A novel amino-oligosaccharide isolated from the culture of Streptomyces strain PW638 is a potent inhibitor of α-amylase

Article abstract of DOI:10.1016/j.carres.2011.06.005

A novel amino-oligosaccharide, named SF638-1, was isolated from the culture filtrate of the Streptomyces strain PW638. Its chemical structure was determined by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and two-dimensional nuclear magnetic resonance spectroscopy. The novel compound was a mixed inhibitor of human pancreatic α-amylase, with a K_i value in the same order of magnitude as that of the α-amylase inhibitor, acarbose. SF638-1 inhibited starch hydrolysis and glucose transfer in vitro, and suppressed postprandial blood glucose elevation in vivo. These results suggest that SF638-1 may be a potent antidiabetic agent.

Full text of DOI:10.1016/j.carres.2011.06.005

Carbohydrate Research 346 (2011) 1898–1902
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
A novel amino-oligosaccharide isolated from the culture of Streptomyces
strain PW638 is a potent inhibitor of a-amylase
Peng Meng a, Yuanqiang Guo a, Qi Zhang a, Jie Hou a, Fang Bai a, , Peng Geng b, , Gang Bai a,c
⇑
⇑
a College of Pharmacy, Nankai University, 94 Weijin Road, Tianjin 300071, People’s Republic of China
b Tianjin State Laboratory of Cellular and Molecular Immunology, Basic Medical College, Tianjin Medical University, 22 Qixiangtai Road, Tianjin 300070, People’s Republic of China
c College of Life Sciences, Nankai University, Tianjin 300071, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel amino-oligosaccharide, named SF638-1, was isolated from the culture filtrate of the Streptomyces
strain PW638. Its chemical structure was determined by electrospray ionization tandem mass spectrom-
etry (ESI-MS/MS) and two-dimensional nuclear magnetic resonance spectroscopy. The novel compound
Received 19 April 2011
Received in revised form 31 May 2011
Accepted 6 June 2011
was a mixed inhibitor of human pancreatic
a-amylase, with a Ki value in the same order of magnitude as
Available online 13 June 2011
that of the -amylase inhibitor, acarbose. SF638-1 inhibited starch hydrolysis and glucose transfer
a
in vitro, and suppressed postprandial blood glucose elevation in vivo. These results suggest that
SF638-1 may be a potent antidiabetic agent.
Keywords:
a-Amylase inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
Amino-oligosaccharide
SF638-1
Streptomyces
Diabetes
1. Introduction
a
-amylase. We have previously isolated and purified six amino-
oligosaccharides (acarviostatins I03, II03, III03, IV03,8 II23, and
II1310
from the culture filtrate of Streptomyces coelicoflavus
ZG0656.
We had investigated the inhibitory effect of the GIB638 com-
plex (extracted from Streptomyces sp. PW638) on
-amylase.11 Its
potent inhibitory activity encouraged us to purify compounds from
this complex. Here, we describe the isolation of a novel -amylase
a-Amylase enzymes (EC 3.2.1.1) are highly evolutionarily
)
conserved in nature, being present in organisms ranging from
microbes to mammals. They catalyze the hydrolysis of
a
-(1?4)-
maltodextrins.1 In humans, the pancreatic
responsible for cleaving large malto-oligosaccharides to smaller
oligosaccharides, which are substrates for intestinal -glucosi-
D
-glycosidic linkages of starch, glycogen, and various
a
a
-amylase (HPA)2 is
a
a
inhibitor designated as SF638-1. The bioactivity of the new amino-
oligosaccharide was also tested using in vitro and in vivo enzymol-
ogy techniques.
dases. This digestion process is important for glucose absorption
from the intestine to the blood, and in fact, HPA activity has been
correlated to postprandial blood glucose levels.3–5 Indeed, inhibi-
2. Results and discussion
tors of
a-amylase have been successfully used in the treatment
of diseases such as diabetes and obesity where control of blood
glucose level is essential.6 Arguably, the most studied inhibitor of
The complex GIB638 (524.6 mg, half-maximal inhibitory
concentration (IC50) = 654.53 lg/mL) was isolated from the culture
a
-amylase is the naturally occurring and commercially available
drug, acarbose,7 which has a Ki value in the low nanomolar
filtrate of Streptomyces strain PW638 as described in Section 3. It
was then separated by semi-preparative high performance liquid
chromatography (HPLC) to afford SF638-1. Initially, a reversed-
phase HPLC was used, giving a fraction at 12.8 min with inhibitory
range.2,8
A great number of medicinal drugs have been derived from
natural products. The richness and diversity of the secondary
metabolism of Streptomyces have made these organisms valuable
sources of antibiotics and other bioactive molecules.9 A key
research focus of our group is the search for bioactive compounds
activities against HPA. This fraction (150.6 mg, IC50 = 33.15 lg/mL)
was further separated on a cation-exchange HPLC. A chromato-
graphic peak with strong inhibitory activity against HPA was
collected at 3.7 min. Although three other chromatographic peaks
were evident at later time points, none of these contained signifi-
cant inhibitory activity against HPA. After salt-removal and
lyophilization, the compound, SF638-1, was obtained from the
that inhibit enteric enzymes including
a-glucosidase and
⇑
Corresponding authors. Tel.: +86 22 23508371 (F.B.); +86 22 23542649 (P.G.).
E-mail addresses: baifang1122@nankai.edu.cn (F. Bai), gengpeng_2001@163.com
3.7 min fraction (45 mg, IC50 = 5.06 lg/mL). The purity of SF638-1
(P. Geng).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.06.005

P. Meng et al. / Carbohydrate Research 346 (2011) 1898–1902
1899
was determined by positive ion mode electrospray ionization mass
spectrometry (ESI-MS) analysis. Detailed procedures are listed in
Section 3.
we concluded that the ions at m/z 505 ([MꢀH2O+2H]+) and m/z
487 ([Mꢀ2H2O+2H]+) indicated the ordinal loss of one and two
water molecules from the parent ion, respectively. The daughter
ion at m/z 343 (Y2 and B2) was due to the loss of one glucose unit
from the reducing or non-reducing end of the original molecule.
The product ion at m/z 325 ([Y2ꢀH2O]+ and [B2ꢀH2O]+) was consis-
tent with the further loss of one water molecule from fragments Y2
and B2. These features of the MS/MS spectrum are predictive of the
SF638-1 structure outlined in Figure 1.
2.1. Structural determination of SF638-1
SF638-1 was obtained as a white amorphous powder. The
molecular formula was determined to be C18H35NO16 based on
data from combined positive high-resolution electrospray ioniza-
tion mass spectrometry (HR-ESI-MS) (Anal. Found [M+H]+
522.2206, Calcd 522.2201) and nuclear magnetic resonance
(NMR). The infrared (IR) spectrum of SF638-1 was characterized
by broad bands at 3371 and 1023 cmꢀ1. Complete acidic hydrolysis
followed by monosaccharide analysis using the 1-phenyl-
3-methyl-5-pyrazolone (PMP) pre-column derivatization HPLC
method revealed that SF638-1 was composed of two structural
SF638-1 was also tested using 1H, 13C, total correlation spec-
troscopy (TOCSY), heteronuclear single quantum correlation
(HSQC), and heteronuclear multiple bond correlation (HMBC)
NMR techniques (see Supplementary data). Three proton signals
at d 5.12 (overlapped), 4.56 (d, 0.6H, J1,2 = 8.1 Hz), and 3.26 (t,
0.6H, Jvic = 8.1 Hz) were correlated to carbon signals at d 92.1,
95.9, and 75.4, respectively, in the HSQC spectrum, a typical char-
acteristic of a reducing terminal glucose unit. One methine signal
at dC 70.3/dH 3.42 (overlapped) for C-4 indicated one glucose unit
at the non-reducing terminus. The configuration of the glycosidic
units,
D-glucose and an unknown moiety. According to data
describing its behavior in MS and NMR (methods described in
detail below), the unknown moiety was confirmed as a special
amino-containing saccharide.
bond between ring A and B was confirmed to be a-(1?4) because
The positive full-scan ESI-MS of SF638-1 showed a strong
[M+H]+ signal at m/z 522. The ESI tandem mass spectrometry
(MS/MS) spectrum from [M+H]+ is shown in Figure 1. We analyzed
the structure of SF638-1 in accordance with the fragmentation pat-
terns of this amino-oligosaccharide in the ESI-MS/MS spectrum,
namely that (1) cleavage occurred on every C-1-oxygen bond;
and (2) each abundant ion in ESI positive-ion mode possessed at
least one nitrogen-containing moiety.12 As a result of this analysis
a methine signal at about dC 78.2/dH 3.26 (overlapped) for A-4 indi-
cated that its hydroxyl group was glycosylated. Furthermore, an
anomeric C-1 methine on ring B at dC 100.4/dH 5.28 (m) with a
ꢁ3.0 Hz proton coupling constant suggested that its reducing hy-
droxyl group was glycosylated in the
a
-form. The connectivity be-
tween rings A and B was also established as
a
-(1?4) by the HMBC
experiment, which revealed correlation of H-C(A4) with C(B1) and
H-C(B1) with C(A4). The configuration of the bond between ring B
Figure 1. Positive ESI-MS/MS fragmentation and spectrum of [M+H]+ of SF638-1 at m/z 522.

1900
P. Meng et al. / Carbohydrate Research 346 (2011) 1898–1902
Table 2
and part C was determined to be N-(1?4) because a methine sig-
nal at dC 69.8/dH 2.84 (t, Jvic 8.4 Hz) for B-4 went to a higher field
than would be expected of a normal O-linked carbon atom. This
suggests attachment to a nitrogen atom, compared with the N-
pseudoglycosidic bond in the reference compound, acarviostatin
I03.8 An anomeric C-1 methine on part C at dC 76.4/dH 3.24 (over-
lapped) was also upfield shifted, revealing a nitrogen atom connec-
tion. Finally, the putative N-(1?4) connection between ring B and
part C was also based on HMBC correlation of H-C(B4) with C(C1)
and H-C(C1) with C(B4).
Effects of a-amylase inhibitors
Compounds
Rat
IC50
a
(
-amylase
Human pancreatic a-amylase
a
l
g/mL)
Type of inhibition (Kib
(lM))
SF638-1
Acarbose
5.06 0.21
1.54 0.15
Mixed(1.68 0.23)
Mixed(1.47 0.14)
a
IC50 is the half-maximal inhibitory concentration.
Ki is the inhibition constant, defined as [E][I]/[EI].
b
The complete proton and carbon signal assignments of SF638-1,
aided by TOCSY, HSQC, and HMBC experiments are shown in
Table 1, in comparison with reported data for trestatin,13 butyta-
tins M03 and M13,14 acarviostatin I03,8 and maltotriose.15 Accord-
ing to the structure elucidations, SF638-1 (Fig. 1) was determined
to be a novel compound.
2.2. Assay for kinetic measurement of SF638-1 inhibitory
activity against a-amylase
We initially investigated the inhibitory effects of SF638-1 and
acarbose on -amylase derived from rat small intestine. As shown
in Table 2, SF638-1 was a potent inhibitor of rat -amylase with an
IC50 (5.06 g/mL) comparable to that of acarbose (1.54 g/mL).
a
a
l
l
A Lineweaver–Burk plot of 1/v (velocity) versus 1/[S] (substrate
concentration) (see Supplementary data) was constructed to
determine the type of HPA inhibition induced by SF638-1. The series
of lines intersect to the left of the vertical axis and above the horizon-
tal axis, indicating that these inhibitors were mixed inhibitors in
which both the enzyme–inhibitor complex (EI) and the enzyme–
substrate–inhibitor complex (ESI) were formed. From the intersec-
tion of lines in Dixon plots of 1/v versus [I] (inhibitor concentration)
(Fig. 2), the Ki values (dissociation constant of EI) of acarbose and
SF638-1 against HPA were determined to be 1.47 and 1.68 lM,
respectively (Table 2), suggesting that they have similar inhibitory
effects.
2.3. Inhibition of starch hydrolysis and glucose transfer by
SF638-1 in vitro using the everted intestinal model
The inhibition of
in vitro using the rat everted intestinal sac system. Similar to
acarbose, SF638-1 (3 g/mL) significantly inhibited starch hydroly-
a-amylase by SF638-1 was further tested
l
sis (P <0.01), glucose production (P <0.01), and glucose transfer to
blood (P <0.01) (Table 3). These results indicated that SF638-1
Figure 2. Dixon plots showing the kinetics of starch hydrolysis by HPA in the
could inhibit
glucose level.
a-amylase within the intestine to reduce blood
presence of (A) acarbose and (B) SF638-1. Initial concentrations of starch for lines in
each plot are: (s) 0.5%, (N) 0.25%, (h) 0.1%, and (ꢀ) 0.05%; v is defined as lg maltose
equivalent/mL/min.
2.4. Suppressive effect of SF638-1 on postprandial blood glucose
elevation in normal mice
administration (Table 4). However, compared with the control
group, blood glucose levels in mice treated with either acarbose
or SF638-1 were significantly (P <0.01) reduced at 30 min after
starch loading. These results demonstrate that SF638-1 effectively
suppresses the postprandial elevation in blood glucose level
We administered starch orally in normal mice to test the effects
of SF638-1 on blood glucose elevation. Blood glucose level in the
control group reached a maximum value at 30 min after oral starch
Table 1
1H and 13C NMR data of SF638-1 in D2Oa
Position
dC
dH
5.12
3.56
3.96
3.26
3.93
Position
dC
dH
4.56
3.26
3.75
3.65
3.61
Position
dC
dH
5.28
3.61
3.73
2.84
3.83
Position
dC
dH
3.24
3.57
3.66
3.42
3.83
A1a
A2a
A3a
A4a
A5a
A6a
92.1
72.1
74.9
78.2
71.6
61.1
A1b
A2b
A3b
A4b
A5b
A6b
95.9
75.4
77.3
77.6
69.2
61.1
B1
B2
B3
B4
B5
B6
100.4
72.1
74.7
69.8
72.0
61.1
C1
C2
C3
C4
C5
C6
76.4
73.1
74.3
70.3
72.0
61.1
ꢁ3.85
ꢁ3.90
ꢁ3.85
ꢁ3.85
a
Assignments are supported by TOCSY and HSQC experiments.

P. Meng et al. / Carbohydrate Research 346 (2011) 1898–1902
1901
Table 3
Inhibition of starch hydrolysis and glucose transfer by inhibitors using the in vitro everted intestinal model
Group
Dose (mg/mL)
Starch hydrolysis E/E0 (%)
Glucose production E/E0 (%)
Glucose transfer E/E0 (%)
Control
Acarbose
SF638-1
—
100
100
100
1.5
0.75
1.5
3.0
45.6 9.4**
75.2 11.7**
67.6 5.2**
51.3 8.6**
68.8 14.8**
87.7 12.1*
72.5 7.1**
70.4 9.3**
54.8 25.2**
79.6 20.5*
76.6 18.7**
59.4 14.7**
Results are mean SD, n = 5.
*
P <0.05 compared with the control group.
P <0.01 compared with the control group.
**
Table 4
Suppressive effect of SF638-1 on postprandial blood glucose elevation in normal rats after starch loading
Group
Dose (mg/kg)
Blood glucose level (mM)
0 min
30 min
60 min
90 min
Control
Acarbose
SF638-1
—
4.32 0.29
4.17 0.37
4.36 0.25
4.20 0.17
4.22 0.41
14.32 0.91
11.41 1.96**
12.21 1.58*
11.63 0.48**
11.57 1.77**
9.24 0.77
10.10 1.32
8.78 1.63
8.53 1.30
9.70 1.74
7.50 0.22
7.56 0.79
7.26 0.36
7.06 0.82
8.62 0.93
1.5
0.75
1.5
3.0
Results are mean SD, n = 8.
*
P <0.05 compared with the control group.
P <0.01 compared with the control group.
**
induced by oral administration of starch in normal mice, and may
have wider relevance to the treatment of diabetes.
3.3. Animals
This study was carried out using 72 Kunming mice (18–22 g,
6 weeks old) and Wistar rats (120–140 g, 4 weeks old) from the
Experimental Animal Center of Academy of Military Medical Sci-
ences (Beijing, China). Animals were maintained in a clean room
at a temperature of 23–26 °C and a relative humidity of 50–60%
with a 12 h light-dark cycle. Both male and female mice were used,
with each mouse housed in a separate cage with food and water
freely available. The experimental protocol was approved by the
Animal Ethics Committee of Nankai University, in accordance with
Principles of Laboratory Animal Care and Use in Research (Ministry
of Health, Beijing, China).
3. Experimental
3.1. General methods
Optical rotation was measured on a Perkin–Elmer 241 MC
polarimeter. IR spectrum was recorded on a Bio-Rad FTS 6000
spectrometer and as KBr pellets. MS was conducted on a Waters
Q-TOF Premier with ESI source. NMR experiments were conducted
on a Mercury 300BB instrument at ambient temperature. The D2O
solutions were used, with sodium 4,4-dimethyl-4-silapentatane-
5-sulfonate (DSS), as the external standard, and NMR spectra were
recorded in 5 mm tubes at 300.07 MHz for 1H and 75.46 MHz for
13C spectra. Semi-preparative HPLC was carried out on a Shimadzu
series instrument.
Two-dimensional NMR spectra were recorded at 25 °C on a
Bruker AVANCE 300 using a z-gradient triple resonance probe.
The parameters for the phase-sensitive 1H–1H TOCSY spectra were
as follows: spectral width, 4807.7 Hz; acquisition time, 0.213 s;
relaxation delay, 1.0 s; spin-locking pulse, 60 ms; and 16 scans
for each of the 160 increments. 1H–13C HSQC experiments were ac-
quired with a spectral width of 4807.7 Hz in the F2 (1H) dimension
and 1798.5 Hz in the F1 (13C) dimension; acquisition time, 0.17 s;
relaxation delay, 2 s; and 32–128 scans per increment (256 incre-
ments). The 1H–13C HMBC spectra were acquired with spectral
widths of 4807.7 Hz in the F2 (1H) dimension and 18115.9 Hz in
the F1 (13C) dimension; acquisition time, 0.213 s; relaxation delay,
1.0 s; mixing time, 65 ms; and 32 scans per increment (256
increments).
3.4. Preparation of the GIB638 complex
The culture filtrate (about 7 L) of Streptomyces sp. PW638,
fermented with Gause’s No. 1 synthetic medium for seven days
in a 10 L fermenter (Biotech-10BG2), was concentrated in vacuo
to ꢁ500 mL. This was then passed through a series number
201 ꢂ 4 macroporous resin column (300 ꢂ 40 mm, Chemical Plant
of Nankai University, Tianjin, China) to partly remove pigments,
followed by further filtration through a series number 001 ꢂ 7
cation exchange resin column (300 ꢂ 40 mm, Chemical Plant of
Nankai University, Tianjin, China). This column was washed with
water, eluted with 0.1 M ammonia, and fractions with inhibitory
activities against HPA were collected. Ethanol (ꢁ9-fold volume)
was added to the concentrated elution, and the supernatant was
discarded after centrifugation. The pellet was lyophilized to give
the GIB638 complex.
3.5. Purification of SF638-1
The GIB638 complex was dissolved in water, filtered through a
3.2. Strain and enzymes
0.45
phase HPLC using a stainless steel column filled with Kromasil
m) at 25 °C. The mobile phase was
MeCN/water (10:90) at a flow rate of 5 mL/min with UV detection
at 205 nm. The fraction containing inhibitory activities against HPA
was collected at 12.8 min, and this was further separated on a
lm membrane, and separated by semi-preparative reversed
Streptomyces strain PW638 was isolated from soil at Fujian
C18 (250 ꢂ 10 mm, i.d.,10
l
province, China, in 2005. Rat
intestinal acetone powder (Sigma Aldrich Japan Co.). Active human
pancreatic -amylase (HPA) was derived by over-expression in a
Pichia pastoris expression system (GS115) in our laboratory.2
a-amylase was prepared from rat
a

1902
P. Meng et al. / Carbohydrate Research 346 (2011) 1898–1902
Waters type S5-SCX semi-preparative HPLC at 25 °C. The mobile
phase was water/ammonia/acetic acid (1000:8:8). Four chromato-
graphic peaks were evident, and the peak at 3.7 min (containing
inhibitory activities against HPA) was collected. A Sephadex G-25
column (eluted with water) was used finally to remove the salts
and yielded, after lyophilization, 45 mg of the SF638-1. The purity
of SF638-1 was determined by positive ion mode ESI-MS analysis.
This test demonstrated a pure preparation with few contaminants.
was separated from the collected blood, and the concentration of
glucose in this plasma was measured.
Results were expressed as mean SD (standard deviation).
Statistical significance of differences between groups was analyzed
using Dunnett’s multiple comparison test based on analysis of
variance test (ANOVA).
3.9. SF638-1
White amorphous powder: ½a D18
ꢃ
+107 (c 0.1, water); UV (water):
end absorption; IR mmax (KBr): 3371, 2936, 1560, 1419, 1361, 1023,
3.6. Assay for rat a-amylase inhibition
576 cmꢀ1; HR-ESI-MS (pos.): m/z 522.2206 [M+H]+ (C18H35NO16
requires 522.2201). For ESI-MS/MS (pos.), see Figure 1; for 1H
and 13C NMR data, see Table 1; for 1H, 13C, TOCSY, HSQC, and HMBC
spectra, see Supplementary data.
The inhibitory effect on rat intestinal
gated using methods described previously.16 Briefly, the assay mix-
ture consisted of soluble starch substrate (200 L, 1 mg / mL) and
100 l of either acarbose or SF638-1. The mixture was pre-
incubated for 5 min at 37 °C, and the reaction was initiated by add-
ing 100 L of crude enzyme solution (as -amylase, 5 U/mL). This
a-amylase was investi-
l
l
l
a
4. Conclusion
mixture was incubated for 30 min at 37 °C, and the reaction was
terminated by heating for 5 min in a boiling water bath. Lugol’s
iodine solution [50 ll, containing distilled water (94%), potassium
iodide (4%) and iodine (2%)] was added to the assay mixture to
label any remaining substrate, and absorbance was measured at
630 nm using a microplate reader (BIO-RAD 680). The IC50 values
were determined using GraphPad Prism 5 statistics software.
SF638-1 was produced by Streptomyces sp. PW638. The com-
pound is a novel amino-oligosaccharide acting as a mixed noncom-
petitive inhibitor of HPA. SF638-1 inhibited HPA with a Ki value in
the same order of magnitude as that of acarbose. Furthermore,
SF638-1 showed a suppressive effect on starch hydrolysis and
postprandial blood glucose elevation in both in vitro and in vivo
tests. We conclude that SF638-1 may have potential as a novel
therapeutic for the treatment of diabetes.
3.7. Kinetics of HPA inhibition by SF638-1
Kinetic analysis of a-amylase inhibition by the sample was per-
formed using a modified version of the method of Geng et al.8 The
enzyme and test compounds were incubated with increasing con-
centrations of soluble amylose (0.05–0.25%, w/v). The substrate
Acknowledgments
This work was supported by grants from the National Natural
Science Foundation of China (21002052 and 30900015), the
Tianjin Municipal Science and Technology Commission, China
(10JCYBJC14300 and 11JCYBJC09300) and the Fundamental
Research Funds for the Central Universities of China (65011161).
solution (100
(acarbose or SF638-1) were pre-incubated at 37 °C for 5 min before
reactions were started by adding 50 L of HPA enzyme solution
(1 U/mL). A 50 L sample was taken every 5 min, and the reaction
was stopped by adding 50 L of 3 M NaOH, followed by addition of
75 L of 3,5-dinitrosalicylic acid (DNS) and boiling for 5 min prior
lL), containing various concentrations of inhibitors
l
l
l
l
Supplementary data
to determination of absorbance at 490 nm using a microarray read-
er. Curves of product amount (in terms of glucose equivalents) ver-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.06.005.
sus time (in minutes) were obtained. The initial velocities (
then determined from the slope of the linear portion (within
30 min) of the curve. Kinetics of -amylase inhibition by the sam-
vi) were
a
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The effects of SF638-1 on postprandial blood glucose elevation
in mice were determined as described by Geng et al.18 Mice were
subjected to an overnight fast and orally loaded simultaneously
with starch (4 g/kg) and SF638-1 (various concentrations). Blood
samples (20
lL) were collected from the tail vein of each mouse
before and 30, 60, and 90 min after administration. The plasma