Chinese

Journal of
Natural
Chinese Journal of Natural Medicines 2015, 13(3): 0208−0214 Medicines

Determination of α-glucosidase inhibitors from Scutellaria baicalensis

using liquid chromatography with quadrupole time of flight tandem
mass spectrometry coupled with centrifugal ultrafiltration
YANG Jun-Ran, LUO Jian-Guang, KONG Ling-Yi*

State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nan-
jing 210009, China
Available online 20 Mar. 2015

[ABSTRACT] The present study aimed at identifying potential lead compounds for diabetes mellitus drug discovery. We developed a
novel method involving centrifugal ultrafiltration separation subsequent liquid chromatography with quadrupole time of flight tandem
mass spectrometry (LC-Q/TOF-MS/MS) determination to screen α-glucosidase inhibitors in complex Scutellaria baicalensis Georgi
(SBG) extract. By adding a second filter to the screening process, the level of non-specific binding of Compounds 1, 3, 10 and 11 was
significantly decreased, and the level of non-specific binding of Compounds 5 and 15 also was reduced. As a result, five flavonoids
identified as baicalein, baicalein, wogonin, chrysin, and oroxylin A, were rapidly found to interact with α-glucosidase and possess
potent anti-α-glucosidase activity in vitro. Specific binding of ligands to α-glucosidase was demonstrated though the proposed method
and the ligands could be ranked in order of affinity for α-glucosidase, which were corresponded to the order of inhibitory activity in
vitro. In conclusion, our results indicated that the developed method is a rapid and effective screening method for rat intestinal
α-glucosidase inhibitors from complex herbal medicines such as SBG.
[KEY WORDS] LC-Q/TOF-MS/MS; Centrifugal ultrafiltration; Flavonoids; Scutellaria baicalensis; α-Glucosidase inhibitors
[CLC Number] R917 [Document code] A [Article ID] 2095-6975(2015)03-0208-07

agents based on the interaction of the compounds with intes-

Introduction
The radix of Scutellaria baicalensis Georgi (SBG), a
perennial herb of the Labiatae family, is a well-known, widely
used medicinal plant in China. Its main chemical constituents
are flavonoids which have been demonstrated to have anti-
bacterial [1], anti-inflammatory [2], and radical scavenging
activities [3]. Although anti-type 2 diabetes drugs can cure the
hyperglycemia-induced mitochondrial damage in streptozoto-
cin-induced diabetes Wistar rats [4], no studies have been car-
ried out to investigate the potentials of anti-α-glucosidase
[Received on] 30-Mar.-2014
[Research funding] This work was supported by the National Key
Scientific and Technological Special Projects (2012ZX09103-
101-007), the Program for Changjiang Scholars and Innovative Re-
search Team in University (PCSIRT-IRT1193), and the Project
Funded by the Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD).
[*Corresponding author] Tel/Fax: 86-25-83271405, E-mail: cpu_
lykong@126.com
These authors have no conflict of interest to declare.
Published by Elsevier B.V. All rights reserved
tinal α-glucosidase.
α-Glucosidase (AGH, EC3.2.1.20), located in the brush-
border surface membrane of the small intestinal cells, is an
exo-type carbohydrate enzyme that catalyzes the liberation of
α-glucose from the non-reducing end of the carbohydrates. The
inhibition of intestinal α-glucosidase can delay the digestion
and absorption of carbohydrates and, consequently, suppress
postprandial hyperglycemia. Many studies have used a broad
range of natural [5] or synthetic compounds [6] with in vitro
AGH inhibitory activity. However, most of these studies were
done by using microbial AGH, not mammalian AGH. Com-
mercially available AGHs may be divided into two families,
types I (microbial) and types II (mammals), on the basis of the
differences in primary structure [7]. Since microbial
α-glucosidase inhibitors (AGIs) may not necessarily inhibit
mammalian AGH and vice versa [8], the study of AGIs from
mammalian sources may be a better model to design and de-
velop anti-AGH agents.
The conventional methods for screening bioactive
compounds from Traditional Chinese Medicines (TCMs)
are both time-consuming and inefficient, requiring isola-

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 YANG Jun-Ran, et al. / Chin J Nat Med, 2015, 13(3): 208−214
tion and purification of the chemical constituents, fol-
lowed by evaluation of their bioactivity in vivo or in vitro
[9]
. Moreover, most of the screening techniques in vitro are
based on optical or radioactive detection, which may be
affected by matrix interference, especially when complex
samples are analyzed. Therefore, the methods using spec-
trometry coupled with bioactivity screening have emerged
as an important analytical tool in the identification and
characterization of biologically active compounds for pro-
tein targets of interest [10-13]. Centrifugal ultrafiltration
hyphenated techniques (LC-MS or GC-MS) have attracted
much attention worldwide [14]. The centrifugal ultrafiltra-
tion technique allows the removal of solutes of low mo-
lecular weight from the chamber, retaining solutes of high
molecular weight (i.e., enzyme and enzyme-ligand), in-
cluding the analytes, after centrifugation through a suitable
molecular weight cutoff [15]. This procedure permits a
minimal consumption of sample, and is a simple, rapid and
easily applicable technique in a routine analytical labora-
tory, providing a satisfactory separation. However, one-filter
centrifugal ultrafiltration can result in high non-specific
binding that might interfere with the detection of specifi-
cally bound ligands [16-17]. Consequently, there is still room
for improvement for this technique.
In the present study, a method for the analysis of AGIs in S.
baicalensis was developed using LC-Q/TOF-MS/MS coupled
with two-filter centrifugal ultrafiltration. As a result, five poten-
tial active candidate compounds, which could interact with rat
intestinal α-glucosidase, were detected and identified. Their
inhibitory activity assays were subsequently confirmed in vitro.
Experimental
Chemicals and reagents
S. baicalensis roots were obtained from Xiansheng
pharmacy store in Nanjing, Jiangsu Province, China, in July,
2011. The reference compounds baicalein, wogonin, chrysin,
oroxylin A, and baicalin were isolated by silica gel column
chromatography and then by preparative RP-HPLC from S.
baicalensis in our laboratory. The purity of these com-
pounds was over 98%, and they were identified by spectral
data (UV, MS, NMR) compared those reported in litera-
ture [18-19]. The chemical structures of the five compounds
are shown in Fig. 1.
Fig. 1 Chemical structures of the five flavonoids identified
from enzyme-ligands mixture
– 209 –
Acetonitrile was of HPLC grade and purchased from
Merck (Darmstadt, Germany) and ultrapure water was pre-
pared by Thermo Scientific Barnstead D8611 Easypure II
(Thermo, Waltham, USA). Rat intestine acetone powder was
purchased from Sigma Chemical Co. Ltd. (St. Louis, MO,
USA). Acarbose was obtained from the National Institute
for the Control of Pharmaceutical and Biological Products
(Beijing, China). Glucose assay kit was obtained from Nan-
jing Jiancheng Bioengineering Institute (Nanjing, China).
Maltose, KH2PO4, and Na2HPO4 used for α-glucosidase
inhibitory activity assaywere all analytical grade reagents
purchased from Nanjing Chemical Reagent Corporation
(Nanjing, China).
LC-Q/TOF-MS/MS analysis
The LC analysis was performed on an Agilent 1200
HPLC system (Agilent Technologies, Santa Clara, CA, USA),
equipped with a dual pump, a degasser, a column oven, an
autosampler and a diode array detector. The separations were
carried out using a Zorbax SB C18 column (250 mm × 4.6 mm
i.d., 5 μm) and an Alltima C18 guard column (4.6 mm × 7.5
mm i.d., 5μm). The column temperature was kept at 25 °C.
The mobile phase was consisted of 0.2% aqueous acetic acid
(A) and acetonitrile (B); the eluting gradient was as follows:
0−5 min, 15% B; 5−15 min, 20% B; 15−20 min, 20%−25% B;
20−35 min, 25% B; 35−50 min, 25%−33% B; 50−60 min,
37% B; 60−70 min, 33%−53% B; 70−80 min, 53% B. The
flow rate was set at 0.8 mL·min−1 and the injection volume
was 15 μL. The chromatogram was acquired at 280 nm.
The LC analysis of rat intestinal α-glucosidase ligands
(baicalin, baicalein, wogonin, chrysin and oroxylin A) in
spiked samples was performed as described above, except for
the mobile phase conditions. The LC was carried out with an
isocratic solvent system consisting of 0.2% aqueous acetic
acid (A) and methanol (B) at a flow rate of 1 mL·min−1. Other
parameters were adopted as follows: injection volume, 15 μL;
Column temperature, 30 °C; and monitored wavelength, 280
nm. The isocratic solvents of 45% methanol were used for
baicalin, and 65% methanol for baicalein, wogonin, chrysin,
and oroxylin A.
The LC-Q/TOF-MS/MS analysis was performed on an
Agilent 1200 Series LC system (Agilent Technologies)
equipped with a binary pump, an auto plate-sampler, a ther-
mostatically controlled column compartment, a diode array
detector, and an Agilent G6520 quadrupole time-of-flight
tandem mass spectrometer. The negative ion ESI-MS and
ESI-MS/MS experiments were conducted using conditions as
follows: skimmer, 65 V; drying gas (N2) flow, 8 L·min−1; dry
temperature, 350 °C; and nebulizer (N2) pressure, 45 psi. Full
scan data acquisition was performed for m/z of 100−1 200 in
the negative ion mode. All of the operations, acquisition, and
analysis of data were monitored by Agilent LC-Q/TOF-MS
Mass Hunter Acquisition Software Version B.04.00 and op-
erated under Mass Hunter Qualitative Analysis Software Ver-
sion B.04.00 (Agilent Technologies).
 YANG Jun-Ran, et al. / Chin J Nat Med, 2015, 13(3): 208−214
Sample preparation
The roots of S. baicalensis (100 g) were extracted with
95% ethanol (500 mL) for 3 h at 85 °C and then filtered. The
filtrate was evaporated to afford a residue (5.3 g). The crude
extract (20.0 mg, drying weight) was dissolved in methanol (1
mL), and filtered through a 0.45-µm microporous membrane.
The filtrate was then used for centrifugal ultrafiltration analy-
sis. Five purified flavonoids (50 μmol·L−1, 100 μmol·L−1)
were dissolved in methanol, and filtered through a 0.45-µm
microporous membrane, respectively. The solutions were
used for the relative ranking of binding affinity analysis by
LC-Q/TOF-MS/MS coupled with a centrifugal ultrafiltration
method.
Centrifugal ultrafiltration procedure
For the centrifugal ultrafiltration experiment, 2 μL of
20.0 mg·mL−1 SBG sample solution or 3.75 mmol·L−1 of a
purified compound was incubated for 2 h at 37 °C with 60
μL of α-glucosidase (1.07 mg·mL−1) in 10 mmol·L−1 am-
monium acetate buffer (pH 6.8), in a total volume of 150
μL. After incubation, each mixture was filtered through a
Microcon (Millipore, Bedford, MA, USA) YM-10 cen-
trifugal filter containing a regenerated cellulose ultrafiltra-
tion membrane with 10 000 MW cutoff by centrifugation
at 10 000 × g for 15 min at 4 °C. Using the modified
method of Mulabagal et al [20], the ultrafiltration filter was
first washed three times by centrifugation with 200 μL of
ammonium acetate buffer (pH 6.8) at 4 °C to remove the
unbound compounds. Subsequently, the washed en-
zyme-ligand solution was transferred into a new filter,
proceeding the same operation. Finally, the ultrafiltrate
was dried under vacuum, and then reconstituted in metha-
nol (100 μL) for analysis. For comparison, the control
experiments with denatured enzyme were carried out be-
fore each screening experiment.
Rat intestinal α-glucosidase inhibitory activity
Rat intestinal α-glucosidase inhibitory activity was
measured as described previously with a slight modification
[21]
. In brief, 10 μL of the test sample in DMSO solution was
reconstituted in 100 μL of 0.1 mol·L−1 phosphate buffer (pH
6.8) in a 96-well microplate and 20 μL of the enzyme solution
added, and the mixture was incubated at 37 °C for 10 min.
The buffer and substrate (5 mmol·L−1 maltose in 0.1 mol·L−1
phosphate buffer) were added to the mixture and incubated at
37 °C for 20 min. The reaction mixtures heated for 3 min in a
boiling water bath to stop the reaction. The concentration of
glucose released from the reaction mixtures was determined
using a glucose assay kit based on the glucose oxidase me-
thod. Individual blanks for test samples were prepared to
correct for background absorbance where the substrate was
replaced with buffer. The control blank contained DMSO (10
μL) in place of the test samples. Acarbose was used as a posi-
tive control. Inhibitory activity was calculated by the follow-
ing equation:
Inhibitory activity (%) = 100 × [(ODcontrol − ODcontrol blank)
– 210 –
− (ODsample − ODsample blank)] /(ODcontrol − ODcontrol blank)
The IC50 value was defined as the concentration of
α-glucosidase inhibitor that inhibited 50% of α-glucosidase
activity.
Results and Discussion
Bio-affinity screening of ligands to a target enzyme is
mainly based on the interaction between the ligands and the
active site of the enzyme. The ultrafiltration membrane retains
the α-glucosidase and the enzyme-ligands complexes, but
allows unbound, low molecular weight compounds to wash
away from the chamber. When specific binding to rat intesti-
nal α-glucosidase occurred, the peak area of the ligand was
greater in the chromatogram corresponding to incubation with
active rat intestinal α-glucosidase than that of sample incu-
bated with inactive rat intestinal α-glucosidase. Background
noise due to non-specific binding of compounds to the ul-
trafiltration membrane was minimized by introducing a sec-
ond filter before the ligand-enzyme dissociation step; the
specific binding ligands were then identified by LC-Q/TOF-
MS/MS.
Centrifugal ultrafiltration
The use of the ultrafiltration technique for screening can
result in high non-specific binding that might interfere with
the detection of specifically bound ligands. By replacing the
ultrafiltration filter before the ligand dissociation and elution
step with a second filter, the level of non-specific binding was
decreased. Fig. 2 shows the ultrafiltration a-glucosidase
screening results for the extract of SBG obtained using
one-filter centrifugal ultrafiltration. As shown in Fig. 2, peaks
1, 3, 10, and 11 were barely detected with centrifugal ultrafil-
tration in the chromatogram (solid line), and Compounds 5
and 15 were also decreased. Therefore, the two-filter method
was used as a tool to screen the ligands of a-glucosidase from
SBG.
Fig. 2 Comparison of specific and non-specific binding of
SBG constituents after incubation with active rat intestinal
α-glucosidase using one-filter and two-filter ultrafiltration
(solid line: two-filter ultrafiltration; dashed line: one-filter
ultrafiltration)
 YANG Jun-Ran, et al. / Chin J Nat Med, 2015, 13(3): 208−214

Screening bioactive components from SBG

The chromatographic conditions were optimized to
achieve a complete separation of the nineteen SBG constitu-
ents in the extract; the baseline separations are shown in Fig.
3a. After the incubation with rat intestinal α-glucosidase and
two-filter affinity purification, the trapped ligands in SGB
were analyzed by LC-Q/TOF-MS/MS. The LC-chroma-
togram for the experiment is shown in Fig. 3b. Enhancement
of the HPLC peak areas in the experimental incubations (dash
lines) indicated the specific binding of ligands to rat intestinal
α-glucosidase. As shown in Fig. 3b, five trapped ligands
(Compounds 5, 13, 15, 16, and 18) from SBG showed spe-
cific binding to rat intestinal α-glucosidase. Compounds 1, 2,
3, 4, 6, 7, 8, 9, 10, 11, 14, 17, and 19 were not regarded as rat
intestinal α-glucosidase ligands because they could not be
distinguished from the control sample in two-filter centrifugal
ultrafiltration screening assay.
Identification of inhibitors in the S. baicalensis extract
The LC-Q/TOF-MS/MS analysis was carried out to
identify compounds that were bound in the rat intestinal
α-glucosidase assays. The mass spectral data of the SBG
extract and the ultrafiltrate were obtained in the negative
ion mode. Detailed fragmentation ions and their rela-
tionships with the molecular structure of each compound
were analyzed by high-resolution Q/TOF-MS/MS, and
the results are listed in Table 1 and Fig. 4. By comparing
the proposed product ions, UV spectra data, measured Fig. 3 LC chromatogram of the extract of S. baicalensis
monitored at 280 nm: (a) extract of S. baicalensis, (b) extract
and theoretical masses, measurement error and proposed
of S. baicalensis incubated with rat intestinal α-glucosidase
natural losses with those of the corresponding reference after performing ultrafiltration (solid lines: control experi-
compounds and the literature [18, 19] , the five ligands were ments incubated with denatured rat intestinal α-glucosidase;
identified as the flavonoids baicalin, baicalein, wogonin, dashed lines: experiments incubated with active rat intestinal
chrysin, and oroxylin A. α-glucosidase), (c) the amplification of Fig. 3b

Table 1 LC-Q/TOF-MS/MS accurate product ion data for the identification of five rat intestinal α-glucosidase ligands from SBG
Peak λmax (nm) Measured m/z Fragment ions Theory m/z Formula [M – H]– Error (ppm) Identificationa
445.077 7 [M – H]– 445.077 6 C21H17O11 –0.25
269.045 3 [M – H – Glc acid]– 269.045 5 C15H9O5 1.06 Baicalein-7-O-Glu acidb
5 222, 276, 316
241.051 0 [M – H – Glc acid – CO]– 241.050 6 C14H9O4 –1.39 (Baicalin)
223.040 6 [M – H – Glc acid – CO – H2O]– 223.040 1 C14H7O3 –2.38
269.045 7 [M – H]– 269.045 5 C15H9 O5 –0.68
251.034 3 [M – H – H2O]– 251.035 0 C15H7O4 2.54
5,6,7-Trihydroxy flavone
13 276, 323 241.050 1 [M – H – CO]– 241.050 6 C14H9O4 2.40
(Baicalein)
223.040 2 [M – H – H2O – CO]– 223.040 1 C14H7O3 –0.38
195.045 0 [M – H – H2O – 2CO]– 195.045 2 C13H7O2 0.69
283.061 7 [M – H]– 283.061 2 C16H11O5 –1.62
268.031 8 [M – H – CH3]– 268.031 7 C15H8O5 –0.26 5,7-Dihydroxy-8-methx
15 222, 274, 324
240.042 5 [M – H – CH3 – CO]– 240.042 8 C14H8O4 1.34 oy flavone (Wogonin)
163.003 8 [M – H – CH3 – C7H5O]– 163.003 7 C8H3O4 –0.86
253.050 7 [M – H]– 253.050 6 C15H9 O4 –0.45
5,7-Dihydroxy flavone
16 268, 315 209.061 0 [M – H – CO2]– 209.060 8 C14H9O2 –0.80
(Chrysin)
145.029 2 [M – H – C6H4O2]– 145.029 5 C9H5O2 2.42
283.061 4 [M – H]– 283.061 2 C16H11O5 –0.88
5,7-Dihydroxy-6-metho
18 222, 270, 318 268.038 1 [M – H – CH3]– 268.037 7 C15H8O5 –1.42
xy flavone (Oroxylin A)
239.035 7 [M – H – CH3 – COH]– 239.035 0 C14H7O4 –2.88
a
Identification results obtained by comparing the retention time, UV spectra, and MS data with authentic standards.
b
glu acid: glucuronic acid

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Fig. 4 Q/TOF-MS/MS in the negative ion mode spectra of Compounds 5 (a), 13(b), 15 (c), 16 (d), and 18 (e)

Compound 5 represented a main constituent of SBG. It fragments gave the ions consistent with baicalin. Thus, com-
exhibited [M − H]− ion at m/z 445.077 7, and its MS-MS pound 5 was identified as baicalin and this identification was

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 YANG Jun-Ran, et al. / Chin J Nat Med, 2015, 13(3): 208−214

confirmed by comparison with a pure standard. Compound 13
displayed the [M − H]¯ ion at m/z 269.045 7. The MS-MS
fragments were similar to that of Compound 5, showing
[M − H − H2O]¯at m/z 251.034 3 and [M − H − CO]¯at m/z
241.050 1. On comparison with the standard, Compound
13 was confirmed as baicalein. Compound 16 yielded an
[M − H]¯ ion at m/z 253.050 7. The UV spectrum showed
maximum absorption bands at 268 and 315 nm. The
MS-MS spectra gave ions of [M − H − 45]¯ at m/z 209.061
0 and [M − H − 108]¯ at m/z 145.029 2. The fragments and
retention time in HPLC of Compound 16 suggested a ten-
tative assignment as chrysin. Compounds 15 and 18 both
exhibited [M − H]¯ ions at m/z 283.161. The MS fragments
and retention time in HPLC of Compound 15 were consis-
tent with the known compound wogonin. The MS spectra
of Compound 18 were almost identical to that of Com-
pound 15. However, its retention time supported its possi-
ble identification as oroxylin A.
Evaluation of relative binding affinity of the inhibitors
To further investigate their binding affinity, the five puri-
fied compounds (baicalin, baicalein, wogonin, chrysin, and
oroxylin A) were evaluated for their inhibitory activity of rat
intestinal α-glucosidase, respectively. Incubations of rat intes-
tinal α-glucosidase were carried out with two equimolar con-
centrations of each compound (50 μmol·L−1, 100 μmol·L−1)
using LC-Q/TOF-MS/MS combined with centrifugal ultrafil-
tration based binding assay as described above. The relative
binding affinity of the ligands in the SBG coupled towards rat
intestinal α-glucosidase was compared using the values of
“enrichment factors”, as defined by Liu et al.[16] The standard
curves for the five ligands were obtained by LC-MS and
showed excellent linearity with correlation coefficients (r2)
exceeding > 0.999 (Table 2). The enrichment values of the
five compounds are shown in Table 3, and were used to rank
the relative binding affinities of these flavonoids. As shown in
Table 3, the enrichment factors for baicalin, baicalein, wogo-
nin, chrysin, and oroxylin A were 38.44% ± 2.91%, 62.07% ±
3.50%, 46.99% ± 1.81%, 30.00% ± 3.43%, and 26.96% ±
1.95% at a test concentration of 50 μmol·L−1, resepctively. At
the concentration of 100 μmol·L−1 for each ligand, the
enrichment factors for baicalin, baicalein, wogonin, chrysin
and oroxylin A were 17.99% ± 1.95%, 37.24% ± 2.13%,
29.55% ± 1.61%, 15.75% ± 2.14% and 14.39% ± 3.20%,
respectively. These results showed that enrichment factors
were dependent upon the relative concentrations of ligand and
enzyme. Additionally, the affinity rank order for binding to rat
intestinal α-glucosidase was baicalein > wogonin > baicalin >
chrysin ≥ oroxylin A, which corresponded to the in vitro
enzyme assay results. This work showed that the devel-
oped method can be used to rapidly rank the order of the
binding of the ligands with respect to their affinity for
high throughput screening of rat intestinal α-glucosidase
inhibitors.
α-Glucosidase inhibitory activity of the ligands in vitro
The inhibitory activity of the five compounds (baicalin,
baicalein, wogonin, chrysin, and oroxylin A) against rat
intestinal α-glucosidase was subsequently conducted in vitro.
As shown in Table 4, baicalein showed significantinhibitory
activity, and baicalin, and wogonin had moderate inhibitory
activity, while chrysin and oroxylin A also had a subtle ef-
fects. Therefore, the inhibitory activity decreased in the
following order: baicalein > wogonin > baicalin > chrysin>

Table 2 The calibration curves, the scope of linearity, LOQ and LOD of screening of Compounds 5, 13, 15, 16, and 18 from S. bai-
calensis
Sample Calibration curves Scope (μg·mL−1) r LOQ (μg·mL−1) LOD (μg·mL−1)
Baicalin y = 24.249 x + 12.528 1.45–66.9 0.999 9 1.43 0.49
Baicalein y = 62.211 x – 9.819 3 0.84–40.5 0.999 4 0.54 0.19
Wogonin y = 65.704 x + 9.120 2 1.78–42.6 0.999 4 0.26 0.11
Chrysin y = 62.780 x + 14.859 1.43–38.1 0.999 0 0.27 0.07
Oroxylin A y = 33.922 x – 4.362 1 0.57–42.6 0.999 6 0.57 0.14

Table 3 Enrichment factors a for ligands (5, 13, 15, 16, and 18) Table 4 Inhibitory activities of the screening ligands (Compounds
to rat intestinal α-glucosidase (mean ± SD, n = 3) 5, 13, 15, 16, and 18) against rat intestinal α-glucosidase (mean ± SD,
n = 3)
Peak Sample 50 μmol·L−1 100 μmol·L−1
Peak Samples Inhibition a (%) IC50 (μmol·L−1)
5 Baicalin 38.44 ± 2.91 17.99 ± 1.95
5 Baicalin 33.34 ± 2.92 98.04 ± 4.16
13 Baicalein 62.07 ± 3.50 37.24 ± 2.13
13 Baicalein 66.67 ± 1.37 38.19 ± 1.82
15 Wogonin 46.99 ± 1.81 29.55 ± 1.61 15 Wogonin 42.62 ± 2.19 78.04 ± 2.96
16 Chrysin 30.00 ± 3.43 15.75 ± 2.14 16 Chrysin 31.33 ± 3.29 164.07 ± 5.6
18 Oroxylin A 26.96 ± 1.95 14.39 ± 3.20 18 Oroxylin A 25.65 ± 3.95 269.8 ± 7.84
a
a
Enrichment factor = (amount of compound specifically bound) Inhibition by 50 μmol·L−1 flavonoids, the IC50 of positive drug
/ (total amount of compound incubation) × 100% (acarbose) was at concentration of 3.5 μmol·L−1

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 YANG Jun-Ran, et al. / Chin J Nat Med, 2015, 13(3): 208−214
oroxylin A. The above results indicated that the ligands from
SBG identified by centrifugal ultrafiltration method were all
anti-α-glucosidase agents, and that the inhibitory activity of
these flavonoids against rat intestinal α-glucosidase corre-
sponded to the results from the LC-Q/ TOF-MS/MS binding
affinity assay.
Conclusions
An LC-Q/TOF-MS/MS method coupled with centrifugal
ultrafiltration was developed and successfully applied to as-
sess the presence of rat intestinal α-glucosidase inhibitors in a
multicomponent S. baicalensis extract. This procedure pro-
vided a better tool for rapid screening and identifying AGIs
from complex plant extract matrix, compared with the com-
monly used bioassay-guided column separation technique.
The availability of such a system allows a rapid identification
of natural α-glucosidase inhibitors for pharmacological inves-
tigation.
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Cite this article as: YANG Jun-Ran, LUO Jian-Guang, KONG Ling-Yi. Determination of α-glucosidase inhibi-
tors from Scutellaria baicalensis using liquid chromatography with quadrupole time of flight tandem mass spec-
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