Two new triterpenoid saponins from the root of Platycodon grandiflorum

Article abstract of DOI:10.1248/cpb.c12-00713

Two new triterpenoid saponins, named platycodon A (3-O-β-D- glucopyranosyl-16-O-β-D-glucopyranosyl-2β,3β,16β, 21β-tetrahydroxyolean-12-en-28-oic acid) and platycodon B (3-O-β-D-glucopyranosyl16-O-β-D-xylopyranosyl-2β,3β, 16β,21β-tetrahydroxyolean-12-en-28-oic acid) were isolated from the roots of Platycodon grandiflorum. The structures of these compounds were elucidated by means of various spectroscopic analyses. Compounds 1 and 2 were tested in HepG-2, A549 and DU145 human cancer cell lines and showed remarkable cytotoxic activities against HepG-2 and A549 cancer cell lines with IC ₅₀ values ranging from 4.9 to 9.4μM, but they displayed weak cytotoxic activities against DU145 cancer cell line with IC₅₀ values greater than 10μm.

Full text of DOI:10.1248/cpb.c12-00713

Note
January 2013
101
Chem. Pharm. Bull. 61(1) 101–104 (2013)
Two New Triterpenoid Saponins from the Root of Platycodon
grandiflorum
Guoxu Ma, Wenjie Guo, Lizi Zhao, Qingxia Zheng, Zhaocui Sun, Jianhe Wei, Junshan Yang, and
Xudong Xu*
Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of
Education, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of
Medical Sciences; Beijing 100193, China.
Received August 13, 2012; accepted September 13, 2012
Two new triterpenoid saponins, named platycodon A (3-O-β-d-glucopyranosyl-16-O-β-d-glucopyrano-
syl-2β,3β,16β,21β-tetrahydroxyolean-12-en-28-oic acid) and platycodon B (3-O-β-d-glucopyranosyl-16-O-β-d-
xylopyranosyl-2β,3β,16β,21β-tetrahydroxyolean-12-en-28-oic acid) were isolated from the roots of Platycodon
grandiflorum. The structures of these compounds were elucidated by means of various spectroscopic analyses.
Compounds 1 and 2 were tested in HepG-2, A549 and DU145 human cancer cell lines and showed remark-
able cytotoxic activities against HepG-2 and A549 cancer cell lines with IC50 values ranging from 4.9 to
9.4µm, but they displayed weak cytotoxic activities against DU145 cancer cell line with IC50 values greater
than 10µm.
Key words Platycodon grandiflorum; saponin; platycodon A; platycodon B; cytotoxic activity
The roots of Platycodon grandiflorum A. DC. (Campanu- anomeric carbon signals at δ 106.5 and 108.1, four oxygenated
laceae), which are called Jiegeng in Chinese, are widely used methane carbon signals at δ 66.1, 79.0, 81.4 and 83.3, and one
1
in traditional Chinese medicine for the treatment of cough, carboxylic carbon signal at δ 179.6. H- and 13C-NMR spec-
excessive phlegm, chest congestion, pyocutaneous disease tra suggested that compound 1 was an oleanolic triterpenoid
(suppurative disease), sore throat, upper respiratory tract saponin derivative. In conjunction with analysis of the het-
infections, and tonsillitis.1,2) Previous chemical and pharma- eronuclear multiple quantum coherence (HMQC) spectrum,
cological studies revealed that triterpenoid saponins are the 1H- and 13C-NMR data for the aglycon of compound 1 were
primary components in P. grandiflorum,3,4) and these saponins assigned as shown in Table 1. The presence of the hydroxyl
1
have been reported to have significant biological activities group at C-21 was suggested by the H-NMR signal at δ 3.42
such as antitumor, antimicrobial, and anti-inflammatory ac- (1H, m), the 13C-NMR signal at δ 79.0 and heteronuclear mul-
tivities.5–8) With the aim of identifying new anti-tumor agents tiple bond coherence (HMBC) correlations as follows: both of
from this plant, we examined its ethanol extract. In this paper, the methyl protons H3-29 (δ 1.21) and H3-30 (δ 1.35) with C-18
we describe the isolation and characterization of two new (δ 55.6), C-19 (δ 34.6), C-21 (δ 79.0) and C-22 (δ 44.9) carbons;
triterpenoid saponins, platycodon A (1) and platycodon B H-22α (δ 2.42) and H-22β (δ 1.26) with C-21 (δ 79.0) carbon.
(2) from Platycodon grandiflorum (Fig. 1), and their in vitro In the HMBC spectrum, proton H-22α (δ 2.42) correlated with
cytotoxic activities against HepG-2, A549 and DU145 human C-16 (δ 83.3) carbon, which revealed a hydroxyl group located
cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5- at C-16 (δ 83.3). This conclusion could be confirmed by the
diphenyltetrazolium bromide (MTT) assay method.
correlations as H-15 (δ 3.47, 2.15) correlated with C-16 (δ 83.3)
Compound 1, a white amorphous powder, [α]D20+0.007° and C-27 (δ 26.9) carbons, respectively. Other cross peaks of
(c=0.63, MeOH). Electrospray ionization (ESI)-MS gave two methyl protons, H3-23 (δ 1.31) and H3-24 (δ 1.17), cor-
the molecular ion peak at m/z 827 [M−H]− in negative ion- related with C-3 (δ 81.4) carbon and showed a hydroxyl group
mode. The fragments {m/z 665 [M−162−H]−} and {m/z 503 located at C-3 (δ 81.4). The HMBC correlations are shown
[M−162−162−H]−} suggested that 1 had two hexose units. in Fig. 2. Compared with the NMR data of the compounds
Its molecular formula was assigned as C42H68O16 based on reported previously, the aglycon of compound 1 was the same
high resolution (HR)-ESI-MS (m/z 827.4432, [M−H]−) and as Platycogenic acid C.9,10) In the nuclear Overhauser effect
analysis of the NMR spectra. The IR spectrum suggested spectrosocpy (NOESY) spectrum, the β configuration of the
the presence of a hydroxyl group (3380cm−1), carbonyl group hydroxyl group at H-21 was identified from the correlations as
1
(1677 cm−1) and double bond (1648cm−1). H-NMR spectrum follows: H-21 (δ 3.42) with H-29 (δ 1.21); β configuration of the
showed the presence of two anomeric proton signals at δ 5.03 hydroxyl group at C-16 was identified from the correlation be-
(d, J=7.8Hz) and δ 5.08 (d, J=7.8Hz), seven tertiary methyl tween H-16 (δ 4.34) and H-27 (δ 1.26). Therefore, the aglycon
groups (δ 1.00, 1.17, 1.21, 1.26, 1.31, 1.34, 1.35) and one ole- of 1 was established as 2β,3β,16 β,21β-tetrahydroxyolean-12-
finic proton (δ 5.57, brs). The 13C-NMR spectrum displayed ene-28-oic acid.11)
42 carbons including 12 carbons for sugar units and 30 car-
From the residual of acid hydrolysis, glucose was de-
bons for the aglycon. In combination with the distortionless termined by means of TLC analysis and the absolute
enhancement by polarization transfer (DEPT) spectrum, there configuration was identified as D type compared with au-
were seven sp3 carbons at δ 16.2, 17.3, 18.1, 18.4, 26.9, 28.8, thentic monosaccharide by GC. The β configurations of
and 30.1, two sp2-hybridized carbons at δ 123.2 and 142.6, two the two d-glucoses were determined by their coupling con-
stants (3JH-1,H-2=7.0–8.0Hz). Total correlated spectroscopy
(TOCSY) and HMQC spectra of compound 1 established the
The authors declare no conflict of interest.
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: xdxu@implad.ac.cn

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Vol. 61, No. 1
Fig. 1. Structures of Compounds 1 and 2
(DU145) cell lines. The results (Table 2) indicate that com-
pounds 1 and 2 showed remarkable cytotoxic activities against
HepG-2 and A549 cancer cell lines with IC50 values ranging
from 4.9 to 9.4µm, but displayed weak cytotoxic activities
against DU145 cancer cell line with IC50 values greater than
10 µm. It should be noted that the significant cytotoxic activi-
ties of compounds 1 and 2 against HepG-2 and A549 may be
due to their free carboxylic group at C-28.12) Early studies on
the cytotoxicity of similar compounds against several human
cancer cell lines have reached the same results.13,14)
Fig. 2. Key HMBC Correlations of Compound 1
Experimental
General Experimental Procedures Optical rotations
1
spin-coupling network of each sugar. Their H- and 13C-NMR
data were assigned and are presented in Table 1. In the were determined on a Perkin-Elmer 341 digital polarimeter.
HMBC spectrum, two anomeric protons, H-1′ (δ 5.03) and IR spectra were obtained on a Shimadzu FTIR-8400S infrared
H-1″ (5.08), correlated with C-3 (δ 81.4) and C-16 (δ 83.3) spectrometer with KBr pellets. ESI-MS/MS and HR-ESI-MS
carbons, respectively. This conclusion was also confirmed were recorded on a LTQ Orbitrap XL mass spectrometer
by the cross peaks between H-1′ (δ 5.03) and H-3 (δ 4.13); (Thermo Scientific, Boston, U.S.A.). 1D- and 2D-NMR were
and between H-1″ (δ 5.08) and H-16 (δ 4.34) in the NOESY recorded with a 600MHz NMR spectrometer (Varian NMR
spectrum. Accordingly, all of this evidence suggested the System). AB-8 macroporous resin produced by Nankai Chem-
structure of compound 1 was 3-O-β-d-glucopyranosyl-16-O- ical Industry was used. Silica gel (100–200, 200–300 mesh)
β-d-glucopyranosyl-2β,3β,16β,21β-tetrahydroxyolean-12-en-28- for column chromatography, silica gel H for medium pressure
oic acid, which was named platycodon A.
liquid chromatography (MPLC), and silica gel PF254 for TLC
Compound 2, a white amorphous powder, [α]D20 −0.002° were produced by Qingdao Marine Chemistry Ltd., People’s
(c=0.43, MeOH). Acid hydrolysis gave glucose and xylose by Republic of China. Semi-preparative HPLC was performed
means of TLC analysis. ESI-MS gave the molecular ion peak on a LUMTECH instrument with a UV detector at 210nm
at m/z 797 [M−H]− in negative ion-mode. The fragments {m/z and using a C30 column (Develosil Packed ϕ20×100mm,
635 [M−162−H]−} and {m/z 503 [M−162−132−H]−} sug- Nomura Chemistry Ltd., Japan). The GC was carried out on
gested that compound 2 has a hexose unit and a pentose unit. an Agilent 7890A Gas chromatograph (Agilent Technology)
Its molecular formula was assigned as C41H66O15 based on with DB-1701 (30m×0.25mm, i.d.) (J&W) with a hydrogen
HR-ESI-MS (m/z 797.4326, [M−H]−) and analysis of the NMR flame ionization detector (FID) detector. The carrier gas was
spectra. After HMQC, HMBC and TOCSY experiments, N2. The initial temperature was set at 80°C for 5min and then
1H- and 13C-NMR signals were assigned as shown in Table increased to 270°C at a speed of 10°C/min.
1
1. Comparison of the H- and 13C-NMR data of compound 2
Sample Collection The roots of Platycodon grandiflorum
with those of compound 1 revealed that compound 2 had the A. DC were collected at the Institute of Medical Plant Devel-
same aglycon as compound 1. Acid hydrolysis of compound opment and authenticated by Dr. Jianhe Wei (Institute of Me-
2 gave glucose and xylose by means of TLC analysis. The dicinal Plant Development). A voucher specimen (P-1-200647)
absolute configurations of sugars were identified as d-glucose has been deposited at the Herbarium of the Institute of Medic-
and d-xylose by means of GC compared with authentic mono- inal Plant Development, Beijing, People’s Republic of China.
saccharide. In the HMBC spectrum, two anomeric protons
Extraction and Isolation The powder of the roots (4.0kg)
at H-1′ (δ 5.01) and H-1″ (δ 4.86) correlated with C-3 (δ 81.5) was refluxed with 95% ethanol three times. The ethanol
and C-16 (δ 83.4), respectively. Thus, based on the evidence extract was then concentrated in vacuum at 60°C to obtain
of the above results, compound 2 was identified as 3-O- a residue (0.9kg). The residue was suspended in water and
β-d-glucopyranosyl-16-O-β-d-xylopyranosyl-2β,3β,16β,21β- then partitioned with EtOAc. The aqueous layer was chro-
tetrahydroxyolean-12-en-28-oic acid and named Platycodon B. matographed over a macroporous resin AB-8 column eluted
Compounds 1 and 2 were tested for their in vitro cytotoxic with H2O and then 95% EtOH. The 95% EtOH fraction was
activities against human hepatocellular carcinoma (HepG-2), evaporated in vacuo to obtain the residue (213g). The resi-
human lung cancer (A549) and human prostatic cancer due was chromatographed on a silica gel column (solvent,

January 2013
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Vol. 61, No. 1
Table 2. In-Vitro Antiproliferative Activity of Compounds 1 and 2
2 was assessed by the MTT method using HepG-2, A549
and DU145 cancer cell lines. Cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) medium supplied with
10% fetal bovine serum and cultured at a density of 6×
104 cells/mL per well in a 96-well microtiter plate. Then 5 dif-
ferent concentrations of each compound dissolved in dimethyl
sulfoxide (DMSO) were added to each well and compared
to doxorubicin (Sigma, ≥98%) as the positive control. Each
concentration was tested in triplicate. After incubation at 37°C
in 5% CO2 for 48h, 10µL of MTT (4mg/mL) was added to
IC50 (µm)
Compounds
HepG-2
A549
DU145
1
4.9 0.5b)
7.5 1.2
6.9 0.8
9.4 0.3
1.5 0.02
32.5 0.4
45.3 0.6
2.3 0.03
2
Doxorubicina)
0.18 0.01
a) Doxorubicin as the positive control. b) The data represent the mean S.D. of
three independent experiments.
CHCl3–MeOH, 100:1–0:1) to obtain eight fractions (Fr. each well and then the cells incubated for another 4h. Next,
1–Fr. 8). Fraction 7 (10.3g, CHCl3–CH3OH, 2:1) was chro- the liquid in the well was removed and DMSO (200µL) added
matographed on a silica gel column (solvent, CHCl3–MeOH, to each well. The absorbance was recorded on a microplate
5:1–1:1) to obtain five fractions (Frs. A–E). Fraction E (1.2g) reader at a wavelength of 570nm.
was chromatographed on MPLC and then subjected to semi-
preparative HPLC with a gradient of 40% MeOH–H2O to
Acknowledgements We would like to thank Wen-yi He
MeOH on a RP-30 column. Finally, compound 1 (13.6mg) (Institute of Materia Medica, Chinese Academy of Medical
was obtained at a tR of 30.2min using a MeOH–H2O (50:50) Sciences and Peking Union Medical College) for measurement
system, while compound 2 (18.9mg) was obtained at a tR of of the NMR spectra. This work was supported by the National
39.1min using a MeOH–H2O (56:44) system.
Natural Science Foundation of China (No. 30772728).
Platycodon A (1): White amorphous powder: [α]D20 +0.007°
(c=0.63, MeOH), IR (KBr) cm−1: 3380, 2950, 1677, 1205,
1060. ESI-MS m/z: 827 [M−H]−, 665, 503. HR-ESI-MS (nega-
tive ion mode) m/z: 827.4431 ([M−H]−, Calcd for C42H67O16:
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