Biologically active arborinane-type triterpenoids and anthraquinones from Rubia yunnanensis

Article abstract of DOI:10.1021/np2002918

Twelve new arborinane-type triterpenoids (1-12) and four new anthraquinones (13-16), together with 50 known compounds, were isolated from the roots of Rubia yunnanensis. The structures of 1-16 were elucidated by spectroscopic data analysis and chemical methods. All compounds were evaluated for their cytotoxic, antibacterial, and antifungal activities. Rubiyunnanol C (5) is the first example of an arborinane-type triterpenoid with a double bond at C-8-C-9.

Full text of DOI:10.1021/np2002918

ARTICLE
pubs.acs.org/jnp
Biologically Active Arborinane-Type Triterpenoids and
Anthraquinones from Rubia yunnanensis
Jun-Ting Fan,†,‡ Bin Kuang,†,‡ Guang-Zhi Zeng,*,† Si-Meng Zhao,†,‡ Chang-Jiu Ji,† Yu-Mei Zhang,† and
Ning-Hua Tan*,†
†State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650201, People’s Republic of China
‡Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
S Supporting Information
b
ABSTRACT: Twelve new arborinane-type triterpenoids (1ꢀ12)
and four new anthraquinones (13ꢀ16), together with 50 known
compounds, were isolated from the roots of Rubia yunnanensis.
The structures of 1ꢀ16 were elucidated by spectroscopic data
analysis and chemical methods. All compounds were evaluated
for their cytotoxic, antibacterial, and antifungal activities. Ru-
biyunnanol C (5) is the first example of an arborinane-type
triterpenoid with a double bond at C-8ꢀC-9.
he plant genus Rubia belongs to the family Rubiaceae, which
consists of about 70 species with commercial, economic, and
1,3,6-trihydroxy-2-methyl-9,10-anthraquinone-3-O-(60-O-acetyl)-
β-D-glucopyranoside,30 1,3,6-trihydroxy-2-methyl-9,10-anthraqui-
none-3-O-α-L-rhamnopyranosyl-(1f2)-β-D-glucopyranoside,16
1,3,6-trihydroxy-2-methyl-9,10-anthraquinone-3-O-(60-O-acetyl)-
β-D-xylopyranosyl-(1f2)-β-D-glucopyranoside,31 1,3,6-trihy-
droxy-2-methyl-9,10-anthraquinone-3-O-(30-O-acetyl)-α-L-rham-
nopyranosyl-(1f2)-β-D-glucopyranoside,32 (2S,3S,4R,9E)-1,3,4-
trihydroxyl-2-[(20R)-20-hydroxytetracosanoylamino]-9-octadecene,33
5,7,20-trihydroxy-6-methoxyflavone,34 (+)-lariciresinol,35 (+)-
isolariciresinol,35 (ꢀ)-secoisolariciresinol,35 vladinol D,36 (+)-
pinoresinol,37 4-hydroxy-3-prenylbenzoic acid,38 6-cis-doco-
senamide,39 1-O-hexadecanolenin, squalene,40 β-sitosterol,
and daucosterol, by comparing their spectroscopic data with
those reported in the literature. Furthermore, all isolated com-
pounds were tested for their cytotoxicity against three human
cancer cell lines and for antibacterial and antifungal activities. In
this paper, the isolation, structure elucidation, and biological
evaluation of these compounds are described.
T
medicinal importance. Previous phytochemical investigations of
Rubia species have shown that this genus is a source of cyclic
hexapeptides, anthraquinones, and arborinane-type triterpenoids.1,2
Rubia yunnanensis (Franch.) Diels, known as “Xiao-Hong-Shen”,
is a perennial climbing herb native to mainland China. Its roots
have a long history of use in folk medicine to treat cancer, vertigo,
insomnia, rheumatism, tuberculosis, menstrual disorders, and
contusions3 and are used as an alternative for Rubia cordifolia, a
well-known traditional Chinese medicine listed in the Chinese
Pharmacopoeia. Previous studies on R. yunnanensis also demon-
strated the presence of cyclic hexapeptides,4,5 anthraquinones,6ꢀ8
and arborinane-type triterpenoids.6,9ꢀ13 In a recent study on this
species, our group reported a series of bioactive cyclic hexapep-
tides.14,15 In the present report, 12 new arborinane-type triter-
penoids (1ꢀ12) and four new anthraquinones (13ꢀ16), to-
gether with 50 known compounds, were isolated from the roots
of the title plant. The 50 known compounds were identified as
rubiarbonol K (17),9 rubiarbonol L (18),9 rubiarbonol G (19),10
1,3,6-trihydroxy-2-methyl-9,10-anthraquinone (20),16 3-hydroxy-
2-hydroxymethyl-9,10-anthraquinone (21),17 1,3,6-trihydroxy-
2-methyl-9,10-anthraquinone-3-O-(60-O-acetyl)-α-L-rhamnopy-
ranosyl-(1f2)-β-D-glucopyranoside (22),16 2-methoxy-1,4-naph-
thoquinone (23),18 rubiarbonol A,19 rubiarbonol F,19 rubianol-c,13
rubianol-d,13 rubianol-e,13 rubiarbonone A,10 rubiarbonone B,11
rubiarbonone C,11 rubiarbonone E,6,12 rubianoside I,13 rubiano-
side A,6,9 rubiarboside C,6,9 rubiarboside G,12 ursolic acid,20 4-
epihederagenin,21 maslinic acid,22 spathodic acid,23 lanosta-9(11),
24-dien-3-one,24 parkeol,25 rubianthraquinone,6 xanthopurpurin,26
1,6-dihydroxy-2-methyl-9,10-anthraquinone,27 rubiadin,28
2-hydroxymethyl-9,10-anthraquinone,29 1-hydroxy-2-meth-
yl-9,10-anthraquinone,16 2-carbomethoxy-9,10-anthraquinone,8
’ RESULTS AND DISCUSSION
Compound 1 was obtained as a white powder with a positive
specific rotation ([α]D23 +1.7). Its molecular formula, C32H52O5,
was deduced by HRESIMS (m/z 539.3707 [M + Na]+), indicating
seven degrees of unsaturation. The IR spectrum showed absorp-
tion bands for hydroxy (3423 cmꢀ1), carbonyl (1728 cmꢀ1), and
1
olefinic (1641 cmꢀ1) groups. The H NMR spectrum of 1
(Table 1) displayed characteristic resonances for two secondary
methyls (δH 0.94, 1.08), five tertiary methyls (δH 1.05, 1.14, 1.17,
1.20, 1.37), an oxygenated methylene (δH 4.08, 4.17), three
Received: April 5, 2011
Published: October 05, 2011
r
Copyright
2011 American Chemical Society and
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American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
Chart 1
oxygenated methines (δH 3.45, 5.28, 5.01), and one olefinic
proton (δH 5.51). The 13C NMR spectrum of 1 (Table 2)
exhibited 32 carbons, including a trisubstituted double bond (δC
118.7, 146.1) and an O-acetyl group (δC 170.5, 22.0), together
with seven methyls, eight methylenes (one oxygenated), eight
methines (three oxygenated), and five quaternary carbons.
Comparison of the NMR data of 1 with those of rubiarbonol
trisubstituted double bond (δH/δC 5.77/120.0, δC 142.4) in the
downfield region of 2 and the replacement of a hydroxymethy-
lene group in rubiarbonol A by a tertiary methyl (δH/δC 0.84/
16.0) at C-17. The double bond was placed between C-7 and
C-8, as determined by HMBC correlations of H-7 (δH 5.77) with
C-5, C-6, C-9, and C-14 and of H-11, H-6, and CH3-26 with C-8
1
(δC 142.4). In addition, the observed 1Hꢀ H COSY correlation
A
19 revealed that both compounds are based on an arborinane-
of H-7 with H-6 and NOE correlations of H-7 with H-15α and
H-15β also supported the position of this double bond. The
location of the tertiary methyl (CH3-28) at C-17 was confirmed
by HMBC correlations of CH3-28 with C-16, C-17, C-21, and
C-18. Furthermore, the β-orientation of CH3-28 was deduced
from the NOE correlation between CH3-28 and CH3-27
(Figure 2). Accordingly, the structure of 2 (rubiyunnanol A)
was established as 3β,19α-dihydroxyarbor-7,9(11)-diene.
type triterpenoid skeleton. The only difference found was the
presence of an additional acetate group in 1. The downfield shift
of the H-7 proton signal (δH 5.28) and HMBC correlations of
H-7 with the acetate carbonyl carbon, C-6, C-14, and C-8 enabled
the acetate unit to be placed at C-7. The relative configuration of
1 was deduced from the analysis of its ROESY spectrum
(Figure 1). The observed NOE correlations of H-3/H-5 and
CH3-23, H-5/H-7, H-7/CH3-26, and H-18/H-21 and CH3-26
indicated that H-3, H-5, H-7, H-18, H-21, CH3-23, and CH3-26
are cofacial and assigned as α-oriented. In turn, the cross-peaks of
H-8/CH3-25 and CH3-27 and H-19/CH3-27 and H-28 indicated
the β-orientation of H-8, H-19, H-28, CH3-25, and CH3-27.
From the above evidence, the structure of 1 was established as
7β-acetoxy-3β,19α,28-trihydroxyarbor-9(11)-ene (rubiarbonol
A 7-acetate).
Compound 3 exhibited the same molecular formula C30-
H48O2 as 2, as established by HRESIMS at m/z 463.3544
[M + Na]+. Analysis of the 1H and 13C NMR data of 3 (Tables 1
and 2) showed a close structural resemblance to 2, with the
compounds differing in the locations of a trisubstituted double
bond (δH/δC 5.34/119.8, δC 159.7) and a hydroxy group. The
olefinic proton signal at δH 5.34 was assigned to H-19, as
deduced by the HMBC correlations of H-19 with C-20, C-13,
1
1
Compound 2 gave the molecular formula C30H48O2, based on
HRESIMS (m/z 463.3540 [M + Na]+), requiring seven degrees
of unsaturation. The 1H and 13C NMR data of 2 (Tables 1 and 2)
were similar to those of rubiarbonol A except for the presence of a
C-17, and C-21 and Hꢀ H COSY correlations of H-19 with
H-20α and H-20β. In addition, cross-peaks of CH3-28, CH3-27,
H-16, H-19, H-20, and H-12 with the olefinic carbon signal at δC
159.7 (C-18) in the HMBC spectrum further supported the
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Journal of Natural Products
ARTICLE
1
Table 1. H NMR Data of Compounds 1ꢀ7 in Pyridine-d5 (δ in ppm, J in Hz)
position
1b
2a
3a
4a
5a
6b
7a
1α
1β
1.44, overlap
1.73, overlap
1.92, overlap
1.53, m
1.55, m
1.52, overlap
1.79, overlap
2.00, overlap
2.54, m
7.26, d (10.5)
6.15, d (10.5)
6.73, s
2.02, m
1.73, overlap
2.00, overlap
2.13, overlap
1.97, overlap
3.47, m
2
1.97, overlap
3
3.45, dd (10.5, 5.0) 3.50, t (7.6)
3.50, dd (9.6, 5.6) 3.50, dd (9.6, 6.0)
5
1.10, m
1.30, m
2.23, m
1.11, overlap
2.26, overlap
2.00, overlap
3.94, m
1.11, overlap
2.02, m
1.82, overlap
2.24, overlap
2.03, overlap
4.11, m
1.85, dd, (12.8, 2.0)
2.24, m
6α
6β
2.31, m
2.30, dd (12.0, 2.4) 2.64, overlap
1.73, overlap
5.28, m
2.00, overlap
4.04, m
2.73, overlap
2.03, overlap
4.16, m
7
5.77, brd (3.6)
8
2.63, brd (10.0)
5.51, brd (5.5)
2.50, m
2.40, brd (9.6)
5.50, brd (5.6)
2.26, overlap
1.91, overlap
2.41, brd (10.0)
5.39, brd (5.6)
1.79, overlap
1.52, overlap
2.78, brd (14.4)
1.91, m
2.55, brd (9.0)
5.43, brd (6.0)
2.24, overlap
2.66, overlap
5.75, brd (5.6)
2.56, m
11
5.53, brs
2.58, brs
4.85, m
12α
12β
15α
15β
16α
16β
18
2.73, overlap
2.45, dd (18.0, 5.5)
1.92, overlap
1.58, brd (14.5)
1.44, overlap
1.98, brd (14.0)
2.22, d (10.0)
5.01, m
3.14, dd (14.0, 9.2) 1.74, overlap
1.45, brd (13.2) 3.07, brd (14.0)
1.78, m 2.00, overlap
2.90, brd (14.4)
2.13, overlap
1.66, m
2.64, brd (14.5)
2.69, m
1.94, dd (14.5, 3.0) 2.03, overlap
1.51, td (13.5, 3.5) 1.56, overlap
1.61, brd (12.4) 1.73, overlap
1.72, m
1.52, overlap
1.70, brd (12.8)
1.97, overlap
1.74, overlap
2.36, d (10.0)
6.05, m
2.03, overlap
2.34, d (10.0)
5.19, m
1.97, overlap
1.61, dd (12.4, 7.2) 2.39, d (9.6)
19
4.46, m
5.34, brs
2.26, overlap
1.91, overlap
1.43, m
1.64, m
1.25, s
1.37, overlap
1.19, overlap
1.79, overlap
0.92, overlap
1.37, overlap
1.25, s
5.08, m
20α
20β
21
2.11, overlap
2.57, m
1.97, overlap
2.08, m
2.13, overlap
2.64, overlap
1.52, m
1.82, overlap
2.71, m
2.16, overlap
2.66, overlap
1.56, overlap
2.16, overlap
1.26, s
1.51, m
1.38, overlap
1.38, overlap
1.24, s
1.41, m
22
2.11, overlap
1.20, s
2.13, overlap
1.12, s
2.03, overlap
1.22, s
23
24
1.05, s
1.16, s
1.11, s
1.11, s
1.10, s
1.03, s
1.08, s
25
1.14, s
1.13, s
1.20, s
1.20, s
1.27, s
1.26, s
1.33, s
26
1.17, s
1.16, s
1.10, s
1.18, s
1.89, s
1.22, s
1.28, s
27
1.37, s
0.97, s
1.17, s
0.92, s
1.38, s
1.35, s
1.45, s
28a
28b
29
4.17, d (11.5)
4.08, d (11.5)
1.08, d (6.5)
0.94, d (6.5)
2.16, s
0.84, s
1.07, s
0.79, s
4.18, dd (11.2, 2.8) 4.23, d (11.5)
4.22, d (11.6)
4.09, d (11.6)
1.08, overlap
0.96, d (6.4)
4.02, brd (11.2)
1.04, d (6.4)
0.93, d (6.4)
3.92, d (11.5)
0.98, d (6.5)
0.89, d (6.5)
0.91, d (6.0)
0.84, overlap
0.91, d (6.4)
0.88, d (6.4)
0.89, d (6.4)
0.85, d (6.4)
30
OAc-7
OAc-19
2.16, s
a Recorded at 400 MHz. b Recorded at 500 MHz.
occurrence of a double bond between C-18 and C-19. Moreover,
HMBC correlations of H-7 with C-6, C-8, and C-14 as well as
NOE correlations of H-7 with H-5 and CH3-26 demonstrated
the hydroxy group to be linked to C-7 and having a β-orientation
(Figure 2). Accordingly, the structure of 3 (rubiyunnanol B) was
elucidated as 3β,7β-dihydroxyarbor-9(11),18-diene.
indicating seven degrees of unsaturation. The IR spectrum
showed the absorption bands for hydroxy (3440 cmꢀ1) and
conjugated carbonyl (1656, 1651 cmꢀ1) groups, and the UV
spectrum displayed an absorption at 251 nm, characteristic of the
presence of an α,β-unsaturated carbonyl chromophore in the
molecule. The 13C NMR spectroscopic data (Table 2) showed
the presence of 30 carbon signals due to one tetrasubstituted
double bond (δC 141.8, 162.1), one ketone carbon (δC 202.1),
seven methyls, eight methylenes (one oxygenated), seven
methines (three oxygenated), and five quaternary carbons.
Comparison of the 1D- and 2D-NMR data of 5 with those of
rubiarbonol A suggested that their structures are closely related.
The main differences were that a characteristic trisubstituted
double bond at C-9ꢀC-11 in conventional arborinane-type
triterpenoids was absent in 5, while a tetrasubstituted double
bond, a carbonyl group, and an additional hydroxy group
were present. HMBC correlations of H-5, H-6α, and H-6β with
the carbonyl carbon indicated that the latter group occurs at C-7.
In addition, the position of the additional hydroxy group at C-11
was deduced by correlations of H-11 with H-12α and H-12β in
Compound 4 was shown to have the molecular formula
C30H50O2, by HRESIMS (m/z 465.3710 [M + Na]+), indicating
1
two mass units more than 3. Comparison of the H and 13C
NMR data (Tables 1 and 2) with those of 3 suggested that the
only difference was the absence of the trisubstituted double bond
between C-18 and C-19 and the appearance of methine (δH/δC
1.61/52.5) and methylene (δH/δC 1.37/20.7) substituents in 4.
These changes implied that the additional methine and methy-
lene signals are located at C-18 and C-19, respectively, as confirmed
by HMBC correlations of H-18 (δH 1.61) with C-17, C-19, C-21,
1
C-27, and C-28, as well as the 1Hꢀ H COSY correlation of H-18
with H-19. Thus, the structure of 4 (19,28-didehydroxyrubiar-
bonol A) was proposed as 3β,7β-dihydroxyarbor-9(11)-ene.
Compound 5, a white powder, gave the molecular formula
C30H48O5 from its HRESIMS (m/z 487.3419 [M ꢀ H]ꢀ),
1
the 1Hꢀ H COSY spectrum combined with HMBC correlations
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Journal of Natural Products
ARTICLE
Table 2. 13C NMR Data of Compounds 1ꢀ12 in Pyridine-d5 (δ in ppm, J in Hz)
position
1a
2a
3a
4a
5a
6a
7a
8a
9b
10b
11a
12a
1
36.9
28.6
77.8
39.6
48.2
28.8
74.6
45.6
146.1
39.5
118.7
37.6
38.2
40.1
32.7
33.4
48.8
59.8
70.6
43.4
58.1
30.8
28.6
16.2
22.0
17.5
16.9
62.6
23.5
23.7
36.3
28.8
78.1
39.3
49.5
23.7
120.0
142.4
146.5
38.1
116.5
38.5
37.6
41.3
29.0
36.6
44.4
59.2
70.1
42.1
57.7
30.7
28.9
16.7
23.3
23.8
17.6
16.0
22.2
23.2
36.9
28.8
78.0
39.5
49.3
33.9
73.0
48.6
147.3
39.9
116.2
35.1
39.8
38.5
30.9
37.7
46.2
159.7
119.8
35.6
62.8
29.3
28.7
16.5
21.7
17.8
22.5
19.9
22.6
23.1
37.1
28.8
78.0
39.5
49.0
33.9
72.2
49.5
148.4
40.0
116.5
36.4
37.6
39.8
32.0
36.8
42.7
52.5
20.7
28.4
59.9
31.1
28.8
16.5
22.1
16.9
15.7
14.2
22.3
23.2
34.8
28.6
77.5
39.8
51.6
39.0
202.1
141.8
162.1
41.8
63.1
46.7
41.8
42.4
29.1
33.4
48.9
60.5
70.9
43.8
57.9
30.6
28.1
16.2
19.9
23.6
20.1
63.0
23.3
23.6
155.4
125.4
203.8
44.1
46.4
33.1
70.6
48.9
143.4
42.1
117.5
36.4
38.0
40.4
33.1
33.5
48.4
56.3
74.7
40.4
57.5
30.2
25.1
22.0
22.2
17.3
16.3
63.2
23.1
23.3
126.0
145.7
200.8
44.2
46.3
33.3
70.6
49.2
144.0
41.3
118.0
37.6
38.4
40.5
33.2
33.3
49.0
60.0
70.7
43.5
58.1
30.8
25.6
22.4
23.7
17.5
16.8
62.9
23.5
23.7
43.0
70.2
88.8
41.3
48.4
33.2
71.9
48.9
146.6
40.6
117.5
37.2
38.2
40.1
32.4
32.6
47.3
59.6
70.0
42.7
57.5
31.2
28.3
18.0
22.7
17.3
16.7
64.9
23.0
23.5
36.8
27.4
89.1
39.6
49.1
33.6
72.2
49.3
147.5
39.5
117.2
37.3
38.2
40.1
32.4
32.7
47.4
59.7
70.0
42.7
57.5
31.2
28.2
17.0
22.0
17.3
16.7
65.0
23.0
23.6
42.9
70.1
88.3
41.3
48.3
33.2
71.9
48.9
146.5
40.6
117.6
37.2
38.2
40.1
32.4
32.6
47.3
59.6
70.0
42.7
57.5
31.1
28.3
18.0
22.7
17.3
16.6
64.9
23.0
23.5
36.7
27.4
89.0
39.6
49.2
33.6
72.5
49.6
147.0
39.5
117.0
35.7
38.6
39.5
32.8
29.8
61.1
61.0
69.2
42.1
55.7
32.0
28.2
17.0
21.8
16.6
16.2
206.4
21.8
23.2
36.8
27.3
89.0
39.7
49.1
33.6
72.1
49.4
147.6
39.4
117.4
37.7
38.4
40.3
33.1
33.4
49.1
60.1
70.8
43.6
58.1
30.8
28.2
16.8
22.0
17.3
16.8
63.0
23.5
23.7
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Glc-
10
106.3
75.7
78.5
71.4
75.1
65.2
107.0
75.6
78.6
71.8
77.2
70.4
106.2
75.6
78.6
71.5
77.2
70.1
107.1
75.6
78.6
71.7
77.2
70.4
105.2
83.3
78.4
71.5
78.4
62.7
20
30
40
50
60
Glc-
100
200
300
400
500
600
OAc-2
105.5
75.3
78.5
71.8
78.5
62.8
105.2
75.3
78.4
71.8
78.4
62.9
171.6
22.3
105.5
75.3
78.5
71.8
78.6
62.8
106.0
77.2
78.2
71.7
78.0
62.8
171.0
21.6
OAc-7
170.5
22.0
OAc-19
OAc-28
OAc-60
170.9
21.8
170.8
21.1
170.9
21.2
170.8
21.1
171.1
20.9
a Recorded at 100 MHz. b Recorded at 125 MHz.
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of H-11 with C-10 and C-13. Furthermore, the tetrasubstituted
double bond present between C-8 and C-9 was determined by
HMBC correlations of H-6α, H-11, CH3-26/C-8 and of H-11,
OH-11, H-12β, H-5, CH3-25/C-9 and was found to be con-
jugated with the carbonyl group (Figure 1). Thus, the planar
structure of 5 was established.
The relative configurations at C-3, C-5, C-18, C-19, C-21,
C-28, CH3-23, CH3-25, CH3-26, and CH3-27 in 5 were estab-
lished as being the same as those in rubiarbonol A by a ROESY
experiment (Figure 1). The α-orientations of H-3, H-5, H-18,
H-21, CH3-23, and CH3-26 were established by NOE correla-
tions of H-3/H-5 and CH3-23 and H-18/H-21 and CH3-26, and
the β-orientations of H-19, H-28, and CH3-27 were deduced by
NOE correlations of CH3-27/H-19 and H-28. Moreover, OH-11
was assigned as α-oriented, as confirmed by NOE correlations of
H-11 with CH3-25 and CH3-27. Accordingly, the structure of 5
(rubiyunnanol C) was elucidated as 3β,11α,19α,28-tetrahydroxy-
arbor-8-en-7-one. Compound 5 is the first example of an
arborinane-type triterpenoid without a double bond at C-9ꢀC-11.
Compound 6 was isolated as white crystals, and its molecular
formula C32H48O5 was established by HRESIMS (m/z 535.3397
[M + Na]+), implying nine degrees of unsaturation. The NMR
spectroscopic data of 6 (Tables 1 and 2) were similar to those of
rubiarbonone E,6,12 except for the appearance of an additional
acetate group (δH/δC 2.16/21.8, δC 170.9) in 6. The HMBC
correlations observed from H-19 (δH 6.05) to the acetate car-
bonyl carbon, C-13, and C-18 indicated that the acetate group
is connected to C-19 (Figure 2). Therefore, compound 6
(rubiarbonone E 19-acetate) was established as 19α-acetoxy-
7β,28-dihydroxyarbor-1,9(11)-dien-3-one.
Compound 7 gave a molecular formula of C30H46O5 by
HRESIMS at m/z 509.3247 [M + Na]+, 16 mass units higher
than that of rubiarbonone E, in accordance with the presence of
an additional hydroxy group. Detailed comparison of its 1H and
13C NMR data (Tables 1 and 2) with those of rubiarbonone E
strongly supported the similarity in their structures in rings BꢀE.
In ring A, an additional hydroxy group was assigned at C-2, as
deduced from HMBC correlations of H-1 (δH 6.73) with C-2,
C-5, C-9, CH3-25, and the C-3 carbonyl group. Further evidence
was obtained from the cross-peaks of H-1 with H-11 and CH3-25
1
Figure 1. Selected 1Hꢀ H COSY, HMBC, and NOE correlations of 1
and 5.
1
Figure 2. Selected 1Hꢀ H COSY, HMBC, and NOE correlations of 2, 3, 6, 7, 11, 12, and 16.
2073
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1
Table 3. H NMR Data of Compounds 8ꢀ12 in Pyridine-d5 (δ in ppm, J in Hz)
position
8a
9b
10b
11b
12b
1α
1β
1.65, m
1.52, overlap
1.73, brd (13.0)
2.55, m
1.64, t (12.0)
2.05, m
1.53, m
1.73, m
1.33, m
2.13, overlap
5.67, m
1.58, overlap
2.36, overlap
1.89, overlap
3.31, m
2α
2β
5.64, m
2.56, overlap
1.97, m
1.98, m
3
3.61, d (10.0)
1.11, m
3.37, dd (11.5, 4.0)
0.96, m
3.61, d (10.0)
1.05, m
3.36, dd (11.5, 4.0)
0.95, m
5
0.97, m
6α
6β
2.23, overlap
1.90, overlap
3.99, overlap
2.39, overlap
5.42, brd (5.6)
2.54, brd (18.0)
2.39, overlap
2.80, brd (14.4)
1.90, overlap
1.55, overlap
1.90, overlap
2.31, overlap
4.69, m
2.20, overlap
1.89, overlap
3.97, overlap
2.36, brd (9.5)
5.45, brd (6.0)
2.44, brd (19.0)
2.29, m
2.19, overlap
1.87, overlap
3.91, overlap
2.36, overlap
5.41, brd (6.0)
2.51, m
2.17, m
2.20, m
1.87, overlap
3.95, overlap
2.23, overlap
5.48, brd (6.0)
2.47, brd (18.5)
2.23, overlap
2.90, m
1.89, overlap
3.99, m
7α
8
2.46, overlap
5.42, brd (5.0)
2.60, overlap
2.46, overlap
2.80, brd (13.5)
2.01, overlap
1.58, overlap
2.01, overlap
2.36, overlap
5.10, m
11
12α
12β
15α
15β
16α
16β
18
2.36, overlap
2.76, m
2.78, brd (14.5)
1.89, overlap
1.52, overlap
1.89, overlap
2.27, d (9.5)
4.64, m
1.87, overlap
1.54, overlap
1.87, overlap
2.27, d (9.5)
4.66, m
1.87, overlap
1.33, overlap
2.63, m
2.56, overlap
5.02, m
19
20α
20β
21
2.13, overlap
2.23, overlap
1.55, overlap
1.55, overlap
1.38, s
2.12, m
2.12, m
2.30, m
2.13, overlap
2.60, overlap
1.58, overlap
2.13, overlap
1.31, s
2.20, overlap
1.52, overlap
1.52, overlap
1.28, s
2.19, overlap
1.54, overlap
1.54, overlap
1.34, s
1.66, m
1.33, overlap
1.27, s
22
23
24
1.15, s
1.05, s
1.13, s
1.03, s
1.17, s
25
1.21, s
1.08, s
1.17, s
1.03, s
1.08, s
26
1.29, s
1.24, s
1.23, s
1.24, s
1.33, s
27
1.11, s
1.07, s
1.08, s
0.93, s
1.40, s
28a
28b
29
4.62, d , (12.0)
4.30, d , (12.0)
0.98, d (5.6)
0.87, d (5.6)
4.58, d (12.0)
4.28, d (12.0)
0.96, d (6.0)
0.85, d (6.0)
4.59, d (12.0)
4.27, overlap
0.96, d (6.0)
0.85, d (6.0)
9.98, s
4.17, overlap
4.10, overlap
1.08, overlap
0.95, d (6.5)
0.86, d (6.5)
0.77, d (6.5)
30
Glc-
10
20
30
40
4.97, d (8.0)
3.99, overlap
4.23, m
4.88, overlap
3.97, overlap
4.22, overlap
4.12, overlap
4.12, overlap
4.88, overlap
4.34, overlap
4.93, overlap
3.91, overlap
4.17, overlap
4.17, overlap
4.08, m
4.90, overlap
3.95, overlap
4.19, m
4.92, d (7.5)
4.24, overlap
4.32, overlap
4.17, overlap
3.94, overlap
4.51, overlap
4.42, m
4.07, overlap
4.07, overlap
4.92, brd (11.6)
4.86, dd (11.6, 4.4)
4.08, overlap
4.14, m
50
60a
60b
Glc-
100
200
300
4.93, overlap
4.27, overlap
4.90, overlap
4.34, m
5.13, d (8.0)
4.06, m
5.10, d , (7.5)
4.02, m
5.16, d (8.0)
4.08, overlap
4.26, overlap
4.26, overlap
3.95, overlap
4.53, dd (12.0, 2.0)
4.38, m
5.37, d (7.5)
4.10, overlap
3.94, overlap
4.32, overlap
4.24, overlap
4.51, overlap
4.37, dd (12.0, 5.0)
4.22, overlap
4.22, overlap
3.97, overlap
4.51, brd (12.0)
4.34, overlap
4.22, m
400
500
4.17, overlap
3.97, m
600a
600b
OAc-2
OAc-28
OAc-60
4.53, brd (12.0)
4.34, dd (12.0, 5.5)
2.45, s
2.31, overlap
2.07, s
2.06, s
2.05, s
2.01, s
a Recorded at 400 MHz. b Recorded at 500 MHz.
2074
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Journal of Natural Products
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Table 4. H and 13C NMR Data of Compounds 13ꢀ16 (δ in ppm, J in Hz)
1
13a
14b
15c
16d
position
δH
δC
position
δH
δC
position
δH
δC
position
δH
δC
1
162.3
117.6
162.4
107.3
1
2
3
4
161.8
123.7
161.4
106.1
1
2
3
4
156.7 1, 10
105.4 2, 20
108.5 3a, 30a 2.70, m
146.4 3b, 30b 2.49, m
131.2 4, 40
123.1 4a, 40a
129.5 5, 50
126.8 6, 60
124.1 7, 70
125.9 8, 80
8a, 80a
198.3
2
3.82, brd (13.2) 47.4
3
7.96, s
37.7
4
7.20, s
7.50, d (2.5)
7.43, overlap
7.43, overlap
4a
131.8 4a
133.9 4a
5.39, m
68.2
5
110.5
164.0
5
6
7
8
113.1
164.8
5
6
7
8
8.66, m
149.5
6
7.56, overlap
7.56, overlap
8.57, m
8.19, brd (7.6) 126.9
7
7.39, dd (8.5, 2.5) 120.5
8.10, d , (8.5) 129.1
7.19, dd (8.4, 2.4) 121.9
7.62, brt (7.6)
7.36, brt (7.6)
134.0
127.5
8
8.09, d (8.4)
129.9
8a
126.1 8a
185.7
123.7 8a
8.25, brd (7.6) 127.2
131.9
9
9
186.3 Glc-
111.3 10
181.9 20
135.4 30
40
100.6 50
73.3 60a
75.8 60b
9a
108.7 9a
181.8 10
135.0 10a
5.74, d (7.5)
4.49, m
103.7
10
10a
CH3-2
75.3
4.41, overlap
4.41, overlap
4.14, m
78.7
2.04, s
8.2
56.1 10
Glc-
71.4
OCH3-6 3.93, s
OH-1 13.20, s
5.10, d (7.2)
3.44, overlap
3.44, overlap
3.19, m
79.1
20
30
40
50
60a
60b
4.59, dd (12.0, 2.0) 62.5
4.41, overlap
70.0 COOCH3-2 3.75, s
52.4
3.73, m
74.3 OH-1
63.6
12.09, s
171.5
4.38, brd (12.0)
4.05, dd (12.0, 7.8)
CH2OH-2 4.63, d (11.4)
4.54, d (11.4)
OAc-60
51.0
170.6
20.6
2.06, s
OH-1
13.39, s
a 1H at 500 MHz and 13C at 100 MHz in DMSO-d6. b 1H at 600 MHz and 13C at 150 MHz in DMSO-d6. c 1H at 500 MHz and 13C at 125 MHz in
pyridine-d5. d 1H at 400 MHz and 13C at 100 MHz in pyridine-d5.
in the ROESY spectrum (Figure 2). Therefore, the structure of 7
(2-hydroxyrubiarbonone E) was assigned as 2,7β,19α,28-tetra-
hydroxyarbor-1,9(11)-dien-3-one.
with analogous data of rubiarboside G12 suggested that 9 is an
arborinane-type triterpenoid diglycoside with two glucopyrano-
syl units. The addition of an extra acetate functionality in 9 was
the only difference determined. The acetate group was located at
C-28 by the apparent downfield shift of C-28 (δC 65.0) in 9 as
well as HMBC correlations of H-28 with the acetate carbonyl
carbon, C-16, C-17, C-18, and C-21. Moreover, the H-100 signal at
δH 5.13 showed a HMBC correlation with C-60 (δC 70.4), in
support of a C-1fC-6 linkage of the two glucose moieties. The
HMBC correlation between H-10 and C-3 suggested that the
sugar unit is attached to C-3. Furthermore, the sugar obtained
from acid hydrolysis was identified as D-glucose by GC analysis.
Thus, the structure of 9 was established as 28-acetoxy-
3β,7β,19α-trihydroxyarbor-9(11)-en-3-O-β-D-glucopyranosyl-
(1f6)-β-D-glucopyranoside (rubiarboside G 28-acetate).
Compound 10 gave a molecular formula of C46H74O17 as es-
tablished by HRESIMS (m/z 933.4635 [M + Cl]ꢀ). The exa-
mination of 1H and 13C NMR spectroscopic data (Tables 3 and 2)
revealed that 10 is an analogue of 9, containing an additional
acetate group. The acetate group was assigned to C-2 because the
methylene signals (δH/δC 1.98, 2.55/27.4) at C-2 in 9 changed
to an oxygenated methine signal (δH/δC 5.64/70.1) in 10. This
assignment was further confirmed by HMBC correlations of H-2
(δH 5.64) with the acetate carbonyl carbon, C-1, and C-3. In
The molecular formula of compound 8 was determined as
C42H66O13 from the HRESIMS (m/z 777.4425 [M ꢀ H]ꢀ).
1
Analysis of the H and 13C NMR spectroscopic data indicated
that 8 is an arborinane-type triterpenoid glycoside with a glu-
copyranose unit. The NMR data of 8 (Tables 2 and 3) were very
similar to those of rubiarboside C,6 except for signals of a
glucopyranosyl unit and an acetate unit. The downfield shift of
C-60 (δC 65.2) in 8 suggested that the acetate group is attached to
the C-60 position of the glucose, which was confirmed by HMBC
correlations from H-60 to the acetate carbonyl carbon. The β-
anomeric configuration for the glucose was determined from the
large 3JH1,H2 coupling constant (J = 8.0 Hz). Acid hydrolysis of 8
yielded D-glucose, which was determined by GC analysis of its
corresponding trimethylsilylated L-cysteine adduct. Therefore,
compound 8 was established as 2α,28-diacetoxy-3β,7β,19α-
trihydroxyarbor-9(11)-en-3-O-(60-O-acetyl)-β-D-glucopyranoside
(rubianol-e 3-O-(60-O-acetyl)-β-D-glucopyranoside).
Compound 9 was assigned a molecular formula of C44H72O15
from its HRESIMS (m/z 839.4799 [M ꢀ H]ꢀ). Two anomeric
signals (δH/δC 4.88/107.0, 5.13/105.5) (β form) observed in
the 1H and 13C NMR spectra (Tables 3 and 2) and comparison
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Journal of Natural Products
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addition, H-2 displayed NOE correlations with CH3-24 and
CH3-25, indicating the acetate group to be α-oriented. Therefore,
compound 10 was characterized as 2α,28-diacetoxy-3β,7β,19α-
trihydroxyarbor-9(11)-en-3-O-β-D-glucopyranosyl-(1f6)-β-D-
glucopyranoside (2α-acetoxy-28-acetylrubiarboside G).
Table 5. IC50 Values (μM) of Active Compounds against
Three Human Cancer Cell Lines
compound
A549
HeLa
SMMC-7721
2
3
6
9
9.8
NAa
5.3
7.9
2.2
2.2
3.0
4.2
NA
0.6
NA
NA
NA
NA
NA
NA
NA
4.3
Compound 11 exhibited a [M ꢀ H]ꢀ ion peak at m/z 795.4513
in its HRESIMS, corresponding to the molecular formula C42-
H68O14. Comparison of the NMR spectroscopic data of 11
(Tables 2 and 3) with those of rubiarboside G showed many
similarities except that the hydroxymethylene group at C-17 was
missing and a formyl group (δH/δC 9.98/206.4) was present in
11. This was supported by the significant downfield shift of C-17
(δC 61.1), as well as key correlations of the formyl proton (H-28)
with C-16, C-17, and C-18 and of H-18, H-21 with the formyl
carbonyl carbon (C-28) in the HMBC spectrum. The β-orienta-
tion of the formyl group was deduced from NOE correlations of
H-28 with H-19 and H-22 (Figure 2). Thus, compound 11 was
elucidated as 28β-formyl-3β,7β,19α-trihydroxyarbor-9(11)-en-
3-O-β-D-glucopyranosyl-(1f6)-β-D-glucopyranoside
8.2
NA
NA
7.0
19
21
NA
NA
NA
0.01
22
23
paclitaxel
camptothecin
a NA: IC50 > 10 μM.
0.003
Table 6. MIC50 Values (μM) of Active Compounds against
Staphylococcus aureus and Candida albicans
(rubiarboside G 28-al).
Compound 12 was found to have the same molecular formula,
C42H70O14, as rubiarboside G, as established by its HRESIMS
(m/z 797.4706 [M ꢀ H]ꢀ). Analysis of the 1D- and 2D-NMR
spectroscopic data of 12 (Tables 2 and 3) and comparison with
those of rubiarboside G suggested that both compounds possess
the same aglycone with two glucopyranosyl units at C-3, differing
only in the sequence of the two sugar units. The significant
downfield shift of C-20 (δC 83.3) and the upfield shift of C-60 (δC
62.7) in 12 implied the (1f2) linkage of the two glucose units, as
confirmed by the HMBC correlations of H-100 with C-20 and of
H-20 with C-100 (Figure 2). Moreover, acid hydrolysis of 12 affor-
ded D-glucose by GC analysis. The structure of 12 was therefore
established as 3β,7β,19α,28-tetrahydroxyarbor-9(11)-en-3-O-β-
D-glucopyranosyl-(1f2)-β-D-glucopyranoside (rubiarbonol A 3-O-
β-D-glucopyranosyl-(1f2)-β-D-glucopyranoside).
Compound 13 was isolated as yellow needles, and its molec-
ular formula wasestablished as C16H12O5 on the basis of HRESIMS
(m/z 307.0578 [M + Na]+), indicating 11 degrees of unsatura-
tion. The IR absorption bands indicated the presence of hydroxy
(3409 cmꢀ1), carbonyl (1659 cmꢀ1), and aromatic (1621 and
1593 cmꢀ1) groups. The UV spectrum of 13 exhibited absorp-
tions maxima at 276, 337, and 415 nm, suggesting an anthraqui-
none as the basic structure. The 1H and 13C NMR spectroscopic
data of 13 (Table 4) were closely related to those of 1,3,6-trihy-
droxy-2-methyl-9,10-anthraquinone,16 except for the presence of
a methoxy group (δH/δC 3.93/56.1) at the C-6 position in 13.
This deduction was confirmed by the HMBC correlation of the
methoxy proton signal (δH 3.93) with C-6, as well as NOE corre-
lations of the methoxy proton signal with H-5 and H-7. There-
fore, the structure of 13 was elucidated as 1,3-dihydroxy-6-
methoxy-2-methyl-9,10-anthraquinone.
compound
S. aureus
C. albicans
19
20
23
NAa
21.5
NA
0.1
10.8
NA
16.0
ampicillin
miconazole nitrate
a NA: MIC50 > 25 μM.
0.9
and C-3. Acid hydrolysis of 14 gave D-glucose as a sugar residue.
Consequently, compound 14 was determined as 1,3,6-trihy-
droxy-2-hydroxymethyl-9,10-anthraquinone-3-O-(60-O-acetyl)-
β-D-glucopyranoside.
Compound 15 was isolated as a pale yellow powder. Its
molecular formula was established as C18H20O9 due to the
quasi-molecular ion peak at m/z 379.1030 ([M ꢀ H]ꢀ) in the
HRESIMS, requiring nine degrees of unsaturation. The 13C
NMR spectroscopic data of 15 (Table 4) displayed 18 carbon
signals corresponding to 10 aromatic carbons, a methyl ester
(δH/δC 3.75/52.4), and signals arising from a β-glucopyranosyl
moiety. The above data obtained indicated that 15 is a naphtho-
hydroquinone glycoside derivative like rubinaphthin A,7 with a
methyl ester group. In the HMBC spectrum, the methyl ester
proton signal showed correlations with the ester carbonyl carbon
(δC 171.5) and C-2. Acid hydrolysis of 15 produced D-glucose as
determined by GC analysis. Accordingly, the structure of 15 was
assigned as 2-carbomethoxy-1,4-naphthohydroquinone-4-O-β-
D-glucopyranoside (rubinaphthin A methyl ester).
Compound 16, a white powder, gave the molecular formula
C20H18O4 from the positive-mode HRESIMS (m/z 345.1111
[M + Na]+), indicating 12 degrees of unsaturation. The 13C
NMR spectrum (Table 4) displayed 10 carbon signals, including
a methylene, six methines (four aromatic and one oxygenated)
and three quaternary carbons (one carbonyl and two aromatic).
Accordingly, compound 16 was presumed to be a dimer with a
symmetrical structure. In the 1H NMR spectrum, four mutually
coupled aromatic proton signals resonated at δH 8.19 (brd, 7.6),
7.62 (brt, 7.6), 7.36 (brt, 7.6), and 8.25 (brd, 7.6), which sug-
gested that 16 possesses a 1,2-disubstituted benzene ring. The
carbonyl signal at δC 198.3 (C-1) and the hydroxymethine group
at δC 68.2 (C-4) were assigned at C-8a and C-4a, respectively,
Compound 14 was obtained as a pale yellow powder, and its
molecular formula was indicated as C23H22O12 by HRESIMS
(m/z 489.1021 [M ꢀ H]ꢀ). The 1H and 13C NMR spectra of 14
(Table 4) showed 14 carbon signals of the anthraquinone skeleton,
a hydroxymethyl (δH/δC 4.54, 4.63/51.0), an acetate group, and
a β-glucopyranosyl unit. Compound 14 was assigned a similar
structure to 1,3,6-trihydroxy-2-methyl-9,10-anthraquinone-3-
O-(60-O-acetyl)-β-D-glucopyranoside30 by comparison of their
NMR data. The only difference was the replacement of the
methyl group at C-2 by the hydroxymethyl group in 14. This was
confirmed by HMBC correlations of CH2OH-2 with C-2, C-1,
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Journal of Natural Products
ARTICLE
which were supported by HMBC correlations from H-8 to C-1,
C-4a, and C-6 and from H-5 to C-4, C-7, and C-8a. Moreover, the
Fr.A (1.5 kg) was subjected to silica gel CC eluted with a gradient of
petroleum etherꢀEtOAc (1:0ꢀ0:1), to obtain six major fractions (Fr.A-1
to Fr.A-6). Fr.A-2 (130 g) was further chromatographed over silica gel
using petroleum etherꢀEtOAc (150:1ꢀ100:1) to yield 1-hydroxy-2-
methyl-9,10-anthraquinone (15 mg) and 2-carbomethoxy-9,10-anthra-
quinone (2 mg). Fr.A-3 (70 g) gave 23 (28 mg) and lanosta-9(11),24-
dien-3-one (40 mg) after repeated chromatography over Sephadex LH-
20 (CHCl3ꢀMeOH, 1:1) and silica gel (petroleum etherꢀMe2CO,
70:1). Ursolic acid (131 mg) and β-sitosterol (120 mg) were obtained by
recrystallization in CHCl3 from Fr.A-4 directly. Fr.A-5 (78 g) was
applied to silica gel eluting with CHCl3ꢀMeOH (50:1ꢀ20:1) and
was then separated over Sephadex LH-20 (CHCl3ꢀMeOH, 1:1) and
then a RP-18 column using MeOHꢀH2O (80ꢀ100%), to yield four
subfractions (Fr.A-5-1 to Fr.A-5-4). Fr.A-5-2 (270 mg) was separated by
semipreparative HPLC (ACNꢀH2O, 90%) to obtain compounds 2
(5 mg), 3 (4 mg), 4 (11 mg), and 17 (9 mg). Fr.A-5-4 (1 g) was further
purified on Sephadex LH-20 (CHCl3ꢀMeOH, 1:1) and then applied to
silica gel (CHCl3ꢀMe2CO, 20:1) to give parkeol (44 mg).
1
1Hꢀ H COSY correlations of H-2/H-3/H-4 and HMBC corre-
lations of H-3/C-4a, C-1, C-2, and C-4 revealed the linkage of
C-1/C-2/C-3/C-4. In addition, the connection of the two parts
of the dimer was concluded unambiguously to be at C-2/C-20. In
the ROESY spectrum, H-2 showed a correlation with H-4, which
indicated that H-2 and H-4 are cofacial and were randomly
assigned as α-oriented (Figure 2). Accordingly, the structure of
16 was determined as 4R0S0,40R0S0-dihydroxy-2R0S0,20R0S0-bi-
naphthalene-1,10-dione.
Compounds 17 and 18, named rubiarbonol K and rubiarbonol
L, respectively, were initially isolated from R. yunnanensis by Zou
and co-workers,9 but their NMR data were not reported. Their
1H and 13C NMR spectroscopic data were determined (Table S1,
Supporting Information).
All compounds isolated were evaluated for their cytotoxicity
against three human cancer cell lines, and the active com-
pounds are included in Table 5. Antibacterial activity against
Staphylococcus aureus and antifungal activity against Candida
albicans of all compounds were tested using the turbidimetric
method41 with the MIC data shown for the active compounds
as shown in Table 6.
The remainder of the cyclopeptide-containing fraction after the
isolation of all cyclic hexapeptides14,15 was combined and named Fr.B
(500 g). Fr.B was chromatographed on a silica gel column eluted with
CHCl3ꢀMeOH (30:1ꢀ8:2) to afford five fractions (Fr.B-1 to Fr.B-5).
Fr.B-1 (50 g) was separated by silica gel CC (petroleum etherꢀMe2CO,
10:1ꢀ1:1) and then by Sephadex LH-20 (CHCl3ꢀMeOH, 1:1) to give
13 (5 mg), 21 (24 mg), rubianthraquinone (42 mg), 2-hydroxymethyl-
9,10-anthraquinone (2 mg), 6-cis-docosenamide (28 mg), and squalene
(8 mg). Fr.B-2 (156 g) was chromatographed over silica gel using
petroleum etherꢀMe2CO (5:1ꢀ0:1) and then Sephadex LH-20 (CHCl3ꢀ
MeOH, 1:1) to give three subfractions (Fr.B-2-1 to Fr.B-2-3). Fr.B-2-1
(11 g) was purified by repeated silica gel CC (CHCl3ꢀMeOH, 30:1) to
obtain 16 (12 mg), 19 (1.3 g), rubiarbonone C (32 mg), and 1-O-
hexadecanolenin (17 mg). Fr.B-2-2 (33 g) was separated by RP-18 gel
(MeOHꢀH2O, 60ꢀ80%) and silica gel (CHCl3ꢀMe2CO, 5:1) to yield
20 (899 mg), xanthopurpurin (28 mg), 1,6-dihydroxy-2-methyl-9,10-
anthraquinone (5 mg), rubiadin (10 mg), and 5,7,20-trihydroxy-6-meth-
oxyflavone (15 mg). Fr.B-2-3 (68 g) was further subjected to passage
over RP-18 gel (MeOHꢀH2O, 50ꢀ80%), followed by repeated silica
gel CC (CHCl3ꢀMeOH, 20:1), to give 18 (9 mg), rubianol-e (74 mg),
rubiarbonone B (11 mg), 4-epihederagenin (18 mg), maslinic acid
(25 mg), spathodic acid (7 mg), and two mixtures. Compound 1 (10 mg)
and rubianol-c (7 mg) were purified by semipreparative HPLC (ACNꢀ
H2O, 60%) from one mixture, and compound 6 (18 mg) and rubiarbo-
none A (38 mg) were purified by semipreparative HPLC (ACNꢀH2O,
65%) from the other mixture. Fr.B-3 (65 g) was subjected to RP-18 gel
CC eluting with MeOHꢀH2O (40ꢀ80%), followed by column chro-
matography over Sephadex LH-20 (CHCl3ꢀMeOH, 1:1) and silica gel
(CHCl3ꢀMe2CO, 3:1), to give 5 (15 mg), rubiarbonol A (594 mg),
(+)-lariciresinol (85 mg), (+)-isolariciresinol (63 mg), (ꢀ)-secoisolari-
ciresinol (100 mg), (+)-pinoresinol (34 mg), and a mixture. The mixture
was finally purified by semipreparative HPLC (MeOHꢀH2O, 60%)
to yield 7 (4 mg) and rubiarbonone E (25 mg). Fr.B-4 (89 g) was
further separated by Sephadex LH-20 (CHCl3ꢀMeOH, 1:1) and
then silica gel CC (CHCl3ꢀMeOH, 15:1) to obtain rubiarbonol F
(52 mg), rubianol-d (2 mg), (2S,3S,4R,9E)-1,3,4-trihydroxyl-2-[(20R)-
20-hydroxytetracosanoylamino]-9-octadecene (20 mg), vladinol D (6 mg),
and 4-hydroxy-3-prenylbenzoic acid (6 mg). Fr.B-5 (52 g) was subjected
to RP-18 gel (MeOHꢀH2O, 40ꢀ60%) and Sephadex LH-20 (CHCl3ꢀ
MeOH, 1:1) column chromatography to provide three fractions (Fr.B-
5-1 to Fr.B-5-3). Compound 15 (13 mg) was purified from Fr.B-5-2
by passage over a RP-18 column (MeOHꢀH2O, 50%). Compound 8
(39 mg), rubiarboside C (300 mg), daucosterol (210 mg), and a
mixture were obtained from Fr.B-5-3 by purification over RP-18
(MeOHꢀH2O, 40ꢀ50%) and silica gel (CHCl3ꢀMeOH, 10:1) col-
umns. The mixture was further separated by semipreparative HPLC
’ EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were ob-
tained on an X-4 micromelting point apparatus. Optical rotations were
measured with a Horiba SEPA-300 polarimeter. UV spectra were
obtained using a Shimadzu UV-2401A spectrophotometer. IR spectra
were obtained by a Tenor 27 spectrophotometer using KBr pellets. 1D
and 2D NMR spectra were recorded on Bruker AM-400, DRX-500, or
AV-600 spectrometers with TMS as internal standard. Mass spectra
were recorded on a VG Autospec-3000 spectrometer or an API QSTAR
time-of-flight spectrometer. GC analysis was performed on an Agilent
Technologies HP5890 gas chromatograph with a 30QC2/AC-5 quartz
capillary column (30 mm ꢁ 0.32 mm, 0.25 μm); detection, FID. Analytical
or semipreparative HPLC was performed on an Agilent 1100 liquid
chromatograph with a Zorbax Eclipse-C18 (4.6 mm ꢁ 150 mm;
9.4 mm ꢁ250 mm) column. Column chromatography was performed
using silica gel (200ꢀ300 mesh, Qingdao Yu-Ming-Yuan Chemical
Co. Ltd., Qingdao, People’s Republic of China), Sephadex LH-20
(Pharmacia Fine Chemical Co., Uppsala, Sweden), and Lichroprep
RP-18 gel (40ꢀ63 μM, Merck, Darmstadt, Germany). Fractions were
monitored by TLC, and spots were visualized by heating silica gel plates
sprayed with 10% H2SO4 in EtOH.
Plant Material. The roots of R. yunnanensis were purchased in
September 2007 from the Yunnan Lv-Sheng Pharmaceutical Co. Ltd.,
Kunming, People’s Republic of China. The material was identified by
Prof. Su-Gong Wu at Kunming Institute of Botany. A voucher specimen
(No. Wu20070905) has been deposited in the Herbarium of Kunming
Institute of Botany, Chinese Academy of Sciences.
Extraction and Isolation. The air-dried and powdered roots of
R. yunnanensis (100 kg) were extracted with MeOH (3 ꢁ 100 L) under
reflux. After removal of the solvent under reduced pressure, the MeOH
extract (21 kg) was suspended in H2O and partitioned successively with
EtOAc and n-BuOH to give an EtOAc-soluble portion (6.4 kg) and an
n-BuOH-soluble portion (8 kg). The EtOAc part (6.4 kg) was subjected
to silica gel column chromatography eluting with CHCl3ꢀMeOH (1:0,
95:5, 9:1, 8:2, 0:1) to afford a fraction in which cyclopeptides were
absent (1.5 kg, CHCl3, Fr.A) and a cyclopeptide-containing fraction
(1.2 kg, CHCl3ꢀMeOH, 95:5, 9:1, 8:2, Fr.B).
2077
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Journal of Natural Products
ARTICLE
(CH3CNꢀH2O, 48%) and yielded rubianoside I (60 mg) and rubiano-
side A (8 mg).
(4.04) nm; IR (KBr) νmax 3547, 3519, 2975, 2954, 2926, 2900, 1706,
1658, 1377, 1272, 1025, 842 cmꢀ1; 1H and 13C NMR data, see Tables 1
and 2; positive-mode FABMS m/z 513 (13) [M + H]+; positive-mode
HRESIMS m/z 535.3397 [M + Na]+ (calcd for C32H48O5Na, 535.3399).
2-Hydroxyrubiarbonone E (7): white powder; [α]2D3 +19.7 (c 0.19,
MeOH); UV (MeOH) λmax (logε) 257 (3.65), 262 (3.66), 324 (3.28) nm;
The n-BuOH layer (8 kg), named Fr.C, was separated using a
macroporous adsorption resin D101 and eluted with a gradient of
MeOHꢀH2O (0ꢀ60%). The fractions eluted with MeOHꢀH2O (20ꢀ
60%, 1.3 kg) were combined and subjected to silica gel CC. Gradient
elution with CHCl3ꢀMeOHꢀH2O (9:1:0.1ꢀ7:3:0.3) gave Fr.C-1
through Fr.C-5. Fr.C-2 (270 g) was further chromatographed over a
silica gel column using EtOAcꢀMeOH (9:1ꢀ8:2), followed by passage
over RP-18 gel (MeOHꢀH2O, 10ꢀ60%), to furnish four subfractions
(Fr.C-2-1 to Fr. C-2-4). Subfractions Fr.C-2-2 (35 g) and Fr.C-2-4
(23 g) were respectively separated over RP-18 gel (MeOHꢀH2O, 30ꢀ
60%) followed by Sephadex LH-20 eluted with CHCl3ꢀMeOH (1:1) and
then purified by semipreparative HPLC (40% MeOH and 35% CH3CN) to
yield 14 (9 mg) and 1,3,6-trihydroxy-2-methyl-9,10-anthraquinone-3-
O-(60-O-acetyl)-β-D-glucopyranoside (140 mg), and 9 (48 mg) and 10
(29 mg), respectively. Fr.C-4 (85 g) was also chromatographed over RP-
18 (MeOHꢀH2O, 20%ꢀ50%) and Sephadex LH-20 (CHCl3ꢀMeOH,
1:1) to give subfractions Fr.C-4-1 to Fr.C-4-5. Compounds 11 (30 mg)
and 12 (15 mg) were isolated from Fr.C-4-1 by semipreparative HPLC
(CH3CNꢀH2O, 30ꢀ33%). 1,3,6-Trihydroxy-2-methyl-9,10-anthraqui-
none-3-O-(60-O-acetyl)-β-D-xylopyranosyl-(1f2)-β-D-glucopyranoside
(22 mg) and 1,3,6-trihydroxy-2-methyl-9,10-anthraquinone-3-O-(30-O-
acetyl)-α-L-rhamnopyranosyl-(1f2)-β-D-glucopyranoside (15 mg)
were purified from Fr.C-4-3 by semipreparative HPLC (MeOHꢀ
H2O, 30ꢀ35%). Fr.C-5 (35 g) was applied to a silica gel column,
eluting with EtOAcꢀMeOH (8:2ꢀ7:3), and then to RP-18 (MeOH-
H2O, 20ꢀ30%) and Sephadex LH-20 (MeOH) columns to obtain 22
(140 mg), 1,3,6-trihydroxy-2-methyl-9,10-anthraquinone-3-O-α-L-rhamno-
pyranosyl-(1f2)-β-D-glucopyranoside (90 mg), and rubiarboside G
(120 mg).
IR (KBr) νmax 3441, 2952, 2933, 1676, 1641, 1631, 1383, 1209, 1060 cmꢀ1
;
1H and 13C NMR data, see Tables 1 and 2; positive-mode ESIMS m/z 509
[M + Na]+; positive-mode HRESIMS m/z 509.3247 [M + Na]+ (calcd for
C30H46O5Na, 509.3242).
Rubianol-e 3-O-(60-O-acetyl)-β-D-glucopyranoside (8): white pow-
der; [α]2D5 ꢀ25.2 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 203
(3.68) nm; IR (KBr) νmax 3449, 3445, 2971, 2952, 2874, 1726, 1371,
1255, 1082, 1039, 888 cmꢀ1; 1H and 13C NMR data, see Tables 3 and 2;
negative-mode FABMS m/z 777 (100) [M ꢀ H]ꢀ; negative-mode
HRESIMS m/z 777.4425 [M ꢀ H]ꢀ (calcd for C42H65O13, 777.4425).
Rubiarboside G 28-acetate (9): white powder; [α]1D6 ꢀ21.7 (c 0.36,
MeOH); UV (MeOH) λmax (log ε) 250 (3.56), 256 (3.59), 261
(3.45) nm; IR (KBr) νm1ax 3426, 2942, 2872, 1737, 1631, 1373, 1245,
1078, 1038, 535 cmꢀ1; H and 13C NMR data, see Tables 3 and 2;
negative-mode FABMS m/z 840 (100) [M]ꢀ; negative-mode HRE-
SIMS m/z 839.4799 [M ꢀ H]ꢀ (calcd for C44H71O15, 839.4792).
2α-Acetoxy-28-acetylrubiarboside G (10): white powder; [α]1D6 ꢀ27.9
(c 0.32, MeOH); UV (MeOH) λmax (log ε) 250 (3.37), 256 (3.39) nm;
IR (KBr) νmax 3427, 2947, 2935, 1722, 1639, 1631, 1373, 1258, 1077,
1
1041 cmꢀ1; H and 13C NMR data, see Tables 3 and 2; negative-mode
FABMS m/z 897 (48) [M ꢀ H]ꢀ; negative-mode HRESIMS m/z
933.4635 [M + Cl]ꢀ (calcd for C46H74O17Cl, 933.4614).
Rubiarboside G 28-al (11): white powder; [α]1D6 ꢀ41.7 (c 0.31,
MeOH); UV (MeOH) λmax (log ε) 257 (3.74), 261 (3.74) nm; IR
(KBr) νmax 3427, 2948, 2874, 1703, 1639, 1345, 1075, 1039, 535 cmꢀ1
;
1H and 13C NMR data, see Tables 3 and 2; negative-mode FABMS
m/z 795 (100) [M ꢀ H]ꢀ; negative-mode HRESIMS m/z 795.4513
[M ꢀ H]ꢀ (calcd for C42H67O14, 795.4530).
Rubiarbonol A 7-acetate (1): white powder; [α]2D3 +1.7 (c 0.22,
MeOH); UV (MeOH) λmax (log ε) 203 (3.74) nm; IR (KBr) νmax 3423,
2971, 2951, 2870, 1728, 1641, 1460, 1444, 1377, 1248, 1209, 1028 cmꢀ1
;
1H and 13C NMR data, see Tables 1 and 2; positive-mode ESIMS m/z
539 [M + Na]+; positive-mode HRESIMS m/z 539.3707 [M + Na]+
(calcd for C32H52O5Na, 539.3712).
Rubiarbonol A 3-O-β-D-glucopyranosyl-(1f2)-β-D-glucopyrano-
side (12): white powder; [α]1D6 ꢀ9.0 (c 0.34, MeOH); UV (MeOH)
λmax (log ε) 251 (3.42), 256 (3.46), 261 (3.31) nm; IR (KBr) νmax 3425,
2944, 2873, 1637, 1373, 1079, 1037, 591 cmꢀ1; 1H and 13C NMR data,
see Tables 3 and 2; negative-mode FABMS m/z 797 (100) [M ꢀ H]ꢀ;
negative-mode HRESIMS m/z 797.4706 [M ꢀ H]ꢀ (calcd for C42H69-
O14, 797.4687).
Rubiyunnanol A (2): white powder; [α]1D6 +23.2 (c 0.27, CHCl3); UV
(MeOH) λmax (log ε) 244 (3.97) nm; IR (KBr) νmax 3440, 2930, 2886,
2867, 1637, 1470, 1452, 1382, 1374, 1087, 1037, 990 cmꢀ1; H and
1
13C NMR data, see Tables 1 and 2; positive-mode ESIMS m/z 463
[M + Na]+; positive-mode HRESIMS m/z 463.3540 [M + Na]+ (calcd
for C30H48O2Na, 463.3552).
1,3-Dihydroxy-6-methoxy-2-methyl-9,10-anthraquinone (13): yel-
lowish needles (CHCl3); mp 250ꢀ251 °C; UV (MeOH) λmax (log ε)
276 (4.42), 337 (3.73), 415 (3.70) nm; IR (KBr) νmax 3409, 2923, 1659,
1621, 1593, 1433, 1370, 1323, 1229, 1123, 1016, 757, 586 cmꢀ1; 1H and
13C NMR data, see Table 4 ; EIMS m/z 284 (100) [M]+; positive-mode
HRESIMS m/z 307.0578 [M + Na]+ (calcd for C16H12O5Na, 307.0582).
1,3,6-Trihydroxy-2-hydroxymethyl-9,10-anthraquinone-3-O-(60-O-
acetyl)-β-D-glucopyranoside (14): pale yellow powder; [α]2D5 ꢀ50.8
(c 0.08, DMSO); UV (MeOH) λmax (log ε) 219 (4.29), 275 (4.40), 302
(4.01), 427 (3.60) nm; IR (KBr) νmax 3525, 3417, 2922, 1712, 1624,
1598, 1580, 1478, 1378, 1306, 1282, 1122, 1082 cmꢀ1; 1H and 13C NMR
data, see Table 4; negative-mode ESIMS m/z489 [M ꢀ H]ꢀ; negative-mode
HRESIMS m/z 489.1021 [M ꢀ H]ꢀ (calcd for C23H21O12, 489.1033).
Rubinaphthin A methyl ester (15): pale yellow powder; [α]1D5 ꢀ73.0
(c 0.34, MeOH); UV (MeOH) λmax (log ε) 260 (4.32), 276 (3.44), 312
(3.46), 357 (3.71) nm; IR (KBr) νmax 3422, 2928, 1670, 1637, 1602,
1454, 1442, 1379, 1345, 1248, 1094, 1075, 771 cmꢀ1; 1H and 13C NMR
data, see Table 4; negative-mode FABMS m/z 379 (77) [M ꢀ H]ꢀ;
negative-mode HRESIMS m/z 379.1030 [M ꢀ H]ꢀ (calcd for C18H19-
O9, 379.1029).
Rubiyunnanol B (3): white needles (CHCl3); mp 247ꢀ248 °C; [α]D16
+25.8 (c 0.32, CHCl3); UV (MeOH) λmax (log ε) 251 (3.03), 256
(3.06) nm; IR (KBr) νmax 3431, 2968, 2939, 2872, 2831, 1639, 1471,
1453, 1375, 1095, 1077, 1027, 809 cmꢀ1; 1H and 13C NMR data, see
Tables 1 and 2; positive-mode ESIMS m/z 463 [M + Na]+; positive-
mode HRESIMS m/z 463.3544 [M + Na]+ (calcd for C30H48O2Na,
463.3552).
19,28-Didehydroxyrubiarbonol A (4): white powder; [α]1D6 +39.7
(c 0.28, CHCl3); UV (MeOH) λmax (log ε) 250 (2.59), 255 (2.60) nm;
IR (KBr) νmax 3423, 2941, 2886, 2869, 1639, 1470, 1454, 1380, 1373,
1031 cmꢀ1; 1H and 13C NMR data, see Tables 1 and 2; positive-mode
ESIMS m/z 465 [M + Na]+; positive-mode HRESIMS m/z 465.3710
[M + Na]+ (calcd for C30H50O2Na, 465.3708).
Rubiyunnanol C (5): white powder; [α]1D8 ꢀ28.9 (c 0.35, MeOH);
UV (MeOH) λmax (log ε) 251 (3.74) nm; IR (KBr) νmax 3440, 2954,
2935, 2871, 1656, 1651, 1379, 1025 cmꢀ1; 1H and 13C NMR data, see
Tables 1 and 2; negative-mode FABMS m/z 487 (100) [M ꢀ H]ꢀ;
negative-mode HRESIMS m/z 487.3419 [M ꢀ H]ꢀ (calcd for C30H47-
O5, 487.3423).
4R0S0,40R0S0-Dihydroxy-2R0S0,20R0S0-binaphthalene-1,10-dione (16):
white powder; [α]2D3 ꢀ38.6 (c 0.24, pyridine); UV (MeOH) λmax (log ε)
251 (4.46), 287 (3.65) nm; IR (KBr) νmax 3351, 2865, 1683, 1600, 1468,
Rubiarbonone E 19-acetate (6): white crystals (CHCl3); mp 259ꢀ
260 °C; [α]2D3 ꢀ4.6 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 225
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Journal of Natural Products
ARTICLE
1456, 1342, 1268, 1224, 1076, 1045, 1018, 970, 773, 764, 707 cmꢀ1; 1H and
13C NMR data, see Table 4; positive-mode ESIMS m/z 345 [M + Na]+;
positive-mode HRESIMS m/z345.1111 [M + Na]+ (calcd for C20H18O4Na,
345.1102).
’ AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: 86-871-5223800. E-mail: nhtan@mail.kib.ac.cn,
gzh_zeng@mail.kib.ac.cn.
Acid Hydrolysis of Compounds 8ꢀ12, 14, and 15. Com-
pounds 8ꢀ12 (6 mg) and 14 and 15 (4 mg) were hydrolyzed with 2 M
HCl in 1,4-dioxane (1:1, 4 mL) under reflux at 80 °C for 6 h, respec-
tively. Each reaction mixture was extracted with CHCl3 three times
(2 mL ꢁ 3). The aqueous layer was neutralized with 2 M NaOH and
then dried to give one or more monosaccharides. The dried powders
were dissolved in pyridine (2 mL), L-cysteine methyl ester hydrochloride
(1.5 mg) was added, and the mixture was heated at 60 °C for 1 h.
Thereafter, trimethylsilylimidazole (1.5 mL) was added to the reaction
mixture in ice-cold water and kept at 60 °C for another 30 min. An
aliquot (4 μL) of the supernatant was directly subjected to GC analysis
under the following conditions: column temperature 180ꢀ280 °C;
programmed increase, 3 °C/min; carrier gas N2 (1 mL/min); injector
and detector temperature 250 °C, split ratio 1:50. The configurations of
D-glucose for 8ꢀ12, 14, and 15 were determined by comparing the
retention times with the derivatives of authentic samples (D-glucose:
18.20 min, L-glucose: 18.79 min).
’ ACKNOWLEDGMENT
This work was supported by the National Natural Science
Foundation of China (30725048, U1032602, 91013002),
the National New Drug Innovation Major Project of China
(2011ZX09307-002-02), the National Basic Research Program
of China (2009 CB522300), the Fund of the Chinese Academy
of Sciences (KSCX2-YW-R-177, West Light Program), the
Innovative Group Program from the Science and Technology
Department of Yunnan Province (2008OC011), and the Fund of
the State Key Laboratory of Phytochemistry and Plant Resources
in West China, Kunming Institute of Botany, Chinese Academy
of Sciences. We thank Mrs. O. Olubanke and Dr. A. H. Adebayo
for proofreading the manuscript.
Cytotoxicity Assays. The cytotoxicity of the test compounds
against the A549 (human lung adenocarcinoma), HeLa (human cervical
carcinoma), and SMMC-7721 (human hepatocellular carcinoma)
cancer cell lines was measured using a sulforhodamine B (SRB) assay
as described in the literature.42 Paclitaxel and camptothecin were
used as positive controls. Briefly, cells were plated in 96-well culture
plates for 24 h and then treated with serial dilutions of all compounds,
with a maximum concentration of 20 μg/mL. After being incubated
for 48 h under a humidified atmosphere of 5% CO2 at 37 °C, cells
were fixed with 25 μL of ice-cold 50% trichloroacetic acid and
incubated at 4 °C for 1 h. After washing with distilled water and
air-drying, the plate was stained for 15 min with 100 μL of 0.4% SRB
(Sigma) in 1% glacial acetic acid. The plates were washed with 1%
acetic acid and air-dried. For reading the plate, the protein-bound dye
was dissolved in 100 μL of 10 mM Tris base. The absorbance was
measured at 560 nm on a microplate spectrophotometer (Molecular
Devices SpectraMax 340, MWG-Biotech, Inc., Sunnyvale, CA, USA).
All tests were performed in triplicate, and results are expressed as
IC50 values.
’ REFERENCES
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Supporting Information. Copies of 1D and 2D NMR
b
spectra and physical data of compounds 1ꢀ18. This material is
available free of charge via the Internet at http://pubs.acs.org.
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