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Citation: Luo, Q.-J.; Zhou, W.-C.; Liu,
X.-Y.; Li, Y.-J.; Xie, Q.-L.; Wang, B.;
Liu, C.; Wang, W.-M.; Wang, W.;
Zhou, X.-D. Chemical Constituents
and α-Glucosidase Inhibitory,
Antioxidant and Hepatoprotective
Activities of Ampelopsis grossedentata.
Molecules 2023,28, 7956. https://
doi.org/10.3390/molecules28247956
Academic Editors: Valeria Naponelli
and Elena Ferrari
Received: 8 November 2023
Revised: 29 November 2023
Accepted: 1 December 2023
Published: 5 December 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
molecules
Article
Chemical Constituents and α-Glucosidase Inhibitory,
Antioxidant and Hepatoprotective Activities of
Ampelopsis grossedentata
Qu-Jing Luo 1, Wen-Chao Zhou 1, Xin-Yi Liu 1, Ya-Jie Li 1, Qing-Ling Xie 1, Bin Wang 1, Chao Liu 1,2,
Wen-Mao Wang 1,2, Wei Wang 1,* and Xu-Dong Zhou 1, *
1TCM and Ethnomedicine Innovation & Development International Laboratory, School of Pharmacy,
Hunan University of Chinese Medicine, Changsha 410208, China; ishtar61@163.com (Q.-J.L.);
zhouwenchao6815@163.com (W.-C.Z.); 18182086610@163.com (X.-Y.L.); 17872355957@163.com (Y.-J.L.);
xieql12@126.com (Q.-L.X.); 004146@hnucm.edu.cn (B.W.); 18674403333@163.com (C.L.);
13907442657@163.com (W.-M.W.)
2Zhangjiajie Meicha Technology Research Center, Hunan Qiankun Biotechnology Co., Ltd.,
Zhangjiajie 427099, China
*Correspondence: wangwei402@hotmail.com (W.W.); xudongzhou999@hnucm.edu.cn (X.-D.Z.);
Tel.: +86-731-8845-8240 (W.W.); +86-731-8845-8240 (X.-D.Z.); Fax: +86-8845-8227 (W.W.);
+86-731-8845-8240 (X.-D.Z.)
Abstract:
Ampelopsis grossedentata is a valuable medicinal and edible plant, which is often used as a
traditional tea by the Tujia people in China. A. grossedentata has numerous biological activities and
is now widely used in the pharmaceutical and food industries. In this study, two new flavonoids
(
1
–
2
) and seventeen known compounds (
3
–
19
) were isolated and identified from the dried stems and
leaves of A. grossedentata. These isolated compounds were characterized by various spectroscopic
data including mass spectrometry and nuclear magnetic resonance spectroscopy. All isolates were
assessed for their
α
-glucosidase inhibitory, antioxidant, and hepatoprotective activities, and their
structure–activity relationships were further discussed. The results indicated that compound
1
exhibited effective inhibitory activity against
α
-glucosidase, with an IC
50
value of 0.21
µ
M. In
addition, compounds
1
–
2
demonstrated not only potent antioxidant activities but also superior
hepatoprotective properties. The findings of this study could serve as a reference for the development
of A. grossedentata-derived products or drugs aimed at realizing their antidiabetic, antioxidant, and
hepatoprotective functions.
Keywords:
Ampelopsis;Ampelopsis grossedentata; flavonoids;
α
-glucosidase inhibitory; antioxidant;
hepatoprotective activity
1. Introduction
Ampelopsis grossedentata, locally called “Meicha” or “Tocha”, is a species of perennial
woody vine, mainly distributed in southern China. In folk, A. grossedentata is a valuable
medicinal and edible plant and is always used as a traditional herbal tea which is made
from its leaves and tender stem. As a medicinal plant, A. grossedentata has been used
for centuries for various therapeutic purposes, to prevent and treat symptoms such as
colds, fevers, sore throats, and toothaches [
1
,
2
]. This plant is known as a rich source of
flavonoids, especially dihydromyricetin. Previous phytochemical study of A. grossedentata
mainly includes flavonoids, polyphenols, steroids, terpenes, and volatile components [
1
,
2
].
Furthermore, it has been discovered to have a wide range of biological activities, including
antioxidant [
3
,
4
], liver protection [
5
,
6
], antidiabetic [
7
,
8
], antitumor [
9
,
10
], antiviral [
11
],
anti-inflammatory [12], and antimicrobial [13] effects.
Currently, A. grossedentata gets increasing attention due to its long history of use and
diverse bioactivities. It has become popular through its use in dietary supplements and
Molecules 2023,28, 7956. https://doi.org/10.3390/molecules28247956 https://www.mdpi.com/journal/molecules
Molecules 2023,28, 7956 2 of 13
other functional products because of its great taste and abundant health benefits. Although
about 40 compounds have been found in it, its biological activity has so far been attributed
to its most abundant flavonoid, dihydromyricetin. Due to our efforts to search for natural
products with novel structures and various biological activity [
14
,
15
], nineteen compounds
(
1
–
19
), including two new flavonoids named meichasu A–B (
1
–
2
) and seventeen other
known compounds, were isolated from the alcoholic extract of A. grossedentata leaves in the
present work (Figure 1). Among them, nine compounds were isolated from A. grossedentata
for the first time. Moreover, the biological properties of all the isolates were investigated,
revealing
α
-glucosidase inhibitory, antioxidant, and hepatoprotective capacities. The results
indicated that compound
1
exhibited potent inhibitory activity against
α
-glucosidase, and
compounds
1
–
2
showed stronger antioxidant activities than the positive control ascorbic
acid. Herein, the details of the isolation, structure identification, and bioactivity of these
compounds are presented.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 13
Currently, A. grossedentata gets increasing aention due to its long history of use and
diverse bioactivities. It has become popular through its use in dietary supplements and
other functional products because of its great taste and abundant health benefits. Alt-
hough about 40 compounds have been found in it, its biological activity has so far been
aributed to its most abundant flavonoid, dihydromyricetin. Due to our efforts to search
for natural products with novel structures and various biological activity [14,15], nineteen
compounds (1–19), including two new flavonoids named meichasu A–B (1–2) and seven-
teen other known compounds, were isolated from the alcoholic extract of A. grossedentata
leaves in the present work (Figure 1). Among them, nine compounds were isolated from
A. grossedentata for the first time. Moreover, the biological properties of all the isolates
were investigated, revealing α-glucosidase inhibitory, antioxidant, and hepatoprotective
capacities. The results indicated that compound 1 exhibited potent inhibitory activity
against α-glucosidase, and compounds 1–2 showed stronger antioxidant activities than
the positive control ascorbic acid. Herein, the details of the isolation, structure identifica-
tion, and bioactivity of these compounds are presented.
Figure 1. Structures of isolated compounds from the leaves of A. grossedentata.
Figure 1. Structures of isolated compounds from the leaves of A. grossedentata.
2. Results and Discussion
2.1. Structure Elucidation
Meichasu A (compound
1
) was obtained as an amorphous gum. The molecular for-
mula C
22
H
16
O
13
with 15 degrees of unsaturation (Figure 2) was indicated by the
[M + H]+
peak at m/z489.0664 (calcd for C
22
H
17
O
13+
, 489.0664) in the HR-ESI-MS and supported by
the
13
C-NMR data (Table 1). IR absorptions at 3388 cm
−1
and 1636 cm
−1
suggested the pres-
Molecules 2023,28, 7956 3 of 13
ence of hydroxyl and carbonyl groups. The
1
H-NMR spectrum of compound
1
exhibited the
presence of 1,2,3,5-tetrasubstituted aromatic rings, showing two sets of two-proton singlets
[
δH
6.97 (2 H, s);
δH
6.52 (2 H, s)], which were attributed to pyrogallol moiety together with
an aromatic proton at
δH
5.96 (1H, s). Meanwhile, a vicinal coupling system of two methines
was indicated by the signals at
δH
5.84 (d, J= 11.4 Hz) and
5.31 (d, J= 11.4 Hz)
. The
13
C
NMR and DEPT spectrum of compound
1
exhibited 18 carbon resonances corresponding to
the above portions and seven quaternary carbons including two carbonyl carbons (
δC
193.4,
166.8) and five aromatic quaternary carbons (Table 1). These spectroscopic data indicated
that compound
1
belonged to the number of compounds with flavanonol skeleton similar
to compound
19
. A comparison of their data suggested that the difference between them
is one additional hydroxyl group at C-6 in compound
1
, which was further confirmed by
a detailed analysis of the 2D NMR spectra (Figure 2). In addition, the galloyl group was
proved to be linked at C-3 from the HMBC cross-peak of H-3 with its carbonyl at δC166.8.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 13
2. Results and Discussion
2.1. Structure Elucidation
Meichasu A (compound 1) was obtained as an amorphous gum. The molecular for-
mula C22H16O13 with 15 degrees of unsaturation (Figure 2) was indicated by the [M + H] +
peak at m/z 489.0664 (calcd for C22H17O13+, 489.0664) in the HR-ESI-MS and supported by
the 13C-NMR data (Table 1). IR absorptions at 3388 cm−1 and 1636 cm−1 suggested the pres-
ence of hydroxyl and carbonyl groups. The 1H-NMR spectrum of compound 1 exhibited
the presence of 1,2,3,5-tetrasubstituted aromatic rings, showing two sets of two-proton
singlets [δH6.97 (2 H, s); δH6.52 (2 H, s)], which were aributed to pyrogallol moiety to-
gether with an aromatic proton at δH5.96 (1H, s). Meanwhile, a vicinal coupling system of
two methines was indicated by the signals at δH5.84 (d, J = 11.4 Hz) and 5.31 (d, J = 11.4
Hz). The 13C NMR and DEPT spectrum of compound 1 exhibited 18 carbon resonances
corresponding to the above portions and seven quaternary carbons including two car-
bonyl carbons (δC193.4, 166.8) and five aromatic quaternary carbons (Table 1). These spec-
troscopic data indicated that compound 1 belonged to the number of compounds with
flavanonol skeleton similar to compound 19. A comparison of their data suggested that
the difference between them is one additional hydroxyl group at C-6 in compound 1,
which was further confirmed by a detailed analysis of the 2D NMR spectra (Figure 2). In
addition, the galloyl group was proved to be linked at C-3 from the HMBC cross-peak of
H-3 with its carbonyl at δC166.8.
Figure 2. Key HMBC and 1H-1H COSY correlations of compounds 12.
The stereochemistry of C-2 and C-3 in compound 1, was determined by the coupling
constant and the electronic circular dichroism (CD) spectra. The large coupling constant
(J = 11.4 Hz) between H-2 and H-3 in 1H NMR spectrum also revealed they were trans-
oriented (see the Supplementary Materials). Meanwhile, the positive Coon effect at 300–
340 nm indicated a 2R configuration. Taking the above into account, the absolute config-
uration at C-2 and C-3 could be determined to be 2R, 3R. Thus, the structure of compound
1 was established as shown above.
Meichasu B (compound 2) was assigned a molecular formula of C21H20O13 determined
on the basis of its positive HRESIMS [M + NH4] + ion peak at m/z 498.1209 (calcd 498.1242)
and the negative ion peak [M − H]− at m/z 479.0851 (calcd 479.0831). The IR spectrum
showed absorption bands for hydroxyl (3364 cm−1) and carbonyl (1651 cm−1) groups. De-
tailed analysis for 1D and 2D NMR data (Table 1) indicated that compound 2 possessed a
flavonol skeleton with an ABX coupling system in B ring [δH7.33, (1 H, d, 2.1 Hz), 7.31 (1
H, dd, 8.3, 2.1 Hz), 6.91 (1 H, d, 8.3, Hz)]. In addition, the presence of a rharmnosyl moiety
was evidenced by the characteristic signals (δC103.5, 73.3, 72.1,72.0, 71.9, 17.7) combined
with the 1H-1H COSY and HMBC spectra. These NMR data above implied the similarity
of compound 2 to the known flavonol, glycoside quercitrin (compound 18) [16], except for
Figure 2. Key HMBC and 1H-1H COSY correlations of compounds 1–2.
Table 1.
The
1
H NMR (600 MHz) and
13
C NMR (151 MHz) data of the samples Meichasu A (
1
) and
Meichasu B (2) (in CD3OD) (δin ppm, J in Hz).
No. 1 2
δCδHδCδH
2 82.9 5.31, d, (11.4) 159.3 --
3 74.2 5.84, d, (11.4) 136.2 --
4 193.4 -- 179.6 --
5 165.4 -- 163.1 --
6 164.2 -- 166.3 --
7 169.9 -- 166.3 --
8 96.6 5.96, s 158.6 --
9 169.1 -- 158.5 --
10 102.1 -- 105.8 --
10127.9 -- 122.9 --
20107.8 6.52, s 116.9 7.33, d (2.1)
30147.0 -- 146.4 --
40135.1 -- 149.8 --
50147.0 -- 116.4 6.91, d (8.3)
600 107.8 6.52, s 122.8 7.31, dd (8.3, 2.1)
100 120.4 -- 103.5 5.35, d (1.7)
200 110.4 6.97, s 71.9 4.22, dd (3.4, 1.7)
300 146.4 -- 72.1 3.75, dd (9.6, 3.4)
400 140.2 -- 73.2 3.34, t (9.6)
500 146.4 -- 72.0 3.42, dq (9.6, 6.1)
600 110.4 6.97, s 17.7 0.94, d (6.1)
700 166.8 --
Molecules 2023,28, 7956 4 of 13
The stereochemistry of C-2 and C-3 in compound
1
, was determined by the cou-
pling constant and the electronic circular dichroism (CD) spectra. The large coupling
constant (
J= 11.4 Hz
) between H-2 and H-3 in
1
H NMR spectrum also revealed they were
trans-oriented
(see the Supplementary Materials). Meanwhile, the positive Cotton effect
at 300–340 nm indicated a 2Rconfiguration. Taking the above into account, the absolute
configuration at C-2 and C-3 could be determined to be 2R, 3R. Thus, the structure of
compound 1was established as shown above.
Meichasu B (compound
2
) was assigned a molecular formula of C
21
H
20
O
13
determined
on the basis of its positive HRESIMS [M + NH
4
]
+
ion peak at m/z498.1209 (calcd 498.1242)
and the negative ion peak [M
−
H]
−
at m/z479.0851 (calcd 479.0831). The IR spectrum
showed absorption bands for hydroxyl (3364 cm
−1
) and carbonyl (1651 cm
−1
) groups.
Detailed analysis for 1D and 2D NMR data (Table 1) indicated that compound
2
possessed
a flavonol skeleton with an ABX coupling system in B ring [
δH
7.33, (1 H, d, 2.1 Hz), 7.31
(
1 H
, dd, 8.3, 2.1 Hz), 6.91 (1 H, d, 8.3, Hz)]. In addition, the presence of a rharmnosyl
moiety was evidenced by the characteristic signals (
δC
103.5, 73.3, 72.1,72.0, 71.9, 17.7)
combined with the
1
H-
1
H COSY and HMBC spectra. These NMR data above implied the
similarity of compound
2
to the known flavonol, glycoside quercitrin (compound
18
) [
16
],
except for the presence of two additional hydroxyls at C-6 and C-8. Their chemical shifts
were determined by comparing the
13
C NMR data with those of quercitrin and the result
of modeling in ChemDraw software. Moreover, in the HMBC spectra, the cross-peak
between
δH5.35 (H-10)
and
δC
136.2 (C-3) demonstrated the rharmnosyl group was attached
at C-3 (Figure 2). The coupling constant (J= 1.7 Hz) of the anomeric proton indicated a
α-rharmnosyl moiety. Thus, the structure of compound 2was determined as shown.
In addition, the structures of the known compounds were identified as (2R,3R)-
Dihydromyricetin (
3
) [
17
], Quercetin (
4
) [
18
], Myricetin (
5
) [
19
], Naringenin (
6
) [
20
], Kaempferol
(
7
) [
18
], lunularic acid (
8
) [
21
], lunularin (
9
) [
22
], (2S,3S)-Dihydromyricetin (
10
) [
23
], 3,5,7-
Trihydroxychromone (
11
) [
24
], Taxifolin (
12
) [
25
], Myricitrin (
13
) [
18
], Afzelin (
14
) [
26
], Phlo-
ridzin (
15
) [
27
], 2-O-[1-(3-Methylbutyryl) phloroglucin-ol]-
β
-D-glucopyranoside (
16
) [
28
],
5,7,3
0
,4
0
,5
0
-Pentahydroxy flavanone (
17
) [
29
], Quercitrin (
18
) [
16
], and (2R,3R)-3,4-Dihydro-5,7-
dihydroxy-4-oxo-2-(3,4,5-trihydroxyphenyl)-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate
(19) [30] by comparing their spectroscopic data with those reported in the literature.
2.2. α-Glucosidase Inhibition Assay
The inhibitory effects of compounds
1
–
19
against
α
-glucosidase was assessed by
determining their IC
50
values, with comparison of that of the positive control acarbose.
The compounds with lower IC
50
values exhibited greater enzymatic inhibition. As a result
(Table 2), compounds
1
,
4
,
5
,
10
, and
19
showed strong
α
-glucosidase inhibitory activity
with IC
50
values ranging from 0.21 to 1.88
µ
M, which were mainly concerned with two
types of skeletons: flavonols and dihydroflavonols. Among them, these dihydroflavonols
(
1
,
10
, and
19
) showed better effects than the flavonols (
4
,
5
). After them, compounds
7
,
8
,
9
,
11
,
16
, and
17
showed good inhibitory activities against
α
-glucosidase with IC
50
values
between 2.90 and 10.91
µ
M. In addition, the other compounds showed weak activities with
less than 50% inhibition of α-glucosidase at a concentration of 20 µM.
Comparing the dihyroflavonols (
1
,
3
,
10
,
12
, and
19
), one can observe that only com-
pounds
1
and
19
, which have a galloyl substituent at the C-3 position, exhibited strong
α
-glucosidase inhibitory activity with IC
50
values of 0.21 and 1.59
µ
M, respectively. At the
same time, the weak inhibitory activity of compound
3
, which has no galloyl substituent
at all, indicates that the presence of a galloyl substituent at the C-3 position can greatly
enhance the inhibitory activity against
α
-glucosidase, which verified the previous find-
ings [
31
]. Although compounds
3
and
10
were optical isomers to each other, compound
10
exhibited significantly stronger inhibitory activity against
α
-glucosidase, which may
indicate that compound
10
has a better steric configuration to bind more effectively to the
active site of
α
-glucosidase than compound
3
. According to the order of the activities of
Molecules 2023,28, 7956 5 of 13
these flavonols (compounds
4
,
5,
and
7
), showing
4
>
5
>
7
, the number of hydroxyl groups
in B ring may also be an important factor enhancing α-glucosidase inhibition activity.
Table 2. α-glucosidase inhibitory activities of compounds 1–19 from A. grossedentata.
Compound IC50 a(µM) Compound IC50 a(µM)
10.21 ±0.01 11 6.04 ±0.26
2>20 12 >20
3>20 13 >20
41.88 ±0.08 14 >20
51.83 ±0.05 15 >20
6>20 16 10.91 ±0.51
72.90 ±0.12 17 4.18 ±0.11
810.32 ±0.31 18 >20
99.42 ±0.51 19 1.59 ±0.03
10 1.69 ±0.05 Acarbose b0.06 ±0.01
a
Data were represented as the mean value
±
SD, n = 4. Values accompanied by different letters are significantly
different (p≤0.05). bAcarbose was employed as the positive control.
Compounds
13
,
14
,
15,
and
18
at all inhibited
α
-glucosidase, minimally or not, which
was in line with previous research that attached glycosyl groups to the C-3 position within
ring C, reducing the potency to inhibit α-glucosidase [32].
2.3. Antioxidant Effect
Three methods, DPPH, ABTS, and FRAP assay, were used to evaluate antioxidant
activities of all the isolates
in vitro
, and the results are shown in Table 3. The results of these
three experiments showed that compounds
1
–
5
,
10
,
12
,
13,
and
17
–
19
exhibited stronger
antioxidant activity than the positive drug, ascorbic acid, and the activity of compound
7
was similar to that of ascorbic acid. The other compounds had weaker or no significant
antioxidant activities. This finding once again confirms that flavonoid compounds have
good antioxidant activities. In the DPPH assay, compound
19
exhibited the strongest
antioxidant activity with an IC
50
value of 17.31
µ
M, while in the ABTS assay, compound
4
showed the strongest activity with an IC
50
value of 2.76
µ
M. In contrast to the DPPH and
ABTS assays, in the FRAP assay, the greater the concentration of ferrous ions, the better the
antioxidant activity of these isolates. Compound
19
showed the strongest activity with a
FRAP value of 2.79 µmol/mL.
The new compounds
1
and
2
both exhibited strong antioxidant activities. Compound
1
was more active than compound
2
in DPPH and ABTS assays, whereas the opposite
result was observed in FRAP assays, which may be due to different mechanisms of each
reaction. Comparing the results of flavonols (
4
,
5,
and
7
) and flavonol glycosides (
2
,
13
,
14
,
and
18
), the flavonols showed roughly higher antioxidant activity than the latter. The order
of the activities of the flavonol glycosides with one single rhamnose was
2>13 >18 >14
,
suggesting that the more hydroxyl groups there are, the better the activity is, which is
consistent with previous findings [
33
]. The above comparison also showed that the activities
of these glycosides were weaker than that of aglycones, which indicated the presence of
sugar groups may decrease the antioxidant activity of flavonoids. Considering the isomers
of compounds
3
and
10
, the results showed that the activity of compound
3
was stronger
than those of compound
10
, indicating that the configuration of compound
3
was more
advantageous. In addition, the comparison of the activities of these three compounds (
1
,
3,
and
19
) implied the presence of galloyl was vital for the effects, and the presence of extra
hydroxyl in the A-ring seemed not necessarily to increase the activities.
Molecules 2023,28, 7956 6 of 13
Table 3. Antioxidant inhibitory activities of compounds 1–19 from A. grossedentata.
Compound
Antioxidant Activity
DPPH Assay IC50 a
(µM)
ABTS Assay IC50 a
(µM)
FRAP Assay a
(µMOL/ML)
132.52 ±0.16 4.43 ±0.02 1.70 ±0.00
244.42 ±0.63 6.73 ±0.03 1.96 ±0.01
328.26 ±0.38 5.91 ±0.11 1.71 ±0.01
425.54 ±0.63 2.76 ±0.04 2.67 ±0.01
529.75 ±0.59 3.39 ±0.07 1.72 ±0.01
6>80 >20 0.07 ±0.00
7>80 19.91 ±0.46 0.85 ±0.01
8>80 >20 0.13 ±0.01
9>80 >20 0.09 ±0.00
10 43.50 ±0.36 5.01 ±0.11 1.43 ±0.01
11 >80 >20 0.15 ±0.00
12 39.21 ±0.18 7.36 ±0.08 1.32 ±0.01
13 35.03 ±0.28 6.88 ±0.13 1.69 ±0.02
14 >80 >20 0.07 ±0.00
15 >80 >20 0.17 ±0.01
16 >80 >20 0.24 ±0.01
17 43.05 ±0.94 10.25 ±0.13 1.18 ±0.01
18 63.51 ±0.97 9.96 ±0.16 1.35 ±0.02
19 17.31 ±0.31 2.19 ±0.01 2.79 ±0.01
ascorbic acid b77.93 ±0.87 19.01 ±0.31 0.57 ±0.01
a
Data were represented as the mean value
±
SD, n = 4. Values followed by different letters are significantly
different (p≤0.05). bAscorbic acid was employed as the positive control.
2.4. Hepatoprotective Activity
Since previous studies have shown that A. grossedentata displayed good properties
in liver protection [
5
,
6
], an APAP-induced HepG2 cell injury model was established to
evaluate the protective effect of the isolated compounds at a concentration of 20
µ
M on
HepG2 cells, and the hepatoprotective drug bicyclol was used as a positive control. The
results (Figure 3) demonstrated that compounds
1
,
2
,
4
,
6
,
7
, and
12
displayed significant
protective effect against acetaminophen-induced HepG2 cell injury, with a corresponding
increase in cell viability from 68.24
±
1.34% to 84.93
±
0.58%, 88.46
±
0.63%, 84.90
±
1.21%,
83.7
±
0.67%, 87.08
±
0.60%, and 85.43
±
1.22%. All these parameters were higher than in
the preparation of cell viability positive control of 83.01
±
0.82%. Compounds
3
,
5
,
8
–
11,
and
13
also exhibited moderate hepatoprotective activities, while compounds
13
–
18
did
not show an obvious liver protection effect. These results again demonstrate the superior
hepatoprotective activity of flavonoids.
Molecules 2023, 28, x FOR PEER REVIEW 7 of 13
the preparation of cell viability positive control of 83.01 ± 0.82%. Compounds 3, 5, 8–11,
and 13 also exhibited moderate hepatoprotective activities, while compounds 13–18 did
not show an obvious liver protection effect. These results again demonstrate the superior
hepatoprotective activity of flavonoids.
Figure 3. Hepatoprotective activity of compounds 1–19 (20 µM). Note: n = 3, mean ± SD. Compared
with the control: ### p < 0.001; compared with the APAP: ** p < 0.01 and *** p < 0.001.
The flavonols (compounds 4, 5, and 7) all exhibited strong hepatoprotective activity,
while their corresponding flavonol glycosides (compounds 13, 14, and 18) were essentially
inactive. This result indicated that the presence of glycosyl at C-3 in flavonols can signifi-
cantly reduce their hepatoprotective activities. Furthermore, compound 19, which is a gal-
loyl derivative of compound 3 by C-3, exhibited stronger activity than compound 3, indi-
cating that the presence of galloyl may enhance the hepatoprotective activity.
Among all the compounds, compound 2 was the most effective in increasing the sur-
vival rate of HepG2 cells. Compared with the inactive compound 18, the difference be-
tween the two was that compound 2 had two additional hydroxyl groups on its A-ring. In
addition, compound 1 exhibited beer activity compared to compound 19, despite the fact
that the only structural difference between the two compounds is that compound 1 has an
extra hydroxyl group on its A-ring. These results suggested that the number of hydroxyl
groups on the A-ring appears to affect the activities. Furthermore, by comparing com-
pounds 4–5 with 7, 3 with 12, 6 with 17, we also found that the number of hydroxyls on
the B-ring has a significant impact on the hepatoprotective activity.
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were measured with a Rudolf AUTOPOL III polarimeter (USA).
Circular dichroism (CD) spectra were recorded on a JASCO J-1500-150 spectropolarime-
ter. UV spectra (methanol) were measured with a PerkinElmer Lambda 650 spectropho-
tometer. IR spectra were obtained on a PerkinElmer Frontier MIR spectrophotometer. In
addition, 1D and 2D NMR spectra were recorded on Bruker Avance-600 NMR spectrom-
eter with TMS as internal standard. Unless stated otherwise, all chemical shifts (δ) are
reported in ppm relative to the solvent signals, and coupling constants are reported in
Her. HR-ESIMS data were acquired on a Waters Xevo G2-S QTof mass spectrometer.
Semi-preparative HPLC was conducted on an Agilent 1260 Infinity II HPLC system
with an Agilent Pursuit XRs 10 C18 column (250 × 10 mm, 5 µm). Column chromatography
(CC) was performed with silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical
✱✱✱
✱✱✱
✱✱✱
✱✱✱
✱✱✱
✱✱✱
✱✱
✱✱✱
✱✱✱
✱✱✱
✱✱✱
✱✱✱
✱✱✱
✱✱✱
Figure 3.
Hepatoprotective activity of compounds
1
–
19
(20
µ
M). Note: n = 3, mean
±
SD. Compared
with the control: ### p< 0.001; compared with the APAP: ** p< 0.01 and *** p< 0.001.
Molecules 2023,28, 7956 7 of 13
The flavonols (compounds
4
,
5,
and
7
) all exhibited strong hepatoprotective activity,
while their corresponding flavonol glycosides (compounds
13
,
14,
and
18
) were essentially
inactive. This result indicated that the presence of glycosyl at C-3 in flavonols can signif-
icantly reduce their hepatoprotective activities. Furthermore, compound
19
, which is a
galloyl derivative of compound
3
by C-3, exhibited stronger activity than compound
3
,
indicating that the presence of galloyl may enhance the hepatoprotective activity.
Among all the compounds, compound
2
was the most effective in increasing the
survival rate of HepG2 cells. Compared with the inactive compound
18
, the difference
between the two was that compound
2
had two additional hydroxyl groups on its A-ring.
In addition, compound
1
exhibited better activity compared to compound
19
, despite the
fact that the only structural difference between the two compounds is that compound
1
has an extra hydroxyl group on its A-ring. These results suggested that the number of
hydroxyl groups on the A-ring appears to affect the activities. Furthermore, by comparing
compounds
4
–
5
with
7
,
3
with
12
,
6
with
17
, we also found that the number of hydroxyls
on the B-ring has a significant impact on the hepatoprotective activity.
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were measured with a Rudolf AUTOPOL III polarimeter (USA).
Circular dichroism (CD) spectra were recorded on a JASCO J-1500-150 spectropolarimeter.
UV spectra (methanol) were measured with a PerkinElmer Lambda 650 spectrophotometer.
IR spectra were obtained on a PerkinElmer Frontier MIR spectrophotometer. In addition,
1D and 2D NMR spectra were recorded on Bruker Avance-600 NMR spectrometer with TMS
as internal standard. Unless stated otherwise, all chemical shifts (
δ
) are reported in ppm
relative to the solvent signals, and coupling constants are reported in Hertz. HR-ESIMS
data were acquired on a Waters Xevo G2-S QTof mass spectrometer.
Semi-preparative HPLC was conducted on an Agilent 1260 Infinity II HPLC system
with an Agilent Pursuit XRs 10 C18 column (250
×
10 mm, 5
µ
m). Column chromatography
(CC) was performed with silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical
Group Co., Shandong, China), ODS RP-18 gel (40–63 mm, Merck, Darmstadt, Germany),
Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and Macroporous adsorbent resin AB-8
(0.3–1.25 mm, Tianjin City Guang Fu Tech. Development Co., Ltd., Tianjin, China). Thin-
layer chromatography (TLC) was employed to monitor the CC fractions, with visualization
achieved through the application of 1% vanillin in H2SO4as a spraying reagent.
3.2. Chemicals and Reagents
DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2
0
-azinobis(3-ethylbenzthiazoline-6-
sulphonic acid)), TPTZ (2,4,6-tris(2-pyridyl)-S-triazine), and ascorbic acid were obtained
from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China).
α
-Glucosidase was
purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). p-NPG(p-
nitrophenyl-
α
-D-glucopyranoside), acarbose, acetaminophen, bicyclol, anhydrous sodium
carbonate, ferric chloride, and potassium persulfate were purchased from Shanghai Aladdin
Biochemical Technology Co., Ltd. (Shanghai, China). Methanol and acetonitrile (HPLC-
grade) were purchased from Sigma-Aldrich (Wuxi) Life Science & Tech. Co., Ltd. (Wuxi,
China). Purified water was purchased from China Resources C’estbon Beverage (China)
Co., Ltd. (Changsha, China). Anhydrous ethanol, dimethyl sulfoxide (DMSO), and all
other chemicals of analytical grade were purchased from Sinopharm Chemical Reagent
Co., Ltd. (Shanghai, China).
3.3. Plant Material
The dried leaves of A. grossedentata were collected from Zhangjiajie (29
◦
20
0
13
00
N,
110
◦
18
0
52
00
E), Hunan Province, China, in July 2020, and identified by associate professors,
Xu-Dong Zhou (Hunan University of Chinese Medicine, Changsha, China) and Wen-Mao
Wang (Zhangjiajie Meicha Technology Research Center, Zhangjiajie, China). The samples
Molecules 2023,28, 7956 8 of 13
were stored in the TCM and Ethnomedicine Innovation & Development International
Laboratory, Hunan University of Chinese Medicine, Hunan Province.
3.4. Extraction and Isolation
The extract (1.0 kg, 21.3% yield) from leaves and stems of A. grossedentata (4.7 kg)
was provided by Zhangjiajie Meicha Technology Research Center of Hunan Qiankun
Biotechnology Co., Ltd. (Zhangjiajie, China), which was obtained by reflux extraction with
95% ethanol. Firstly, the extract was dissolved in 90% ethanol and left at room temperature
(25
◦
C) to recrystallize to obtain the main constituent dihydromyricetin (compound
3
with
large amounts). Then, a large amount of dihydromyricetin crystals were removed by
suction filtration and then washed repeatedly with distilled water (3
×
50 mL) 3 times
to obtain filtrate. Secondly, the filtrate was concentrated until there was no ethanol in it,
redissolved with a small amount of water, and then added to the macroporous adsorption
resin (AB-8) CC for separation. The volume ratio of extract and macroporous adsorbent
resin was 1:10, and 15 fractions (Fr.1-Fr.15) were obtained by gradient elution of H
2
O-EtOH
(100:0 →5:95, v/v).
Fr.9 was dissolved in methanol and separated by Sephadex LH-20 CC (eluting with
MeOH) to obtain Fr.9-1-Fr.9-14. A part of Fr.9-9 was then purified by semi-preparative
HPLC (MeOH-H
2
O, 50:50, 3 mL/min, 9.7 min) to yield compound
1
(5.3 mg). The other
part was repeatedly eluted twice with Sephadex LH-20 CC eluting with MeOH, to obtain
compound
10
(4.2 mg). Compounds
11
(2.6 mg) and
12
(27.3 mg) were obtained by elution
of Fr.9-5 on silica gel column with CH
2
Cl
2
-EtOAc (100:0
→
20:80, v/v). The Fr.9-5 was
then separated using a semi-preparative HPLC (MeOH-H
2
O, 50:50, 3 mL/min, 8.6 min) to
give compounds
2
(10.1 mg) and
13
(16.7 mg). Fr.9-3 was separated by Sephadex LH-20
CC eluting with MeOH, and then further eluted by silica gel column chromatography
(DCM-EtOAc, 2:1 →1:10, v/v) to yield compound 15 (14.5 mg).
Fr.13 was chromatographed by Sephadex LH-20 (MeOH) to obtain Fr.13-1-Fr.13-17,
and then Fr.13-12, Fr.13-13, Fr.13-9, Fr.13-11 were purified by Sephadex LH-20 (MeOH),
respectively. Thus, compounds
4
(26.7 mg),
5
(24.8 mg),
6
(4.5 mg), and
7
(5.0 mg) were
obtained. Fr.13-6 was separated by Sephadex LH-20 in MeOH and then purified by semi-
prepared HPLC (MeOH-H
2
O, 54:46, 3 mL/min) to afford a mixture of compounds
8
and
9
. The mixture was further purified by Sephadex LH-20 in MeOH and then compounds
8
(2.3 mg) and 9(1.4 mg) were obtained, separately.
Fr.12 was subjected to Sephadex LH-20 (MeOH) to obtain Fr.12-1-Fr.12-19. Fr.12-7 was
purified by Sephadex LH-20 (MeOH) and then separated by semi-preparate HPLC (MeOH-
H
2
O, 52:48, 3 mL/min) to yield compound
14
(3.4 mg). Fr.12-4 was purified by silica gel
column chromatography (DCM-EtOAc, 40:1
→
1:5, v/v) to yield compound
16
(5.7 mg).
Fr.12-12 was subjected to silica gel CC (DCM-EtOAc, 50:1
→
1:1, v/v) to obtain compound
17
(3.1 mg). Fr.12-8 was separated by silica gel column chromatography (DCM-EtOAc,
50:1
→
1:10, v/v) and further purified by semi-preparative HPLC (MeOH-H2O, 60:40,
3 mL/min
) to obtain compound
18
(23.0 mg). Fr.12-15 was eluted twice by silica gel CC
(DCM-EtOAc, 20:1
→
1:5, v/v), and then isolated by semi-preparative HPLC (MeOH-H
2
O,
55:45, 3 mL/min) to obtain compound 19 (38.8 mg).
Meichasu A (
1
): yellow, amorphous gum;
[α]25
D
+ 14.03 (c0.05, MeOH); UV (MeOH)
λmax
(log
ε
): 210 (2.86), 293 (2.93) nm; CD (MeOH)
λmax
(
∆ε
): 280 (
−
0.2),
330 (+0.8) nm
; IR
(KBr)
νmax
3388, 2925, 1636, 1210, 1034 cm
−1
;
1
H and
13
C NMR data (CD
3
OD, 600/
151 MHz
),
see Table 1; HRESIMS m/z489.0664 [M + H]+(calcd for C22 H17O13+, 489.0664).
Meichasu B (
2
): yellow, amorphous gum;
[α]25
D−
147.1 (c0.15, MeOH); UV (MeOH)
λmax
(log
ε
): 204 (2.86), 256 (2.91), 350 (2.97) nm; IR (KBr)
νmax
3364, 1651, 1201,
1089 cm−1
;
1
H and
13
C NMR data (CD
3
OD, 600/151 MHz), see Table 1; HRESIMS m/z 498.1209
[M + NH
4
]
+
(calcd for C
21
H
24
NO
13+
, 498.1242) and m/z479.0851 [M
−
H]
−
(calcd for
C21H19O13−, 479.0831).
Molecules 2023,28, 7956 9 of 13
3.5. α-Glucosidase Inhibitory Assay
The
α
-glucosidase inhibitory activities of isolated compounds were evaluated by
using a method previously described, but with minor modifications [
34
]. The experiment
was conducted using a 96-well plate, and each reaction had a total volume of 200
µ
L.
All isolated compounds and acarbose were dissolved and diluted in DMSO (Dimethyl
sulfoxide), p-NPG (p-nitrophenyl-
α
-D-glucopyranoside) and enzymes were dissolved in
PBS (Phosphate-buffered saline). Briefly, 98
µ
L of PBS (0.1 M, pH 6.8) was added to each
well. Then, 2
µ
L of different concentrations of sample solutions and 25
µ
L of
α
-glucosidase
solution (0.25 U/mL) were added. After shaking and mixing slightly, the 96-well plate
was placed in a 37
◦
C constant-temperature incubator and incubated for 20 min. Next,
25 µL
of p-NPG (4 mM) was added to the plate to initiate the reaction, and it was further
incubated in a 37
◦
C constant-temperature incubator for 15 min. Then, 50
µ
L of Na
2
CO
3
(0.2 M) solution was quickly added to each well to terminate the reaction. The absorbance
was measured at 405 nm using a microplate reader, and the results were obtained from a
minimum of three independent experiments. Acarbose was used as positive control. The
experiment was divided into 4 groups: group A was the sample group containing enzyme,
group B replaced the enzyme with PBS, group C replaced the sample or acarbose with
DMSO, and group D was the blank control group without enzyme and sample, and other
reagents were consistent with group A.
The percentage inhibition rate (%) of
α
-glucosidase for each test sample was
calculated as
:
Inhibition rate (%) = [1 −(ODA−ODB)/(ODC−ODD)] ×100%
The IC50 values were calculated by using GraphPad Prism 9.0 software.
3.6. Measurement of Antioxidant Activity
3.6.1. DPPH Assay
The DPPH free radical scavenging capacity of isolated compounds was determined
according to a modified method [
35
]. The experiment was conducted using a 96-well plate,
and each well had a total volume of 200
µ
L. All isolated compounds and positive drug were
dissolved and diluted in DMSO and DPPH (2,2-Diphenyl-1-picrylhydrazyl) was dissolved
in anhydrous ethanol. Briefly, 190
µ
L of a working solution of DPPH (0.2 mM) was added to
each well. Then, 10
µ
L of a sample solution of different concentrations (1.6, 1.2, 0.8, 0.4, 0.2,
and 0.1 mM) was added, mixed well, and left to incubate in the dark at room temperature
for 60 min. After that, the absorbance values of each well were measured at 517 nm using
a microplate reader and the results were obtained from a minimum of
three independent
experiments. Ascorbic acid was used as the positive control. DMSO was used to replace
the sample solution as a blank and absolute ethanol was used to replace the DPPH solution
as a control.
The percentage free radical scavenging rate (%) of each test sample for DPPH was
calculated as:
Free radical scavenging rate (%) = [Ablank −(Asample −Acontral)] ÷Ablank ×100%
3.6.2. ABTS Assay
The ABTS free radical scavenging capacity of all isolated compounds was measured,
the procedure followed a method with minor modifications [
36
]. The experiment was
conducted using a 96-well plate, and each well had a total volume of 200
µ
L. Equal
volumes of ABTS (2,2
0
-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)) solution (7 mM)
and potassium persulfate solution (2.45 mM) were mixed evenly and left to react at room
temperature for 12 h in the dark to obtain ABTS radical cation. The mixture was then diluted
with anhydrous methanol to achieve an absorbance value of about 0.7
±
0.02 units at
405 nm
. Then, 190
µ
L of ABTS working solution was added to each well, followed by
10 µL
samples of varying concentrations (0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and
0.00625 mM
) that
Molecules 2023,28, 7956 10 of 13
were dissolved and diluted with DMSO. After mixing well, the samples were incubated
at room temperature for 6 min away from light. The subsequent experimental method is
basically consistent with the DPPH assay, the only difference is that ABTS assay detects the
absorbance value at 405 nm.
The percentage radical cation scavenging rate (%) of each test sample for ABTS was
calculated as follows:
Radical cation scavenging rate (%) = [Ablank −(Asample −Acontral)] ÷Ablank ×100%
The IC50 values were calculated by using GraphPad Prism 9.0 software.
3.6.3. FRAP Assay
The total antioxidant capacity of the samples was measured by the FRAP (ferric
reducing ability of plasma) assay, with slight modifications [
37
]. The acetate buffer (0.3 M,
pH 3.6), TPTZ (2,4,6-tris(2-pyridyl)-S-triazine) solution (10 mM) in 40 mM HCl and ferric
chloride aqueous solution (10 mM) were uniformly mixed at a volume ratio of 7:1:1 to obtain
the FRAP working solution. FRAP working solution was preheated for 10 min in a constant
temperature incubator at 37
◦
C before use. The experiment was conducted using a 96-well
plate, and each well had a total volume of 204
µ
L. Then, 180
µ
L of FRAP working solution
was added to each well, followed by 6
µ
L of sample solution (0.68 mM), and finally 18
µ
L of
distilled water. Then, the mixture was stirred well and left to react at room temperature for
10 min. The absorbance values of each well were measured at 593 nm using a microplate
reader and the results were obtained from at least three independent experiments. The
sample solution was replaced with DMSO as a blank solution and ascorbic acid was used as
a positive control. Different concentrations of FeSO
4·
7H
2
O (0.075–0.00078
µ
mol/mL) were
used to establish the standard curve. The antioxidant capacity of the sample was expressed
as the Fe
2+
concentration (
µ
mol/mL) required to achieve the same absorbance change
value. The final result was calculated by GraphPad Prism 9.0 software and presented as
mean ±standard deviation (SD) of µmol Fe2+ per milliliter.
3.7. Hepatoprotective Activity
HepG2 cells were maintained in DMEM medium supplemented with 10% fetal bovine
serum, 100 U/mL penicillin G, and 100
µ
g/mL streptomycin. The cells were seeded in
culture flasks and placed in a 5% CO
2
incubator at 37
◦
C for routine cultivation. The
hepatoprotective activities of the isolated samples were tested according to the modified
method [
38
]. To evaluate the effect of test samples on cell viability, a CCK-8 assay was
performed. The experiment was conducted in 96-well plates, and 100
µ
L of HepG2 cells
in logarithmic growth phase (8
×
10
5
cells/mL) were seeded in each well and cultured
for 24 h. After that, samples (final concentration of 20
µ
M) and acetaminophen (APAP,
final concentration of 6 mM) were added at one time to incubate for another 48 h. Then,
10
µ
L CCK-8 was added to each well and incubated for 1 h, followed by measuring the
absorbance at a wavelength of 450 nm using a microplate reader.
4. Conclusions
In the present study, a comprehensive phytochemical investigation was conducted
to isolate 19 compounds from the traditional dietary tea, A. grossedentata. Their structures
were characterized through extensive spectroscopic analysis (NMR and HR-ESIMS). These
included two previously undescribed flavonoids, which were named meichasu A–B (
1
–
2
),
and seven compounds that were isolated for the first time from A. grossedentata. Based on
the traditional applications of the tea and the structural characteristics of these isolates,
the
α
-glucosidase inhibitory, antioxidant, and hepatoprotective activities of the isolated
compounds were evaluated. The results showed that compound
1
exhibited potent in-
hibitory activity against
α
-glucosidase, with an IC
50
value of 0.21
µ
M. The antioxidant and
hepatoprotective abilities of compounds
1
–
2
were found to be stronger than those of the
positive control drug, indicating their broad biological activities.
Molecules 2023,28, 7956 11 of 13
A. grossedentata, as a traditional herb, is a precious and rich resource for discovering
bioactive natural products. These research findings have enriched the knowledge of the
chemical diversity of A. grossedentata, reflecting the potential
α
-glucosidase inhibitory,
antioxidant, and hepatoprotective effects of the flavonoids contained in this herbal tea, and
that these active compounds may have potential therapeutic implications for the treatment
of diabetes and liver damages.
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/molecules28247956/s1, Figure S1.
1
H NMR spectrum of meichasu
A (
1
) (CD
3
OD, 600 MHz).; Figure S2.
13
C NMR spectrum of meichasu A (
1
) (CD
3
OD, 125 MHz).;
Figure S3. DEPT 135
◦
spectrum of meichasu A (
1
) (CD
3
OD, 125 MHz).; Figure S4. HSQC spectrum of
meichasu A (
1
).; Figure S5. HMBC spectrum of meichasu A (
1
); Figure S6.
1
H-
1
H COSY spectrum
of meichasu A (
1
).; Figure S7. HR-ESIMS spectrum of meichasu A (
1
).; Figure S8. UV spectrum of
meichasu A (
1
).; Figure S9. IR spectrum of meichasu A (
1
).; Figure S10. CD spectrum of meichasu
A (
1
).; Figure S11.
1
H NMR spectrum of meichasu B (
2
) (CD
3
OD, 600 MHz).; Figure S12.
13
C NMR
spectrum of meichasu B (
2
) (CD
3
OD, 125 MHz).; Figure S13. DEPT 135
◦
spectrum of meichasu B (
2
)
(CD
3
OD, 125 MHz).; Figure S14. HSQC spectrum of meichasu B (
2
).; Figure S15. HMBC spectrum
of meichasu B (
2
).; Figure S16.
1
H-
1
H COSY spectrum of meichasu B (
2
).; Figure S17. HR-ESIMS
spectrum of meichasu B (
2
).; Figure S18. UV spectrum of meichasu B (
2
).; Figure S19. IR spectrum of
meichasu B (2).
Author Contributions:
Conceptualization, X.-D.Z. and Q.-J.L.; methodlogy, X.-Y.L. and Y.-J.L.; soft-
ware, X.-D.Z. and Q.-J.L.; investigation, Q.-J.L. and W.-C.Z.; resources, C.L. and W.-M.W.; validation,
Q.-J.L.; formal analysis, X.-D.Z. and B.W.; data curation, Q.-J.L., Q.-L.X. and W.-C.Z.; writing & origi-
nal draft preparation, Q.-J.L.; writing---review and editing, X.-D.Z. and Q.-J.L.; funding acquisition,
X.-D.Z., C.L. and B.W.; supervision, X.-D.Z. and W.W.; administration, W.W. All authors have read
and agreed to the published version of the manuscript.
Funding:
This research was funded by the Scientific research project of Hunan Provincial Department
of Education (20C1397), Special Scientific and Technological Project for Comprehensive Utilization
of Ampelopsis grossedentata Resources of Hunan Qiankun Biotechnology Co., Ltd., the discipline
construction program (2022A2-19) and Innovation Training program (2021YX11, 202286 & 2023CX71)
for students in Hunan University of Chinese Medicine.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: There are no conflicts of interests to declare.
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