Fitoterapia 105 (2015) 234239

Contents lists available at ScienceDirect

Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Depside derivatives with anti-hepatic brosis and anti-diabetic activities

from Impatiens balsamina L. owers
Qian Li a, Xiaoshu Zhang a, Jiaqing Cao a, Zhenghong Guo a, Yuntian Lou a, Meng Ding a, Yuqing Zhao a,b,
a
b

Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, PR China
Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China

a r t i c l e

i n f o

Article history:
Received 4 June 2015
Received in revised form 6 July 2015
Accepted 7 July 2015
Available online 13 July 2015
Keywords:
Impatiens balsamina L
Anti-hepatic brosis
Anti-diabetic activity

a b s t r a c t
Eighteen compounds (118), seven new (39) and eleven previously reported (1, 2, and 1018), were isolated
from the owers of Impatiens balsamina (Linn). The structures of the isolated compounds were elucidated using
different spectroscopic methods, including NMR (1D and 2D), UV, IR, and HRESIMS. Analysis of the bioassay
results showed the compounds had notable anti-hepatic brosis activity against murine Hepatic Stellate Cells
(t-HSC/Cl-6) and anti-diabetics activity against -glucosidase. Specically, new compounds 7, 8, 9 showed significant inhibitory activity on t-HSC/Cl-6 cells with IC50 values of 42.12, 109.2, and 34.04 g/mL respectively, while
the IC50 values of positive control Silymarin and Fufang Biejia Ruangan Pian were 202.34 and 231.56 g/mL,
respectively. In addition, compounds 2, 4, 7, 8, 10, 11, 17, and 18 exhibited excellent -glucosidase inhibitory
activity. Among these compounds, 7 exhibited the highest activity with an IC50 value of 0.72 g/mL, while the
IC50 value of the positive control acarbose was 3.36 g/mL. This is the rst study evaluating the anti-hepatic brosis and anti-diabetic activities of compounds isolated from the owers of I. balsamina.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Impatiens balsamina Linn. (Balsaminaceae) is an annual herb grown
as an ornamental garden plant. It is commonly known as garden balsam
and in Bangladesh, it is called Dopati. In Chinese medicinal practices,
the aerial parts of I. balsamina are used to treat articular rheumatism,
beriberi, bruises, pain, and swelling [1]. In particular, the owers are
used to treat lumbago, neuralgia, burns, and scalds [2]. Chemically,
this plant has been characterized by the presence of naphthoquinones,
coumarins, phenolic acids, avonoids, anthocyanidins, steroids, and
peptides [35]. Recent studies have revealed that the plant extracts
exhibit anticancer, antimicrobial, antinociceptive, and antidermatitic
activities [69]. Previously reported studies on the ower have predominantly focused on evaluating the bioactivity of the crude extract, the biological potentials of individually isolated pure chemical components
have not been systematically evaluated so far.
In the present work, eighteen compounds 118 (seven new and
eleven known; see Fig. 1) have been isolated from the owers of
I. balsamina through repeated column chromatography (silica gel,
sephadex LH-20, ODS and polyamide) and semi-preparative HPLC. The
isolation and characterization of the new compounds are described in

Corresponding author at: Department of Traditional Chinese Materia Medica,

Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenhe District, Shenyang
110016, Liaoning, PR China.
E-mail address: yqz2016@163.com (Y. Zhao).

http://dx.doi.org/10.1016/j.tote.2015.07.007
0367-326X/ 2015 Elsevier B.V. All rights reserved.

this paper. In addition, all the obtained compounds have been assessed
for their activity against t-HSC/Cl-6 cells and -glucosidase.
2. Experimental
2.1. General experimental procedures
Column chromatography (cc): silica gel (SiO2: 300400 mesh,
Qingdao Marine Chemical Group, Co.); ODS (300400 mesh, Agela Technologies Co.); Macroporous resin D 101 (Tianjin Chemical Co.); Polyamide (Sinopharm) and Sephadex LH-20 (Pharmacia, Co.). IR spectra on a
Bruker IFS-55 infrared spectrophotometer were recorded in MeOH
and KBr disks respectively. NMR spectra were recorded with a Bruker
ARX-600 (1H, 600 MHz; 13C, 150 MHz) spectrometer in DMSO with
tetramethylsilane as internal standard. High-resolution electrospray ionization mass spectra (HRESIMS) were recorded on an Agilent 1100 LC
MSD TOF (time-of-ight) system [ionization mode, positive; nebulizing
gas (N2) pressure, 35 psi; drying gas (N2) ow, 12 L/min, temp, 325 C;
capillary voltage, 3000 V; fragmentor voltage, 225 V]. The analytical
grade solvents were used for extraction and the mobile phase, include
ethanol, petroleum ether, dichloromethane, ethyl acetate and n-butanol
(Tianjing DaMao Chemical Reagents Co. Tianjing, China). HPLC grade
methanol was from Concord Chemical Reagents Co. (Tianjin, China).
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and DMSO were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). The cell culture reagents were from Gibco/Invitrogen
(Grand Island, NY, USA). pNPG (p-nitrophenyl--D-glucopyranoside)

Q. Li et al. / Fitoterapia 105 (2015) 234239

235

Fig. 1. Structures of compounds 118.

and -glucosidase (Type 1, from baker yeast) were purchased from

Sigma-Aldrich Co. (St. Louis, MO, USA) All other chemical agents were
analytical grade.
2.2. Plant material
The owers of I. balsamina L. were purchased from Nanjing Zelang
Phar. CO. Ltd. (Nanjing, China) in November 2010. The voucher specimen of this herb (No. 20101002) was identied by Prof. Jincai Lu of
Shenyang Pharmaceutical University.
2.3. Extraction and isolation
The owers of I. balsamina L. (10.0 kg) were extracted with 75%
ethanol (3 120 L, 2 h each) at 78 C. The extract was concentrated in

vacuum and suspended in water (8 L), then partitioned with petroleum

ether, dichloromethane, ethyl acetate and n-butanol and 100 g, 320 g,
120 g, 300 g of soluble fractions were obtained after concentration of
the solvents under vacuum.
The ethyl acetate fraction (120 g) was subjected to a silica gel column chromatography (CC), using a gradient of petroleum etherethyl
acetate (PE-EtOAc) as eluant to provide twelve fractions (Fr. 1Fr. 12)
based on TLC analysis. Fr. 8 was repeatedly chromatographed on a silica
gel (200 to 300 mesh) column using CH2Cl2MeOH then subjected to
Sephadex LH-20 CC with CH2Cl2MeOH (1:1, V/V) as the eluent to
obtain subfraction Fr. 82 and compound 17. Fr. 82 was subjected to
a silica gel CC, using a gradient of CH2Cl2MeOHH2O (7:4:1, V/V/V)
to give compounds 1 and 18. Fr. 6 was repeatedly chromatographed
on a silica gel column using PE-acetone and subjected to Sephadex
LH-20 CC with MeOH as the eluent to yield compounds 10 and 11.

236

Q. Li et al. / Fitoterapia 105 (2015) 234239

Compound 6: white powder, UV (CH3OH) max log () 263 (2.42);

IR, v max (cm 1 ) 3419, 1713, 1607, 1514, 1263, 1073, 1465, 763;
1
H-NMR and 13 C-NMR data, see Table 1; and HRESIMS at m/z
545.1604 [M + Na]+ (calcd. for C25H30O12Na 545.1629).
Compound 7: white powder, UV (CH3OH) max log () 263 (2.42);
IR, v max (cm 1 ) 3419, 1713, 1607, 1514, 1263, 1073, 1465, 763;
1
H-NMR and 13 C-NMR data, see Table 2; and HRESIMS at m/z
383.1155 [M + Na]+ (calcd. for C19H20O7Na 383.1101).
Compound 8: yellow powder, UV (CH3OH) max log () 269 (2.43);
IR, v max (cm 1 ) 3386, 2255, 1712, 1604, 1514, 1262, 1078, 1003;
1
H-NMR and 13 C-NMR data, see Table 3; and HRESIMS at m/z
797.1912 [M + Na]+ (calcd. for C36H38O19Na 797.1905).
Compound 9: orange yellow powder, UV (CH3OH) max log () 263
(2.42); IR, vmax (cm1) 3419, 1713, 1607, 1514, 1263, 1073; 1H-NMR
and 13C-NMR data, see Table 2; and HRESIMS at m/z 369.0787
[M + Na]+ (calcd. for C14H18O10Na 369.0792).

The n-butanol fraction (300 g) was applied to a macroporous resin D

101 column eluted with EtOHH2O in gradient. The 70% EtOHH2O eluate (300 g) was then subjected to polyamide CC with MeOHH2O as the
eluent to obtain ve fractions (AE) based on TLC analysis. Fraction A
was subjected to a silica gel CC, using a gradient of CH2Cl2MeOH as eluant to provide nine fractions (A1A9). A6 was subjected to Sephadex
LH-20 CC with MeOH as the eluent to afford compound 12 and fraction
A63, which was further puried by preparative HPLC with MeOHH2O
(37:63, V/V) to yield compounds 4 and 5. A7 was further separated by
preparative HPLC with MeOHH2O (35:65, V/V) to get compounds 3
and 13. A8 was subjected to ODS CC eluted with MeOHH2O to afford
compound 14. Fraction B was subjected to a silica gel CC, using a gradient of CH2Cl2MeOH as eluent to provide eight fractions (B1B8).
Fraction B6 was chromatographed on Sephadex LH-20 CC with MeOH
as the eluent and subjected to ODS CC eluted with MeOHH2O to obtain
compounds 2, 6 and 7. B7 was subjected to a silica gel CC, using a gradient of CH2Cl2MeOHH2O (7:3:1, V/V/V) and puried by preparative
HPLC with MeOHH2O (45:55, V/V) to yield compound 15. Fraction C
was subjected to a silica gel CC with a gradient of CH2Cl2MeOH as eluent to provide nine fractions (C1C9). C9 was subjected to a silica gel CC,
using a gradient of CH2Cl2MeOHEtOAcH2O (2:2:4:1, V/V/V/V) and
was also puried by preparative HPLC with MeOHH2O (50:50, V/V)
to yield compounds 8, 9 and 16.
Compound 3: white needle crystal, UV (CH3OH) max log () 263
(2.42); IR, vmax (cm 1) 3419, 1713, 1607, 1514, 1263, 1073, 1465,
763; 1H-NMR and 13C-NMR data, see Table 1; and HRESIMS
489.1017 [M + Na]+ (calcd. for C21H22O12Na 489.1003).
Compound 4: white needle crystal, UV (CH3OH) max log () 263
(2.42); IR, vmax (cm 1) 3419, 1713, 1607, 1514, 1263, 1073, 1465,
763; 1H-NMR and 13C-NMR data, see Table 1; and HRESIMS at m/z
503.1170 [M + Na]+ (calcd. for C22H24O12Na 503.1160).
Compound 5: white needle crystal, UV (CH3OH) max log () 263
(2.42); IR, vmax (cm 1) 3419, 1713, 1607, 1514, 1263, 1073, 1465,
763; 1H-NMR and 13C-NMR data, see Table 1; and HRESIMS at m/z
517.1339 [M + Na]+ (calcd. for C23H26O12Na 517.1316).

2.4. Acid hydrolysis and HPLC analysis

The absolute conguration of the sugar units were determined by
the method of Tanaka et al. [10]. Compounds 39 afforded D-glucose
(tR = 11.45 min).
2.5. Cell viability assay
The cell viability was assessed with the MTT assay [12]. The murine
Hepatic Stellate Cells (t-HSC/Cl-6) were plated in 96-well plates
(1 104 cells/well) for overnight, then cells were treated by compounds
at various concentrations for another 48 h. After removing all the
left liquid in the wells, 100 L of fresh medium with MTT reagent
(5 g/mL) was added to each well for 3 h of incubation at 37 C. After
the incubation period, the formazan crystal was dissolved with DMSO,
and the reduction of cell viability was determined at 490 nm using a
microplate reader. For this experiment, 18 compounds were dissolved
in DMSO as a mother solution with 0.1 M concentration.

Table 1
1
H [ in ppm, multiplicity (J in Hz)] and 13C NMR ( in ppm) spectroscopic data of compounds 36.
Position

Compound 3a
H

1
2
3
4
5
6
7

6.35 (1H, d, J = 1.88)

6.54 (1H, d, J = 1.88)
3.58 (1H, s)
3.39 (1H, s)

8
1
2, 6
3, 5
4
7
1
2
3
4
5
6

7.94 (2H, d, J = 8.68)

6.92 (2H, d, J = 8.68)

4.75 (1H, d, J = 7.04)

3.27 (1H, m)
3.23 (1H, m)
3.22 (1H, m)
3.32 (1H, m)
3.73 (1H, d, J = 10.96)
3.55 (1H, s)

Compound 4a
C
108.72
150.57
103.84
157.24
100.92
157.13
29.26
172.83
119.53
132.42
115.86
162.93
163.87
101.88
73.56
77.25
69.82
76.84
60.92

OCH3-8
OCH2-8
CH2
CH2
CH3
a

400 MHz for 1H NMR and 150 MHz for 13C NMR.

6.32 (1H, d, J = 2.2)

6.52 (1H, d, J = 2.2)
3.63 (1H, s)
3.45 (1H, s)

7.99 (2H, d, J = 8.76)

6.90 (2H, d, J = 8.68)

4.72 (1H, d, J = 7.5)

3.22 (1H, m)
3.20 (1H, m)
3.19 (1H, m)
3.27 (1H, m)
3.70 (1H, d, J = 8.00)
3.51 (1H, d, J = 8.00)
3.47 (3H, s)

Compound 5a
C
107.80
150.30
103.68
157.27
100.82
156.87
28.69
171.35
119.09
132.13
115.63
162.81
163.50
101.67
73.30
77.09
69.57
76.44
60.65

6.32 (1H, d, J = 2.2)

6.52 (1H, d, J = 2.2)
3.63 (1H, s)
3.47 (1H, s)

7.89 (2H, d, J = 8.80)

6.88 (2H, d, J = 8.68)

4.72 (1H, d, J = 7.28)

3.27 (1H, m)
3.24 (1H, m)
3.21 (1H, m)
3.30 (1H, m)
3.70 (1H, d, J = 10.76)
3.53 (1H, m)

Compound 6a
C
107.83
150.31
103.69
157.21
100.78
156.84
28.82
170.91
118.94
132.12
115.62
163.01
163.44
101.66
73.30
77.08
69.54
76.39
60.62

6.33 (1H, d, J = 2.2)

6.52 (1H, d, J = 2.2)
3.65 (1H, s)
3.45 (1H, s)

7.90 (2H, d, J = 8.72)

6.90 (2H, d, J = 8.72)

4.72 (1H, d, J = 7.20)

3.23 (1H, m)
3.24 (1H, m)
3.19 (1H, m)
3.29 (1H, m)
3.71 (1H, d, J = 10.96)
3.52 (1H, m)

C
107.88
150.28
103.61
157.14
100.83
156.83
28.72
170.92
119.19
132.09
115.53
162.70
163.42
101.64
73.30
77.07
69.59
76.39
60.64

51.44
3.91 (2H, qui, J = 7.08)

60.04

0.99 (3H, t, J = 7.08)

13.89

3.85 (2H, tri)

1.34 (2H, qui)
1.15 (2H, sex)
0.77 (3H, tri)

63.87
30.05
18.46
13.46

Q. Li et al. / Fitoterapia 105 (2015) 234239

Table 2
1
H [ in ppm, multiplicity (J in Hz)] and 13C NMR ( in ppm) spectroscopic data of compounds 7 and 9.
Compound 7a
C

Position H

1
2
3
4
5
6
7

105.22
150.72
100.85
156.93
99.85
156.79
28.79

1
2
3
4
5
6
7

8
1
2, 6
3, 5
4
7
OCH2-8

170.91
119.34
7.89 (1H, d, J = 8.80) 132.06
6.89 (1H, d, J = 8.80) 115.54
162.71
163.55
3.87 (2H, tri)
63.68

8
1
2
3
4
5
6

CH2
CH2
CH3

1.36 (2H, qui)

1.17 (2H, sex)
0.78 (3H, tri)

according to the formula: [[1 (ODtest ODblank)] / (control ODtest

control ODblank)] 100%.

Compound 9b

Position H

237

6.08 (1H, d, J = 2.2)

6.24 (1H, d, J = 2.2)
3.35 (2H, s)

107.08
156.14
5.84 (1H, s)
99.30
159.65
5.97 (1H, s)
95.87
156.21
3.48 (1H, d, J = 7.08)
34.61
3.37 (1H, s)
178.92
4.54 (1H, d, J = 7.26) 102.60
3.34 (1H, m)
73.43
3.18 (1H, m)
76.31
3.11 (1H, m)
69.59
3.15 (1H, m)
77.25
3.63 (1H, m) 3.47
60.85
(1H, m)

30.09
18.50
13.52

400 MHz for 1H NMR and 150 MHz for 13C NMR.
600 MHz for 1H NMR and 150 MHz for 13C NMR.

2.6. -Glucosidase inhibitory assay

The -glucosidase inhibitory assay was performed according to
a previously described method, with slight modication, in which
acarbose was used as positive control [11]. Thirty microliter of the
-glucosidase solution (2 units mL1, 0.1 mol/L potassium phosphate
buffer, pH 6.8) was pre-mixed with 20 L of sample solution at different
concentrations incubated at 37.5 C for 5 min, after that, 150 L of pnitrophenyl glucopyranoside (pNPG, 10 mmol/L) as substrate in
800 L of 0.1 mol L 1 potassium phosphate buffer (pH 6.8) was
added to the mixture to start the reaction, and the reaction mixture
was incubated at 37.5 C for another 30 min, followed by addition of
2 mL of 1 mol/L Na2CO3 solution to terminate the reaction. The amount
of released product (p-nitro phenol) was measured at 405 nm used by
microplate reader (varioskan ash-3001, Thermo Scientic, USA) to
estimate the enzymatic activity. The inhibitory rate (%) was calculated

3. Results and discussion

Compound 3 was obtained as white needle crystals. Its molecular
formula was deduced to be C21H22O12 by HRESIMS mass spectrum
at m/z 489.1017 [M + Na]+ (calcd. for C21H22O12Na 489.1003). The IR
spectrum exhibited absorptions that were attributed to hydroxyl
(3419 cm 1), ester carbonyl (1713 cm 1), and phenyl (1607, 1514,
1465, and 763 cm1) groups. The splitting pattern in the 1H-NMR and
13
C-NMR spectrum (Table 1) at lower eld indicated that compound 3
exhibits two isolated aromatic spin systems. Signals at H 6.92 (2H, d,
J = 8.68 Hz, H-3, 5) and 7.94 (2H, d, J = 8.68 Hz, H-2, 6), and C
115.86 2, 132.42 2, along with that for the phenyl carboxyl group
(C 163.87), suggested the presence of a p-hydroxybenzoic acid moiety
[13]. The other moiety was indicated by the 1,2,4,6-tetrasubstituted AX
spin system; signals attributed to this system were observed at H 6.54
(1H, d, J = 1.88 Hz, H-5) and 6.35 (1H, d, J = 1.88 Hz, H-3) in the proton
spectrum and at C 157.24, 157.13, and 150.57 (three oxygenated aromatic carbons) in the carbon spectra. In addition, a methylene group
(H 3.58 and 3.39; C 29.26) and a carboxyl group (C 172.83, C-8)
were also observed. Taken together, these data led to the establishment
of the 2,4,6-trihydroxy-phenylacetic acid structural moiety in 3. The
HMBC correlations (Fig. 2) between the signals of the methylene proton
signals at H 3.58 and 3.39 with carbons (C 108.72, 150.57, 157.13, and
172.83 corresponding to C-1, C-2, C-6, and the carbonyl carbon C-8, respectively) provided additional support for the assigned structure. An
upeld shift of 3.8 ppm for C-7 in compound 3, when compared to
the chemical shift of the free carboxyl carbon of 4-hydroxybenzoic
acid, provided indirect evidence that C-7 is the site of esterication
in the 4-hydroxybenzoic acid [14]. Thus, these results established the
presence of an ester linkage between C-2 and C-7 atoms. The complete
NMR data of 3 were consistent with that of compound 1, [15] except
that 3 contained an additional monosaccharide. HPLC analysis of
the acid-hydrolyzed 3 suggested that the monosaccharide was Dglucose. In addition, the conguration was prompted by the large
coupling constant observed for the anomeric proton (H 4.75, d, J =
7.04 Hz). Moreover, the linkages were established on the basis of HMBC
correlations between H-1 (H 4.75) and C-4 (C 157.24). Therefore,
the structure of 3 was established unambiguously as 2-O-(4-

Table 3
1
H [ in ppm, multiplicity (J in Hz)] and 13C NMR ( in ppm) spectroscopic data of compounds 8.
Compound 8a
Position
1
2
3
4
5
6
7

Position

4
5
6

3.27 (1H, m)

6.34 (1H, d, J = 1.96)

107.10
150.51
103.82
157.52
100.76
157.18
28.82

1
2
3
4

169.96
119.14

5
6

132.46
115.64
162.87
163.83
101.93
73.30
76.25

6-1
6-2
6-3
6-4
6-5, 6-9
6-6, 6-8
6-7

6.54 (1H, d, J = 1.96)

3.86 (1H, d, J = 17.12)
3.39 (1H, s)

8
1
26
35
4
7
1
2
3
a

7.92 (2H, d, J = 8.72)

6.87 (2H, d, J = 8.72)

4.74 (1H, d, J = 7.04)

3.27 (1H, m)
3.22 (1H, m)

400 MHz for 1H NMR and 150 MHz for 13C NMR.

3.72 (1H, d, J = 10.88)

3.54 (1H, m)
5.39 (1H, d, J = 8.16)
3.13 (1H, m)
3.29 (1H, m)
3.22 (1H, s)
3.55 (1H, m)
4.40 (1H, d, J = 10.88)
4.14 (1H, m)
6.38 (1H, d, J = 15.84)
7.53 (1H, d, J = 15.84)
7.49 (2H, d, J = 8.68)
6.77 (2H, d, J = 8.68)

C
69.61
77.20
60.73
94.71
72.43
76.51
69.66
74.8
63.38
166.67
114.02
145.13
125.16
130.45
115.89
159.96

238

Q. Li et al. / Fitoterapia 105 (2015) 234239

Fig. 2. Key HMBC corrections of compounds 3 and 8.

hydroxybenzoyl)-4-O--D-glucopyranosyl-6-hydroxyphenylacetic
acid.
Compound 4 was obtained as white needle crystal. It was assigned
the molecular formula C22H24O12 as indicated by the HRESIMS
at m/z 503.1170 [M + Na]+ (calcd. for C22H24O12Na 503.1160). The
1
H- and 13C-NMR spectra (Table 1) agreed with those of 3, except for
an additional methoxy group H 3.47 (3H, s), C 51.44. In the HMBC,
this methoxy H 3.47 was correlated to C-8 (C 171.35). Consequently,
compound 4 was determined to be methyl 2-O-(4-hydroxybenzoyl)4-O--D-glucopyranosyl-6-hydroxyphenylacetate.
Compound 5 gave a molecular formula C23H26O12 on the basis of
the positive HRESIMS at m/z 517.1339 [M + Na]+ (calcd. for
C23H26O12Na 517.1316). The 1H and 13C NMR experiments (Table 1)
were similar to literature values for compound 3 except for an additional ethoxy group [H 3.91 (2H, q, J = 7.08 Hz), C 60.04, OCH2-8; H
0.99 (3H, t, J = 7.08 Hz), C 13.89, CH3]. The attachment of this ethoxy
group unit to C-8 was established through HMBC correlations of OCH2-8
(H 3.91) to C-8 (C 170.91). Accordingly, compound 5 was identied as
ethyl
2-O-(4-hydroxybenzoyl)-4-O--D-glucopyranosyl-6hydroxyphenylacetate.
Compound 6 exhibited the molecular formula C25H30O12 as evidenced by its positive HRESIMS at m/z 545.1604 [M + Na]+ (calcd.
for C25H30O12Na 545.1629). The signals (Table 1) were consistent with
3 except for an additional butoxy group [H 3.85,(2H, tri), C 63.73,
OCH2-8; H 1.34,(2H, qui), C 29.98, CH2; H 1.15 (2H, sex), C 18.46,
CH2; H 0.77 (3H, tri), C 13.48, CH3]. HMBC correlations of OCH2-8 (H
3.85) to C-8 (C 170.92) were observed. Therefore compound 6 was determined as butoxy 2-O-(4-hydroxybenzoyl)-4-O--D-glucopyranosyl6-hydroxyphenylacetate.
Compound 7 exhibited a [M + Na]+ peak at m/z 383.1155 in the
HRESIMS, corresponding to the molecular formula C19H20O7 (calcd.
for C19H20O7Na 383.1101). The spectroscopic data (Table 2) of the
aglycone were similar to those of compound 6, except the lack of the
-D-glucopyranose attached to C-4. Compound 7 was deduced as
butoxy 2-O-(4-hydroxybenzoyl)-4,6-dihydroxyphenylacetate.
Compound 8 gave a molecular formula C36H38O19 on the basis of
the positive HRESIMS at m/z 797.1912 [M + Na]+ (calcd. for
C36H38O19Na 797.1905). Its 1H- and 13C-NMR spectrum (Table 3) exhibited characteristic proton signals of an AABB-system benzene [H 7.92
(2H, d, J = 8.72 Hz, H-2,6), 6.87 (2H, d, J = 8.72 Hz, H-3, 5); C
132.46 2 (C-2, 6), 115.64 2 (C-3, 5)], an AB-system aromatic
ring [H 6.34 (1H, d, J = 1.96 Hz, H-3), 6.54 (1H, d, J = 1.96 Hz, H-5);
C 103.82 (C-3), 100.76 (C-5)], a methylene [H 3.39 (1H, s), 3.86
(1H, s,); C 28.82 (C-7)], and two carbonyls [C 169.96 (C-8), 163.83
(C-7)], along with a -D-glucose. These data indicated that the molecular structure was close to that of 3. Its spectrum also exhibited a 6-Op-coumaroyl--glucose moiety at H 7.49 (2H, d, J = 8.68 Hz, H-6-5,
6-9), 6.77 (2H, d, J = 8.68 Hz, H-6-6, 6-8), 6.38 (1H, d, J =
15.84 Hz, H-6-2), 7.53 (1H, d, J = 15.84 Hz, H-6-3); C 130.45
(C-6-5, 6-9), 115.89 (C-6-6, 6-8), 114.02 (C-6-2) 145.13 (C-6-3)
and a -D-glucose, the conguration was prompted by the large

coupling constant of the anomeric proton (H 5.39, d, J = 8.16 Hz)

[13]. The HMBC experiment (Fig. 2) revealed long range coupling
from H-6 (H 4.14) to C-6-1 (C 166.67), H-6-2 (H 6.38) to C-6-1
(C 166.67), C-6-3 (C 145.13) and H-6-2 (H 7.53) to C-6-1 (C
166.67), C-6-2 (C 114.02), C-6-5 (C 130.45), C-6-9 (C 130.45) reinforced the structure of this moiety. The HMBC correlations from H-1
(H 5.39) to C-8 (C 169.96), indicating the 6-O-p-coumaroyl--glucose
moiety attached to C-8. So compound 8 was identied as (6-O-pcoumaroyl)--D-glucopyranosyl-2-O-(4-hydroxybenzoyl)-4-O--Dglucopyranosyl-6-hydroxyphenylacetate.
Compound 9 was isolated as a yellow powder (MeOH). Its molecular
formula was determined as C14H18O10 by HRESIMS at m/z 369.0787
[M + Na]+ (calcd. for C14H18O10Na 369.0792). The NMR data of compound 9 was similar to that of compound 3 (Table 2). The difference
was the absence of the signals for p-hydroxybenzoic acid moiety
which found to be replaced by hydroxy. The above evidences allowed
compound 9 to be determined as 4-O--D-glucopyranosyl-2,6dihydroxyphenylacetic acid.
Besides the new compounds, the known compounds (Fig. 1)
were identied as 1,2-O-(4-dihydroxy-benzoyl)-2,4,6-trihydroxyphenylacetic acid (1), [15] methyl 2-O-(4-hydroxybenzoyl)-2,4,6trihydroxyphenylacetate (2), [16] kaempferol (10), quercetin (11),
kaempferol-3-O-glucoside (12), kaempferol-3-O-rutinoside (13),
rutin (14), [17] kaempferol 3-O--rhamnoside-7,4-di-O--galactoside
(15) [18] 6-methoxykaemferol-3-O--D-glucosyl(1 2)--Dglucopyranosyl-(6-(E)-caffeoyl)-7-O--D-glucopyranoside (16), [19]
dihydromyricetin (17), and myricetin (18) [20] All isolated compounds
were assayed for their activity against self-activated t-HSC/Cl-6 cells
(Table 4). New compounds 7, 8, and 9 inhibited t-HSC/Cl-6 cells
with IC50 values of 42.12, 109.20, and 34.04 g/mL, respectively,
which were signicantly lower than those observed for the positive
controls, Silymarin and Fufang Biejia Ruangan Pian (IC50 = 231.56
and 202.34 g/mL, respectively). The inhibitory activities of compound
Table 4
IC50 values of compounds 118 for inhibition t-HSC/Cl-6 cells, values are the mean of
triplicate experiments.
Compounds

IC50 (g/mL)a
Hepatic brosis

Compounds

IC50 (g/mL)a
Hepatic brosis

1
2
3
4
5
6
7
8
9
10

42.66
N100
N100
N100
N100
N100
42.12
109.20
34.04
N100

11
12
13
14
15
16
17
18
Silymarinb
Fufang Biejia Ruangan Pianb

N100
N100
53.51
69.54
76.23
86.43
24.33
15.91
202.34
231.56

a
IC50 value was calculated from the least-squares regression equations in the plot of the
logarithm of three graded concentrations vs.% inhibition g/mL.
b
Silymarin and Fufang Biejia Ruangan Pian was used as positive control.

Q. Li et al. / Fitoterapia 105 (2015) 234239

Table 5
IC50 values of compounds 118 for inhibition of -glucosidase, values are the mean of
triplicate experiments.
Compounds

IC50 (g/mL)a

Compounds

IC50 (g/mL)a

1
2
3
4
5
6
7
8
9
10

N50
9.15
N50
17.50
N50
N50
0.72
10.53
22.26
5.20

11
12
13
14
15
16
17
18
Acarboseb

2.24
21.52
24.9
11.91
N50
N50
3.29
1.65
3.36

a
IC50 value was calculated from the least-squares regression equations in the plot of the
logarithm of three graded concentrations vs.% inhibition.
b
Acarbose was used as a positive control.

7 was higher than those of compounds 36, and 8, demonstrating that

glycosylation of the hydroxyl(s) on the 4-position signicantly decreased the inhibitory activity. Compound 9 exhibited more excellent
activity than 38, indicating that the presence of p-hydroxybenzoic
acid moiety on the 2-position may decrease the activity. Compounds
1318 also showed signicant anti-hepatic brosis activity, in particular, the compounds 17 and 18 efciently inhibited t-HSC/Cl-6 cells
with IC50 values of 24.33 and 15.91 g/mL, respectively.
All isolated compounds were also assayed for their ability to inhibit
-glucosidase (Table 5). The new compounds 4, 7, and 8 exhibited potent -glucosidase inhibitory activities. Among these three compounds,
7 (IC50 = 0.72 g/mL) had the highest activity, which was followed by
that of 8 (IC50 = 10.35 g/mL) and 4 (IC50 = 17.50 g/mL). Flavonoids
11 and 18 showed inhibitory activities with IC50 values of 2.24 and
1.65 g/mL, respectively, which were more signicant than the activity
of the positive control, acarbose (IC50 = 3.36 g/mL). Compounds 10,
11, 17, and 18 exhibited higher inhibitory activities than those of
compounds 1216, suggesting that the presence of hydroxyl(s) on the
3- and/or 7-position(s) is required for the activity. This conclusion is
consistent with the results reported in previous studies [21].
In summary, this article describes the rst study investigating the
activity of compounds isolated from the owers of I. balsamina towards
the treatment of hepatic brosis and diabetics. Our results reveal that
depsides, which are present in the owers of I. balsamina, exhibit potential anti-hepatic brosis and anti-diabetic activities.
Acknowledgments
The research was supported by E&T Modern Center for Natural
Products of Liaoning Province of China (No. 2008402021) and the
Construction of R&D Institute of State Original New Drug at Benxi of

239

Liaoning Province (2010ZX09401-304-105B). We are grateful to the

Analytical Center of Shenyang Pharmaceutical University for identication of the measurements of NMR, IR and HRESIMS.
References
[1] Jiangsu, Dictionary of Chinese Crude Drugs, Shanghai Scientic Technological
Publishers, Shanghai, 1977.
[2] A. Ghani, Medicinal Plants of Bangladesh: Chemical Constituents and Uses, Second
ed. The Asiatic Society of Bangladesh, Dhaka, 2003.
[3] B.A. Bohm, G.H.N. Towers, A study of phenolic compounds in impatiens, Can. J. Bot.
40 (1962) 677683.
[4] K. Thevissen, I.E.J.A. Francois, L. Sijtsma, A. Van Amerongen, W.M.M. Schaaper, R.
Meloen, T. Posthuma-Trumpie, W.F. Broekaert, B.P.A. Cammue, Antifungal activity
of synthetic peptides derived from Impatiens balsamina antimicrobial peptides
Ib-AMP1 and Ib-AMP4, Peptides 26 (2005) 11131119.
[5] X. Yang, D.K. Summerhurst, S.F. Koval, C. Ficker, M.L. Smith, M.A. Bernards, Isolation
of an anti-microbial compound from Impatiens balsamina L. using bioassay-guided
fractionation, Phytother. Res. 15 (2001) 676680.
[6] Y.C. Wang, Y.H. Lin, Anti-gastric adenocarcinoma activity of 2-methoxy-1,4naphthoquinone, an anti-Helicobacter pylori compound from Impatiens balsamina
L, Fitoterapia 83 (2012) 13361344.
[7] I.E.J.A. Franois, G.I. Dwyer, M.F.C.D. Bolle, et al., Processing in transgenic Arabidopsis
thaliana plants of polyproteins with linker peptide variants derived from the
Impatiens balsamina antimicrobial polyprotein precursor, Plant Physiol. Biochem.
40 (2002) 871879.
[8] M.Z. Imam, N. Nahar, S. Akter, M.S. Rana, Antinociceptive activity of methanol
extract of owers of Impatiens balsamina, J. Ethnopharmacol. 142 (2012) 804810.
[9] V.A. Motz, C.P. Bowers, L.M. Young, D.H. Kinder, The effectiveness of jewelweed,
Impatiens capensis, there lated cultivar I. balsamina and the component, lawsone
in preventing post poison ivy exposure contact dermatitis, J. Ethnopharmacol. 143
(2012) 314318.
[10] F.J. Tu, Y. Dai, Z.H. Yao, X.L. Wang, X.S. Yao, Flavonol glycosides from Epimedium
pubescens, Chem. Pharm. Bull. 11 (2011) 13171321.
[11] T. Li, X.D. Zhang, Y.W. Song, A microplate-based screening method for
alphaglucosidase inhibitors, Chin. J. Clin. Pharmacol. Ther. 10 (2005) 11281134.
[12] T.J. Mosmann, Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 5563.
[13] D.C.F.G. Denise, V.A. Leila, Acylsteryl glycosides from Pithecellobium cauliorum,
Phytochemistry 9 (1998) 13651367.
[14] L.Y. Deng, C.H. Yuan, L. Pan, H.Y. Chai, W.J. Keller, A.D. Kinghorn, Antioxidant and
quinone reductase-inducing constituents of black choke berry (Aronia melanocarpa)
fruits, J. Agric. Food Chem. 60 (2012) 1155111559.
[15] M. Hillenbrand, J. Zapp, H. Becker, Depsides from the petals of Papaver rhoeas, Planta
Med. 70 (2004) 380382.
[16] X.W. Yang, Y.L. Li, S.M. Li, Y.H. Shen, J.M. Tian, Z.J. Zhu, L. Feng, L. Wu, S. Lin, N. Wang,
Y.H. Liu, W.D. Zhang, Mono- and sesquiterpenoids, avonoids, lignans, and other
miscellaneous compounds of Abies georgei, Planta Med. 77 (2011) 742748.
[17] Y.L. Li, J. Li, N.L. Wang, X.S. Yao, Flavonoids and a new polyacetylene from Bidens
parviora Willd, Molecules 13 (48) (2008) 19311941.
[18] L.O.A. Manguro, I. Ugi, P. Lemmen, R. Hermann, Flavonol glycosides of Warburgia
ugandensis leaves, Phytochemistry 64 (2003) 891896.
[19] K.H. Lee, K.M. Park, K.R. Kim, J. Hong, H.C. Kwon, K.R. Lee, Three new avonol glycosides from the aerial parts of Tetragonia tetragonoides, Heterocycles 75 (2008)
419426.
[20] C.C. Shen, Y.S. Chang, L.K. Hott, Nuclear magnetic resonance studies of 5,7-dihydroxy
avonoids, Phytochemistry 34 (1993) 843845.
[21] Y.H. Wang, L.M. Xiang, C.H. Wang, C. Tang, X.J. He, Antidiabetic and Antioxidant
effects and phytochemicals of mulberry fruit (Morus alba L.) polyphenol enhanced
extract, PLoS One 8 (2013) 111.