Platycoside N: A new oleanane-type triterpenoid saponin from the roots of platycodon grandiflorum

Article abstract of DOI:10.3390/molecules15128702

A new oleanane-type triterpenoid saponin, named platycoside N (1), together with six known saponins, was isolated from the roots of Platycodon grandiflorum. On the basis of acid hydrolysis, comprehensive spectroscopic data analyses and comparison with the spectral data of the known compounds, its structure was elucidated as 3-O-β-Dglucopyranosyl-( 1→6)-β-D- glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-en-28-oic acid 28-O-β-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside. The six known compounds were platycodin D (2), deapioplatycodin D (3), platycodin D ₃ (4), deapioplatycodin D₃ (5), platycoside E (6) and deapioplatycoside E (7).

Full text of DOI:10.3390/molecules15128702

Molecules 2010, 15, 8702-8708; doi:10.3390/molecules15128702
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molecules
ISSN 1420-3049
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Article
Platycoside N: A New Oleanane-Type Triterpenoid Saponin
from the Roots of Platycodon grandiflorum
Wei Li 1,†, Wei Zhang 1,†, Lan Xiang 2, Zi Wang 1, Yi-Nan Zheng 1,*, Ying-Ping Wang 3,*,
Jing Zhang 1 and Li Chen 4
1
College of Chinese Material Medicine, Jilin Agricultural University, Changchun 130118, China;
E-Mail: liwei7727@126.com (W.L.)
2
School of Pharmacy, Shangdong University, Jinan 250012, China
3
Institute of Special Wild Economic Animals and Plant, CAAS, Jilin 132109, China
4
Norman Bethune College of Medicine, Jilin University, Changchun 130021, China
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mail: zhenyinan@tom.com (Y.N. Z.);
E-Mail: yingpingw@126.com (Y.P.W.) Tel.: +86-432-66513091(Y.P.W.);
Fax: +86-432-66513409(Y.P.W.).
Received: 13 September 2010; in revised form: 22 November 2010 / Accepted: 25 November 2010 /
Published: 30 November 2010
Abstract: A new oleanane-type triterpenoid saponin, named platycoside N (1), together
with six known saponins, was isolated from the roots of Platycodon grandiflorum. On the
basis of acid hydrolysis, comprehensive spectroscopic data analyses and comparison with
the spectral data of the known compounds, its structure was elucidated as 3-O-β-D-
glucopyranosyl-(1→6)-β-D-glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-en-28-oic
acid 28-O-β-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside. The six known
compounds were platycodin D (2), deapioplatycodin D (3), platycodin D3 (4), deapio-
platycodin D3 (5), platycoside E (6) and deapioplatycoside E (7).
Keywords: platycoside N; Platycodon grandiflorum; triterpenoid saponin

Molecules 2010, 15
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1. Introduction
The roots of Platycodon grandiflorum A. DC (Campanulaceae), Platycodi Radix, are traditionally
used as food and a herbal medicine in the treatment of a wide range of diseases, including bronchial
asthma, hepatic fibrosis, bone disorders [1-4], hypercholesterolemia and hyperlipidemia [5]. The
principal bioactive constituents of this herb are triterpenoid saponins (platycosides), which exhibit a
variety of pharmacological activities, such as anti-inflammatory [6,7], anti-obesity [8-11], anti-cancer
[12-15] and hypoglycemic effects [16,17]. To date, more than 30 saponins have been isolated from this
plant [18-25]. In order to find more bioactive compounds, we have now studied the chemical
constituents of P. platycodiflorum, and in this paper, we report the presence in this species of a new
oleanane-type triterpenoid saponin, named platycoside N, together with six known compounds, from
the roots of Platycodon grandiflorum. (Figure 1)
Figure 1. Chemical structures of compounds 1-7.
2. Results and Discussion
Platycoside N (1) was a white amorphous powder, and its molecular formula C53H86O24 was
determined based on the HR-ESI-MS spectra. The oleanane-type triterpenoid saponin nature of
compound 1 was revealed through analysis of its spectral features. The IR spectrum exhibited
absorptions at 3,425 cm−1 (OH), 1,647 cm−1 (ester carbonyl), and 1,616 cm−1 (double bond). Six
methyl groups (δ 0.89 × 2, 0.98, 1.17, and 1.58 × 2) and one olefinic proton (δ 5.46, br s) of the
aglycon were observed in the 1H-NMR spectrum. The 13C-NMR spectrum showed that the aglycon had
six methyl carbons at δ 16.0, 18.6, 17.7, 27.2, 33.3, and 24.8, two olefinic carbons at δ 123.1 (CH) and
144.5 (C), one oxymethylene and three oxymethine carbons at δ 66.6, and 70.1, 74.2 and 83.1,
respectively, and one carbonyl carbon at δ 176.0 (Table 1). The information of the 1H-NMR spectrum

Molecules 2010, 15
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13
coupled with the C-NMR spectrum indicated that 1 had 2β,3β,16α,23-tetrahydroxyolean-12-en-28-
oic acid (polygalacic acid) as an aglycon [20]. The 13C-NMR spectrum showed 53 signals, of which 30
were assigned to a triterpenoid moiety and 23 to the saccharide portion. The downfield shift of C-3 (δ
83.1) and for the upfield shift of C-28 (δ 176.0), revealed that the sugar moieties were attached to the
aglycon at these two positions. The 1H and 13C-NMR spectra of 1 exhibited four anomeric protons at δ
5.10 × 2 (2H, d, J = 7.5 Hz), 6.27(1H, d, J = 2.5 Hz ), 5.68 (1H, br s) ppm and carbons at δ 106.7 × 2,
1
93.9, 101.5 (Table 1). In the H-NMR spectrum, one methyl signal at δ 1.59 (3H, d, J = 5.5 Hz)
belonging to rhamnose was observed. In addition, the monosaccharides were identified as glucose,
rhamnose and arabinose by TLC and a combination of DEPT, HMQC and HMBC experiments. Acid
hydrolysis of 1 also gave glucose, arabinose and rhamnose in a ratio of 2:1:1 respectively, as
confirmed by GC analysis of the respective trimethylsilyl derivatives [20]. The 1H- and 13C-NMR and
2D-NMR analysis indicated that all the monosaccharides of 1 were in pyranose forms. The β-anomeric
configurations of the D-gulucose units were determined by its 3JH1,H2 coupling constants (7.5 Hz). The
α-anomeric configurations of the L-arabinose and L-rhamnose were determined by the broad singlet of
their anomeric protons [24]. The linkages between sugar moieties and C-3 of the aglycon were
corroborated through HMBC experiments, i.e., H-1 (δ 5.10) of the terminal glucose correlated with
C-6 (δ 70.8) of the inner glucose, and H-1 (δ 5.10) of the inner glucose correlated with C-3 (δ 83.1) of
the sapogenin. The linkages of sugar moieties at C-28 were established based on HMBC correlations
between H-1 (δ 5.68) of rhamnose and C-2 (δ 75.3) of arabinose, and H-1 (δ 6.27) of arabinose and
C-28 (δ 176.0) of aglycone (Figure 2). On the basis of all the above evidence, platycoside N (1) was
identified as 3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-2β,3β,16α,23-tertahydroxyolean-12-
en-28-oic acid 28-O-β-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside.
Table 1. 13C-NMR data of compound 1 in pyridine–d5 (δ ppm).
Position δ C
Position δ C
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
46.8
3-O-Glc
70.1
83.1
42.4
47.5
20.5
33.6
40.2
47.5
37.2
24.4
123.1
144.5
42.4
36.2
74.2
48.6
41.5
1
2
3
4
5
106.7
75.3
78.7
72.1
76.0
70.8
6
Glc
1′
2′
3′
4′
5′
6′
Ara
1
106.7
74.9
78.7
71.6
78.7
62.3
93.9
75.3
70.8
2
3

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Table 1. Cont.
19
20
21
22
23
24
25
26
27
28
29
30
47.2
31.0
36.2
32.2
66.6
16.0
18.6
17.7
27.2
176.0
33.3
24.8
4
5
Rha
1
2
3
4
5
6
66.6
63.7
101.5
71.6
72.7
73.1
68.7
18.6
Figure 2. The key HMBC correlations of compound 1 (from H to C).
The six known saponins were identified as platycodin D (2), deapio platycodin D (3), platycodin D3
(4), deapio platycodin D3 (5), platycoside E (6) and deapio platycoside E (7) through comparison of
their UV, IR, NMR and MS data with literature values [25,26].
3. Experimental
3.1. General
ESI-MS (negative mode) measurements were carried out on an Agilent 1100 series LC/MSD Trap
SL mass spectrometer. HR-ESI-MS (positive and negative modes) was analyzed on a Bruker
FT-ICRMS spectrometer. IR spectra were recorded on an IR-47 spectrometer. NMR spectra were
recorded on a Bruker Avance DRX 400 NMR spectrometer using TMS as internal standard, and
chemical shifts δ were given in ppm. Silica gel (200–300 mesh) for column chromatography and silica

Molecules 2010, 15
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gel G for TLC were purchased from Qingdao Marine Chemical Factory, Qingdao, China.
AB-8 macroporous resin was purchased from Tianjin Nankai factory. Preparative HPLC was
performed on a Waters 600 liquid chromatography instrument with a UV detector, monitored at 210
nm using a C18 column (Zorbax Eclipse XDB, 250 mm × 9 mm; 10 μm)
3.2. Plant material
The roots of P. grandiflorum were purchased at Changchun Guangfulu market in Changchun-city of
Jilin province, China and identified by Prof. Yi-Nan Zheng, College of Chinese Material Medicine,
Jilin Agricultural University. A voucher specimen (No.20050116) has been deposited in the herbarium
of the same college.
3.3. Extraction and isolation
Dry and powdered roots of P. grandiflorum (2.0 Kg) were refluxed three times with 30 L of 70%
methanol, 3 h each time. Extracts were concentrated, suspended in water and sequentially partitioned
with ethyl acetate and n-butanol. The n-butanol fraction was subjected to macroporous resin AB-8
column and eluted sequentially with water, 30% ethanol and 70% ethanol. The 30% ethanol elution
was repeatedly chromatographed on a reverse-phase column, and eluted with aqueous methanol,
affording three fractions A-C. Fraction A was purified by HPLC to afford compounds 1 (23 mg), 2
(15 mg) and 3 (40 mg). Fraction B and C gave 4 (32 mg), 5 (22 mg), 6 (16 mg) and 7 (15 mg).
Platycoside N (1): White amorphous powder; IR (KBr) cm−1: 3425, 2947, 1647, 1616, 1114; ESI-MS
1
m/z: 1105 [M-H]-, HR-ESI-MS m/z 1105.5408 [M-H]- (Calcd for C53H85O24, 1105.5431). H-NMR
(400 MHz, pyridine-d5) δ: 0.89 × 2, 0.98, 1.17, 1.58 × 2 (each 3H, s, CH3 of C-26, C-29, C-30, C-24,
C-25, C-27), 1.59 (3H, d, CH3 of rhamnose), 4.36 (1H, d, J = 3.0Hz, H-3), 3.82, 4.60 (each 1H, d, H-
23), 4.72(1H, m, H-2), 5.10 × 2 (each 1H, d, J = 7.5 Hz, H-1′ and H-1of glucose), 5.68 (1H, br s, H-1
of rhamnose), 6.27 (1H, br s, H-1 of arabinose), 5.46 (1H, br s, H-12) 5.03 (1H, br s, H-16). 13C-NMR
(100 MHz, pyridine-d5) data: see Table 1.
3.4. Acid hydrolysis of 1
Compound 1 (2.0 mg) was refluxed with 4.0 M HCl (5.0 mL) for 1 h at 95 °C, and the reaction
mixture was extracted with ethyl acetate. The aqueous layer was then adjusted to pH 7.0 with
NaHCO3. After evaporating to dryness, the sugar mixture was dissolved in pyridine and developed on
silica gel TLC [CHCl3-MeOH-H2O (7:3:0.5, lower phase), n-BuOH-AcOH-H2O (4:1:5, upper phase).
Three spots were seen on the TLC after spraying with 4% α-naphthol-EtOH-5% H2SO4. Through
comparison with authentic sugar standards (purchased from Sigma), it was found that compound 1
possessed D-glucose, L-rhamnose and L-arabinose units.
4. Conclusions
In summary, we have isolated a new oleanane-type triterpenoid saponin, named platycoside N (1),
together with six known saponins from the roots of Platycodon grandiflorum.

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Acknowledgements
This work was supported by the grant from Key Project in the National Science & Technology
Pillar Program during the Eleventh Five-Year Plan Period (2007BAI38B03); Jilin Science &
Technology Development Plan (20100938).
References and Notes
1. Lee, K.J.; Kim, J.Y.; Jung, K.S.; Choi, C.Y.; Chung, Y.C.; Kim, D.H.; Jeong, H.G. Suppressive
effects of Platycodon grandiflorum on the progress of carbon tetrachloride-induced hepatic
fibrosis. Arch. Pharm. Res. 2004, 27, 1238-1244.
2. Choi, J.H.; Hwang,Y.P.; Lee, H.S.; Jeong, H.G. Inhibitory effect of Platycodi Radix on
ovalbumin-induced airway inflammation in a murine model of asthma. Food Chem. Toxicol. 2009,
47, 1272-1279.
3. Jeong, H.M.; Han, E.H.; Jin, Y.H.; Hwang, Y.P.; Kim, H.G.; Park, B.H.; Kim, J.Y.; Chung, Y.C.;
Lee, K.Y.; Jeong, H.G. Saponins from the roots of Platycodon grandiflorum stimulate osteoblast
differentiation via p38 MAPK- and ERK-dependent RUNX2 activation. Food Chem. Toxicol.
2010, 48, 3362-3368.
4. Lee, E.B. Pharmacological studies on Platycodon grandiflorum A. DC. IV. A comparison of
experimental pharmacological effects of crude platycodin with clinical indications of platycodi
radix. Yakugaku Zasshi 1973, 93, 1188-1194.
5. Kim, K.S.; Ezaki, O.; Ikemoto, S.; Itakura, H. Effects of Platycodon grandiflorum feeding on
serum and liver lipid concentrations in rats with diet-induced hyperlipidemia. J. Nutr. Sci.
Vitaminol. 1995, 41, 485-491.
6. Shin, C.Y.; Lee, W.J.; Lee, E.B.; Choi, E.Y.; Ko, K.H. Platycodin D and D3 increase airway
mucin release in vivo and in vitro in rats and hamsters. Planta Med. 2002, 68, 221-225.
7. Kim, Y.P.; Lee, E.B.; Kim, S.Y.; Li. D.; Ban, H.S.; Lim, S.S.; Shin, K.H.; Ohuchi, K. Inhibition
of prostaglandin E2 production by platycodin D isolated from the root of Platycodon
grandiflorum. Planta Med. 2001, 67, 362-364.
8. Han, L.K.; Xu, B.J.; Kimura, Y.; Zheng, Y.N.; Okuda, H. Platycodi radix affects lipid metabolism
in mice with high fat diet-induced obesity. J. Nutr. 2000, 130, 2760-2764.
9. Han, L.K.; Zheng, Y.N.; Xu, B.J.; Okuda, H.; Kimura, Y. Saponins from platycodi radix
ameliorate high fat diet-induced obesity in mice. J. Nutr. 2002, 132, 2241-2245.
10. Xu, B.J.; Han, L.K.; Zheng, Y.N.; Lee, J.H.; Sung, C.K. In vitro inhibitory effect of triterpenoidal
saponins from Platycodi Radix on pancreatic lipase. Arch. Pharm. Res. 2005, 28, 180-185.
11. Zhao, H.L.; Kim, Y.S. Determination of the kinetic properties of platycodin D for the inhibition of
pancreatic lipase using a 1,2-diglyceride-based colorimetric assay. Arch. Pharm. Res. 2004, 27,
968-972.
12. Kim. M.O.; Moon, D.O.; Choi, Y.H.; Shin, D.Y.; Kang, H.S.; Choi, B.T.; Lee, J.D.; Li, W.; Kim,
G.Y. Platycodin D induces apoptosis and decreases telomerase activity in human leukemia cells.
Cancer Lett. 2008, 261, 98-107.

Molecules 2010, 15
8708
13. Kim, J.Y.; Park, K.W.; Moon, K.D.; Lee, M.K.; Choi, J.; Yee, S.T.; Shim, K.H.; Seo, K.I.
Induction of apoptosis in HT-29 colon cancer cells by crude saponin from Platycodi Radix. Food
Chem. Toxicol. 2008, 46, 3753-3758.
14. Shin, D.Y.; Kim, G.Y.; Li, W.; Choi, B.T.; Kim, N.D.; Kang, H.S.; Choi, Y.H. Implication of
intracellular ROS formation, caspase-3 activation and Egr-1 induction in platycodon D-induced
apoptosis of U937 human leukemia cells. Biomed. Pharmacother. 2009, 63, 86-94.
15. Zhang, L.; Liu, Z.H.; Tian, J.K. Cytotoxic triterpenoid saponins from the roots of Platycodon
grandiflorum. Molecules 2007, 12, 832-841.
16. Lee, H.; Kang, R.; Kim, Y.S.; Chung, S.I.; Yoon, Y. Platycodin D inhibits adipogenesis of 3T3-
L1 cells by modulating kruppel-like factor 2 and peroxisome proliferator-activated receptor
gamma. Phytother. Res. 2009, 24, S161-S167
17. Zheng, J.; He, J.; Ji, B.; Li, Y.; Zhang, X. Antihyperglycemic effects of Platycodon grandiflorum
(Jacq.) A. DC. extract on streptozotocin-induced diabetic mice. Plant Foods Hum. Nutr. 2007, 62,
7-11.
18. Choi, Y.H.; Yoo, D.S.; Choi, C.W.; Cha, M.R.; Kim, Y.S.; Lee, H.S.; Lee, K.R.; Ryu, S.Y.
Platyconic acid A, a genuine triterpenoid saponin from the roots of Platycodon grandiflorum.
Molecules 2008, 13, 2871-2879.
19. Fu, W.W.; Dou, D.Q.; Zhao, C.J.; Shimizu, N.; Pei, Y.P.; Pei, Y.H.; Chen, Y.J.; Takeda, T.
Triterpenoid saponins from Platycodon grandiflorum. J. Asian Nat. Prod. Res. 2007, 9, 35-40.
20. Fu, W.W.; Shimizu, N.; Dou, D.Q.; Takeda, T.; Fu, R.; Pei, Y.H; Chen, Y.J. Five new triterpenoid
saponins from the roots of Platycodon grandiflorum. Chem. Pharm. Bull 2006, 54, 557-560.
21. Kim, Y.S.; Kim, J.S.; Choi, S.U.; Kim, J.S.; Lee, H.S.; Roh, S.H.; Jeong, Y.C.; Kim, Y.K.; Ryu,
S.Y. Isolation of a new saponin and cytotoxic effect of saponins from the root of Platycodon
grandiflorum on human tumor cell lines. Planta Med. 2005, 71, 566-568.
22. Fu, W.W.; Shimizu, N.; Takeda, T.; Dou, D.Q.; Chen, B.; Pei, Y.H.; Chen, Y.J. New A-ring
lactone triterpenoid saponins from the roots of Platycodon grandiflorum. Chem. Pharm. Bull.
2006, 54, 1285-1287.
23. Fu, W.W.; Hou, W.B.; Dou, D.Q.; Hua, H.M.; Gui, M.H.; Fu, R.; Chen, Y.J.; Pei, Y.H. Saponins
of polygalacic acid type from Platycodon grandiflorum (in Chinese). Yao Xue Xue Bao 2006, 41,
358-360.
24. Nikaido, T.; Koike, K.; Mitsunaga, K.; Saeki, T. Two new triterpenoid saponins from Platycodon
grandiflorum. Chem. Pharm. Bull. 1999, 47, 903-904.
25. Li, W.; Xiang, L.; Zhang, J.; Zheng, Y.N.; Han, L.K.; Saito, M. A New Triterpenoid Saponin
from the Roots of Platycodon grandiflorum. Chin. Chem. Lett. 2007, 18, 306-308.
26. He, Z.; Qiao, C.; Han, Q.; Wang, Y.; Ye, W.; Xu, H. New triterpenoid saponins from the roots of
Platycodon grandiflorum. Tetrahedron 2005, 61, 2211-2215.
Sample Availability: Samples of the compounds are available from the authors (liwei7727@126.com).
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