Effect on α-glucosidase inhibition and antioxidant activities of butyrolactone derivatives from Aspergillus terreus MC751

Article abstract of DOI:10.1007/s00044-013-0659-4

Butyrolactone I and II from Aspergillus terreus MC751 as well as three synthetic butyrolactone I derivatives were assessed for α-glucosidase inhibitory and antioxidant activities. Butyrolactone I (1), which has a prenyl side chain and an alpha hydroxy-lactone group, was the most potent α-glucosidase inhibitor and also had antioxidant activities with IC ₅₀ values of 52.17 ± 5.68 and 51.39 ± 3.68 μM, respectively. In contrast, butyrolactone II (2) lacking a prenyl side chain was the most potent antioxidant with an IC₅₀ of 17.64 ± 6.41 μM, but was less active against α-glucosidase. Acetylation of all hydroxyl groups of butyrolactone I significantly decreased both the α-glucosidase inhibitory and antioxidant activity. The prenyl and alpha hydroxy-lactone groups seem to have a synergic effect on the inhibitory activity but not antioxidant activity. This is the first structure-activity relationship report on the α-glucosidase inhibition and antioxidant activity by butyrolactone derivatives.

Full text of DOI:10.1007/s00044-013-0659-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0659-4
ORIGINAL RESEARCH
Effect on a-glucosidase inhibition and antioxidant activities
of butyrolactone derivatives from Aspergillus terreus MC751
•
Rizna Triana Dewi Sanro Tachibana
•
Ahmad Darmawan
Received: 25 December 2012 / Accepted: 29 May 2013
Ó Springer Science+Business Media New York 2013
Abstract Butyrolactone I and II from Aspergillus terreus
MC751 as well as three synthetic butyrolactone I deriva-
tives were assessed for a-glucosidase inhibitory and anti-
oxidant activities. Butyrolactone I (1), which has a prenyl
side chain and an alpha hydroxy-lactone group, was the
most potent a-glucosidase inhibitor and also had antioxi-
dant activities with IC50 values of 52.17 5.68 and
51.39 3.68 lM, respectively. In contrast, butyrolactone
II (2) lacking a prenyl side chain was the most potent
antioxidant with an IC50 of 17.64 6.41 lM, but was less
active against a-glucosidase. Acetylation of all hydroxyl
groups of butyrolactone I significantly decreased both the
a-glucosidase inhibitory and antioxidant activity. The
prenyl and alpha hydroxy-lactone groups seem to have a
synergic effect on the inhibitory activity but not antioxidant
activity. This is the first structure–activity relationship
report on the a-glucosidase inhibition and antioxidant
activity by butyrolactone derivatives.
Introduction
Type 2 diabetes, the most prevalent and serious metabolic
disease in the world today (Kaneto et al., 2010; Shaw et al.,
2010), is characterized by hyperglycemia (Baynes and
Thorpe, 1999; Ceriello, 2012). Intracellular hyperglycemia
induces overproduction of free radicals at the mitochon-
drial level. Hence, overproduction of free radicals is a key
event in the activation of all other pathways involved in the
pathogenesis of diabetic complications (Ceriello, 2012). It
has been shown that antioxidant therapy may help prevent
illnesses caused by oxidative stress by quenching free
radicals, thereby protecting cells and tissues from oxidative
damage (Jung et al., 2008). Inhibition of a-glucosidase is
one approach to suppressing postprandial hyperglycemia,
thereby delaying glucose absorption and controlling the
hyperglycemia in diabetic patients (Gao et al., 2004; Dewi
et al., 2007) and reducing chronic vascular complications
(Meng and Zhou, 2012). Therefore, the ideal antidiabetic
compound should possess both hypoglycemic and antiox-
idant properties (Mayur et al., 2010; Sancheti et al., 2011).
As part of our efforts to isolate potential a-glucosidase
inhibitors and antioxidant compounds from fungi, the ter-
restrial strains, Aspergillus terreus was investigated (Dewi
et al., 2007, 2012a, b). A. terreus has been recovered both
marine and terrestrial sources and is well known for the
production of lovastatin, a cholesterol-lowering agent (Par-
vatkar et al., 2009; Wang et al., 2011). A previous study of A.
terreus MC751 resulted in the isolation of butyrolactone I as
an antidiabetic and antioxidant (Dewi et al., 2012b). Here,
we report the isolation, derivatization, and structural eluci-
dation of butyrolactone derivatives from A. terreus MC751.
The a-glucosidase inhibitory and antioxidant activities, as
well as structure–activity relationship (SAR) studies of
butyrolactone I derivatives are also described.
Keywords Aspergillus terreus MC75 ꢀ
Butyrolactones derivatives ꢀ
a-Glucosidase inhibitory activity ꢀ Antioxidant
R. T. Dewi
The United Graduate School of Agriculture Science, Ehime
University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan
S. Tachibana (&)
Department of Applied Biosciences, Faculty of Agriculture,
Ehime University, 3-5-7 Tarumi, Matsuyama,
Ehime 790-8566, Japan
e-mail: tatibana@agr.ehime-u.ac.jp
R. T. Dewi ꢀ A. Darmawan
Research Center for Chemistry, Indonesian Institute of Sciences,
Kawasan PUSPIPTEK Serpong, Tangerang Selatan,
Banten 15314, Indonesia
123

Med Chem Res
Materials and methods
reversed-phase HPLC, using the isocratic mobile phase of
MeOH/water (30:70) at a flow rate of 1 ml min-1 with
300 nm as the detection wavelength.
General instrument and chemicals
Melting points were measured on a Yanaco micro melting
point apparatus (Yanaco Co., Ltd., Kyoto, Japan) and were
uncorrected. Optical rotation values were measured with a
Jasco P-2100 polarimeter. UV–Vis absorption spectra of
the active compounds in methanol were recorded on a
Hitachi U-1600 spectrophotometer. HPLC was conducted
with Waters (USA), UV–Vis spectrophotometric detector,
and reversed-phase C18 column (Type UG 120, 5 lm, size
4.6 mm ID 9 250 mm). The mass spectra of the com-
pound were measured with high-resolution FAB-MS. The
nuclear magnetic resonance (NMR) spectra were recorded
at 500 MHz for 1H and 125 MHz for 13C on a JEOL JNM-
ECA 500 with TMS as an internal standard. HMQC and
HMBC techniques were used to assign correlations
Fermentation on CzY, extraction, and isolation
Five disks (5 mm) of fungal mycelia were used to inoculate
150 ml of seed medium in an Erlenmeyer flask (500 ml) in
CzY. Each culture was maintained at 25 °C under static
conditions for 15 days. 15-liter cultures were separated
from mycelia by filtration. The filtrate was extracted with
EtOAc (5 9 1 l), and the extract was dried under reduced
pressure to obtain a brown solid (2.18 g). The filtrate
extract was fractioned by column chromatography (CC) on
silica gel using a stepwise gradient from 100 % n-hexane,
to 100 % EtOAc, to 50 % EtOAc in MeOH to obtain nine
fractions (F1–F9). Fractions 4 and 5 were combined and
rechromatographed on a silica gel column eluted using a
stepwise gradient from 80 % n-hexane in EtOAc, to 100 %
EtOAc to give compound 1 (500 mg). F7 (280 mg) was
subjected to CC on silica gel (EtOAc/n-hexane, step gra-
dient elution from 20:80 to 0:100) followed by preparative
TLC (CHCl3:Acetone, 5:1) to give compound 2 (60 mg).
Compound 1: Yellowish gum, UV (MeOH) kmax nm (log
1
between H and 13C signals. The chemical shift values (d)
are given in parts per million (ppm), and coupling constant
(J) in Hz. TLC was run on silica gel 60 F254 pre-coated
plates (Merck 5554) and spots were detected by UV light.
a-Glucosidase [(EC 3.2.1.20)] type I: from Sachar-
omyces cereviceae, p-nitrophenyl a-D-glucopyranoside (p-
NPG), DMSO, 1,1-diphenyl-2-picrylhydrazyl (DPPH),
quercetin dihydrate, and Silica gel (60–200 mesh Wako
gel) were purchased from Wako Pure Chemical Industries,
Ltd (Osaka, Japan). All the solvents used in this study were
purchased from Wako Pure Chemicals and distilled prior to
use.
22:5
e): 307.5 (4.44). ½aꢁD ?68.333 (c = 0.3, MeOH). 1H NMR
and 13C NMR data are presented in Table 2. HRFABMS:
[M?H]? m/z 425.1607, calculated for C24H25O7.
Compound2:Colorlessgum, UV(MeOH)kmax nm(loge):
28:7
1
307.5 (4.17). ½aꢁD ?4.78 (c = 0.45, Acetone). H NMR
(Acetone-d6) d 3.4 (2H, d, J = 14.8, H-6), 3.76 (3H, s,
5-OCH3), 6.59 (2H, d, J = 8.0, H-300,500), 6.69 (2H, d,
J = 8.0, H-20,60), 6.98 (2H, d, J = 8, H-30,50), 7.66 (2H, d,
J = 8.0, H-200,600). 13C NMR (Acetone-d6) d 170.94 (C-5),
168.83 (C-1), 158.91 (C-40), 157.38 (C-400), 139.28 (C-2),
132.33 (C-20 and C-60) 130.12 (C-200 and C-600), 128.06 (C-
100), 124.89 (C-10), 122.78 (C-3), 115.47 (C-30 and C-50),
116.66 (C-300 and C-500), 85.97 (C-4), 53.79 (OCH3), 39.18
(C-6). FAB-MS: [M?H]? m/z 357 for C19H17O7.
Microorganisms and culture conditions
A. terreus MC751 was obtained from the Research Center
for Chemistry-Indonesian Institute of Sciences. The stock
culture of A. terreus MC751 was grown on PDA at 25 °C
for 7 days. Optimization of the culture medium was con-
ducted by culturing the A. terreus MC751 in five different
media: PMP (2.4 % potato dextrose broth, 1 % malt
extract, and 0.1 % peptone), PDB (2.4 % potato and 2 %
dextrose), and ME (1 % glucose, 0.1 % peptone, and 1.5 %
malt extract), each culture broth was incubated at 25 °C
with shaking at 100 rpm for 7 days. Czapek-dox (Cz) (3 %
sucrose, 0.2 % sodium nitrate (NaNO3), 0.1 % K2HPO4,
0.05 MgSO4ꢀ7H2O, 0.05 % KCl, and 0.001 % ferrous
sulfate), incubated for 10 days under static conditions at
25 °C. Medium Cz, with addition of 0.5 % yeast extract
(CzY) incubated for 15 days under static conditions. The
culture broth was prepared by 3 rounds of extraction for
each broth (200 ml) with EtOAc (100 ml), dried by rotary
evaporator, and assessed for a-glucosidase inhibitory and
DPPH radical scavenging activities. The butyrolactone I
content of the extract was investigated by analytical
Preparation of butyrolactone I derivatives
Derivatization of butyrolactone I (1) was conducted by
cyclization and acetylation. Cyclization of 1 was carried out
according to the method of Parvatkar et al. (2009). Com-
pound 1 (90 mg) was dissolved in MeOH (10 ml) containing
conc. HCl (0.2 ml). The mixture was stirred at room tem-
perature for 2 h until complete conversion of compound 1 as
indicated by TLC. The solvent was removed under vacuum
and the resulting residue was extracted with CHCl3. The
CHCl3 soluble fraction was separated by preparative TLC
elution with n-hexane:EtOAC (2:3), to yield compound 3
(60 mg, 67 %). The butyrolactone I acetate was obtained
123

Med Chem Res
treating 212 mg of compound 1 with acetic anhydride in
pyridine. After the usual work-up, compounds 4 (90 mg,
32 %) and 5 (60 mg, 42 %) were obtained.
results were expressed as mean values SDs. Quercetin
was used as reference standard.
Compound 3: Yellowish solid crystal, mp: 93–95 °C,
UV (MeOH) kmax nm (log e): 307.5 (4.92); 226.5 (4.85);
DPPH free radicals scavenging activity
28:7
½aꢁD ?88.73 (c = 0.58, CHCl3). 1H NMR (Acetone-d6) d
The antioxidant activities of compounds 1–5 were evalu-
ated according to the method of Yen and Chen (1995).
Aliquots of samples in MeOH (2 ml) at various concen-
trations were each mixed with 0.5 ml of DPPH 1 mM in
MeOH. All the mixtures were shaken vigorously and left to
stand at room temperature for 30 min in the dark. The
absorbance of the reaction solutions were measured spec-
trophotometrically at 517 nm. IC50 value was the effective
concentration in which DPPH 50 % of radicals were
scavenged and was obtained by interpolation with linear
regression analysis. A lower IC50 value indicated a greater
antioxidant activity.
1.16 (6H, s, H1000,1100), 1.63 (2H, br, J = 6.5, H800), 2.50
(2H, m, H700), 3.36 (2H, J = 15, H6), 3.68 (3H, s, 5-OMe),
6.38 (1H, s, H200), 6.50 (1H, s, H500), 6.56 (1H, d, J = 3.4,
H600), 6.86 (2H, d, J = 9.0, H30, 50), 7.65 (2H, d, J = 9.0,
H20, 60). 13C NMR (Acetone-d6) d 173.52 (C-5), 172.50 (C-
1), 157.98 (C-40), 154.25 (C-400), 144.91 (C-2), 74.95 (0C0 -
900), 126.37 (C-200), 129.61 (C-60 and C-20), 125.47 (C-6 ),
132.99 (C-10), 117.48 (C-300), 130.55 (C-100), 33.75 (C-800),
121.19 (C-3), 116.63 (C-50 and C-30), 123.74 (C-500), 86.75
(C-4), 53.77 (OCH3), 40.01 (C-6), 23.34 (C-700), 27.41 (C-
1000), 27.62 (C-1100). FAB-MS: [M?H]? m/z 425 for
C24H25O7.
Compound 4: Colorless gum, UV (CHCl3) kmax nm (log
20
e): 281 (3.74). ½aꢁD ?7.154 (c = 1.3, CHCl3). FAB-MS:
Results and discussion
[M?H]? m/z 551 for C30H31O10. H NMR and 13C NMR
are presented in Table 2.
1
Isolation and characterization of active compounds
Compound 5: Colorless gum, UV (CHCl3) kmax nm (log
20
e):294 (3.84).½aꢁD ?39.957 (c = 1.15, CHCl3). FAB-MS:
In the previous study it was reported that butyrolactone I
from A. terreus MC751 cultured in Cz broth exhibited
potential activity as an antidiabetic and antioxidant (Dewi
et al., 2012b). In order to enhance the a-glucosidase
inhibitory and antioxidant activities and to increase the
production of the secondary metabolites we initially
examined EtOAc extracts from A. terreus MC751 cultured
in PDB, PMP, ME, Cz, and CzY media as shown in
Table 1. The results revealed that butyrolactone was effi-
ciently produced by the fungal strains when yeast extract
was added to the medium. The increase in butyrolactone I
led to enhanced activities against both a-glucosidase and
DPPH free radicals by the EtOAc extract of CzY compared
to other extracts. In an attempt to isolate and characterize
the constituent compounds, the culture strain was scaled up
to 15 l using CzY under static conditions for 15 days at
25 °C. Purification of the culture filtrate extract using CC
[M?H]? m/z 509, for C28H29O9. H NMR and 13C NMR
are presented in Table 2.
1
Biological activities
a-Glucosidase inhibitory activity
The inhibitory activity for a-glucosidase was assessed as
reported by Kim et al. (2004). Briefly, p-NPG (250 ll,
3 mM) and 495 ll of 100 mM phosphate buffer (pH 7.0)
were added to a tube containing 5 ll of sample dissolved in
DMSO at various concentrations. The reaction mixture was
pre-incubated for 5 min at 37 °C, the reaction was started
by adding 250 ll of a-glucosidase (0.065 U/ml), and the
incubation was continued for 15 min. The reaction was
stopped by adding 1 ml of 0.2 M Na2CO3. The inhibitory
effect on a-glucosidase activity was determined by mea-
suring the amount of p-nitrophenol released at k 400 nm.
Individual blanks for test samples were prepared to correct
background absorbance where the enzyme was replaced
with 250 ll of phosphate buffer. The percent inhibition of
a-glucosidase inhibitory and antioxidant activity was
Table 1 Preliminary experiment on ethyl acetate extracts of A. terreus
MC751
Sample
IC50 (lg/ml)a
Butyrolactone I
(% w/w)a
DPPH
a-GIs
PMP
PDB
ME
Cz
40.13 3.23
48.39 2.21
69.80 5.30
24.05 1.56
17.47 2.63
50.32 2.42
40.39 3.14
[100
21.83
22.05
13.64
34.66
54.31
assessed using the following formula:
%
Inhibi-
tion = (A - B)/A 9 100, where A was the absorbance of
the control reaction and B was the absorbance in the
presence of the sample. The IC50 were calculated from the
main inhibitory values by applying logarithmic regression
analysis. The assays were carried out in triplicate and the
6.45 1.71
4.32 0.64
CzY
a
Data are shown as mean SD from three independent experiments
123

Med Chem Res
and preparative TLC on silica gel led to compound 1 as a
yellowish gum and compound 2 as a colorless gum.
Characterization of the active compounds by spectrometric
methods revealed compound 1 to be fully consistent with
(Parvatkar et al., 2009). Compound 1 was treated with
acetic anhydride in pyridine to give butyrolactone I
2,40,400-triacetate (4) [methyl-4-acetoxy-2-(4-acetoxy-3-
(3-methylbut-2-enyl)benzyl)-3-(4-acetoxyphenyl)-5-oxo-
butyrolactone
I
[(methyl-4-hydroxy-2-(4-hydroxy-3-(3-
2,5-dihydrofuran-2-carboxylate] and butyrolactone
I
methylbut-2-enyl)benzyl)-3-(4-hydroxyphenyl)-5-oxo-2,5-
dihyrofuran-2-carboxylate] (Dewi et al., 2012b), whereas
compound 2 coincided with butyrolactone II [methyl-4-
hydroxy-2-(4-hydroxybenzyl)-3-(4-hydroxyphenyl)-5-oxo-
2,5-dihydrofuran-2-carboxylate] (Nitta et al., 1983; Rao
et al., 2000). In order to verify the structure, compound 1
was treated with alcoholic hydrochloric acid at room
temperature, which afforded compound 3. The molecular
formula of compound 3 was found to be identical with
that of 1 based on FAB-MS (C24H25O7). The MS and
NMR spectral data of compound 3 were fully consistent
with those for Aspernolide A [methyl 2-((2,2-dimethylch-
roman-6-yl)methyl)-4-hydroxy-3-(4-hydroxyphenyl)-5-oxo-
2,5-dihydrofuran-2-carboxylate] reported in the literature
40,400-diacetate (5) [methyl 2-(4-acetoxy-3-(3-methylbut-
2-enyl)benzyl)-3-(4-acetoxyphenyl)-4-hydroxy-5-oxo-2,5-
dihydrofuran-2-carboxylate]. The structures of compound
4 and 5 were determined from MS and NMR (1 and 2D)
data. A comparison of the 1H NMR spectra of com-
pounds 1, 4, and 5 showed a similar pattern. Compounds
1, 4, and 5 showed only minor differences. 1,4-Disub-
stituted and 1,2,4-trisubstituted phenols in compound 1
were shifted to low field signals in compounds 4 and 5
(Table 2), indicating that acetylation occurred. All iso-
lated and derivative compounds from A. terreus MC751
in CzY are shown in Fig. 1. The compounds were
examined for a-glucosidase inhibitory and antioxidant
activities.
1
Table 2 13C and H data of
1 (Acetone-d6)
4 (CDCl3)
5 (Acetone-d6)
butyrolactone 1 (1) and
13C
1H
13C
1H
13C
1H
acetylation derivatives 4 and 5
(500 and 125 Hz, respectively,
dH and dC in ppm, J in Hz)
1
169.14
137.14
127.88
86.07
–
169.46
136.83
126.25
86.62
–
168.69
139.22
127.43
85.82
–
2
–
–
–
3
–
–
–
4
–
–
–
5
169.75
38.58
–
169.38
39.29
–
169.64
38.94
–
6
3.52 d (14.8)
3.68 d (14.8)
3.54 d (14.5)
10
122.28
129.59
116.00
156.53
124.63
134.51
129.25
153.26
115.18
126.48
29.20
–
144.67
123.79
130.35
153.73
131.24
132.60
133.51
149.43
123.00
129.73
29.08
–
126.64
122.38
129.03
151.13
130.68
132.22
129.15
148.23
121.96
129.16
28.62
–
20,60
30,50
40
100
200
300
400
500
600
7.12 d (9.0)
7.35 d (8.8)
7.20 d (8.8)
6.92 d (9.0)
7.58 d (8.8)
7.74 d (8.8)
–
–
–
–
–
–
6.53 dd (3.4)
–
6.73 dd (3.4)
6.78 dd (3.4)
–
–
–
–
–
6.61 d (8.0)
6.65 d (8.0)
3.14 d (6.8)
5.10 b (7.3)
–
6.85 d (8.0)
6.67 d (8.0)
6.74 d (8.0)
3.05 d (6.9)
5.00 br (7.0)
–
6.79 dd (8.0)
700
800
900
3.09 d (6.9)
121.50
131.87
17.75
122.49
134.09
17.88
5.02 br (7.0)
121.37
133.40
17.86
–
1000
1100
5-OCH3
40-OAc
C=O
400-OAc
C=O
2-OAc
C=O
1.66 s
1.61 s
1.67 s
3.86 s
2.24 s
–
1.58 s
25.70
1.70 s
25.88
25.78
1.67 s
53.58
3.78 s
54.42
54.84
3.74 s
20.23
21.34
2.33 s
167.05
20.81
169.28
20.99
–
2.24 s
–
2.25 s
165.56
21.07
169.60
2.30 s
–
169.09
123

Med Chem Res
O
OCH3
1 Butyrolactone I
5
R3O
4"
1"
6
R1=R2=R3=H
1'
OR1
4'
4
2"
4 Butyrolactone I 2,4’,4”-triacetate
R1=R2=R3= CH3CO
O
8"
1
7"
2'
OR2
5 Butyrolactone I 4’,4”-diacetate
R1=R3= CH3CO R2=H
O
10'
11"
O
OCH3
HO
OH
O
2
ButyrolactoneII
OH
O
O
OCH3
O
OH
O
OH
O
3
AspernolidesA
Fig. 1 Structure of butyrolactone I derivatives
Table 3 a-Glucosidase inhibitory and antioxidant activity of com-
pounds 1–5 from A. terreus MC751
Table 4 Inhibition constant (Ki value) and mode of compounds 1, 2,
3, and 5 from A. terreus MC751 against S. cereviceae a-glucosidase
Compound
IC50 (lM)a
Compound
Inhibition mode
Ki (lM)
a-Glucosidase
DPPH
1
Mix inhibition
Noncompetitive
Uncompetitive
Uncompetitive
Mix inhibition
70.51
152.64
235.80
127.88
27.13
2
1
52.17 5.68
96.01 3.70
175.18 5.95
[300
51.39 3.68
17.64 6.41
47.55 3.08
ND
3
2
5
3
Quercetin
4
5
84.18 8.98
14.6 3.72
ND
Quercetin
39.63 5.21
(1–5) and quercetin are tabulated in Table 3. As previously
reported, compound 1 was a potent inhibitor of the a-glu-
cosidase with an IC50 of 52.17 lM (Dewi et al., 2012b). In
contrast, compound 2, which lacks a prenyl side chain,
exhibited less inhibitory activity. Converting the prenyl
side chain to a dihydropyran ring in compound 3 caused a
significant decrease in the inhibitory activity (Table 3).
Hence, it was assumed that the prenyl side chain of com-
pound 1 contributed to the inhibitory effect. However, the
substitution of any hydroxyl group with an acetyl group in
butyrolactone I led to a dramatic reduction in inhibitory
activity in compound 4, a finding consistent with Gao et al.
(2004) which reported that the removal of hydroxyls in
flavonoids decreased a-glucosidase inhibitory activity.
ND not detected
a
IC50 value are shown as mean SD from three independent
experiments
a-Glucosidase inhibitory activity
We investigated the inhibitory activities of compounds 1–5
against a-glucosidase from S. cereviceae. Quercetin was
used as a positive control based on the reports that it is a
phenolic compound with a stronger inhibitory effect on
a-glucosidase from S. cereviceae than acarbose (Tadera
et al., 2006; Li et al., 2009; Jo et al., 2009). The a-glu-
cosidase inhibitory activities of butyrolactone derivatives
123

Med Chem Res
(a)
(a)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
0
0.24
0.12
0
0.12
0.06
1/[S] (mM)-1
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
1/[S](mM)-1
(b)
(b)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
0.2
0.1
0
0.28
0.14
1/[S] (mM)-1
-1.0
-0.5
0.0
0.5
[1/S] (mM)-1
1.0
1.5
Fig. 3 Lineweaver–Burk plots of compounds 3 (a) and 5 (b) against
S. cereviceseae
Fig. 2 Lineweaver–Burk plots of compounds 1 (a) and 2 (b) against
S. cereviceseae a-glucosidase at different concentrations of pNPG
1/V increased with the concentration of compound 2, but
the Km remained constant. The results suggest that com-
pound 2 displayed non-competitive inhibition which indi-
cated that it binds to a site other than the active site of the
a-glucosidase enzyme (Bu et al., 2010). The inhibitory
mechanisms of compounds 3 and 5 are presented in
Fig. 3a, b. The results showed a straight line parallel to the
plot of 1/V versus 1/[S] indicating uncompetitive inhibition
(Takahasi and Miyazawa, 2012). Based on the analysis, an
inhibition constant (Ki) value was obtained (Table 4). This
result indicated that the side chain group in the aromatic
ring of butyrolactone influenced the mechanism of inhibi-
tion. Compound 4 was not examined due to its poor
inhibitory activity against a-glucosidase.
Compound 5, which retained one OH-bond as an alpha
hydroxy-lactone, showed significantly higher activity
against a-glucosidase than compound 4 (Table 3). This is
consistent with our suggestion that the hydroxy-lactone
moiety in butyrolactone I contributed to the inhibitory effect
(Dewi et al., 2012b). Based on the a-glucosidase inhibitory
activities of compounds 1–5, SAR inference could be made.
Compounds 1 and 5 showed stronger activity than the others,
which suggested that the inhibitory effect of these butyro-
lactones was influenced by both the prenyl side chain and
alpha hydroxy-lactone group. The influence of the prenyl
side chain in butyrolactones was reported previously (Par-
vatkar et al., 2009; Cazar et al., 2005); however, no SAR
study of the a-glucosidase inhibitory activity of butyrolac-
tone I derivatives has been reported before.
DPPH free radicals scavenging activity
The inhibitory mechanisms of all the active compounds
were analyzed using Lineweaver–Burk plots. a-Glucosi-
dase solution (0.065 U/ml) was incubated with increas-
ingly higher concentrations of substrate (pNPG) with and
without an inhibitor. The results showed various mecha-
nisms of action (Table 4). Compound 1 exhibited mixed-
inhibition close to non-competitive type (Fig. 2a) consis-
tent with our previous report (Dewi et al., 2012b). The
mechanism action of compound 2 is shown in Fig. 2b. The
A critical feature of antioxidant is the ability to scavenge free
radicals (Jung et al., 2008). Hence, antioxidant activities of
compounds 1–5 were evaluated in the DPPH free radical
scavenging assay. Compounds 1–3 showed potential scav-
enging activity while compounds 4 and 5 did not (Table 3).
The antioxidant activity may originate from the phenolic
groups. Compound 2, which lacks a prenyl side chain, was
the most powerful antioxidant. This result was consistent
with the previous reports (Boiko et al., 2006); Osorio et al.,
123

Med Chem Res
Aspergillus terreus on a-glucosidase activity and postprandial
hyperglycemia. PJBS 10:3131–3135
Dewi RT, Tachibana S, Kazutaka I, Ilyas M (2012a) Isolation of
antioxidant compounds from Aspergillus terreus LS01. JMBT
4:010–014
Dewi RT, Tachibana S, Darmawan A (2012b) Antidiabetic and
antioxidative activities of butyrolactone I from Aspergillus
terreus MC751. WASET 70:882–887
Gao H, Nishioka T, Kawabata J, Kasai T (2004) Structure-activity
relationships for a-glucosidase inhibition of baicalein, 5,6,7-
tryhdroxyflavone: the effect of A-ring substitution. Biosci
Biotechnol Biochem 68:357–369
2012) that a prenylated phenolic group in compound 1
decreased antioxidant activity. Cyclization of the prenyl
group in compound 3 also decreased the antioxidant activity.
In the case of compounds 4 and 5, replacement of an OH
group with an acetyl group dramatically decreased the
antioxidant activity (Boiko et al., 2006; Osorio et al., 2012).
Hence, it was concluded that the absence of a prenyl side
chain increased the activity and acetylation appeared to be
detrimental to the antioxidant activity.
Jo SH, Ka EH, Lee HS, Jang HD, Kwon YI (2009) Comparison of
antioxidant potential and rat intestinal a-glucosidases inhibitory
activities of quercetin, rutin, and isoquercetin. IJARNP 2:52–60
Jung JY, Lee IK, Seok SJ, Lee HJ, Kim YH, Yun BS (2008)
Antioxidant polyphenols from the mycelia culture of the
medicinal fungi Inonotus xeranticus and Phellinus linteus.
J App Microbiol 104:1824–1832
Kaneto H, Katakani N, Matsuhisa M, Matsuoka T (2010) Role of
reactive oxygen species in the progression of type 2 diabetes and
atherosclerosis. Mediat Inflamm 2010:1–11
Kim YM, Wang MH, Rhee HI (2004) A novel a-glucosidase inhibitor
from pine bark. Carbohydr Res 339:715–717
Li YQ, Zhou FC, Gao F, Bian JS, Shan F (2009) Comparative
evaluation of quercetin, isoquercetin and rutin as inhibitor of a-
glucosidase. J Agric Food Chem 57:11463–11468
Mayur B, Sandesh S, Shruti S, Yum SS (2010) Antioxidant and a-
glucosidase inhibitory properties of Carpesium abrotanoides L.
J Med Plant Res 4:1547–1553
Meng P, Zhou X (2012) a-Glucosidase inhibitory effect of a
bioactivity guided fraction GIB-638 from Streptomyces fradiae
PWH638. Med Chem Res 21:4422–4429
Nitta K, Fujita N, Yoshimura T, Arai K, Yamamoto Y (1983)
Metabolic products of Aspergillus terreus. IX1) biosynthesis of
butyrolactone derivatives isolated from strains IFO 8835 and
4100. Chem Pharm Bull 31:1528–1533
Conclusion
In conclusion, two butyrolactone compounds (1 and 2), and
three butyrolactone I derivatives (3, 4, and 5) were studied
for a-glucosidase inhibitory and antioxidant activities.
Butyrolactone I, which has a prenyl side chain and alpha
hydroxy-lactone, was found to be the most potent antidi-
abetic. In contrast, butyrolactone II (2) lacking a prenyl
side chain was the most potent antioxidant with an IC50 of
17.64 6.41 lM, but was less active against the a-glu-
cosidase enzyme. Acetylation of all hydroxyl groups of
butyrolactone I (1) significantly decreased both the activity
against a-glucosidase and the antioxidant activity. The
prenyl and alpha hydroxy-lactone groups appear to have a
synergic effect on the a-glucosidase inhibitory activity but
not the antioxidant activity. This is the first report on the
SAR of butyrolactone derivatives against a-glucosidase
inhibitory and DPPH radical scavenging activities.
´
Osorio M, Aravena J, Vergara A, Tabirga L, Baeza E, Catalan K,
Gonzales C, Carvajal M, Carrasco H, Espinoza L (2012)
Acknowledgments The authors are grateful to Sofa Fajriah M.Sc.
and Megawati S.Si. from the Research Center for Chemistry-Indo-
nesian Institute of Sciences, for NMR spectral measurements. We also
thank Dr. Satoshi Yamauchi of the Faculty of Agriculture, Ehime
University for measurements of optical rotation and Dr. Kazutaka Itoh
for support and assistance with this research.
´
synthesis and DPPH radical scavenging activity of prenylated
phenol derivatives. Molecules 17:556–570
Parvatkar RR, D’Souza C, Tripathi S, Naik CG (2009) Aspernolides
A and B, butenolides from a marine-derived fungus Aspergillus
terreus. Phytochemistry 70:128–132
Rao KV, Sadhukhan AK, Veerender M, Mohan EVS, Dhanvantri SD,
Sitaramkumar S, Babu MJ, Vyas K, Reddy OG (2000) Butyro-
lactones from Aspergillus terreus. Chem Pharm Bull 48:559–562
Sancheti S, Sancheti S, Bafna M, Seo SY (2011) 2,4,6-Trihydroxy-
benzaldehyde as a potent antidiabetic agent alleviates postpran-
dial hyperglycemia in normal and diabetic rats. Med Chem Res
20:1181–1187
References
Baynes JW, Thorpe SR (1999) Perspective in diabetes, role of
oxidative stress in diabetic complications, a new perspective on
an old paradigm. Diabetes 48:1–9
Shaw JE, Sicree RA, Zimmet PZ (2010) Diabetes atlas, Global
estimates of the prevalence of diabetes for 2010 and 2030.
Diabetes Res Clin Pract 87:4–14
Boiko MA, Terakh EI, Prosenco AE (2006) Relationship between the
electrochemical and antioxidant activities of alkyl-subtituted
phenols. Kinet Catal 47:677–681
Tadera K, Minami Y, Takamatsu K, Matsuoka T (2006) Inhibitor of
a-glucosidase and a-amylase by flavonoids. J Nutr Sci Vitaminol
52:149–153
Takahasi T, Miyazawa M (2012) Synthesis and structure-activity
relationships of serotonin derivatives effect on a-glucosidase
inhibition. Med Chem Res 21:1762–1770
Wang Y, Zheng J, Liu P, Wang w, Zhu W (2011) Three new
compounds from Aspergillus terreus PT06-2 grown in a high salt
medium. Marine Drugs 9:1368–1378
Yen GC, Chen HY (1995) Antioxidant activity of various tea extracts
in relation to their antimutagenicity. J Agric Food Chem
43:27–32
Bu T, Liu M, Zheng L, Guo Y, Lin X (2010) a-Glucosidase inhibition
and the in vivo hypoglycemic effect of butyl-isobutyl-ptalate
derived from the Laminaria japonica rhizoid. Phytother Res
24:1588–1591
Cazar ME, Hirschmann GS, Astudillo L (2005) Antimicrobial
butyrolactone I derivatives from the Ecuadorian soil fungus
Aspergillus terreus Thorn. var terreus. Word J Microbiol
Biotechnol 21:1067–1075
Ceriello A (2012) The emerging challenge in diabetes: the ‘‘metabolic
memory’’. Vasc Pharmacol 57:133–138
Dewi RT, Iskandar Y, Hanafi M, Kardono LBS, Angelina M,
Dewijanti DI, Banjarnahor SDS (2007) Inhibitory effect of koji
123