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Journal of Separation Science
Screening and separation of α-amylase inhibitors from Solanum nigrum with
amylase-functionalized magnetic graphene oxide combined with high-speed
counter-current chromatography
Yin Cen1, Aiping Xiao2, Xiaoqing Chen1,*, Liangliang Liu2,*
College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan
410083, China
Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha Hunan
410205, China
Corresponding author:
Xiaoqing Chen, Tel: +86-731-88830833. E-mail: [email protected] (X.Q. Chen)
Liangliang Liu, Tel: +86-731-88998525. E-mail: [email protected] (L.L. Liu)
Received: 03 30, 2017; Revised: 09 19, 2017; Accepted: 10 11, 2017
This article has been accepted for publication and undergone full peer review but has not been through
the copyediting, typesetting, pagination and proofreading process, which may lead to differences
between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201700333
This article is protected by copyright. All rights reserved.
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Journal of Separation Science
Abbreviation: GO, graphene oxide; MGO, magnetic graphene oxide; MGO-amylase,
α-amylase functionalized magnetic graphene oxide; VSM, vibrating sample magnetometry;
APTES, 3-aminopropyltriethoxysilane; NHS, N-Hydroxysuccinimide; EDC,
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HSCCC, high-speed
counter-current chromatography; HEMW, n-hexane/ethyl acetate/methanol/water; K value,
partition coefficient.
A screening method using α-amylase-functionalized magnetic graphene oxide combined
with high-speed counter-current chromatography was proposed and utilized to screen and
separate α-amylase inhibitors from extract of Solanum nigrum. The
α-amylase-functionalized magnetic graphene oxide was characterized and found to
demonstrate satisfactory structure, magnetic response (24.5 emu/g) and reusability
(retained 90% of initial activity after five cycles). The conditions for the screening with
α-amylase functionalized magnetic graphene oxide were optimized and set at pH 7.0 and
25°C. As a result, two potent flavonoid compounds, apigenin-7-O-glucuronide (1) and
astragalin (2), were separated and collected through high-speed counter-current
chromatography and subjected to high-performance liquid chromatography analysis with
purity higher than 90% (according to HPLC data), which were identified as α-amylase
inhibitors. These results suggested that utilization of α-amylase functionalized magnetic
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Journal of Separation Science
graphene oxide in the rapid screening and isolation bioactive compounds from complex
natural products is a feasible and environmentally friendly method.
Keywords: Αlpha-amylase; High-speed counter-current chromatography; Magnetic
graphene oxide; Traditional Chinese medicine
1. Introduction
Traditional Chinese Medicines are widespread herbal sources and regarded as
“inexhaustible source” containing varieties of phytochemicals [1]. By means of extensive
investigation on Traditional Chinese Medicines, much information concerning the discovery
of bioactive compounds and new chemical entities could be obtained [2]. However,
traditional techniques are found to be laborious, time-consuming and inefficient in the
analysis of complex natural products [3]. Up to now, the development of new separation
analytical method has prompted the discovery of active components in Traditional Chinese
Medicines. For instance, hyphenated techniques and high-speed counter-current
chromatography (HSCCC) target separation method greatly improved the separation
efficiency [4–7].
The utilization of nanoparticles in numerous areas has attracted wide interests in
scientific studies because of their specific properties including good biocompatibility.
Magnetic nanoparticles are a class of nanoparticles possessing magnet properties, which
could be conveniently isolated from reaction mixture and repetitively used [8]. Magnetic
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Journal of Separation Science
nanoparticles, such as Fe3O4 nanoparticles, possess such advantages as high affinity to
proteins, facile decoration with functional groups and easy isolation from reaction mixture
[9, 10]. On the basis of these separation advantages, problematic and tedious procedures in
filtrations and centrifugations are generally omitted. In recent years, an increase in the use
of graphene oxide (GO) in many fields has been witnessed, as GO demonstrated as a facial
material with large specific surface area and preferable physical and chemical properties
[11, 12]. The fabrication of GO and Fe3O4 retained the advantages of each material and this
composite material thus has been widely applied in the immobilization of enzyme and
catalyst [13].
HSCCC, as a liquid–liquid partition chromatography without support, is characterized as
no irreversible adsorption, high resolution and low cost, and thus has been increasingly
implemented in analytical-scale isolation protocols for the recovery of bioactive herbal
medicines [14, 15]. The utilization of HSCCC in analyzing complex herbal medicine extracts
exhibited such advantages as high sample-loading capacity and high sample recovery. By
means of collecting essential data, target-guided HSCCC can perform accurate preparative
fractionations [16]. Nowadays, it has been used in isolating many Traditional Chinese
Medicines and natural product extracts such as grape seeds, cacao beans, Cordyceps
militaris, Phaffia rhodozyma and so on [17–22].
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Journal of Separation Science
As the enzymes relevant to diabetes, α-amylase targeted specifically α-D-(1–4)-glycosidic
bonds, participating in the hydrolysis of starch into tiny glucose units [23]. The control of
postprandial hyperglycemia has been considered to be efficient in the treatment of diabetes
[24]. Therefore, suppression of α-amylase activity may slow down the sugar adsorption in
human body which is believed to be helpful in the treatment of type 2 diabetes mellitus.
Solanum nigrum is one kind of plant belonging to the family of Solanaceae, which is
widespread in temperate and tropical climate zones of Europe, Asia and America. The leaves
of Solanum nigrum are abundant in alkaloids. Solanum nigrum possessing abundant
bioactive compounds including flavonoids and steroidal alkaloids has raised scientific
concern these years. Subsequently, functions such as anti-cancer, antioxidation,
anti-inflammation have been found [25, 26]. Recently, we have reported that the ethyl
acetate fraction of Solanum nigrum showed anti-diabetes potential due to its bioactive
content which can be bound to α-amylase, and thus inhibit the activities of enzyme.
Herein, a facile analytical method based on α-amylase functionalized magnetic graphene
oxide (MGO-amylase) targeted by HSCCC by HPLC–MS was established to rapid screen and
isolate potent α-amylase inhibitors. This is the first time to use MGO-amylase in screening
α-amylase inhibitors from extracts of Solanum nigrum. As a result, MGO-amylase was
successfully synthesized and characterized by using TEM and vibrating sample
magnetometry (VSM). The as-prepared material exhibited good capabilities to screen
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Journal of Separation Science
potential α-amylase from extracts of Solanum nigrum and satisfactory reusability. Two
potential α-amylase inhibitors identified as apigenin-7-O-glucuronide and astragalin were
screened out.
2. Materials and Methods
2.1. Chemicals
Flake graphite (≤100 mesh) was obtained from Nanjing XFNANO Materials Tech (Nanjing,
China). α-Amylase (≥10 units/mg, from porcine pancreas) was purchased from
Sigma–Aldrich (City, State abbreviation, USA). Solanum nigrum was purchased from Hunan
Tianjian Chinese Medicine Pieces (Changsha, China). Acetonitrile of HPLC grade was
purchased from Tedia (Phoenix, AZ, USA). Ultrapure water was obtained from an ELGA
water purification system (Veolia, Germany). All of other chemicals were of analytical grade
and obtained from Sinopharm Chemical Reagent (Shanghai, China).
2.2. Preparation of α-amylase-functionalized magnetic graphene oxide
2.2.1. Synthesis of graphene oxide
Graphene oxide was synthesized by a modified method initially developed by Hummer
[27]. Flake graphite (0.3 g), condensed H3PO4 (4 mL), and H2SO4 (36 mL) were mixed and
stirred in an ice bath. Next, KMnO4 (1.5 g) was slowly added to it. The mixed solution was
stirred at 50°C for 12 h. After that, ice water (130 mL) was slowly added to it, followed by
another slow addition of 30% H2O2 (3 mL). Then, the reaction mixture was filtered and
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Journal of Separation Science
washed with HCl aqueous solution (1 M) and water respectively until pH was close to 6.
Finally, the resulting graphite oxide precipitate was dried in vacuum at 60°C for 8 h.
2.2.2. Synthesis of amino Fe3O4 nanoparticles
Ferric chloride (1.30 g), PEG 6000 (1.00 g) and sodium acetate (3.60 g) were respectively
weighed and dissolved in ethylene glycol (40 mL) under mechanical stirring. The mixture was
then sealed in an autoclave and heated at 180°C for 6 h. Then, the autoclave was cooled
down to room temperature and the corresponding Fe3O4 nanoparticles were poured out,
washed with water and ethanol, and finally dried under vacuum. Under continuous
mechanical stirring, Fe3O4 nanoparticles (100 mg) were transferred into ethanol (200 mL).
APTES (3 mL) was then added dropwise to the solution and the mixture was stirred at room
temperature for 6 h. Finally, the prepared amino Fe3O4 nanoparticles were washed with
ethanol and dried under vacuum.
2.2.3. Synthesis and characterization of α-amylase-functionalized magnetic graphene
GO (2 mg/mL, 10 mL) solution was ultrasonicated for 1 h and added to a solution (10 mL)
containing EDC (10 mg/mL) and NHS (6 mg/mL). The resulting solution was shaken at room
temperature for 30 min. Subsequently, amino Fe3O4 nanoparticles (20 mg) and amylase
solution (1 mg/mL, 5 mL) were added into the suspension. The mixture was shaken at room
temperature for 1 h. When the reaction reached saturation, excess solution was removed by
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Journal of Separation Science
fixing MGO-amylase with a magnet. The MGO-amylase was washed with phosphate buffer
solution (10mM, pH 7.4) for three times and dispersed in buffer for further use. TEM was
utilized to confirm the surface morphology of materials with a Tecnai-G20 transmission
electron microscope (FEI, Hillsboro, Oregon, USA). Magnetic properties of the prepared
materials were measured at room temperature on a vibration sample magnetometer
VSM7407 (Lake Shore, Westerville, OH, USA).
2.3. Preparation of Solanum nigrum extracts
Dried Solanum nigrum (50.0 g) was decocted and extracted thrice (each for 1 h) with
ethanol solution (90% v/v, 300 mL) at 90°C. Subsequently, the combined ethanol extracts
were evaporated to dryness under reduced pressure and then dissolved in ultrapure water
(100 mL). According to the polar order, same volume of petroleum ether, ethyl acetate and
n-butanol were used respectively to extract the essential ethanol extracts. After that, the
ethyl acetate fraction of Solanum nigrum extract was evaporated to remove the solvent and
suspended in ultrapure water (100 mL) to form aqueous solution. Finally, the solution was
filtered and stored at 4°C for further study.
2.4. HPLC conditions
Agilent 1260 Infinity HPLC system (Agilent Technologies, USA) with a Waters XBridgeTM
C18 reversed-phase column (250 mm×4.6 mm i.d., 5 μm) was used for HPLC separation. The
temperature was maintained at 25°C. The mobile phase consisted of solvent A (0.4% v/v
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Journal of Separation Science
acetic acid in water) and solvent B (0.4% v/v acetic acid in acetonitrile), which was delivered
at 0.8 mL/min with gradient elution program as following: 0–5 min, 10% B; 5–30 min,
10–30% B; 30–60 min, 30–60% B; 60–70 min 60–90% B; 70–90 min 90% B. The diode array
detector was fixed to scan from 200 to 400 nm and the representative chromatogram was
recorded at 254 nm.
2.5. Α-amylase activity assay
Enzyme activity was spectrophotometrically determined by using soluble starch as the
substrate. Enzyme (1 mL) and soluble starch (2 mL) were mixed and preincubated at 25°C
for 3 min. Subsequently, to the mixture was added 3,5-dinitrosalicylic acid (1 mL) to cease
the reaction after being boiled for 10 min and the absorbance was then measured at 540
nm. Control experiments were conducted under the same conditions while the enzyme was
replaced by an equivalent volume of distilled water. All experiments were performed three
2.6. Screening of α-amylase inhibitors from Solanum nigrum
The screening assay was conducted using MGO-amylase as the fishing medium according
to the following procedure. The first step was incubation, in which MGO-amylase (20 mg)
and Solanum nigrum extracts (1 mL) were mixed up and shaken at 25°C for 30 min.
Subsequently, the magnetic separation was carried out by an external magnet, and
MGO-amylase, along with the potential bound inhibitors were collected. Finally, the elution
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Journal of Separation Science
step was conducted to wash off the binders. The process was carried on by adding methanol
(1 mL) and shaken for 5 min. The supernatant was filtered and stored in refrigerator before
2.7. High-speed counter-current chromatography target guided separation
2.7.1. Selection of two-phase solvent system
Given that the selection of two-phase solvent system is essential for HSCCC separation, a
suitable solvent system would guarantee the separation of target compounds according to
coefficient partition (K values). K values of solvent system were determined by the following
method: a small amount of sample was dissolved in solvent system and shaken vigorously to
reach phase equilibrium. Subsequently, two phases of the solution were separated
respectively and evaporated to yield residues. Thereafter, the residues of two phases were
dissolved in methanol solution (2.0 mL) and filtered for HPLC analysis. As mentioned in
Equation (1). K value of the target compound was calculated by the peak area of upper
phase (S1) divided by the corresponding peak area of lower phase (S2).
K = S1 / S2 (1)
2.7.2. Preparation of two-phase solvent systems and sample solution
In this study, the solvent systems composed of n-hexane/ethyl acetate/methanol/water
(HEMW) at different volume ratios were studied to meet separation requirements. Each
part of solvent was mixed and shaken vigorously. After reaching equilibrium, the separated
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Journal of Separation Science
two phases were degassed by ultrasound respectively. For the preparation of HSCCC
separation, 200 mg of the ethyl acetate extract of Solanum nigrum (200 mg) was suspended
in solution containing upper phase and lower phase (1:1, v/v, 5.0 mL).
2.7.3. High-speed counter-current chromatography separation
HSCCC separation was utilized on a TBE-300A HSCCC apparatus (Tauto Biotechnique
Company, China) with three PTFE multilayer coil columns connected in series (inner
diameter of the tubing: 1.6 mm, total column volume: 260 mL) and a 20 mL sample loop.
The revolution radius or the distance between the holder axis and the central axis of the
centrifuge (R) was 5 cm. β values of the multilayer coil ranged from 0.5 at the internal
terminal to 0.8 at the external terminal (β = r/R, where r is the distance from the coil to the
holder shaft). The revolution speed was regulated in the range between 0 and 1000 rpm by
a speed controller. In HSCCC separation, the upper organic phase was used as the stationary
phase. HSCCC analysis was conducted according to the following procedures: fulfill the
multilayer coil column with the upper phase solution, rotate the column at 900 rpm and
smoothly pump the lower phase into the column at a flow rate of 2.0 mL/min to reach the
equilibrium (as a symbol of the emergence of the mobile phase front). Next, the sample
solution (5.0 mL) was loaded into the devices. The temperature was persistently set at 25°C
and the effluent was monitored at 254 nm with a TBD-2000 UV detector and manually
collected in every 5 min according to the chromatographic profile. About 300 mL of upper
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Journal of Separation Science
phase solvents and 500 mL of lower phase solvents were used in the separation. After
separation, the column was stopped and nitrogen gas was utilized to blow out the solution
remained in the column. The obtained fractions from HSCCC separation were numbered and
stored for further HPLC analysis to identify the target compounds.
2.8. Identification of α-amylase inhibitors
The screened and separated potential α-amylase inhibitors from Solanum nigrum extract
were subsequently analyzed by HPLC–MS. The HPLC analysis was performed under the
afore-mentioned conditions. Agilent 6460 Triple Quadrupole LC–MS (Agilent Technologies
Inc., USA) was used for the mass analysis. An ESI interface was used and worked in positive
ionization mode. The mass detection mode was set as the full scan mode from 100 to 1000
m/z. 1H NMR spectra were recorded with an AVANCE III 400 M spectrometer (Bruker
Corporation, Karlsruhe, Germany) operating at 400 MHz, while deuterated DMSO was used
as the solvent, and chemical shifts (δ, ppm) were reported with reference to
3. Results and discussion
3.1. Characterization of α-amylase functionalized magnetic graphene oxide
TEM images of Fe3O4 nanoparticles, GO and MGO-amylase are shown in Fig. 1. It can be
seen in Fig. 1a that the round Fe3O4 nanoparticles showed the average diameter of 400 nm,
which was in consistence with the reported values. The sheet structure with wrinkled edge
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Journal of Separation Science
of GO could also be found in Fig. 1b. Through the combination of GO and Fe3O4
nanoparticles and the functionalization of amylase, Fe3O4 nanoparticles could be observed
to be bound with GO steadily in Fig. 1c and 1d.
The magnetization curves of prepared magnetic nanomaterials were characterized by
vibration sample magnetometer and the magnetization curves are shown in Fig. S1. As a
result, the maximum saturation magnetizations of MGO (31.5 emu/g) and MGO-amylase
(24.5 emu/g) were lower than that of Fe3O4 nanoparticles (74.9 emu/g). The lowered
maximum saturation magnetizations of MGO and MGO-amylase were caused by the
existence of GO and enzyme on MGO-amylase. However, the acceptable magnetic property
of MGO-amylase was retained for isolation.
3.2. Effects of pH and temperature on the activities of α-amylase-functionalized magnetic
graphene oxide
Enzymes are sensitive to temperature and solution environment. Therefore, the activities
of MGO-amylase at different pH and temperatures were evaluated respectively to confirm
the optimum conditions for screening α-amylase inhibitors. As shown in Fig. S2, the effect of
pH value on the activity of MGO-amylase was examined in a pH range from 5.0 to 9.0. It was
found that MGO-amylase exhibited the optimum activity at pH 7.0. The effect of
temperature on the activity of MGO-amylase was also evaluated. Different temperatures
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Journal of Separation Science
from 5 to 65°C were measured and the results of relative activities are shown in Fig. S3.
According to the results, MGO-amylase showed the highest activity at 25°C.
3.3. Reusability of α-amylase functionalized magnetic graphene oxide
The recycling of MGO-amylase was important to reduce the costs in the applications. The
reusability of MGO-amylase was demonstrated by conducting a series of activity assays.
Subsequently, the MGO-amylase was separated from the reaction system by using an
external magnet and the absorbance of supernatant was measured after one cycle. Then
the corresponding MGO-amylase was reused and poured into another solution to start a
new cycle. As shown in Fig. S4, about 90% of the initial activity was still retained after five
cycles though the activity of MGO-amylase decreased slightly. It revealed that MGO was
sufficient for the immobilization of enzyme and the appropriate reusability of MGO-amylase
made it preferable for the screening of α-amylase inhibitors.
3.4. Screening of α-amylase inhibitors from Solanum nigrum
The inhibitory activities of petroleum ether, ethyl acetate and n-butanol parts of Solanum
nigrum were tested. It demonstrated that the ethyl acetate fraction of Solanum nigrum
exhibited potent amylase inhibitory activity with IC50 value at 61.3 μg/mL, indicating that
this fraction was rich in amylase inhibitors. However, IC50 values of the other two fractions
exceeded 1000.0 μg/mL. Obviously, it would be meaningful to screen and separate active
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Journal of Separation Science
components from the ethyl acetate fraction of Solanum nigrum. Thus, this fraction was
stored for further investigation on the screening of α-amylase inhibitors.
Utilization of MGO-amylase in screening active compounds is of great benefit such as high
efficiency and reusability. When the ethyl acetate fraction of Solanum nigrum was incubated
with MGO-amylase, the potential inhibitors would bind to MGO-amylase, which would be
directly separated from the extract solution by an external magnet and then released for
further analysis. Control experiment was conducted following the standard procedure by
using inactivated MGO-amylase. As shown in Fig. 2, two peaks observed in the
chromatogram of MGO-amylase were not identified in the chromatogram of inactivated
MGO-amylase, suggesting that they could be potential α-amylase inhibitors.
3.5. Optimization of high-speed counter-current chromatography solvent system
A suitable two-phase system plays a significant role in HSCCC analysis. The criterion on a
suitable solvent system is K values for compounds falling into a reasonable range of 0.5 to
2.0 and meanwhile taking a short settling time. Referring to golden rules proposed by
Yoichiro Ito [28], HEMW system was frequently used in separating compounds with medium
polarities [29]. Based on the polarity and chemical properties of the target compounds in
this study, the HEMW system was chosen for HSCCC separation. Different volume ratios of
four solvents (3:5:3:5, 1:5:1:5, 0:4:1:5 and 0.5:5:0.5:5) were investigated and the results are
shown in Table S1. According to the results, the HEMW system at a volume ratio of 3:5:3:5
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Journal of Separation Science
was unable to recognize 1 and K value of 1 in HEMW system at a volume ratio of 1:5:1:5 was
lower than 0.5. The HEMW system at a volume ratio of 4:1:5 showed large K values of
compounds, which was unsuitable for HSCCC analysis. However, when the volume ratio was
adjusted to be 0.5:5:0.5:5(v/v/v/v), adequate K values of target compounds 1 and 2 were
acquired and thus it was selected as the solvent system for the separation of the two target
Apart from solvent system, the flow rate was set at 2 mL/min to guarantee the efficiency
of separation and the adequate peak resolution of HSCCC analysis. The revolution speed
also has an impact on solid phase retention. Higher revolution speed would increase solid
phase retention and shorten analysis process, but it may cause emulsification [30].
Therefore, a rotary speed of 900 rpm was utilized. Under the selected HEMW system
(0.5:5:0.5:5, v/v/v/v), two target compounds were separated and collected within 150 min.
3.6. High-speed counter-current chromatography target guided separation
To separate the target compounds screened by the previous experiments, HSCCC
separation was conducted and the representative HSCCC chromatogram of Solanum nigrum
extracts is presented in Fig. 3. Fraction 1 and 2 are collected for further HPLC analysis. The
corresponding chromatographic profiles of collected target fractions are illustrated in Fig.
S5. Ultimately, 3.8 mg of fraction 1 and 2.2 mg of fraction 2 were obtained respectively from
50.0 g of dried Solanum nigrum samples after HSCCC separation. By virtue of peak area
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Journal of Separation Science
normalization method, both of the two target fractions were found to be of high purity
(>90%) and subjected to further identification.
3.7. Identification of target compounds
Two target compounds were characterized to confirm their structures by HPLC–MS and
the related UV and MS data are shown. The UV spectra of 1 and 2 exhibited two absorbance
bands at around 250 and 355 nm. The MS spectrum of 1 showed a protonated molecular ion
[M+H]+ at 447 m/z and a fragment ion at 269 m/z, which were in agreement with the
reported value [31]. Therefore, compound 1 was identified as apigenin-7-O-glucuronide (1)
[32]. A protonated molecular ion [M+H]+ at 449 m/z and a fragment ion [M+H-162]+ at 287
m/z could be observed in the MS spectrum of 2. The fragment ion at 287 m/z could be due
to the loss of a sugar moiety. As the obtained data were in agreement with literature, 2 was
identified as astragalin (2) [33]. The chemical structures of these compounds are shown in
Fig. S6. The data of two compounds are shown in the Supporting Information.
4. Conclusion
In this study, a screening and separation method of α-amylase inhibitors from Solanum
nigrum extract with MGO-amylase integrated with HSCCC was established. The prepared
MGO-amylase was thoroughly characterized and observed to demonstrate satisfactory
structure and magnetic response. The optimum conditions for the screening with
MGO-amylase were set at pH 7.0 and 25°C. As a result, two potential α-amylase inhibitors
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Journal of Separation Science
were successfully screened out and isolated under the HEMW system (0.5:5:0.5:5, v/v/v/v),
which were identified as apigenin-7-O-glucuronide and astragalin. The results illustrated
that the proposed method provided a feasible way for the discovery of bioactive
compounds from Traditional Chinese Medicines.
This work was supported by the National Natural Science Foundation of China
The authors have declared no conflict of interest.
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Figure captions:
Fig. 1. The TEM images of (a) Fe3O4 nanoparticles, (b) GO and (c and d) MGO-amylase.
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Journal of Separation Science
Fig. 2. The chromatograms of (a) Solanum nigrum extract, (b) Eluent after screening with
α-amylase and (c) with denatured α-amylase.
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Journal of Separation Science
Fig. 3. HSCCC chromatogram of Solanum nigrum extract.
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