A new triterpene glycoside from the stems of Akebia quinata

Full text of DOI:10.1002/bkcs.10002

Note
DOI: 10.1002/bkcs.10002
BULLETIN OF THE
H.J. Ko et al.
KOREAN CHEMICAL SOCIETY
A New Triterpene Glycoside from the Stems of Akebia quinata
Hae Ju Ko,† Joo Hee Lee,‡ Yeong Shik Kim,‡ Je Hyun Lee,§ and Eun-Rhan Woo†,
*
†BK-21 Plus Project Team, College of Pharmacy, Chosun University, Gwangju 501-759, South Korea.
*E-mail: wooer@chosun.ac.kr
‡College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742,
South Korea
§Department of Korean Medicine, Dongguk University, Geongju 780-714, South Korea
Received June 19, 2014, Accepted September 1, 2014, Published online January 5, 2015
Keywords: Akebia quinata, Lardizabalaceae, Triterpene glycoside
Akebiae caulis, dried stems of Akebia quinata (Lardizabalaceae),
is an important crude drug used mainly as a diuretic agent for
the treatment of edema and rheumatic pain.1,2 Previous phyto-
chemical investigations resulted in the isolation of triterpenes,
triterpene glycosides, and phenylethanoid glycosides.3–5
Regarding the biological activity of A. quinata, only the cyto-
toxic effect of oleanane disaccharides has been reported so
far.6 The anti-inflammatory activity of this plant has not been
explored yet. We have recently reported the isolation of lignan
glycoside derivatives with an anti-inflammatory effect from
this plant.7 In continuing research on this source, a new triter-
pene glycoside (1) and seven known triterpene glycosides
(2–8) were further isolated from the n-BuOH soluble fraction.
The structure of 1 was identified on the basis of 1D and 2D
NMR, including 1H-1H COSY, HSQC, HMBC, and NOESY
spectroscopic analyses. Here we report the isolation, structure
elucidation of the new triterpene glycoside (1), and the inhib-
itory effects of all isolates on the NO production in lipopoly-
saccharides (LPS)-stimulated RAW 264.7 cells (Figure 1).
Compound 1 was obtained as a white amorphous powder,
tertiary methyl groups at δC 16.3, 17.9, 18.9, and 26.4 (CH3-
24, 25, 26, and 27), two oxygenated carbons at δC 77.5 (C-3)
and 71.6 (C-23), an olefinic carbon at δC 143.8 (C-12) and
123.4 (C-13), an exomethylene carbon at δC 107.6 (C-29),
and a carbonyl carbon at δC 176.2 (C-28) were assigned to a
30-noroleanane type of triterpene.8 Another 18 carbon signals
were assigned to three sugar moieties. Based on the 1H and 13
C
NMR data, the structure of aglycone of 1 was closely related to
quinatic acid, which was isolated from the callus tissue of
A. quinata8 except for the different substituent of C-23 in 1.
Meanwhile, three anomeric proton signals at δH 6.23 (1H, d,
J = 7.6 Hz, glc-H10), 5.88 (1H, br s, Rha-H1000), and 4.98
(1H, d, J = 7.6 Hz, glc-H100) showed HMQC correlations with
anomeric carbon signals at δC 96.1 (glc-C10), 103.0 (Rha-
C1000), and 105.3 (glc-C100), indicating that 1 possessed
three sugar units (Table 1).9,10 Acid hydrolysis of 1 with
1M HCl in MeOH produced aglycone, as well as L-rhamnose
and D-glucose as the sugar moieties. These sugars were iden-
tified by direct high-performance liquid chromatography
(HPLC) analysis of the hydrolysate, which was performed
on an aminopropyl-bonded silica gel column, and direct
comparison with an authentic sample using co-TLC (sugars
of Compound 1was confirmed by HPLC. And compound 1
direct comparison with an authentic sample using co-
TLC.).4 Furthermore, the configuration of the glucopyranosyl
and rhamnopyranosyl were assigned to be β– and α–, respec-
tively, according to the coupling constants of the anomeric
protons.11 In addition, the upfield chemical shift of C-
28 (δC 176.2) indicated that sugar moieties should be
located at C-28 in 1. The sequence of sugar residues of
1 was assigned unambiguously on the basis of HMBC
spectra. The location of the sugar chain at C-28 was identified
as O-α-L-rhamnopyranosyl-(1!4)-O-β-D-glucopyranosyl-
(1!6)-O-β-D-glucopyranosyl from the following HMBC
correlations: H-1000 of rhamnose (δH 5.88)with C-400 of glucose
(δC 78.6), H-100 of glucose (δH 4.98) with C-60 of glucose (δC
69.7), and H-10 of glucose (δH 6.23) with C-28 of aglycone (δC
176.2). Furthermore, the HMBC correlation between H-29
and C-19/C-21 indicated that 20(29)-exomethylene group
was connected at C-20. The relative configuration of the
25
½αꢀD + 19.4ꢁ (c 0.77, MeOH). Its molecular formula was iden-
tified as C47H74O18 by the high-resolution electrospray ioni-
zation time-of-flight mass spectrometry (HR-ESI-TOF-MS)
data at m/z 949.4770 [M + Na]+ (C47H74O18Na, calcd. for
949.4773). In the infrared (IR) spectrum, absorption bands
for hydroxyl (3350 cm−1) and carbonyl (1730 cm−1) groups
1
were observed. The H-NMR spectrum of 1 (Table 1) dis-
played signals due to four tertiary methyl groups at δH 0.81,
1.00, 1.14, and 1.16 (each 3H, s, H-24, 25, 26, 27), an olefinic
proton at δH 5.47 (1H, br s, H-12), an oxygenated methine pro-
ton at δH 3.69 (1H, br s, H-3), an exomethylene proton each at
δH 4.68 and 4.79 (each 1H, s, H-29a, and H-29b), one hydro-
xymethyl proton at δH 3.67 (1H, d, J = 10.6 Hz, H-23a) and
3.87 (1H, d, J = 10.6 Hz, H-23b), and three anomeric protons
at δH 6.23 (1H, d, J = 7.6 Hz, H-10), 5.88 (1H, br s, H-1000), and
4.98 (1H, d, J =7.6 Hz, H-100). The spectral features and phys-
icochemical properties of 1 suggested that 1 would be a triter-
pene glycoside. The 13C-NMR spectrum displayed 47 carbon
signals; 29 of them were assigned to the aglycone part, and the
others to the oligosaccharide. In the 13C-NMR spectrum, four
Bull. Korean Chem. Soc. 2015, Vol. 36, 356–359
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Wiley Online Library
356

BULLETIN OF THE
Note
KOREAN CHEMICAL SOCIETY
29
30
29
12
12
25
25
O
C
O R3
28
28
O
3
27
HO
3
O
7
27
7
O
HO
O
23
24
CH2OH
O
R2O
23
O
1'
OH
24
O
glc'
1''
HO
OH
HO
HO
R1
R2
R3
glc''
2
3
4
5
6
7
8
Rha
H
H
Rha
Rha
H
H
Glc
H
GlcUA
Glc
GlcUA
H
Rha(1 → 4)-Glc(1 → 6)-Glc-
Rha(1 → 4)-Glc(1 → 6)-Glc-
Rha(1 → 4)-Glc(1 → 6)-Glc-
Rha(1 → 4)-Glc(1 → 6)-Glc-
Rha(1 → 4)-Glc(1 → 6)-Glc-
Rha(1 → 4)-Glc(1 → 6)-Glc-
Rha(1 → 4)-Glc(1 → 6)-Glc-
O
HO
1'''
1
H3C
HO
O
rha'''
HO
OH
Rha
Figure 1. Structures of compounds 1–8 from A. quinata.
Table 1. 1H- (600 MHz) and 13C-NMR (150 MHz) data of 1 in pyridine-d5 (δ in ppm)
Position
δC
δH
Position
δC
δH
6.23 (d, J = 8.1)
4.12 (dd, J = 9.0, 8.1)
4.22 (dd, J = 9.0, 9.0)
4.31 (dd, J = 9.0, 9.0)
4.08 (m)
4.65 (dd, J = 11.7, 3.0)
4.29 (m)
4.98 (d, J = 7.8)
3.91 (dd, J = 9.2, 7.8)
4.12 (dd, J = 9.2, 9.2)
4.36 (dd, J = 9.2, 9.2)
3.63 (dd, J = 9.2, 3.2)
4.18 (br d, J = 11.9)
4.07 (dd, J = 11.9, 3.2)
5.88 (br s)
4.66 (br d, J = 3.3)
4.53 (dd, J = 9.3, 3.3)
4.31 (dd, J = 9.3, 9.3)
4.91 (dd, J = 9.3, 6.2)
1.73 (d, J = 6.2)
1
2
3
4
5
6
7
8
33.7
26.9
77.5
43.8
41.0
18.5
33.1
40.4
48.3
37.6
23.8
123.4
143.8
42.5
28.5
24.1
47.7
47.9
42.0
148.7
30.4
38.0
71.6
18.9
16.3
17.9
26.4
176.1
107.6
1.40 (2H, m)
Glc-10
20
96.1
74.1
79.0
71.2
78.3
69.6
1.85 (1H, m), 1.11 (1H, m)
3.94 (1H, br s)
30
40
1.69 (1H, m)
1.72 (2H, m)
1.35 (2H, m)
50
60a
60b
Glc-100
200
105.3
75.6
76.1
78.5
76.9
61.6
9
1.93 (1H, m)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
300
1.96 (2H, m)
5.48 (1H, br s)
400
500
600a
600b
Rha-1000
2000
1.17 (1H, m), 2.31 (1H, m)
2.00 (2H, m)
103.0
72.9
73.1
74.3
70.6
18.7
3000
3.14 (dd, J =14.5, 4.0)
4000
2.19 (2H, m)
5000
6000
2.18 (1H, m), 2.08 (1H, m)
2.02 (1H, m),1.75 (1H, m)
3.87, 3.67 (each, 1H, d, J = 10.5)
0.81 (3H, s)
1.00 (3H, s)
1.14 (3H, s)
1.16 (3H, s)
4.74 (1H, br s), 4.68 (1H, br s)
Assignments were based on 2D NMR including COSY, HMQC and HMBC. Well-resolved couplings are expressed with coupling patterns and coupling
constants in Hertz in parentheses.
Bull. Korean Chem. Soc. 2015, Vol. 36, 356–359
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
357

BULLETIN OF THE
Note
KOREAN CHEMICAL SOCIETY
29
12
25
O
O
28
3
27
HO
HO
O
O
H
1'
OH
CH2OH
CH2OH
O
O
23
O
24
O
OH
O
glc'
glc'
1''
O
HO
HO HO
HO
OH
HO
HO
OH
glc''
glc''
HO
O
O
HO
1'''
H3C
HO
H3C
HO
O
O
rha'''
rha'''
HO
HO
OH
OH
Figure 2. Key HMBC and NOESY correlations of compound.
hydroxyl groups at C-3 and C-23 was proposed from the
NOESY experiment (Figure 2) and from the comparison of
theobservedandreportedNMRdata.3,12 Inthe1H-NMRspec-
trum, the coupling pattern of H-3 (δH 3.69, br s) supported the
β-orientationofH-3. Inaddition, intheNOESYspectrum, cor-
relations between H-3 and Me-24, Me-24, and Me-25/Me-26
indicated that these are on the same side (β). Based on these
results, the relative configuration of the hydroxyl groups at
C-3 andC-23wereidentified asthe α form. Accordingly, com-
pound 1 was identified as 3α,23α-dihydroxy-30-norolean-
12,20(29)-dien-28-oic acid 28-O-α-L-rhamnopyranosyl-
(1!4)-O-β-D-glucopyranosyl-(1!6)-O-β-D-glucopyranosyl
ester, named, akeqintoside E, as shown in Figure 1.
The structures of the other known compounds (2–8) were
identified as akeboside Sth (2),13 begoniifolide A (3),14 akebia
saponin PJ2 (4),15 3β-[(O-GlcUA-(1–3)-O-[Rha-(1–2)]-Ara)
oxy]olean-12-en-28-oic acid O-Rha-(1–4)-Glc-(1–6)-Glc
ester (5),4 kalopanax saponin D (6),13 3β-[(O-GlcUA-(1–3)-
Ara)oxy]olean-12-en-28-oic acid O-Rha-(1–4)-Glc-(1–6)-
Glc ester (7),4 and akebia saponin PJ3 (8)5 by comparing their
spectroscopic data with those reported in the literature. All iso-
lated compounds were examined for their inhibitory effects on
LPS-induced NO production in RAW 264.7 cells. The IC50
values of the isolated compounds (1–8) were more than
100 μM, while that for compound 8 was 65.59 μM (data not
shown). All isolated triterpene glycosides (1–8) were inactive.
heteronuclear multiple quantum coherence (HMQC) and
HMBC experiments, were recorded on a Varian VNMRS
600 MHz spectrometer (KBSI-Gwangju center) operating at
600 (1H) and 150 MHz (13C), respectively, with chemical
shifts given in ppm (δ). TLC was carried out on precoated Kie-
selgel 60 F254 (art. 5715, Merck, Germany) and RP-18 F254
s
(art. 15389, Merck, Germany) plates. Column chromatogra-
phy (CC) was performed on silica gel 60 (40–63 and
63–200 μM, Merck, Germany), Diaion HP 20 (75–150 μM,
Mitsubishi Chemical Co., USA), and Sephadex LH-20
(25–100 μM, Sigma, Sweden). Silver carbonate (Ag2CO3,
Aldrich Co., Koenigs–Knorr glucosylation, USA), D-(+)glu-
cose (C6H12O6, Sigma), and L-(+)rhamnose (C6H12O5,
Sigma) were used as neutralization reagents, and standard
sugar on acid hydrolysis experiment, respectively. HPLC
was performed using a Waters HPLC system equipped with
Waters 600 Q-pumps, an ELSD detector, and a Capcell Pak
(NH2) UG 80 column (250 × 4.6 mm, 5 μM, Shiseido, Japan)
(flow rate 1.0 mL/min, solvent (MeCN/H2O = 17:3)). Low-
pressure liquid chromatography was carried out over a Merck
Lichroprep Lobar®-A RP-18 (240 × 10 mm) column with an
FMI QSY-0 pump (ISCO).
Plant Material. The stems of A. quinata were collected in
Gyeongju, Gyeongbuk province, Korea, in August 2011
and identified by Professor J.H. Lee, one of the authors.
Avoucher specimen (CSU-877-17) is deposited intheHerbar-
ium of the College of Pharmacy, Chosun University.
Experimental
Extraction and Isolation. The air-dried stems of A. quinata
(11 kg) were cut and extracted with MeOH three times for 4 h
at 80 ꢁC. The resultant MeOH extract (480 g) was suspended
in water (1.5 l × 3) and then partitioned sequentially with equal
volumes of dichloromethane, ethyl acetate, and n-butanol.
Each fraction was evaporated in-vaccuo to yield the residues
of CH2Cl2 (45.2 g), EtOAc (11.0 g), n-BuOH (57.0 g), and
General Procedures. Optical rotations were measured using
an Autopol-IV polarimeter. IR spectra were recorded on an
IMS 85 (Bruker). HR-ESI-MS spectra were obtained on a
Q-TOF (Synapt HDMS system, Waters, USA) mass spec-
trometer. NMR spectra, including NOESY, COSY,
Bull. Korean Chem. Soc. 2015, Vol. 36, 356–359
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
358

BULLETIN OF THE
Note
KOREAN CHEMICAL SOCIETY
water(150.3 g)extracts. Then-BuOHsolublefraction(22.0 g)
was subjected to CC over a Diaion HP 20 column and eluted
with H2O/MeOH (100:0 ! 0:100) gradient system to yield six
subfractions (B1–B6). Subfraction B5 (2.6 g) was purified by
Sephadex LH 20 CC (MeOH/H2O, 1:1 ! 2:1) to yield three
subfractions (B51–B53). Subfraction B51 (1.6 g) was purified
byLichroprepRP 18 CC(MeOH/H2O, 3:2) toyield10 subfrac-
tions (B511–B5110). Subfraction B514 (0.14 g) was purified
by silica gel CC (CHCl3/MeOH/H2O, 4:1:0.2 ! 2:1:0.2) to
yield 8 (85.2 mg). Subfraction B515 (0.13 g) was purified by
silica gel CC (CHCl3/MeOH/H2O, 2:1:0.2) to yield 3 (18.7
mg), 4 (46.6 mg), and 1 (9.3 mg). In addition, subfraction
B516 (0.13 g) was purified by silica gel CC (CHCl3/MeOH/
H2O, 2:1:0.2) to yield 5 (13.1 mg) and 6 (20.7 mg). Subfraction
B517 (0.13 g), and B519 (0.12 g) were then purified by silica
gel CC (CHCl3/MeOH/H2O, 2:1:0.2 and 3:1:0.2) to yield 2
(35.9 mg) and 7 (22.4 mg), respectively.
remaining after Griess assay were used for cell viability with
the MTT-based colorimetric assay.
Acknowledgments. This work was supported by a grant
(12172MFDS989) from the Ministry of Food and Drug
Safety, Govt. ofKorea, in2013–2014. WethankDr. K.D. Park
at the Korea Basic Science Institute for Gwangju Center for
help in obtaining the NMR spectra.
Supporting Information. Spectral data of compound 1, gen-
eral experimental procedures, and the isolation details are
available upon request from the corresponding author.
References
1. D. K. Ahan, Illustrated Book of Korean Medicinal Herbs,
Kyo-Hak Publishing Co, Korea, 1998, p. 384.
2. D. Bensky, S. Clavey, E. Stöger, Chinese Herbal Medicine:
Materia Medica, 3rd ed., Eastland Press, Seattle, U.S.A.,
2004, p. 283.
3. H. Gao, Z. Wang, Phytochemstry 2006, 67, 2697.
4. Y. Mimaki, S. Doi, M. Kuroda, A. Yokosuka, Chem. Pharm.
Bull. 2007, 55, 1319.
5. Y. Mimaki, M. Kuroda, A. Yokosuka, H. Hiroshi, M. Fukush-
ima, Y. Sashida, Chem. Pharm. Bull. 2003, 51, 960.
6. H.-J. Jung, C. O. Lee, K.-T. Lee, J. Choi, H.-J. Park, Biol. Pharm.
Bull. 2004, 27, 744.
7. H.-G. Jin, A. R. Kim, H. J. Ko, S. K. Lee, E.-R. Woo, Chem.
Pharm. Bull. 2014, 62, 288.
8. A. Ikuta, H. Itokawa, Phytochemistry 1988, 27, 3809.
9. W. Li, Y. Ding, Y. N. Sun, X. T. Yan, S. Y. Yang, C. W. Choi,
J. Y. Cha, Y. M. Lee, Y. H. Kim, Chem. Pharm. Bull. 2013,
61, 471.
10. S.-G. Li, X.-J. Huang, M.-M. Li, M. Wang, R.-B. Feng,
W. Zhang, Y.-L. Li, Y. Wang, W.-C. Ye, Chem. Pharm. Bull.
2014, 62, 35.
11. S. M. Sang, A. Lao, H. C. Wang, Z. Chen, Phytochemistry 1999,
52, 1611.
12. M. Kuroda, T. Shizume, Y. Mimaki, Chem. Pharm. Bull. 2014,
62, 92.
Acidic Hydrolysis of Compound 1. Compound 1 (3 mg) was
dissolved in 1N HCl (1 mL) and MeOH (1 mL), and refluxed
at 90 ꢁC for 90 min.16 The reaction solution was evaporated
under reduced pressure, and the hydrolysate was extracted
with EtOAc (3 × 3 mL). The aqueous fraction was neutralized
with Ag2CO3 and filtered. The filtrate was then concentrated
under reduced pressure. The residue was compared with a
standard sugar using TLC (CHCl3:MeOH:H2O, 6/4/1), which
revealed the sugar to be D-(+)glucose (Rf = 0.26), and L-(+)
rhamnose (Rf = 0.46) in 1. In addition, HPLC of the sugar frac-
tion showed the presence of L-rhamnose and D-glucose; tR
(min) 7.6 and 15.0, respectively.
25
Akeqintoside E (1). Colorless gum; ½αꢀD +19.4ꢁ (c 0.77,
MeOH); IR (KBr) νmax 3350, 2950, 2830, 1730, 1660,
1
1452, 1115, 1032 cm−1; H-NMR and 13C-NMR data, see
Table 1; HR-ESI-TOF-MS (positive mode) m/z: 949.4770
[M + Na]+ (calcd. for C47H74O18Na, 949.4773).
Measurement of NO Production and Cell Viability Assay.
Nitric oxide production was determined by measuring the
amount of nitrite from cell culture supernatant as previously
described.17 Briefly, RAW 264.7 cells (1 × 105 cells/well)
were cultured in a flat-bottom 96-well microtiter plate in quad-
ruplicate for 12 h. Thereafter, 100 μL of the medium was
replaced with fresh medium containing either compound
and 1 μg/mL of LPS (Sigma Chemical Co., St. Louis, MO,
USA), and further cultured for 24 h. The culture supernatant
was collected at the end of culture for nitrite assay, which
was used as a measure of NO production. The culture super-
natant (100 μL) was mixed with equal volume of Griess rea-
gent at room temperature for 10 min. The absorbance was
measured at 540 nm by a microplate reader. The cells
13. C.-J. Shao, R. Kasai, J. D. Xu, O. Tanaka, Chem. Pharm. Bull.
1989, 37, 311.
14. X. Liao, B.-G. Li, L.-S. Ding, Y.-J. Pan, Y.-Z. Chen, Acta
Pharm. Sin. 2000, 35, 821.
15. X.-C. Li, D.-J. Wang, S.-G. Wu, C.-R. Yang, Phytochemistry
1990, 29, 595.
16. M. R. Kim, H.-I. Moon, J. H. Chung, Y. H. Moon, K.-S. Hahm,
E.-R. Woo, Chem. Pharm. Bull. 2004, 52, 1466.
17. S. Khan, E. M. Shin, R. J. Choi, Y. H. Jung, J. Kim, A. Tosun,
Y. S. Kim, J. Cell. Biochem. 2011, 112, 2179.
Bull. Korean Chem. Soc. 2015, Vol. 36, 356–359
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
359