Subscriber access provided by UNIV OF NEBRASKA - LINCOLN
Article
#-Glucosidase Inhibition and Antihyperglycemic Activity of Phenolics from the Flowers of Edgeworthia gardneri Yan-Yan Ma, Deng-Gao Zhao, Ai-Yu Zhou, Yu Zhang, Zhiyun Du, and Kun Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03081 • Publication Date (Web): 04 Sep 2015 Downloaded from http://pubs.acs.org on September 11, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 32
Journal of Agricultural and Food Chemistry
α-Glucosidase Inhibition and Antihyperglycemic Activity of Phenolics from the Flowers of Edgeworthia gardneri Yan-Yan Ma,
†, ┴
Deng-Gao Zhao, †, ┴ Ai-Yu Zhou, † Yu Zhang, ‡ Zhiyun Du,∗, † and
Kun Zhang∗, †, ‡
†
College of Light Industry and Chemical Engineering, Guangdong University of
Technology, Guangzhou 510006, People’s Republic of China. ‡
School of chemistry and environment engineering, Wuyi University, Jiangmen
529020, People’s Republic of China.
Author Contributions ⊥
These authors contributed equally and should be considered co-first-authors.
*corresponding authors E-mail:
[email protected] (Zhiyun Du), Tel/fax: +86-20-39323363; e-mail:
[email protected] (Kun Zhang), Tel/fax: +86-20-39323363.
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
ABSTRACT
2
The flowers of Edgeworthia gardneri are consumed as an herbal tea in Tibet with
3
potential health benefits. To complement the current knowledge regarding the
4
chemical composition and antihyperglycemic activity of the flower of E. gardneri,
5
two new phenolics, Gardnerol A and B (1 and 2), along with nineteen known
6
phenolics were isolated from the flower of E. gardneri. All isolates were evaluated
7
for their inhibitory activity against α-glucosidase. Compound 5, identified as the
8
major constituent of the flower of E. gardneri, showed a significant α-glucosidase
9
inhibitory activity and acted as a competitive inhibitor. The oral administration of
10
compound 5 at a dose of 300 mg/kg significantly reduced the postprandial blood
11
glucose levels of normal and STZ-induced diabetic mice. Furthermore, compound 5
12
significantly decreased the fasting blood glucose levels in STZ-induced diabetic
13
mice.
14
KEYWORDS
15 16
Edgeworthia gardneri, phenolics, α-glucosidase inhibitor, antihyperglycemic activity, herbal tea
17
2
ACS Paragon Plus Environment
Page 2 of 32
Page 3 of 32
Journal of Agricultural and Food Chemistry
18
INTRODUCTION
19
Diabetes mellitus (DM), a common and complex metabolic disease, is
20
characterized by abnormally high blood glucose levels (hyperglycemia) due to insulin
21
resistance and deficiency.1 Glycemic control is an effective therapy for diabetes,
22
minimizing the risk of long-term complications from the disease.2 α-Glucosidase, a
23
critical enzyme for the digestion of carbohydrates, catalyzes the cleavage of
24
absorbable monosaccharides, starting from disaccharides and oligosaccharides. Thus,
25
α-glucosidase inhibitors reduce postprandial hyperglycemia by slowing the digestion
26
of carbohydrates in the intestines.3 However, classic α-glucosidase inhibitors, such as
27
acarbose and miglitol, also cause gastrointestinal side effects.4, 5 The consumption of
28
natural α-glucosidase inhibitors derived from plant-based foods or supplements offers
29
an attractive strategy to control postprandial hyperglycemia due to their low cost and
30
low incidence of major undesirable side effects.6−9 Many types of fruits, vegetables
31
and drinks, including strawberries, blueberries, broccoli sprouts, green peppers, beer
32
hops, and green tea extracts were shown to display α-glucosidase inhibitory
33
activity.10−13
34
The flower of Edgeworthia gardneri Wall. Meisn., named “Lv-Luo-Hua” in
35
Chinese, has been used to prepare an herbal tea that is commonly consumed as a
36
health beverage in Tibet.14,15 E. gardneri is mainly distributed in Eastern Tibet and
37
the Northwest Yunnan province. It is claimed that drinking the herbal tea of E.
38
gardneri can alleviate the severity of many disorders, such as diabetes and
39
hyperlipidemia.14,
15
Previous studies have demonstrated that the extracts of the
3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 32
40
flower of E. gardneri exhibit many medicinal activities, in particular,
41
antihyperglycemic
42
α-glucosidase.15−18 Nevertheless, most constituents of E. gardneri are still evaluated
43
from complex mixtures. Furthermore, to the best of our knowledge, the
44
α-glucosidase inhibition and antihyperglycemic activity of constituents from E.
45
gardneri have yet to be investigated systematically.
activity
and
significant
inhibitory
activity
against
46
In the present work, we report the isolation and biological activity of phenolics
47
from the flower of E. gardneri. This is the first report on the α-glucosidase inhibition
48
and antihyperglycemic activity of phenolics from the flower of E. gardneri, both in
49
vitro and in vivo.
50
MATERIALS AND METHODS
51
Chemicals and Plant Materials. All organic solvents used in the study, such as
52
petroleum ether (PE), chloroform (CHCl3), ethyl acetate (EtOAc), n-butanol (BuOH),
53
methanol (CH3OH), and dimethyl sulfoxide (DMSO), were of analytical grade.
54
Methanol-d4
55
Saccharomyces cerevisiae, p-nitrophenyl-α-glucopyranoside (PNPG), streptozotocin
56
(STZ), and acarbose were purchased from Sigma-Aldrich (St. Louis, MO, USA).
57 58
(99.8%),
dimethyl
sulfoxide-d6
(99.9%),
α-glucosidase
from
The flowers of E. gardneri were purchased from Xizang Sheng−Qi− Bao−Jian−Pin, Co., Ltd. (Tibet, China).
59
Animals. Kunming mice (4−6 weeks old) were obtained from the Experimental
60
Animal Center of Guangdong Province (Guangzhou, China). The use of mice was
61
reviewed and approved by the Ethics Committee for Animal Experimentation of the
4
ACS Paragon Plus Environment
Page 5 of 32
Journal of Agricultural and Food Chemistry
62
Guangdong University of Technology (Guangzhou, China) and was in accordance
63
with the National Institutes of Health Guide for the Care and Use of Laboratory
64
Animals.
65
To induce diabetes, the mice were treated with a single intraperitoneal injection of
66
streptozotocin (100 mg/kg) dissolved in citrate buffer (pH 4.5) under fasting
67
conditions. The blood glucose level was monitored on day 7 from the tail vein using a
68
one-touch glucometer (Lifescan, Inc., Milpitas, CA). Mice with fasting blood glucose
69
levels ≥16.0 mmol/L were classified as diabetic mice. General Experimental Procedures. Melting points were determined on an X-4
70 71
digital display micromelting point apparatus and are uncorrected. Optical rotations ([α]
72
25 D
73
Thermo Nicolet 6700 FT-IR spectrometer. UV spectra were recorded on a
74
PerkinElmer Lambda 25Shimadzu 160 UV/VIS Spectrometer. The HRESIMS spectra
75
were recorded on an Agilent 6210 series LC/MSD TOF from Agilent Technologies.
76
NMR spectra were acquired on a Bruker AVANCE HD III-400 using TMS as an
77
internal standard. The X-ray diffraction data were collected on a SuperNova, Dual,
78
Eos diffractometer; the structure was solved with the Superflip program using charge
79
flipping and refined with the ShelXL program (using graphite-monochromated Mo K
80
α radiation).Silica gel (200-300 mesh, Qingdao Marine Chemical Factory, China),
81
macroreticular resin (D-101, Sinopharm Chemical Reagent Co., Ltd., Shanghai,
82
China), Sephadex LH-20 gel (GE Healthcare, Uppsala, Sweden), and MCI gel
83
(CHP20P, 75-150 µm, Mitsubishi Chemical Industries Ltd., Tokyo, Japan) were used
) were measured on a Perkin−Elmer 341 polarimeter. IR spectra were obtained using
5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
84
for column chromatography. Preparative HPLC was carried out on a Shimadzu
85
LC-6AD instrument with an SPD-20A detector, using an YMC-Pack ODS-A column
86
(250 mm×20 mm, 5µm). TLC was performed using Merck precoated plates (Si gel 60
87
F254, Germany) of 0.25 mm thickness. The spots on TLC were detected with 254 and
88
365 nm UV light and visualized by spraying with 98% H2SO4/C2H5OH (5:95, v/v)
89
followed by heating. The absorbances in the enzymatic assay were determined at 405
90
nm using a Bio-Rad Model 680 microplate reader.
91
Extraction and Isolation. The dried power of the flower of E. gardneri (12.0 kg)
92
was extracted three times with MeOH (40L) for 7 days each at room temperature. The
93
combined extracts were concentrated under reduced pressure, and the residue (862 g)
94
was partitioned into H2O and extracted with petroleum ether, EtOAc, and n-BuOH,
95
successively.
96
The EtOAc fraction (118 g) was subjected to column chromatography (CC) over
97
silica gel (petroleum ether/EtOAc, 40:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 1.5:1, 1:1,
98
and 0:1, v:v) to give nine major fractions (A−I). Fraction A (1.2 g) was
99
chromatographed over MCI (MeOH/H2O, 9:1) to yield two fractions, A1 and A2.
100
Fraction A2 (231 mg) was subjected to Sephadex LH-20 (CHCl3/MeOH, 1:1) to yield
101
compound 6 (22 mg). Fraction B (2 g) was purified by CC over MCI (MeOH/H2O,
102
9:1) to yield two major fractions, B1 and B2. Fraction B1 (506 mg) was run again by
103
CC over Sephadex LH-20 (CHCl3/MeOH, 1:1) to obtain compounds 7 (16 mg), 8 (31
104
mg), and 9 (27 mg). Fraction B2 (700 mg) was also run again by CC over Sephadex
105
LH-20 (CHCl3/MeOH, 1:1) to obtain 10 (123 mg). Fraction C (1.1 g) was purified by
6
ACS Paragon Plus Environment
Page 6 of 32
Page 7 of 32
Journal of Agricultural and Food Chemistry
106
CC over MCI (MeOH/H2O, 9:1) to yield one fraction C1, and fraction C1 (638 mg)
107
was separated by CC over Sephadex LH-20 (CHCl3/MeOH, 1:1) to obtain compounds
108
1 (14 mg), 11 (56 mg), and 12 (21 mg). Fraction E (1.1 g) was purified by CC over
109
MCI (MeOH/H2O, 9:1) to yield one fraction E1, and fraction E1 was recrystallized
110
from methanol to yield compound 3 (34 mg). Fraction G (1.6 g) was applied to MCI
111
(MeOH/H2O, 9:1) to give three major fractions (G1−G3). Fraction G1 (300 mg) was
112
subjected to CC over Sephadex LH-20, eluted with MeOH/H2O (1:1) and finally
113
purified by prep-HPLC (MeOH/H2O, 4:6) to yield compound 13 (11 mg). The
114
Fraction H (1.8 g) was subjected to CC over MCI (MeOH/H2O, 9:1) to yield three
115
fractions, H1−H3. Fraction H1 (1.5 g) was subjected to CC over Sephadex LH-20,
116
eluted with MeOH-H2O (1:1) and then purified by prep-HPLC (MeOH/H2O, 4:6) to
117
yield compound 14 (30 mg). Fraction I (82 g) was chromatographed over MCI
118
(MeOH/H2O, 9:1) to yield three fractions, I1−I3. Fraction I1 (61 g) was recrystallized
119
from methanol to yield compound 5 (53 g) and a mother liquor (3.6 g). The mother
120
liquor was subjected to CC over silica gel (CHCl3/MeOH, 10:1, 5:1, 3:1, 0:1) to yield
121
two subfractions, I1.1 and I1.2. Fraction I1.2 (1.4 g) was purified by CC over
122
Sephadex LH-20 (CHCl3/MeOH, 1:1) to yield compound 15 (18 mg). Fraction I2 (2.3
123
g) was subjected to CC over Sephadex LH-20 (MeOH/H2O, 1:1) to yield two
124
subfractions, I2.1 and I2.2. Fraction I2.2 (1.4 g) was purified by CC over Sephadex
125
LH-20 (CHCl3/MeOH, 1:1) to yield compound 16 (45 mg).
126
The n-BuOH fraction (118 g) was subjected to CC over D-101 macroreticular resin
127
with an H2O/EtOH gradient (1:0, 8:1, 5:1, 3:1, 1.5:1, 1:1, 0:1) to yield four major
7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
128
fractions (1−4). Fraction 2 was subjected to CC over silica gel (CHCl3/MeOH, 4:1,
129
3:1, 2:1, 0: 1) to yield two fractions, 2.1 and 2.2. Fraction 2.2 (2.6 g) was purified by
130
CC over Sephadex LH-20 (CHCl3/MeOH, 1:1) to obtain compounds 17 (19 mg) and
131
18 (31 mg). Fraction 3 (42 g) was evaluated by TLC and compared with flavonoid
132
standards, including standards for compounds 5, 10, 11, 15, and 16, and the spots
133
were visualized by spraying them with a 5% H2SO4/EtOH solution followed by
134
heating. The TLC results showed that the spots displayed by fraction 3 were similar to
135
those of flavonoids (compounds 5, 15, and 16). Moreover, the content of compound 5
136
was the highest in this fraction. Fraction 3 (14 g) was subjected to CC over silica gel
137
(CHCl3/MeOH, 4:1, 3:1, 2:1, 0: 1) to yield three fractions, 3.1−3.3. Fraction 3.1 (2.6 g)
138
was purified by CC over Sephadex LH-20 (CHCl3/MeOH, 1:1) to obtain two
139
subfractions, 3.1.1 and 3.1.2. Fraction 3.1.2 (827 mg) was run again by CC over
140
Sephadex LH-20 (CHCl3/MeOH, 1:1) to obtain compounds 17 (19 mg) and 18 (31
141
mg). Fraction 3.2 (3.1 g) was applied to CC over Sephadex LH-20 (CHCl3/MeOH,
142
1:1) to yield two subfractions, 3.2.1 and 3.2.2. Fraction 3.2.1 (216 mg) was purified
143
by prep-HPLC (MeOH/H2O, 3:7) to yield compound 4 (22 mg). Fraction 3.2.2 (3.1 g)
144
was chromatographed over Sephadex LH-20 (CHCl3/MeOH, 1:1) to yield crude 19
145
(34 mg) and 20 (11 mg), and then they were separately recrystallized from MeOH.
146
Fraction 3.3 (2.9 g) was subjected to CC over Sephadex LH-20 (CHCl3/MeOH, 1:1)
147
to obtain crude 2 (41 mg) and compound 21 (8 mg), and then compound 2 was
148
recrystallized from MeOH.
149
Structural Elucidation of New Products. Gardnerol A (1): colorless crystals
8
ACS Paragon Plus Environment
Page 8 of 32
Page 9 of 32
Journal of Agricultural and Food Chemistry
150
(MeOH); mp 191−192°C; IR (KBr) νmax 3228, 1693, 1589, 1513, 1433, 1031, 836,
151
792, 456 cm−1; UV (MeOH) λmax (log ε) 208 (4.40), 212 (4.41), 285 (3.90), 322 (4.12)
152
nm; 1H (DMSO-d6 and methanol-d4, 400 MHz) and
153
methanol-d4, 100 MHz) data, see Table 1; HRESIMS m/z 357.0970 [M + H]+ (calcd
154
for C19H17O7, 357.0969).
155
13
C NMR (DMSO-d6 and
Gardnerol B (2): with power (CHCl3-MeOH); [α]25D −10.5 (c 0.38, MeOH); IR (KBr)
156
νmax 3407, 3078, 2923, 1743, 1721, 1611, 839, 723 cm−1; UV (MeOH) λmax (log ε) 214
157
(4.26), 252 (3.62), 286 (3.93), 308 (3.98) nm; 1H (DMSO-d6 and methanol-d4, 400
158
MHz) and
159
HRESIMS m/z 343.0811 [M + H]+ (calcd for C18H15O7, 343.0812).
13
C NMR (DMSO-d6 and methanol-d4, 100 MHz) data, see Table 2;
160
X-ray Analysis. The measurement was collected on a SuperNova, Dual, Eos
161
diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The
162
structure of compound 1 was solved by direct method (SHELXS-2008).
163
Crystal Data for 1. Gardnerol A (1) was crystallized from CHCl3/ CH3OH (1:1) to
164
give colorless crystals. A single crystal of dimensions 0.35 × 0.31 × 0.24 mm3 was
165
used for X-ray measurements. Crystal data: C19H16O7, space group P 1 21/c 1, a =
166
14.7352(17) Å, b = 8.7127(9) Å, c = 14.2158(12) Å, α = 90.00°, β = 114.431(12) °, γ
167
= 90.00°, V = 1661.6(3) Å3, Z = 4, Dcalc = 1.424 g/cm3, R1 = 0.0713, wR2 = 0.2056.
168
The supplementary crystallographic data for 1 reported in this paper has been
169
deposited at the Cambridge Crystallographic Data Centre, under the reference
170
numbers CCDC 1403668. Copies of the data can be obtained, free of charge, on
171
application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, fax:
9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
172
Page 10 of 32
+44 1223 336033 or e-mail: data_
[email protected].
173
α-Glucosidase Inhibition. The α-glucosidase inhibition was assessed according to
174
the slightly modified method of Jeon et al.19 The α-glucosidase (0.1 U/mL) and
175
substrate (p-NPG, 1.0 mM) were dissolved in potassium phosphate buffer (0.1 M, pH
176
6.7), and all samples were dissolved in DMSO. The inhibitor (10 µL) was
177
preincubated with α-glucosidase (40 µL) at 37 °C for 10 min, and then the substrate
178
(50 µL) was added to the reaction mixture. The enzymatic reaction was performed at
179
37 °C for 30 min. The reaction was then terminated by the addition of Na2CO3 (1 M,
180
100 µL). All samples were analyzed in triplicate with five different concentrations
181
near the IC50 values, and the absorbance at 405 nm was determined using a microplate
182
reader. The inhibition percentage (%) was calculated by the following equation:
183
Inhibition (%) = [(OD
184
OD control blank)] × 100.
185
control−
OD
control blank)
− (OD
sample−
OD
sample blank)/
(OD
control−
Type of α-Glucosidase Inhibition. The mode of inhibition of α-glucosidase was
186
investigated
with
increasing
concentrations
of
substrate
(4-nitrophenyl
187
α-D-glucopyranoside) and compound 5. Then, the inhibition type was determined by
188
a Lineweaver−Burk plot according to Michaelis−Menten kinetics. Origin (version 8.0)
189
software was used for plotting the results.
190
Oral Sucrose Tolerance Test (OSTT). The fasting normal and STZ-induced
191
diabetic mice were orally administered with compound 5 (150 and 300 mg/kg of
192
body weight), acarbose (5 mg/kg), or the control and after 30 min and were given a
193
sucrose solution (3 g/kg of body weight). Each group has six mice. Compound 5 and
10
ACS Paragon Plus Environment
Page 11 of 32
Journal of Agricultural and Food Chemistry
194
acarbose were suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na). The
195
control mice were administered with the same volume of 0.5% CMC-Na solution.
196
The tail vein glucose concentrations were measured with a glucometer at 0, 0.5, 1.0,
197
1.5, 2.0 and 3.0 hours after the sucrose load.
198
Hypoglycemic Activity Assay. The fasting normal and STZ-induced diabetic mice
199
(n=6 for each group) were orally administered with compound 5 (150 and 300 mg/kg
200
of body weight), glibenclamide (10 mg/kg), or the control (0.5% CMC-Na). The tail
201
vein glucose concentrations were measured with a glucometer at 0, 1.5, 3, 5, 7 and 9
202
hours after administration.
203
Statistical Analysis. The data were expressed as the mean ± SEM and were
204
analyzed using SPSS (version 19.0) statistical software (SPSS, Chicago, IL, USA).
205
The statistical significance of the differences (p < 0.05) between the mean values of
206
the treatment and control groups were obtained from a one-way analysis of variance
207
(ANOVA) followed by Tukey’s or Dunnett’s test.
208
RESULTS AND DISCUSSION
209
Isolation of Compounds from the Flower of E. gardneri. The extract was
210
subjected to repeated column chromatography over silica gel, Sephadex LH-20, and
211
ODS to yield 21 compounds (Figure 1). Compounds 1 and 2 were identified as new
212
compounds. The 19 known compounds were identified as edgeworic acid (3),20
213
8-(3-(2,4-benzenediol)-propionic acid methyl ester)-coumarin-7-β-D-glucoside (4),
214
tiliroside (5), 4-hydroxybenzoic acid (6), ferulic acid (7), 4- hydroxybenzaldehyde (8),
215
trans-p-hydroxycinnamic acid (9), kaempferol (10), quercetin (11), caffeic acid (12),
11
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
(13),
(-)-secoisolariciresinol
(14),
isoquercetin
Page 12 of 32
216
(+)-lariciresinol
(15),
217
kaempferol-3-O-β-D-glucoside (16), rutin (17), kaempferol-3-rutinoside (18), syringin
218
(19), coniferin (20), and zingerone 4-O-β-D-glucopyranoside (21).
219
Structure Elucidation of the Two New Compounds. Gardnerol A (1) was
220
obtained as colorless crystals after crystallization from CHCl3–CH3OH. The
221
molecular formula of 1 was established as C19H16O7 from the [M + H] + ion at m/z
222
357.0907 (calcd. for C19H17O7, 357.0967) in the HRESIMS. The IR spectrum
223
exhibited strong absorption bands at 3228 (OH), 1693 (conjugated ester C=O), and
224
1619, 1582, and 1513 (aromatic, C=C) cm-1. The UV spectrum of 1 showed the
225
characteristic maxima of a coumarin structure at 231, 252, and 325 nm. The 1H NMR
226
spectrum exhibited the typical signals in the aromatic region associated with H-3 at δH
227
6.18 (d, J=9.6 Hz), H-4 at δH 7.88 (d, J=9.6 Hz), H-5 at 7.37 (d, J=8.8 Hz), and H-6 at
228
δH 6.95 (d, J=8.8 Hz, H-6) of a 7, 8-disubstituted coumarin. The HMBC correlations
229
from the hydroxy proton at δH 10.63 to C-6, C-7, and C-8 confirmed that the hydroxy
230
group was connected to C-7 (Figure 2). Additionally, the aromatic region of the 1H
231
NMR spectrum clearly showed the presence of an ABX spin system [δH 6.35 (d, J =
232
2.0 Hz, H-2′), δH 6.93 (d, J = 8.4 Hz, H-5′), and δH 6.27 (dd, J = 8.4, 2.0 Hz, H-6′)],
233
indicative of a trisubstituted aromatic ring, which was confirmed by proton-coupling
234
patterns and 1H-1H COSY correlations (Figure 2). The HMBC correlations of the
235
hydroxy proton at δH 9.37 with C-2′, C-3′, and C-4′ supported the assignment that the
236
hydroxy was attached to C-3′. In addition, one singlet at δH 3.52 (3H, s, –COOCH3)
237
and two mutually coupled triplets at δH 2.66 (2H, t, J=8.0 Hz, H-7′) and δH 2.46 (2H, t,
12
ACS Paragon Plus Environment
Page 13 of 32
Journal of Agricultural and Food Chemistry
238
J=8.0 Hz, H-8′) evidenced the presence of a methyl propionate side chain. The
239
location of this side chain at C-4′ was determined on the basis of crosspeaks from H-7′
240
to C-3′, C-4′, and C-5′ and from H-4′ to C-7′ in the HMBC spectrum. The linkage of
241
C-1′ to C-8 by one atom of oxygen was deduced from the above-mentioned reasoning
242
and the molecular weight. Thus, the structure of 1 was defined as methyl
243
3-(2-hydroxy-4-O-(7-hydroxycoumarinyl) phenyl) propanoate. An X-ray diffraction
244
analysis corroborated this proposed structure (Figure 3).
245
Gardnerol B (2) was obtained as a white powder. The HRMS data of compound 2
246
showed a molecular ion at m/z 527.1173 [M + Na]+ (calcd. 527.1160), which was 162
247
mass units (i.e., C6H10O5) more than that of compound 3 {m/z 343.0811, [M + H]+}.
248
Correspondingly, the 1H and 13C NMR data of 2 were similar to those of 3 except for
249
the presence of an additional glucopyranosyl moiety.
250
anomeric proton signal at δH 5.09 as a doublet with a coupling constant of 7.6 Hz
251
indicated the presence of the β-glucopyranosyl moiety. The HMBC correlation
252
between the anomeric proton (H-1′′′, δH 5.09) and C-7 (δC 154.7) indicated that the
253
β-glucopyranosyl moiety was attached to C-7 (Figure 2). Furthermore, to confirm the
254
structure of 2, compound 2 was hydrolyzed under acidic conditions. The sugar residue
255
of 2 was identified as D-(+)-glucose by TLC comparison with an authentic sample
256
and by its optical rotation value (see Supporting Information). On the basis of the
257
above analyses, the structure of compound 2 was identified as edgeworic
258
acid-7-O-β-D- glucoside.
259
20
The observation of the
α-Glucosidase Inhibition. The IC50 values of compounds 1− −21 and extracts from
13
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
260
the flower of E. gardneri are shown in Table 3. In accordance with the previous report,
261
the flower extract exhibited a significant inhibitory activity with an IC50 value of 267
262
µg/mL. Among these compounds, compound 11 was the most active, with an IC50
263
value of 5.1 µg/mL. Additionally, compound 10 showed a potent inhibitory activity
264
with an IC50 value of 56.2 µg/mL. Compound 5, the most abundant compound in the
265
extract, showed a moderate activity, with an IC50 value of 202 µg/mL. Compounds 8,
266
12–13, and 16–18 also showed moderate activities with IC50 values of 486, 957, 279,
267
179, 272, and 253 µg/mL, respectively. Unfortunately, compounds 2, 3, 7, 14, and
268
19–21 showed no activities at the maximum concentration tested (3000 µg/mL).
269
Previous literature reported that flavonoids with 7, 3, 3′, and 4′ hydroxy groups
270
showed significant α-glucosidase inhibitory activity.21 Our results revealed that the 3′
271
hydroxy groups are responsible for increased activity because compounds 10 and 11,
272
with a 3′ hydroxy group, were more active than compounds 5 and 15–18, without a 3′
273
hydroxy group. Compounds 2 and 3 showed significantly less activity than
274
compounds 1 and 4. This result suggests that the methyl ester group has an impact on
275
the inhibitory activity. When compound 4 was compared to compound 1, the
276
inhibitory activity was reduced because of the absence the hydroxy groups at the C-5
277
position.
278
Types of α-Glucosidase Inhibition. To clarify the α-glucosidase inhibition mode
279
of compound 5, which was the most abundant compound in the extract,
280
Lineweaver-Burk plots were generated (Figure 4).22 As shown in Figure 4, the value
281
of vertical axis intercept (1/Vmax) remained unchanged with the increase of the
14
ACS Paragon Plus Environment
Page 14 of 32
Page 15 of 32
Journal of Agricultural and Food Chemistry
282
concentrations of compound 5, indicating that compound 5 was a competitive
283
inhibitor. According to Michaelis−Menten kinetics, the value of inhibition constant
284
(Ki) is 0.473 mM.
285
Antihyperglycemic Effects of Compound 5 on Oral Sucrose Tolerance in
286
Normal and STZ -induced Diabetic Mice. The oral sucrose tolerance test (OSTT) is
287
usually performed to evaluate the efficacy of a drug in inhibiting intestinal
288
α-glucosidase in vivo.23 Compound 5 was a major constituent of the extract of E.
289
gardneri, and 53 g of compound 5 were obtained from the extract. In addition,
290
compound 5 showed significant α-glucosidase inhibitory activity in vitro. Thus,
291
compound 5 was evaluated for its antihyperglycemic effects using an OSTT in both
292
normal and STZ-induced diabetic mice. In comparison with the vehicle, the oral
293
administration of compound 5 at a dose of 300 mg/kg significantly (p < 0.05) reduced
294
the postprandial blood glucose level of normal mice (Figure 5A). The
295
antihyperglycemic effect was observed at 30, 60, and 90 min after sucrose loading and
296
was compared with that of acarbose (5 mg/kg). In contrast, the 150 mg/kg dose did
297
not show a significant decrease in glycemia throughout the experiment. The OSTT
298
was repeated in STZ-induced diabetic mice. These results were similar to those for
299
normal mice (Figure 5B). According to the results of the OSTT, compound 5 inhibited
300
the activity of intestinal α-glucosidase.
301
The Hypoglycemic Effect of Compound 5 on Normal and STZ-induced 24, 25
302
Diabetic Mice. According to previous methods,
the hypoglycemic activity of
303
compound 5 was evaluated in both normal and STZ-induced diabetic mice. As shown
15
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 16 of 32
304
in Figure 6, compound 5, which had decreased fasting glucose levels in STZ-induced
305
diabetic mice, did not lower the fasting glucose levels in normal mice. In diabetic
306
mice, the oral administration of compound 5 at 150 and 300 mg/kg at both doses
307
significantly decreased the fasting glucose levels when compared with the
308
vehicle-treated groups (p < 0.05). The dose at 300 mg/kg resulted in a significant
309
decrease in glucose level at 1.5 h (−9.7%), 3 h (−10.9%), 5 h (−18.5%), 7 h (−31.5%),
310
and 9 h (−37.8%). After an administration of 150 mg/kg of compound 5, the glucose
311
level decreased by 4.7% at 1.5 h, 10.5% at 3 h, 20.2% at 5 h, 24.4% at 7 h, and 36.4%
312
at 9 h. Because compound 5 displayed a hypoglycemic effect in diabetic but not in
313
normal mice, in contrast with glibenclamide, these results indicate that compound 5
314
may not act directly via insulin liberation. Previous studies suggested that kaempferol,
315
the
316
hyperglycemia-impaired pancreatic β-cell viability and insulin-secretory function.26
317
Therefore, the hypoglycemic action of compound 5 probably involves the protection
318
of pancreatic β-cell survival and function. Further studies are necessary to
319
demonstrate the mechanisms by which compound 5 decreased the fasting glucose
320
levels in STZ-induced diabetic mice.
hydrolysis
products
of
compound
5,
improved
the
chronic
321
In summary, two new phenolics, along with 19 known phenolics, were isolated
322
from the flower of E. gardneri. Their inhibitory activity against α-glucosidase from
323
Saccharomyces cerevisiae was evaluated, and the data showed that the flower of E.
324
gardneri is a rich source of natural α-glucosidase inhibitors. Compound 5, the most
325
abundant compound in the extract, showed a significant α-glucosidase inhibitory
16
ACS Paragon Plus Environment
Page 17 of 32
Journal of Agricultural and Food Chemistry
326
activity and acted as a competitive inhibitor of α-glucosidase. Furthermore, compound
327
5 is effective in vivo for reducing the fasting and postprandial blood glucose levels in
328
STZ-induced diabetic mice. In conclusion, the present study complements the current
329
knowledge about the chemical composition and antihyperglycemic activity of the
330
flower of E. gardneri, and it provides scientific evidence to substantiate the use of this
331
flower for traditional therapeutic and dietary uses.
332 333
334
Acknowledgment. This work was supported by the National Natural Science Foundation of China (No.21402030, 21402031 and 21272043). Supporting Information Available: Acid hydrolysis procedure, the HRESIMS,
335
IR, 1H,
13
336
available free of charge via the Internet at http://pubs.acs.org.
C, and 2D NMR spectrum of compounds 1 and 2. This information is
17
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
337
REFERENCES
338
1. Wild, S.; Roglic, G.; Green, A.; Sicree, R.; King, H. Global Prevalence of
339
Diabetes: Estimates for the year 2000 and projections for 2030. Diabetes Care 2004,
340
27, 1047–1053.
341
2. Blonde, L. Benefits and risks for intensive glycemic control in patients with
342
diabetes mellitus. Am. J. Med. Sci. 2012, 343, 17−20.
343
3. Bolen, S.; Feldman, L.; Vassy, J.; Wilson, L.; Yeh, H. C.; Marinopoulos, S.; Wiley,
344
C.; Selvin, E.; Wilson, R.; Bass, E. B.; Brancati, F. L. Systematic review: comparative
345
effectiveness and safety of oral medications for type 2 diabetes mellitus. Ann. Intern.
346
Med. 2007, 147, 386−399.
347
4. Lebovitz, H. E. α-Glucosidase inhibitors. Endocrinol. Metab. Clin. North Am. 1997,
348
26, 539−551.
349
5. Fujisawa, T.; Ikegami, H.; Inoue, K.; Kawabata, Y.; Ogihara, T. Effect of two
350
α-glucosidase inhibitors, voglibose and acarbose, on postprandial hyperglycemia
351
correlates with subjective abdominal symptoms. Metabolism 2005, 54, 387−390.
352
6. Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N. Antidiabetic
353
Indian plants: a good source of potent amylase inhibitors. Evidence-Based
354
Complement. Altern. Med. 2011, 2011, 810207.
355
7. Benalla, W.; Bellahcen, S.; Bnouham, M. Antidiabetic medicinal plants as a source
356
of α-glucosidase inhibitors. Curr. Diabetes Rev. 2010, 6, 247−254.
357
8. Yilmazer-Musa, M.; Griffith, A. M., Michels, A. J.; Schneider, E.; Frei, B.
358
Grape seed and tea extracts and catechin 3-gallates are potent inhibitors of α-amylase
18
ACS Paragon Plus Environment
Page 18 of 32
Page 19 of 32
Journal of Agricultural and Food Chemistry
359
and α-glucosidase activity. J. Agric. Food Chem. 2012, 60, 8924−8929.
360
9. Li, Y. Q.; Zhou, F. C.; Gao, F.; Bian, J. S.; Shan, F. Comparative evaluation of
361
quercetin, isoquercetin and rutin as inhibitors of α-glucosidase. J. Agric. Food Chem.
362
2009, 57, 11463−11468.
363
10. McDougall, G. J.; Shpiro, F.; Dobson, P.; Smith, P.; Blake, A.; Stewart, D.
364
Different polyphenolic components of soft fruits inhibit α-amylase and α-glucosidase.
365
J. Agric. Food Chem. 2005, 53, 2760−2766.
366
11. da Silva Pinto, M.; Kwon, Y.-I.; Apostolidis, E.; Lajolo, F. M.; Genovese, M. I.;
367
Shetty, K. Functionality of bioactive compounds in Brazilian strawberry (Fragaria ×
368
ananassa Duch.) cultivars: evaluation of hyperglycemia and hypertension potential
369
using in vitro models. J. Agric. Food Chem. 2008, 56, 4386−4392.
370
12. Gonçalves, R.; Mateus, N.; de Freitas, V. Inhibition of α-amylase activity by
371
condensed tannins. Food Chem. 2011, 125, 665−672.
372
13. Liu, M.; Yin, H.; Liu, G.; Dong, J.J.; Qian, Zh.H.; Miao, J.L. Xanthohumol, a
373
Prenylated Chalcone from Beer Hops, Acts as an α-Glucosidase Inhibitor in Vitro. J.
374
Agric. Food Chem. 2014, 62, 5548−5554.
375
14. Xu, P.; Xia, Z.; Lin, Y. Chemical constituents from Edgeworthia gardneri
376
(Thymelaeaceae) Biochem. Syst. Ecol. 2012, 45, 148−150.
377
15. Wang Q.-Y.; Xu, H.-Y.; Xu, Z.-H.; Lu, Z.-M.; Liu, M.; Shi, J.-S. Hypoglycemic
378
effect of water extracts from Edgeworthia gardneri (Wall.) Meissn on type 2 diabetic
379
mice. Nat. Prod. Res. Dev. 2014, 26, 1385−1388.
380
16. Geng, Y.; Yang, H.-M.; Xu, H.-Y.; Shi, J.-S. α-Glucosidase inhibitory activity of
19
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
381
the alabastrum of Edgeworthia gardneri (Wall.) Meissn. Journal of Food Science
382
and Biotechnology 2013, 32(9), 967−971.
383
17. Gao, D.; Zhang, Y.-L.; Xu, P.; Lin, Y.-X.; Yang, F.-Q.; Liu, J.-H.; Zhu, H.-W.;
384
Xia, Z.-N. In vitro evaluation of dual agonists for PPARγ/β from the flower of
385
Edgeworthia gardneri (wall.)Meisn. J. Ethnopharmacol. 2015, 162, 14−19.
386
18. Li, S.-S.; Gao, Z.; Feng, X.; Hecht, S. M. Biscoumarin derivatives from
387
Edgeworthia gardneri that inhibit the lyase activity of DNA polymerase β. J. Nat.
388
Prod. 2004, 67, 1608−1610.
389
19. Jeon, S. Y.; Oh, S.; Kim, E.; Imm, J. Y. α-Glucosidase Inhibiton and
390
Antiglycation Activity of Laccase-Catalyzed Catechin Polymers. J. Agric. Food Chem.
391
2013, 61, 4577−4584.
392
20. Li, X. N.; Tong, S. Q.; Cheng, D. P.; Li, Q. Y.; Yan, J. Z. Coumarins from
393
Edgeworthia chrysantha. Molecules 2014, 19: 2042−2048.
394
21. Escandón-Rivera, S.; González-Andrade, M.; Bye, R.; Linares, E.; Navarrete, A.;
395
Mata, R. α-Glucosidase Inhibitors from Brickellia cavanillesii. J. Nat. Prod. 2012,
396
75, 968−974.
397
22. Yan, J.; Zhang, G.; Pan, J.; Wang, Y. α-Glucosidase inhibition by luteolin:
398
Kinetics, interaction and molecular docking. Int. J. Biol. Macromol. 2014, 64,
399
213−223.
400
23. Brindis, F.; Rodríguez, R.; Bye, R.; Gonzalez-Andrade, M.; Mata, R.
401
(Z)-3-Butylidenephthalide from Ligusticum porteri, an α-Glucosidase Inhibitor. J.
402
Nat. Prod. 2011, 14, 314−320.
20
ACS Paragon Plus Environment
Page 20 of 32
Page 21 of 32
Journal of Agricultural and Food Chemistry
403
24. Nuñez-López, A. M.; Paredes-López, O.; Reynoso-Camacho, R. Functional and
404
Hypoglycemic Properties of Nopal Cladodes (O. ficusindica) at Different Maturity
405
Stages Using in Vitro and in Vivo Tests. J. Agric. Food Chem. 2013, 61,
406
10981−10986.
407
25. Narváez-Mastache, M. J.; Garduño-Ramírez, L. M.; Alvarez, L.; Delgado, G.
408
Antihyperglycemic Activity and Chemical Constituents of Eysenhardtia platycarpa. J.
409
Nat. Prod. 2006, 69, 1687−1691.
410
26.
411
hyperglycemia-impaired pancreatic beta-cell viability and insulin secretory function.
412
Eur. J. Pharmacol. 2011, 670, 325–332.
Zhang,
Y.;
Liu,
D.
Flavonol
kaempferol
21
ACS Paragon Plus Environment
improves
chronic
Journal of Agricultural and Food Chemistry
413
Figure captions
414
Figure 1. Structures of compounds 1−21.
415
Figure 2. Key 1H-1H COSY and HMBC correlations for compounds 1 and 2.
416
Figure 3. X-ray structure of compound 1.
417
Figure 4. (A) Lineweaver–Burk plots of the reaction of α-glucosidase at different
418
concentrations of substrate and compound 5. (B) A partially enlarged view of Figure
419
4A.
420
Figure 5. Effects of compound 5 on blood glucose levels in normal (A) and STZ
421
-induced diabetic mice (B) using the OSTT. Data are the means ±SEM for 6 mice in
422
each group. * P < 0.05 by one-way ANOVA with post-hoc test compared with
423
control.
424
Figure 6. Hypoglycemic effect of compound 5 on normal (A) and STZ-induced
425
diabetic mice (B). Data are the means ±SEM for 6 mice in each group. * P < 0.05 by
426
one-way ANOVA with post-hoc test compared with control.
427
22
ACS Paragon Plus Environment
Page 22 of 32
Page 23 of 32
Journal of Agricultural and Food Chemistry
Table 1. 1H and 13C NMR Data for Compound 1 in Methanol-d4 and DMSO-d6 Methanol-d4 position
δC
2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 9′C-OMe 7-OH 3′-OH
162.7, C 112.3, CH 146.1, CH 114.1, C 126.2, CH 115.0, CH 156.0, C 130.4, C 149.9, C 158.8, C 103.2, CH 157.3, C 122.1, C 131.3, CH 106.8, CH 26.5, CH2 35.1, CH2 175.8, C 51.9, CH3
DMSO-d6
δH mult (J in Hz) 6.19, d (9.6) 7.88, d (9.6) 7.37, d (8.8) 6.95, d (8.8)
6.35, d (2.4)
6.93, d (8.4) 6.27, dd (8.4, 2.4) a (2.80, m), b (2.80, m) a (2.56, m), b (2.56, m) 3.62, s
δC 159.7, C 111.7, CH 144.7, CH 112.2, C 125.3, CH 113.6, CH 154.2, C 128.2, C 148.3, C 156.8, C 101.5, CH 155.9, C 120.2, C 130.1, CH 105.3, CH 24.8, CH2 33.5, CH2 172.9, C 51.1, CH3
23
ACS Paragon Plus Environment
δH mult (J in Hz) 6.18, d (9.6) 7.94, d (9.6) 7.40, d (8.8) 6.93, d (8.8)
6.24, d (2.4)
6.89, d (8.4) 6.19, dd (8.4, 2.4) a (2.66, m), b (2.66, m) a (2.46, m), b (2.46, m) 3.52, s 10.63, s 9.37, s
Journal of Agricultural and Food Chemistry
Page 24 of 32
Table 2. 1H and 13C NMR Data for Compound 2 in Methanol-d4 and DMSO-d6 Methanol-d4 position
δC
2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′
162.2, C 114.7, CH 145.6, CH 116.3, C 126.0, CH 114.1, CH 154.7, C 132.4, C 149.3, C 158.7, C 103.8, CH 157.1, C 122.9, C 131.4, CH 107.3, CH 26.5, CH2 35.8, CH2 178.7, C 101.8, CH 74.6, CH 77.8, CH 70.9, CH 78.2, CH 62.2, CH2
DMSO-d6
δH mult (J in Hz) 6.28, d (9.6) 7.90, d (9.6) 7.46, d (8.8) 7.29, d (8.8)
6.39, d (2.4)
6.96, d (8.4) 6.31, dd (8.4, 2.4) a (2.79, m), b (2.79, m) a (2.53, m), b (2.53, m) 5.09, d (7.6) 3.37, overlap 3.45, overlap 3.37, overlap 3.45, overlap a 3.86, m b 3.69, dd (12.0, 5.2)
δC 159.5, C 113.5, CH 144.4, CH 114.3, C 125.1, CH 112.5, CH 153.4, C 130.2, C 148.3, C 156.9, C 102.4, CH 155.8, C 121.0, C 130.0, CH 105.7, CH 25.1, CH2 34.6, CH2 175.1, C 100.2, CH 73.0, CH 76.7, CH 69.4, CH 77.2, CH 60.5, CH2
24
ACS Paragon Plus Environment
δH mult (J in Hz) 6.34, d (9.6) 8.05, d (9.6) 7.40, d (8.8) 6.93, d (8.8)
6.33, d (2.4)
6.94, d (8.4) 6.27, dd (8.4, 2.4) a (2.65, m), b (2.65, m) a (2.40, m), b (2.40, m) 5.07, d (7.6) 3.16, overlap 3.25, m 3.13, overlap 3.36, m a 3.67, m b 3.45, dd (12.0, 5.2)
Page 25 of 32
Journal of Agricultural and Food Chemistry
Table 3. α-Glucosidase Inhibition by Extract and Compounds 1−21a Extract 1 2 3 4 5 6 7 8 9 10 11 a
IC50(µg/mL) 267±19 517±24 > 2000b > 2000 897±59 202±12 1200±134 > 2000 486±45 541±32 56.2±4.1 5.1±0.3
12 13 14 15 16 17 18 19 20 21 Acarbose
IC50(µg/mL) 957±36 279±14 > 2000 1233±87 179±9.2 272±15 253±19 > 2000 > 2000 > 2000 465±37
Values are the means ± SEM from at least three independent experiments. b Exceeds
maximum concentration tested.
25
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
5
6
4
4a
Page 26 of 32
3
Glc O
2
7
R1
8a O 1
8
O 1'
2'
O
R1 OH
O
O
O OH
6'
R2
R2
5'
HO 3'
4'
HO
7'
R2
8' 9'
O
R2=OCH3 R2=OH R2=OH R2=OCH3
HO
1'
20 R1=H 7 R1=OCH3 R2=OH 9 R1=H R2=OH 21 R =H 1 12 R1=OH R2=OH
R2=
OH
R2= O
HO
O
O OH 4'
OH H
O H
OH
HO OH
7 5 4 OH O
OH
19 R1=OCH3 R2=
R1
H 8
R2 1 O
O
6
O
1 R1=OH 2 R1=O-Glc 3 R1=OH 4 R1=O-Glc
OH
R1
HO
HO O
O
13
14 OH
5 R1=O-S1 10 R1=OH 11 R1=OH 15 R1=O-Glc 16 R1=O-Glc 17 R1=O-S2 18 R1=O-S2
R2=H R2=H R2=OH R2=OH R2=H R2=OH R2=H
O O
O
OH HO
HO
O
OH
OH
HO
OH
OH Glc
O
O
O
S1
Figure 1
26
ACS Paragon Plus Environment
OH HO OH S2
OH OH
Page 27 of 32
Journal of Agricultural and Food Chemistry
OH HO
O
O
O
O
O
O
O
O HO
OH OH
HO
O
HO
O
O 1
H-1H COSY
OH
HMBC
Figure 2
27
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 3
28
ACS Paragon Plus Environment
Page 28 of 32
Page 29 of 32
Journal of Agricultural and Food Chemistry
Figure 4
29
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 5
30
ACS Paragon Plus Environment
Page 30 of 32
Page 31 of 32
Journal of Agricultural and Food Chemistry
Figure 6
31
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Table of Contents Graphic
32
ACS Paragon Plus Environment
Page 32 of 32