Two new oleanane-type triterpenoids from platycodi radix and anti-proliferative activity in HSC-T6 Cells

Article abstract of DOI:10.3390/molecules171214899

Two new oleanane-type triterpenoids, named platycodonoids A and B (1, 2), together with five known saponins, including platycodin D (3), deapioplatycodin D (4), 3-O-β-D-glucopyranosyl polygalacic acid (5), 3-O-β-D- glucopyranosyl platycodigenin (6) and polygalacin D (7), were isolated from the roots of Platycodon grandiflorum. On the basis of spectral data and chemical evidence, the structures of the new compounds were elucidated as 2β,3β,23,24-tetrahydroxy-28-nor-olean-12-en-16-one (1) and 2β,3β,23,24- tetrahydroxy-28-nor-olean-12-en-16-one-3-O-β-D- glucopyranoside (2). Compounds 1-7 were evaluated for their in vitro anti-proliferative activity against the HSC-T6 cell line.

Full text of DOI:10.3390/molecules171214899

Molecules 2012, 17, 14899-14907; doi:10.3390/molecules171214899
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Two New Oleanane-Type Triterpenoids from Platycodi Radix
and Anti-proliferative Activity in HSC-T6 Cells
Qin Zhan 1, Feng Zhang 1, Lianna Sun 2, Zhijun Wu 1 and Wansheng Chen 1,*
1
Department of Pharmacy, Changzheng Hospital, Second Military Medical University,
415 Fengyang Road, Shanghai 200003, China; E-Mails: zhanqin5@yahoo.com.cn (Q.Z.);
fengzhangky@yahoo.com.cn (F.Z.); wuzhijun999@sina.com (Z.W.)
2
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
China; E-Mail: sssnmr@yahoo.com.cn
* Author to whom correspondence should be addressed; E-Mail: chenwanshengsmmu@yahoo.com.cn;
Tel./Fax: +86-21-8188-6181.
Received: 9 November 2012; in revised form: 29 November 2012 / Accepted: 30 November 2012 /
Published: 13 December 2012
Abstract: Two new oleanane-type triterpenoids, named platycodonoids A and B (1, 2),
together with five known saponins, including platycodin D (3), deapioplatycodin D (4),
3-O-β-D-glucopyranosyl polygalacic acid (5), 3-O-β-D-glucopyranosyl platycodigenin (6)
and polygalacin D (7), were isolated from the roots of Platycodon grandiflorum. On the
basis of spectral data and chemical evidence, the structures of the new compounds were
elucidated as 2β,3β,23,24-tetrahydroxy-28-nor-olean-12-en-16-one (1) and 2β,3β,23,24-
tetrahydroxy-28-nor-olean-12-en-16-one-3-O-β-D-glucopyranoside (2). Compounds 1–7
were evaluated for their in vitro anti-proliferative activity against the HSC-T6 cell line.
Keywords: Platycodon grandiflorum; oleanane-type triterpenoid; triterpenoid saponin
1. Introduction
Platycodon grandiflorum A. DC. is a perennial plant in the family Campanulaceae which grows
widely in East Asia. Platycodi Radix, the root of P. grandiflorum, has been used in traditional Oriental
medicine as an expectorant for pulmonary disease and a remedy for respiratory disorders [1,2]. Platycodi
Radix has recently been reported to exhibit many pharmacological activities, including anti-cancer
properties [3–8], atopic dermatitis-like skin lesions treating [9–11], anti-skin photoaging effects [12],

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antiobesity and glucose metabolism regulation [13–17], anti-atherosclerotic [18], and anti-hyperlipidemic [19]
activities. Chemical investigation of Platycodi Radix revealed that triterpenoid saponins were the main
chemical components, and more than 55 triterpenoid saponins have been isolated from Platycodi Radix to
date [20,21]. Based on the structures of the aglycones, the triterpenoid saponins are classified into three
types: the platycodigenin type, platycogenic acid A lactone type and polygalacic acid type [22]. It has
been previously demonstrated that Platycodi Radix showed protective effects against acute ethanol,
acetaminophen-, carbon tetrachloride-, thioacetamide and cholestasis-induced hepatotoxicity or hepatic
injury in mice and inhibited the progress of hepatic fibrosis in rats [23–30]. In our preliminary
pharmacological study, the 70% EtOH extract of Platycodi Radix was also found to exhibit significant
protective activities against liver fibrosis in rats. In a continued effort to search for possible hepatoprotective
component from this herb, an investigation of Platycodi Radix was undertaken, and this has led to the
isolation of two new triterpenoids [an aglycone and its saponin, named platycodonoids A (1) and B (2)
(Figure 1)], which possess a rare 28-nor-oleanane-type with a C-16 keto group in the aglycone structure.
Besides, five known saponins were isolated and identified as platycodin D (3) [31], deapio-platycodin D
(4) [21], 3-O-β-D-glucopyranosyl polygalacic acid (5) [32], 3-O-β-D-glucopyranosyl platycodigenin (6) [33]
and polygalacin D (7) [32,34], by comparison of their IR, NMR and MS data with literature values. To
the best of our knowledge, it was first time triterpenoids such a platycodonoids A and B having a
28-nor-oleanane-type skeleton are reported from the genus Platycodon.
Figure 1. Structures of compounds 17.
2. Results and Discussion
The air-dried roots of P. Grandiflorum were extracted three times with 70% EtOH under reflux. The
combined extract was chromatographed over a macroporous adsorbing resin column and partitioned as
described in the Experimental section. After repeated column chromatography, two new compounds 1
and 2 and five known saponins 3–7 were isolated and identified.
Compound 1 was obtained as white amorphous powder. Its molecular formula was assigned to be
C29H46O5 based on the HRESIMS spectrum (m/z 497.3243 [MNa], calcd. for C29H46O5Na, 497.3243).
The IR spectrum exhibited absorptions at 3415, 2947, 1711, and 1385 cm−1 assignable to hydroxyl,
methyl, ketone and methylene functions, respectively. The 1H-NMR spectrum of 1 showed the following

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signals: five tertiary methyl groups at H 0.78, 0.84, 0.96, 1.14, and 1.63 (each 3H, s); two pairs of
germinal oxygenated protons at H 4.20, 4.21, 4.87, 5.20 (each, d, J = 10.8 Hz); two oxygenated protons
at H 4.39 (d, J = 3.6 Hz) and 4.58 (dt, J = 7.2, 3.6 Hz); and an olefinic proton at H 5.38 (t, J = 3.6 Hz). The
13C-NMR spectrum revealed 29 carbon signals, which were further classified by DEPT and HSQC
experiments as five methyls, ten methylenes (two oxygenated), six methines (two oxygenated), five
quaternary carbons, one trisubstituted double bond (C 123.3 and 142.9), and one keto carbonyl (C
214.0) (Table 1). The aforementioned data implied that 1 was a nor-oleanane-type triterpenoid with four
hydroxyls. In the HMBC spectrum, two pairs of germinal oxygenated protons at H 4.20, 4.21, 4.87, 5.20
(H2-23, H2-24) showed correlations to C-3 (C 75.2), C-4 (C 48.1) and C-5 (C 48.5), indicating that two
hydroxyls were linked at C-23 and C-24. The two remaining hydroxyls were placed at C-2 (C 72.0) and
C-3, which were deduced by the HMBC correlations from the proton H-2 to C-1 (C 44.7), C-3, and C-4,
and from H-3 (H 4.39) to C-1, C-2, C-3, C-4, C-23 (C 64.1) and C-24 (C 64.7) (Figure 2). The small
coupling constant (J2,3 = 3.6 Hz) of H-2 and H-3 indicated the axial position of H-3 and the equatorial
position of H-2, which was further confirmed by a NOESY correlation between H-2 and H-3. Moreover,
the chemical shifts H2-15 at H 2.55 (d, J = 14.4 Hz), 1.93 (d, J = 14.4 Hz) and C-15 at C 47.2 were quite
different from that reported for oleanane-type triterpenoid [platycoside O, H-15, H 1.81 (dd, J = 15.0,
3.0 Hz), 2.55 (d, J = 12.0 Hz), C-15 (C 36.1)] [21]. These results suggested the keto group (C 214.0)
was at C-16, which was confirmed by the HMBC spectrum correlations from H2-15, H-17 (H 1.65),
H-18 (H 2.79) and H-22 (H 1.34, 2.14) to C-16 (C 214.0). The 1H-1H COSY spectrum of 1 indicated the
presence of partial structures (Figure 2). Consequently, by comparison with the structure of platycoside
O [21], compound 1 was determined to be 2β,3β,23,24-tetrahydroxy-28-nor-olean-12-en-16-one, and
named platycodonoid A.
Compound 2 was obtained as white amorphous powder. It gave an [MNa] peak at m/z 659.3728 in
HRESIMS spectrum corresponding to the molecular formula of C35H56O10 (calcd. for C35H56O10Na,
659.3771), which showed a unit of C6H10O5 more than that of compound 1. The 1D-NMR spectra of 2
displayed similarities to those of 1, except for an additional sugar unit. An anomic proton signal at H 5.15
(H-1', d, J = 7.8 Hz), an oxygenated methylene (H2-6') at H 4.35 (t, J = 6.0 Hz) and 4.59 (d, J = 10.8 Hz),
four oxymethine protons in the range H 3.504.50 in the 1H-NMR spectrum suggested that 2 contained
13
a sugar moiety. In accordance, the C-NMR spectrum displayed six carbon signals at C 63.0, 72.0,
75.6, 79.0, 79.0, 106.6. The subsequent acid hydrolysis of 2 gave glucose only, which was analyzed by gas
chromatography as glucitol acetate [35]. The linkage of this β-D-glucopyranose (J = 7.8 Hz) (Table 1) was
confirmed by the HMBC correlation between H-1' and C-3. Therefore, the structure of platycodonoid B
(2) was elucidated as 2β,3β,23,24-tetrahydroxy-28-nor-olean-12-en-16-one 3-O-β-D-glucopyranoside.
Figure 2. Key 1H-1H COSY (bold lines) and HMBC (H→C) correlations of compound 1.

Molecules 2012, 17
14902
1
13
Table 1. H-NMR (600 MHz) and C-NMR (150 MHz) data for compounds 1 and 2
(pyridine-d5, H in ppm, J in Hz).
1
2
Position
C
H
C
H
1
44.7 2.33 (1H, dd, 13.8, 3.0), 1.30 (1H, m) 45.0 2.08 (1H, m), 1.57 (1H, m)
2
72.0 4.58 (1H, dt, 7.2, 3.6)
75.2 4.39 (1H, d, 3.6)
48.1
70.0 4.76 (1H, m)
3
86.4 4.61 (1H, brs)
4
47.8
5
48.5 1.82 (1H, brd, 12.0)
19.2 1.98 (1H, m), 1.83 (1H, brs)
33.5 1.53 (1H, m), 0.91 (1H, m)
40.1
48.4 1.85 (1H, d, 12.0)
6
19.4 1.92 (1H, m ), 1.61 (1H, m)
7
33.4 1.47 (1H, t, 13.8), 1.18 (1H, m)
8
40.3
9
47.3 2.49 (1H, t, 5.4)
37.3
47.3 2.53 (1H, t, 5.4)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
29
30
1'
37.7
24.2 2.05 (1H, m), 0.84(1H, m)
123.3 5.38 (1H, t, 3.6)
142.9
24.2 2.04 (1H, m), 0.88 (1H, m)
123.6 5.42 (1H, brs)
142.9
47.1
46.9
47.2 2.55 (1H, d, 14.4), 1.93 (1H, d, 14.4) 47.1 2.58 (1H, d, 14.4), 1.96 (1H, d, 14.4)
214.0
213.8
48.1 1.65 (1H, m)
45.1 2.79 (1H, m)
47.0 1.40 (1H, t, 13.2), 1.17 (1H, m)
31.3
48.3 1.71 (1H, m)
45.2 2.83 (1H, m)
47.3 1.47 (1H, t, 13.2), 1.21(1H, m)
31.4
35.0 1.09 (2H, m)
21.5 2.14 (1H, m), 1.34 (1H, m)
35.0 1.09 (2H, m)
21.6 2.17 (1H, m), 1.38 (1H, m)
64.1 4.87 (1H, d, 10.8), 4.20 (1H, d, 10.8) 63.8 4.99 (1H, d, 10.8), 4.11 (1H, d, 11.4)
64.7 5.20 (1H, d, 10.8), 4.21 (1H, d, 10.8) 63.8 4.80 (1H, d, 10.8), 4.26 (1H, d, 10.8)
17.9 0.96 (3H, s)
17.6 1.63 (3H, s)
27.3 1.14 (3H, s)
33.6 0.78 (3H, s)
23.7 0.84 (3H, s)
18.3 0.99 (3H, s)
18.0 1.55 (3H, s)
27.2 1.17 (3H, s)
33.5 0.85 (3H, s)
23.8 0.89 (3H, s)
106.6 5.15 (1H, d, 7.8)
75.6 4.05 (1H, t, 8.1)
79.0 4.18 (1H, m)
2'
3'
4'
72.0 4.18 (1H, m)
5'
79.0 3.98 (1H, m)
6'
63.0 4.59 (1H, d, 10.8), 4.35 (1H, t, 6.0)
The origin of compounds 1 and 2 (Scheme 1) was proposed to be the oleanane-type triterpenoids i
(compound 37). Decarboxylation at C-28 would produce a key intermediate ii, which could undergo an
oxidation at C-16 to yield iii (1 and 2).

Molecules 2012, 17
Scheme 1. The plausible biogenetic origin of compounds 1 and 2.
14903
Compounds 1–7 were evaluated for their anti-proliferative activities against the Hepatic Stellate Cell
(HSC)-T6 line using the 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay [36].
Colchicine was used as positive control in this study (IC50 value < 10 μM). Among the tested compounds,
compounds 1, 3, 4 and 7 were the most potent, showing IC50 values of 5.27, 1.77, 8.24 and 1.04 μM,
respectively. The IC50 values for the remaing compounds, 2, 5 and 6, were 69.63, 8150.23, and 13.36 μM,
respectively. It was reported that saponins from Platycodi Radix prevented the increase in the serum
levels of hepatic enzyme markers (alanine aminotransferase and aspartate aminotransferase) and
reduced oxidative stress, such as glutathione content and lipid peroxidation, in the liver in a
dose-dependent manner [23–30]. The reason why compounds 1, 3, 4 and 7 delayed the formation of liver
fibrosis need to be further studied. In the structure-activity relationship of these oleanane-type
triterpenoids, the presence of a free carboxyl functional group at C-28 seemed not to be related to the
hepatoprotective activity (compounds 5 and 6). When forming C-28 glycosides, the presence of the
glycosides affected the activity, and the number of monosaccharides in the sugar moiety increased the
activity (compounds 3, 4 and 7). In contrast, when came to the 28-nor-oleanane-type triterpenoids 1 and
2, the aglycone was more active than its corresponding glycoside.
3. Experimental
3.1. General
Optical rotations were measured with Perkin-Elmer 341 polarimeter. UV and IR spectra were recorded
on Shimadzu UV-2550 and Perkin-Elmer 577 (using KBr disks) spectrophotometers, respectively.
1
NMR spectra were acquired on a Bruker Avance III (600 MHz for H-NMR, ppm relative to TMS)
spectrometer. ESIMS spectra were made on an Agilent 1200 series HPLC and interfaced to an Agilent
6410 triple-quadrupole mass spectrometer equipped with an electrospray ionization source, and
HRESIMS spectra were made on an Agilent 1290 series HPLC and interfaced to an Agilent 6538 UHD
Accurate-Mass Q-TOF LC/MS (Agilent Corporation, Wilmington, DE, USA). GC-MS was conducted
on a Thermo Finnigan Trace GC apparatus using an L-Chirasil-Val column (25 m × 0.32 mm, i.d.).
Semi-preparation RP-HPLC isolation was achieved with an Agilent 1200 instrument with refractive
index detector (RID) using a YMC 5 μm C8 column (250 mm × 10 mm). Methanol for semi-preparative
HPLC was of HPLC-grade (Merck, Darmstadt, Germany). Column chromatography: silica gel
(200300 mesh); macroporous adsorbing resin (D-101, ZTC-1, 0.31.2 mm, Tianjin Zhentiancheng
Science & Technology Co., Ltd., Tianjin, China); sephadex LH-20 gel (4070 μm, Amersham
Pharmacia Biotech AB, Uppsala, Sweden); silica gel H (Qingdao Haiyang Chemical Co. Ltd., Qingdao,
China). All solvents for column chromatography and acid hydrolysis were of analytical grade (Shanghai

Molecules 2012, 17
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Chemical Reagents Company, Ltd., Shanghai, China). Spots of compounds on TLC were developed
using 10% H2SO4-EtOH solution.
3.2. Plant Material
Platycodi Radix was collected from Taihe, Anhui Province, China, in September 2010 and was
identified by Professor Hanming Zhang of School of Pharmacy, Second Military Medical University.
A voucher specimen (No. 20100921) was deposited at the Department of Pharmacognosy, School of
Pharmacy, Second Military Medical University.
3.3. Extraction and Isolution
Platycodi Radix was air-dried (10 kg) and extracted three times with 70% EtOH (50 L × 3 times)
under reflux. The combined extract was concentrated in vacuo and suspended in water. The aqueous
layer was chromatographed over a macroporous adsorbing resin column eluting with H2O, 30% EtOH,
60% EtOH and 95% EtOH. The 95% EtOH-eluted fraction (30 g) was applied to a silica gel column
(CHCl3-MeOH, 50:1 to 10:1, v/v) and purified by semi-preparative HPLC (MeOH-H2O, 4:1) to give
compound 1 (10.4 mg). The 60% EtOH-eluted fraction (120 g) was chromatographed on silica gel
column eluting with a CHCl3-MeOH gradient (30:1 to 2:1, v/v) to afford five subfractions (A-E).
Subfraction B (20 g) was chromatographed on a silica gel column (CHCl3-MeOH, 10:1 to 5:1, v/v),
followed by Sephadex LH-20 column (MeOH-H2O, 1:1), and finally separated by semi-preparative
HPLC (MeOH-H2O, 3:2) to yield compound 2 (9.3 mg). By the same procedure, compounds 3 (20.0 mg),
4 (8.7 mg), 5 (11.2 mg), 6 (9.4 mg) and 7 (7.6 mg) were obtained from subfractions CE.
3.4. Characterization of Compound 1 and Compound 2
Compound 1: white amorphous powder; [α]2D2.0 44.3 (c 0.174, MeOH); IR (KBr) max 3396, 2943, 2908,
1705, 1639, 1452, 1429, 1381, 1248, 1074, 1043, 898.7 cm−1; 1H-NMR and 13C-NMR data see Table 1;
ESIMS m/z 497.4 [MNa]; HRESIMS m/z 497.3243 [MNa] (calcd. for C29H46O5Na, 497.3243).
Compound 2: white amorphous powder; [α]2D2.0 59.9 (c 0.128, MeOH); IR (KBr) max 3415, 2947, 1711,
1456, 1433, 1385, 1365, 1134, 1047, 696, 594 cm−1; 1H-NMR and 13C-NMR data see Table 1; ESIMS
m/z 659.5 [MNa]; HRESIMS m/z 659.3728 [MNa] (calcd. for C35H56O10Na, 659.3771).
3.5. Acid Hydrolysis of Compound 2
Compound 2 (3.0 mg) was refluxed with 1M HCl (dioxane-H2O, 1:1, 2 mL) at 90 °C for 3 h in a water
bath. After dioxane was removed, the solution was extracted with EtOAc (2 mL × 3 times). After
evaporating to dryness, the monosaccharide portion was analyzed by gas chromatography after
conversion of the hydrolysates into corresponding alditol acetates. Only D-glucose was detected. The
EtOAc portion was washed with H2O and evaporated to yield the aglycone. The aglycone was identified
by TLC together with compound 1.

Molecules 2012, 17
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3.6. In Vitro Inhibitory Activity on Cell Proliferation
Tested compounds 17 were dissolved in DMSO (final concertration, 0.1%). Inhibitory activity of
compounds 17 against HSC-T6 cell line was evaluated by the MTT assay [36]. Briefly, cells at the
exponential growth phase were harvested and seeded into a flatbottom 96-well plate. A total of 90 μL
containing 5 × 104 cells was added to each well of the plate and incubated for 24 h in a 5% humidified
CO2 at 37 C. HSC-T6 cells were treated with vehicle or compounds at concerntration of 0.01, 0.1, 1,
10, 100 and 1000 μg/mL. After 48 h of incubation at 37 C, 20 μL/well, MTT was then added and the
plate was again incubated at 37 C for 4 h. Reduction of MTT to formazan was measured in an ELISA
plate reader at 570 nm. Inhibitory activity of compounds 17 on cell proliferation (% of control) was
calculated as 100 × (absorbance of treated compound—absorbance of background light)/(absorbance of
control—absorbance of background light). Data were expressed as the mean of the three independent
experiment. Colchicin was used as a positive control.
4. Conclusions
In conclusion, the phytochemical investigation of the roots extract of Platycodi Radix afforded two
new triterpenoids, platycodonoids A (1) and B (2), together with five known triterpenoids: platycodin D
(3), deapio platycodin D (4), 3-O-β-D-glucopyranosyl polygalacic acid (5), 3-O-β-D-glucopyranosyl
platycodigenin (6) and polygalacin D (7). The structures of the compounds were elucidated on the basis
of spectral analysis and chemical evidence and literature comparisons in the case of the known ones.
Compounds 1, 3, 4 and 7 exhibited significant hepatoprotective activities against HSC-T6 cell lines in vitro
(IC50 value < 10 μM).
Acknowledgments
We gratefully acknowledge financial support of this work by National Natural Science Foundation of
China (81073030) and The National Key Technology R & D Program in the 11th Five year Plan of
China (2008BA151B00-2).
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Sample Availability: Samples of the compounds 17 are available from the authors.
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