Three new lignan glycosides with IL-6 inhibitory activity from Akebia quinata

Article abstract of DOI:10.1248/cpb.c13-00668

Three new lignan glycosides, akeqintoside A [(7S,8S)-7,8-dihydro-8- hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol 2′-O-β-D-glucopyranoside] (1), akeqintoside B [(7R,8R)-7,8-di-hydro- 8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-(9′-methoxy- 7′-propenyl) benzofuran 2′-O-β-D-glucopyranoside] (2), and akequintoside C [7R*,8R*-dihydroxy-7-(4-hydroxy-3-methoxyphenyl)- glycerol 9-O-β-D-(6′-O-caffeoyl)-glucopyranoside] (3) were isolated from Akebia quinata along with five known compounds, syringin (4), vanilloloside (5), salidroside (6), 3,4-dihydroxyphenylethyl alcohol 8-O-β-D- glucopyranoside (7), and calceolarioside B (8). The structures of the compounds were identified based on one dimensional (1D)- and 2D-NMR, including ¹H-¹H correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond connectivity (HMBC) and nuclear Overhauser effect spectroscopy (NOESY) spectroscopic analyses. The inhibitory activity of these isolated compounds against interleukin-6 (IL-6) production in tumor necrosis factor-alpha (TNF-α) stimulated MG-63 cells was also examined.

Full text of DOI:10.1248/cpb.c13-00668

288
Note
Chem. Pharm. Bull. 62(3) 288–293 (2014)
Vol. 62, No. 3
Three New Lignan Glycosides with IL-6 Inhibitory Activity from Akebia
quinata
,a
Hong-Guang Jin,a,c A Ryun Kim,a Hae Ju Ko,a Sang Kook Lee,b and Eun-Rhan Woo*
a College of Pharmacy, Chosun University; Gwangju 501–759, Republic of Korea: b College of Pharmacy, Natural
c
Products Research Institute, Seoul National University; Seoul 151–742, Republic of Korea: and College of Pharmacy,
Jilin Medical College; Jilin 132013, China.
Received August 22, 2013; accepted December 24, 2013
Three new lignan glycosides, akeqintoside
3-methoxyphenyl)-1ꢀ-benzofuranpropanol 2ꢀ-O-β-D-glucopyranoside] (1), akeqintoside
A
[(7S,8S)-7,8-dihydro-8-hydroxymethyl-7-(4-hydroxy-
[(7R,8R)-7,8-di-
B
hydro-8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1ꢀ-(9ꢀ-methoxy-7ꢀ-propenyl) benzofuran 2ꢀ-O-β-D-
glucopyranoside] (2), and akequintoside C [7R*,8R*-dihydroxy-7-(4-hydroxy-3-methoxyphenyl)-glycerol 9-O-
β-D-(6ꢀ-O-caffeoyl)-glucopyranoside] (3) were isolated from Akebia quinata along with five known compounds,
syringin (4), vanilloloside (5), salidroside (6), 3,4-dihydroxyphenylethyl alcohol 8-O-β-D-glucopyranoside (7),
and calceolarioside B (8). The structures of the compounds were identified based on one dimensional (1D)-
1
and 2D-NMR, including H–1H correlation spectroscopy (COSY), heteronuclear single quantum coherence
(HSQC), heteronuclear multiple bond connectivity (HMBC) and nuclear Overhauser effect spectroscopy
(NOESY) spectroscopic analyses. The inhibitory activity of these isolated compounds against interleukin-6
(IL-6) production in tumor necrosis factor-alpha (TNF-α) stimulated MG-63 cells was also examined.
Key words lignan glycoside; interleukin-6 (IL-6) inhibitory effect; Lardizabalaceae; Akebia quinata
Akebia quinata DECAISENE (Lardizabalaceae) is a creeping the IR spectrum, absorption bands for hydroxyl (3380cm−1)
woody vine that is widely distributed in East Asia, includ- and aromatic ring (1605, 1518cm−1) groups were observed.
1
ing Korea, China, and Japan.1) Traditionally, its dried stem The H-NMR spectrum (Table 1) of 1 showed 1,3,4-trisub-
is used mainly as a diuretic agent for the treatment of edema stituted aromatic protons at δH 6.97 (1H, d, J=2.0Hz, H-2),
and rheumatic pain.2,3) Previous phytochemical investigations 6.85 (1H, dd, J=2.0, 8.0Hz, H-6) and 6.78 (1H, d, J=8.0Hz,
resulted in the isolation of triterpenes, triterpene glycosides, H-5), 1,2,4,5-tetrasubstituted aromatic protons at δH 6.92 (1H,
and phenylethanoid glycosides.4–6) Regarding the biological s, H-3′) and 6.82 (1H, s, H-6′), hydroxyl propyl protons at δH
activity of A. quinata, only the cytotoxic effect of oleanane di- 3.57 (2H, t, J=6.5Hz, H-9′), 2.62 (2H, t, J=6.5Hz, H-7′) and
saccharides has been reported so far.7) The anti-inflammatory 1.82 (2H, m, H-8′), hydroxyl methyl protons at δH 3.84 (1H,
activity of this plant has not been explored yet. In an ongoing m, H-9a) and 3.76 (1H, m, H-9b), an oxygenated methine pro-
investigation into anti-inflammatory compounds from this ton at δH 5.52 (1H, d, J=6.0Hz, H-7), a methine proton at δH
plant, the methanol extract of A. quinata was investigated. 3.52 (1H, m, H-8), methoxyl protons at δH 3.82 (3H, s, OCH3),
By means of repeated column chromatography using silica in addition to a glucosyl anomeric proton at δH 5.01 (1H, d,
gel, Sephadex LH-20, and LiChroprep RP-18, three new lig- J=8.0Hz, H-1″). Acid hydrolysis of 1 in refluxing 1N-HCl/
nan glycosides, akequintoside A (1), akequintoside B (2), and MeOH afforded D-(+)-glucose which was detected by direct
akequintoside C (3), along with five known compounds, were comparison with an authentic sample using co-TLC.14) Fur-
isolated. The structures of the known compounds were identi- thermore, the configuration of the glucosidic linkage was de-
fied as syringin (4),8,9) vanilloloside (5),10) salidroside (6),11) termined to be β based on the coupling constant (J=8.0 Hz).15)
3,4-dihydroxyphenylethyl alcohol 8-O-β-D-glucopyranoside In the heteronuclear multiple bond correlation (HMBC) exper-
(7),12) and calceolarioside B (8),13) by comparing their spec- iment, long-range correlations (Fig. 2) were observed between
troscopic data with those reported in the literature (Fig. 1). each of H-2/H-6 and C-7, H-6′ and C-7′. These results indi-
Furthermore, these five known compounds were isolated from cated that 1 had two phenylpropanoid units.16,17) Significant
this plant for the first time. The inhibitory activity of the iso- HMBC correlations were also observed between H-7/C-5′ and
lated compounds against interleukin-6 (IL-6) production in H-8/C-4′. Therefore, it could be concluded that two phenylpro-
tumor necrosis factor-alpha (TNF-α) stimulated MG-63 cells panoids formed a 7,8-dihydro-8-hydroxymethyl-7-phenyl-1′-
was examined. This paper reports the isolation and structural benzofuranpropanol skeleton.16,17) In the 1H–1H-correlation
characterization of these compounds and their inhibitory ac- spectroscopy (COSY) spectrum, the oxymethine proton at δH
tivities against IL-6 production.
5.52 showed coupling with H-8, in addition methylene protons
at δH 2.62 showed coupling with H-8′ and H-9′. The glycosidic
linkage was identified at C-2′ by nuclear Overhauser enhance-
Results and Discussion
Akequintoside A (1) was obtained as a white amorphous ment spectroscopy (NOESY) experiment and HMBC cor-
powder, [α]D25 −35.1 (MeOH). Its molecular formula was relations as shown in Fig. 2. The HMBC correlation between
identified as C25H32O11 by positive mode high resolution elec- H-1″ and C-2′ indicated that the glucopyranosyl moiety was
trospray ionization mass spectrometry (HR-ESI-MS) data at connected at C-2′. The relative configuration of H-7 and H-8
m/z 531.1847 [M+Na]+ (Calcd for C25H32O11Na: 531.1842). In was identified as cis form from the distinct NOE correlation
between H-7 and H-8, H-8 and H-2/H-6, also based on the
reported NMR data.17,18) The absolute stereochemistry at C-7
The authors declare no conflict of interest.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: wooer@chosun.ac.kr

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289
Fig. 1. The Structures of Compounds 1–8 from A. quinata
1
1
and C-8 were assigned to be both S, on the basis of negative 71.5 (C-4″), 62.6 (C-6″). The H-, 13C-NMR and H–1H COSY
Cotton effect at 246 and 291nm in its circular dichroism (CD) spectral data indicated that compound 2 was a dihydrobenzo-
spectrum.17,18) Accordingly, the structure of 1 was identified furan-type lignan glucoside formed by two phenylpropanoid
1
as (7S,8S)-7,8-dihydro-8-hydroxymethyl-7-(4-hydroxy-3-meth- units.19,20) Closer examination of the H- and 13C-NMR data
oxyphenyl)-1′-benzofuranpropanol 2′-O-β-D-glucopyranoside, revealed the structure of 2 to be quite similar to that of 1
named akeqintoside A, as drawn in Fig. 1.
except for the presence of an additional methoxyl group at δC
Akequintoside B (2) was obtained as a white amorphous 58.1(δH 3.35) and an olefinic bond [δC 134.1 (δH 6.56, H-7′); δC
powder, [α]D25 −50.4 (MeOH). Its molecular formula was iden- 124.8 (δH 6.18, H-8′)] in 2. Long-range correlations between
tified as C26H32O11 based on positive mode HR-ESI-MS data each of the two methoxyl protons at δH 3.35, 3.82 and δC 74.4
at m/z 543.1841 [M+Na]+ (Calcd for C26H32O11Na: 543.1843). (C-9′), 149.3 (C-3), respectively, indicated the position of the
In the IR spectrum, absorption bands for hydroxyl (3370cm−1) methoxyl groups at C-10′ and C-3. Meanwhile, the coupling
and aromatic ring (1610, 1520cm−1) groups were observed. constant of the anomeric proton (J=7.5Hz) suggested that the
1
The H-NMR spectrum (Table 1) of 2 showed five aromatic sugar was β form.15) These observations suggested that the
protons at δH 7.18 (1H, s, H-3′), 7.07 (1H, s, H-6′), 6.98 (1H, structure of 2 was dehydrodiconiferyl alcohol 9′-methoxy-2′-
d, J=2.0Hz, H-2), 6.86 (1H, dd, J=2.0, 8.5Hz, H-6) and 6.78 O-β-D-glucopyranoside.21) The relative configuration of C-7
(1H, d, J=8.5Hz, H-5), an oxygenated methine proton at and C-8 was characterized by a NOESY experiment, which
δH 5.56 (1H, d, J=6.0Hz, H-7), a methine proton at δH 3.54 showed distinct NOE correlations between H-7 and H-8, H-8
(1H, m, H-8), a glucosyl anomeric proton at δH 5.04 (1H, d, and H-2/H-6. These observations suggested that the relative
J=7.5Hz, H-1″), hydroxymethyl protons at δH 3.79–3.83 (2H, configurations at C-7 and C-8 are in a cis arrangement. In ad-
m), disubstituted trans double bond protons at δH 6.56 (1H, dition, the CD cotton effect of 2 was entirely opposite to that
d, J=15.5Hz, H-7′) and 6.18 (1H, dt, J=6.5, 15.5Hz, H-8′), of 1, suggesting that the aglycone of 2 is the enantiomer of 1.
two methoxyl protons at δH 3.82 and 3.35, in addition to a The CD spectrum of 2 showed a positive Cotton effect at 246
glucosyl anomeric proton at δH 5.04 (1H, d, J=7.5Hz, H-1″). and 291nm, which indicated the absolute configurations of 2
Furthermore, acid hydrolysis of 2 in refluxing 1N-HCl/MeOH to be 7R,8R-configurations.17,18) Accordingly, the structure of
afforded D-(+)-glucose which was detected by direct compari- 2 was identified as (7R,8R)-7,8-dihydro-8-hydroxymethyl-7-
son with an authentic sample using co-TLC.14) The 13C-NMR (4-hydroxy-3-methoxyphenyl)-1′-(9′-methoxy-7′-propenyl) ben-
and HSQC spectral data (Table 1) displayed 26 carbon sig- zofuran 2′-O-β-D-glucopyranoside, named akeqintoside B, as
nals, including one oxygenated methine carbon at δC 90.0 drawn in Fig. 1.
(C-7), olefinic carbons at δC 134.1 (C-7′) and 124.8 (C-8′), one
Akequintoside C (3) was obtained as a white amorphous
oxygenated methylene carbon at δC 64.9 (C-9), and D-glucosyl powder, [α]D25 −26.7 (MeOH). Its molecular formula was iden-
carbons at δC 103.0 (C-1″), 78.4 (C-5″), 77.9 (C-3″), 75.1 (C-2″), tified as C25H30O13 by positive mode HR-ESI-MS data at m/z

290
Vol. 62, No. 3
Table 1. 1H- and 13C-NMR Data of 1, 2 and 3
1
2
3
No.
δH
δC
δH
δC
δH
δC
1
134.4
110.9
149.3
147.9
116.4
120.2
89.6
134.2
110.8
149.3
148.0
116.4
120.2
90.0
134.6
111.9
148.9
147.1
115.9
121.1
75.6
2
3
4
5
6
7
8
9
6.97 (d, 2.0)
6.98 (d, 2.0)
6.98 (d, 2.0)
6.78 (d, 8.0)
6.85 (dd, 2.0, 8.0)
5.52 (d, 6.0)
3.52 (m)
6.78 (d, 8.5)
6.72 (d, 8.0)
6.86 (dd, 2.0, 8.5)
5.56 (d, 6.0)
3.54 (m)
6.79 (dd, 2.0, 8.0)
4.56 (d, 6.0)
55.2
55.0
3.91 (dt, 3.0, 6.0)
3.62 (dd, 6.0, 10.0)
4.01 (dd, 3.0, 10.0)
75.6
3.76 (m)
65.0
3.79 (m)
64.9
72.8
3.84 (m)
3.83 (m)
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
1″
2″
3″
4″
5″
6″
137.2
142.6
118.1
130.7
147.6
119.7
32.9
132.6
142.9
116.7
131.3
149.5
118.2
134.1
124.8
74.4
127.8
115.2
147.0
150.0
116.6
123.3
147.4
114.9
169.2
7.04 (d, 2.0)
6.92 (s)
7.18 (s)
6.77 (d, 8.0)
6.82 (s)
7.07 (s)
6.94 (d, 2.0, 8.0)
7.56 (d, 16.0)
6.27 (d, 16.0)
2.62 (t, 6.5)
1.82 (m)
6.56 (d, 15.5)
35.8
6.18 (dt, 6.5, 15.5)
4.05 (d, 6.5)
3.57 (t, 6.5)
62.3
3.35 (s)
58.1
5.01 (d, 8.0)
102.9
75.0
77.9
71.5
78.3
62.6
5.04 (d, 7.5)
103.0
75.1
4.33 (d, 7.5)
105.2
75.3
77.8
71.8
75.6
64.7
3.45 (dd, 8.0, 8.5)
3.44 (dd, 8.5, 8.5)
3.40 (dd, 8.5, 8.5)
3.39 (ddd, 2.5, 5.5, 8.5)
3.68 (dd, 5.5, 12.0)
3.87 (dd, 2.5, 12.0)
3.82 (s)
3.45 (dd, 8.0, 8.5)
3.44 (dd, 8.5, 8.5)
3.40 (dd, 8.5, 8.5)
3.40 (ddd, 2.5, 5.5, 8.5)
3.68 (dd, 5.5, 12.0)
3.84 (dd, 2.5, 12.0)
3.82 (s)
3.26 (dd, 7.5, 9.0)
3.39 (dd, 9.0, 9.0)
3.36 (dd, 9.0, 9.0)
3.52 (ddd, 2.0, 6.0, 9.0)
4.30 (dd, 6.0, 12.0)
4.50 (dd, 2.0, 12.0)
3.82 (s)
77.9
71.5
78.4
62.6
OCH3
56.6
56.6
56.5
600MHz, CD3OD; chemical shifts in ppm relative to TMS; coupling constants (J) in Hz.
561.1581 [M+Na]+ (Calcd for C25H30O13Na: 561.1584). In the (1H, dd, J=2.0, 12.0Hz, H-6″a), 4.30 (1H, dd, J=6.0, 12.0Hz,
IR spectrum, absorption bands for hydroxyl (3400cm−1), car- H-6″b) [ δC 105.2 (C-1″), 77.8 (C-3″), 75.6 (C-5″), 75.3 (C-2″),
bonyl (1700cm−1), and aromatic ring (1513, 1435cm−1) groups 71.8 (C-4″), 64.7 (C-6″)], and acid hydrolysis experiment sup-
1
were observed. The H-NMR spectrum (Table 1) of 3 showed ported the presence of D-glucopyranose.8,15) The coupling con-
two sets of ABX aromatic protons at δH 7.04 (1H, d, J=2.0Hz, stant (J=7.5Hz) of the anomeric proton of D-glucose indicated
H-2′)/6.98 (1H, d, J=2.0Hz, H-2), 6.94 (1H, dd, J=2.0, 8.0Hz, it to be the β-form.15) The glycosidic linkage was established
H-6′)/6.79 (1H, dd, J=2.0, 8.0Hz, H-6), 6.77 (1H, d, J=8.0Hz, by a HMBC experiment and comparison of the reported NMR
H-5′)/6.72 (1H, d, J=8.0Hz, H-5), two oxymethine protons data.8,15) The downfield shift of C-6′ and the HMBC correla-
at δH 4.56 (1H, d, J=6.0Hz, H-7) and 3.91 (1H, dt, J=3.0, tion between H-6″ and C-9′ confirmed that the caffeoyl group
6.0Hz, H-8), one oxymethylene protons at δH 4.01 (1H, dd, was attached to C-6″ of glucose in 3. In addition, the HMBC
J=3.0, 10.0Hz, H-9a) and 3.62 (1H, dd, J=6.0, 10.0Hz, H-9b), correlation between H-1″ and C-9 suggested that glucose was
disubstituted trans double bond protons at δH 7.56 (1H, d, attached to C-9 of the guaiacylglycerol moiety. Furthermore,
J=16.0Hz, H-7′) and 6.27 (1H, d, J=16.0Hz, H-8′), and one long-range correlations were observed between the following
methoxyl group at δH 3.82 (3H, s). In the 13C-NMR spectrum protons and carbons (H-2/H-6 and C-7; H-7 and C-9; H-9 and
1
(Table 1), 25 carbon signals appeared besides those of the C-1″; H-6″ and C-3″/C-9′; H-7′ and C-2′/C-6′/C-9′). In the H–
sugar unit, including one carbonyl carbon at δC 169.2 (C-9′), 1H COSY spectrum, the glycosylated proton of H-9 showed
two oxygenated methine carbons at δC 75.6 (C-7) and 75.6 couplings with H-8 and H-7 (Fig. 2). The relative configura-
(C-8), olefinic carbons at δC 147.4 (C-7′) and 114.9 (C-8′), and tion of 3 was proposed from a NOESY experiment (Fig. 2),
one oxygenated methylene carbon at δC 72.8 (C-9). Based on and a comparison of the observed and reported NMR data.8,16)
1
the H- and 13C-NMR data, the structure of 3 was closely re- In the NOESY spectrum, correlations between H-1″, assigned
lated to guaiacylglycerol 4-O-β-D-glucopyranoside, which was an α-orientation, and H-8, H-7 indicated that these are on the
isolated from Osmanthus asiaticus8) except for the presence of same side (α). These NOE correlations suggested the relative
a caffeoyl group in the sugar unit and the different location configuration at C-7 and C-8 as shown in 3. Accordingly,
of the sugar in 3. The signals from the sugar unit appeared at the structure of 3 is proposed to be 7R*,8R*-dihydroxy-7-(4-
δH 4.33 (1H, d, J=7.5Hz, H-1″), 3.26 (1H, dd, J=7.5, 9.0Hz, hydroxy-3-methoxyphenyl)-glycerol 9-O-β-D-(6′-O-caffeoyl)-
H-2″), 3.39 (1H, dd, J=9.0, 9.0Hz, H-3″), 3.36 (1H, dd, J=9.0, glucopyranoside, named akequintoside C.
9.0Hz, H-4″), 3.52 (1H, ddd, J=2.0, 6.0, 9.0Hz, H-5″), 4.50
IL-6 is a cytokine, originally identified as a T-cell derived

March 2014
291
1
Fig. 2. Key H–1H COSY (—) HMBC (H→C), and NOESY Correlations of 1, 2 and 3
Fig. 3. Inhibitory Effect of Compounds 1–8 against IL-6 Production in TNF-α Sitimulated MG-63 Cells
MG-63 cells (3×104) were incubated for 24h. Cultures were incubated with or without compounds (100µg/mL) for 30min and then stimulated with TNF-α (10ng/mL)
for 24h. IL-6 in the supernatant was measured by ELAISA as described in Experimental. Results are expressed as the mean S.E. from three different experiments. BAY
11–7085 was used as a positive control. *p<0.05 or **p<0.01 compared with TNF-α treated value.
factor that regulates B-cell growth and differentiation.22) tion, and inhibitory activity of 8 isolates, including three new
Human IL-6 is an important component of the inflammatory compounds and five known compounds, from A. quinata.
cascade. Dysregulation of IL-6 production has been impli-
cated in a variety of inflammatory/autoimmune disease states,
Experimental
including rheumatoid arthritis, cardiac myxoma, Castleman’s
General Experimental Procedures Optical rotations
disease, and mesangial proliferative glomerulonephritis.22) The were measured using an Autopol-IV polarimeter. UV spectra
proinflammatory cytokines IL-1 and TNF-α markedly stimu- were obtained on a Shimadzu UV/Visible Spectrophotometer.
late the production IL-6.23)
IR spectra were recorded on an IMS 85 (Bruker). CD spectra
The inhibitory activity of the isolated compounds (1–8) were recorded on a JASCO J-810 spectropolarimeter. HR-
against IL-6 production in TNF-α stimulated MG-63 cells ESI-MS was obtained on a Q-TOF (Synapt HDMS system,
was examined. Among them, compounds 2, 3 and 8 showed Waters, U.S.A.) mass spectrometer. NMR spectra, including
moderate inhibitory activity against IL-6 production in TNF-α NOESY, COSY, heteronuclear multiple quantum coherence
stimulated MG-63 cells, while compounds 1 and 4–7 showed (HMQC) and HMBC experiments, were recorded on a Var-
no activity (Fig. 3, Table 2).
ian VNMRS 600 MHz spectrometer (KBSI-Gwangju center)
In conclusion, this paper reports the isolation, characteriza- operating at 600MHz (1H) and 150MHz (13C), respectively,

292
Vol. 62, No. 3
Table 2. Inhibitory Effect of Compounds 1–8 against IL-6 Production in
TNF-α Stimulated MG 63 Cells
B36). Subfraction B31 (0.79g) was then purified by Lichroprep
RP 18 CC (MeOH–H2O, 1:3), followed by preparative HPLC
eluted with MeOH–H2O (27:75) to give 4 (1.6mg), and 2
(1.6mg). Subfracton B33 (0.44g) was purified by Lichroprep
RP 18 CC (MeOH–H2O, 1:3.3) to give 3 (4.2mg), 1 (2.5mg),
and 8 (20.5mg).
Treatment
None
IL-6 (pg/mL)
Inhibition (%)
52.6 9.0
—
TNF-α
250.6 4.3
—
Akeqintoside A (1): Amorphous powder (MeOH); [α]D20
−35.1° (c=0.12, MeOH); UV λmax (MeOH) nm (logε) 217
(4.23), 289 (3.61), 328 (4.07); IR νmax (film) cm−1: 3400, 1702,
1630, 1590, 1516, 1438, 1150; CD (MeOH, c 4.92×10−6 M): 246
(−5685.0), 291 (−7818.9) nm; HR-ESI-MS (positive mode)
m/z: 531.1847 [M+Na]+ (Calcd for C25H32O11Na, 531.1842);
1H-NMR (CD3OD for 600MHz): see Table 1; 13C-NMR
(CD3OD for 150MHz): see Table 1.
BAY 11–7085
30.2 2.1**
390.9 7.8*
182.9 5.4**
175.4 8.6**
228.0 4.9*
213.0 2.3**
218.0 6.2**
285.7 11.1*
142.8 6.3**
87.9**
0.0*
1
2
3
4
5
6
7
8
27.3**
30.1**
9.09**
15.3**
13.07**
0.0*
43.18**
Akeqintoside B (2): Amorphous powder (MeOH); [α]D27
−50.4° (c=0.77, MeOH); UV λmax (MeOH) nm (logε) 218
(4.22), 289 (3.63), 328 (4.00); IR νmax (film) cm−1: 3405, 1700,
1630, 1593, 1517, 1440, 1152; CD (MeOH, c 3.07×10−6 M):
246 (+0.48), 291 (+0.35) nm; HR-ESI-MS (positive mode)
m/z: 543.1841 [M+Na]+ (Calcd for C26H32O11Na, 543.1843);
MG-63 cells (3×104) were incubated for 24h. Cultures were incubated with or
without compounds (100µg/mL) for 30min and then stimulated with TNF-α (10ng/
mL) for 24h. IL-6 in the supernatant was measured by ELAISA as described in Ex-
perimental. Results are expressed as the mean S.E. from three different experiments.
BAY 11–7085 was used as a positive control. *p<0.05 or **p<0.01 compared with
TNF-α treated value.
with chemical shifts given in ppm (δ). Semi-preparative HPLC 1H-NMR (CD3OD for 600MHz): see Table 1; 13C-NMR
was performed using a Waters HPLC system equipped with (CD3OD for 150MHz): see Table 1.
Waters 600 Q-pumps, a 996 photodiode array detector, and
Akeqintoside C (3): White amorphous solid; [α]D28 −26.7°
a YMC-Pack ODS-A column (250×10mm i.d., 5µm), flow (c=0.21, MeOH); UV λmax (MeOH) nm (logε) 218 (4.25), 289
rate 4.0mL/min. TLC was carried out on precoated Kieselgel (3.60), 328 (4.05); IR νmax (film) cm−1: 3405, 1702, 1633, 1590,
60F254 (art. 5715, Merck) and RP-18F254s (art. 15389, Merck) 1513, 1435, 1150; HR-ESI-MS (positive mode) m/z: 561.1581
plates. Column chromatography was performed on silica [M+Na]+ (Calcd for C25H30O13Na: 561.1584); 1H-NMR
gel 60 (40–63 and 63–200µm, Merck), MCI gel CHP20P (CD3OD for 600MHz): see Table 1; 13C-NMR (CD3OD for
(75–150 µm, Mitsubishi Chemical Co.), and Sephadex LH-20 150MHz): see Table 1.
(25–100 µm, Sigma). Silver carbonate (Ag2CO3, Aldrich Co.)
Acidic Hydrolysis of 1–3 Compounds 1–3 (1mg each)
and D-(+)-glucose (C6H12O6, Sigma) were used as neutraliza- were dissolved in 1N HCl (1mL) and MeOH (1mL) and
tion reagent and standard sugar on acid hydrolysis experiment, refluxed at 90°C for 90min.14) The reaction solution was
respectively. BAY 11–7085 (Sigma) was used as a positive evaporated under reduced pressure, and the hydrolysate was
control on IL-6 bioassay. Low pressure liquid chromatogra- extracted with EtOAc (3mL×3). The aqueous fraction was
phy was carried out over a Merck Lichroprep Lobar®-A RP-18 neutralized with Ag2CO3, filtered, and the filtrate was con-
(240×10mm) column with a FMI QSY-0 pump (ISCO).
centrated under reduced pressure. The residue was compared
Plant Materials The stems of A. quinata were collected with standard sugar using TLC (CHCl3–MeOH–H2O, 6:4:1),
in Gyeongju, Gyeongbuk province, Korea, in August 2011 which showed the sugar to be D-(+)-glucose (Rf=0.13) in 1–3.
and identified by Dr. J. H. Lee, Professor of the department of
Bioassay of Human IL-6 IL-6 bioassay was carried out
Korean Medicine, Dongguk University. A voucher specimen using a slight modification of an established method.24) Briefly,
(CSU-877-17) was deposited in the Herbarium of the College 500µL of the MG-63 cells (3×104 cells/mL) in Dulbecco’s
of Pharmacy, Chosun University.
modified Eagle’s medium (DMEM) containing 10% fetal
Extraction and Isolation The air-dried stem of A. qui- bovine serum (FBS) were dispensed into a 24-well plate; the
nata (11kg) were cut and extracted with MeOH three times culture was incubated for 24h at 37°C. Then, 5µL of TNF-α
for 4h at 80°C. The resultant MeOH extract (480g) was sus- (10ng/mL), 5µL of BAY 11–7085 (10ng/mL), and 5µL of the
pended in water (1.5L×3) and then partitioned sequentially dimethyl sulfoxide (DMSO) with or without the compounds
with equal volumes of dichloromethane, ethyl acetate, and (100 µg/mL) were added. After incubation at 37°C with 5%
n-butanol. Each fraction was evaporated in vaccuo to yield the CO2 for 24h, the medium was stored at −20°C until mea-
residues of CH2Cl2 (45.2g), EtOAc (11.0g), n-BuOH (57.0g), surement. The IL-6 content of the medium was measured in
and water (150.3g) extracts. The n-BuOH soluble fraction an enzyme-linked immunosorbent assay (ELISA) procedure.
(57.0g) was subjected to column chromatography (CC) over a 96-well plates were coated with 100µL of purified rat anti-
diaion HP 20 column and eluted with H2O–MeOH (100:0→ human IL-6 monoclonal antibody in 0.1M NaHCO3 (pH 9.6)
0:100) gradient system. The fractions were combined based by overnight incubation at 4°C. The wells were blocked with
on their TLC pattern to yield subfractions designated B1–B6. 200µL of 3% bovine serum albumin (BSA) in phosphate
Fraction B2 (3.47g), containing 5, 6, and 7 was purified by buffered saline (PBS) for 2h at room temperature (RT) and
MCI gel CC (MeOH–H2O, 1:9→2:8), followed by Sephadex then incubated with 100µL of specific antibody for 2h at RT.
LH 20 CC (MeOH–H2O, 1:20), and finally by silica gel CC One hundred microliters of horseradish peroxidase (HRP)
(CHCl3–MeOH–H2O, 4:1:0.2→2:1:0.2) to give 5 (3.2mg), conjugated rabbit anti-goat immunoglobulin G (IgG) (1:1000
6 (3.0mg), and 7 (1.2g). Fraction B3 (5.6g) was subjected to dilution) was added to each well and incubated for 2h at RT.
silica gel CC eluting with a CHCl3–MeOH–H2O (6:1:0.1→ One hundred microliters of 3,3′,5,5′-tetramethyl-benzidine
2:1:0.1) in gradient system to give six subfractions (B31– (TMB) substrate solution was added and incubated for 10min

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293
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at RT. The color reaction was stopped with 50µL of 0.4N HCl
and the optical density was read at 450nm using a Microplate
Reader (Molecular Devices Co., Ltd., U.S.A.).
12) Park H.-J., Lee M.-S., Lee K.-T., Sohn H.-C., Han Y.-N., Miyamoto
K., Chem. Pharm. Bull., 47, 1029–1031 (1999).
Acknowledgments This research was supported by a
Grant (12172MFDS989) from Ministry of Food and Drug
Safety in 2013. We thank Dr. K. D. Park, and Mr. S.-T. Hong
at the Korea Basic Science Institute for Gwangju Center for
their aid in obtaining the NMR and mass spectra.
13) Damtoft S., Jensen S. R., Phytochemistry, 37, 441–443 (1994).
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Lang F. A., Iinuma M., Tanaka T., Phytochemistry, 65, 207–213
(2004).
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