α-Glucosidase inhibition and antihyperglycemic activity of flavonoids from Ampelopsis grossedentata and the flavonoid derivatives

Article abstract of DOI:10.1016/j.bmc.2016.02.018

The dried leaves and stems of Ampelopsis grossedentata have been used as a health tea and herbal medicine for hundreds of years in China. The study was aimed at searching for novel α-glucosidase inhibitors among the richest components of A. grossedentata and their derivatives. Three known major components (1-3) were isolated by recrystallization process and six new derivatives (4-9) were obtained by etherification of the bioactive flavonoid. All compounds were evaluated for their inhibitory activities against α-glucosidase (from Saccharomyces cerevisiae). As a result, compound 9 showed a significant α-glucosidase inhibitory activity with IC₅₀ value of 9.3 μM and acted as a competitive inhibitor with the value of the inhibition constant (K_i) being 10.3 μM. The oral administration of compound 9 at a dose of 50 mg/kg significantly reduced the post prandial blood glucose levels of normal and streptozotocin (STZ)-induced diabetic mice. Furthermore, compound 9 significantly decreased the fasting blood glucose levels in STZ-induced diabetic mice.

Full text of DOI:10.1016/j.bmc.2016.02.018

Accepted Manuscript
α-Glucosidase inhibition and antihyperglycemic activity of flavonoids from
Ampelopsis grossedentata and the flavonoid derivatives
Jia Chen, Yuechan Wu, Jianwei Zou, Kun Gao
PII:
S0968-0896(16)30097-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.02.018
BMC 12820
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 December 2015
6 February 2016
9 February 2016
Please cite this article as: Chen, J., Wu, Y., Zou, J., Gao, K., α-Glucosidase inhibition and antihyperglycemic activity
of flavonoids from Ampelopsis grossedentata and the flavonoid derivatives, Bioorganic & Medicinal Chemistry
(2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.02.018
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α-Glucosidase inhibition and antihyperglycemic activity of
flavonoids from Ampelopsis grossedentata and the flavonoid
derivatives
Jia Chen, Yuechan Wu, Jianwei Zou, Kun Gao
School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang
University, Ningbo, 315100, People’s Republic of China
Abstract: The dried leaves and stems of Ampelopsis grossedentata have been used as a health tea and
herbal medicine for hundreds of years in China. The study was aimed at searching for novel
α-glucosidase inhibitors among the richest components of A. grossedentata and their derivatives. Three
known major components (1-3) were isolated by recrystallization process and six new derivatives (4-9)
were obtained by etherification of the bioactive flavonoid. All compounds were evaluated for their
inhibitory activities against α-glucosidase (from Saccharomyces cerevisiae). As a result, compound 9
showed a significant α-glucosidase inhibitory activity with IC50 value of 9.3 μM and acted as a
competitive inhibitor with the value of the inhibition constant (Ki) being 10.3 µM. The oral
administration of compound 9 at a dose of 50 mg/kg significantly reduced the post prandial blood
glucose levels of normal and streptozotocin (STZ)-induced diabetic mice. Furthermore, compound 9
significantly decreased the fasting blood glucose levels in STZ-induced diabetic mice.
Keywords: Ampelopsis grossedentata; flavonoids; α-Glucosidase inhibitor; antihyperglycemic
activity; health tea.
*Corresponding author. Tel.: +86-574-88229598; Fax: +86-574-88130130.
E-mail address: gaok@nit.net.cn (K. Gao)
1

1. Introduction
Diabetes mellitus is a chronic metabolic disease, which has become a serious global problem. In
2010 an estimated 285 million people had diabetes and within the next 20 years this number is
expected to almost double.1 Many antidiabetic therapies focus on decreasing the level of blood glucose,
for glycemic control is an effective therapy for diabetes.2, 3 α-Glucosidase, a critical enzyme for the
digestion of carbohydrates, catalyzes the cleavage of absorbable monosaccharides, starting from
disaccharides and oligosaccharides. Thus, α-glucosidase inhibitors reduce postprandial hyperglycemia
by slowing the digestion of carbohydrates in the intestines.4 Although a number of synthetic medicines
are available, drugs of a natural origin have also aroused great interest.5-8 The consumption of natural
and natural analog α-glucosidase inhibitors derived from plant-based foods or supplements offers an
attractive strategy to control postprandial hyperglycemia due to their low cost and low incidence of
major undesirable side effects.
A. grossedentata belongs to the genus of Ampelopsis Michx. (family: Vitaceae) and is a wild plant
growing in mountainous areas of southern China. Its dried leaves and stems, called Vine Tea or Mao
Yan Mei, have been consumed as a health tea and herbal medicine for hundreds of years.9 As recorded
in the Chinese Materia Medica, vine tea carries out several functions such as clearing away heat,
promoting diuresis and blood circulation and removing channel obstructions.10 Several studies have
demonstrated that vine tea possesses many beneficial pharmacological properties including
anti-inflammatory,11 antioxidative,9, 12 antihypertensive, hepatoprotective and antiviral activities.13, 14 It
is also claimed that drinking the herbal tea of A. grossedentata can alleviate the severity of many
disorders, such as diabetes and hyperlipidemia.15 However, to the best of our knowledge, the
α-glucosidase inhibition and antihyperglycemic activity of constituents from A. grossedentata and their
derivatives have yet to be deeply investigated.
2

In the present paper, work was undertaken to deal with the purification of the major components
1-3 (Figure 1) and the structural modification of bioactive component 1 from A. grossedentata, to
hopefully obtain a better understanding of the potential bioactivity of flavonoids and their analogs.
Figure 1. The structures of three known major components 1-3 and six new flavonoid derivatives 4-9.
2. Results and Discussion
2.1. Chemistry
The chemical structures of compounds 1-9 are reported in Figure 1. Three known compounds
were recognized as myricetin (1), dihydromyricetin (2) and myricitrin (3) by comparison with literature
values.16
The synthetic routes for the compounds 4-9 are outlined in Figure 2.
3

Figure 2. General procedures for the synthesis of new flavonoid derivatives 4-9. Reaction conditions:
(a) TsCl, DCM, KOH or Et3N, 0℃, 2h to 5h; (b) EGTs-n (n = 1-4), K2CO3, DMF, 110℃, 3h to 5h; (c)
TsCl, DCM, pyridine, 0℃; (d) AEGTs-1, K2CO3, DMF, 110℃, 5h; (e) TsCl, DCM, Et3N, 0℃to room
temperature, 12h; (f) AEGTs-2, K2CO3, DMF, 110℃, 3h;
Ethylene glycol ditosylates were prepared from the corresponding polyethylene glycols (PEGs) or
amino polyethylene glycol (APEGs) according to previous methods with some modifications.17, 18 The
etherification of myricetin (1) was carried out by the method of an improved Williamson ether
synthesis using K2CO3 as a base in the solvent system of N,N-dimethylformamide (DMF). The reaction
4

mixtures were purified by CC or semi-preparative HPLC to give the desired myricetin derivatives 4-9.
The purity of the products (4-9) was analyzed by HPLC.
The structures of 4-9 were elucidated on a comprehensive analysis of their spectroscopic data.
Compounds 4-9 are polar molecules and need a higher temperature at 275 ºC (General experimental
procedures) to gasify in the HRESIMS for all the structures of these compounds contain the ethoxyl
hydroxyl and five phenolic hydroxyl groups. The conditions for HRESIMS are easy to make ethoxyl
hydroxyl group lose a molecule of water; thus only [M-H2O+H]+ signals for these compounds are
observed in the HRESIMS spectra (Supplementary Materials).
Compound 4 has a [M-H2O+H]+ ion peak at m/z 345.0602 in the HRESIMS corresponding to a
molecular formula C17H14O9 with eleven degrees of unsaturation, being C2H4O2 more than myricetin
(1). The 1H NMR spectrum showed the presence of a hydroxyl proton [δ 12.42 (1H, br s)], two sets of
meta-phenyl protons assigned to two phenyl groups [δ 7.33 (1H, s), 7.24 (1H, s), 6.43 (1H, s) and 6.20
(1H, s)] and an ethoxyl hydroxyl [δ 4.31 (2H, m) and 4.29 (2H, m)]. The signals of 4 were similar to
those of 1 16, being one ethoxyl hydroxyl group more than myricetin. Accordingly, the 13C NMR
spectrum displayed a total of 17 carbon resonances, which were 2 carbon resonances more than
myricetin and was also assigned to an ethoxyl hydroxyl group [δ 64.2 and 64.1]. The location of the
ethoxyl hydroxyl group at the C-4' position could be determined on the basis of the characteristic
downfield shifts of the carbon resonances for C-4' (δ 136.7), when compared to that of myricetin (δ
126.0, C-4').16 The assignment of 1H NMR and 13C NMR for compounds 5-9 was done in the same way
by comparison of NMR data with that of myricetin.
2.2. α-Glucosidase inhibition
The α-glucosidase (from Saccharomyces cerevisiae) inhibition was assessed according to a
slightly modified method of Jeon et al.19 The IC50 values of compounds 1−9 are shown in Table 1.
5

Among the major components, compound 1 showed a good α-glucosidase inhibitory activity with an
IC50 value of 319.3 μM, and other abundant compounds 2 and 3 showed moderate activities with IC50
values of 633.9 and 837.7 μM, respectively. The activity of flavonoid 2 being lower than that of 1
revealed that the double band of C ring in the molecular is an essential requirement for the activity.
Moreover, the α-glucosidase inhibition of 3 was also less sensitive than that of 1. The weak activity of
3 could be caused by the absence of the essential hydroxyl group at C ring which may be responsible
for the activity.
Table 1 α-Glucosidase inhibitory activity of compounds 1-9.
Compounds
IC50 (μM ± SEM)a
319.3 ± 22.9
633.9 ± 24.9
837.7 ± 22.0
84.5 ± 8.6
1
2
3
4
43.8 ±1.5
5
233.1 ± 12.0
463.1 ± 15.8
41.1 ± 2.3
6
7
8
9
9.3 ± 0.4
Acarbose
720.3 ± 57.5
aIC50 values are shown as mean ± SEM from three independent experiments.
Based on a reservation of both the double band and a hydroxyl group of C ring, the natural
flavonoid 1 was modified. As a result, new flavonoid derivatives 4-7 were obtained. The introduction
of some polyethylene glycol in C-4 of the B ring caused an increased activity in the biological assay.
The structure-activity relationship among compounds 4-7 suggested that the most active flavonoid
would display two ethylene glycols, for compound 5 was found to be the most active with IC50 value of
43.8 μM. Unfortunately, while the number of introduced ethylene glycols was up to four, a decreased
activity in the biological assay ensued, for compound 7 showed a moderate activity with IC50 values of
463.1 μM.
Interestingly, the introduction of nitrogen substituted groups including N-tosyl group at C-4′ in
6

flavonoid were also beneficial to the α-glucosidase inhibitory activities. For example, the B ring of
compound 8, replaced by a nitrogen substituted group at C-4′, showed a potent inhibitory activity with
an IC50 value of 41.1 μM. Additionally, compound 9, by introducing an N-tosyl group in the B ring,
was the most active, with an IC50 value of 9.3 μM. The result was also supported by a
computer-assisted docking (CAD) study, which suggests that it is not only the 8-OH of compound 9
that forms a hydrogen bond with the ASP349 in the active site, but also the N-tosyl group of compound
9 that forms hydrogen bonds with the LYS155 and SER244 in the active sites (Supplementary
Materials).
2.3. Type of α-glucosidase inhibition
To clarify the α-glucosidase inhibition mode of compound 9, which was the analog of the most
abundant compound in the extract, Lineweaver−Burk plots were generated.20 As shown in Figure 3,
the value of the vertical axis intercept (1/Vmax) remained unchanged with the increase of the
concentrations of compound 9, indicating that compound 9 was a competitive inhibitor. According to
Michaelis−Menten kinetics, the value of the inhibition constant (Ki) is 10.3 μM.
Figure 3. (a) Lineweaver−Burk plots of the reaction of α-glucosidase at different concentrations of
substrate and compound 9. (b) Partially enlarged view of panel A.
2.4. Antihyperglycemic effects of compound 9 on oral sucrose tolerance in normal and
STZ-induced diabetic mice.
7

The OSTT is usually performed to evaluate the efficacy of a drug in inhibiting intestinal
α-glucosidase in vivo.21 Compound 9 was the most active among the evaluated flavonoids, for it
showed a significant α-glucosidase inhibitory activity in vitro. Thus, compound 9 was further evaluated
for its antihyperglycemic effects using an OSTT in both normal and STZ-induced diabetic mice. In
comparison to the vehicle, the oral administration of compound 9 at a dose of 100 and 50 mg/kg
significantly (p < 0.05) reduced the postprandial blood glucose level of normal mice (Figure 4a). The
antihyperglycemic effect was observed at 30, 60 and 90 min after sucrose loading and was compared to
that of acarbose (5 mg/kg). In contrast, the 25 mg/kg dose did not show a significant decrease in
glycemia throughout the experiment. The OSTT was repeated in STZ-induced diabetic mice. These
results were similar to those for normal mice (Figure 4b). According to the results of the OSTT,
compound 9 inhibited the activity of intestinal α-glucosidase.
Figure 4. Effects of compound 9 on blood glucose levels in (a) normal and (b) STZ-induced diabetic
mice using the OSTT. Data are the means ± SEM for six mice in each group. p < 0.05 by one-way
ANOVA with a post-hoc test compared to the control.
2.5. Hypoglycemic effect of compound 9 on normal and STZ-induced diabetic mice.
According to previous methods,22, 23 the hypoglycemic activity of compound 9 was evaluated in
both normal and STZ-induced diabetic mice. As shown in Figure 5, compound 9, which had decreased
fasting glucose levels in STZ-induced diabetic mice, did not lower the fasting glucose levels in normal
8

mice. In diabetic mice, the oral administration of compound 9 at doses of 100 and 50 mg/kg
significantly decreased the fasting glucose levels when compared to the vehicle treated groups (p <
0.05). The dose at 100 mg/kg resulted in a significant decrease in the glucose level at 1.5 h (− 37.0 %),
3 h (− 45.1 %), 5 h (−60.0 %), 7 h (− 65.4 %) and 9 h (− 67.3 %). After an administration of 50 mg/kg
of compound 9, the glucose level decreased by 27.8 % at 1.5 h, 35.9 % at 3 h, 47.3 % at 5 h, 59.6 % at
7 h and 61.5 % at 9 h. Because compound 9 displayed a hypoglycemic effect in diabetic mice but not in
normal mice, in contrast with glibenclamide, these results indicate that compound 9 may not act
directly via insulin liberation.
Figure 5. Hypoglycemic effect of compound 9 on (a) normal and (b) STZ-induced diabetic mice. Data
are the means ± SEM for six mice in each group. p < 0.05 by one-way ANOVA with a post-hoc test
compared to the control.
3. Conclusions
In the present study, myricetin (1), dihydromyricetin (2) and myricitrin (3) were major
components obtained from leaves and stems of A. grossedentata, and flavonoid derivatives (4–9) were
newly semi-synthesized from naturally occurring myricetin. It is particularly noteworthy that
compound 9 acted as a competitive α-glucosidase inhibitor with IC50 values of 9.3 μM. This is the first
report that myricetin analogs showed significant α-glucosidase inhibitory activity. Compound 9 will
9

enter into the architectural diversity of the antihyperglycemic activity flavonoids family. Moreover, the
result suggested that the introduction of ethylene glycol and N-tosyl group moieties in myricetin are
beneficial for α-glucosidase inhibitory activities. This is also a first-time report on the introduction of
ethylene glycol and N-tosyl group moieties in naturally occurring myricetin.
The result of Lee’s group suggested that the 4'-OH function plays an important role in anti-α
glucosidase. 24 While this function is masked in compound 9, the activity is better in the study, because
the α-glucosidase used in the two experiments were not identical, just of 40% sequence similarity
(Supplementary Materials); thus it was possible that the interaction mode for compound 9 differed from
those reported.
Previous studies suggested that rutin, the analog of compound 9, improved the chronic
hyperglycemia impaired pancreatic β-cell viability and insulinsecretory function.25 Therefore, the
hypoglycemic action of compound 9 probably is involved in the protection of pancreatic β-cell survival
and function.
Although compound 9 showed a better α-glucosidase inhibitory activity than that of acarbose, the
result of in vivo study indicated that acarbose has a stronger hypoglycemic effect than the prepared
compound 9. This might be because many enzyme systems are related to the hypoglycemic effect and
acarbose is not the only α-glucosidase inhibitor, there are other enzymes inhibitors too, which also
reduce the rate of digestion of complex carbohydrates.26 Further studies are necessary to demonstrate
the mechanisms by which compound 9 decreased the fasting glucose levels in STZ-induced diabetic
mice.
4. Materials and methods
4.1. Chemicals and plant materials
10

All organic solvents used in the study, such as petroleum ether (PE), chloroform (CHCl3), ethyl
acetate (EtOAc), methanol (CH3OH) and N,N-dimethylformamide (DMF), were of an analytical grade.
Methanol-d4 (99.8%), dimethyl sulfoxide-d6 (99.9%) and α-glucosidase from Saccharomyces cerevisiae,
p-nitrophenyl-α-glucopyranoside (PNPG), streptozotocin (STZ) and acarbose were purchased from
Sigma-Aldrich (St. Louis, MO, USA).
The leaves of A. grossedentata were purchased from Zhangjiajie Maoyanmei Group Co., Ltd.
(Hunan, China).
4.2. Animals
Kunming mice (4−6 weeks old) were obtained from the Experimental Animal Center of Zhejiang
Province (China). The use of mice was reviewed and approved by the Ethics Committee for Animal
Experimentation of the Ningbo University (Zhejiang, China) and was in accordance with the National
Institutes of Health Guide for the Care and Use of Laboratory Animals.
To induce diabetes, the mice were treated with a single intra-peritoneal injection of streptozotocin
(100 mg/kg) dissolved in citrate buffer (pH 4.5) under fasting conditions. The blood glucose level was
monitored on day 7 from the tail vein using a one-touch glucometer (Lifescan, Inc., Milpitas, CA).
Mice with fasting blood glucose levels of ≥16.0 mmol/L were classified as diabetic mice.
4.3. General experimental procedures
IR spectra were recorded on a Nicolet NEXUS 670 FT-IR spectrometer. UV detections were
measured on a Shimadzu UV-260 spectrophotometer. NMR spectra were recorded on Varian
Mercury-300BB NMR (300 MHz) and Varian Mercury plus-400 (400 MHz) spectrometers with TMS
as an internal standard. High Resolution Electro Spray Ionization Mass Spectroscopy (HRESIMS) data
were measured on a Bruker Daltonics APEX II 47e spectrometer and acquired in positive-ion mode,
using source voltage of 3.98 kV, capillary temperature of 275 °C, sheath gas flow rate of 2 arb, sweep
11

gas flow rate of 1 arb. Silica gel (200-300 mesh) and CHP20P MCI gel (75–150 μm, Mitsubishi
Chemical Industries, Ltd.) were used for column chromatography (CC). Silica gel GF254 (10-40 μm)
used for thin-layer chromatography (TLC) were supplied by Qingdao Marine Chemical Factory,
Qingdao, P. R. China. Spots were detected on TLC under UV light or by heating after spraying with 5%
H2SO4 in C2H5OH (v/v). Semipreparative and analytical High Performance Liquid Chromatography
(HPLC) were carried out on Waters equipment (1525 pump and 2996 photodiode array detector) with a
YMC-Pack ODS-A column (250 × 10 mm or 250 × 4.6 mm, 5 μm). The absorbances in the enzymatic
assay were determined at 405 nm using a Bio-Rad Model 680 microplate reader.
4.4. General procedures for extraction and isolation of the major components
Air-dried leaves and stems (5.0 kg) of A. grossedentata were extracted three times (each for 7
days) with 45% ethanol (40 L) at room temperature. The ethanol filtrates were collected and
concentrated under reduced pressure by a rotary evaporator at 40 ºC to dryness, producing 1500 g of
ethanol extract. The extract was subjected to CC (D101-macroporous absorption resin) with a gradient
of EtOH:H2O (10:90, 20:80, 40:60, 70:30, 90:1) as eluent, and five fractions (Fr.1-Fr.5) were collected
according to TLC analysis. Fr.2 (50 g) was recrystallized from EtOAc:MeOH (4:1) to afford compound
1 (40 g). Fr.3 (500 g) was fractionated using the same procedure (MCI gel CC, eluted with 40 % EtOH
in H2O), leading to the appearance of crude crystals of 2 (450 g), then compound 2 (400 g) was finally
obtained by recrystallizing with hot water. The remaining Fr.3 was recrystallized from MeOH to yield
compound 3 (10 g).
4.5. General procedures for the synthesis of flavonoid derivatives
4′-O-(2-hydroxy)-ethyl myricetin (4): 4 was obtained from myricetin (509 mg, 1.6 mmol),
ethylene glycol ditosylate (296 mg, 0.8 mmol), K2CO3 (276 mg, 2.0 mmol) and DMF (30 mL),
according to the general procedure, as a yellow amorphous powder (262 mg, yield: 45%); UV (MeOH)
12


max(log ) 364 (5.92), 253 (4.36) nm; IR (K Br)max: 3540, 3446, 3334, 2933, 1657, 1617, 1596, 1506,
1
1102 cm1. H NMR (DMSO-d6, 400 MHz): δ 12.42 (1H, br s, 5-OH), 7.33 (1H, s, H-2′), 7.24 (1H, s,
H-6′), 6.43 (1H, s, H-8), 6.20 (1H, s, H-6), 4.31 (2H, m, H-1″), 4.29 (2H, m, H-2″); 13C NMR
(DMSO-d6, 100 MHz): δ 176.0 (C-4), 164.2 (C-7), 160.8 (C-5), 156.2 (C-9), 146.3 (C-3′), 145.8 (C-5′),
143.9 (C-2), 136.5 (C-3), 134.2 (C-4′), 122.7 (C-1′), 108.0 (C-6), 107.8 (C-2′), 103.1 (C-10), 98.3
(C-6), 93.5 (C-8), 64.2 (C-1''), 64.1 (C-2''); HRESIMS m/z 345.0602 [M-H2O+H]+ (calcd for
[C17H13O8], 345.0605).
4′-O-[2-(2-hydroxy ethoxyl) hydroxy]-ethyl myricetin (5): 5 was obtained from myricetin (509 mg,
1.6 mmol), diethylene glycol ditosylate (332 mg, 0.8 mmol), K2CO3 (276 mg, 2.0 mmol) and DMF (30
mL), according to the general procedure, as a yellow amorphous powder (236 mg, yield: 36%); UV
(MeOH) max(log ) 364 (2.56), 253 (2.40) nm; IR (K Br)max: 3397, 2948, 1654, 1021cm1; 1H NMR
(DMSO-d6, 400 MHz): δ 12.39 (1H, br s, 5-OH), 7.42 (1H, s, H-2'), 7.31 (1H, s, H-6'), 6.43 (1H, s,
H-8), 6.20 (1H, s, H-6), 4.38 (2H, br s, H-1"), 4.17 (2H, br s, H-2"), 3.84 (2H, br s, H-3"), 3.39 (2H, br
s, H-4")；13C NMR (DMSO-d6, 100 MHz): δ 176.1 (C-4), 164.4 (C-7), 160.7 (C-5), 156.3 (C-9), 152.4
(C-3'), 150.6 (C-5'), 145.4 (C-2), 141.0 (C-4'), 136.8 (C-3), 125.7 (C-1'), 112.2 (C-6'), 109.8 (C-2'),
103.0 (C-10), 98.4 (C-6), 93.5 (C-8), 73.1 (C-3"), 72.9 (C-2"), 72.4 (C-1"), 72.1 (C-4"); HRESIMS
(positive) m/z 389.0865 [M-H2O+H]+ (calcd for [C19H17O9], 389.0867).
4′-O-{2-[2-(2-hydroxy ethoxyl)-hydroxy ethoxyl] hydroxy}-ethyl myricetin (6): 6 was obtained
from myricetin (509 mg, 1.6 mmol), triethylene glycol ditosylate (367 mg, 0.8 mmol), K2CO3 (276 mg,
2.0 mmol) and DMF (30 mL), according to the general procedure, as a brownish amorphous powder
(156 mg, yield: 22%); UV (MeOH) λmax(logε)：339 (2.53), 260 (2.41) nm；IR (K Br)max: 3410,
2925, 1649, 1197, 1043 cm1；1H NMR (DMSO-d6, 400 MHz): δ12.67 (1H, br s, 5-OH), 6.70 (2H, s,
H-2', 6'), 6.35 (1H, s, H-8), 6.18 (1H, s, H-6), 4.28 (2H, br s, H-1"), 3.97 (2H, d, J = 4.4Hz, H-2"), 3.65
13

(2H, br s, H-6"), 3.32 (2H, br s, H-3"), 3.22 (2H, br s, H-4"), 3.04 (2H, d, J=4.4Hz, H-5")；13C NMR
(DMSO-d6,100 MHz): δ 177.8 (C-4), 165.3 (C-7), 161.4 (C-5), 159.1 (C-9), 156.7 (C-2), 150.3
(C-3',5'), 136.7 (C-4'), 136.0 (C-3), 123.9 (C-1'), 108.3 (C-2',6'), 104.1 (C-10), 98.9 (C-6), 93.8 (C-8),
72.2 (C-5"), 71.8 (C-2"), 69.7 (C-1"), 69.1 (C-3"), 69.0 (C-4"), 68.3 (C-6"); HRESIMS (positive) m/z
433.1127 [M-H2O+H] (calcd for [C21H21O10], 433.1129).
4′-O-{{2-{2-[2-(2-hydroxy ethoxyl) hydroxy ethoxyl]-hydroxy ethoxyl} hydroxy}}-ethyl
myricetin (7): 7 was obtained from myricetin (509 mg, 1.6 mmol), tetraethylene glycol ditosylate (402
mg, 0.8 mmol), K2CO3 (276 mg, 2.0 mmol) and DMF (30 mL), according to the general procedure, as a
yellow amorphous powder (200 mg, yield: 25%); UV (MeOH) max(log ) 345 (2.54), 265 (2.42) nm;
IR (K Br)max: 3043, 2925, 1655, 1198, 1043 cm1. 1H NMR (DMSO-d6, 400 MHz): δ 12.64 (1H, br s,
5-OH), 7.06 (2H, s, H-2', 6'), 6.32 (1H, s, H-8), 6.15(1H, s, H-6), 4.27 (2H, br s, H-1"), 4.13 (2H, br s,
H-2"), 3.69 (2H, t, J=4.8Hz, H-8"), 3.50 (2H, br s, H-3"), 3.45 (2H, t, J=4.8Hz, H-4"), 3.37 (4H, m,
H-5", 6"), 3.29 (2H, m, H-7"); 13C NMR (DMSO-d6, 100 MHz): δ 177.8 (C-4), 165.7 (C-7), 161.3
(C-5), 156.5 (C-9), 156.0 (C-2), 150.1 (C-3', 5'), 137.0 (C-4'), 136.6 (C-3), 124.4 (C-1'), 108.3 (C-2', 6'),
103.7 (C-10), 99.0 (C-6), 93.6 (C-8), 71.5 (C-7"), 71.0 (C-2"), 70.4 (C-1"), 70.2 (C-3"), 70.1 (C-4"),
70.0 (C-5"), 69.5 (C-6"), 69.0 (C-8"); HRESIMS m/z 477.1389 [M-H2O+H] (calcd for [C23H25O11],
477.1391).
4′-O-(2-N,N-hydroxy ethyl-phenyl)-ethyl myricetin (8): 8 was obtained from myricetin (509 mg,
1.6 mmol), K2CO3 (276 mg, 2.0 mmol), N,N-bis[2-(p-toluenesulfonyloxy) ethyl]-N-phenylamine (468
mg, 0.8 mmol) and DMF (30 mL), according to the general procedure, as a yellow amorphous powder
(398 mg, yield: 52%); UV (MeOH) max(log ) 363 (2.56), 260.4 (2.42) nm; IR (K Br)max: 3378, 2925,
2372, 1655, 1594, 1118 cm1. 1H NMR (DMSO-d6, 400 MHz): δ 12.39 (1H, br s, 5-OH), 7.23 (2H, s,
H-2', 6'), 7.13 (2H, t, J = 7.8 Hz, H-7", 9"), 6.74 (2H, d, J = 8.3 Hz, H-6", 10"), 6.36 (1H, s, H-8), 6.18
14

(1H, s, H-6), 4.09 (2H, t, J = 6.8 Hz, H-2"), 3.72 (2H, t, J = 6.8 Hz, H-1"), 3.56 (2H, t, J = 6.8Hz,
H-3"), 3.44 (2H, t, J=6.8Hz, H-4"); 13C NMR (DMSO-d6, 100 MHz): δ 176.0 (C-4), 164.9 (C-7), 160.7
(C-5), 156.3 (C-9), 150.5 (C-3', 5'), 147.9 (C-5"), 145.7 (C-2), 136.7 (C-4'), 136.0 (C-3), 129.1 (C-1'),
125.9 (C-7", 9"), 115.4 (C-8"), 111.5 (C-6", 10"), 107.2 (C-2', 6'), 102.8 (C-10), 98.4 (C-6), 93.3 (C-8),
68.7 (C-1"), 58.1 (C-4"), 53.0 (C-3"), 50.3 (C-2"); HRESIMS m/z 482.1456 [M-H2O+H] (calcd for
[C25H24NO9], 482.1446).
4′-O-(2-N,N-hydroxy ethyl-toluene-4-sulfonyl)-ethyl myricetin (9): 9 was obtained from myricetin
(509 mg, 1.6 mmol), K2CO3 (276 mg, 2.0 mmol), N,O,O'-Tri(toluene-4-sulfonyl)diethanolamine (454
mg, 0.8 mmol) and DMF (30 mL), according to the general procedure, as a light yellow amorphous
powder (402 mg, yield: 45%); UV (MeOH) max(log ) 364 (2.56) nm; IR (K Br)max: 3397, 2923,
1
1656, 1160, 1048 cm1. H NMR (DMSO-d6, 400 MHz): δ 12.39 (1H, br s, 5-OH), 7.63 (2H, d, J =
8.0Hz, H-6", 10"), 7.47 (1H, s, H-2'), 7.38 (2H, d, J = 8.0Hz, H-7", 9"), 7.31 (1H, s, H-6'), 6.43 (1H, s,
H-8), 6.20(1H, s, H-6), 4.41 (2H, br s, H-1"), 4.23 (2H, br s, H-2"), 3.44(4H, br s, H-3", 4"), 2.36(3H, s,
H-11"); 13C NMR (DMSO-d6, 100 MHz): δ176.1 (C-4), 164.5 (C-7), 160.7 (C-5), 156.2 (C-9), 152.5
(C-3'), 150.6 (C-5'), 145.2 (C-2), 143.3 (C-8"), 140.9 (C-5"), 136.9 (C-4'), 135.1 (C-3), 129.9 (C-7",
9"), 126.9 (C-6", 10"), 126.1 (C-1'), 111.9 (C-6'), 110.2 (C-2'), 103.0 (C-10), 98.4 (C-6), 93.5 (C-8),
73.2 (C-1"), 72.7 (C-4"), 52.2 (C-3"), 51.9 (C-2"), 20.9 (C-11"); HRESIMS m/z 542.1113 [M-H2O+H]
(calcd for [C26H24NO10S], 542.1115).
4.6. α-Glucosidase inhibition
The α-glucosidase inhibition was assessed according to a slightly modified method of Jeon et al.19
The α-glucosidase (0.1 U/mL) and substrate (p-NPG, 1.0 mM) were dissolved in potassium phosphate
buffer (0.1 M, pH 6.7), and all samples were dissolved in DMSO. The inhibitor (10 μL) was
preincubated with α-glucosidase (40 μL) at 37 °C for 10 min, and then the substrate (50 μL) was added
15

to the reaction mixture. The enzymatic reaction was performed at 37 °C for 30 min and the reaction
was then terminated by the addition of Na2CO3 (1 M, 100 μL). All samples were analyzed in triplicate
with five different concentrations near the IC50 values, and the absorbance at 405 nm was determined
using a microplate reader. The inhibition percentage (%) was calculated by the following equation:
Inhibition (%) = [(ODcontrol− ODcontrol blank) − (ODsample− ODsample blank)/ (ODcontrol−ODcontrol blank)] × 100.
4.7. Type of α-glucosidase inhibition
The mode of inhibition of α-glucosidase was investigated with increasing concentrations of
substrate (4-nitrophenyl α-D-glucopyranoside) and compound 9. Then, the inhibition type was
determined by a Lineweaver−Burk plot according to Michaelis−Menten kinetics. Origin (version 8.0)
software was used for plotting the results.
4.8. Oral sucrose tolerance test (OSTT)
The fasting normal and STZ-induced diabetic mice were orally administered with compound 9
(25, 50 and 100 mg/kg of body weight), acarbose (5 mg/kg), or the control and, after 30 min, were
given a sucrose solution (3 g/kg of body weight). Each group had six mice. Compound 9 and acarbose
were suspended in 0.5 % sodium carboxy methyl cellulose (CMC−Na). The control mice were
administered with the same volume of 0.5 % CMC−Na solution. The tail vein glucose concentrations
were measured with a glucometer at 0, 0.5, 1.0, 1.5, 2.0 and 3.0 h after the sucrose load.
4.9. Hypoglycemic activity assay
The fasting normal and STZ-induced diabetic mice (n = 6 for each group) were orally
administered with compound 9 (25, 50 and 100 mg/kg of body weight), glibenclamide (10 mg/kg), or
the control (0.5 % CMC−Na). The tail vein glucose concentrations were measured with a glucometer at
0, 1.5, 3, 5, 7 and 9 h after administration.
4.10. Statistical analysis
16

The data were expressed as the mean ± standard error of the mean (SEM) and were analyzed
using SPSS (version19.0) statistical software (SPSS, Chicago, IL). The statistical significance of the
differences (p < 0.05) between the mean values of the treatment and control groups were obtained from
a one-way analysis of variance (ANOVA) followed by Tukey’s or Dunnett’s test.
Acknowledgment
This work was funded by the National Natural Science Foundation of China (No. 21172196 and
No. 21272211) and Natural Science Foundation of Zhejiang and Ningbo (No. LQ16B020002 and No.
2015A610272).
Supplementary data
HPLC chromatograms, UV, HRESIMS, IR and NMR spectra for compounds 4-9 are available.
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α-Glucosidase inhibition and antihyperglycemic activity of flavonoids from Ampelopsis
grossedentata and the flavonoid derivatives
Jia Chen, Yuechan Wu, Jianwei Zou, Kun Gao*
School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo, 315100,
People’s Republic of China
20