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Quickly Screening for Potential #-Glucosidase Inhibitors from Guava Leaves Tea by Bio-Affinity Ultrafiltration Coupled with HPLC-ESI-TOF/MS Method Lu Wang, Yufeng Liu, You Luo, Kuiying Huang, and Zhenqiang Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05280 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on February 1, 2018
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Journal of Agricultural and Food Chemistry
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Quickly
Screening
for
Potential
2
Inhibitors from Guava Leaves Tea by Bio-Affinity
3
Ultrafiltration Coupled with HPLC-ESI-TOF/MS
4
Method
α-Glucosidase
5
Lu Wang†, Yufeng Liu†, You Luo†, Kuiying Huang‡, Zhenqiang Wu†*
6
† School of Biology and Biological Engineering, Guangdong Provincial Key
7
Laboratory of Fermentation and Enzyme Engineering, South China University of
8
Technology, Guangzhou 510006, P. R. China
9
‡ Guangzhou Institute of Microbiology, Guangzhou 510663, P. R. China
10 11 12 13 14 15 16 17 18 19 20 21 22
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ABSTRACT: Guava leaves tea (GLT) has a potential anti-hyperglycemic effect.
24
Nevertheless, it is unclear which compound plays a key role in reducing blood sugar.
25
In this study, GLT extract (IC50 = 19.37 ± 0.21 µg/mL) exhibited a stronger inhibitory
26
potency against α-glucosidase than did acarbose (positive control) at IC50 = 178.52 ±
27
1.37 µg/mL. To rapidly identify the specific α-glucosidase inhibitor components from
28
GLT, an approach based on bio-affinity ultrafiltration combined with high
29
performance liquid chromatography coupled to electrospray ionization-time of
30
flight-mass spectrometry (BAUF-HPLC-ESI-TOF/MS) was developed. Under the
31
optimal bio-affinity ultrafiltration conditions, eleven corresponding potential
32
α-glucosidase inhibitors with high affinity degrees (ADs) were screened and identified
33
from the GLT extract. Quercetin (IC50 = 4.51 ± 0.71 µg/mL) and procyanidin B3 (IC50
34
= 28.67 ± 5.81 µg/mL) were determined to be primarily responsible for the
35
anti-hyperglycemic effect, which further verified the established screening method.
36
Moreover, structure-activity relationships were discussed. In conclusion, the
37
BAUF-HPLC-ESI-TOF/MS method could be applied to determine the potential
38
α-glucosidase inhibitors from complex natural products quickly.
39
Keywords: guava leaves tea, α-glucosidase inhibitors, quick screening, bio-affinity
40
ultrafiltration, HPLC-ESI-TOF/MS
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INTRODUCTION
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Diabetes mellitus is a serious chronic endocrine metabolic disorder characterized by
47
high blood glucose levels. Based on the results of a World Health Organization (WHO)
48
survey, 90%-95% of over 400 million diabetes mellitus patients worldwide have type
49
2 diabetes mellitus
50
medicinal plants or food matrices have attracted increasing interest in the treatment
51
and prevention of diabetes due to its efficiency and low toxicity
52
one of the main carbohydrate hydrolysis enzymes, is responsible for the cleavage of
53
oligosaccharides and disaccharides into monosaccharides suitable for absorption in
54
the small intestine 6,7. α-Glucosidase inhibitors can diminish the absorption of glucose
55
and thus, reduce postprandial blood glucose levels
56
inhibitors have been considered a first line therapy by the International Diabetes
57
Federation (IDF) and the American Association of Clinical Endocrinologists (AACE)
58
11,12
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natural products is an increasingly interesting and challenging research area for the
60
management of diabetes mellitus.
1,2
. Currently, carbohydrate hydrolysis enzyme inhibitors from
3-5
. α-Glucosidase,
8-10
. Natural α-glucosidase
. Thus, the rapid screening of α-glucosidase inhibitors from complex systems of
61
Conventional bio-assay guided approaches for screening bioactive components
62
from complex extracts require multiple-step extractions and separation procedures by
63
organic solvents, which are inefficient and environmental unfriendly 13,14. However,
64
decomposition, irreversible adsorption, and dilution effects of the isolated substances
65
typically leads to false positives events with correspondingly high failure risks. To
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rapidly identify and isolate specific bioactive compounds, a combinatorial method of
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bio-affinity ultrafiltration and high performance liquid chromatography coupled to
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electrospray ionization-time of flight-mass spectrometry (BAUF-HPLC-ESI-TOF/MS)
69
has been developed to identify potential novel bioactive compounds 15. In this assay,
70
the bio-active molecules (ligands) were firstly combined with α-glucosidase (receptor)
71
to form the ligand-receptor complexes, then the ultrafiltration centrifugation separates
72
the formed complexes under the optimal conditions, and the ligands released from the
73
complexes
74
HPLC-ESI-TOF/MS analysis. BAUF-HPLC-ESI-TOF/MS has been applied to screen
75
and identify a number of novel bioactive components from complex extracts systems
76
at early drug discovery stages without requiring tedious isolation and purification
77
steps. For example, Chen et al. (2016) used bio-affinity ultrafiltration technology with
78
DNA Top I (topoisomerase I) as a drug target to successfully isolate specific alkaloids
79
with potential anti-cancer activity from Lycoris radiata16. Ma et al. (2017) rapidly
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screened out potential α-amylase inhibitors from Rhodiola rosea by affinity
81
ultrafiltration coupled with UPLC-TOF/MS based on a metabolomic method
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Guava (Psidium guajava L.) belongs to the Myrtaceae family and is widely cultivated
83
in tropical and subtropical environments. Guava leaves tea (GLT) is a commercial
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product manufactured by freshly guava leaves. The manufacturing process included
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four stages: plucking, solar withering, indoor withering, and guava leaves tea product.
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Pharmacology reports have confirmed that the crude extracts of guava leaves
87
possessed
88
concentrated on evaluating the total α-glucosidase inhibition activity of complex
could
strong
be
readily
identified
anti-hyperglycemic
and
effects
subsequently
18-21
.
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While
quantified
previous
by
17
.
studies
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systems or pure chemicals isolated from guava leaves extracts, the specific bioactive
90
components responsible for the anti-hyperglycemic effects of GLT have not yet been
91
elucidated to date.
92
In the present work, α-glucosidase was selected as a drug target to establish the
93
BAUF-HPLC-ESI-TOF/MS assay method. Optimal bio-affinity ultrafiltration assay
94
conditions were investigated by metabolomic method. The developed method was
95
used to rapidly identify and recognize major α-glucosidase inhibitors from guava
96
leaves tea extracts. The present work provided useful information for rapidly
97
recognizing and identifying hypoglycemic components from complex medicinal
98
products.
99
MATERIALS AND METHODS
100
Chemicals
101
α-Glucosidase
102
p-nitrophenyl-α-D-glucopyranoside
103
compounds and acarbose (≥ 99.8%) were purchased from Sigma-Aldrich (St. Louis,
104
MO, USA). p-Hydroxycinnamic acid was used as the internal standards. Formic acid,
105
dimethylsulphoxide (DMSO) and acetonitrile (ACN) solvents were purchased from
106
Fisher Scientific (HPLC grade, 99.9%, Waltham, MA, USA). 0.5 mL centrifugal
107
filters devices in different sizes (10 kDa, 30 kDa, and 50 kDa) were purchased from
108
Millipore Co. Ltd. (Bedford, Massachusetts, USA). The deionized water was purified
109
by a Milli-Q water purification system from Millipore (Bedford, Massachusetts, USA).
from
Saccharomyces (p-NPG,
cerevisiae ≥
99.8%),
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(≥ standard
99.8%), phenolics
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Other analytical-grade reagents were obtained from Sigma-Aldrich (St. Louis,
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Missouri, USA).
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Tea Material and Extraction
113
Guava leaves tea was provided by the Jiangmen Nanyue Guava Tea farmer
114
cooperative (Jiangmen, Guangdong, China) and authenticated by a specialist, Peibiao
115
Liu (General manager of Jiangmen Nanyue Guava Tea Farmer Cooperative, Jiangmen,
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China). One gram (dry mass, DM) of GLT powder was extracted with 10 mL of 70%
117
methanol by ultrasonic extraction (320 W, 40°C) for 30 min. The extracts was filtered
118
using a 0.45 µm Whatman No. 1 filter paper (Maidstone, UK). The filtrates were
119
evaporated under vacuum at 45°C until dry. The dried powder was dissolved by
120
adding 20 mL dimethylsulphoxide.
121
Total α-Glucosidase Inhibitory Assay
122
Evaluation of α-glucosidase inhibitory potency was based on a previous methodwith
123
some modifications 22. 100 µL of 1 U/mL α-glucosidase mixed with 100 µL of extract
124
dilutions (1, 5, 15, 25, 40 and 50 µg/mL) was incubated at 37°C for 10 min. Instead of
125
the extract dilutions, 100 µL of phosphate buffer (PBS buffer, 0.01 M, pH = 6.8) was
126
used as an enzyme control, and 100 µL of 0.01 M phosphate buffer acted as the
127
extract control. Next, 100 µL of a p-NPG (5 mM in PBS buffer) was added to the
128
above mixture. The mixtures was incubated at 37°C for 20 min and terminated by
129
adding 500 µL of a 1 M Na2CO3 solution. Mixture absorbance was determined at 405
130
nm in a 96-well plates. The enzyme inhibition activity was calculated using the
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following Eq. 1:
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α-Glucosidase inhibitory potency (%) =
A1 - A0)( ( - B1 - B0) ×100 A1 - A0
(Eq. 1)
133
where A1, A0, B1, and B0 represent the absorbance of the blank test group (containing
134
PBS buffer and enzyme), the blank control group (containing PBS buffer only), the
135
sample test group (containing sample extracts, PBS buffer, and enzyme), and the
136
sample control group (containing sample extracts and PBS buffer), respectively.
137
Bio-Affinity Ultrafiltration (BAUF) Conditions
138
The screening procedure was conducted based on the previous reports, with slight
139
modifications
140
dimethylsulphoxide. α-Glucosidase was dissolved in 10 mM PBS buffer (pH = 6.8).
141
100 µL of 2.0 mg/mL sample extracts were reacted with 200 µL of α-glucosidase (1
142
U/mL, 5 U/mL, 10 U/mL) at 37°C for 30 min. An inactivated α-glucosidase
143
(incubated at 100°C for 10 min) was used as the blank group in the same way. The
144
reaction mixtures were ultra-filtered through centrifugal filter devices in different
145
sizes (10, 30, and 50 kDa) and centrifuged at 10,000×g for 10 min to intercept the
146
α-glucosidase-ligand complexes at room temperature. The unbound components in
147
the complexes were washed three times using 200 µL of 10 mM PBS buffer (pH = 6.8)
148
by centrifugation. Afterward, the α-glucosidase-ligand complexes were incubation
149
with 70% ACN for 10 min, the ligands were released from the complexes by
150
centrifuged at 10,000×g for 10 min, which was repeated twice. Then the combined
151
filtrates were evaporated under vacuum at 37°C until dry. Finally, the dryness were
152
re-dissolved in 200 µL of 70% ACN and analyzed by the HPLC-ESI-TOF/MS.
16,17
. Briefly, the tested GLT extracts were dissolved in 5%
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HPLC-ESI-TOF/MS Analysis
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The Agilent 1200 HPLC system was fitted with a Zorbax Eclipse Plus C18 column
155
(250 mm × 4.6 mm, 5 µm, Agilent, USA) and an ultra-high resolution micro TOF-QII
156
mass spectrometer with 20,000 FWHM of mass resolution (maXis, Bruker, Billerica,
157
MA, USA). An binary solvent was consisted of 0.1% formic acid-H2O (v/v) as solvent
158
A and acetonitrile as solvent B with the following gradient program: 0-5 min with
159
15% B; 5-10 min with 15 to 20% B; 10-20 min with 20 to 25% B; 20-30 min with 25
160
to 35% B; 30-40 min with 35 to 50% B; 40-55 min with 80% B; 45-50 min with 15%
161
B. The injection volume was 20 µL. The flow rate was maintained at 0.8 mL/min and
162
the UV detection wavelength was performed by scanning from 200 to 600 nm at 30°C
163
column temperature. Conditions for MS operation were based on our previously work
164
23
165
fragmentation and some reference data 23,24.
166
Principle Components Analysis (PCA)
167
MarkerLynx XS software (Waters, Milford, MA, USA) was used to regulate and
168
normalize the origin HPLC-ESI-TOF/MS data. The parameters were set as follow:
169
2% percent of peak baseline noise value, one second of peak width value at 10%
170
height, 100 counts of marker intensity threshold value, and 5% of a noise elimination
171
threshold value with the retention of isotopic peak. The mass tolerance value was set
172
at 4.0 ppm. Next, the obtained data were further analyzed using IBM SPSS 17.0
173
statistical software (Milford, MA, USA). PCA was performed to detect clustering
174
trends of samples (with α-glucosidase) and blank (with inactivated α-glucosidase).
. All compounds were identified by their mass spectra, distribution patterns of ion
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Calculation of Affinity Degree (AD) for Selected Potential α-Glucosidase
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Inhibitors
177
Using the optimal bio-affinity ultrafiltration-HPLC method, the α-glucosidase
178
inhibitors from GLT extracts were selected based on the above described procedure.
179
According to variations in the peak areas before and after incubation with
180
α-glucosidase, the affinity degree (AD) was defined as the interaction ability between
181
the ligands and α-glucosidase. A higher affinity degree represented a stronger
182
inhibitory ability for α-glucosidase. The AD was calculated based on Eq. 2:
183
AD(%) =
A1 − A2 × 100% A0
(Eq. 2)
184
where A1, A2, and A0 represent the peak areas of selected compounds obtained from
185
incubations of the GLT extract with activated, inactivated and without α-glucosidase.
186
Statistical Analysis
187
All results presented in this paper were the average of three independent assays and
188
were expressed as the mean ± standard deviation (SD). The results were analyzed by
189
one-way analysis of variance (ANOVA). Significant differences were determined by
190
Duncan’s multiple range tests or by independent sample T-tests when necessary.
191
Samples with p < 0.05 were considered statistically significant in all cases. Statistical
192
analyses were conducted using IBM SPSS version 17.0 (SPSS Inc., Chicago, IL, USA)
193
software package for Windows.
194
RESULTS AND DISCUSSION
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Total α-Glucosidase Inhibitory Potency of GLT extracts
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Managing postprandial plasma glucose levels is important in the early management of
197
diabetes 22. Natural α-glucosidase inhibitors from medicinal plant extracts play an
198
important role in decreasing postprandial hyperglycemia 24,4,5. In this work, different
199
concentrations of GLT extracts were used to investigate total inhibitory activity
200
against α-glucosidase in vitro. Acarbose served as a positive control. As shown in
201
Figure 1AB, the GLT extracts showed remarkably higher α-glucosidase inhibitory
202
activity with an IC50 at 19.37 ± 0.21 µg/mL when compared with acarbose at 178.52 ±
203
1.37 µg/mL. The GLT extracts were confirmed to have potential anti-hyperglycemic
204
effects and were enriched in natural α-glucosidase inhibitory molecules. Consequently,
205
there is an urgent need to rapidly recognize and identify special natural α-glucosidase
206
inhibitors from GLT.
207
Optimal Conditions of Bio-Affinity Ultrafiltration
208
In the present work, α-glucosidase was selected as the drug target (receptor), and the
209
potential bioactive compounds from GLT extracts were considered the ligands. The
210
ligand-receptor complexes formed by interactions between α-glucosidase and the
211
ligands were separated by centrifugal ultrafiltration. A schematic of the
212
BAUF-HPLC-ESI-TOF/MS assay method is shown in Fig. 2. PCA was used as a
213
screening tool to explore profile changes in the metabolome in GLT extracts before
214
and after reactions with α-glucosidase. To determine the optimal affinity ultrafiltration
215
conditions, the concentrations of enzymes and sizes of centrifugal ultrafiltration filter
216
devices in affinity ultrafiltration assays were investigated17. Fig. 3A shows the effects
217
of α-glucosidase concentration on its metabolomic profile. The differences between
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the metabolomic profiles of the GLT extract samples (with an activated α-glucosidase
219
group) and blanks (with an inactivated α-glucosidase group) were more evident with
220
increasing enzyme concentration. The degrees of separation in the PCA analysis
221
corresponded with α-glucosidase inhibitory ability. The results indicated that 10 U/mL
222
of α-glucosidase showed the largest differences between the samples and blanks.
223
Hence, 10 U/mL of α-glucosidase was selected as the optimal inhibitory concentration
224
for the following experiments.
225
In the bio-affinity ultrafiltration assays, membrane filter size is a very important
226
factor for separating α-glucosidase ligands. Fig. 3B shows the effects of molecular
227
membrane filter size on the metabolomic profiles of GLT extract samples and blanks.
228
The results showed that 30 kDa membrane filters resulted in a good separation
229
performance between samples and blanks. Ma et al. (2017) reported that two types of
230
complexes
231
α-amylase-inhibitors) in membrane filters during the separation process
232
work, because a 10 kDa filter was not suitable for separating the two types of
233
complexes, the retention of α-glucosidase-inhibitor complexes was dominant for 30
234
kDa filters, and the retention of the two types of complexes was weak for 50 kDa
235
filters. Therefore, 30 kDa filters were used for the formed ligand-receptor complexes.
236
In conclusion, 10 U/mL of α-glucosidase and 30 kDa membrane filters were selected
237
as the optimal bio-affinity ultrafiltration conditions used in the following experiments.
238
Separation of Potential α-Glucosidase Inhibitors from GLT Extract
exist
with
different
retention
rates
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(macromolecule
and
17
. In this
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239
After incubating with α-glucosidase and optimal bio-affinity ultrafiltration, the bound
240
ligands in the GLT extracts were released by 70% acetonitrile solution and analyzed
241
by HPLC-TOF/MS. The 11 bioactive compounds incubated with α-glucosidase in the
242
GLT extracts showed higher bio-affinity ability when compared with the inactivated
243
control group. These results indicated that these 12 constituents showed specific
244
binding toward α-glucosidase. Therefore, these 12 constituents were considered major
245
potential α-glucosidase ligands (Fig. 4). It is worth noting that the major
246
α-glucosidase ligands in GLT extracts were clearly described for the first time.
247
Based on variations in peak areas before and after incubation with α-glucosidase,
248
the affinity degree (AD) of 12 potential inhibitors with α-glucosidase are listed in
249
Table 1. Compound 12 possessed the greatest affinity degree (18.86 ± 0.28%),
250
followed by compounds 3 (8.54 ± 0.15%), 8 (7.47 ± 0.09%), 7 (6.56 ± 0.13%), 4
251
(5.32 ± 0.02%), 6 (5.18 ± 0.08%), 5 (4.96 ± 0.11%), 2 (4.75 ± 0.14%) 10 (3.93 ±
252
0.07%), 11 (3.15 ± 0.02%), 9 (2.31 ± 0.11%) and 1 (1.07 ± 0.03%). As expected, there
253
were very obvious differences in the ADs among the selected compound.
254
Theoretically, the differences among the ADs may be due to different competitive
255
binding relationships between the bioactive constituents with α-glucosidase.
256
Identification of Potential α-Glucosidase Inhibitors
257
The 12 compounds from the GLT extracts with different affinity degrees on
258
α-glucosidase were identified by comparing their HPLC retention times and m/z
259
fragmentation patterns of quasi-molecular ions based on reference data 25. The HPLC
260
retention time, the corresponding MS/MS data and the identification of the labeled
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peaks are shown in Table 1. As shown in Table 1 and Fig. 4, compound 1 was
262
identified as gallic acid based on its UV/Vis absorption spectrum (215 nm and 270 nm)
263
and the main [M+H]+ ion at m/z 171.1221. Compound 2 was determined to be
264
L-epicatechin according to the UV/Vis absorption spectrum (256 nm and 280 nm) and
265
the main ions [M+H]+ ion at m/z 291.3121. Compound 3 was likely procyanidin B3
266
based on the parent [M+H]+ ions at m/z 579.1520 [M+H]+ and the [M-C15H10O7]+ ion
267
at m/z 462.3567 and the [C15H10O7+H]+ ion at m/z 303.0512. Compounds 4 and 5
268
were two isomers from the parent [M+H]+ ion at m/z 465.3610 that produced two
269
main ions fragmentations at 303.0501 [C15H10O7+H]+ and 163.1221 [M-C15H10O7]+.
270
Compounds 4 and 5 were identified as hyperoside and isoquercitrin after comparing
271
with the standards, respectively. Three isomers of quercetin glucoside (compounds 6,
272
7, and 8) were characterized by the parent ion m/z 435.0901, which produced two
273
main ions at 303.0501 and 133.2510. Wang et al. (2017)25 reported that compounds 6,
274
7
275
quercetin-3-O-α-L-arabinopyranoside and avicularin. Compound 9 can be identified
276
as quercitrin based on the parent ion m/z 449.0984 [M+H]+ that produced two main
277
ions at 303.0510 [C15H10O7+H]+ and 146.1037 [M-C15H10O7]+. Compound 10 was
278
likely kaempferol-3-arabofuranoside based on the parent ion m/z 419.0984 [M+H]+,
279
which produced the two main ions at 287.0563 [C15H10O6+H]+ and 133.2036
280
[M-C15H10O6]+. Compound 11 was likely a flavonoid compound based on the UV/Vis
281
absorption spectrum (210 nm, 284 nm and 355 nm) and the parent ion m/z 573.1625
and
8
were
likely
quercetin-3-O-β-D-xylopyranoside,
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[M+H]+, which produced the two main ions at 315.0721 and 259.0975. Compound 12
283
was likely quercetin based on the main ion at 303.0501 [C15H10O7+H]+.
284
Structure-Activity
285
α-Glucosidase
286
Phenolics and flavonoid compounds are known secondary metabolites of plant
287
matrices and have shown well pharmacological effects, such as anti-diabetic,
288
anti-oxidant, anti-inflammatory, and anticancer activities
289
compounds structure according to substituent groups largely leads to their differences
290
in bio-activities 30. The chemical structures of the identified potential α-glucosidase
291
inhibitors from the GLT extracts are shown in Fig. 5. As shown in Table 1, three
292
representative compounds, quercetin with an AD at 18.86%, procyanidin B3 at 8.56%
293
and avicularin at 7.47%, exhibited notably stronger affinity to α-glucosidase than the
294
other selected compounds. The IC50 values of the compound were: quercetin (IC50 =
295
4.51 ± 0.71 µg/mL), L-epicatechin (IC50 = 45.56 ± 0.11 µg/mL), procyanidins B3
296
(IC50 = 28.67 ± 5.81 µg/mL), hyperoside (IC50 = 55.31 ± 4.17 µg/mL), isoquercitrin
297
(IC50 = 42.94 ± 3.11 µg/mL), quercetin-3-O-α-L-arabinopyranoside (IC50 = 41.81 ±
298
5.12 µg/mL), quercetin-3-O-β-D-xylopyranoside (IC50 = 44.78 ± 2.62 µg/mL),
299
avicularin (IC50 = 21.84 ± 3.82 µg/mL), quercitrin (IC50 = 43.27 ± 2.17 µg/mL),
300
kaempferol-3-O-arabofuranoside (IC50 = 58.19 ± 3.32 µg/mL) and gallic acid (IC50 =
301
348.63 ± 2.93 µg/mL) (Fig. 6). Quercetin and procyanidins B3 showed the highest
302
inhibitory effects on α-glucosidase. By contrast, gallic acid had the lowest inhibitory
303
capacity. In previous studies, the structure-activity relationships have been known to
Relationships
between
Phenolics/Flavonoids
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and
26-29
. The diversity of
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involve the following characteristics: (1) flavonoid hydroxylation improved their
305
inhibitory effects on α-glucosidase; (2) flavonoid glycosylation reduced their
306
inhibitory activity; and (3) procyanidins exhibited strong inhibitory activity against
307
α-glucosidase 26,27. Because quercetin possessed a high number of hydroxy groups,
308
especially 3’- and 4’-dihydroxyl groups in the B-ring and 3-OH in the C-ring, which
309
are important structures that contribute toward inhibiting α-glucosidase activity 31.
310
Wang et al. (2010) also confirmed that the glycosylation of quercetin significantly
311
reduced the inhibitory activity against α-glucosidase
312
replaced by different glycosides, the ability for flavonol glycosides to inhibit
313
α-glucosidase activity was significantly lower than that of quercetin. Procyanidin B3
314
(catechin dimer) differed from other natural polyphenols due to their polymeric nature
315
26,27
316
inhibitory activity against carbohydrate hydrolysis enzymes and could be used as lead
317
compounds for the development of antidiabetic therapeutics
318
showed the lowest affinity degree to α-glucosidase, which was consistent with the
319
reports described by Xiao et al. (2013) 26. This may have been due to the number and
320
structure of OH groups in the phenolic compounds. In this study, quercetin and
321
procyanidin B3 were primarily responsible for the anti-hyperglycemic effects of GLT,
322
which also further verified the established screening method. Additionally, the
323
structure-activity relationships revealed that the hydroxylation of flavonoids improved
324
the α-glucosidase inhibitory effect, and the glycosylation of hyroxyl groups on
325
flavonoids
21
. Because 3-hydroxy was
. Hakamata et al. (2006) reported that procyanidin isomers showed stronger
decreased
the
inhibitory
effect.
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32
. However, gallic acid
Consequently,
the
Journal of Agricultural and Food Chemistry
326
BAUF-HPLC-ESI-TOF/MS method could be used as a valuable high-throughput
327
screening platform for the rapid screening of natural α-glucosidase inhibitors from
328
complex medicinal plant extracts.
329
AUTHOR INFORMATION
330
Corresponding author:
331
20-39380663; E-mail:
[email protected] 332
Funding
333
The work was supported by the Science and Technology Project of Guangdong
334
Province, China (2016A020210011 and 2017B020207003) and the Special fund for
335
Agricultural Science and Technology Research Project of Jiangmen City, China
336
(20150160008347)
337
Notes
338
The authors declare no competing financial interest
339
ABBREVIATIONS USED
340
GLT, guava leaves tea; DM, dried mass; PCA, principal components analysis; IDF,
341
International Diabetes Federation; AACE , American Association of Clinical
342
Endocrinologists; BAUF-HPLC-TOF/MS, bio-affinity ultrafiltration and high
343
performance liquid chromatography time-of-flight coupled with mass spectrometry;
344
AD, Affinity degree
345
References
346
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*
Zhenqiang Wu, Tel: (+86) 20-39380663; Fax: (+86)
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Figure captions
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Figure 1. The half-maximal inhibitory concentrations (IC50) of the guava leaves tea
470 471 472
extract (A) and the positive control acarbose (B) on α-glucosidase in vitro Figure 2. A schematic diagram of BAUF-HPLC-ESI-TOF/MS assay to screen for potential α-glucosidase inhibitors from guava leaves tea extract
473
Figure 3. The effect of α-glucosidase concentration (A) and the size of membrane
474
filter (B) on its PCA scores plot before and after its reaction with α-glucosidase
475
by centrifugal ultrafiltration (Samples-1U indicates sample reaction with 1 U/mL
476
α-glucosidase through 30 kDa the membrane filter, Blanks-1U indicates sample
477
reaction with the inactivated 1 U/mL α-glucosidase by 30 kDa the membrane
478
filter; Samples-5U indicates sample reaction with 5 U/mL α-glucosidase through
479
30 kDa the membrane filter, Blanks-5U indicates sample reaction with the
480
inactivated 5 U/mL α-glucosidase by 30 kDa the membrane filter; Samples-10U
481
indicates sample reaction with 10 U/mL α-glucosidase by 30 kDa the membrane
482
filter, Blanks-10U indicates sample reaction with the inactivated 10 U/mL
483
α-glucosidase by 30 kDa the membrane filter; Samples-10K indicates sample
484
reaction with 10 U/mL α-glucosidase by 10 kDa the membrane filter,
485
Samples-30K indicates sample reaction with 10 U/mL α-glucosidase by 30 kDa
486
the membrane filter, Samples-50K indicates sample reaction with 10 U/mL
487
α-glucosidase by 50 kDa the membrane filter, Blanks-10K indicates sample
488
reaction with 10 U/mL inactivated α-glucosidase by 10 kDa the membrane filter,
489
Blanks-30K indicates sample reaction with 10 U/mL inactivated α-glucosidase
490
by 30 kDa the membrane filter, Blanks-50K indicates sample reaction with 10
491
U/mL inactivated α-glucosidase by 50 kDa the membrane filter; 1-3 indicate
492
three parallel tests)
493
Figure 4. The HPLC chromatograms (280 nm) of the chemical constituents in the
494
guava leaves tea extract obtained by ultrafiltration. The blue solid line represents
495
HPLC profiles of guava leaves tea extract without ultrafiltration; the red and
496
black lines represent HPLC profiles of guava leaves tea extract with activated
497
and inactivated α-glucosidase by ultrafiltration, respectively. IS, internal 21
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498 499 500
standards (p-hydroxycinnamic acid) Figure 5. The chemical structures of the selected potential α-glucosidase inhibitors in the guava leaves tea extracts
501
Figure 6. The half-maximal inhibitory concentrations (IC50) of the screened
502
individual α-glucosidase inhibitor from guava leaves tea extract. Different letters
503
(a-f) means statistically significant differences at p < 0.05.
504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 22
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522 523 524 Peak No.
Retention
Table 1 Identification of potential α-glucosidase inhibitors from GLT extract using HPLC-ESI-TOF/MS method and their bio-affinity degrees. Notes: “ND” Not identified; SD, Standard deviation; AD, Affinity degree λmax (nm)
time (min)
Molecular ion
MS (m/z)
Mw
Formula
Compounds
(m/z)
Affinity degree
Reference
(AD, ± SD %) +
1
3.73
215, 270
169.2101[M+H]
2
14.05
256, 280
291.0876[M+H]+ +
171.1221 291.0876
170
C7H6O5
Gallic acid
1.07 ± 0.03
Standard
290
C15H14O6
L-epicatechin
4.75 ± 0.14
Standard
578
C30H26O12
Procyanidin B3
8.54 ± 0.15
Standard
3
17.31
254,354
579.1520 [M+H]
579.1520, 462.3567,
4
17.89
256, 351
465.3610 [M+H]+
303.0501, 163.1221
465
C21H20O12
Hyperoside
5.32 ± 0.02
Standard
256, 351
+
303.0501, 163.1221
465
C21H20O12
Isoquercitrin
4.96 ± 0.11
Standard
+
301.0512
5
18.09
465.3610 [M+H]
6
19.78
254, 359
435.0901 [M+H]
303.0490, 133,1412
434
C20H18O11
Quercetin-3-O-β-D-xylopyranoside
5.18 ± 0.08
Standard
7
20.41
254, 356
435.0930 [M+H]+
303.0509,133.2510
434
C20H18O11
Quercetin-3-O-α-L-arabinopyranoside
6.56 ± 0.13
Standard
253, 357
+
303.0511, 133.1526
434
C20H18O11
Avicularin
7.47 ± 0.09
Standard
+
449.1194, 303.0510,
449
C21H20O11
Quercitrin
2.31 ± 0.11
Standard
418
C20H18O10
Kaempferol-3-arabofuranoside
3.93 ± 0.07
Standard
572
C28H28O13
ND
3.15 ± 0.02
Unknown
302
C15H10O7
Quercetin
18.86 ± 0.28
Standard
8 9
21.29 22.05
262,391
435.0940 [M+H] 449.1194[M+H]
146.1037 10
25.76
257, 363
419.0984 [M+H]+
419.0984, 287.0563,
11
29.17
210, 284,
573.1624 [M+H]+
573.1624, 315.0721,
133.2036
355 12
32.78
254, 364
259.0975 +
303.0516 [M+H]
303.0516
525 526
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Figure 2. A schematic diagram of BAUF-HPLC-ESI-TOF/MS assay to screen for potential α-glucosidase inhibitors from guava leaves tea extract 399x249mm (300 x 300 DPI)
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Figure 3. The effect of α-glucosidase concentration (A) and the size of membrane filter (B) on its PCA scores plot before and after its reaction with α-glucosidase by centrifugal ultrafiltration (Samples-1U indicates sample reaction with 1 U/mL α-glucosidase through 30 kDa the membrane filter, Blanks-1U indicates sample reaction with the inactivated 1 U/mL α-glucosidase by 30 kDa the membrane filter; Samples-5U indicates sample reaction with 5 U/mL α-glucosidase through 30 kDa the membrane filter, Blanks-5U indicates sample reaction with the inactivated 5 U/mL α-glucosidase by 30 kDa the membrane filter; Samples-10U indicates sample reaction with 10 U/mL α-glucosidase by 30 kDa the membrane filter, Blanks-10U indicates sample reaction with the inactivated 10 U/mL α-glucosidase by 30 kDa the membrane filter; Samples-10K indicates sample reaction with 10 U/mL α-glucosidase by 10 kDa the membrane filter, Samples-30K indicates sample reaction with 10 U/mL α-glucosidase by 30 kDa the membrane filter, Samples-50K indicates sample reaction with 10 U/mL α-glucosidase by 50 kDa the membrane filter, Blanks-10K indicates sample reaction with 10 U/mL inactivated α-glucosidase by 10 kDa the membrane filter, Blanks-30K indicates sample reaction with 10 U/mL inactivated α-glucosidase by 30 kDa the membrane filter, Blanks-50K indicates sample reaction
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with 10 U/mL inactivated α-glucosidase by 50 kDa the membrane filter; 1-3 indicate three parallel tests) 399x629mm (300 x 300 DPI)
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Graphical abstract 254x190mm (300 x 300 DPI)
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