Bioactive 3,4-seco-triterpenoids from the fruits of Acanthopanax sessiliflorus

Article abstract of DOI:10.1021/np3002173

Eight new 3,4-seco-lupane triterpenes and glycosides, acanthosessiligenins I and II (1, 3) and acanthosessiliosides A-F (2, 4-8), as well as six known 3,4-seco-lupane triterpenes (9-14) were isolated from an ethanolic extract of Acanthopanax sessilif lorus fruits. The chemical structures of 1-8 were determined by spectroscopic data interpretation. All isolated compounds were tested for their cytotoxicity against six human cancer cell lines and their ability to inhibit LPSinduced nitric oxide production in RAW 264.7 macrophages.

Full text of DOI:10.1021/np3002173

Article
pubs.acs.org/jnp
Bioactive 3,4-seco-Triterpenoids from the Fruits of Acanthopanax
sessiliflorus
Dae-Young Lee,† Kyeong-Hwa Seo,† Dong-Sung Lee,‡ Youn-Chul Kim,‡ In-Sik Chung,† Gye-Won Kim,§
Dae-Sung Cheoi,⊥ and Nam-In Baek*,†
†Graduate School of Biotechnology and Department of Oriental Medicinal Materials and Processing, Kyung Hee University, Yongin
446-701, Republic of Korea
‡Standardized Material Bank for New Botanical Drugs, College of Pharmacy, Wonkwang University, Iksan 570-749, Republic of
Korea
§Brewing Research Center, Hankyung National University, Ansung 456-749, Republic of Korea
⊥Jeongseon Agricultural Extension Center, Jeongseon 233-852, Republic of Korea
S
* Supporting Information
ABSTRACT: Eight new 3,4-seco-lupane triterpenes and glycosides,
acanthosessiligenins I and II (1, 3) and acanthosessiliosides A−F (2, 4−
8), as well as six known 3,4-seco-lupane triterpenes (9−14) were isolated
from an ethanolic extract of Acanthopanax sessiliflorus fruits. The
chemical structures of 1−8 were determined by spectroscopic data
interpretation. All isolated compounds were tested for their cytotoxicity
against six human cancer cell lines and their ability to inhibit LPS-
induced nitric oxide production in RAW 264.7 macrophages.
Acanthopanax sessiliflorus (Rupr. et Maxim) Seem, belonging to
the plant family Araliaceae, is distributed widely in Korea,
mainland China, and Japan. Acanthopanax species are used
commonly in traditional oriental medicine to treat rheumatoid
arthritis, diabetes, tumors, hypertension, and cerebrovascular
diseases.1,2 Previous phytochemical research has resulted in the
isolation of lupane triterpene glycosides from the leaves and
twigs of Acanthopanax species3,4 and 3,4-seco-lupane triterpenes
from the leaves of A. divaricatus and A. senticosus.5,6
Triterpenoids and lignans are thought to be the active
constituents of plants in this genus.7−9 However, most
phytochemical and biological studies have focused mainly on
the leaves, bark, and roots of Acanthopanax species, and only a
few studies have dealt with the fruits.
We report herein the isolation of eight new 3,4-seco-lupane
triterpenes (1, 3) and glycosides (2, 4−8) from the fruits of A.
RESULTS AND DISCUSSION
sessiliflorus, together with six known compounds (9−14). The
present search for bioactive constituents from Korean medicinal
plants revealed that an ethanol extract of A. sessiliflorus fruit
showed marginal cytotoxicity when evaluated against human
cancer cells (data not shown). In addition, investigations of the
extracts and components from Acanthopanax species have
demonstrated previously evidence for anti-inflammatory effects
by these substances.10,11 Therefore, the isolated compounds 1−
14 were evaluated for their cytotoxic activity against a small
panel of human cancer cell lines and were also tested for their
anti-inflammatory potential using lipopolysaccharide (LPS)-
induced nitric oxide production inhibition in RAW 254.7
macrophage cells.
■
A 70% ethanolic extract of dried A. sessiliflorus fruits was
suspended in H2O and extracted successively with EtOAc and
n-BuOH. The EtOAc- and n-BuOH-soluble fractions were
concentrated under reduced pressure to produce a residue that
was subjected to multiple chromatographic steps, using Diaion
HP-20, Sephadex LH-20, silica gel, and reversed-phase C18
silica gel, yielding compounds 1−14.
Comparison of the NMR and MS data with reported values
led to identification of the known compounds as 22α-
Received: March 20, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
dx.doi.org/10.1021/np3002173 | J. Nat. Prod. XXXX, XXX, XXX−XXX

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a
1
Table 1. H NMR Spectroscopic Data of Compounds 1−8
position
1
2
3
4
5
1α
1β
1.72 (m)
2.20 (m)
1.55 (m)
1.25 (m)
1.81 (m)
4.72 (dd, 2.4, 11.6)
2.50 (dd, 8.4, 11.6)
3.49 (dd, 2.8, 14.0)
1.78 (d, 10.8)
4.84 (dd, 2.4, 11.6)
2.68 (dd, 8.4, 11.6)
3.65 (dd, 2.8, 14.0)
1.78 (d, 10.8)
4.88 (dd, 5.2, 13.2)
2.64 (dd, 8.0, 11.2)
3.65 (dd, 2.4, 14.6)
1.90 (d, 10.4)
2α
2β
2.47 (m)
2.42 (m)
9α
11α
11β
18
1.38 (m)
1.25 (m)
4.00 (ddd, 6.2, 10.8, 11.6)
4.08 (ddd, 6.4, 10.8, 11.8)
4.18 (m) overlap
2.62 (dd, 10.8, 11.2)
1.80 (dd, 9.6, 9.6)
3.47 (ddd, 4.4, 9.6, 9.6)
1.72 (m)
1.71 (m)
1.81 (dd, 11.8, 11.8)
3.36 (ddd, 5.2, 10.8, 11.2)
2.10 (m)
2.60 (dd, 11.2, 11.8)
19
3.66 (ddd, 4.6, 10.8, 11.2)
3.31 (ddd, 4.4, 10.8, 11.2)
2.08 (m)
3.58 (ddd, 4.4, 10.8, 11.2)
22α
22β
23a
23b
24
4.80 (brd, 4.4)
5.07 (d, 2.0)
4.83 (d, 2.0)
1.72 (s)
1.36 (m)
1.40 (m)
1.45 (m)
4.77 (brd, 5.2)
1.15 (s)
4.93 (brs)
4.75 (brs)
1.62 (s)
1.09 (s)
1.16 (s)
1.37 (s)
1.29 (s)
0.91 (s)
1.05 (s)
1.69 (s)
4.77 (brs)
4.61 (brs)
3.61 (s)
1.37 (s)
1.38 (s)
25
1.12 (s)
1.00 (s)
1.29 (s)
1.31 (s)
26
1.21 (s)
1.06 (s)
1.14 (s)
1.21 (s)
27
0.80 (s)
0.66 (s)
1.13 (s)
1.25 (s)
29
2.03 (s)
1.77 (s)
1.69 (s)
1.94 (s)
30a
30b
OCH3
1′
4.90 (brs)
4.79 (brs)
3.65 (s)
4.79 (brs)
4.75 (brs)
4.78 (brs)
4.92 (brs)
4.62 (brs)
4.67 (brs)
3.61 (s)
3.60 (s)
6.32 (d, 8.0)
6.39 (d, 8.0)
4.16 (dd, 9.2, 9.2)
4.27 (dd, 9.2, 9.2)
4.34 (dd, 9.2, 9.2)
4.01 (m)
6.41 (d, 8.0)
4.13 (dd, 7.6, 8.0)
4.22 (dd, 7.6, 7.6)
4.28 (dd, 7.6, 7.6)
4.03 (m)
2′
4.11 (dd, 8.0, 8.0)
4.27 (dd, 7.6, 8.0)
4.33 (dd, 7.6, 8.0)
4.00 (m)
3′
4′
5′
6′α
6′β
4.41 (dd, 2.0, 12.0)
4.32 (dd, 4.8, 12.0)
6
4.43 (dd, 2.0, 12.0)
4.39 (dd, 4.4, 12.0)
4.43 (dd, 2.8, 12.0)
4.35 (dd, 4.8, 12.0)
8
position
7
1α
1β
4.28 (dd, 4.4, 12.4)
4.47 (dd, 3.6, 11.2)
2.56 (m)
3.70 (dd, 3.2, 7.6)
2α
2β
2.54 (dd, 2.4, 14.0)
2.75 (m)
2.81 (brd, 14.4)
2.80 (m)
3.08 (dd, 8.4, 14.4)
2.74 (d, 9.2)
9α
11α
11β
18
1.68 (m)
1.80 (m)
1.82 (m)
1.20 (m)
4.54 (ddd, 8.8, 9.2, 9.2)
1.77 (dd, 9.6, 10.8)
3.47 (ddd, 5.6, 10.8, 11.2)
2.25 (m)
2.70 (dd, 10.8, 11.2)
2.53 (dd, 11.2, 11.6)
19
3.57 (ddd, 4.4, 10.8, 11.2)
3.53 (ddd, 4.8, 11.2, 11.6)
22α
22β
23a
23b
24
1.59 (m)
4.77 (brd, 4.4)
1.11 (s)
4.75 (brd, 4.4)
1.08 (s)
5.10 (brs)5.00 (brs)
1.30 (s)
0.93 (s)
0.99 (s)
1.04 (s)
1.82 (s)
4.96 (brs)
4.80 (brs)
1.39 (s)
1.06 (s)
1.17 (s)
1.20 (s)
1.98 (s)
5.00 (brs)
4.80 (brs)
1.85 (s)
0.97 (s)
1.14 (s)
1.13 (s)
1.90 (s)
4.99 (brs)
4.66 (brs)
25
26
27
29
30a
30b
OCH3
1′
6.35 (d, 8.0)
6.47 (d, 8.0)
6.44 (d, 8.0)
2′
4.11 (dd, 8.0, 8.4)
4.27 (dd, 8.4, 8.8)
4.30 (dd, 8.8, 8.8)
4.01 (m)
4.19 (dd, 8.0, 8.4)
4.32 (dd, 8.4, 8.4)
4.37 (dd, 8.4, 8.4)
4.05 (m)
4.18 (dd, 8.0, 8.4)
4.30 (dd, 8.4, 8.4)
4.35 (dd, 8.4, 8.4)
4.06 (m)
3′
4′
5′
6′α
6′β
4.43 (dd, 2.8, 12.0)
4.35 (dd, 4.8, 12.0)
4.46 (dd, 2.0, 12.0)
4.38 (dd, 4.4, 12.0)
4.47 (brd, 11.2)
4.34 (dd, 3.6, 11.2)
a
1H NMR data were measured in pyridine-d5 at 400 MHz. Chemical shift (δ) are in ppm, and coupling constants (J in Hz) are given in parentheses.
The assignments were based on DEPT, COSY, NOESY, HSQC, and HMBC experiments.
B
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a
Table 2. 13C NMR Spectroscopic Data of Compounds 1−8
position
1
2
3
4
5
6
7
8
1
34.9
28.8
174.2
147.9
50.5
26.2
33.3
39.7
41.3
43.4
22.1
25.1
38.6
40.9
30.1
27.2
62.9
44.3
47.9
151.7
42.3
75.6
113.7
23.6
20.5
16.5
15.0
178.8
19.5
110.4
51.5
31.5
30.3
178.6
147.4
50.0
26.4
331
86.6
38.0
173.5
79.3
56.2
18.1
34.9
42.3
48.9
46.3
67.2
36.9
37.2
42.1
29.9
32.3
55.4
48.3
47.0
150.5
30.6
36.3
24.2
32.0
18.5
17.3
14.6
179.0
19.1
109.7
51.1
87.2
38.7
173.3
79.4
56.1
18.8
35.5
42.9
48.9
47.0
67.7
37.0
37.7
42.8
29.7
31.0
57.0
49.6
47.4
150.5
30.5
36.8
25.0
32.4
19.3
18.0
15.3
174.8
19.6
110.1
51.2
95.5
74.4
78.7
71.2
79.5
62.3
87.2
38.3
173.4
79.3
56.1
18.8
35.5
42.9
49.0
47.0
67.8
37.0
37.4
42.8
29.8
26.7
63.0
44.1
47.5
150.6
41.9
74.8
24.8
32.6
19.1
17.9
15.2
174.7
19.3
110.7
51.0
95.6
74.3
78.7
71.2
79.4
62.3
84.5
38.1
170.9
81.6
56.1
19.0
34.6
43.3
42.7
47.9
23.8
25.7
38.8
41.6
30.6
31.3
56.6
49.8
47.8
151.4
32.9
37.6
24.6
32.6
18.8
17.0
15.0
178.8
19.6
110.0
85.5
38.4
175.8
82.4
56.2
18.8
34.4
43.2
42.7
47.7
23.8
25.5
38.6
41.6
29.8
26.7
62.9
44.2
47.6
151.4
41.7
74.9
24.7
32.7
19.2
17.0
14.9
174.8
19.3
110.6
70.5
38.8
173.1
147.7
49.5
25.1
32.3
41.7
44.1
44.2
75.3
33.5
35.0
42.1
28.9
26.6
62.9
44.1
47.7
150.5
41.6
74.8
113.8
23.4
19.0
17.8
13.8
174.8
18.7
111.1
2
3
4
5
6
7
8
39.5
41.0
43.2
21.9
24.9
38.5
40.7
30.0
31.9
56.6
49.7
47.7
151.1
32.7
37.3
114.0
23.6
20.2
16.3
14.9
174.8
19.6
110.0
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
OCH3
1′
95.5
74.1
78.4
71.0
79.3
62.2
96.3
74.5
78.3
71.2
79.4
62.4
95.5
74.3
78.8
71.1
79.7
62.1
95.5
74.3
78.8
71.0
79.5
62.1
2′
3′
4′
5′
6′
a
13C NMR data were measured in pyridine-d5 at 100 MHz. The assignments were based on DEPT, COSY, NOESY, HSQC, and HMBC
experiments.
hydroxychiisanogenin (9),12 3,4-seco-lupan-20(30)-ene-3,28-
dioic acid (10),13 (1R)-1,4-epoxy-11α,22α-hydroxy-3,4-seco-
lupan-20(30)-ene-3,28-dioic acid (11),14 (+)-divaroside
(12),15 chiisanoside (13),5 and 22α-hydroxychiisanoside
(14).12
signals for three tertiary methyl protons [δH 1.21 (H-26), 1.12
(H-25), 0.80 (H-27)], an oxygenated methine proton [δH 4.80
(brd, J = 4.4 Hz, H-22)], and a methoxy proton (δH 3.65) were
observed. The 13C NMR spectrum of 1 (Table 2) supported by
DEPT experiments indicated the presence of 31 carbons
including two carbonyl carbons [δC 174.2 (C-3) and 178.8 (C-
28)], two olefinic quaternary carbons [δC 151.7 (C-20), 147.9
(C-4)], two exomethylene carbons [δC 113.7 (C-23), 110.4 (C-
30)], an oxygenated methine carbon [δC 75.6 (C-22)], a
methoxy carbon (δC 51.5), five methyl carbons [(δC 23.6 (C-
24), 20.5 (C-25), 19.5 (C-29), 16.5 (C-26), 15.0 (C-27)], and
Compounds 1−8 showed bands at 3462 to 3342, 1746 to
1710, and 1665 to 1640 cm−1 in the FT-IR spectrum,
suggesting the presence of hydroxy group, carbonyl group,
and double-bond absorptions. Of these, compound 1 was
obtained as a white, amorphous powder. The molecular
formula was determined to be C31H48O5 from the pseudomo-
lecular ion peak [M − H]− at m/z 499.3401 (calcd for
1
18 other carbon signals. Several cross-peaks in the H−1H
1
C31H47O5, 499.3423) in the negative HRFABMS. In the H
COSY spectrum confirmed a key connection among the proton
signals (Figure 1). The HMBC spectrum showed crucial long-
range correlations between H-23a, H-23b/C-5; H-2a, H-2b/C-
3; and H-1a/C-3 (Figure 1). On further analysis of the HSQC
and DEPT 135 spectra of 1, the assignments of the proton and
carbon NMR signals (Tables 1 and 2) were confirmed
unambiguously. Therefore, 1 was assigned as a 3,4-seco-
lupane-type triterpenoid. In addition, a long-range correlation
NMR spectrum (Table 1) the observation of signals for two
allyl methyl protons [δH 1.72 (H-24) and 2.03 (H-29)] located
at sp2 carbons and four olefinic methine protons at δH 5.07 (J =
2.0 Hz, H-23a), 4.83 (J = 2.0 Hz, H-23b), 4.90 (brs), and 4.79
(brs), of which the chemical shifts and small coupling constants
(or broad singlet) were typical of two exomethylene units,
confirmed the presence of two isopropenyl moieties. Also,
C
dx.doi.org/10.1021/np3002173 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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11. Furthermore, the stereostructure of 3 was confirmed by a
NOESY experiment (Figure 1), which showed a correlation
between H-1 and H3-24, H3-25, as well as between H-11 and
H3-25, H3-26. These NOE correlations indicated that the
oxygen at C-1 is β-oriented and that the hydroxy at C-11 is α-
configured. Also, the long-range correlations between the
methoxy protons signal (δH 3.61) and the carbonyl carbon
signal at C-3 (δC 173.5) in the HMBC spectrum revealed that
the methoxy group is affixed to C-3. Consequently, the
structure of 3 (acanthosessiligenin II) was determined as (1R)-
1,4-epoxy-11α-hydroxy-3,4-seco-lup-20(30)-ene-3,28-dioic acid
3-methyl ester.
1
Figure 1. Key H−1H COSY, HMBC, and NOESY correlations of
compounds 1 and 3.
Compound 4, a white powder, showed a pseudomolecular
ion peak [M + H]+ at m/z 679.4003 in the positive HRFABMS,
which was 162 mass units larger than that of 3 and consistent
with a pseudomolecular formula of C37H59O11 (calcd for
between the signals of the methoxy protons (δH 3.65) and the
C-3 carbonyl carbon (δC 174.2) was also evident. The coupling
constant between H-22 and H-21 was 4.4 Hz, indicating that
OH-22 is α-oriented.12 Therefore, the structure of compound 1
was determined as 22α-hydroxy-3,4-seco-lupa-4(23),20(30)-
diene-3,28-dioic acid 3-methyl ester, which has previously
been unreported, and this compound was named acanthosessi-
ligenin I.
1
C37H59O11, 679.4057). A comparison of the H and 13C NMR
spectra showed that they are quite similar to those of 3 except
for the presence of proton and carbon resonances for an
additional β-glucopyranosyl group (Tables 1 and 2). This
finding was confirmed by acid hydrolysis of 4, which yielded the
same aglycone [TLC, Rf 0.55 (RP-18 F254s), acetone−H2O
(4:1)] as that of 3 and D-glucose [TLC, Rf 0.40 (silica gel F254),
CHCl3−MeOH−H2O (65:35:10)]. The glucose moiety was
determined to be attached to C-28 by observation of the
glycosylation effect of the 13C NMR resonance of C-28 relative
to that in 3 (δC 174.8 in 4 vs δC 179.0 in 3) and from the long-
range correlations of H-1′ (δH 6.39, d, J = 8.0 Hz) with C-28
(δC 174.8) observed in the HMBC spectrum. Therefore, the
structure of 4 (acanthosessilioside B) was determined as (1R)-
1,4-epoxy-11α-hydroxy-3,4-seco-lup-20(30)-ene-3,28-dioic acid
3-methyl ester 28-O-β-D-glucopyranoside.
Compound 2 was isolated as a white powder, and its
molecular formula was established as C36H56O9 from the
pseudomolecular ion peak [M + H]+ at m/z 633.3637 (calcd
for C36H57O9, 633.4002) in the positive HRFABMS. Its 1H and
13C NMR spectra (Tables 1 and 2) were similar to those of 1,
with the exception of proton and carbon resonances for an
additional sugar moiety and the lack of a methoxy group and an
oxygenated methine moiety at the C-3 and C-22 position,
respectively. The quaternary carbon signal for C-17 (δC 56.6)
shifted 6.3 ppm upfield compared with 1 and 22α-
hydroxychiisanoside (14),12 suggesting that C-22 of 2 is a
methylene group. As for the monosaccharide unit, an anomeric
proton signal at δH 6.32 (d, J = 8.0 Hz) and a carbon signal at
δC 95.5 (C-1′) as well as the oxygenated methine and
methylene carbon signals at δC 79.3 (C-5′), 78.4 (C-3′), 74.1
(C-2′), 71.0 (C-4′), and 62.2 (C-6′) suggested the presence of a
β-glucopyranosyl group. The connectivity between the
glucopyranosyl unit (C-1′) and the C-28 of the aglycone was
verified by the presence of a cross-peak between δH 6.32 (H-1′)
and δC 174.8 (C-28) in the HMBC spectrum as well as the
chemical shift of the anomeric carbon signal (δC 95.5).
Therefore, the structure of 2 (acanthosessilioside A) was
determined to be 3,4-seco-lupa-4(23),20(30)-diene-3,28-dioic
acid 28-O-β-D-glucopyranoside.
Compound 5 was isolated as a white powder, and its
molecular formula was established as C37H58O12 from the
pseudomolecular ion [M + H]+ at m/z 695.3936 (calcd for
1
C37H59O12, 695.4006) in the positive HRFABMS. The H and
13C NMR spectra (Tables 1 and 2) of 5 were similar to those of
4 except that the methylene signals due to C-22 in 4 were
replaced by those of an oxygenated methine (δH 4.77 and δC
74.8) in 5. The molecular weight of 5 was 16 Da more than that
of 4, indicating the presence of an additional hydroxy group. In
5, the chemical shifts of the carbon signals of C-17 (δC 63.0)
and C-21 (δC 41.9) were shifted downfield by 6.0 and 11.4
ppm, respectively, and that of C-18 (δC 44.1) was shifted
upfield by 5.5 ppm when compared with 4 and isochiisano-
side,12 suggesting that C-22 is hydroxylated. This conclusion
was supported by the HMBC spectrum, which showed a long-
range correlation between the proton signal of H-22 (δH 4.77)
and the carbon signals of C-18 (δC 44.1), C-19 (δC 47.5), and
C-28 (δC 174.7). The coupling constant between H-22 and H-
21 was 5.2 Hz, indicating that OH-22 is α-oriented.12,14
Consequently, the structure of compound 5 (acanthosessilio-
side C) was determined as (1R)-1,4-epoxy-11α,22α-dihydroxy-
3,4-seco-lup-20(30)-ene-3,28-dioic acid 3-methyl ester 28-O-β-
D-glucopyranoside.
Compound 3 was isolated as white, needle-like crystals. The
positive HRFABMS of 3 showed a [M + H]+ ion peak at m/z
517.3307, in accordance with a pseudomolecular ion of
1
C31H49O6 (calcd for C31H49O6, 517.3529). The H and 13C
NMR spectra (Tables 1 and 2) exhibited signals for five tertiary
methyl groups (δH 1.37, 1.29, 1.09, 1.05, 0.91), an allyl methyl
group (δH 1.69), an exomethlyene group (δH 4.77, 4.61) due to
an isopropenyl moiety, two oxygenated methine groups [δH
4.72 (dd, J = 2.4, 11.6 Hz, H-1), δC 86.6 (C-1); δH 4.00 (ddd, J
= 6.2, 10.8, 11.6 Hz, H-11), δC 67.2 (C-11)], a methoxy group
(δH 3.61, δC 51.1), and two carbonyl groups [δC 173.5 (C-3),
179.0 (C-28)]. When compared with a previously reported
compound, isochiisanoside,12 3 was found to lack a sugar
moiety at the C-28 position but to possess an additional
methoxy group. The oxygenated methine proton (H-11) of 3
appeared at δH 4.00 (ddd, J11,12eq = 6.2 Hz, J11,12ax = 11.6 Hz,
J9,11 = 10.8 Hz), coupled with H-9 [δH 1.78 (d, J = 10.8 Hz)].
These data indicated a diaxial arrangement between H-9 and H-
11 and an equatorial confirmation of the hydroxy group at C-
Compound 6, a white powder, showed a pseudomolecular
ion peak [M − H]− at m/z 647.3744 in the negative
HRFABMS, and the mass spectrometric data were consistent
with the pseudomolecular formula of C36H55O10 (calcd for
C36H55O10, 647.3795). The 13C NMR and DEPT spectra
indicated the presence of 36 carbons, including six methyls,
nine methylenes, six methines, eight quaternary carbons, and
one glycosyl moiety (Tables 1 and 2). The 13C NMR signals for
D
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C-9 and C-12 (δC 42.7 and 25.7) were shifted upfield by 6.3
and 11.3 ppm, respectively, and those of C-10 and C-13 shifted
downfield by 0.9 and 1.4 ppm, respectively, when compared
with the 13C NMR signals of 5 and isochiisanoside.12 This
indicated that C-11 of 6 is not oxygen-bearing. When compared
with a previously isolated compound, 3,4-seco-lupan-20(30)-
ene-3,28 dioic acid (10),13 compound 6 was determined to be a
monoglycoside of 10. The proton and carbon signals due to the
sugar moiety suggested the presence of a β-glucopyranosyl
group (Tables 1 and 2). The correlation between δH 6.35 (H-
1′) and δC 170.9 (C-3) in the HMBC spectrum and the
chemical shift of the anomeric carbon signal (δC 96.3)
supported the presence of a glucopyranosyl group at C-3.
Therefore, the chemical structure of compound 6 (acantho-
sessilioside D) was determined as (1R)-1,4-epoxy-3,4-seco-lup-
20(30)-ene-3,28-dioic acid 3-O-β-D-glucopyranoside.
coupling constant (J = 8.0 Hz) of the anomeric proton signals
in the 1H NMR spectra (Table 1) of these glycosides suggested
the β-configuration of the glucopyranosyl moiety in each case.
All isolated compounds (1−14) were tested for their
biological activity. All compounds were inactive when tested
against a small cancer cell line panel (IC50 values <10 μM). As
shown in Table 3, compounds 1−4 and 6 showed moderate
Table 3. Inhibitory Effects of Compounds 1−14 against LPS-
Induced NO Production in RAW264.7 Macrophage Cells
a
b
compound
IC50 (μM)
cell viability (%)
1
2
3
4
6
11.3
18.3
47.0
20.6
11.9
6.5
88.8 3.7
93.5 2.0
91.1 3.4
93.0 5.7
95.2 1.2
84.6 2.5
Compound 7 was isolated as a white powder, and its
molecular formula was established as C36H56O11 from the
pseudomolecular ion peak [M + H]+ at m/z 665.3994 (calcd
c
aminoguanidine
a
The IC50 value of each compound was defined as the concentration
1
(μM) that caused 50% inhibition of NO production in LPS-activated
for C36H57O11, 665.3901) in the positive HRFABMS. The H
and 13C NMR spectra of 7 were found to be similar to those of
6 (Tables 1 and 2), with the exception of the proton and
carbon resonances for an oxygenated methine group (δH 4.77,
brd, J = 4.4 Hz and δC 74.9) in 7. A hydroxy group could be
positioned at C-22 from the correlation between the proton
signal of H-22 and the carbon signals of C-18 (δC 44.2), C-19
(δC 47.6), and C-28 (δC 174.8) in the HMBC spectrum. The J
value and the configuration of H-22 were in good agreement
with those of 5 and other reported data.12,14 Furthermore, the
molecular weight of 7 was 16 Da more than 6, indicating the
presence of an additional hydroxy group. The glucose moiety
was deduced to be attached to C-28 via a glycosidic linkage,
from the glycosidation-induced shift of the 13C NMR resonance
of C-28 relative to that in 6 (δC 178.8 in 6 vs δC 174.8 in 7) and
from a long-range correlation of H-1′ (δH 6.47, d, J = 8.0 Hz)
with C-28 (δC 174.8) observed in the HMBC spectrum.
Therefore, the structure of compound 7 (acanthosessilioside E)
was determined as (1R)-1,4-epoxy-22α-hydroxy-3,4-seco-lup-
20(30)-ene-3,28-dioic acid 28-O-β-D-glucopyranoside.
RAW 264.7 macrophage cells. Compounds 5 and 7−14 were inactive
b
(IC50 > 50 μM). Cell viability was expressed as a percentage (%) of
c
the LPS-only treatment group. Positive control. The results are
averages of three independent experiments, and the data are expressed
as means SD.
potencies in inhibiting NO production and had no influence on
cell viability. All other compounds were inactive (IC50 > 50
μM) against LPS-induced NO production in RAW 264.7
macrophages.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
obtained using a Fisher-John’s melting point apparatus with a
microscope. Optical rotations were measured on a JASCO P-1010
digital polarimeter. 1H, 13C, and 2D NMR spectra were recorded on a
Varian Unity INOVA AS 400 FT-NMR instrument, and chemical
shifts are given in δ (ppm) based on tetramethylsilane (TMS) as
internal standard. IR spectra were run on a Perkin-Elmer Spectrum
One FT-IR spectrometer. HRFABMS were obtained using a JEOL
JMS-700 mass spectrometer. A Shimadzu gas chromatograph (GC-
14B) equipped with an on-column injection system and flame
ionization detector (FID) was used. Silica gel 60 (Merck, 230−400
mesh), LiChroprep RP-18 (Merck, 40−63 μm), and Sephadex LH-20
(Amersham Biosciences) were used for column chromatography
(CC). Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm)
and precoated RP-18 F254s plates (Merck) were used for analytical
thin-layer chromatography. Spots were visualized by spraying with
10% aqueous H2SO4 solution, followed by heating.
Compound 8 was isolated as a white powder, and its
molecular formula was established as C36H54O11 from the
pseudomolecular ion peak [M + H]+ at m/z 663.3753 (calcd
1
for C36H55O11, 663.3744) in the positive HRFABMS. The H
and 13C NMR spectra of 8 (Tables 1 and 2) suggested it to be a
monoglycoside of 22α-hydroxychiisanogenin (9).12 The sugar
moiety was determined to be β-glucopyranoside from a
hemiacetal proton signal at δH 6.44 (d, J = 8.0 Hz) and a
hemiacetal carbon signal, four oxygenated methine carbon
signals, and an oxygenated methylene carbon signal (Table 2).
The signal at δC 95.5 suggested that 8 has a 28-O-glycosidic
linkage through an ester bond, which was confirmed from a
long-range correlation of H-1′ with C-28 (δC 174.8) observed
in the HMBC spectrum. Thus, the structure of compound 8
(acanthosessilioside F) was determined as (1R)-1α,11α,22α-
trihydroxy-3,4-seco-lupa-4(23),20(30)-diene-3,28-dioic acid
3,11-lactone 28-O-β-D-glucopyranoside.
Plant Material. The fruits of A. sessiliflorus were obtained in
August 2009 from the Jeongseon Agricultural Extension Center,
Jeongseon, Korea, and were identified by Prof. Dae-Keun Kim, College
of Pharmacy, Woo Suk University, Jeonju, Korea. A voucher specimen
(KHU090809) is preserved at the Laboratory of Natural Products
Chemistry, Kyung Hee University, Yongin, Korea.
Extraction and Isolation. The air-dried fruits of A. sessiliflorus (10
kg) were powdered and extracted three times for 24 h with 36 L of
aqueous 70% EtOH at room temperature. After concentration in
vacuo, the EtOH extract (2012 g) was suspended in H2O (3 L) and
then partitioned successively with EtOAc (3 L × 3) and n-BuOH (3
L), followed by concentration, to give EtOAc (E, 118 g), n-BuOH (B,
284 g), and water (1610 g) fractions. Fraction E (100 g) was subjected
to silica gel CC (15 × 21 cm) using a gradient of CHCl3−MeOH
(15:1 → 10:1 → 5:1 → 3:1 → 1:1, 2.8 L each) to yield 14 fractions
(E1 to E14). Fraction E3 [36.3 g, elution volume/total volume (Ve/
Vt) 0.15−0.33] was subjected to silica gel CC [6 × 16 cm, CHCl3−
EtOAc (7:1, 5.5 L)] to give five subfractions (E3-1 to E3-5). CC [silica
The monosaccharide obtained after aqueous acid hydrolysis
of each glycoside (2, 4−8) was identified as glucose by TLC
comparison with an authentic sample [TLC, Rf 0.40 (silica gel
F254), CHCl3−MeOH−H2O (65:35:10)]. The absolute config-
uration was determined to be D based on GC analysis of a chiral
derivative of the monosaccharide obtained by hydrolysis of each
compound (see Experimental Section). The relatively large
E
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gel (3.5 × 16 cm), n-hexane−EtOAc (1:1, 3 L)] of subfraction E3-3
(1.80 g, Ve/Vt 0.34−0.53) gave 19 subfractions (E3-3-1 to E3-3-19).
Subfraction E3-3-7 (184 mg, Ve/Vt 0.30−0.41) was separated by CC
[RP-18 (3.5 × 5.5 cm), acetone−H2O (3:2, 1.6 L)] to give compound
3 [19.7 mg, Ve/Vt 0.46−0.65, TLC (RP-18 F254s) Rf 0.55, acetone−
H2O (4:1)] and compound 10 [12.2 mg, Ve/Vt 0.87−0.92, TLC (RP-
18 F254s) Rf 0.25, acetone−H2O (4:1)]. Subfraction E3-3-9 (95 mg,
Ve/Vt 0.49−0.53) was chromatographed [RP-18 (3.5 × 6.5 cm),
acetone−MeOH−H2O (1:1:4, 0.8 L)] to give compound 9 [38.9 mg,
Ve/Vt 0.52−0.76, TLC (RP-18 F254s) Rf 0.40, acetone−MeOH−H2O
(1:1:1)]. Fraction E8 (8.98 g, Ve/Vt 0.59−0.67) was fractionated using
silica gel CC [4 × 12 cm, CHCl3−MeOH−H2O (16:3:1 → 13:3:1,
each 3.7 L)] and gave nine subfractions (E8-1 to E8-9). Subfraction
E8-4 (1.85 g, Ve/Vt 0.45−0.58) was purified using CC [RP-18 (3.5 ×
6.5 cm), MeOH−H2O (3:1, 1.2 L)] and gave compound 2 [93 mg,
Ve/Vt 0.84−0.98, TLC (RP-18 F254s) Rf 0.40, MeOH−H2O (5:1)] and
compound 6 [13.5 mg Ve/Vt 0.65−0.70, TLC (RP-18 F254s) Rf 0.45,
MeOH−H2O (5:1)]. Subfraction E8-5 (1.22 g, Ve/Vt 0.59−0.68) was
fractionated using Sephadex LH 20 CC [3 × 50 cm, MeOH−H2O
(4:1, 1.8 L)] and yielded five subfractions (E8-5-1 to E8-5-5).
Purification of subfraction E8-5-1 (280 mg, Ve/Vt 0.01−0.25) using
CC [RP-18 (3 × 6 cm), MeOH−H2O (1:1, 0.6 L)] gave compound
11 [35 mg, Ve/Vt 0.77−0.85, TLC (RP-18 F254s) Rf 0.40, MeOH−
H2O (2:1)]. Fraction E9 (5.80 g, Ve/Vt 0.68−0.72) was fractionated
using silica gel CC [5 × 18 cm, CHCl3−EtOH−H2O (16:3:1 →
13:3:1 → 10:3:1, each 3.2 L)] and gave four subfractions (E9-1 to E9-
4). Subfraction E9-1 (1.25 g, Ve/Vt 0.01−0.30) was separated by RP-
18 (3 × 6 cm) CC using MeOH−H2O (3:1, 1.5 L) as eluent and was
further purified by RP-18 CC (2.5 × 5 cm), eluting with MeOH−H2O
(2:1), to give compound 4 [23 mg, Ve/Vt 0.54−0.61, TLC (RP-18
F254s) Rf 0.30, MeOH−H2O (3:1)]. Subfraction E9-2 (2.45 g, Ve/Vt
0.31−0.68) was chromatographed over RP-18 (5 × 5.5 cm), eluting
with MeOH−H2O (1:1 → 2:1, each 1.8 L), to provide 11 subfractions
(E9-2-1 to E-9-2-11). Subfraction E9-2-7 (140 mg, Ve/Vt 0.66−0.78)
was purified over silica gel (3 × 15 cm) and eluted with CHCl3−
MeOH (3:1, 0.8 L) to give compound 1 [21 mg, Ve/Vt 0.22−0.32,
TLC (silica gel F254) Rf 0.55, CHCl3−MeOH−H2O (13:3:1)].
Fraction E10 (6.75 g, Ve/Vt 0.72−0.78) was fractionated using silica
gel CC [7 × 15 cm, CHCl3−MeOH−H2O (17:3:1 → 15:3:1 →
13:3:1, each 3.2 L)] and gave 10 subfractions (E10-1 to E10-10).
Subfraction E10-6 (624 mg, Ve/Vt 0.75−0.82) was separated by RP-18
(3.5 × 9 cm) CC, eluting with MeOH−H2O (2:3, 1.7 L), to obtain
compound 7 [8 mg, Ve/Vt 0.78−0.81, TLC (RP-18 F254s) Rf 0.50,
MeOH−H2O (3:1)]. Subfraction E10-4 (1.64 g, Ve/Vt 0.35−0.60) was
chromatographed over a RP-18 column (4.5 × 10 cm), eluted with
MeOH−H2O (1:1.5, 2 L), and further purified by RP-18 (2.5 × 7 cm)
CC [E10-4-14 (34 mg, Ve/Vt 0.76−0.81)], using MeOH−H2O (2:1,
0.4 L) as eluent, to afford compound 5 [10 mg, Ve/Vt 0.50−1.00, TLC
(RP-18 F254s) Rf 0.30, MeOH−H2O (2:1)]. Fraction E12 (10.56 g, Ve/
Vt 0.84−0.91) was fractionated using silica gel CC [CHCl3−EtOH−
H2O, 17:3:1 → 12:3:1 → 9:3:1 (each 3.5 L)] and gave 25 subfractions
(E12-1 to E12-25). Subfraction E12-6 (559 mg, Ve/Vt 0.23−0.28) was
subjected to RP-18 CC [3.5 × 4.5 cm, MeOH−H2O (1:3 → 1:2 →
1:1, each 0.8 L)] to give 18 fractions (E12-6-1 to E12-6-18) and was
further purified by RP-18 CC [E12-6-12 (115 mg, Ve/Vt 0.54−0.70)],
using EtOH−MeOH−H2O (1:1:3, 2.4 L) as eluent, to yield
compound 8 [12 mg, Ve/Vt 0.50−0.60, TLC (RP-18 F254s) Rf 0.60,
EtOH−MeOH−H2O (1:1:1)].
compound 12 [35 mg, Ve/Vt 0.87−0.92, TLC (RP-18 F254s) Rf 0.25,
MeOH−H2O (2:1)]. Fraction B2-7 (4.95 g, Ve/Vt 0.45−0.52) was
chromatographed over RP-18 (4 × 9 cm), eluting with MeOH−H2O
(1:1, 4 L), and gave 10 subfractions (B2-7-1 to B2-7-15). Subfraction
B2-7-4 (1.05 g, Ve/Vt 0.25−0.51) was separated by RP-18 (4 × 7.5
cm) CC, eluting with MeOH−H2O (1:1, 1.8 L), to obtain compound
14 [350 mg, Ve/Vt 0.32−0.60, TLC (RP-18 F254s) Rf 0.55, MeOH−
H2O (2:1)].
Acanthosessiligenin I (1): white, amorphous powder; mp 230−232
°C; [α]20D −24.1 (c 0.5, MeOH); IR (CaF2 window) νmax 3360, 1732,
1
1648 cm−1; H and 13C NMR data, see Tables 1 and 2; negative
HRFABMS m/z 499.3401 [M − H]− (calcd for C31H47O5, 499.3423).
Acanthosessilioside A (2): white powder; mp 242−243 °C; [α]20
−70.5 (c 0.5, MeOH); IR (CaF2 window) νmax 3345, 1744, 1640 cm−1D;
1H and 13C NMR data, see Tables 1 and 2; positive HRFABMS m/z
633.3956 [M + H]+ (calcd for C36H57O9, 633.4002).
Acanthosessiligenin II (3): white needles; mp 236−237 °C; [α]20
D
−18.2 (c 0.7, MeOH); IR (CaF2 window) νmax 3350, 1740, 16548
cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRFABMS
m/z 517.3307 [M + H]+ (calcd for C31H49O6, 517.3529).
Acanthosessilioside B (4): white powder; mp 248−250 °C; [α]20
+34.6 (c 0.5, MeOH); IR (CaF2 window) νmax 3342, 1746, 1648 cm−1D;
1H and 13C NMR data, see Tables 1 and 2; positive HRFABMS m/z
679.4003 [M + H]+ (calcd for C37H59O11, 679.4057).
Acanthosessilioside C (5): white powder; mp 252−253 °C; [α]20
+66.8 (c 0.5, MeOH); IR (CaF2 window) νmax 3351, 1741, 1651 cm−1D;
1H and 13C NMR data, see Tables 1 and 2; positive HRFABMS m/z
695.4006 [M + H]+ (calcd for C37H59O12, 695.4006).
Acanthosessilioside D (6): white powder; mp 247−248 °C; [α]20
+21.0 (c 0.5, MeOH); IR (CaF2 window) νmax 3462, 1710, 1641 cm−1D;
1H and 13C NMR data, see Tables 1 and 2; negative HRFABMS m/z
647.3744 [M − H]− (calcd for C36H55O10, 647.3795).
Acanthosessilioside E (7): white powder; mp 255−256 °C; [α]20
−45.2 (c 0.5, MeOH); IR (CaF2 window) νmax 3420, 1714, 1645 cm−1D;
1H and 13C NMR data, see Tables 1 and 2; positive HRFABMS m/z
665.3892 [M + H]+ (calcd for C36H57O11, 665.3901).
Acanthosessilioside F (8): white powder; mp 259−260 °C; [α]20
+19.2 (c 0.5, MeOH); IR (CaF2 window) νmax 3380, 1740, 1655 cm−1D;
1H and 13C NMR data, see Tables 1 and 2; positive HRFABMS m/z
663.3753 [M + H]+ (calcd for C36H55O11, 663.3744).
Acid Hydrolysis of 2 and 4−8 and Determination of the
Absolute Configuration of the Monosaccharide Components.
Each compound (5 mg) was hydrolyzed with 2 mL of 2 N HCl in
H2O for 6 h at 80 °C, followed by neutralization with 2 mL of 2 N
NaOH in H2O and then extracted with CHCl3. The aqueous layer was
concentrated under a vacuum to give a residue of the sugar fraction.
The residue was dissolved in pyridine (100 μL), and then 0.1 M L-
cysteine methyl ester hydrochloride (150 μL) was added. After
reacting at 60 °C for 90 min, the reaction mixture was dried under a
vacuum. For derivatization, 100 μL of N-methyl-N-(trimethylsilyl)
trifluoroacetamide was added, and the mixture incubated at 37 °C for
30 min. Then, the mixture was subjected to GC analysis under the
following conditions: capillary column, DB-5 (30 m × 0.32 mm × 0.25
μm); detector, FID; detector temperature, 280 °C; injector temper-
ature, 250 °C; carrier, N2 gas (20.4 mL/min); oven temperature, 170−
250 °C with a rate of 5 °C/min, with 1 μL of each sample injected
directly into the inject port (splitless mode). By comparing the
retention time (tR) of the monosaccharide derivative with the standard
sample (D-glucose, Sigma), the absolute configuration of the
monosaccharide in 2 and 4−8 was confirmed to be D-glucose (tR
12.67 min).
Fraction B was chromatographed on a column prepared with a
highly porous polymer, Diaion HP-20 (12 × 45 cm), and successively
eluted with H2O and MeOH to give two fractions (B1 and B2).
Fraction B2 (73.40 g) was subjected to silica gel (12 × 15 cm) CC
using a gradient of CHCl3−MeOH−H2O [7:3:1 (8 L) → 65:35:10
(12 L)] to yield 11 fractions (B2-1 to B2-11). Fraction B2-4 (3.50 g,
Ve/Vt 0.31−0.36) was subjected to RP-18 (5 × 7 cm) CC elution with
MeOH−H2O [1.5:1 (3.2 L) → 2:1 (2.8 L) → 4:1 (3.6 L)] to give six
subfractions (B2-4-1 to B2-4-6). Subfraction B2-4-1 (913 mg, Ve/Vt
0.01−0.34) was purified over RP-18 (4 × 7.5 cm) CC, eluting with
MeOH−H2O (3:2, 2.2 L), to give compound 13 [250 mg, Ve/Vt
0.34−0.56, TLC (RP-18 F254s) Rf 0.60, MeOH−H2O (2:1)] and
Cytotoxicity Assay. Cell culture and cytotoxic assays against
human colon adenocarcinoma (HCT-116), human breast adenocarci-
noma (MCF-7), human breast adenocarcinoma (SK-BR-3), human
ovarian adenocarcinoma (SK-OV-3), human cervix adenocarcinoma
(HeLa), human hepatoma (HepG2), and human melanoma (SK-
MEL-5) were performed employing the MTT [3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich] assay as
described in the literature.16 The reference substance used was
F
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Journal of Natural Products
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paclitaxel, which exhibited IC50 values of 0.05, 0.90, 0.11, 0.40, and
0.15 uM for the HCT-116, MCF-7, SK-BR-3, SK-OV-3, and SK-MEL-
5 cell lines, respectively.
(16) Lee, D. Y.; Yoo, K. H.; Chung, I. S.; Kim, J. Y.; Chung, D. K.;
Kim, D. K; Kim, S. H.; Baek, N. I. Arch. Pharm. Res. 2008, 31, 830−
833.
Measurement of NO Production and Cell Viability. This assay
was carried out as previously described.17 Briefly, RAW 264.7
macrophages were harvested and seeded in 96-well plates (1 × 104
cells/well) for measurement of NO production. The plates were
pretreated with various concentrations of samples for 30 min and
incubated with LPS (1 μg/mL) for 24 h. The amount of NO was
determined by the nitrite concentration in the cultured RAW264.7
macrophage supernatants using the Griess reagent. The cell viability
was evaluated by MTT reduction. Aminoguanidine (Sigma), a well-
known NOS inhibitor, was tested as a positive control.
(17) Jung, L. H.; Lee, D. Y.; Cho, J. G.; Lee, S. M.; Kang, H. C.; Seo,
W. D.; Kang, H. W.; Kim, J. Y.; Baek, N. I. J. Kor. Soc. Appl. Biol. Chem.
2011, 54, 865−870.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR, 13C NMR, and HRFABMS spectra of 1−8 are
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +82-31-201-2661. Fax: +82-31-201-2157. E-mail:
nibaek@khu.ac.kr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Jeongseon-gun (20100903) and
the Ministry for Food, Agriculture, Forestry and Fisheries
(311025-03-1-SB010), Republic of Korea.
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