Three new steroidal glycosides from roots of Reineckia carnea

Article abstract of DOI:10.1080/14786419.2012.658797

Two new spirostanols and a new furostanol, reinocarnoside A (1), B (2) and C (3), were isolated from the roots of Reineckia carnea, together with two known compounds, (25S)-1β,3β,4β-trihydroxyspirostan-5β-yl-O- β-D-glucopyranoside (4), kitigenin-5β-O-β-D-glucopyranoside (5). The structures of three new compounds were elucidated by spectroscopic methods including 1-D NMR, 2-D NMR and MS spectrums, and their anticancer activities were evaluated by MTT method.

Full text of DOI:10.1080/14786419.2012.658797

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Three new steroidal glycosides from
roots of Reineckia carnea
a
a
a
a
Qian Wang , Qune Hou , Zhiyong Guo , Kun Zou , Yanhong
a
a
a
a
Xue , Nianyu Huang , Fan Cheng & Yuan Zhou
a
Hubei Key Laboratory of Natural Products Research and
Development, College of Chemistry and Life Science , China Three
Gorges University , Yichang 443002 , P.R. China
Published online: 03 Feb 2012.
To cite this article: Qian Wang , Qune Hou , Zhiyong Guo , Kun Zou , Yanhong Xue , Nianyu
Huang , Fan Cheng & Yuan Zhou (2013) Three new steroidal glycosides from roots of Reineckia
carnea , Natural Product Research: Formerly Natural Product Letters, 27:2, 85-92, DOI:
10.1080/14786419.2012.658797
To link to this article: http://dx.doi.org/10.1080/14786419.2012.658797
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Natural Product Research, 2013
Natural Product Research
Vol. 27, No. 2, 85–92, http://dx.doi.org/10.1080/14786419.2012.658797
2
012, 1–8, iFirst
Three new steroidal glycosides from roots of Reineckia carnea
Qian Wang, Qune Hou, Zhiyong Guo, Kun Zou*, Yanhong Xue, Nianyu Huang,
Fan Cheng and Yuan Zhou
Hubei Key Laboratory of Natural Products Research and Development, College of Chemistry and
Life Science, China Three Gorges University, Yichang 443002, P.R. China
(
Received 8 July 2011; final version received 2 October 2011)
Two new spirostanols and a new furostanol, reinocarnoside A (1), B (2) and C (3),
were isolated from the roots of Reineckia carnea, together with two known
compounds,
(25S)-1�,3�,4�-trihydroxyspirostan-5�-yl-O-�-D-glucopyranoside
(4), kitigenin-5�-O-�-D-glucopyranoside (5). The structures of three new com-
pounds were elucidated by spectroscopic methods including 1-D NMR, 2-D NMR
and MS spectrums, and their anticancer activities were evaluated by MTT method.
Keywords: Reineckia carnea; steroidal glycosides; structural elucidation; antican-
cer activity; NMR
1
. Introduction
Reineckia carnea (Andr.) Kunth, an evergreen medicinal herb, is a monotypic genus of
Reineckia from family Liliaceae (syn. yudaicao, guanyincao, etc. in Chinese), and
R. carnea is only distributed in China and Japan. In China, it is mainly distributed around
Yangzi valley, and is an important medical plant to Miao nationality for haemostasia,
relieving cough, clearing away the lung-heat, detoxification and relieving the inflammation
of the pharynx (Fu, 2002). During the investigation of new anti-inflammatory and
anticancer activity metabolites from the plants around Yangzi valley, we found the crude
extracts from two plants, Tupistra chinensis Baker and R. carnea, showed strong anti-
inflammatory activity in vitro, and we have isolated many new furostanols with anti-
inflammatory and anticancer activity from the plant of T. chinensis (Guo et al., 2009; Xu
et al., 2007; Zou, Wang, Du, Li, & Tu, 2006; Zou, Wang et al., 2007; Zou, Wu et al., 2007;
Zou et al., 2009). Investigation of bioactive constituents from the dried rhizomes of R.
carnea, has resulted in the isolation of three new compounds, reinocarnoside A (1), B (2)
and C (3), together with two known compounds, (25S)-1�,3�,4�-trihydroxyspirostan-
5
�-yl-O-�-D-glucopyranoside (4), kitigenin-5�-O-�-D-glucopyranoside (5). In this article,
the structural elucidation and anticancer activity of reinocarnoside A (1), B (2) and C (3)
from R. carnea are reported (Figure 1).
2
. Results and discussion
A total of 1.5 kg dried rhizomes of R. carnea, collected in Changyang of Hubei Province,
were powdered and extracted with 70% EtOH, and the 70% EtOH extract was partitioned
*
Corresponding author. Email: kzou@ctgu.edu.cn
ISSN 1478–6419 print/ISSN 1478–6427 online
*
Corresponding author. Email: kzou@ctgu.edu.cn
�
http://dx.doi.org/10.1080/14786419.2012.658797
2012 Taylor & Francis
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28 6 Q Q. .WW aa nn gg ee tt aa ll ..
21
27
2
7
2
1
25
s
O
23
s
1
7
25
R
11
23
O
19
13
OH
HO
O
O
1
11
17
1
9
13
O
O
1
Ara
9
HO
1
5
O
Glc
OH
9
1
5
5
7
HO
HO
3
OH
O
7
3
O
3
5
OH
1
2
R=OH
R=H
OH
O
O
OH
OH
Rha
OH
OH
OH
OH
Figure 1. The structures of compounds 1–3.
successively with petroleum ether, ethyl acetate and n-butanol. The n-butanol fraction was
purified by repeated column chromatography, including silica gel, Sephadex LH-20
column, reverse phase silica gel chromatography and HPLC to afford three new and two
known compounds.
Reinocarnoside A (1), which was obtained as white powder, possessed the molecular
formula of C H O which was determined on the basis of HR–ESI–MS data with
3
3
54 10,
m/z[M þ H] 611.7838 and [M þ Na]þ 633.7656 (Calcd 611.7835 and 633.7650, respec-
tively) and NMR data. It gave a positive Liebermann–Burchard reaction and negative
Ehrlich reaction (Wu, 2007), which suggested that 1 was spirostanol-type compound.
Compound 1 was hydrolysed with 2 mol/L HCl to afford D-glucose by PC test only. The
þ
1
H-NMR spectrum showed two singlet methyl signals and two doublet methyl signals at
0.80 (s, 18-CH , 3H), 1.10 (s, 19-CH , 3H), 1.16 (d, J ¼ 7.6 Hz, 21-CH , 3H) and 1.06 (d,
�
3
3
3
13
J ¼ 7.8 Hz, 27-CH 3H), respectively. In C-NMR spectrum of compound 1, the carbon
3
,
resonance at � 109.7 (C-22) obviously was the hemiketal carbon signal. These characteristic
proton and carbon signals displayed that the aglycone of compound 1 might be a
1
3
spirostanol-type structure. The C-NMR and DEPT experiments disclosed that there
were 4 quaternary carbons, 14 methines, 11 methenes and 4 methyls in compound 1.
Except for the carbon signals at � 66.8 (C-3), 80.9 (C-16), 62.1 (C-17) and 64.7 (C-26), there
were seven oxygenated carbons signals at � 71.9 (C-1), 87.6 (C-5), 74.4 (Glu-2), 77.1
(
Glu-3), 70.0 (Glu-4), 76.8 (Glu-5), 61.1 (Glu-6), in addition, the anomeric carbon signal at
95.6 (Glu-1), implying that a glucose existed and two substituted hydroxyl groups in the
�
1
3
aglycone in reinocarnoside A (1). Comparing the C-NMR data of A, B, C, D and E rings
from reinocarnoside A to the reference compound tupichigenin E (Pan, Chang, Wei, &
Wu, 2003), we found that they were exactly similar, besides glycosidation shift and
deuterated solvent effects. Thus, the aglycone of reinocarnoside A was determined as
spirostanol-1ꢀ,3ꢀ,5ꢀ,26-tetraol. The configuration of anomeric proton [� 4.95
H
(
d, J ¼ 7.8 Hz, 1H), � 95.6] from glucose was confirming by the coupling constant of
C
anomeric proton and acid hydrolysis. The coupling constant of anomeric proton from
glucose was 7.8 Hz, which indicated that the anomeric hydroxyl group was ꢀ configura-
tion, and only ꢀ-D-glucose was test in acid hydrolysis procedure by PC. The linkage of ꢀ-D-
glucose to the aglycone of reinocarnoside A was proved by the HMBC and HMQC
experiments, and there was a cross-correlation signal from � 4.95 (d, J ¼ 7.8 Hz, Glu-1H,
1
H) to � 87.6 (C-5) in HMBC spectrum. The absolute configuration of 27-Me was
determined as S, because of the difference of chemical shifts between H-26a and H-26b

NN aa t tuu r raa ll PPr or od du uc ct tR Re se es ae ra cr hc h 8 73
Figure 2. The key HMBC and NOE correlations of the new compounds 1–3.
and the proton shift of 27-Me (Agrawal, 2005). Therefore, the structure of compound 1
was identified as (25S)-spirostanol-1�,3�,5�,26-tetraol-5�-D-glucopyranoside (Figure 2).
Reinocarnoside B (2) was obtained as white powder and its molecular formula was
þ
C H O which was determined on the basis of HR–ESI–MS data with m/z [M þ H]
3
3
54 9,
þ
5
data. It gave a positive Liebermann–Burchard reaction and negative Ehrlich reaction (Wu,
95.7844 and [M þ Na] 617.7662 (Calcd 595.7840 and 617.7666, respectively) and NMR
1
007), which displayed that 2 was spirostanol-type compound. The H-NMR spectrum
2
showed two singlet methyl signals and two doublet methyl signals at ꢀ 1.37 (s, 18-CH3,
3
2
ꢀ
H), 1.00 (s, 19-CH , 3H), 1.43 (d, J ¼ 7.6 Hz, 21-CH , 3H) and 1.32 (d, J ¼ 6.5 Hz,
3
3
1
7-CH , 3H), respectively. In C-NMR spectrum of compound 2, a carbon resonance at
3
3
110.4 (C-22) was the hemiketal carbon signal apparently. These characteristic proton and
carbon signals suggested that the aglycone of 2 might be spirostanol-type structure. The
1
3
C-NMR and DEPT experiments showed that there were 4 quaternary carbons, 13
methines, 12 methenes and 4 methyls in compound 2. Except for the carbon signals at
66.5 (C-3), 81.8 (C-16), 64.2 (C-17) and 67.6 (C-26), there were six oxygenated carbons
ꢀ
signals at ꢀ 90.2 (C-5), 75.2 (Glu-2), 79.4 (Glu-3), 71.2 (Glu-4), 78.8 (Glu-5), 63.8 (Glu-6),
in addition, the anomeric carbon signal at ꢀ 102.3 (Glu-1), implying that a glucose existed
and one substituted hydroxyl group in the aglycone of reinocarnoside B (2). Comparing
the NMR data of reinocarnoside B (2) to those of reinocarnoside A (1), we found that they
were exactly similar, besides only one additional oxygenated carbon signal in compound 1,
which indicated that 2 was a analogue of compound 1. Further comparing the NMR data
of 1 and 2 carefully, the aglycone of compound 2 was identified as spirostanol-3�,5�,26-
triol. Similarly, the results of the acid hydrolysis procedure and analysis of NMR data
showed the structure of compound 2 was (25S)-spirostanol-3�,5�,26-triol-5�-D-
glucopyranoside.
Compound 3 was obtained as a white amorphous powder. Its positive Molish reaction,
Liebermann–Burchard reaction and Ehrlich reaction indicated its furostanol-type skeleton
(
Wu, 2007). The molecular formula of C H O was established by HR–ESI–MS, which
44 72 19
þ
showed a quasi-molecular ion peak at m/z 874.0408 (Calcd for [M þ H] 874.0402) and
þ
13
8
95.4663 (Calcd for [M þ Na] 895.4667). The C-NMR spectrum and DEPT experiment
indicated the presence of 44 carbon signals in compound 3, including 5 methyls, 12
methenes, 23 methines and 4 quaternary carbons. Five methyl groups of compound 3 were
observed at ꢀ 0.90 (s, 3H) (H-18), 1.27 (s, 3H) (H-19), 1.64 (s, 3H) (H-21), 1.03 (d, J ¼ 6.0 Hz,
3
H) (H-27) and 1.71 (d, J ¼ 6.5 Hz, 3H) (Rha-6), indicating that there was a 6-desose in
compound 3. Four protons of oxygenated methines or methylenes were found at ꢀ 4.13 (m,
H), 4.63 (m, 1H), 4.98 (m, 1H), 3.48 (t, J ¼ 9.2 Hz, 1H), 4.10 (dd, J ¼ 10.0, 9.2 Hz, 1H) in the
1

48 8 Q Q. .WW aa nn gg ee tt aa ll ..
aglycone moiety. The above proton signals were correlated to their corresponding carbons
at � 83.4 (C-1), 78.6 (C-3), 85.7 (C-16) and 75.2 (C-26) in HMQC spectrum of compound 3,
respectively. These characteristic proton and carbon resonance signals, combined with the
2
two sp carbon signals at � 153.1 (C-22) and 105.0 (C-20), suggested that the aglycone of
C
compound 3 might be a 20(22)-en-furostanol-type steroid.
A total of 17 carbon signals of two hexoses and a pentaose were observed at � 101.9
(
Ara-1), 73.2 (Ara-2), 73.6 (Ara-3), 69.8 (Ara-4), 68.9 (Ara-5); 98.6 (Rha-1), 71.9 (Rha-2),
7
7
1.1 (Rha-3), 73.3 (Rha-4), 69.4 (Rha-5), 17.9 (Rha-6) and 104.8 (Glu-1), 74.3 (Glu-2),
1
3
8.2 (Glu-3), 71.8 (Glu-4), 77.8 (Glu-5), 62.9 (Glu-6) in the C-NMR spectrum. In the
HMQC spectrum, the three anomeric protons at � 4.61 (d, J ¼ 7.0 Hz, 1H), 5.44 (br s, 1H)
and 4.25 (d, J ¼ 8.0 Hz, 1H) were crossed with three anomeric carbons at � 101.9, 98.6 and
1
04.8, respectively. Acid hydrolysis of 3 with 2.0 mol/L HCl afforded to three monosac-
charides, ꢀ-D-glucose, ꢁ-L-rhamnose and ꢀ-D-arabinose, which can be tested by PC. The
HMBC correlations from the anomeric proton of arabinose [4.61 (d, 7.0H z, 1H) (Ara-
1
H)] to 83.4 (C-1) and from 4.13 (m, 1H) (H-1) to the anomeric carbon of arabinose 101.9
C-1, Ara), from 5.44 (br s, 1H) (H-1, Rha) to 78.6 (C-3) and from 4.63 (m, 1H) (H-3) to
8.6 (C-1, Rha) and from 4.25 (d, J ¼ 8.0 Hz, 1H) (H-1, Glc) to 75.2 (C-26) and from 3.48
(
9
(
t, J ¼ 9.2 Hz, 1H) (H-26a) to 104.8 (C-1, Glc) disclosed that the glycosidation positions
were at C-1, C-3 and C-26 in the aglycone of compound 3, respectively. Moreover, the
positions of glucose linked to the aglycone were confirmed by the correlations of NOE
NMR experiments, from 4.13 (m, 1H) (H-1) to [4.61 (d, J ¼ 7.0 Hz, 1H) (H-1, Ara)], from
4
.63 (m, 1H) (H-3) to 5.44 (br s) (H-1, Rha) and from 3.49 (t, J ¼ 9.2 Hz, 1H) (H-26a) to
.25 (d, J ¼ 8.0 Hz, 1H) (H-1, Glc). The configurations of anomeric protons from glucose
4
and arabinose were determined as ꢀ by the coupling constants, and the configuration of
anomeric protons from rhamnose was ꢁ by the broad singlet peak type in proton NMR
spectrum. The configuration of aglycone moiety was determined by comparing the NMR
data of aglycone from 3 to (25S)-26-O-ꢀ-D-glucopyranoyl-furost-1ꢀ,3ꢀ,26-triol-3-O-ꢀ-D-
glucopyranoside (Zou et al., 2009). The absolute configuration of C-25 was proved by
proton chemical shifts of H-26 and 27-Me (Agrawal, 2005). Thus, the structure of 3 was
identified as (25S)-26-O-ꢀ-D-glucopyranoyl-20(22)-en-furost-1ꢀ,3ꢀ,5ꢀ,26-tetraol-1-O-ꢀ-D-
arabinopyranoside-3-O-ꢁ-L-rhamnopyranoside.
Compounds 4 and 5 were determined as (25S)-1ꢀ,3ꢀ,4ꢀ-trihydroxyspirostan-5ꢀ-yl-3-
O-ꢀ-D-glucopyranoside, kitigenin-5ꢀ-O-ꢀ-D-glucopyranoside (5) by comparing their NMR
data to the references (Kanmoto, Mimaki, Nikaido, Koike, & Ohmoto, 1994; Shen
et al., 2003).
The anticancer activity of isolated compounds 1–5 were evaluated by MTT method,
and the results showed that compounds 1–4 displayed cytotoxic activity to A549 cell line in
different levels, but showed no significant cytotoxic activity to Caski, HepG 2 and MCF-7
cell lines (Table 1).
a
Table 1. The results of compounds 1–5 inhibiting cancer cell lines.
Cell line
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
A549
Caski
HepG 2
MCF-7
20
50
4100
4100
50
4100
4100
4100
40
70
80
4100
30
4100
4100
4100
4100
4100
4100
4100
a
Notes: The inhibiting activity to cancer cell lines were recoded with IC and the units of IC50
5
0
�1
was mg mL
.

NN aa t tuu r raa ll PPr or od du uc ct tR Re se es ae ra cr hc h 8 95
3
3
. Experimental
.1. General experimental procedure
Column chromatography was carried out with silica gels (Qingdao Ocean Chemical
Company, 200–300 meshes). TLC was performed with percolated silica gel GF-254 plates.
The detection of spots was done at UV 254 nm or by spraying with 95% H SO . IR
2
4
spectrum was recorded on Nicolet FT360 spectrometer. Optical rotations were measured
on a Perkin Elmer 241 spectropolarimeter using a 10-cm cell tube. Mass spectra (ESI–MS,
HRESI–MS) were measured using a fast atom bombardment mode on aVG AUTO Spec-
3
DECA XP plus mass spectrometer, respectively, and ions were given in m/z. The H-,
00 mass spectrometer and using an electron spraying mode on a Finnigan-MAT LCQ
1
1
3
C- and 2-D-NMR spectrum were recorded on a Bruker 400 spectrometer in methanol-d4
using tetramethylsilane as an internal standard. Chemical shifts were given in ppm and
scalar coupling reported in hertz.
3
.2. Plant material
The rhizomes of R. carnea were collected from Changyang in Hubei Province in July 2006
and identified by Dr Faju Chen and Yubing Wang. A voucher specimen (no. 2006071802)
was deposited at Hubei Key Laboratory of Natural Products Research and Development,
China Three Gorges University, Yichang, China.
3
.3. Extraction and isolation
The dry roots of R. carnea (1.5 kg) were powdered and refluxed with 70% ethanol
(
6 L � 5), and the extract was evaporated in vacuo to yield a residue, which was freeze-dried
to afford a powder (250 g). Then, it was suspended in water and extracted with petroleum-
ether, ethyl acetate and n-butanol successively. The n-butanol extract (95 g) was dissolved
in water (2.0 L), and subjected to macroporous resin (AB-8) column chromatography
eluting with a gradient ethanol–water system (100% water ! 100% ethanol). The 70%
ethanol eluate (5 g) was purified by repeated Rp-C18 silica gel column chromatography
eluted with a gradient acetonitrile–water system (100% water ! 60% acetonitrile) to give
1
2 fractions. Fractions 5 and 10 were combined, respectively, and re-purified by Sephadex
LH-20 gel chromatography. Fractions 15 (0.5 g) was further purified by repeated
preparative HPLC eluted with gradient solvent (45% ! 80% acetonitrile/water (V/V)
within 30 min, 2.0 mL/min, detection at UV 203 nm), and then repeated semi-preparative
HPLC eluted with gradient solvent (45% ! 60% acetonitrile/water (V/V) within 30 min,
1
.5 mL/min, detection at UV 203 nm) to afford compounds 3 (15.5 mg, t ¼ 15 min) and 2
R
(
10.8 mg, t ¼ 25 min).
R
Fractions 20 (0.2 g) was further purified by repeated preparative HPLC eluted with
gradient solvent (45% ! 70% acetonitrile/water (V/V) within 35 min, 1.5 mL/min,
detection at UV 203 nm), and then repeated semi-preparative HPLC eluted with gradient
solvent (45% ! 70% acetonitrile/water (V/V) within 35 min, 1.5 mL/min, detection at UV
2
03 nm) to give compounds 1 (30 mg, t ¼ 25 min), 4 (20 mg, t ¼ 15 min) and 5 (10 mg,
R
R
t ¼ 10 min).
R
3
.4. Acid hydrolysis
A total of 2.0 mg of 1, 2 and 3 were hydrolysed with 2.0 mol/L HCl, respectively. The
hydrolysed products were tested by paper chromatography.

69 0 Q Q. .WW aa nn gg ee tt aa ll ..
3
.5. Inhibiting cancer cell line assay
The cytotoxic activity to A549, Caski, HepG 2 and MCF-7 were screened by MTT
method.
3
.5.1. Compound 1
�
20
D
�
White powder, m.p. 176–177 C, ½�ꢀ ꢁ80.2 (c 0.09, CH OH); IR (kBr, ꢀ ): 3430, 2921,
3
max
ꢁ1
þ
þ
1
6
1
2
7
1
4
556 and 1320 cm ; HR–ESI–MS data with m/z [M þ H] 611.7838 and [M þ Na]
1
33.7656 (Calcd 611.7835 and 633.7650, respectively); H (400 MHz, CD OD-d ): 4.15 (H-
3 4
, m, 1H), 2.57 (H-2a, m, 1H), 2.05 (H-2b, m, 1H), 4.41 (H-3, m, 1H), 2.35 (H-4a, m, 1H),
.19 (H-4b, m, 1H), 1.94 (H-6a, m, 1H), 1.47 (H-6b, m, 1H), 1.45 (H-7a, m, 1H), 1.01 (H-
b, m, 1H), 1.65 (H-8, m, 1H), 1.15 (H-9, m, 1H), 1.35 (H-11, m,2H), 0.98 (H-12a, m,
H),1.69 (H-12b, m, 1H), 1.04 (H-14, m, 1H), 1.97 (H-15a, m, 1H), 1.43 (H-15b, m, 1H),
.50 (H-16, m, 1H), 1.76 (H-17, m, 1H), 1.10 (H-18, s, 3H), 0.80 (H-19, s, 3H), 2.25 (H-20,
m, 1H), 1.16 (H-21, d, 7.6, 3H), 1.92 (H-23a, m, 1H), 1.85 (H-23b, m, 1H), 1.72 (H-24a, m,
H), 1.42 (H-24b, m, 1H), 1.86 (H-25, m, 1H), 3.47 (H-26a, dd, 7.8, 9.3, 1H); 4.07 (H-26b,
m, 1H), 1.06 (H-27, d, 6.5, 3H), 4.95 (Glu H-1, d, 7.8, 1H), 4.03 (Glu H-2, m, 1H), 4.25
1
(
Glu H-3, m, 1H), 4.28 (Glu H-4, m, 1H), 3.95 (Glu H-5, m, 1H), 4.54 (Glu H-6a, m, 1H),
1
.30 (Glu H-6b, m, 1H); C (100 MHz, CD OD-d ): 71.9 (C-1), 25.6 (C-2), 66.8 (C-3),
3
4
2
1
1
6
5
3 4
7.9 (C-4), 87.6 (C-5), 31.2 (C-6), 24.1 (C-7), 34.2 (C-8), 43.8 (C-9), 42.8 (C-10), 20.9 (C-
1), 39.6 (C-12), 40.3 (C-13), 55.8 (C-14), 26.4 (C-15), 80.9 (C-16), 62.1 (C-17), 15.5 (C-18),
3.3 (C-19), 42.1 (C-20), 15.0 (C-21), 109.7 (C-22), 25.4 (C-23), 23.8 (C-24), 27.1 (C-25),
4.7 (C-26), 16.1 (C-27), 95.6 (Glu-1), 74.4 (Glu-2), 77.1 (Glu-3), 70.0 (Glu-4), 76.8 (Glu-
), 61.1 (Glu-6).
3
.5.2. Compound 2
�
20
D
�
White powder, m.p. 180–181 ; ½�ꢀ ꢁ85.2 (c 0.08, CH OH); IR (kBr, ꢀmax): 3435, 2920,
3
ꢁ1
þ
þ
1
560 and 1323 cm ; HRESI–MS, m/z[M þ H] 595.7844 and [M þ Na] 617.7662 (Calcd
1
95.7840 and 617.7666, respectively); H (400 MHz, CD OD-d ): 1.69 (H-1a, m, 1H), 0.98
5
3
4
(
1
(
H-1b, m, 1H), 2.58 (H-2a, m, 1H), 2.03 (H-2b, m, 1H), 4.39 (H-3, m, 1H), 2.37 (H-4a, m,
H), 2.16 (H-4b, m, 1H), 1.96 (H-6a, m, 1H), 1.48 (H-6b, m, 1H), 1.47 (H-7a, m, 1H), 1.00
H-7b, m, 1H), 1.66 (H-8, m, 1H), 1.17 (H-9, m, 1H), 1.38 (H-11, m, 2H), 0.99 (H-12a, m,
1
4
H), 1.70 (H-12b, m, 1H), 1.02 (H-14, m, 1H), 1.95 (H-15a, m, 1H), 1.45 (H-15b, m, 1H),
.48 (H-16, m, 1H), 1.77 (H-17, m, 1H), 1.37 (H-18, s, 3H), 1.00 (H-19, s, 3H), 2.25 (H-20,
m, 1H), 1.43 (H-21, d, 7.6, 3H), 1.90 (H-23a, m, 1H), 1.88 (H-23b, m, 1H), 1.70 (H-24a, m,
H), 1.43 (H-24b, m, 1H), 1.84 (H-25, m, 1H), 3.45 (H-26a, dd,7.8, 9.3, 1H); 4.09 (H-26b,
m, 1H), 1.32 (H-27, d, 6.5, 3H), 4.88 (Glu H-1, d, 7.8, 1H), 4.00 (Glu H-2, m, 1H), 4.27
1
(
4
Glu H-3, m, 1H), 4.25 (Glu H-4, m, 1H), 3.96 (Glu H-5, m, 1H), 4.57 (Glu H-6a, m, 1H),
1
3
.32 (Glu H-6b, m, 1H); C (100 MHz, CD OD-d ): 29.3 (C-1), 39.4 (C-2), 66.5 (C-3),
3 4
4
0.1 (C-4), 90.2 (C-5), 47.2 (C-6), 26.4 (C-7), 35.5 (C-8), 45.6 (C-9), 41.8 (C-10), 21.2
(
(
(
C-11), 40.6 (C-12), 41.6 (C-13), 56.2 (C-14), 34.5 (C-15), 81.8 (C-16), 64.2 (C-17), 18.0
C-18), 16.5 (C-19), 38.8 (C-20), 16.8 (C-21), 110.4 (C-22), 31.9 (C-23), 26.2 (C-24), 31.5
C-25), 67.6 (C-26), 19.4 (C-27), 102.3 (Glu-1), 75.2 (Glu-2), 79.4 (Glu-3), 71.2 (Glu-4),
7
8.8 (Glu-5), 63.8 (Glu-6).
3
.5.3. Compound 3
�
20
�
White amorphous powder; m.p. 201–202 , ½�ꢀ ꢁ98.0 (c 0.1, CH OH); IR (kBr, ꢀmax):
D
HR–ESI–MS m/z 874.0408 (Calcd for
[M þ H] 874.0402) and 895.4663 (Calcd for [M þ Na] 895.4667); H-NMR (400 MHz,
CD OD-d ): 4.13 (H-1, m, 1H), 2.58 (H-2a, br d, 14.4, 1H), 2.06 (H-2b, br d, 14.4, 1H),
3
ꢁ
1
3
428, 2925, 1559 and 1318 cm
;
þ
þ
1
3
4

NN aa t tuu r raa ll PPr or od du uc ct tR Re se es ae ra cr hc h 9 17
4
.63 (H-3, m, 1H), 1.93 (H-4a, m, 1H), 1.75 (H-4b, m, 1H), 2.40 (H-5, m, 1H), 1.29 (H-6a,
m, 1H), 1.16 (H-6b, m, 1H), 1.72 (H-7a, m, 1H), 0.99 (H-7b, m, 1H), 1.60 (H-8, m, 1H),
.20 (H-9, m, 1H), 1.31 (H-11a, m, 1H), 1.21 (H-11b, m, 1H), 1.72 (H-12a, m, 1H), 1.07
H-12b, dd, 5.5, 10.8, 1H), 1.08 (H-14, m, 1H), 2.00 (H-15a, m, 1H), 1.44 (H-15b, m, 1H),
.98 (H-16, m, 1H), 1.96 (H-17, m, 1H), 0.90 (H-18, s, 3H), 1.27 (H-19, s, 3H), 1.64 (H-21,
s, 3H), 1.89 (H-23a, m, 1H), 1.45 (H-23b, m, 1H), 2.23 (H-24a, m, 1H), 2.18 (H-24b, m,
H), 1.94 (H-25, m, 1H), 3.48 (H-26a, t, 9.2, 1H), 4.10 (H-26b, dd, 10.1, 9.2, 1H), 1.03
H-27, d, 6.0, 3H), 4.61 (Ara H-1, d, 7.0, 1H), 3.59 (Ara H-2, m, 1H), 3.70 (Ara H-3, m,
H), 4.03 (Ara H-4, m, 1H), 3.49 (Ara H-5a, m, 1H), 3.98 (Ara H-5b, m, 1H), 5.44 (Rha
H-1, br s, 1H), 4.60 (Rha H-2, br s, 1H), 4.46 (Rha H-3, m, 1H), 4.27 (Rha H-4, t, 7.8, 1H),
.83 (Rha H-5, m, 1H), 1.71 (Rha H-6, d, 6.5, 1H), 4.25 (Glc H-1, d, 8.0, 1H), 3.98
Glc H-2, m), 1H, 26 (Glc H-3, m, 1H), 4.25 (Glc H-4, m, 1H), 3.98 (Glc H-5, m, 1H), 4.40
1
(
4
1
(
1
4
(
(
(
(
(
(
(
1
3
Glc H-6a, m, 1H), 4.57 (Glc H-6b, m, 1H); C (100 MHz, CD OD-d ): 83.4 (C-1), 27.7
3
4
C-2), 78.6 (C-3), 35.1 (C-4), 36.5 (C-5), 27.1 (C-6), 27.3 (C-7), 34.5 (C-8), 44.6 (C-9), 43.2
C-10), 22.4 (C-11), 40.3 (C-12), 41.0 (C-13), 56.0 (C-14), 32.8 (C-15), 85.7 (C-16), 65.8
C-17), 17.3 (C-18), 14.9 (C-19), 105.0 (C-20), 11.9 (C-21), 153.1 (C-22), 32.1 (C-23), 24.1
C-24), 33.7 (C-25), 75.2 (C-26), 17.2 (C-27), 101.9 (Ara-1), 73.2 (Ara-2), 73.6 (Ara-3), 69.8
Ara-4), 68.9 (Ara-5), 98.6 (Rha-1), 71.9 (Rha-2), 71.1 (Rha-3), 73.3 (Rha-4), 69.4 (Rha-5),
1
(
7.9 (Rha-6), 104.8 (Glc-1), 74.3 (Glc-2), 78.2 (Glc-3), 71.8 (Glc-4), 77.8 (Glc-5), 62.9
Glc-6).
4
. Conclusions
In conclusion, five steroidal compounds, including three new compounds – reinocarnoside
A, B and C, were isolated from the roots of R. carnea, and the structure of reinocarnoside
A, B and C were identified as (25S)-spirostanol-1�,3�,5�,26-tetraol-5�-D-glucopyranoside
(
1), (25S)-spirostanol-3�,5�,26-triol-5�-D-gluco-pyranoside (2) and (25S)-26-O-�-D-gluco-
pyranosyl-20(22)-en-furost-1�,3�,5�,26-tetraol-1-O-�-D-arabinoside-3-O-ꢀ-L-rhnosamno-
side (3) by NMR and MS spectrums. The anticancer activity of isolated compounds 1–5
was evaluated by MTT method, and the results showed that compounds 1–4 displayed
cytotoxic activity to A549 cell line in different levels, but showed no significant cytotoxic
activity to Caski, HepG 2 and MCF-7 cell lines.
Acknowledgements
Financial support for this study from the National Natural Science Foundations of China
(
(
30870254) and Innovation Team Program of Natural Science Foundations of Hubei Province
2007ABC008) are gratefully acknowledged.
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89 2 Q Q. .WW aa nn gg ee tt aa ll ..
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