18.69
7.41
84.7
1.2
1.3
80.4
1.6
1.8
76.3
2.5
2.8
68.0
2.9
3.0
67.1
2.9
3.0
11.82
12.8
16.1
18.4
26.1
27.0
Table 3
Inhibitory effects of isolated compounds 2–5 on
a
-glucosidase
g/mL)
Reactant (h)
IC50 valuea
(
l
Compound
IC50 valuea
(
lM)
Type of inhibition
1
2
3
6
52.1 1.7
27.7 1.5
11.3 1.3
18.2 1.6
25.1 1.8
1
2
3
4
51.7 1.4
0.16 0.2
0.14 0.2
176.6 2.2
300.1 3.5
NTc
Noncompetitive
Noncompetitive
NTc
12
Acarboseb
a
b
c
All compounds were examined in triplicate experiments.
Used as positive control.
NT is not tested.
100
14
12
10
8
A
1.5 μΜ
1.0 μΜ
0 μΜ
B
80
60
40
20
0
6
4
2
0
-0.002 -0.001
0
0.001 0.002 0.003 0.004 0.005 0.006
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Concentration (μM)
Compound 2, 1/[S, μM]
14
12
10
8
1.5 μΜ
1.0 μΜ
0 μΜ
B
6
4
2
0
-0.002 -0.001
0
0.001 0.002 0.003 0.004 0.005 0.006
Compound 3, 1/[S, μM]
Figure 3. Effects of compounds 2 and 3 on the activity of
a-glucosidase. (A) Dose dependent inhibition of a-glucosidase activity (compound 2, ꢀ; compound 3, N). (B)
Lineweaver-Burk plot of PNP release by -glucosidase in the presence of compounds 2 and 3 in phosphate buffer (pH 7.0) at 37 °C.
a
biphenyl bond.24 Therefore, the structure of compound 2 was as-
signed as gambiriin D,25 which is a new thermal transformation
product of (+)-catechin.
All pure isolates obtained in the present investigation were
evaluated for their anti-diabetic activity against the -glucosi-
dase26 using acarbose as the positive control. The
-glucosidase
a
a

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T. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1621–1624
inhibitory activity of structurally modified catechin derivatives 2
and 3 were found to be significantly improved over that of their
parent (+)-catechin (1) (Table 2). Catechin [60–8]-catechin (3), with
C–C linkage between C-60 of B-ring for the upper unit and C-8 of B-
References and notes
1. Anselmino, M. J. Diabetes Complicat. 2009, 23, 427.
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3. Israili, Z. H. Am. J. Ther. 2011, 18, 117.
ring for the lower unit, displaying the most potent
inhibitory activity, in concentration-dependent manner
(Fig. 3A), and with an IC50 value of 0.14 0.2 M. While, the iso-
lated (+)-epicatechin (4) showed relatively weak inhibitory activity
with an IC50 value of 176.6 2.2 M. As shown in Figure 3B, the
inhibition kinetics of -glucosidase of gambiriin D (2) and catechin
[60–8]-catechin (3) analyzed by Lineweaver–Burk plots showed
compounds 2 (ki, 0.25 M) and 3 (ki, 0.24 M) were noncompeti-
a-glucosidase
4. Asano, N.; Tomioka, E.; Kizu, H.; Matsui, K. Carbohydr. Res. 1994, 253, 235.
5. Benalla, W.; Bellahcen, S.; Bnouham, M. Curr. Diabetes Rev. 2010, 6, 247.
6. Mata, R.; Cristians, S.; Escandón-Rivera, S.; Juárez-Reyes, K.; Rivero-Cruz, I. J.
Nat. Prod. 2013, 76, 468.
7. Arts, I. C.; van de Putte, B.; Hollman, P. C. J. Agric. Food Chem. 2000, 48, 1752.
8. Corder, R.; Mullen, W.; Khan, N. Q.; Marks, S. C.; Wood, E. G.; Carrier, M. J.;
Crozier, A. Nature 2006, 444, 566.
9. Ito, H.; Kobayashi, E.; Li, S. H.; Hatano, T.; Sugita, D.; Kubo, N.; Shimura, S.; Itoh,
Y.; Tokuda, H.; Nishino, H.; Yoshida, T. J. Agric. Food Chem. 2002, 50, 2400.
10. Pallarès, V.; Fernández-Iglesias, A.; Cedó, L.; Castell-Auví, A.; Pinent, M.;
Ardévol, A.; Salvadó, M. J.; Garcia-Vallvé, S.; Blay, M. Free Radical Biol. Med.
2013, 60, 107.
11. Jeon, S. Y.; Oh, S.; Kim, E.; Imm, J. Y. J. Agric. Food Chem. 2013, 61, 4577.
12. Liu, H.; Zou, T.; Gao, J. M.; Gu, L. Food Chem. 2013, 141, 488.
13. Kim, T. J.; Silva, J. L.; Jung, Y. S. Food Chem. 2011, 126, 116.
14. Yoo, H. H.; Park, J. H. Food Chem. 2012, 133, 998.
a
l
l
a
l
l
tive inhibitors because an increasing concentration of substrate re-
sulted in a family of lines which declined with a common intercept
on the x-axis.27
Thermal processing of pure (+)-catechin was carried out at
121 °C for five different times 1, 2, 3, 6, and 12 h. In order to esti-
mate the relationship between activity and composition of the
reactants, the relative contents of the active compounds was deter-
mined by HPLC (Table 2). While no significant production of 2 and
3, as the most active compounds, were observed at the reacted
mixtures, there was a maximal production of gambiriin D (2) and
catechin [60–8]-catechin (3) after 3 h thermal treatment. As the
reaction time increased, the production of relatively inactive (+)-
epicatechin slightly increased, and reached a maximum of 27% rel-
ative content in the 12 h reactant. On the other hand, the decreas-
ing of (+)-catechin content was readily detected by thermal
processing (Table 2).
15. Bae, J. S.; Kim, T. H. Bioorg. Med. Chem. Lett. 2012, 21, 793.
16. Park, C. H.; Chung, B. Y.; Lee, S. S.; Bai, H. W.; Cho, J. Y.; Jo, C.; Kim, T. H. Bioorg.
Med. Chem. Lett. 2013, 23, 1099.
17. A sample solution of (+)-catechin (1.0 g) in H2O (1.0 L) in chapped vials was
autoclaved at 121 °C for 1, 2, 3, 6, and 12 h, respectively. The inhibitory
activities against a-glucosidase are summarized in Table 3 and the 3 h heating
reactant exhibited the most enhanced inhibitory activity when compared to
parent (+)-catechin. The unreacted parent compound (1) (603 mg) after
heating treatment for 3 h was removed by crystallization and the dried
reactant was directly subjected to column chromatography over a YMC GEL
ODS AQ 120-50S column (1.1 cm i.d. ꢃ 33 cm) with aqueous MeOH, to yield
pure compounds 2 (4.3 mg, tR 18.7 min), 3 (4.5 mg, tR 7.3 min), and 4 (63.7 mg,
tR 11.7 min). HPLC analysis was carried out on a YMC-Pack ODS A-302 column
(4.6 mm i.d. ꢃ 150 mm; YMC Co., Ltd) and the solvent system consisted of a
linear gradient that started with 10% (v/v) MeCN in 0.1% HCOOH/H2O
(detection: UV 280 nm; flow rate: 1.0 ml/min; at 40 °C), increased to 80%
MeCN over 23 min, and then increased to 100% MeCN over 5 min.
18. Sang, S.; Cheng, X.; Stark, R. E.; Rosen, R. T.; Yang, C. S.; Ho, C. T. Bioorg. Med.
Chem. Lett. 2002, 10, 2233.
19. Taniguchi, S.; Kuroda, K.; Doi, K.; Inada, K.; Yoshikado, N.; Yoneda, Y.; Tanabe,
M.; Shinata, T.; Yoshida, T.; Hatano, T. Yakugaku Zasshi 2007, 127, 1291.
20. Cui, C. B.; Tezuka, Y.; Kikuchi, T.; Nakano, H.; Tamaoki, T.; Park, J. H. Chem.
Pharm. Bull. 1992, 40, 889.
21. Stobiecki, M.; Popenda, M. Phytochemistry 1994, 37, 1707.
22. Liang, Y. H.; Ye, M.; Yang, W. Z.; Qiao, X.; Wang, Q.; Yang, H. J.; Wang, X. L.; Guo,
D. A. Phytochemistry 2011, 72, 1876.
During the last decade, structure modification of (+)-catechin to
improve its bioactivity has been performed using microbial and
enzymatic transformations.11,28,29 In addition, previously studies
have revealed that catechin oligomers from natural products are
major inhibitors against
a
-glucosidase.30,31 In this investigation,
the structural modification of (+)-catechin using heating process-
ing showed potentially enhanced anti-diabetic activity against
a-glucosidase and this finding indicated that replacement of the
A-ring at the C-8 of (+)-catechin is correlated with an increase in
biological activity.
23. Slade, D.; Ferreira, D.; Marais, J. P. Phytochemistry 2005, 66, 2177.
24. Tanaka, T.; Mine, C.; Watarumi, S.; Fujioka, T.; Kunihide, M.; Zhang, Y. J.; Kouno,
I. J. Nat. Prod. 2002, 65, 1582.
The results of the current study verify that natural (+)-catechin
(1) is transformed into a new modified products, gambiriin D (2),
along with catechin [60–8]-catechin (3), (+)-epicatechin (4), and
the structure of new compound was elucidated by interpretation
of spectroscopic data. The catechin dimers 2 and 3 exhibited more
25. Gambiriin D (2): Yellow amorphous powder, ½a D25
ꢀ
ꢁ 134:3ꢂ (c 0.1, MeOH); UV
(MeOH) kmax (loge): 280 (2.90), 230 (sh) (3.86), 206 (4.20) nm; CD (MeOH) De:
212 (ꢁ0.49), 241 (+0.11), 282 (ꢁ0.49) nm; 1H and 13C NMR, see Table 1; ESIMS
m/z 601 [M+Na]+, HRFABMS m/z 601.1317 [M+Na]+ (calcd for C30H26O12Na,
601.1322).
26. Assay of a-glucosidase inhibitory activity: a previously reported method with a
minor modification (Kim, Y. M.; Wang, M. H.; Rhee, H. I. Carbohydr. Res. 2004,
339, 715.) was used for the evaluation of the ability of the compounds to inhibit
potent anti-diabetic activity against a-glucosidase than the parent
(+)-catechin. The biotransformation of (+)-catechin by heat pro-
cessing may be a valuable and convenient strategy for structural
modification and the enhancement of activity. More systematic
structural modifications will be performed for the further develop-
ment activity improvement of (+)-catechin.
a
-glycosidase. Briefly,
a
-glucosidase (1
l
L; EC 3.2.1.20) was incubated in
100 mmol Lꢁ1 potassium phosphate buffer (pH 6.8). Sample solution (1
lL)
was premixed with 94 l
L of 100 mmol Lꢁ1 potassium phosphate buffer (pH
6.8). After incubation at 37.5 °C for 20 min, substrate (3 mmol Lꢁ1 p-NPG) was
added to initiate the reaction. The reaction mixture was incubated at 37.5 °C
for 30 min, 100 l
L of 0.1 mol Lꢁ1 Na2CO3 was then added to stop the reaction.
The amount of released p-nitrophenol was measured at 410 nm using a UV
microplate reader (Infinite F200; Tecan Austria GmBH, Grödig, Austria). The
half-maximal inhibitory concentration (IC50) value was calculated by linear
regression analysis of activity under the assay conditions. Acarbose was used
as a positive control and all assays were carried out in triplicate. Kinetic
parameters were determined using the Lineweaver-Burk double-reciprocal
plot at increasing concentrations of substrate and inhibitor.
Acknowledgments
This work was supported by Blue-Bio Industry Regional Innova-
tion Center (RIC08-06-07) at Dongeui University as a RIC program
under the Ministry of Trade, Industry and Energy and Busan city.
27. Woo, H. S.; Kim, D. W.; Curtis-Long, M. J.; Lee, B. W.; Lee, J. H.; Kim, J. Y.; Kang, J.
E.; Park, K. H. Bioorg. Med. Chem. Lett. 2011, 15, 6100.
28. Jiang, H. Y.; Shii, T.; Matsuo, Y.; Tanaka, T.; Jian, Z. H.; Kouno, I. Food Chem.
2011, 129, 830.
Supplementary data
29. López-Serrano, M.; Barceló, A. R. J. Agric. Food Chem. 2002, 50, 1218.
30. Toshima, A.; Matsui, T.; Noguchi, M.; Qiu, J.; Tamaya, K.; Miyata, Y.; Tanaka, T.;
Tanaka, K. J. Sci. Food Agric. 2010, 90, 1545.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
01.027.
31. Wang, H.; Liu, T.; Song, L.; Huang, D. J. Agric. Food Chem. 2012, 60, 3098.