Stilbenes with Potent Protein Tyrosine Phosphatase-1B Inhibitory Activity from the Roots of Polygonum multiflorum

Article abstract of DOI:10.1021/acs.jnatprod.9b00777

Seven new stilbene glycosides including three dimers (1-3) and four monomers (4-7) were isolated from the roots of Polygonum multiflorum along with nine previously identified stilbenes (8-16). In addition, two deglucosylated stilbenes, 2a and 3a, were also obtained as new dimeric stilbenes. The structures of the purified phytochemicals were elucidated by interpreting their spectroscopic data (NMR, HRMS, and ECD). To the best of our knowledge, this represents the first isolation of a phenylpropanoid (C₆-C₃) substituted with a stilbene unit (7) from the Polygonaceae family. In an in vitro enzyme assay with human recombinant protein tyrosine phosphatase-1B (PTP1B), compounds 2-5 showed weak PTP1B inhibition with an IC₅₀ value range of 27.4-37.6 μM, while three deglucosylated stilbenes 2a, 3a, and 8a exhibited IC₅₀ values of 2.1, 1.9, and 12.1 μM, respectively. The inhibition modes and binding mechanism of selected inhibitors (2a and 3a) were investigated using kinetic methods and molecular docking simulations.

Full text of DOI:10.1021/acs.jnatprod.9b00777

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Article
Stilbenes with Potent Protein Tyrosine Phosphatase-1B Inhibitory
Activity from the Roots of Polygonum multiflorum
Thi-Thuy An Nguyen,⊥ Manh Tuan Ha,⊥ Se-Eun Park, Jae Sue Choi, Byung Sun Min,*
and Jeong Ah Kim*
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ABSTRACT: Seven new stilbene glycosides including three dimers (1−3) and four monomers (4−7) were isolated from the roots
of Polygonum multiflorum along with nine previously identified stilbenes (8−16). In addition, two deglucosylated stilbenes, 2a and
3a, were also obtained as new dimeric stilbenes. The structures of the purified phytochemicals were elucidated by interpreting their
spectroscopic data (NMR, HRMS, and ECD). To the best of our knowledge, this represents the first isolation of a phenylpropanoid
(C6−C3) substituted with a stilbene unit (7) from the Polygonaceae family. In an in vitro enzyme assay with human recombinant
protein tyrosine phosphatase-1B (PTP1B), compounds 2−5 showed weak PTP1B inhibition with an IC50 value range of 27.4−37.6
μM, while three deglucosylated stilbenes 2a, 3a, and 8a exhibited IC50 values of 2.1, 1.9, and 12.1 μM, respectively. The inhibition
modes and binding mechanism of selected inhibitors (2a and 3a) were investigated using kinetic methods and molecular docking
simulations.
rotein tyrosine phosphatase-1B (PTP1B), an intracellular
attention as novel PTP1B inhibitors, because they tend to be
Pnonreceptor member of the PTP superfamily, has more efficacious in cells with high structural diversity when
compared to synthetic compounds.13
emerged as a new drug target for therapeutic purposes in
Stilbenes, a class of plant phenols, have attracted biomedical
interest for their intricate structures and diverse biological
activities.14,15 The structure of stilbenes is characterized by the
presence of a 1,2-diphenylethylene nucleus; therefore, this class
of compounds can be divided into two categories: monomeric
and oligomeric stilbenes.15 These subtypes are produced by
coupling homogeneous or heterogeneous monomeric stilbenes
with a variety of molecular skeletons. However, stilbenes are
not widely distributed in plants, and phytochemical inves-
tigations have focused solely on a few families, including the
Gnetaceae, Leguminosae, Polygonaceae, and Vitaceae.15
Stilbene glycosides, the main active constituent in the rhizomes
of Polygonum multiflorum Thunb. (Polygonaceae),16 were
reported to possess antioxidative,17,18 anti-inflammatory,19 and
antiatherosclerotic activities.20 Polygonum multiflorum, a
Korean medicinal herb, has been historically used as an
recent years. PTP1B plays a well-established role in down-
regulating signaling in response to insulin and leptin.1 In
insulin signaling, PTP1B dephosphorylates activated insulin
receptors and insulin receptor substrates to attenuate the
cellular response to insulin binding.1 In the leptin signaling
pathway, PTP1B has the function of binding and dephosphor-
ylation for Janus kinase 2, which is normally phosphorylated in
response to leptin binding to the leptin receptor to trigger a
feeling of satiety.2 Knockout studies in mice have shown that
PTP1B-deficient mice exhibit increased insulin sensitivity and
obesity resistance.3,4 These findings have motivated scientists
to develop PTP1B inhibitors as potential therapies for diabetes
and obesity.1,5 Furthermore, recent findings have suggested
that inhibiting PTP1B may have important applications in the
treatment of cancer and inflammation.6−9 In addition, PTP1B
has recently emerged as a regulator of a variety of processes
within the central nervous system, which may have
implications in treating neurological disorders.10−12 However,
discovering selective PTP1B inhibitors with ideal pharmaco-
logical properties continues to be a challenge because of the
low selectivity and the highly charged nature of the PTP1B
catalytic domain.13 Natural products have recently gained
Received: August 14, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
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A

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Figure 1. Chemical structures of compounds 1−16, 2a, 3a, and 8a.
antiallergy, antitumor, antibacterial, hemostatic, spasmolytic,
and analgesic agent.21 A previous study revealed that a MeOH
extract of P. multiflorum inhibited PTP1B inhibitory activity
with an IC50 value of 10.2 μg/mL.22 However, there is
currently no information regarding the PTP1B inhibitory
activity of the chemical constituents of P. multiflorum. Thus, in
the present study, the in vitro PTP1B inhibitory activity of 16
stilbenes including seven new stilbene glycosides isolated from
P. multiflorum was investigated. Interestingly, three deglucosy-
lated stilbenes (2a, 3a, and 8a) showed more potent PTP1B
inhibition than their corresponding precursors (2, 3, and 8). In
addition, enzyme kinetic analysis and molecular modeling were
performed to characterize the interactions between the active
stilbenes (2a and 3a) and PTP1B, as well as their inhibitory
mechanisms.
833.2271 [M + Na]+ (calcd for C40H42O18Na, 833.2269).
The UV spectrum of 1 showed absorption maxima at 310 and
235 nm. The IR spectrum of 1 exhibited absorption bands
attributable to hydroxy (3322 cm−1) and aromatic
1
(1510 cm−1) residues. The H NMR data of 1 (Table 1)
revealed the typical signals of a trans-disubstituted double
bond [δH 7.33 (1H, d, J = 16.8 Hz, H-8′) and 6.85 (1H, d, J =
16.8 Hz, H-7′)], two para-disubstituted benzene rings [δH 7.28
(2H, d, J = 8.5 Hz, H-2,6) and 6.78 (2H, d, J = 8.5 Hz, H-3,5)
for ring A1 and 7.33 (2H, d, J = 8.5 Hz, H-2′,6′) and 6.72 (2H,
d, J = 8.5 Hz, H-3′,5′) for ring B1], a meta-tetrasubstituted
benzene ring [δH 6.25 (1H, d, J = 2.9 Hz, H-12) and 5.89 (1H,
d, J = 2.9 Hz, H-14) for ring A2], a pentasubstituted benzene
ring [δH 6.39 (1H, s, H-12′) for ring B2], and two aliphatic
proton signals [δH 5.52 (1H, d, J = 3.1 Hz, H-8) and 5.40 (1H,
d, J = 3.1 Hz, H-7)]. In addition, signals corresponding to two
anomeric protons [δH 4.60 (2H, d, J = 8.0 Hz)] were also
RESULTS AND DISCUSSION
1
evident in the H NMR spectrum. The 13C NMR and DEPT
■
A methanol extract of P. multiflorum roots was suspended in
H2O and successively partitioned with n-hexane, CH2Cl2,
EtOAc, and n-BuOH. The EtOAc fraction (310.5 g) was
separated by repeated column chromatography over silica gel,
MCI gel, Sephadex LH-20, and semipreparative HPLC to
afford seven new stilbene glycosides named polygonibenes A−
G (1−7), along with nine known analogs (8−16) (Figure 1).
Polygonibene A (1) was obtained as a brown powder. Its
molecular formula was established as C40H42O18 by the
HRFABMS sodium adduct molecular ion peak at m/z
spectra of 1 showed the presence of 40 carbon signals that
could be classified into 24 aromatic carbons, two olefinic
carbons, two methine carbons, and 12 carbons of two sugar
moieties. Through acid hydrolysis and subsequent TLC
analysis of the monosaccharide derivative, the sugar units of
1 were identified as D-glucose. The D-glucose moieties were
3
suggested to be the β-configuration due to the large J1,2 value
of the anomeric protons. The analysis of the above
spectroscopic data, combined with the molecular formula,
suggested that 1 is a dimeric stilbene glucoside consisting of
B
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1
Table 1. H NMR (500 MHz) and 13C NMR (125 MHz) Data of Polygonibenes A−C (1−3) in Methanol-d4
1
2
3
position
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
1
2
3
4
5
6
7
8
134.2
128.7
116.2
158.5
116.2
128.7
93.9
133.9
128.7
116.2
158.6
116.2
128.7
93.7
132.8
129.2
116.1
158.5
116.1
129.2
95.0
7.28, d (8.5)
6.78, d (8.5)
7.27, d (8.6)
6.79, d (8.6)
7.27, d (8.6)
6.78, d (8.5)
6.78, d (8.5)
7.28, d (8.5)
5.40, d (3.1)
5.52, d (3.1)
6.79, d (8.6)
7.27, d (8.6)
5.64, d (6.5)
5.56, d (6.5)
6.78, d (8.5)
7.27, d (8.6)
5.47, d (7.8)
5.51, d (7.8)
49.5
49.6
50.4
9
139.8
137.3
151.6
103.6
156.2
106.5
131.1
129.4
116.3
158.3
116.3
129.4
134.6
121.0
131.1
138.9
152.1
97.8
133.3
138.3
151.6
103.7
156.3
106.3
132.6
124.2
138.8
160.6
110.3
129.0
130.3
121.9
133.6
137.8
152.1
103.7
156.0
102.8
107.8
75.5
133.3
138.6
151.7
103.5
156.3
106.9
132.7
125.1
138.3
161.0
110.1
128.5
130.3
122.4
133.8
137.9
152.0
103.6
155.9
102.8
107.7
75.4
10
11
12
13
14
1′
2′
3′
4′
5′
6.25, d (2.9)
5.89, d (2.9)
6.29, d (2.9)
6.18, d (2.9)
7.30, d (1.5)
6.29, d (2.9)
6.08, d (2.9)
7.26, d (1.5)
7.33, d (8.5)
6.72, d (8.5)
6.72, d (8.5)
7.33, d (8.5)
6.85, d (16.8)
7.33, d (16.8)
6.85, d (8.3)
6.85, d (8.3)
6′
7′
8′
9′
7.42, dd (8.3, 1.5)
6.95, d (16.5)
7.64, d (16.5)
7.48, dd (8.3, 1.5)
6.94, d (16.4)
7.67, d (16.4)
10′
11′
12′
13′
14′
Glc1-1″
2″
3″
4″
5″
6″
6.39, s
6.26, d (2.8)
6.25, d (2.8)
159.1
119.2
107.1
75.5
78.0
70.6
6.61, d (2.8)
4.64, d (7.0)
6.59, d (2.8)
4.34, d (7.5)
4.60, d (8.0)
a
a
a
3.40
3.43
3.44
3.39
3.38
3.38
a
a
3.32, m
77.9
70.9
78.2
62.3
77.8
71.0
78.2
62.3
a
a
a
3.40
3.47
78.2
61.8
2.89, m
3.25, m
3.21, m
3.39, dd (12.0, 2.5)
3.48 (12.0, 4.5)
4.60, d (8.0)
3.56, m
3.43, m
3.51, t (7.5)
3.27, m
3.75, dd (12.0, 2.5)
3.66, dd (12.0, 4.5)
4.50, d (7.9)
3.56, m
3.70, dd (12.0, 2.0)
3.62, dd (12.0, 4.5)
4.49, d (7.9)
3.53, m
Glc2-1‴
2‴
3‴
4‴
5‴
107.8
75.5
77.9
70.9
78.2
62.1
108.0
75.5
77.9
70.6
78.3
81.8
108.1
75.4
78.0
70.9
78.2
62.3
a
a
3.44
3.47
3.40
a
3.48, m
3.27, m
3.79, dd (12.0, 2.5)
3.68, dd (12.0, 4.5)
3.19, m
a
6‴
3.76, dd (12.0, 2.5)
3.70, dd (12.0, 4.5)
3.44
3.58, dd (12.0, 4.5)
a
Overlapped.
two (E)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside units
connected together through a dihydrofuran bridge (C ring) to
form a dimeric stilbene skeleton, which is common in natural
oligomeric stilbenes.23 The NMR data of 1 (Table 1) and ε-
viniferin were similar,23 except for the absence of two protons
at positions 10 and 10′ as well as the appearance of two D-
glucose moieties in 1. This suggested that 1 resulted from the
replacement of two protons at positions 10 and 10′ in ε-
viniferin by two O-D-glucosyl groups, respectively. This
inference was confirmed by the HMBC correlations (Figure
2) from H-1″ (Glc1, δH 4.60) to C-10 (δC 137.3) and H-1‴
(Glc2, δH 4.60) to C-10′ (δC 138.9). In addition, the ε-
viniferin skeleton in 1 was further supported by the HMBC
correlations (Figure 2) from H-7 (δH 5.40) to C-14′ (δC
119.2)/C-2 (δC 128.7), H-8 (δH 5.52) to C-13′ (δC 159.1)/C-
10 (δC 137.3)/C-14 (δC 106.5), H-7′ (δH 6.85) to C-2′ (δC
129.4), and H-8′ (δH 7.33) to C-10′ (δC 138.9)/C-14′ (δC
119.2). The trans orientation of H-7 and H-8 in the furan ring
was confirmed by the NOESY correlations (Figure 2) between
H-7 (δH 5.40) and H-14 (δH 5.89) and between H-8 (δH 5.52)
and H-2 (δH 7.28). The absolute configurations of the
dihydrobenzofuran skeleton in 1 at C-7 and C-8 were
determined via ECD data, which were examined in the range
220−240 nm.24,25 The ECD spectrum of 1 showed a positive
Cotton effect at 239 nm, opposite to that of (−)-ε-viniferin
(7R,8R) (Figure S8.1, Supporting Information), indicating that
the absolute configurations of 1 at C-7 and C-8 are 7S and
8S.24,25 Thus, the structure of 1 was assigned as shown as a
new dimeric stilbene glycoside (Figure 1) and was named
polygonibene A.
C
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Figure 2. Key COSY, HMBC, and NOESY correlations of polygonibenes 1−7.
A molecular formula of C40H42O18 was established for
polygonibene B (2, a brown powder with a positive optical
rotation) based on the sodium adduct molecular ion peak at
m/z 833.2273 [M + Na]+ (calcd for C40H42O18Na, 833.2269)
= 8.3, 1.5 Hz, H-6′)] for ring B1 and an AX coupling system
[δH 6.61 (1H, d, J = 2.8 Hz, H-14′) and 6.26 (1H, d, J = 2.8
Hz, H-12′)] for ring B2. The 13C NMR and DEPT spectra of 2
showed the presence of 40 carbon signals including 24
aromatic carbons, two olefinic carbons, two sp3 methine
carbons, and 12 carbons of two D-glucopyranosyl moieties. By
performing the same method as used for 1, both of the sugar
moieties in 2 were determined as β-D-glucose. Comparison of
the NMR data of 2 (Table 1) with those of δ-viniferin26
revealed their close structural similarities, except for the
replacement of two protons at positions 10 and 10′ in δ-
viniferin by O-D-glucose moieties in 2. This observation was
confirmed by the HMBC correlations (Figure 2) from H-1″
(Glc1, δH 4.64) to C-10 (δC 138.3) and H-1‴ (Glc2) (δH
1
in the HRFABMS. Similar to 1, the H NMR spectrum of 2
also revealed the presence of a trans-disubstituted double bond
[δH 7.64 (1H, d, J = 16.5 Hz, H-8′) and 6.95 (1H, d, J = 16.5
Hz, H-7′)], a para-disubstituted benzene ring [δH 7.27 (2H, d,
J = 8.6 Hz, H-2,6) and 6.79 (2H, d, J = 8.6 Hz, H-3,5)] for ring
A1, a meta-tetrasubstituted benzene ring [δH 6.29 (1H, d, J =
2.9 Hz, H-12) and 6.18 (1H, d, J = 2.9 Hz, H-14)] for ring A2,
two aliphatic protons [δH 5.64 (1H, d, J = 6.5 Hz, H-7) and
5.56 (1H, d, J = 6.5 Hz, H-8)], and two anomeric protons [δH
4.64 (1H, d, J = 7.0 Hz, H-1″) and 4.50 (1H, d, J = 7.9 Hz, H-
1‴)]. In addition, the remaining aromatic proton signals
belonged to an ABX coupling system [δH 7.30 (1H, d, J = 1.5
Hz, H-2′), 6.85 (2H, d, J = 8.3 Hz, H-5′), and 7.42 (1H, dd, J
1
4.50) to C-10′ (δC 137.8). Assignments of all H and 13C
NMR signals were accomplished by interpreting HMQC and
HMBC spectra. The relative configuration of 2 was defined via
D
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1
Table 2. H NMR (500 MHz) and 13C NMR (125 MHz) Data of Polygonibenes D−G (4−7)
a
a
a
b
4
5
6
7
position
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
1
2
3
4
5
6
1′
2′
3′
4′
5′
6′
α
133.4
137.6
152.1
103.5
155.9
102.3
130.4
129.0
116.3
158.1
116.3
129.0
121.7
129.7
107.4
73.6
133.4
137.6
152.0
103.5
155.9
102.3
130.4
129.0
116.3
158.1
116.3
129.0
121.7
129.7
107.5
73.7
133.4
137.9
152.0
103.5
155.9
102.2
130.5
128.9
116.4
158.1
116.4
128.9
121.9
129.6
107.7
75.4
133.8
142.0
151.4
105.1
151.5
116.0
130.1
129.1
116.4
158.8
116.4
129.1
120.4
136.1
107.8
75.4
6.29, d (2.8)
6.68, d (2.8)
6.31, d (2.8)
6.70, d (2.8)
6.29, d (2.8)
6.67, d (2.8)
6.68, s
7.48, d (8.6)
6.82, d (8.6)
7.49, d (8.6)
6.82, d (8.6)
7.42, d (8.6)
6.80, d (8.6)
7.18, d (8.6)
6.73, d (8.6)
6.82, d (8.6)
7.48, d (8.6)
7.76, d (16.5)
6.97, d (16.5)
4.70, d (7.9)
3.77, m
5.09, t (9.5)
3.77, m
3.54, m
6.82, d (8.6)
7.49, d (8.6)
7.79, d (16.5)
6.98, d (16.5)
4.75, d (7.9)
3.86, m
5.22, t (9.4)
3.86, m
3.60, m
6.80, d (8.6)
7.42, d (8.6)
7.74, d (16.5)
6.95, d (16.5)
4.60, d (7.7)
3.63, m
3.58, m
3.55, m
3.68, m
6.73, d (8.6)
7.18, d (8.6)
7.32, d (16.8)
6.34, d (16.8)
4.60, d (7.9)
3.53, m
3.42, m
3.48, m
3.19, m
3.57, m
β
1″
2″
3″
4″
5″
6″
78.7
69.0
77.8
62.0
78.8
69.2
77.8
62.1
77.6
70.9
75.2
63.7
77.9
70.8
78.2
61.9
3.87, dd (11.8, 2.7)
3.81, dd (11.8, 4.3)
3.86, m
4.45, dd (12.0, 1.4)
4.38, dd (12.0, 5.5)
8.08, s
1‴
2‴
3‴
4‴
5‴
6‴
7‴
8‴
171.0
21.2
127.5
111.3
148.8
150.2
116.1
124.0
146.1
116.0
161.9
135.4
111.9
149.6
146.9
116.8
120.6
40.1
2.07, s
7.38, d (1.8)
6.77, d (2.0)
6.89, d (8.2)
6.79, d (8.2)
7.18, dd (8.2, 1.8)
7.65, d (15.9)
6.46, d (15.9)
6.53, dd (8.2, 2.0)
4.53, dd (5.9, 1.9)
3.01, dd (15.5, 6.2)
2.91, dd (15.5, 2.1)
39.8
9‴
OCH3-3‴
167.7
56.4
170.3
56.4
3.94 (s)
3.80 (s)
a
b
In acetone-d6. In methanol-d4.
the NOESY spectrum. The NOESY correlations (Figure 2)
between H-7 (δH 5.64) and H-14 (δH 6.18), as well as H-8 (δH
5.56) and H-2 (δH 7.27), revealed a trans relationship between
H-7 and H-8. The absolute configurations of 2 at C-7 and C-8
were deduced from the absolute configuration of its
deglucosylated stilbene (2a) via ECD data analysis. The
ECD spectrum of 2a displayed a positive Cotton effect at
239 nm that was opposite to that of (−)-δ-viniferin (7R,8R)
(Figure S8.2, Supporting Information), indicating that the
absolute configurations of 2a are 7S and 8S.24,25 Subsequently,
the absolute configurations of 2 were determined as 7S and 8R.
Thus, the structure of 2 (polygonibene B) was assigned as
shown in Figure 1 and is a new dimeric stilbene glycoside.
Polygonibene C (3) was isolated as a brown powder with a
negative optical rotation. The molecular formula of 3 was
determined to be the same as 2 (C40H42O18) based on the
HRFABMS sodium adduct molecular ion peak at m/z
833.2272 [M + Na]+ (calcd for C40H42O18Na, 833.2269).
Compounds 2 and 3 were obtained by HPLC chromato-
graphic purification with different retention times under the
same conditions [2 (tR = 27.0 min) and 3 (tR = 32.0 min)].
The structural features of 3, including 1D NMR and HMBC
correlations, were similar to those of 2 (Table 1 and Figure 2).
However, the optical rotation and ECD data of 3 differ from
those of 2, indicating that 3 is a stereoisomer of 2. In addition,
acid hydrolysis of 2 and 3 formed the aglycone compounds 2a
and 3a, respectively. The H NMR spectra of 2a and 3a
1
(Experimental Section) were also similar, but their optical
rotations and ECD data (Figure S8.2, Supporting Information)
were opposite. This further supported that compounds 2 and 3
contain enantiomeric aglycones. The relative configuration of
C-7/8 was determined to be trans according to the NOESY
correlations (Figure 2) between H-7 (δH 5.47) and H-14 (δH
6.08), as well as H-8 (δH 5.51) and H-2 (δH 7.27). The 3a
ECD data were similar to those of (−)-δ-viniferin (7R,8R)
(Figure S8.2, Supporting Information), indicating that 3a and
(−)-δ-viniferin have the same absolute configurations
(7R,8R).24,25 Consequently, the absolute configurations of 3
were determined as 7R and 8S. Based on the above analysis,
the structure of 3, a new dimeric stilbene glycoside
(polygonibene C), was assigned as shown in Figure 1.
Polygonibene D (4) was isolated as a brown powder. The
HRFABMS of 4 showed a protonated molecular ion peak at
m/z 449.1445 [M + H]+ (calcd for C21H25O10, 449.1458),
corresponding to the molecular formula C21H24O10. Similar to
1
8, the H NMR spectrum of 4 also showed the typical signals
of an (E)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside de-
rivative, with a trans-disubstituted double bond at δH 7.76 (1H,
d, J = 16.5 Hz, H-α) and 6.97 (1H, d, J = 16.5 Hz, H-β), two
aromatic rings with one AABB coupling system at δH 7.48 (2H,
E
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d, J = 8.6 Hz, H-2′,6′) and 6.82 (2H, d, J = 8.6 Hz, H-3′,5′),
one AX coupling system at δH 6.68 (1H, d, J = 2.8 Hz, H-6)
and δH 6.29 (1H, d, J = 2.8 Hz, H-4), and an anomeric proton
at δH 4.70 (1H, d, J = 7.9 Hz, H-1″) (Table 2). In addition, the
signals of an acetoxy group [δH 2.07 (3H, s); δC 171.0
(−OCO), 21.2 (−CH3)] were also observed in the 1D
NMR spectra of 4. The 13C NMR spectrum of 4 in
combination with DEPT analysis revealed the signals of 21
carbons that included one carbonyl carbon, eight sp2, four sp3
methines, one sp3 methylene carbon, six quaternary carbons,
and one methyl carbon. Inspection of the NMR data of 4
indicated that this compound is structurally quite similar to 8,
except for the replacement of a hydroxy group with an acetoxy
group in the glucose moiety. The position of the acetoxy group
at C-3″ was verified by the HMBC correlation between H-3″
(δH 5.09) and carbonyl carbon (δC 171.0) (Figure 2), as well
as the downfield shift of C-3″ (δC 78.7) and the upfield shift of
C-2″ (δC 73.6), in 4 compared with those of 8 [δC 77.5 (C-
3″), 75.0 (C-2″)].27 Therefore, the structure of 4 (poly-
gonibene D) was elucidated as (E)-2,3,5,4′-tetrahydroxystil-
bene-2-O-(3″-O-acetyl)-β-D-glucoside, a new monomeric
stilbene glycoside.
16.8 Hz, H-β), a para-disubstituted benzene ring at δH 7.18
(2H, d, J = 8.6 Hz, H-2′,6′) and 6.73 (2H, d, J = 8.6 Hz, H-
3′,5′), a pentasubstituted benzene ring at δH 6.68 (1H, s, H-4),
and an anomeric proton at δH 4.60 (1H, d, J = 7.9 Hz, H-1″).
In addition, the proton signals of a trisubstituted benzene ring
at δH 6.77 (1H, d, J = 2.0 Hz, H-2‴), 6.79 (1H, d, J = 8.2 Hz,
H-5‴), and 6.53 (1H, dd, J = 8.2, 2.0 Hz, H-6‴), a methoxy
group at δH 3.80 (3H, s, OCH3-3‴), three aliphatic proton
signals at δH 4.53 (1H, dd, J = 5.9, 1.9 Hz, H-7‴), 3.01 (1H,
dd, J = 15.5, 6.2 Hz, H-8‴), and 2.91 (1H, d, J = 15.5, 2.1 Hz,
H-8‴), and the carbon signal of a carbonyl group at δC 170.3
(C-9‴) suggested the presence of an aromatic 3-methoxy-4-
hydroxy system substituted by a δ-lactone moiety, which was
similar to the phenylpropanoid (C6−C3) moiety of gnetu-
montanins C−G.30,31 The 13C NMR and DEPT spectra of 7
(Table 2) revealed the presence of 30 carbon atoms including
a carbonyl carbon, 10 sp2 methines, 6 sp3 methines, 2 sp3
methylenes, 10 quaternary carbons, and one methoxy carbon.
The β-linkage of the glucosyl moiety was deduced from the 7.9
Hz coupling constant of the anomeric proton signal at δH 4.60.
The above data suggested that 7 is a stilbene glucoside
consisting of an (E)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-
glucoside unit and a ferulic acid unit connected together
through a δ-lactone bridge. This was confirmed along with the
location of the δ-lactone moiety by the HMBC correlations
from H-7‴ (δH 4.53)/H2-8‴ (δH 3.01, 2.91)/H-4 (δH 6.68)/
H-α (δH 7.32) to C-6 (δC 116.0) and H-7‴ (δH 4.53)/H-4 (δH
6.68) to C-5 (δC 151.5) (Figure 2). The absolute configuration
of 7 at C-7‴ was determined by analyzing the ECD data. In a
previous study, the 7‴S configuration of the phenylpropanoid
(C6−C3) moiety in apocynin B was characterized by a positive
Cotton effect at 233 nm ([θ] +7590).32 In the present
investigation, the ECD spectrum of 7 exhibited a positive
Cotton effect at 229 nm (+4.61), indicating that the absolute
configuration of 7 at C-7‴ is also in the S form. Based on the
above analysis, the structure of 7 (polygonibene G) was
assigned as shown in Figure 1.
The HRESIMS of polygonibene E (5, a brown powder)
exhibited a sodium adduct molecular ion peak at m/z 605.1636
[M + Na]+ (calcd for C30H30O12Na, 605.1635), corresponding
1
to the molecular formula C30H30O12. A comparison of the H
and 13C NMR spectra of 5 with those of 4 indicated that the
difference was only in the signals belonging to the substituent
at C-3″ of the glucose moiety (Table 2). These signals were
attributable to the replacement of the acetoxy group in 4 with a
feruloyl group [δH 7.38 (1H, d, J = 1.8 Hz, H-2‴), 6.89 (1H, d,
J = 8.2 Hz, H-5‴), 7.18 (1H, dd, J = 8.2, 1.8 Hz, H-6‴), 7.65
(1H, d, J = 15.9 Hz, H-7‴), 6.46 (1H, d, J = 15.9 Hz, H-8‴),
and 3.94 (3H, s, OCH3-3‴); δC 127.5 (C-1‴), 111.3 (C-2‴),
148.8 (C-3‴), 150.2 (C-4‴), 116.1 (C-5‴), 124.0 (C-6‴),
146.1 (C-7‴), 116.0 (C-8‴), 167.7 (C-9‴), and 56.4 (OCH3-
3‴)] in 5.28 This was confirmed by the HMBC correlations
from H-7‴ to C-2‴/C-6‴/C-9‴, H-8‴ to C-1‴, H-3″ to C-9‴,
and H-1″ to C-2 (Figure 2). Therefore, the structure of 5
(polygonibene E) was elucidated as (E)-2,3,5,4′-tetrahydrox-
ystilbene-2-O-(3″-O-feruloyl)-β-D-glucoside, a new monomeric
stilbene glycoside.
The nine known compounds were identified as (E)-2,3,5,4′-
tetrahydroxystilbene-2-O-β-D-glucoside (8),27 (E)-2,3,5,4′-tet-
rahydroxystilbene-2-O-(6″-O-β-D-glucopyranoside)-β-D-gluco-
side (9),33 (E)-2,3,5,4′-tetrahydroxystilbene-2-O-(6″-O-ace-
tyl)-β-D-glucoside (10),21 (E)-2,3,5,4′-tetrahydroxystilbene-2-
O-(2″-O-galloyl)-β-D-glucoside (11),21 (E)-2,3,5,4′-tetrahy-
droxystilbene-2-O-(2″-O-feruloyl)-β-D-glucoside (12),28 re-
sveratrol (13),34 (E)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-xy-
loside (14),35 (E)-2,3,5,4′-tetrahydroxystilbene-2-O-α-L-rham-
n o s i d e ( 1 5 ) , a n d 3 , 5 - d i h y d r o x y - 2 - [ 2 - ( 4 -
hydroxyphenyl)acetyl]benzoic acid (16)36 (Figure 1) based
on the analysis of their observed and reported spectroscopic
Polygonibene F (6) was obtained as a brown powder with
the molecular formula C21H22O10 based on the HRFABMS
protonated molecular ion peak at m/z 435.1288 [M + H]+
(calcd for C21H23O10, 435.1291). The 1H and 13C NMR
spectra of 6 (Table 2) were similar to those of 8 except for
additional signals due to a formyl group (HCOO−) [δH 8.08
(1H, s); δC 161.9] in 6.29 The position of the attached formyl
group at C-6″ was verified by the downfield shift of H2-6″ (δH
4.45, 4.38) in 6 compared with those of 8 [δH 3.86 (H2-6″)]
and the HMBC correlations from δH 8.08 (1H, s, HCOO−) to
δC 63.7 (C-6″) and δH 4.45/4.38 (H2-6″) to δC 161.9
(HCOO−) (Figure 2). Thus, the structure of 6 (polygonibene
F) was elucidated as (E)-2,3,5,4′-tetrahydroxystilbene-2-O-(6″-
formyl)-β-D-glucoside, a new monomeric stilbene glycoside.
The molecular formula of polygonibene G (7, a brown
powder) was determined to be C30H30O12 based on its
HRESIMS data [a sodium adduct molecular ion peak at m/z
605.1631 [M + Na]+ (calcd for C30H30O12Na, 605.1635)].
1
data. Herein, the H and 13C NMR data of compound 15 are
established for the first time based on conventional NMR
techniques (1H−1H COSY, HMQC, and HMBC).
The PTP1B inhibitory activity of the isolated compounds
(1−16) and three deglucosylated stilbenes (2a, 3a, and 8a)
were evaluated using p-nitrophenyl phosphate (p-NPP) as a
substrate, and IC50 values were determined by linear regression
analysis (Table 3). As a result, compounds 2−5, 9, and 13,
together with three aglycones (2a, 3a, and 8a), significantly
inhibited PTP1B compared to the non-inhibitor-treated
control group (p < 0.05). The other compounds did not
show significant inhibition under the tested concentration
(IC50 > 50 μM). The new compounds (2−5) showed weak
PTP1B inhibition with an IC50 range of 27.4−37.6 μM, while
1
The H NMR spectrum of 7 (Table 2) also showed proton
signals of a stilbene glycoside: a trans-disubstituted double
bond at δH 7.32 (1H, d, J = 16.8 Hz, H-α) and 6.34 (1H, d, J =
F
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moiety was substituted, showed stronger inhibition than 6 and
9−12, which have other substituent positions. This suggested
that a substituent occurring at position C-3″ on the glucose
moiety might positively influence the PTP1B inhibitory activity
of (E)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside deriva-
tives.
Since the aglycone stilbenes (2a and 3a) showed the
strongest PTP1B inhibition, the mode of inhibition and
inhibition constants (Ki values) of these compounds were
further investigated using two kinetic methods: Lineweaver−
Burk and Dixon plots (Figure S9, Supporting Information). In
the Lineweaver−Burk plot, the lines crossed at the same point
on the x-intercept, and the y-intercept increased as the
inhibitor concentration increased, indicating that both 2a and
3a inhibited PTP1B via noncompetitive mechanisms. The
results of Dixon plots also indicated that PTP1B inhibition was
noncompetitive for 2a and 3a (Figure S9, Supporting
Information) with Ki values of 1.02 and 0.92 μM, respectively
(Table 3).
Table 3. Inhibitory Activity of Compounds 1−16, 2a, 3a,
and 8a against PTP1B
PTP1B inhibition,
Ki
a
b
c
compound
1, 6−16
2
3
4
5
8a
2a
IC50 (μM)
value
inhibition type
>50
e
e
e
e
e
e
e
e
e
e
e
e
32.2 0.46
34.9 0.58
37.6 3.02
27.4 0.02
12.1 0.53
2.1 0.08
1.9 0.04
15.1 1.76
1.02
0.92
e
noncompetitive
noncompetitive
e
3a
sodium
d
orthovanadate
d
ursolic acid
5.0 0.02
e
e
a
The 50% inhibition concentrations (IC50, μM) are expressed as the
b
mean SEM four independent experiments. Determined by Dixon
plot. Determined by Lineweaver−Burk plots. Used as positive
control. Not tested.
c
d
e
To investigate the protein−ligand interaction geometries for
compounds 2a and 3a at a molecular level, molecular docking
simulation of these compounds and the reference ligands
[compound B (an allosteric inhibitor) and ursolic acid (a
catalytic inhibitor)] were performed using AutoDock 4.2
software. The molecular docking models of the deglucosylated
stilbenes (2a and 3a) are illustrated in Figure 3. The binding
energies of 2a, 3a, ursolic acid, and compound B with
interacting residues (H-bond and van der Waals interacting
residues), along with the number of H-bonds, are listed in
Table 4. In a previous study, three α helices [α3 helix
(Glu186−Glu200), α6 helix (Ala264−Ile281), and α7 helix
(Val287−Ser295)] were reported as core regions of the
allosteric site in PTP1B.37 The docking results presented here
show that compound 2a binds to the allosteric site of PTP1B
with a binding energy of −7.01 kcal/mol by forming three
the three deglucosylated stilbenes (2a, 3a, and 8a) exhibited
more potent activity with IC50 values of 2.1, 1.9, and 12.1 μM,
respectively. In comparison, the IC50 values of the positive
controls (ursolic acid and sodium orthovanadate) were 5.0 and
15.1 μM, respectively. This suggested that the presence of
glucose moieties negatively influences the resultant PTP1B
inhibitory activity of stilbene glycosides. This finding is similar
to a previous study, in which the deglucosylated stilbene 8a
exhibited much stronger DPPH radical scavenging activity
(IC50 = 0.4 mM) compared to its corresponding precursor (8,
IC50 = 40 mM).18
In addition, the structure−activity relationships for (E)-
2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside derivatives
(4−6 and 9−12) were also observed in the PTP1B inhibitory
assay. Compounds 4 and 5, in which the C-3″ of the glucose
Figure 3. Inhibition mode of 2a (A) and 3a (C) for the PTP1B allosteric site and 2D ligand interaction diagram of PTP1B allosteric inhibition by
2a (B) and 3a (D).
G
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Table 4. Binding Site Residues and Docking Scores of Compounds 2a, 3a, and Reported Inhibitors of PTP1B Obtained Using
Autodock 4.2
binding
energy
(kcal/mol)
no. of
H-
bonds
H-bond
van der Waals
interacting
residues
electrostatic
interacting
residues
a
interacting
b
b
b
b
b
compound
residues
hydrophobic interacting residues
Ile281 (π−δ), Leu192 (π−alkyl), Phe280 (π−π T-shaped)
2a
3a
−7.01
3
Asp236,
Asn193,
Glu276
−9.35
4
Asn193,
Ala189,
Ser187
Gly277
Phe280 (π−π stacked), Leu192 Pro188 (π−alkyl), Ala189 (π−alkyl Glu276 (π−
and π−σ)
anion)
ursolic acid (cata-
lytic inhibitor)
−8.88
3
3
Arg24, Arg254
Tyr46, Arg24 (π−alkyl)
c
compound B (al-
losteric inhibi-
tor)
−10.85
Phe280,
Lys279,
Glu276
Phe280 (π−alkyl, π−π stacked, and π−π T-shaped), Leu192 (π−δ
and π−alkyl), Phe196 (π−π stacked and π−alkyl), Ile281 (alkyl)
a
b
Estimated binding free energy of the ligand−receptor complex. The number of hydrogen bonds and all amino acid residues from the enzyme−
c
inhibitor complex were determined with the AutoDock 4.2 program. 3-(3,5-Dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonic acid (4-
sulfamoyl-phenyl)-amide.
The EtOAc fraction (310.5 g) was chromatographed with vacuum
liquid chromatography (VLC) on silica gel eluted with a CH2Cl2/
MeOH gradient (2−100% MeOH) to yield four fractions: 52B−E.
Fraction 52C (11.36 g) was subjected to column chromatography
(CC) on MCI gel eluted with a MeOH/H2O gradient (20−100%
MeOH) to obtain 12 fractions: 53A−L. Fraction 53D (2.91 g) was
separated on silica gel CC (CH2Cl2/MeOH/formic acid, 20:1:0.5 →
10:10:0.5) to obtain seven fractions: 56A−G. Fraction 56F (1.33 g)
was chromatographed on MCI gel CC eluted with a mixture of
MeOH/H2O (40−100% MeOH) to obtain 8 (873.6 mg) and 6 (15.2
mg). Fraction 53E (318.5 mg) was chromatographed on a Sephadex
LH-20 column using a gradient solvent system of MeOH/H2O (50−
100% MeOH) and yielded six fractions: 54A−F. Compounds 4 (4.0
mg, tR = 32.0 min) and 10 (7.2 mg, tR = 37.0 min) were obtained
from fraction 54D (28.7 mg) by preparative HPLC (isocratic eluent,
45% MeOH in H2O, 6 mL/min, 45 min). Fraction 54E (35.6 mg)
was rechromatographed by preparative HPLC with an isocratic eluent
(55% MeOH in H2O, 6 mL/min, 30 min) to obtain 14 (10.7 mg, tR =
18.0 min) and 15 (7.2 mg, tR = 20.0 min). Fraction 53G (948.4 mg)
was chromatographed on MCI gel CC eluted with a MeOH/H2O
gradient (55−100% MeOH) to obtain five fractions: 60A−E.
Compound 12 (30.6 mg) was purified from fraction 60D with
Sephadex LH-20 CC eluted with a mixture of MeOH/H2O (1:1).
Fraction 60E was passed over a Sephadex LH-20 column eluted with
MeOH−H2O (50%) to obtain 13 (31.8 mg) and fraction 60E3.
Compound 7 (3.1 mg, tR = 53.0 min) was obtained from fraction
60E3 by preparative HPLC with a MeOH/H2O gradient (40−60%
MeOH, 6 mL/min, 60 min). Fraction 53H (330.9 mg) was
fractionated on a Sephadex LH-20 column (MeOH/H2O, 1:1) to
obtain 5 (20.8 mg) and fractions 66A−D. Compound 16 (8.1 mg, tR
= 20.5 min) was obtained from fraction 66B (30.6 mg) by preparative
HPLC (isocratic eluent, 60% MeOH in H2O, 6 mL/min, 35 min).
Fraction 52D (183.2 g) was separated on silica gel VLC eluted with a
CH2Cl2/MeOH/H2O gradient (9:1:0.1 → 7:3:0.3) to obtain four
fractions: 67A−D. Fraction 67D (10.2 g) was subjected to MCI gel
CC eluted with a MeOH/H2O gradient (20−100% MeOH) to obtain
12 fractions: 67D1−12. Fraction 67D9 (314.2 mg) was further
chromatographed with preparative HPLC (isocratic eluent, 45%
MeOH in H2O, 6 mL/min, 40 min) to obtain 2 (70.5 mg, tR = 27.0
min) and 3 (57.9 mg, tR = 32.0 min). Compound 11 (30.1 mg, tR =
16.5 min) was obtained from fraction 67D6 (94.1 mg) by preparative
HPLC with an isocratic eluent of MeOH/H2O (45% MeOH, 6 mL/
min, 25 min). Fraction 67D7 (4.4 g) was separated on silica gel CC
(CH2Cl2/MeOH/H2O, 6.5:1:0.1) to obtain three fractions: 69A−C.
Fraction 69C (256.2 mg) was further subjected to preparative HPLC
(isocratic eluent, 40% MeOH in H2O, 6 mL/min, 30 min) to obtain 1
(7.4 mg, tR = 19.0 min) and 9 (7.1 mg, tR = 21.5 min).
strong hydrogen bond interactions via the hydroxy groups at
positions 4, 13, and 13′ of compound 2a with Glu276, Asn193,
and Asp236 residues (Figure 3B). In addition, 2a shared the
same allosteric residues, including Leu192 (in the α3 helix)
and Phe280 (in the α7 helix), with the reference allosteric
inhibitor (compound B) via hydrophobic interactions.
Similarly, at the allosteric site, the hydroxy groups at positions
10′, 11′, 10, and 4 of compound 3a showed four hydrogen-
bonding interactions with three residues (Asn193, Ala189, and
Ser187) in the α3 helix of PTP1B (Figure 3D), with −9.35
kcal/mol binding energy. Interestingly, hydrophobic inter-
actions were observed between ring B of both 2a and 3a and
the allosteric residues Phe280 and Leu192. In addition, the
benzene rings D of 3a interacted with Glu276 via π−anion
electrostatic interaction and Pro188 and Ala189 residues via
hydrophobic interactions. A van der Waals interaction was also
viewed between compound 3a and the Gly277 residue in the
α6 helix. These interactions were critical in blocking the
interactions between α7 and α3−α6, preventing closure of the
catalytic WPD loop and stabilizing PTP1B in an open, inactive
conformation.37 The negative binding energies of 2a and 3a
indicated that they have a high affinity and tight binding
capacity for the active site of PTP1B. These in silico results
were concordant with the in vitro kinetic analysis results and
demonstrated that the aglycone stilbene derivatives (2a and
3a) are noncompetitive and allosteric PTP1B inhibitors.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations, IR, UV,
NMR spectra, HRFABMS, HRESIMS, HPLC, and TLC, were carried
out according to previously described procedures (Supporting
Information).38
Plant Material. Roots from P. multiflorum were purchased from a
traditional market in Jeonju-si, Korea, in January of 2018 and
authenticated by one of the authors (B.S.M.). A voucher specimen
(22A-PM) was stored at the Laboratory of Pharmacology in the
College of Pharmacy, Kyungpook National University, Republic of
Korea.
Extraction and Isolation. The dried roots of P. multiflorum (20.0
kg) were extracted and refluxed with 100% MeOH (9 L × 3) at 40 °C
for 4 h. The extract was concentrated in vacuo to obtain a brown
residue (2.44 kg). The residue was suspended in distilled water and
successively partitioned with n-hexane, CH2Cl2, EtOAc, and n-butanol
to afford n-hexane (80.1 g), CH2Cl2 (35.2 g), EtOAc (310.5 g), and n-
butanol (308.7 g) fractions and an aqueous extract.
Polygonibene A (1). Brown powder; [α]2D1.2 + 81.0 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 235 (4.27), 310 (4.17) nm; IR (ATR) νmax
3322, 1610, 1580, 1510, 1320, 1250, 1088 cm−1; ECD (MeOH) λmax
H
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(Δε) 198 (−0.48), 212 (−1.06), 239 (+4.90) nm); 1H NMR
(methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz),
see Table 1; HRFABMS m/z 833.2271 [M + Na]+ (calcd for
C40H42O18Na, 833.2269).
3a (1.2 mg, tR = 29.5 min). Their structures were confirmed by
1
HRESIMS, H NMR, ECD spectra, and optical rotation data.
1
(E)-2,3,5,4′-Tetrahydroxystilbene (8a). Brown powder; H NMR
(acetone-d6, 500 MHz) δH 7.39 (2H, d, J = 8.5 Hz, H-2′,6′), 7.29
(1H, d, J = 16.5 Hz, H-β), 7.01 (1H, d, J = 16.5 Hz, H-α), 6.83 (2H,
d, J = 8.5 Hz, H-3′,5′), 6.56 (1H, d, J = 2.6 Hz, H-6), 6.34 (1H, t, J =
2.6 Hz, H-4); HRESIMS m/z 245.0829 [M + H]+ (calcd for
C14H13O4, 245.0814).
Polygonibene B (2). Brown powder; [α]2D1.2 + 99.8 (c 0.18,
MeOH); UV (MeOH) λmax (log ε) 230 (4.60), 325 (4.46) nm; ECD
(MeOH) λmax (Δε) 204 (+2.48), 221 (−0.22), 239 (+0.48) nm; IR
1
(ATR) νmax 3340, 1620, 1590, 1512, 1330, 1250, 1096 cm−1; H
NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125
MHz), see Table 1; HRFABMS m/z 833.2273 [M + Na]+ (calcd for
C40H42O18Na, 833.2269).
10,10′-Deglucosyl-polygonibene B (2a). Brown powder; [α]D21.2
+34.5 (c 0.02, MeOH); ECD (MeOH) 200 nm (Δε + 1.18), 222 nm
(Δε + 0.07), 240 nm (Δε + 0.95); 1H NMR (methanol-d4, 500 MHz)
δH 7.35 (1H, dd, J = 8.3, 1.8 Hz, H-6′), 7.25 (2H, d, J = 8.5 Hz, H-
2,6), 7.23 (1H, d, J = 16.4 Hz, H-8′), 7.22 (1H, d, J = 1.8 Hz, H-2′),
6.97 (1H, d, J = 16.4 Hz, H-7′), 6.85 (1H, d, J = 8.3 Hz, H-5′), 6.75
(2H, d, J = 8.5 Hz, H-3,5), 6.47 (1H, d, J = 2.7 Hz, H-14′), 6.27 (1H,
d, J = 2.8 Hz, H-12), 6.23 (1H, d, J = 2.7 Hz, H-12′), 5.94 (1H, d, J =
2.8 Hz, H-14), 5.55 (1H, d, J = 6.6 Hz, H-7), 4.93 (1H, d, J = 6.6 Hz,
H-8); HRESIMS m/z 487.1398 [M + H]+ (calcd for C28H23O8,
487.1392).
Polygonibene C (3). Brown powder; [α]2D1.2 −55.5 (c 0.13,
MeOH); UV (MeOH) λmax (log ε) 230 (4.55), 320 (4.40) nm;
ECD (MeOH) λmax (Δε) 205 (−1.32), 218 (+0.58), 236 (−0.61)
nm; IR (ATR) νmax 3328, 1620, 1590, 1510, 1330, 1250, 1098 cm−1;
1H NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125
MHz), see Table 1; HRFABMS m/z 833.2272 [M + Na]+ (calcd for
C40H42O18Na, 833.2269).
Polygonibene D (4). Brown powder; [α]2D1.2 +27.7 (c 0.03,
MeOH); UV (MeOH) λmax (log ε) 235 (3.45), 310 (3.54) nm; IR
(ATR) νmax 3326, 1739, 1620, 1580, 1510, 1320, 1260, 1088 cm−1;
1H NMR (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125
MHz), see Table 2; HRESIMS m/z 449.1445 [M + H]+ (calcd for
C21H25O10, 449.1458).
10,10′-Deglucosyl-polygonibene C (3a). Brown powder; [α]D21.2
−11.2 (c 0.02, MeOH), ECD (MeOH) 209 nm (Δε + 2.76), 239 nm
(Δε −3.35); 1H NMR (methanol-d4, 500 MHz) δH 7.35 (1H, dd, J =
8.3, 1.7 Hz, H-6′), 7.26 (2H, d, J = 8.5 Hz, H-2,6), 7.23 (1H, d, J =
16.4 Hz, H-8′), 7.22 (1H, d, J = 1.7 Hz, H-2′), 6.98 (1H, d, J = 16.4
Hz, H-7′), 6.85 (1H, d, J = 8.3 Hz, H-5′), 6.75 (2H, d, J = 8.5 Hz, H-
3,5), 6.48 (1H, d, J = 2.8 Hz, H-14′), 6.28 (1H, d, J = 2.8 Hz, H-12),
6.24 (1H, d, J = 2.8 Hz, H-12′), 5.95 (1H, d, J = 2.8 Hz, H-14), 5.56
(1H, d, J = 6.6 Hz, H-7), 4.94 (1H, d, J = 6.6 Hz, H-8); HRESIMS m/
z 487.1397 [M + H]+ (calcd for C28H23O8, 487.1392).
PTP1B Inhibitory Assay. The PTP1B inhibitory activity of
compounds 1−16, 2a, 3a, and 8a were evaluated using p-nitrophenyl
phosphate (p-NPP) as a substrate and ursolic acid as a positive
control according to a previously reported procedure.40 IC50 values
were calculated using nonlinear regression analysis in an Excel
(Microsoft Excel 2016, U.S.A.) spreadsheet.
PTP1B Kinetic Assay. The PTP1B kinetic analysis was conducted
using the method previously described by Tuan et al. (Supporting
Information).40
Molecular Docking Simulation. The docking protocol was
carried out with the AutoDock 4.2 program using a previously
described method40 with modifications (Supporting Information).
Statistical Analysis. Statistical analyses were performed as
previously described (Supporting Information).40
Polygonibene E (5). Brown powder; [α]2D1.2 −12.8 (c 0.2, MeOH);
UV (MeOH) λmax (log ε) 225 (4.45), 325 (4.45) nm; IR (ATR) νmax
1
3316, 1740, 1620, 1570, 1512, 1330, 1260, 1140 cm−1; H NMR
(acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz), see
Table 2; HRESIMS m/z 605.1636 [M + Na]+ (calcd for
C30H30O12Na, 605.1635).
Polygonibene F (6). Brown powder; [α]2D1.2+25.2 (c 0.2, MeOH);
UV (MeOH) λmax (log ε) 225 (4.23), 325 (4.35) nm; IR (ATR) νmax
1
3327, 1740, 1620, 1580, 1510, 1330, 1260, 1150 cm−1; H NMR
(acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz), see
Table 2; HRFABMS m/z 435.1288 [M + H]+ (calcd for C21H23O10,
435.1291).
Polygonibene G (7). Brown powder; [α]2D1.2 −46.3 (c 0.04,
MeOH); UV (MeOH) λmax (log ε) 230 (3.98), 290 (3.99) nm; ECD
(MeOH) λmax (Δε) 207 (−1.64), 229 (+4.61), 250 (−1.30) nm; IR
(ATR) νmax 3397, 1741, 1620, 1570, 1510, 1330, 1260, 1088 cm−1;
1H NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125
MHz), see Table 2; HRESIMS m/z 605.1631 [M + Na]+ (calcd for
C30H30O12Na, 605.1635).
(E)-2,3,5,4′-Tetrahydroxystilbene-2-O-α-L-rhamnoside (15).
1
Brown powder; H NMR (acetone-d6, 500 MHz) δH 7.74 (1H, d, J
ASSOCIATED CONTENT
■
= 16.5 Hz, H-α), 7.41 (2H, d, J = 8.6 Hz, H-2′,6′), 6.92 (1H, d, J =
16.5 Hz, H-β), 6.78 (2H, d, J = 8.6 Hz, H-3′,5′), 6.63 (1H, d, J = 2.8
Hz, H-6), 6.24 (1H, d, J = 2.8 Hz, H-4), 4.47 (1H, d, J = 7.9 Hz, H-
1″), 3.57 (1H, t, J = 8.5 Hz, H-3″), 3.42 (1H, m, H-2″), 3.38 (1H, m,
H-5″), 3.14 (1H, td, J = 4.5, 9.1 Hz, H-4″), 1.27 (3H, d, J = 6.1 Hz,
H-6″); 13C NMR (acetone-d6, 125 MHz) δC 158.1 (C-4′), 155.8 (C-
5), 152.1 (C-3), 137.9 (C-2), 133.3 (C-1), 130.5 (C-1′), 129.3 (C-β),
128.9 (C-2′,6′), 121.9 (C-α), 116.3 (C-3′,5′), 107.6 (C-1″), 103.5
(C-4), 102.1 (C-6), 77.7 (C-2″), 76.3 (C-4″), 75.6 (C-3″), 73.5 (C-
5″), 18.1 (C-6″). HRESIMS m/z 413.1208 [M + Na]+ (calcd for
C20H22O8Na, 413.1212).
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b00777.
Experimental methods, 1D and 2D NMR and HRMS
1
spectra of polygonibenes A−G (1−7) and H NMR
spectra of compounds 2a and 3a, ECD spectra of
compounds 1, 2a, 3a, ε-viniferin, and δ-viniferin, and
kinetic studies for selected compounds 2a and 3a (PDF)
Acid Hydrolysis of 2, 3, and 8. Compounds 2, 3, and 8 (30 mg
each) were individually refluxed with 10% HCl (10 mL) for 3 h at 90
°C, and each reaction mixture was extracted with EtOAc. After the
aqueous layer was neutralized with BaCO3, the precipitates were
filtered, and the filtrate was repeatedly evaporated in vacuo to yield a
colorless syrup that was purified by preparative TLC (solvent CHCl3/
MeOH/H2O 8:5:1) (Rf 0.31). According to a previously reported
method,39 the monosaccharide residue was identified as D-glucose.
Each EtOAc layer was washed with aqueous 5% NaHCO3, dried, and
evaporated to yield a brown powder. The resultant residue was
purified by preparative HPLC using MeOH/H2O elution (45%
MeOH, 6 mL/min, 40 min) to obtain the respective deglucosylated
stilbenes 8a (5.2 mg, tR = 25.0 min),18 2a (1.1 mg, tR = 27.5 min), and
AUTHOR INFORMATION
■
Corresponding Authors
Byung Sun Min − Daegu Catholic University, Gyeongbuk,
Republic of Korea; Phone: +82-850-3613;
Email: bsmin@cu.ac.kr; Fax: +82-53-850-3602
Jeong Ah Kim − Kyungpook National University, Daegu,
Republic of Korea; orcid.org/0000-0001-7337-1812;
Phone: +82-53-950-8574; Email: jkim6923@
knu.ac.kr; Fax: +82-53-950-8557
I
https://dx.doi.org/10.1021/acs.jnatprod.9b00777
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
(17) Lv, L.; Gu, X.; Tang, J.; Ho, C. T. Food Chem. 2007, 104,
1678−1681.
Other Authors
Thi-Thuy An Nguyen − Kyungpook National University,
Daegu, Republic of Korea
Manh Tuan Ha − Daegu Catholic University, Gyeongbuk,
Republic of Korea
Se-Eun Park − Pukyong National University, Busan,
Republic of Korea
Jae Sue Choi − Pukyong National University, Busan,
Republic of Korea; orcid.org/0000-0001-9034-8868
(18) Ryu, G.; Ju, J. H.; Park, Y. J.; Ryu, S. Y.; Choi, B. W.; Lee, B. H.
Arch. Pharmacal Res. 2002, 25, 636−639.
(19) Wang, X.; Zhao, L.; Han, T.; Chen, S.; Wang, J. Eur. J.
Pharmacol. 2008, 578, 339−348.
(20) Yao, W.; Fan, W.; Huang, C.; Zhong, H.; Chen, X.; Zhang, W.
Biomed. Pharmacother. 2013, 67, 140−145.
(21) Kim, H. K.; Choi, Y. H.; Choi, J. S.; Choi, S. U.; Kim, Y. S.; Lee,
K. R.; Kim, Y.-K.; Ryu, S. Y. Arch. Pharmacal Res. 2008, 31, 1225.
(22) Hoang, D. M.; Trung, T. N.; Hien, P. T. T.; Ha, D. T.; Van
Luong, H.; Lee, M.; Bae, K. Nat. Prod. Sci. 2010, 16, 239−244.
(23) Ngoc, T. M.; Minh, P. T. H.; Hung, T. M.; Thuong, P. T.; Lee,
I.; Min, B. S.; Bae, K. Arch. Pharmacal Res. 2008, 31, 598.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b00777
̀
(24) Lemiere, G.; Gao, M.; De Groot, A.; Dommisse, R.; Lepoivre,
Author Contributions
J.; Pieters, L.; Buss, V. J. Chem. Soc., Perkin Trans. 1 1995, 1775−1779.
(25) Ito, T.; Masuda, Y.; Abe, N.; Oyama, M.; Sawa, R.; Takahashi,
Y.; Chelladurai, V.; Iinuma, M. Chem. Pharm. Bull. 2010, 58, 1369−
1378.
⊥T.T.A.N. and M.T.H. contributed equally to this article.
Notes
The authors declare no competing financial interest.
(26) Timperio, A. M.; D’Alessandro, A.; Fagioni, M.; Magro, P.;
Zolla, L. Plant Physiol. Biochem. 2012, 50, 65−71.
(27) Kumar, S.; Lee, H. Y.; Liou, J. P. J. Nat. Prod. 2017, 80, 1294−
1301.
ACKNOWLEDGMENTS
■
This research was supported by the National Research
Foundation of Korea (NRF), funded by the Ministry of
S c i e n c e , I C T , a n d F u t u r e P l a n n i n g ( N R F -
2018R1D1A1B07045287), and a grant (18182MFDS214)
from Ministry of Food and Drug Safety in 2018. We are
grateful to the Korea Basic Science Institute (KBSI) for mass
spectrometric measurements.
(28) Chen, W. S.; Zhang, W. D.; Yang, G. J.; Chen, H. S.; Qiao, C.
Z. Chin. Chem. Lett. 2001, 12, 503−506.
(29) Brecker, L.; Mahut, M.; Schwarz, A.; Nidetzky, B. Magn. Reson.
Chem. 2009, 47, 328−332.
(30) Li, X. M.; Lin, M.; Wang, Y. H.; Liu, X. Planta Med. 2004, 70,
160−165.
(31) Wungsintaweekul, B.; Umehara, K.; Miyase, T.; Noguchi, H.
Phytochemistry 2011, 72, 495−502.
REFERENCES
■
381.
(32) Fan, W.; Tezuka, Y.; Xiong, Q.; Hattori, M.; Namba, T.;
Kadota, S. Chem. Pharm. Bull. 1999, 47, 1049−1050.
(33) Pozdeeva, A. V.; Komarova, N. I.; Vasiliev, V. G.; Rogachev, A.
D.; Salakhutdinov, N. F.; Tolstikov, G. A. Mendeleev Commun. 2013,
23, 37−38.
(1) Zhang, S.; Zhang, Z. Y. Drug Discovery Today 2007, 12, 373−
(2) Johnson, T. O.; Ermolieff, J.; Jirousek, M. R. Nat. Rev. Drug
Discovery 2002, 1, 696−709.
(3) Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins,
S.; Loy, A. L.; Normandin, D.; Cheng, A.; Himms-Hagen, J.; Chan, C.
C.; Ramachandran, C.; Gresser, M. J.; Tremblay, M. L.; Kennedy, B.
P. Science 1999, 283, 1544−1548.
(34) Mannila, E.; Talvitie, A.; Kolehmainen, E. Phytochemistry 1993,
33, 813−816.
(35) Sun, Y. N.; Cui, L.; Li, W.; Yan, X. T.; Yang, S. Y.; Kang, J. I.;
Kang, H. K.; Kim, Y. H. Bioorg. Med. Chem. Lett. 2013, 23, 4801−
4805.
(36) Li, X. C.; Liu, C.; Yang, L. X.; Chen, R. Y. J. Asian Nat. Prod.
Res. 2011, 13, 826−830.
(37) Wiesmann, C.; Barr, K. J.; Kung, J.; Zhu, J.; Erlanson, D. A.;
Shen, W.; Fahr, B. J.; Zhong, M.; Taylor, L.; Randal, M.; McDowell,
R. S.; Hansen, S. K. Nat. Struct. Mol. Biol. 2004, 11, 730.
(38) Pham, V. C.; Nguyen, T. T. A.; Vu, T. O.; Cao, T. Q.; Min, B.
S.; Kim, J. A. Phytochem. Lett. 2019, 32, 23−28.
(39) Ngo, Q. M. T.; Lee, H. S.; Nguyen, V. T.; Kim, J. A.; Woo, M.
H.; Min, B. S. Phytochemistry 2017, 141, 147−155.
(40) Ha, M. T.; Seong, S. H.; Nguyen, T. D.; Cho, W. K.; Ah, K. J.;
Ma, J. Y.; Woo, M. H.; Choi, J. S.; Min, B. S. Phytochemistry 2018,
155, 114−125.
(4) Klaman, L. D.; Boss, O.; Peroni, O. D.; Kim, J. K.; Martino, J. L.;
Zabolotny, J. M.; Moghal, N.; Lubkin, M.; Kim, Y. B.; Sharpe, A. H.;
Stricker-Krongrad, A.; Shulman, G. I.; Neel, B. G.; Kahn, B. B. Mol.
Cell. Biol. 2000, 20, 5479−5489.
(5) Yip, S. C.; Saha, S.; Chernoff, J. Trends Biochem. Sci. 2010, 35,
442−449.
(6) Bentires-Alj, M.; Neel, B. G. Cancer Res. 2007, 67, 2420−2424.
(7) Stuible, M.; Doody, K. M.; Tremblay, M. L. Cancer Metastasis
Rev. 2008, 27, 215−230.
́
(8) Julien, S. G.; Dube, N.; Read, M.; Penney, J.; Paquet, M.; Han,
Y.; Kennedy, B. P.; Muller, W. J.; Tremblay, M. L. Nat. Genet. 2007,
39, 338.
́
́
(9) Traves, P. G.; Pardo, V.; Pimentel-Santillana, M.; Gonzalez-
́
Rodríguez, A.; Mojena, M.; Rico, D.; Montenegro, Y.; Cales, C.;
́
Martin-Sanz, P.; Valverde, A. M.; Bosca, L. Cell Death Dis. 2014, 5,
No. e1125.
(10) Vieira, M. N.; Lyra e Silva, N. M.; Ferreira, S. T.; De Felice, F.
G. Front. Aging Neurosci. 2017, 9, 7.
(11) Tautz, L. J. Clin. Invest. 2015, 125, 2931−2934.
(12) Jeong, B.; Choi, S. Y.; Jang, H. S.; Yoo, G.; Kim, S. H.; Kim, J.
H.; Kwon, Y. S.; Roh, J. S.; Yoon, Y.; Shin, S. S.; Yang, H. Nat. Prod.
Sci. 2017, 23, 9−15.
(13) Jiang, C. S.; Liang, L. F.; Guo, Y. W. Acta Pharmacol. Sin. 2012,
33, 1217.
(14) Ha, M. T.; Park, D. H.; Shrestha, S.; Kim, M.; Kim, J. A.; Woo,
M. H.; Choi, J. S.; Min, B. S. Fitoterapia 2018, 131, 119−126.
(15) Shen, T.; Wang, X. N.; Lou, H. X. Nat. Prod. Rep. 2009, 26,
916−935.
(16) Lv, G.; Lou, Z.; Chen, S.; Gu, H.; Shan, L. J. Ethnopharmacol.
2011, 137, 449−456.
J
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