Arch. Pharm. Res.

DOI 10.1007/s12272-014-0357-x

RESEARCH ARTICLE

A new megastigmane glycoside from Akebia quinata

Hong-Guang Jin • A Ryun Kim • Hae Ju Ko •

Eun-Rhan Woo

Received: 20 December 2013 / Accepted: 9 February 2014

Ó The Pharmaceutical Society of Korea 2014

Abstract A new megastigmane glycoside, 8S*,9R*- Introduction

megastigman-3-one-4,6-diene-8,9-diol-9-O-b-D-glucopy-
ranoside, named akequintoside D (1), as well as six known Akebia quinata DECAISENE (Lardizabalaceae) is a creeping
compounds, roseoside II (2), 3-O-caffeoylquinic acid (3), woody vine which is widely distributed in East Asia,
methyl-3-O-caffeoylquinate (4), 3,4,5-trimethoxyphenyl-b- including Korea, China, and Japan (Lee 2003). Its dried
D-glucopyranoside (5), cuneataside D (6), 3,4-dimethoxy- stem is used as a diuretic agent for the treatment of the
phenyl-6-O-(a-L-rhamnopyranosyl)-b-D-glucopyranoside painful urinary dribbling, edema and ascites (Ahn 1998;
(7) were isolated from the stem of Akebia quinata. The Bensky et al. 2004). Previous phytochemical investigations
structures of compounds (1-7) were identified based on 1D resulted in the isolation of triterpenes, triterpene glyco-
and 2D NMR, including 1H–1H COSY, HSQC, HMBC and sides, and phenylethanoid glycosides (Gao and Wang
NOESY spectroscopic analyses. The inhibitory activity of 2006; Mimaki et al. 2003; 2007). Regarding the biological
these isolated compounds against interleukin-6 (IL-6) activity of A. quinata, only the cytotoxic effect of oleanane
production in TNF-a stimulated MG-63 cells was also disaccharides has been reported so far (Jung et al. 2004).
examined. Moreover, the anti-inflammatory activity of this plant has
not been explored in detail.
Keywords Megastigmane glycoside  IL-6 inhibitory In an ongoing investigation into anti-inflammatory
effect  Lardizabalaceae  Akebia quinata compounds from this plant, the methanol extract of A.
quinata was investigated. By means of repeated column
chromatography using silica gel, MCI gel, Sephadex LH-
20, and LiChroprep RP-18, a new megastigmane glycoside,
akequintoside D (1), along with six known compounds were
isolated. The structures of the known compounds were
identified as roseoside II (2), 3-O-caffeoylquinic acid (3),
methyl-3-O-caffeoylquinate (4), 3,4,5-trimethoxyphenyl-
b-D-glucopyranoside (5), cuneataside D (6), and 3,4-dime-
thoxyphenyl-6-O-(a-L-rhamnopyranosyl)-b-D-glucopyranoside
Electronic supplementary material The online version of this (7), by comparing their spectroscopic data with those
article (doi:10.1007/s12272-014-0357-x) contains supplementary reported in the literature (Fig. 1). Furthermore, these six
material, which is available to authorized users.
known compounds were isolated from this plant for the first
H.-G. Jin  A. R. Kim  H. J. Ko  E.-R. Woo (&) time. The inhibitory activity of these isolated compounds
College of Pharmacy, Chosun University, 375 Seosuk-dong, against IL-6 production in TNF-a stimulated MG-63 cells
Dong-gu, Gwangju 501-759, Republic of Korea was examined.
e-mail: [email protected]
This paper reports the isolation and structural charac-
H.-G. Jin terization of these compounds and their inhibitory activities
College of Pharmacy, Jilin Medical College, Jilin 132013, China against IL-6 production.

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 H.-G. Jin et al.

10
11 12
1 6 8 HO
O 1' OH HO
3 5 OH O OH
OH O OH OH
O 13 6' O
O
OH
OH
1 2

R2
HO COOR
R1 R3
O
OH
HO O
HO
OH O OH
OH OH
O
OR4

3 R=H 5 R1 = OCH3 R2 = OCH3 R3 = OCH3 R4 = H

4 R = CH3 6 R1 = OCH3 R2 = OH R3 = OH R4 = Rha
7 R1 = OCH3 R2 = OCH3 R3 = H R4 = Rha

Fig. 1 Structures of compounds 1–7

Materials and methods Plant materials

General experimental procedure The stem of A. quinata were collected in Gyeongju, Gy-
eongbuk province, Korea, in August 2011 and identified by
Optical rotations were measured using an Autopol-IV Dr. J. H. Lee, Professor of the department of Korean
polarimeter. IR spectra were recorded on an IMS 85 Medicine, Dongguk University. A voucher specimen
(Bruker). CD spectra were recorded on a JASCO J-810 (CSU-877-17) was deposited in the Herbarium of the
spectropolarimeter. HR-ESI–MS spectra were obtained on College of Pharmacy, Chosun University.
a Q-TOF (Synapt HDMS system, Waters, USA) mass
spectrometer. NMR spectra, including NOESY, COSY, Extraction and isolation
heteronuclear multiple quantum coherence (HMQC) and
HMBC experiments, were recorded on a Varian UNITY The air-dried stem of A. quinata (11 kg) were cut and
INOVA 500 NMR spectrometer (KBSI-Gwangju center) extracted with MeOH three times for 4 h at 80 °C. The
operating at 500 MHz (1H) and 125 MHz (13C), respec- resultant MeOH extract (480 g) was suspended in water
tively, with chemical shifts given in ppm (d). TLC was (1.5 L 9 3) and then partitioned sequentially with equal
carried out on precoated Kieselgel 60 F254 (art. 5715, volumes of dichloromethane, ethyl acetate, and n-butanol.
Merck) and RP-18 F254s (art. 15389, Merck) plates. Col- Each fraction was evaporated in vaccuo to yield the resi-
umn chromatography was performed on silica gel 60 dues of CH2Cl2 (45.2 g), EtOAc (11.0 g), n-BuOH
(40–63 and 63–200 lm, Merck), MCI gel CHP20P (57.0 g), and water (150.3 g) extracts. The n-BuOH soluble
(75–150 lm, Mitsubishi Chemical Co.), and Sephadex LH- fraction (57.0 g) was subjected to column chromatography
20 (25–100 lm, Sigma). Silver carbonate (Ag2CO3, (CC) over a diaion HP 20 column and eluted with H2O/
Aldrich Co.) and (?)-D-glucose (C6H12O6, Sigma) were MeOH (100:0 ? 0:100) gradient system. The fractions
used as neutralization reagent and standard sugar on acid were combined based on their TLC pattern to yield sub-
hydrolysis experiment, respectively. Low pressure liquid fractions designated B1–B6. Fraction B2 (3.47 g) was
chromatography was carried out over a Merck Lichroprep purified by MCI gel CC (MeOH/H2O, 1:9 ? 2:8) to yield
LobarÒ-A RP-18 (240 9 10 mm) column with a FMI four subfractions (B21–B24). Subfraction B23 (0.54 g),
QSY-0 pump (ISCO). containing 3, 5, and 7 was purified by Lichroprep RP 18

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Megastigmane glycoside from Akebia quinata

Table 1 1H,13C NMR Data of No. 1 2

1 and 2
dH dC dH dC

1 40.1 42.6
2 2.37 (2H, s) 54.6 2.15 (1H, d, J = 17.0 Hz) 50.8
2.53 (1H, d, J = 17.0 Hz)
3 202.1 201.3
4 5.96 (1H, s) 127.2 5.86 (1H, s) 127.3
5 159.2 167.4
6 145.1 80.1
7 6.10 (1H, d, J = 9.5 Hz) 135.5 5.87 (1H, d, J = 1.5 Hz) 131.7
8 4.93 (1H, dd, J = 4.0, 9.5 Hz) 71.4 5.87 (1H, d, J = 3.0 Hz) 135.4
9 3.96 (1H, dd, J = 4.0, 6.5 Hz) 79.6 4.42 (1H, qdd, J = 1.5, 3.0, 6.5 Hz) 77.4
10 1.24 (3H, d, J = 6.5 Hz) 15.6 1.29 (3H, d, J = 6.5 Hz) 21.3
11 1.34 (3H, s) 29.8 1.04 (3H, s) 23.6
12 1.38 (3H, s) 29.9 1.03 (3H, s) 24.8
13 2.14 (3H, s) 22.9 1.92 (3H, s) 19.3
10 4.38 (1H, d, J = 7.5 Hz) 103.1 4.34 (1H, d, J = 7.5 Hz) 102.9
20 3.20 (1H, dd, J = 7.5, 9.0 Hz) 75.1 3.21 (1H, dd, J = 7.5, 9.0 Hz) 75.4
30 3.37 (1H, dd, J = 9.0, 9.0 Hz) 78.1 3.38 (1H, dd, J = 9.0, 9.0 Hz) 78.2
1 40 3.29 (1H, dd, J = 9.0, 9.0 Hz) 71.7 3.30 (1H, dd, J = 9.0, 9.0 Hz) 71.8
500 MHz, CD3OD for H and
125 MHz for 13C NMR; 50 3.29 (1H, ddd, J = 2.0, 5.0, 9.0 Hz) 78.0 3.29 (1H, ddd, J = 2.0, 5.0, 9.0 Hz) 78.2
chemical shifts in ppm relative 60 3.67 (1H, dd, J = 5.0, 11.5 Hz) 62.8 3.63 (1H, dd, J = 5.0, 11.5 Hz) 63.0
to TMS; coupling constants 3.86 (1H, dd, J = 2.0, 11.5 Hz) 3.86 (1H, dd, J = 2.0, 11.5 Hz)
(J) in Hz

CC (MeOH/H2O, 1:20), and finally by silica gel CC 3-O-Caffeoylquinic acid (3)

(CHCl3/MeOH/H2O, 4:1:0.2 ? 2:1:0.2) to yield 3
(121.0 mg), 5 (26.5 mg), and 7 (17.0 mg). In addition, Brown powder; [a]20 D -134.6° (MeOH; c 0.44); ESI–MS
subfraction B21 (2.04 g) was purified by Sephadex LH 20 m/z: 353 [M-H]?; IR mmax (film) cm-l: 3,412, 2,928,
CC (MeOH/H2O, 1:20), and finally by silica gel CC 1,689, 1,082, 853; 1H NMR (500 MHz, CD3OD) 7.57 (1H,
(CHCl3/MeOH/H2O, 4:1:0.2 ? 2:1:0.2) to yield 6 d, J = 16.0 Hz, H-70 ), 7.05 (1H, d, J = 2.0 Hz, H-20 ), 6.94
(15.0 mg). Fraction B3 (5.6 g) was subjected to silica gel (1H, dd, J = 2.0, 8.0 Hz, H-60 ), 6.77 (1H, d, J = 8.0 Hz,
CC eluting with a CHCl3/MeOH/H2O (6:1:0.1 ? 2:1:0.1) H-50 ), 6.29 (1H, d, J = 16.0 Hz, H-80 ), 5.38 (1H, dd,
in a gradient system to yield six subfractions (B31–B36). J = 5.0, 10.0 Hz, H-3), 4.13 (1H, dd, J = 3.0, 6.0 Hz,
Subfraction B31 (0.79 g) was then purified by repeated H-5), 3.68 (1H, dd, J = 3.0, 10.0 Hz, H-4), 2.15 (1H, dd,
Lichroprep RP 18 CC (MeOH/H2O, 1:3) to yield 1 J = 3.0, 15.0 Hz, H-6), 2.10 (1H, m, H-2), 2.00 (1H, m,
(4.4 mg), 2 (3.2 mg), and 4 (3.0 mg). H-2), 1.94 (1H, dd, J = 3.0, 15.0 Hz, H-6); 13C NMR
(125 MHz, CD3OD) d: 181.1 (C-7), 169.3 (C-90 ), 149.7 (C-
Akeqintoside D (1) 40 ), 147.0 (C-70 ), 146.9 (C-30 ), 127.9 (C-10 ), 123.1 (C-60 ),
116.6 (C-50 ), 115.7 (C-20 ), 115.2 (C-80 ), 77.9 (C-1), 75.2
Colorless gum; [a]20
D -19.6° (MeOH; c 0.37); HR-ESI–MS (C-4), 73.2 (C-5), 72.8 (C-3), 40.8 (C-2), 39.2 (C-6).
(positive mode) m/z: 409.1841 [M ? Na]? (calcd for C19-
H30O8Na, 409.1838); IR mmax (film) cm-1: 3,410, 2,935, Methyl-3-O-caffeoylquinate (4)
1,655, 1,650, 1,076, 1,035; 1H, and 13C NMR: see Table 1.
Pale brownish powder; [a]20D -30.7° (MeOH; c 0.15); ESI–
Roseoside II (2) MS m/z: 367 [M-H]?; IR mmax (film) cm-l: 3,408, 2,930,
1,672, 1,082, 872; 1H NMR (500 MHz, CD3OD) d: 7.53
Colorless gum; [a]20
D ?82.9° (MeOH; c 0.16); HR-ESI–MS (1H, d, J = 16.0 Hz, H-70 ), 7.04 (1H, d, J = 2.0 Hz, H-20 ),
m/z: 385.1862 [M-H]- (calcd for C19H29O8, 385.1861); 6.94 (1H, dd, J = 2.0, 8.0 Hz, H-60 ), 6.77 (1H, d,
CD (MeOH, c 8.5 9 10-6 M): 241 (?41.37), 318 J = 8.0 Hz, H-50 ), 6.22 (1H, d, J = 16.0 Hz, H-80 ), 5.27
(-54.25) nm; IR mmax (film) cm-1: 3,400, 2,940, 1,653, (1H, dd, J = 5.0, 7.5 Hz, H-3), 4.13 (1H, dt, J = 3.0,
1,076, 1,035; 1H , and 13C NMR: see Table 1. 7.5 Hz, H-5), 3.73 (1H, dt, J = 3.0, 7.5 Hz, H-4), 3.69

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 H.-G. Jin et al.

(3H, s, H-8), 2.20 (1H, dd, J = 3.0, 13.5 Hz, H-6), 2.17 (C-3), 146.3 (C-4), 114.1 (C-5), 109.4 (C-6), 104.4 (C-2),
(2H, dd, J = 7.5, 13.5 Hz, H-2), 2.01 (1H, dd, J = 7.5, 103.5 (C-10 ), 102.3 (C-100 ), 78.1 (C-30 ), 77.1 (C-50 ), 75.1
13.5 Hz, H-6); 13C NMR (125 MHz, CD3OD) d: 175.6 (C- (C-20 ), 74.2 (C-400 ), 72.5 (C-500 ), 72.3 (C-200 ), 71.7 (C-40 ),
7), 168.4 (C-90 ), 150.0 (C-40 ), 147.4 (C-70 ), 147.1 (C-30 ), 70.0 (C-300 ), 68.1 (C-60 ), 57.3 (3-OCH3), 56.7 (4-OCH3),
127.7 (C-10 ), 123.1 (C-60 ), 116.7 (C-50 ), 115.2 (C-20 ), 115.1 18.1 (C-600 ).
(C-80 ), 75.9 (C-1), 72.6 (C-4), 72.3 (C-3), 70.4 (C-5), 53.1
(C-8), 38.2 (C-6), 37.9 (C-2). Acidic hydrolysis of 1

3,4,5-Trimethoxyphenyl-b-D-glucopyranoside (5) Compound 1 (2 mg) was dissolved in 1 N HCl (1 mL) and

MeOH (1 mL) and refluxed at 90 °C for 90 min (Kim et al.
White powder; [a]20 D -22.3° (MeOH; c 0.38); EI-MS m/z: 2004). The reaction solution was evaporated under reduced
346 [M]?; IR mmax (film) cm-l: 3,410, 2,910, 1,650, 1,075; pressure, and the hydrolysate was extracted with EtOAc
1
H NMR (500 MHz, CD3OD) d: 6.49 (2H, s, H-2, 5), 4.81 (3 mL 9 3). The aqueous fraction was neutralized with
(1H, d, J = 7.5 Hz, H-10 ), 3.92 (1H, dd, J = 2.0, 12.0 Hz, Ag2CO3, filtered, and the filtrate was concentrated under
H-60 ), 3.81 (6H, s, 3, 5-OCH3), 3.70 (3H, s, 4-OCH3), 3.66 reduced pressure. The residue was compared with standard
(1H, dd, J = 6.5, 12.0 Hz, H-60 ), 3.33–3.47 (4H, m, H-20 , sugar using TLC (CHCl3:MeOH:H2O, 6/4/1), which
30 , 40 , and 50 ); 13C NMR (125 MHz, CD3OD) d: 156.2 (C- showed the sugar to be (?)-D-glucose (Rf = 0.13) in 1.
1), 154.9 (C-3), 154.9 (C-5), 134.6 (C-4), 103.3 (C-10 ), 96.2
(C-2), 96.2 (C-6), 78.6 (C-50 ), 78.2 (C-30 ), 75.1 (C-20 ), 71.8 Bioassay of IL-6
(C-40 ), 62.9 (C-60 ), 61.4 (4-OCH3), 56.7 (3, 5-OCH3).
IL-6 bioassay was carried out using a slight modification of
Cuneataside D (6) an established method (Kim et al. 2003; Liu et al. 2006).
Briefly, 500 lL of the MG-63 cells (3 9 104 cells/mL) in
White amorphous powder; [a]20 D -60.3° (H2O; c 0.10); DMEM containing 10 % FBS were dispensed into a 24-well
ESI–MS m/z: 471 [M ? Na]?; IR mmax (film) cm-l; 3,400, plate; the culture was incubated for 24 h at 37 °C. Then,
1,618, 1,514, 1,441, 1,250, 1,203, 1,055; 1H NMR 5 lL of TNF-a (10 ng/mL), 5 lL of BAY 11-7085 (10 ng/mL),
(500 MHz, CD3OD) d: 6.74 (1H, d, J = 2.5 Hz, H-5), 6.71 and 5 lL of the DMSO with or without the compounds
(1H, d, J = 8.5 Hz, H-2), 6.58 (1H, dd, J = 2.5, 8.5 Hz, (100 lg/mL) were added. After incubation at 37 °C with
H-6), 4.71 (1H, d, J = 7.5 Hz, H-10 ), 4.71 (1H, d, 5 % CO2 for 24 h, the medium was stored at -20 °C until
J = 1.5 Hz, H-100 ), 4.02 (1H, dd, J = 1.5, 11.0 Hz, H-60 ), measurement. The IL-6 content of the medium was measured
3.83 (3H, s, 3-OCH3), 3.82 (1H, m, H-200 ), 3.57–3.68 (3H, in an ELISA procedure. 96-well plates were coated with
m, H-300 , 500 , and H-50 ), 3.36–3.51 (4H, m, H-20 , 30 , 40 , and 100 lL of purified rat anti-human IL-6 monoclonal antibody
H-400 ), 1.22 (3H, d, J = 6.0 Hz, H-600 ); 13C NMR in 0.1 M NaHCO3 (pH 9.6) by overnight incubation at 4 °C.
(125 MHz, CD3OD) d: 152.6 (C-1), 149.4 (C-3), 143.2 (C- The wells were blocked with 200 lL of 3 % BSA in PBS for
4), 116.2 (C-5), 110.2 (C-6), 104.1 (C-2), 103.9 (C-10 ), 2 h at room temperature (RT) and then incubated with
102.3 (C-100 ), 78.1 (C-30 ), 77.0 (C-50 ), 75.1 (C-20 ), 74.2 (C- 100 lL of specific antibody for 2 h at RT. 100 lL of HRP
400 ), 72.5 (C-500 ), 72.3 (C-200 ), 71.7 (C-40 ), 70.0 (C-300 ), 68.1 conjugated rabbit anti-goat IgG (1:1000 dilution) was added
(C-60 ), 56.6 (3-OCH3), 18.1 (C-600 ). to each well and incubated for 2 h at RT. 100 lL of TMB
(3,30 ,5,50 -tetramethyl-benzidine) substrate solution was
3,4-Dimethoxyphenyl-6-O-(a-L-rhamnopyranosyl)-b-D- added and incubated for 10 min at RT. The color reaction
glucopyranoside (7) was stopped with 50 lL of 0.4 N HCl and the optical density
was read at 450 nm using a Microplate Reader (Molecular
Amorphous solid; [a]20D -56.8° (MeOH; c 1.2); ESI–MS m/z: Devices Co., Ltd., USA).
485 [M ? Na]?; IR mmax (film) cm-l: 3,410, 1,614, 1,506,
1,447, 1,241, 1,213, 1,055; 1H NMR (500 MHz, CD3OD)
d: 6.87 (1H, d, J = 9.0 Hz, H-5), 6.76 (1H, d, J = 2.5 Hz, Results and discussion
H-2), 6.66 (1H, dd, J = 2.5, 9.0 Hz, H-6), 4.75 (1H, d,
J = 7.5 Hz, H-10 ), 4.71 (1H, d, J = 1.5 Hz, H-100 ), 4.02 Repeated column chromatography of the n-BuOH soluble
(1H, dd, J = 1.5, 11.0 Hz, H-60 ), 3.82 (1H, dd, J = 1.5, fraction of the stem of A. quinata yielded a new meg-
10.0 Hz, H-200 ), 3.81 (3H, s, 3-OCH3), 3.78 (3H, s, astigmane glycoside, named akequintoside D (1), as well as
4-OCH3), 3.57–3.69 (3H, m, H-300 , 500 and H-50 ), 3.34–3.55 six known phenolic compounds 2–7 (Fig. 1).
(4H, m, H-20 , 30 , 40 and H-400 ), 1.21 (3H, d, J = 6.0 Hz, Compound 1 was obtained as colorless gum, [a]20 D
H-600 ); 13C NMR (125 MHz, CD3OD) d: 153.9 (C-1), 151.2 -19.6° (MeOH). Its molecular formula was determined to

123
Megastigmane glycoside from Akebia quinata

HO HO

O O
O O

OH OH OH OH
O O
OH OH

OH OH

Fig. 2 Selected 1H–1H COSY (dark line), HMBC (H ? C) corre- Fig. 3 Key NOESY correlations of compound 1
lations of compound 1

be C19H30O8 by HR-ESI–MS data at m/z 409.1841 correlation between H-10 and C-9 suggested that glucose
[M ? Na]? (calcd for C19H30O8Na 409.1838). In the IR was attached to C-9 of megastigmane moiety. Furthermore,
spectrum, the absorption bands for the hydroxyl long-range correlations were observed between the fol-
(3,410 cm-1), and carbonyl (1,655 cm-1) groups were lowing protons and carbons (H-4 and C-2/C-6/C-13; H-7
observed. The 1H NMR spectrum (Table 1) of 1 showed and C-1/C-9; H-8 and C-6/C-9/C-10). In the 1H–1H COSY
four methyl protons at dH 1.24 (3H, d, J = 6.5 Hz, H-10), spectrum, the olefinic proton of H-7 showed couplings with
1.34 (3H, s, H-11), 1.38 (3H, s, H-12) and 2.14 (3H, s, H-8, H-9 and H-10 (Fig. 2). The relative configuration of 1
H-13), two oxymethine protons at dH 4.93 (1H, dd, was proposed from a NOESY experiment (Fig. 3), and a
J = 4.0, 9.5 Hz, H-8) and 3.96 (1H, dd, J = 4.0, 6.5 Hz, comparison of the observed and reported NMR data (Ot-
H-9), two olefinic protons at dH 5.96 (1H, s, H-4) and 6.10 suka et al. 1992; Yajima et al. 2009; Lee et al. 2011). In the
(1H, d, J = 9.5 Hz, H-7), methylene protons at dH 2.37 NOESY spectrum, correlations between H-10 , tentatively
(2H, s, H-2), in addition to a glucosyl anomeric proton at assigned an a-orientation (Lee et al. 2011), and H-9, H-8
dH 4.38 (1H, d, J = 7.5 Hz, H-10 ). Acid hydrolysis of 1 in indicated that these are on the same side (a). These NOE
refluxing 1 N HCl/MeOH afforded (?)-D-glucose which correlations suggested the relative configuration at C-8 and
was detected by direct comparison with an authentic C-9 as shown in Fig. 3. Accordingly, the structure of 1 is
sample using co-TLC (Kim et al. 2004). In the 13C NMR proposed to be 8S*,9R*-megastigman-3-one-4,6-diene-8,9-
spectrum (Table 1), 13 carbon signals appeared besides diol-9-O-b-D-glucopyranoside, named, akequintoside D.
those of sugar unit, which included one carbonyl carbon at Compound 2 was obtained as colorless gum, [a]20 D
dC 202.1 (C-3), two oxygenated methine carbons at dC 71.4 ?82.9° (MeOH), and has the same molecular formula
(C-8) and 79.6 (C-9), two olefinic carbons at dC 127.2 (C- (C19H30O8) as 1 by HR-ESI–MS (m/z 385.1862 [M-H]-).
4) and 135.5 (C-7), four methyl carbons at dC 15.6 (C-10), The 1H, and 13C NMR spectra showed a typical pattern of
29.8 (C-11), 29.9 (C-12) and 22.9 (C-13). These spectral megastigmane glycoside. The 1H NMR spectrum (Table 1)
data indicated that 1 was to be a megastigman derivative of 2 showed four methyls [dH 1.29 (3H, d, J = 6.5 Hz,
(Lee et al. 2011; 2012; Jin et al. 2012). In addition, the H-10), 1.04 (3H, s, H-11), 1.03 (3H, s, H-12), 1.92 (3H, s,
signals from the sugar unit appeared at dH 4.38 (1H, d, H-13)], one oxymethine [dH 4.42 (1H, qdd, J = 1.5, 3.0,
J = 7.5 Hz, H-10 ), 3.20 (1H, dd, J = 7.5, 9.0 Hz, H-20 ), 6.5 Hz, H-9)], three methines [dH 5.86 (1H, s, H-4), 5.87
3.37 (1H, dd, J = 9.0, 9.0 Hz, H-30 ), 3.29 (1H, dd, J = 9.0, (1H, d, J = 1.5 Hz, H-7), 5.87 (1H, d, J = 3.0 Hz, H-8)],
9.0 Hz, H-40 ), 3.29 (1H, ddd, J = 2.0, 5.0, 9.0 Hz, H-50 ), one methylene [dH 2.15 (1H, d, J = 17.0 Hz, H-2a), 2.53
3.67 (1H, dd, J = 5.0, 11.5 Hz, H-60 a), 3.86 (1H, dd, (1H, d, J = 17.0 Hz, H-2b)], and a b-D-glucopyranosyl
J = 2.0, 11.5 Hz, H-60 b) [dC 103.1 (C-10 ), 75.1 (C-20 ), 78.1 moiety [dH 4. 34 (1H, d, J = 7.5 Hz, H-10 )] (Ishimaru et al.
(C-30 ), 71.7 (C-40 ), 78.0 (C-50 ), 62.8 (C-60 )] and acid 1987). In the 13C NMR spectrum (Table 1), 13 carbon
hydrolysis experiment strongly supported the presence of signals appeared beside the b-D-glucose moiety, which
D-glucopyranose (Ishimaru et al. 1987). The coupling included one carbonyl (dC 201.3), one oxygenated methine
constant (J = 7.5 Hz) of the anomeric proton of D-glucose (dC 77.4), three methines (dC 127.3, 131.7, 135.4), four
indicated it to be the b-form (Ishimaru et al. 1987). Based quaternary carbons (dC 42.6, 167.4, 80.1), and four methyls
on the 1H and 13C NMR data, the structure of 1 was closely (dC 21.3, 23.6, 24.8, 19.3). The 1H,13C NMR, HSQC, and
related to reseoside II, which was isolated from Alangium HMBC spectra indicated that compound 2 had the same
premnifolium except for the different locations of hydroxyl planar ‘‘roseoside’’ skeleton (Otsuka et al. 1995). Further-
group and double bond in 1 (Otsuka et al. 1995). The more, the CD spectrum of 2 showed a positive Cotton
glycosidic linkage was established by a HMBC experiment effect at 241 nm and a negative effect at 318 nm, which
and comparison of the reported NMR data. The HMBC indicated the absolute configurations of 2 to be 6S, 9R-

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 H.-G. Jin et al.

Table 2 Inhibitory effect of compounds 1–7 against IL-6 production The structures of five known compounds were identified
in TNF-a stimulated MG 63 cells as 3-O-caffeoylquinic acid (3) (Nishimura et al. 1984; Teng
Treatment IL-6 (pg/mL) Inhibition (%) et al. 2002), methyl-3-O-caffeoylquinate (4) (Ida et al.
1994), 3,4,5-trimethoxyphenyl-b-D-glucopyranoside (5)
None 50.12 ± 1.8 –
(Shimomura et al. 1988), cuneataside D (6), (Chang and
TNF-a 250.6 ± 24.8 – Case 2005) and 3,4-dimethoxyphenyl-6-O-(a-L-rhamno-
Bay 11-7085 30.2 ± 2.1** 87.9** pyranosyl)-b-D-glucopyranoside (7) (Graikou et al. 2005),
**
1 175.4 ± 6.8 30.1** by comparing their spectroscopic data with those reported
**
2 210.5 ± 4.9 15.9** in the literature.
3 233.1 ± 9.8 7.39 IL-6 is a cytokine, originally identified as a T cell
4 213.0 ± 7.0 15.35** derived factor that regulates B-cell growth and differenti-
5 220.5 ± 4.3** 12.5** ation (Hirano et al. 1986). Human IL-6 is an important
6 255.6 ± 4.9 0.0 component of the inflammatory cascade. Dysregulation of
7 213.0 ± 10.7* 14.8* IL-6 production has been implicated in a variety of
MG-63 cells (3 9 104) were incubated for 24 h. Cultures were inflammatory/autoimmune disease states, including rheu-
incubated with or without compounds (100 lg/mL) for 30 min and matoid arthritis, cardiac myxoma, Castleman’s disease, and
then stimulated with TNF-a (10 ng/mL) for 24 h. IL-6 in the super- mesangial proliferative glomerulonephritis (Hirano et al.
natant was measured by ELAISA as described in Materials and
1990). The proinflammatory cytokines IL-1 and TNF-a
Methods. Results are expressed as the mean ± SE from three dif-
ferent experiments. BAY 11-7085 was used as a positive control markedly stimulate the production IL-6 (Van Damme et al.
*P \ 0.05 or **P \ 0.01 compared with TNF-a treated value 1987).
The inhibitory activity of the isolated compounds (1–7)
configurations (Otsuka et al. 1995; Yamano and Ito 2005). against IL-6 production in TNF-a stimulated MG-63 cells
Based on the above results, the structure of 2 was identified was examined. Among them, compound 1 showed mod-
as reseoside II, which was isolated from Vinca rosea and erate inhibitory activity against IL-6 production in TNF-a
Alangium premnifolium (Bhakuni et al. 1974; Otsuka et al. stimulated MG-63 cells, while compounds 2, 4, 5, and 7
1995). showed negligible activity (Fig. 3; Table 2).

Fig. 4 Inhibitory effect of compounds 1–7 against IL-6 production in described in Materials and Methods. Results are expressed as the
TNF-a sitimulated MG-63cells. MG-63 cells (3 9 104) were incu- mean ± SE from three different experiments. BAY 11-7085 was used
bated for 24 h. Cultures were incubated with or without compounds as a positive control. *P \ 0.05 or **P \ 0.01 compared with TNF-a
(100 lg/mL) for 30 min and then stimulated with TNF-a (10 ng/mL) treated value
for 24 h. IL-6 in the supernatant was measured by ELAISA as

123
Megastigmane glycoside from Akebia quinata

In conclusion, this paper reports the isolation, charac- inflammatory response. Archives of Pharmacal Research 26:
terization, and inhibitory activity of 7 isolates, including a 306–311.
Kim, M.R., H.-I. Moon, J.H. Chung, Y.H. Moon, K.-S. Hahm, and E.-
new compound and six known compounds, from A. qui- R. Woo. 2004. Matrix Metalloproteinase-1 inhibitor from the
nata. Fig. 4. stem bark of Styrax japonica S. et Z. Chemical & Pharmaceu-
tical Bulletin 52: 1466–1469.
Acknowledgments This work was supported by research funds Lee, I.K., K.H. Kim, S.Y. Lee, S.U. Choi, and K.R. Lee. 2011. Three
from Chosun University in 2013. new megastigmane glucopyranosides from the Cardamine
komarovii. Chemical & Pharmaceutical Bulletin 59: 773–779.
Lee, S.Y., I.K. Lee, S.U. Choi, and K.R. Lee. 2012. A new
megastigmane glucoside from the aerial parts of Erythronium
japonicum. Natural Product Sciences 18: 166–170.
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