New triterpenoids from the tubers of corydalis ternata: Structural elucidation and bioactivity evaluation

Article abstract of DOI:10.1055/s-0030-1270781

Two new triterpene glycosides, coryternic acid 3-O - D-glucuronopyranoside (1) and coryternic acid 3-O - D-glucuronopyranoside-6-O-methyl ester (2), were isolated from a MeOH extract of the tubers of Corydalis ternata. Acidic hydrolysis of 1 and 2 yielded a new triterpene as their aglycone, coryternic acid (3). The structures of these new compounds were determined through spectral analysis, including extensive 2D-NMR data. In this study we reported that triterpenoids were first isolated from the genus Corydalis. Compound 2 exhibited significant cytotoxicity against the A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines (IC15.16, 17.07, 13.32, and 11.95M, respectively) and significantly reduced NO production in lipopolysaccharide (LPS)-activated microglia/BV-2 cells with an ICvalue of 16.2M without cell toxicity. Georg Thieme Verlag KG Stuttgart - New York.

Full text of DOI:10.1055/s-0030-1270781

Letters 1555
The tubers of Corydalis ternata (Papaveraceae) have been used in
the traditional Korean medicine for the treatment of spasms and
gastric ulcers [1]. C. ternata, well known as Corydalis tuber in Ko-
rea, contains alkaloids such as berberine, coptisine, and proto-
pine as its main chemical constituents [1,2]. Among them, proto-
pine was reported to decrease the glutamate level and increase
the glutamate dehydrogenase (GDH) activity in the brains of rats
[2]. In continuation of the search for bioactive constituents from
Korean medicinal plant sources, we investigated a MeOH extract
of the tubers of C. ternata for its cytotoxic potential based on the
fact that the extract showed considerable cytotoxicity against
four human cancer cell lines in screening procedures [3]. We had
reported the isolation of the cytotoxic alkaloids from the tubers
of C. ternata, recently [3]. In the process of our continuing efforts
to study this source, we further isolated two new triterpene gly-
cosides (1–2), in addition to the identification of a new triterpene
as their aglycone (3), from the MeOH extract of this plant
New Triterpenoids from the
Tubers of Corydalis ternata:
Structural Elucidation and
Bioactivity Evaluation
Ki Hyun Kim1, Il Kyun Lee1, Sang Un Choi2, Jei Hyun Lee3,
Eunjung Moon4, Sun Yeou Kim4, Kang Ro Lee1
1
Natural Products Laboratory, School of Pharmacy,
Sungkyunkwan University, Suwon, Korea
Korea Research Institute of Chemical Technology, Daejeon, Korea
College of Oriental Medicine, Dongguk University, Gyeong Ju, Korea
Graduate School of East-West Medical Science,
2
3
4
Kyung Hee University Global Campus, Gyeonggi-do, Korea
"
Abstract
!
(l Fig. 1) and evaluated the cytotoxicities of 1–3. Moreover, we
also tested the inhibitory activities of 1–3 on neuroinflammation
in the LPS-activated microglia/BV‑2 cell line.
Two new triterpene glycosides, coryternic acid 3-O-β-D-glucuro-
nopyranoside (1) and coryternic acid 3-O-β-D-glucuronopyrano-
side-6′-O-methyl ester (2), were isolated from a MeOH extract of
the tubers of Corydalis ternata. Acidic hydrolysis of 1 and 2
yielded a new triterpene as their aglycone, coryternic acid (3).
The structures of these new compounds were determined
through spectral analysis, including extensive 2D‑NMR data. In
this study we reported that triterpenoids were first isolated from
the genus Corydalis. Compound 2 exhibited significant cytotoxic-
ity against the A549, SK‑OV‑3, SK‑MEL‑2, and HCT-15 cell lines
(IC50 = 15.16, 17.07, 13.32, and 11.95 µM, respectively) and signif-
icantly reduced NO production in lipopolysaccharide (LPS)-acti-
vated microglia/BV‑2 cells with an IC50 value of 16.2 µM without
cell toxicity.
Compound 1 was obtained as a white powder. The molecular for-
mula was established as C37H56O11 from the [M + H]+ peak at m/z
677.3904 (calcd. for C37H57O11: 677.3901) in the HR‑FAB‑MS. The
IR spectrum indicated that 1 possesses hydroxy (3383 cm−1), car-
bonyl (1666 cm−1), and C=C double bond (1642 cm−1) functional
groups. The 1H and 13C NMR spectra of 1 (l Tables 1 and 2) were
"
similar to those of serratagenic acid [4], except for the presence of
an additional OCH3 group [δH 3.70; δC 50.9] and one set of reso-
nances attributable to a β-glucuronopyranosyl unit [δH 4.38 (d,
J = 7.5 Hz); δC 171.2, 105.5, 76.3, 76.3, 73.9, and 71.8] in 1 [5,6].
Comparison of the 13C NMR data of 1 with that of serratagenic ac-
id showed the downfield shift of C-3 (+ 11.5) and upfield shift of
C-2 (− 2.5) in 1, indicating glycosylation at C-3. This linkage was
confirmed by the HMBC correlation between H-1′ (δH 4.38) and
C-3 (δC 89.6). HMBC correlations of H-30 (δH 1.13)/C-29 (δC
177.3) and O-CH3 (δH 3.70)/C-29 (δC 177.3) indicated that the
OCH3 group was located at C-29, and the presence of a carboxyl
group at C-28 was verified by the HMBC correlation of H-18 (δH
Key words
Corydalis ternate · Papaveraceae · triterpenes · cytotoxicity ·
neuroinflammation
"
Abbreviations
!
2.70)/C-28 (δC 179.8) (l Fig. 2). The relative configuration of 1
was confirmed to be identical to that of serratagenic acid in the
NOESY spectrum. The β-orientation of OH-3 was deduced from
the correlations of H-3/H3-23 and H-3/H-5 in the NOSEY spec-
trum, and the methyl ester group at C-29 was reconfirmed by
the NOSEY correlations of H-18/H3-30 and O-CH3/H3-27
SRB: sulforhodamine B
LPS: lipopolysaccharide
NO: nitric oxide
"
Supporting information available online at
(l Fig. 2). Acidic hydrolysis of 1 yielded the aglycone 3 and D-glu-
http://www.thieme-connect.de/ejournals/toc/plantamedica
curonic acid. Extensive studies of the 1D and 2D NMR spectra of 3
led to the identification of a new triterpene as the aglycone, 3β-
hydroxy-olean-12-ene-28,29-dioic acid 29-methyl ester (3, cory-
Fig. 1 Chemical structures of compounds 1–3.
Kim KH et al. New Triterpenoids from… Planta Med 2011; 77: 1555–1558

1556 Letters
Table 1 1H NMR spectral data of compounds 1–3a.
Table 2 13C NMR spectral data of compounds 1–3a.
H
1
2
3
C
1
2
3
1
1.48 m, 1.34 m
1.75 m, 1.45 m
3.17 dd (11.5, 3.5)
0.80 m
1.47 m, 1.33 m
1.77 m, 1.45 m
1.48 m, 1.30 m
1.76 m, 1.40 m
1
2
3
4
5
6
7
8
9
38.3 t
38.3 t
25.5 t
89.6 d
38.7 s
55.5 d
17.8 t
32.5 t
39.1 s
48.2 d
36.4 s
22.8 t
122.8 d
143.3 s
41.3 s
27.4 t
23.0 t
45.6 s
42.5 d
41.9 t
43.6 s
29.8 t
33.6 t
27.0 q
15.5 q
14.5 q
16.2 q
24.8 q
179.8 s
177.3 s
27.2 q
50.8 q
105.6 d
73.8 d
76.0 d
71.7 d
75.2 d
169.9 s
51.3 s
38.6 t
26.6 t
78.5 d
39.3 s
55.5 d
18.3 t
32.8 t
39.3 s
48.3 d
36.9 s
23.0 t
123.0 d
143.5 s
41.5 s
27.6 t
23.3 t
45.8 s
42.8 d
42.1 t
43.8 s
30.1 t
33.8 t
27.4 q
15.1 q
14.7 q
16.4 q
25.1 q
180.0 s
177.6 s
27.5 q
51.1 q
2
25.5 t
89.6 d
38.7 s
55.5 d
17.9 t
32.6 t
39.1 s
48.4 d
36.5 s
22.8 t
122.8 d
143.3 s
41.3 s
27.4 t
23.1 t
45.6 s
42.5 d
41.9 t
43.6 s
29.9 t
33.6 t
27.1 q
15.5 q
14.5 q
16.5 q
25.0 q
179.8 s
177.3 s
27.3 q
50.9 q
105.5 d
73.9 d
76.3 d
71.8 d
76.3 d
171.2 s
3
3.17 dd (12.0, 3.5) 3.15 dd (11.5, 5.0)
5
0.81 m
0.75 m
6
1.55 m, 1.36 m
1.62 m, 1.36 m
1.61 m
1.57 m, 1.34 m
1.62 m, 1.33 m
1.61 m
1.58 m, 1.34 m
1.63 m, 1.34 m
1.62 m
7
9
11
1.92 m, 1.69 m
5.30 br t (1.5)
1.57 m, 1.13 m
1.63 m, 1.33 m
2.70 dd (13.5, 3.5)
1.88 m, 1.63 m
1.92 m, 1.66 m
1.87 m, 1.61 m
1.05 s
1.91 m, 1.67 m
5.29 br t (1.5)
1.58 m, 1.13 m
1.63 m, 1.32 m
1.98 m, 1.69 m
5.30 br t (3.5)
1.59 m, 1.11 m
1.64 m, 1.32 m
12
15
10
16
11
18
2.70 dd (13.5, 3.5) 2.70 dd (14.0, 4.0)
12
19
1.89 m, 1.63 m
1.91 m, 1.65 m
1.88 m, 1.61 m
1.04 s
1.92 m, 1.64 m
1.95 m, 1.66 m
1.90 m, 1.63 m
0.97 s
13
21
14
22
15
23
16
24
0.85 s
0.84 s
0.80 s
17
25
0.95 s
0.94 s
0.94 s
18
26
0.80 s
0.80 s
0.77 s
19
27
1.17 s
1.17 s
1.16 s
20
30
1.13 s
1.13 s
1.13 s
21
29-OCH3
GluA1′
2′
3.70 s
3.69 s
3.69 s
22
4.38 d (7.5)
3.24 t (8.5)
3.37 t (9.0)
3.51 t (9.0)
3.77 br d (8.5)
4.38 d (8.0)
3.24 t (9.0)
3.34 t (9.0)
3.51 t (9.0)
3.82 d (9.5)
3.77 s
23
24
3′
25
4′
26
5′
27
6′-OCH3
28
29
1H NMR run at 500 MHz in CD3OD. Chemical shifts are given in δ values. Proton cou-
a
30
pling constants (J) in Hz are given in parentheses
29-OCH3
GluA1′
2′
3′
ternic acid). D-Glucuronic acid was detected by TLC and identi-
fied based on its optical rotation value [6,7]. Thus, 1 was eluci-
dated as 3β-O-(β-D-glucuronopyranosyl)-olean-12-ene-28,29-
dioic acid 29-methyl ester (coryternic acid 3-O-β-D-glucurono-
pyranoside).
Compound 2 was obtained as a white powder with the molecular
formula C38H58O11 based on the [M + Na]+ peak at m/z 713.3884
(calcd. for C38H58NaO11: 713.3877) in the HR‑FAB‑MS. The 1H and
4′
5′
6′
6′-OCH3
13C NMR run at 125 MHz in CD3OD. Chemical shifts are given in δ values
a
13
"
C NMR spectra of 2 (l Tables 1 and 2) were very similar to those
of 1. The 1H NMR spectrum of 2 displayed the signal for an addi-
tional methoxy group (δH 3.77). The sugar carbon data (l Table 2)
"
revealed a noticeable difference in the δ value of C-6′ (−1.3) in
comparison to the corresponding value in the sugar unit of 1. This
evidence, together with the observation of the HMBC correlation
between O-CH3 (δH 3.77) and C-6′ (δC 169.9), suggested that com-
pound 2 contains a 6′-O-methyl-β-glucuronopyranoside [8].
Acidic hydrolysis and spectral analysis of 2 confirmed the pres-
ence of the 6′-O-methyl-β-D-glucuronopyranoside from the 1H
NMR coupling constant (J = 8.0 Hz) and its optical rotation value
[6–8]. The relative configuration of 2 was confirmed to be identi-
cal to that of 1 in the NOESY spectrum. Thus, 2 was characterized
as 3β-O-(6′-O-methyl-β-D-glucuronopyranosyl)-olean-12-ene-
28,29-dioic acid 29-methyl ester (coryternic acid 3-O-β-D-glu-
curonopyranoside-6′-O-methyl ester). To the best of our knowl-
edge, this is the first study to report triterpenoids being isolated
from the genus Corydalis.
Fig. 2 Key HMBC (→) and NOESY (⇠⇢) correlations of 1.
In this study, the cytotoxicities of 1–3 against the A549, SK‑OV‑3,
SK‑MEL‑2, and HCT15 human cancer cell lines were evaluated us-
ing the sulforhodamine B (SRB) bioassay in vitro [9]. The results
Kim KH et al. New Triterpenoids from… Planta Med 2011; 77: 1555–1558

Letters 1557
tric oxide (NO) production in LPS-activated BV-2 cells. As shown
in l Fig. 3, compound 2 effectively inhibited the NO production
Table 3 Cytotoxicity of compounds 1–3 against four cultured human cancer
cell lines using the SRB assay in vitro.
"
with an IC50 value of 16.2 µM without significant cell toxicity.
Compound 3 also reduced NO levels in the medium; however, it
showed very minimal cell toxicity. Compound 2 might be a po-
tential natural agent for the treatment of various tumors and for
the improvement of neurodegenerative diseases through sup-
pression of neuroinflammation in the brain.
Compound
IC50 (µM)a
A549
SK‑OV‑3
> 30.0
SK‑MEL‑2
HCT-15
1
> 30.0
19.02
13.32
28.71
0.01
20.83
11.95
28.64
0.02
2
15.16
> 30.0
0.01
17.07
> 30.0
0.01
3
Doxorubicinb
a
Materials and Methods
IC50 value of compounds against each cancer cell line, which was defined as the con-
centration (µM) that caused 50% inhibition of cell growth in vitro. b Doxorubicin as a
positive control
!
Coryternic acid 3-O-β‑D-glucuronopyranoside (1): white powder;
mp. 214–215°C; [α]D25: + 58.3 (c 0.25, MeOH); IR (KBr): νmax
=
3383, 2947, 2835, 1666, 1642, 1454, 1026 cm−1; FAB‑MS: m/z =
677 [M + H]+; HR‑FAB‑MS: m/z = 677.3904 [M + H]+ (calcd. for
"
of this assay (l Table 3) showed that compound 2 exhibited sig-
nificant cytotoxicity against the A549, SK‑OV‑3, SK‑MEL‑2, and
HCT-15 cell lines (IC50 = 15.16, 17.07, 13.32, and 11.95 µM, re-
spectively). Neuroinflammation has been implicated in various
neurodegenerative disorders such as Alzheimerʼs and Parkin-
sonʼs disease [10–12]. The inhibitory effects of 1–3 on neuroin-
flammation were also evaluated by assessing cell viability and ni-
1
C37H57O11: 677.3901). H NMR, see l Table 1 and 13C NMR, see
"
"
l Table 2.
Coryternic acid 3-O-β‑D-glucuronopyranoside-6′-O-methyl ester
(2): white powder; mp. 217–218°C; [α]2D5: + 64.2 (c 0.07, MeOH);
IR (KBr): νmax = 3385, 2947, 2835, 1667, 1641, 1453, 1027 cm−1
;
Fig. 3 The effect of compounds 1–3 on cell viabil-
ity (A) and NO production (B) in LPS-activated BV-2
cells. Cell viability was detected by MTT assay, and
NO production was measured by nitrite assay using
a Griess reaction. NOS inhibitor, N-monomethyl-L-
arginine (NMMA), was used as a positive control in
this study. All data are presented as the mean SEM
of three independent experiments. * P < 0.05 and
*** p < 0.001 indicate significant differences com-
pared to treatment with LPS alone.
Kim KH et al. New Triterpenoids from… Planta Med 2011; 77: 1555–1558

1558 Letters
FAB‑MS: m/z = 713 [M + Na]+; HR‑FAB‑MS: m/z = 713.3884 [M +
received September 24, 2010
revised November 30, 2010
accepted January 24, 2011
1
Na]+ (calcd. for C38H58NaO11: 713.3877). H NMR, see l Table 1
"
and 13C NMR, see l Table 2.
"
Coryternic acid (3): white powder; mp. 292–293°C; [α]2D5: + 81.0
(c 0.30, MeOH); IR (KBr): νmax = 3386, 2945, 2835, 1667, 1637,
1454, 1027 cm−1; FAB‑MS: m/z = 501 [M + H]+; HR‑FAB‑MS: m/z =
Bibliography
DOI http://dx.doi.org/10.1055/s-0030-1270781
Published online February 21, 2011
Planta Med 2011; 77: 1555–1558
© Georg Thieme Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
1
501.3585 [M + H]+ (calcd. for C31H49O5: 501.3580). H NMR, see
13
"
"
l Table 1 and C NMR, see l Table 2.
A detailed description of the bioassays is available as Supporting
Information. The positive controls, doxorubicin (purity ≥ 98%)
and N-monomethyl-L-arginine (NMMA, purity ≥ 98%) were pur-
chased from Sigma Corporation.
Correspondence
Prof. Dr. Kang Ro Lee
Natural Products Laboratory,
School of Pharmacy
Sungkyunkwan University
300 Chonchon-dong, Jangan-ku
Suwon 440-746
Supporting information
The spectral data of compounds 1–3, the general experimental
procedures, the isolation details, and details regarding the acidic
hydrolysis of 1–2 and bioassays protocols are available as Sup-
porting Information.
Korea
Phone: + 82312907710
Fax: + 82312907730
krlee@skku.ac.kr
Acknowledgements
!
This study was supported by a grant (09112KFDA890) from the
Korean Food & Drug Administration in 2009. We thank Drs. E.J.
Bang, S.G. Kim, and J.J. Seo at the Korea Basic Science Institute
for their assistance with the NMR spectroscopic and mass spec-
trometric measurements.
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