全 文 ：
Inhibition of protein tyrosine phosphatase 1B by triterpenes isolated from
Potentilla discolor Bge
Zhendong Tuo, Na Li, Jialin Li, Shizhou Qi, Banban Li, Le Zhang, Long Cui*
College of Pharmacy, Beihua University, Jilin 132013, China
Abstract: Seven oleanene triterpenes were isolated from the roots of Potentilla discolor Bge and their structures were identified
as 3-oxoolean-12-en-27-oic acid (1), gypsogenic acid (2), 3α-hydroxyolean-12-en-27-oic acid (3), 3β-hydroxyolean-12-en-27-oic
acid (4), aceriphyllic acid A (5), aceriphyllic acid A methyl ester (6), and oleanolic acid (7). Compounds 1–7 inhibited protein
tyrosine phosphatase 1B (PTP1B) activity, with IC50 values ranging from (7.5±0.5) to (22.7±0.5) μmol/L. Among the isolates,
compounds 1, 2, 3 and 7 from the Potentilla discolor Bge were found to exhibit selective PTP1B inhibitory activity.
Keywords: Potentilla discolor Bge, Protein tyrosine phosphatase 1B, Triterpenes
CLC number: R284 Document code: A Article ID: 1003–1057(2016)3–224–04
Received: 2015-10-25, Revised: 2015-11-28, Accepted: 2015-12-09.
Foundation item: Science and Technology Development Program of
Jilin Province (Grant No. 20150101225JC).
*Corresponding author. Tel.: +86-13604471092, +86-18604498621,
E-mail: cuilong71@163.com
http://dx.doi.org/10.5246/jcps.2016.03.027
1. Introduction
Protein tyrosine phosphatase 1B (PTP1B) is an
important factor in non-insulin-dependent diabetes
mellitus (type-2 diabetes). As a negative regulator of the
insulin-signaling pathway[1–3], PTP1B is a critical non-
transmembrane phosphotyrosine phosphatase in human
cells[4,5]. It has been suggested that inhibition of PTP1B
is an effective therapeutic approach and an attractive
target for the treatment of type-2 diabetes[6–8].
Potentilla discolor Bge (Rosaceae) is a perennial herb
that grows on damp rocks along valleys and is distributed
in the mid-northern area of China. It has been used in
folk medicine for the treatment of dysentery, diarrhea,
leucorrhea, malaria, and hematochezia[9]. Recently,
triterpene glycosides[10] isolated from Potentilla discolor
Bge have found to exhibit many pharmacological
properties including anti-hyperglycemic[11], anti-tumor[12],
and anti-oxidant[13] activity. In this study, we report
the isolation, structure elucidation, and evaluation of
PTP1B inhibitory activity of seven triterpenes which
were obtained from Potentilla discolor Bge.




2. Experimental
2.1. Materials and instruments
Whole samples of Potentilla discolor Bge were
purchased from the herbal medicine association of
Taejon, Korea and were identified by Prof. Ki-hwan Bae,
College of Pharmacy, Chungnam National University.
The voucher specimen was deposited at the College
of Pharmacy, Beihua University, Jilin, China (No.
AR20131023).
NMR spectra were recorded with a Bruker-Avance II
500 (1H NMR 500 MHz, 13C NMR 125 MHz), using
tetramethysilane as an internal standard. All accurate
mass experiments were performed on a Micromass
QTOF2 Mass Spectrometer (Micromass, Wythenshawe,
UK). PTP1B and protein serine/threonine phosphatase 1
(PP1) were purchased from BIOMOL® International LP
(USA). Positive phase silica gel was purchased from
National Medicine Group Chemical Reagent Co.
2.2. Extraction and isolation
The roots of Potentilla discolor Bge (5.0 kg) were
extracted with MeOH at room temperature for two
weeks and the MeOH solution was concentrated to
obtain a crude extract. This extract was suspended
in H2O, partitioned successively with n-hexane, EtOAc,
Short communication
224 Journal of Chinese Pharmaceutical Sciences http://www.jcps.ac.cn

225 Tuo, Z.D. et al. / J. Chin. Pharm. Sci. 2016, 25 (3), 224–227
and BuOH, to afford n-hexane, EtOAc, and BuOH
soluble fractions, respectively. The n-hexane-soluble
fraction (280.0 g, IC50 = 21.3 μg/mL) was subjected to
silica gel column chromatography (50 cm×15 cm) and
eluted with a gradient of n-hexane–EtOAc (20:1 to 0:1,
v/v) to afford 12 fractions (A1–A12). Fraction A10
(3.7 g) was eluted on a silica gel column (40 cm×4.5 cm)
with CHCl3–acetone (8:1, v/v) to afford compound 2
(17.5 mg) and 7 (30.1 mg). Fraction A11 (2.3 g) was
eluted on a silica gel column using CHCl3–MeOH
(40:1 to 5:1, v/v) to give 9 sub-fractions (A11.1–A11.9).
A11.4 (221.0 mg) was further purified by preparative
HPLC (2 mL/min) with a gradient from 60% to 80%
ACN in H2O over 50 min to yield compound 1 (25.5 mg,
tR = 43.1 min). A11.7 (184.3 mg) was further purified
by preparative HPLC (2 mL/min) using a gradient
from 75% to 90% MeOH in H2O over 50 min to get
compounds 5 (7.9 mg, tR = 34.8 min) and 6 (11.7 mg,
tR = 39.5 min). Fraction A12 (1.98 g) was repeatedly
chromatographed on a silica gel, eluting with CHCl3–
MeOH (10:1 to 0:1, v/v) to obtain five subfractions
(A12.1–12.5). Finally, A12.4 (442 mg) was purified by
semi-preparative HPLC (2 mL/min) using a gradient
from 80% to 95% ACN in H2O over 50 min to obtain
compounds 3 (24.0 mg, tR = 11.9 min) and 4 (33.9 mg,
tR = 13.1 min).
2.3. PTP1B and PP1 inhibition assay
The enzyme activity was measured using p-nitrophenyl
phosphate (pNPP) as described previously[6]. To each
well on a 96-well plate (final volume: 100 μL) was
added 2 mmol/L pNPP and PTP1B (0.05–0.1 μg) in a
buffer containing 50 mmol/L citrate (pH 6.0), 0.1 mol/L
NaCl, 1 mmol/L EDTA, and 1 mmol/L dithiothreitol
(DTT), with or without test compounds. Following
incubation at 37 ºC for 30 min, the reaction was
terminated with 1 N NaOH. The amount of produced
p-nitrophenol was estimated by measuring the absorbance
at 405 nm. The non-enzymatic hydrolysis of 2 mmol/L
pNPP was analyzed by measuring the increase in
absorbance at 405 nm without PTP1B enzyme. For the
PP1 assay, PP1 was added in the assay buffer (50 mmol/L
Tris-HCl, 0.1% β-mercaptoethanol, 1 mmol/L EDTA,
1 mmol/L MnCl2, and 20 mmol/L MgCl2, pH 7.6) and
incubated for 30 min. The reaction was stopped by
adding 1 N NaOH, and amount of p-nitrophenol was
determined by measuring the absorbance at 405 nm.
3. Results
Compound 1: C30H46O3; white amorphous powder;
m.p. 209–210 ºC; +173.1 (c 1.2, CHCl3); ESI-MS
m/z: 453 [M-H]+. 1H NMR (500 MHz, CDCl3) δ: 0.82
(3H, s, H-25), 0.85 (3H, s, H-26), 0.87 (3H, s, H-29), 1.03
(3H, s, H-27), 1.05 (3H, s, H-24), 1.06 (3H, s, H-23), 1.07
(3H, s, H-28), 5.73 (1H, br s, H-12). 13C NMR (125 MHz,
CDCl3) δ: 39.3 (C-1), 39.8 (C-2), 218.3 (C-3), 47.4
(C-4), 54.9 (C-5), 19.7 (C-6), 34.3 (C-7), 39.7 (C-8),
49.5 (C-9), 35.8 (C-10), 22.6 (C-11), 126.4 (C-12),
138.1 (C-13), 56.1 (C-14), 22.6 (C-15), 30.0 (C-16),
32.8 (C-17), 46.5 (C-18), 43.6 (C-19), 31.3 (C-20),
35.5 (C-21), 36.8 (C-22), 27.2 (C-23), 22.0 (C-24),
15.8 (C-25), 18.2 (C-26), 181.1 (C-27), 28.5 (C-28),
33.7 (C-29), 23.9 (C-30). Therefore, according to the
literature[14], compound 1 could unambiguously be
determined to be 3-oxoolean-12-en-27-oic acid.
Compound 2: C30H46O5; white amorphous powder;
+52.6 (c 0.46, MeOH); ESI-MS m/z: 485 [M-H]+.
1H NMR (500 MHz, pyridine-d5) δ: 0.96 (3H, s, H-25),
1.02 (3H, s, H-26), 1.20 (3H, s, H-29), 1.30 (3H, s,
H-27), 1.64 (3H, s, H-24), 2.04 (1H, ddd, J1 = 6.1 Hz,
J2 = 6.1 Hz, J3 = 11.6 Hz, H-5), 3.44 (1H, dd, J1 = 6.4 Hz,
J2 = 11.9 Hz, H-18), 3.92 (1H, d, J = 10.4 Hz, H-30β), 3.99
(1H, d, J = 10.4 Hz, H-30α), 4.67 (1H, dd, J1 = 7.9 Hz,
J2 = 7.9 Hz, H-3), 5.51 (1H, br s, H-12).
13C NMR (125 MHz,
pyridine-d5) δ: 39.0 (C-1), 27.7 (C-2), 75.5 (C-3), 54.4
(C-4), 51.9 (C-5), 21.7 (C-6), 33.0 (C-7), 40.1 (C-8),
48.4 (C-9), 36.7 (C-10), 23.9 (C-11), 122.4 (C-12),
144.9 (C-13), 42.2 (C-14), 28.3 (C-15), 23.7 (C-16),
46.6 (C-17), 42.0 (C-18), 46.5 (C-19), 31.0 (C-20),
34.2 (C-21), 33.2 (C-22), 180.3 (C-23), 12.3 (C-24),
16.0 (C-25), 17.3 (C-26), 26.2 (C-27), 178.9 (C-28),
33.2 (C-29), 23.8 (C-30). Therefore, according to
the literature[15], compound 2 could unambiguously be
determined to be gypsogenic acid.
25
D[α]
25
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Tuo, Z.D. et al. / J. Chin. Pharm. Sci. 2016, 25 (3), 224–227 226
Compound 3: C30H48O3; white amorphous powder;
m.p. 211–212 ºC; +114.0 (c 1.0, CHCl3); ESI-MS
m/z: 455 [M-H]+. 1H NMR (500 MHz, CDCl3) δ: 0.82
(3H, s, H-25), 0.84 (3H, s, H-26), 0.85 (3H, s, H-29),
0.86 (3H, s, H-27), 0.92 (3H, s, H-24), 0.95 (3H, s,
H-23), 1.02 (3H, s, H-28), 3.27 (1H, br s, J = 7.0 Hz,
H-18), 5.61 (1H, br s, H-12). 13C NMR (125 MHz,
CDCl3) δ: 36.4 (C-1), 27.2 (C-2), 76.1 (C-3), 39.6
(C-4), 48.7 (C-5), 17.9 (C-6), 33.2 (C-7), 36.5 (C-8),
49.0 (C-9), 36.5 (C-10), 23.2 (C-11), 125.6 (C-12),
137.2 (C-13), 55.4 (C-14), 22.5 (C-15), 25.8 (C-16),
32.6 (C-17), 46.7 (C-18), 43.9 (C-19), 30.8 (C-20),
34.0 (C-21), 35.9 (C-22), 27.8 (C-23), 22.5 (C-24),
16.0 (C-25), 17.7 (C-26), 178.7 (C-27), 27.9 (C-28),
32.5 (C-29), 23.3 (C-30). Therefore, according to the
literature[14], compound 3 could unambiguously be
determined to be 3α-hydroxyolean-12-en-27-oic acid.
Compound 4: C30H48O3; white amorphous powder;
m.p. 222–224 ºC; +119.6 (c 1.1, CHCl3); ESI-MS
m/z: 455 [M-H]+. The 1H NMR and 13C NMR data were
similar to compound 3 except for the configuration
of 3-OH. According to the literature[16], compound 4
was identified as 3β-hydroxyolean-12-en-27-oic acid.
Compound 5: C30H48O4; white powder; m.p. 249–250 ºC;
+105.0 (c 1.09, CHCl3); ESI-MS m/z: 473 [M-H]
+.
1H NMR (500 MHz, CDCl3) δ: 0.50 (3H, s, H-24),
0.83 (3H × 2, s, H-28 and H-30), 0.86 (3H, s, H-29),
0.96 (3H, s, H-25), 0.98 (3H, s, H-26), 2.82 (1H, d,
J = 10.7 Hz, H-23β), 3.32 (1H, d, J = 10.7 Hz, H-23α),
3.47 (1H, br s, H-3), 5.57 (1H, br s, H-12). 13C NMR
(125 MHz, CDCl3) δ: 36.5 (C-1), 25.4 (C-2), 77.8 (C-3),
39.8 (C-4), 47.6 (C-5), 17.4 (C-6), 32.7 (C-7), 35.9 (C-8),
49.5 (C-9), 38.6 (C-10), 23.0 (C-11), 123.1 (C-12),
140.5 (C-13), 58.1 (C-14), 22.9 (C-15), 28.1 (C-16),
31.1 (C-17), 41.5 (C-18), 42.1 (C-19), 33.7 (C-20),
34.9 (C-21), 36.7 (C-22), 68.8 (C-23), 17.4 (C-24),
16.3 (C-25), 19.5 (C-26), 184.2 (C-27), 28.3 (C-28),
33.4 (C-29), 23.4 (C-30). Therefore, according to the
literature[17], compound 5 could unambiguously be
determined to be aceriphyllic acid A.
Compound 6: C31H50O4; white powder; m.p. 244–245 ºC;
+101 (c 1.22, CHCl3); ESI-MS m/z: 485 [M-H]
+.
The 1H NMR and 13C NMR data were similar to compound
5 except for the absence of a methyl group. According
to the literature[17], compound 6 was identified as
aceriphyllic acid A amethyl ester.
Bioactivity-guided fractionation of the n-hexane-soluble
extract (IC50 = 21.3 μg/mL) from the roots of Potentilla
discolor Bge using an in vitro PTP1B inhibitory assay,
yielded seven oleanene triterpenes: 3-oxoolean-12-en-
27-oic acid (1), gypsogenic acid (2), 3α-hydroxyolean-
12-en-27-oic acid (3), 3β-hydroxyolean-12-en-27-oic acid
(4), aceriphyllic acid A (5), aceriphyllic acid A methyl
ester (6) and oleanolic acid (7). The structures of the
isolates (Fig. 1) were determined by the NMR analysis.












4. Discussion
Seven oleanene triterpenes were isolated from the
roots of Potentilla discolor Bge. All the isolates were
assayed for their inhibitory activity against PTP1B
(Table 1). To determine the activity of these compounds,
inhibition of PP1 was evaluated. A known inhibitor,
RK-682, was used as the positive control. Compounds
1–7 inhibited PTP1B activity in a dose dependent
manner with IC50 values ranging from (7.5±0.5) μmol/L to
(22.7±0.5) μmol/L. Among these compounds, compound 1
showed the weakest inhibitory activity, which indicated
that carbonyl group at C-3 in this skeleton might
decrease PTP1B inhibitory activity. Comparison between
compounds 3 and 4, which only differ in hydroxyl
configuration at C-3, suggested that the α-configuration
of hydroxyl group at C-3 might be important for the
inhibition of PTP1B. Compounds 5 and 6 exhibited PP1
inhibitory activity, which suggested that the hydroxyl
group at C-23 might also affect PP1 activity. Among
25
D[α]
25
D[α]
25
D[α]
25
D[α]
Figure 1. Chemical structures of compounds 1–7.
COOH
O
COOH
HO
R1
R3
R1
R2
1 2 R1 = COOH
7 R1 = CH3
3 R1 = R2 = CH3 R3 = COOH
4 R1 = R2 = CH3 R3 = COOH
5 R1 = R2 = CH2OH R3 = COOH
6 R1 = R2 = CH2OH R3 = COOCH3
OH
OH
OH
OH












the isolates, compounds 1, 2, 3 and 7 were found to
exhibit selective PTP1B inhibitory activity.
In this study, we demonstrated that oleanane triterpenoids
from Potentilla discolor Bge possess better PTP1B
inhibition activity. Therefore, further investigation and
optimization of these derivatives might enable the
discovery of new PTP1B inhibitors that are potentially
useful in the treatment of type-2 diabetes.
Acknowledgements
This study was supported by Science and Technology
Development Program of Jilin Province (Grant No.
20150101225JC).
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227 Tuo, Z.D. et al. / J. Chin. Pharm. Sci. 2016, 25 (3), 224–227
Compounds
IC50 (μmol/L)
a
PTP1B PP1
3-Oxoolean-12-en-27-oic acid (1) 22.7±0.5 >100
Gypsogenic acid (2) 15.7±0.5 >100
3α-Hydroxyolean-12-en-27-oic acid (3) 10.1±0.8 >100
3β-Hydroxyolean-12-en-27-oic acid (4) 11.5±0.4 12.6±0.6
Aceriphyllic acid A (5) 16.3±0.4 24.5±0.5
Aceriphyllic acid A methyl ester (6) 7.5±0.5 11.6±0.5
Oleanolic acid (7) 11.8±0.3 >100
RK682b 9.8±0.3 NTc
Table 1. Inhibitory activity of compounds 1−7 against PTP1B and PP1
a IC50 values were determined by regression analyses and represented as
mean±SD of three replicates; b Positive control; c Not tested.
翻白草三萜类成分及其PTP1B抑制活性研究
脱振东, 李娜, 李佳琳, 齐世洲, 李班班, 张乐, 崔龙*
北华大学 药学院, 吉林省 吉林市 132013
摘要: 对翻白草的化学成分进行研究, 从中分离得到7个三萜类化合物, 分别鉴定为3-oxoolean-12-en-27-oic acid (1),
gypsogenic acid (2), 3α-hydroxyolean-12-en-27-oic acid (3), 3β-hydroxyolean-12-en-27-oic acid (4), aceriphyllic acid A (5),
aceriphyllic acid A methyl ester (6)和oleanolic acid (7)。对化合物1–7进行了抑制PTP1B活性的实验, 其IC50范围为(7.5±0.5)
至(22.7±0.5) μmol/L。其中, 化合物1, 2, 3和7表现出对PTP1B的选择抑制性。
关键词: 翻白草, 蛋白酪氨酸磷酸酯酶1B, 三萜类化合物