Triterpene saponins with a-glucosidase and PTP1B inhibitory activities from the leaves of Aralia elata

Article abstract of DOI:10.1016/j.phytol.2018.06.002

Two new triterpene saponins named congmuyenoside V (1) and congmuyenoside VI (2), together with eleven known ones (3-13) were isolated from the leaves of Aralia elata. Structures of compounds 1-13 were determined by comprehensive spectroscopic analyses, principally including 1D, 2D NMR and HRESIMS techniques. In addition, all compounds were evaluated for their inhibitory effects on α-glucosidase and protein tyrosine phosphatase 1B (PTP1B). Compounds 1, 7 and 9-13 exhibited stronger α-glucosidase inhibitory activities than acarbose (the positive control). Simultaneously compounds 1 and 13 also demonstrated significant inhibitory effects on PTP1B.

Full text of DOI:10.1016/j.phytol.2018.06.002

Phytochemistry Letters 26 (2018) 179–183
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Triterpene saponins with a-glucosidase and PTP1B inhibitory activities from
the leaves of Aralia elata
⁎
Yan Zhang, Feng-Ying Han, Jie Wu, Shao-Jiang Song
School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University,
Shenyang, 110016, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Aralia elata
Triterpene saponinsα-glucosidase
PTP1B
Two new triterpene saponins named congmuyenoside V (1) and congmuyenoside VI (2), together with eleven
known ones (3-13) were isolated from the leaves of Aralia elata. Structures of compounds 1-13 were determined
by comprehensive spectroscopic analyses, principally including 1D, 2D NMR and HRESIMS techniques. In ad-
dition, all compounds were evaluated for their inhibitory effects on α-glucosidase and protein tyrosine phos-
phatase 1B (PTP1B). Compounds 1, 7 and 9-13 exhibited stronger α-glucosidase inhibitory activities than
acarbose (the positive control). Simultaneously compounds 1 and 13 also demonstrated significant inhibitory
effects on PTP1B.
1. Introduction
have potential α-glucosidase inhibitiory activity (Yin et al., 2014).
These findings might provide some evidences for the relationship of
Aralia elata, one species of the genus Aralia (Araliaceae), was widely
distributed in northeastern China and rarely in Korea and Japan
Shikov et al., 2016). It has been used as a traditional Chinese herb to
oleanane-type saponins and these two enzymes.
In our continuing research to seek new bioactive constituents from
A. elata, two new oleanane-type triterpene saponins and eleven known
ones were isolated from the leaves of A. elata. Their structures were
demonstrated by spectroscopic methods, and the inhibitory activities of
all compounds against α-glucosidase and PTP1B were tested. Given
their inhibition of T2DM drug targets, triterpene saponins from A. elata
might have great potential.
(
treat diabetes mellitus (Chung et al., 2005), rheumatoid arthritis,
neurasthenia, and hepatitis (Lee and Jeong, 2009; Nhiem et al., 2011;
Tomatsu et al., 2003). Previous phytochemical investigations of A. elata
revealed the presence of triterpene saponins, flavones and poly-
saccharides (Clement and Clement, 2014). As reported, triterpene sa-
ponins with different structure types were regarded as the main
bioactive constituents (Liu et al., 1995; Stavropoulou et al., 2017), most
of which displayed significant biological activities. This made tri-
terpene saponins from A. elata an attractive target.
2. Results and discussion
Compound 1 was isolated as a white amorphous powder from
Type 2 diabetes (T2DM) is a major health-threatening disease
worldwide, which accounts for about 90 percent of diabetes
CH
Molish reactions. The molecular formula of 1 was determined as
48 78
C H O20 with ten degrees of unsaturation, which showed a pseudo
3
OH, and provided a positive result in the Liebermann-Burchard and
(
Bandorowicz-Pikula, 2017). It has been reported that α-glucosidase
+
inhibitors which could inhibit carbohydrates hydrolysis could be used
as anti-diabetic drugs to treat T2DM (Bischoff, 1995). Protein-tyrosine
phosphatase 1B (PTP1B) is a negative regulator of the insulin signaling
pathway, which is also considered as a promising potential therapeutic
target for T2DM (Combs, 2010). Triterpene saponins especially olea-
nane-type saponins have been demonstrated to possess anti-diabetic
effects (Wang et al., 2011). Recently, some triterpenoids derived from
oleanolic acid exhibit potent PTP1B inhibitory effect in studies for
searching new PTP1B inhibitors (Liu et al., 2013). Furthermore, tri-
terpenoid saponins isolated from Gypsophila paniculata were found to
molecular ion peak [M + Na] at m/z 997.4769 (calcd. for 997.4769)
by HRESIMS. The IR absorption bands were observed at 3424, 1077 and
−1
1036 cm , which were characteristic of glycosidic-type structures. The
1
H NMR spectrum showed characteristic signals of an olean-12-ene
skeleton with six tertiary methyl groups at δ
H
0.94 (3H, s, H-24), 0.96
(3H, s, H-25), 1.15 (3H, s, H-26), 1.77 (3H, s, H-27), 0.96 (3H, s, H-29),
2
1.02 ppm (3H, s, H-30) and a sp proton at δ
Two olefinic carbons at δ 122.7 (C-12) and 144.4 (C-13) indicated a
typical △12 pentacyclic triterpene derivative. The C NMR spectrum
showed signals of six angular methyls at δ 13.7, 16.3, 17.6, 24.6, 27.2,
H
5.60 (1H, br s, H-12).
C
13
C
⁎
Corresponding author.
E-mail address: songsj99@163.com (S.-J. Song).
https://doi.org/10.1016/j.phytol.2018.06.002
Received 14 February 2018; Received in revised form 18 April 2018; Accepted 11 June 2018
1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

Y. Zhang et al.
Phytochemistry Letters 26 (2018) 179–183
Fig. 1. Key HMBC and ROESY correlations of compounds 1-2.
3
3.1 on the basis of the HSQC spectrum. Characteristic chemical shifts
at δ 47.3 (C-5), 74.4 (C-16) and 64.3 (C-23) further revealed that the
nucleus of 1 was hederagenin with 16-OH. The chemical shifts of C-3
units. In the HMBC spectrum, the anomeric proton at δ
H
6.31 (glc-H-1)
175.9 (C-28), con-
C
showed correlation with the carbon signal at δ
C
firming a glucose was linked to C-28 through ether linkage. The gly-
cosylation at C-3 of aglycone was confirmed as a glc (1→3)-glc moiety,
(
δ
C
82.0) and C-16 (δ 74.4) indicated an α-orientation of H-3 and a β-
C
orientation of H-16 (Liang et al., 2011). The orientation of H-3, H-16
and the hydroxymethyl at C-23 were also supported by the ROESY
through HMBC correlations of δ
H
5.06 (glc-H-1') to δ
C
82.0 (C-3), δ
H
5.21 (glc-H-1′′) to δ 88.8 (glc-C-3′). According to the relatively large
C
correlations (Fig. 1). The correlation of δ
1.68 (d, 1H, H-5) and δ 0.94 (s, 3H, H-24) with δ
5), which indicated that a hydroxy group was connected to the C-23
carbon. And the correlation of δ 4.23 (d, 1H, H-3) with δ 1.68 (d, 1H,
H-5), which indicated that H-3 was in an α-orientation. The multiplicity
5.27) as a broad singlet and the lack of ROESY correlation
H
3.67, 4.27 (d, 2H, H-23) with
coupling constant (J = 7.8 Hz) and acid hydrolysis of 1, the config-
uration of glucose was identified as D-glucose in β anomeric orientation
of its pyranose form. Based on the above spectral data, the structure of 1
was identified as 3-O-[β-D-glucopyranosyl (1→3)-β-D-glucopyranosyl]-
caulophyllogenin 28-O-β-D-glucopyranosyl ester.
δ
H
H
H
0.96 (s, 3H, H-
2
H
H
of H-16 (δ
H
3
Compound 2, white amorphous powder from CH OH, the Molish
with protons of Me-27 confirmed that 16-OH group was a-oriented. Due
to the glycosylation shift, the downfield shift of C-3 and upfield shifted
of C-28 suggested that 1 was a bidesmoside saponin. The 1H NMR
and Liebermann-Burchard reactions were positive. After acid hydro-
lysis, compound 2 afforded sugar moieties that were identified as D-
glucose based on gas chromatography (GC) analysis of chiral deriva-
tives. And the coupling constants (7.2 or 7.8 Hz) for the anomeric
protons in the 1H NMR spectrum suggested β-configurations for the
spectrum of 1 showed three anomeric proton signals at δ
J = 7.8 Hz, glc-H-1), 5.06 (d, J = 7.8 Hz, glc-H-1′), 5.21 (d, J = 7.8 Hz,
glc-H-1′′), which correlated in the HSQC spectrum to carbons at δ 95.8,
05.4 and 105.9 respectively, revealing the presence of three sugar
H
6.31 (d,
1
13
C
glucopyranosyl moieties. The H and C (Tables 1 and 2) NMR spec-
1
troscopic data were closely related to those of 1 except for the sugar
180

Y. Zhang et al.
Phytochemistry Letters 26 (2018) 179–183
Table 2
C (150 MHz) and 1H (600 MHz) NMR data for sugar moieties of 1-2 in C
D N
5 5
Table 1
C (150 MHz) and 1H (600 MHz) NMR data for aglycone moieties of 1-2 in
a,b
a,b
C
5
D
5
N
.
.
Position
1
2
Position
1
2
δ
C
δ
H
(multi, J in Hz)
δ
C
δ
H
(multi, J in Hz)
δ
C
δ
H
(multi, J in Hz)
δ
C
H
δ (multi, J in Hz)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
38.8
26.0
82.0
43.5
47.6
18.2
33.1
40.1
47.3
36.9
23.8
122.7
144.4
42.0
36.2
74.4
49.1
41.3
47.1
30.8
35.9
32.2
64.3
0.98 (m), 1.47 (m)
1.91 (m), 2.22 (m)
4.23 (d, 9.6)
–
1.68 (d, 11.4)
1.32 (m), 1.67 (m)
1.38 (m), 1.75 (m)
–
1.86 (m)
–
1.93 (m), 1.99 (m)
5.60 (br s)
–
–
1.71 (m), 2.52 (m)
5.27 (br s)
–
3.51 (d, 7.8)
1.32 (m), 2.73 (m)
–
1.20 (m), 2.38 (m)
2.08 (m), 2.35 (m)
3.67 (d, 10.2)
38.7
25.9
83.0
43.5
48.0
18.2
33.1
40.0
47.2
36.9
23.8
122.6
144.4
42.0
36.1
74.4
49.0
41.2
47.1
30.8
35.9
32.2
64.7
0.98 (m), 1.46 (m)
1.90 (m), 2.20 (m)
4.13 (d, 10.2)
–
1.57 (d, 12.0)
1.37 (m), 1.68 (m)
1.33 (m), 1.69 (m)
–
1.84 (m)
–
1.98 (m)
5.56 (br s)
–
–
1.71 (m), 2.52 (m)
5.25 (br s)
–
3.45 (dd, 3.6, 14.4)
1.33 (m), 2.75 (m)
–
1.23 (m), 2.40 (m)
2.12 (m), 2.38 (m)
3.66 (d, 11.4)
4.27 (d, 9.0)
1.06 (s)
0.89 (s)
1.10 (s)
1.73 (s)
–
0.93 (s)
0.98 (s)
3-O-glc
1'
2'
3'
4'
5'
6'
glc
1”
2'
3”
4”
5”
6”
glc
1”'
2”
3”'
4”
5”'
6”'
28-O-glc
1
105.4
75.5
88.8
69.6
77.9
62.4
5.06 (d, 7.8)
4.02 (m)
4.03 (m)
4.09 (m)
3.78 (m)
103.8
79.4
87.6
69.8
77.7
63.1
4.98 (d, 7.2)
4.01 (m)
4.22 (m)
4.01 (m)
3.81 (m)
4.26 (m), 4.50 (m)
4.30 (m), 4.42 (m)
105.9
74.4
78.2
71.5
78.7
62.3
5.21 (d, 7.8)
4.10 (m)
4.21 (m)
4.14 (m)
4.13 (m)
103.7
76.3
78.6
72.1
77.6
62.1
5.72 (d, 7.8)
4.08 (m)
4.24 (m)
4.17 (m)
3.64 (m)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4.06 (m), 4.12 (m)
4.25 (m), 4.42 (m)
105.2
75.4
78.5
71.4
77.9
62.3
5.08 (d, 7.8)
4.02 (m)
4.95 (m)
4.13 (m)
4.13 (m)
4.21 (m), 4.51 (m)
95.8
74.1
78.9
71.1
79.4
62.2
6.31 (d, 7.8)
4.11 (m)
4.24 (m)
4.29 (m)
3.99 (m)
95.8
74.1
78.8
71.0
79.1
62.3
6.29 (d, 7.8)
4.24 (m)
4.24 (m)
4.30 (m)
4.29 (m)
4
.27 (d, 12.0)
2
3
4
5
2
2
2
2
2
2
3
4
5
6
7
8
9
0
13.7
16.3
17.6
27.2
175.9
33.1
24.6
0.94 (s)
0.96 (s)
1.15 (s)
1.77 (s)
–
13.3
16.2
17.5
27.2
175.9
33.1
24.5
6
4.35 (m), 4.40 (m)
4.25 (m), 4.47 (m)
aThese assignments are based on data from the 1H- H COSY, HSQC, HSQC-
1
0.96 (s)
1.02 (s)
TOCSY, HMBC and ROESY experiments.
b
Overlapped 1H-NMR signals are reported without designated multiplicity.
aThese assignments are based on data from the 1H- H COSY, HSQC, HSQC-
1
TOCSY, HMBC and ROESY experiments.
Table 3
b
Overlapped 1H-NMR signals are reported without designated multiplicity.
α-glucosidase and PTP1B inhibitory assay results of compounds 1-13.
Compound
α-glucosidase inhibitory activity
PTP1B inhibitory activity
(IC50, μM)
moieties at C-3. The signals of sugar moieties were assigned through
HSQC, COSY, TOCSY and HSQC-TOCSY. Long-range couplings were
a
a
(IC50, μM)
observed between a proton signal at δ
signal at δ 83.0 (C-3), between a proton signal at δ
and the carbon signal at δ 79.4 (glc-C-2′), and between a proton signal
at δ 5.21 (glc-H-1‴) and the carbon signal at δ 87.6 (glc-C-3′) in the
HMBC spectrum of 2, which suggests glycosylation at C-3 with a glc
1→2)-[glc (1→3)]-glc moiety. Therefore, the structure of compound 2
was elucidated as 3-O-{[β-D-glucopyranosyl(1→2)]-[β-D-glucopyr-
anosyl(1→3)]-β-D-glucopyranosyl}-caulophyllogenin 28-O-β-D-gluco-
pyranosyl ester.
H
4.98 (glc-H-1′) and the carbon
1
2
3
4
5
6
7
8
9
2.96 ± 0.32
6.82 ± 0.45
6.99 ± 0.22
12.82 ± 0.51
19.37 ± 0.23
6.97 ± 0.44
2.98 ± 0.56
12.54 ± 0.66
2.21 ± 0.43
0.87 ± 0.57
2.52 ± 0.48
3.32 ± 0.57
0.73 ± 0.39
4.59 ± 0.41
20.37 ± 0.46
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
C
H
5.72 (glc-H-1″)
C
H
C
(
1
1
1
0
1
2
> 100
> 100
> 100
The other known compounds (3-13) were identified as glucocau-
lophyllogenin (Farias et al., 2010), acutoside A (Voutquenne et al.,
13
11.22 ± 0.33
b
2
003), 3-O-{[β-D-glucopyranosyl (1→2)]-[β-D-glucopyranosyl (1→3)-
Acarbose
Oleanolic acid
ND
b
ND
9.75 ± 0.66
β-D-glucopyranosyl (1→3)]-β-D-glucopyranosyl} oleanolic acid 28-O-β-
D-glucopyranosyl ester (Zhang et al., 2012), aralia-saponin V (Song
et al., 2001), lucynoside E (Magid et al., 2006), congmuyenoside B
a
IC50 values represent the means ± SD of three parallel measurements.
ND means not determined.
b
(
Kuang et al., 1996), 3-O-[β-D-glucopyranosyl (1→3)-β-D-glucopyr-
anosyl (1→3)]-[β-D-glucopyranosyl (1→2)]-β-D-glucopyranosyl heder-
3
. Experimental
agenin 28-O-β-D-glucopyranoside (Zhang et al., 2012), collinsonidin
(
Joshi et al., 1992), congmuyenoside III (Zhang et al., 2013), aralia-
3
.1. General
saponin IV (Song et al., 2000), 3-O-α-L-arabinopyranosyl echinocystic
acid (Yakovishin et al., 1999), respectively, based on the NMR analyses
and comparison with reported literatures.
Column chromatography was performed on a 200–300 mesh silica
gel (Qingdao Marine Chemical Factory, P. R. China). Column chroma-
tography was performed using YMC ODS-A gel (12 nm S-75 μm, YMC
Co., Ltd., Japan) and D101 Macro porous adsorption resin (Shanghai
Hualing Resin Factory, P. R. China). TLC was performed with precoated
silica gel GF254 plates (Qingdao Marine Chemical Factory, P. R. China).
IR spectrum were acquired on Bruker EQUINOX55 spectrometer
All isolated compounds were evaluated for their inhibitory effects
on α-glucosidase and PTP1B. As shown in Table 3, compounds 1, 7 and
9
-13 displayed potent inhibitory effects on α-glucosidase than acarbose
(
the positive control). In addition, compounds 1 and 13 exhibited
moderate inhibitory activities on PTP1B. Compound 13 displayed
stronger inhibitory effect than others on both two enzymes.
181

Y. Zhang et al.
Phytochemistry Letters 26 (2018) 179–183
(
Bruker Co., Karlsruhe, Germany). NMR spectrum were performed on a
subsequently added to the solution to heat at 60 °C for 2 h. Then the
chloro trimethyl silane (0.3 mL) was added to the mixture and stirred
for another 2 h at 60 °C. After the reactions had been performed, the
supernatant was diluted 20 times and analysed by GC under the fol-
lowing conditions: the column temperature was maintained at 80 °C for
5 min, then increased from 80 to 280 °C (25 °C/min) and maintained for
Bruker ARX-300 or 600 spectrometer using TMS as the internal stan-
dard. HRESIMS were measured with a Bruker Daltonics Inc. micro-TOF-
Q spectrometer. HPLC separations were performed on a Hitachi 655-15
series pumping system equipped with a Hitachi L-2490 refractive index
detector using a YMC-Park ODS-A column (250 × 10 mm I.D, S-5 μm,
1
2 nm). GC separations were performed on an Agilent 7890 A Gas
2
5 min; the carrier gas was N (1.4 ml/min); the split ratio was 1/20; the
Chromatograph equipped with a SPH-300 A FID detector using an
Agilent 19091J-413 capillary column (30 m × 320 μm×0.25 μm).
injection temperature was 250 °C; and the injection volume was 1 μL.
The absolute configurations of the monosaccharides of 1 and 2 were
finally determined to be all D-glucose by comparing the retention times
with hose of the standard samples D-glucose (14.489 min) and L-glu-
cose (14.576 min).
3.2. Plant material
The leaves of A. elata were collected from Liaoning Province, China,
in August 2009 and were identified by Prof. Lu Jincai of Shenyang
Pharmaceutical University. A voucher specimen was deposited at the
School of Traditional Chinese Materia Medica (No. 090811).
3.6. α-glucosidase inhibition assay
The stock solutions of positive control and each test substances were
prepared in DMSO and further diluted with PBS to obtain an experi-
mental concentration. Enzyme solution was prepared using α-glucosi-
dase (0.5 U/mL, Sigma, St. Louis, MO, USA) dissolved in 0.2 M po-
tassium phosphate buffer (pH 6.8). The purified compound (80 μL) at
different concentrations was mixed with enzyme solution (80 μL) then
the mixture was incubated at 37 °C for 10 min. The p-nitrophenyl-α-D-
glucopyranoside (40 μL, 1.5 mM) was added to the mixture as start of
3
.3. Extraction and isolation
The air-dried leaves (8 kg) of A. elata were extracted with 60%
ethanol firstly for three times under reflux. The combined solution were
concentrated under vacuum and subjected to macroporous resin D101
column chromatography, eluted with an EtOH-H O gradient. The so-
2
lution eluted with 60% ethanol was evaporated to dryness under va-
cuum to give a residue (800 g). The residue was chromatographied on
2 3
the reaction. After 30 min incubation at 37.0 °C, 0.1 M Na CO (50 μL)
silica gel with a CH
H). Fr. D was subjected to column chromatography on silica gel and
eluted using an increasing MeOH gradient in CH Cl (20–100%), to
provide twelve fractions (fr. D -D12). Fr. D was subjected to ODS
column chromatography and eluted with a MeOH-H O gradient to yield
1 fractions (fr. D5-1-D5-11). Among them, fr. D5-6 was purified by pre-
2
Cl
2
-MeOH gradient to afford eight fractions (fr. A-
was added to tube to terminate. The absorbance of yellow color pro-
duced due to the releasing of p-nitrophenol from the hydrolysis of p-
nitrophenyl-α-D-glucopyranoside. The sample was evaluated through
detecting the absorbance at 405 nm using a BIO-RAD Model 680 mi-
croplate reader. The non-enzymatic hydrolysis of p-nitrophenyl-α-D-
glucopyranoside was corrected by detecting the addition in absorbance
at 405 nm obtained in the absence of α-glucosidase. Acarbose (Sigma,
St. Louis, MO, USA), a common α-glucosidase inhibitor, was used as a
positive control. The inhibition activity was expressed as percentage
inhibition of enzyme activity and was calculated using the following
equation: [1− (Asample−Ablank) / (Acontrol− Ablank)] × 100%. Asample is
the absorbance of the samples, Acontrol is the absorbance of PBS that
replaces the samples, Ablank is the absorbance of PBS that replaces the
samples and the enzyme. The IC50 values were calculated plotting
graphs with percentage inhibition on the y-axis and log concentrations
on x-axis using graph pad prism v 6.0. The IC50 values are reported as
best fit value after normalization of data (n = 3).
2
2
1
5
2
1
2
parative HPLC using MeOH-H O (v/v = 8:2) to give 3 (10 mg), 4
(
148 mg). Fr. D
with a solvent system MeOH-H
). Fr. D9-10 was further separated with silica gel column chromato-
graphy with a solvent system of CH Cl -MeOH-H O (v/v/v = 7:3:0.5)
to obtain 7 (345 mg), 10 (10 mg) and 13 (20 mg). Fr. D9-12 was purified
using Sephadex column chromatography and eluted with MeOH-H O to
give 8 (21 mg). Fr. E was fractionated using column chromatography on
silica gel and eluted with an increasing MeOH gradient in CH Cl
30–100%), led to nine fractions (fr. E -E ). Fr. E was applied to ODS
column chromatograph using a solvent system MeOH-H O to afford
9
was separated using ODS column chromatography
2
O to afford sixteen fractions (fr. D9-1-D9-
1
6
2
2
2
2
2
2
(
1
9
5
2
eighteen fractions (fr. E5-1-E5-19). Of these fractions, fr. E5-9 was sepa-
rated using silica gel column chromatography with a solvent system of
CH
2 (125 mg). Fr. E
with a solvent system MeOH-H
), the eighth fraction was subjected to silica gel column chromato-
graphy eluting with a solvent system of CH Cl -MeOH-H O (v/v/
v = 6:1:0.1) to yield 5 (60 mg), 6 (32 mg), 9 (180 mg) and 11 (205 mg).
2
Cl
2
-MeOH-H
2
O (v/v/v = 8:2:0.25) to give 1 (24 mg), 2 (15 mg) and
was purified using ODS column chromatography
O to obtain fifteen fractions (fr. E6-1-E6-
3.7. PTP1B inhibition assay
1
6
2
Buffer solution (pH 5.5) was composed of 50 mM citrate, 0.1 M
NaCl, 1 mM EDTA and 1 mM dithiothreitol (DTT). Stock solution (in
assay buffer containing 10% DMSO) of the test compounds (1 mM) and
positive control (100 μM) were prepared. Taking suitable aliquots from
the stock solution, five different concentrations were made by dilutions
(50-3.125 μM). The sample (10 μL), 10 mM p-nitrophenyl phosphate
1
5
2
2
2
3.4. Characteristic data of the compounds 1 and 2
(
pNPP) (50 μL) and PTP1B (human recombinant) (40 μL, 0.1 U/mL,
Compound 1: white amorphous powder, IR (KBr) vmax: 3424, 1077
ProSpec-Tany TechnoGene Ltd., Ness Ziona, Israel) were added to each
well of a 96-well plate (final volume: 100 μL). After incubation at 37 °C
for 30 min, the reaction was terminated using 0.1 M NaOH (20 μL). p-
Nitrophenyl, the product generated by dephosphorylation of pNPP, can
be monitored at 405 nm using a BIO-RAD Model 680 microplate reader.
The non-enzymatic hydrolysis of 10 mM pNPP was corrected by de-
tecting the addition in absorbance at 405 nm obtained in the absence of
PTP1B enzyme. The PTP1B inhibitor oleanolic acid was used as a po-
−
1
+
and 1036 cm ; HRESIMS at m/z 997.4769 [M + Na] (calcd. for
1
13
C
48
H
78
O
20Na, 997.4769); H and C NMR, see Tables 1 and 2.
Compound 2: white amorphous powder, IR (KBr) vmax: 3422, 1078
−
1
+
and 1032 cm ; HRESIMS at m/z 1137.5680 [M+H] (calcd. for
1
13
54 89
C H O25, 1137.5687); H and C NMR, see Tables 1 and 2.
3.5. Acid hydrolysis of 1 and 2
sitive control. The equation used was: [(Ablank−Asample
)
Compounds 1 and 2 were heated with 2 M HCl (10 mL) at 95 °C for
h. The reaction mixture was extracted using CHCl (3 × 5 mL). The
3
residue was dissolved in pyridine (2 mL) after aqueous layer was eva-
porated to dryness. L-cysteine methyl ester hydrochloride (5 mg) was
/Ablank]×100%. The IC50 values were calculated plotting graphs with
percentage inhibition on the y-axis and log concentrations on x-axis
using graph pad prism v 6.0. The IC50 values are reported as best fit
value after normalization of data (n = 3).
4
182

Y. Zhang et al.
Phytochemistry Letters 26 (2018) 179–183
Conflict of interest
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biological evaluation of oleanolic acid derivatives as PTP1B inhibitors. Eur. J. Med.
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The authors declared that there are no conflicts of interest.
Acknowledgments
This work was supported by National Natural Science Foundation of
China [grant number 81602992], and the Project of Innovation Team
Foundation from Liaoning Education Department [grant number
LT2015027].
Magid, A.A., Voutquenne, L., Harakat, D., Pouny, I., Caron, C., Moretti, C., Lavaud, C.,
2
006. Triterpenoid saponins from the fruits of Caryocar villosum. J. Nat. Prod. 69,
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S.B., Kim, Y.H., 2011. Oleanane-type triterpene saponins from the bark of Aralia elata
and their NF-kappa B inhibition and PPAR activation signal pathway. Bioorg. Med.
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Appendix A. Supplementary data
Shikov, A.N., Pozharitskaya, O.N., Makarov, V.G., 2016. Aralia elata var. mandshurica
(Rupr. & Maxim.) J. Wen: an overview of pharmacological studies. Phytomedicine
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2018.06.002.
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Song, S.J., Nakamura, N., Ma, C.M., Hattori, M., Xu, S.X., 2000. Four new saponins from
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