Three steroids with unique structural feature of 5β-spirostan-1β, 3β,17α-trihydroxyl from Reineckia carnea

Article abstract of DOI:10.1248/cpb.59.53

A new spirostanol sapogenin and two spirostanol saponins, tentatively named reineckiagenin A (1), reineckiagenoside A (2), and reineckiagenoside B (3), were isolated from the whole plant of Reineckia carnea. By detailed analysis of their 1D and 2D NMR spectra, chemical methods, and by comparison with spectra data of known compounds, the structures of the new steroids were determined to be 25(S)-5β-spirostan-1β,3β,17α-triol (1), 25(S)-5β-spirostan-1β,3β,17α-triol 1-O-β-D- xylopyranoside (2), 25(S)-5β-spirostan-1β,3β,17α-triol 1-O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside (3). Compounds 1, 2, and 3 are the first naturally occurring steroids with unique structural feature of 5β-spirostan-1β,3β,17α-trihydroxyl.

Full text of DOI:10.1248/cpb.59.53

January 2011
Regular Article
Chem. Pharm. Bull. 59(1) 53—56 (2011)
53
b
Three Steroids with Unique Structural Feature of 5 -Spirostan-
b b
a
1 ,3 ,17 -trihydroxyl from Reineckia carnea
Zhong-Quan ZHANG,a,b Jian-Chao CHEN, Jian YAN, and Ming-Hua QIU
a
a
,a
*
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese
Academy of Sciences; Kunming 650204, China: and b Research Institute of Resource Insects, Chinese Academy of Forestry;
Kunming 650224, China. Received July 7, 2010; accepted September 16, 2010; published online October 1, 2010
A new spirostanol sapogenin and two spirostanol saponins, tentatively named reineckiagenin A (1), reinecki-
agenoside A (2), and reineckiagenoside B (3), were isolated from the whole plant of Reineckia carnea. By detailed
analysis of their 1D and 2D NMR spectra, chemical methods, and by comparison with spectra data of known
compounds, the structures of the new steroids were determined to be 25(S)-5b-spirostan-1b,3b,17a-triol (1),
25(S)-5b-spirostan-1b,3b,17a-triol 1-O-b-D-xylopyranoside (2), 25(S)-5b-spirostan-1b,3b,17a-triol 1-O-a-L-
rhamnopyranosyl-(1→2)-b-D-xylopyranoside (3). Compounds 1, 2, and 3 are the first naturally occurring
steroids with unique structural feature of 5b-spirostan-1b,3b,17a-trihydroxyl.
Key words Reineckia carnea; steroid; reineckiagenin A; reineckiagenoside A; reineckiagenoside B
Reineckia carnea (ANDR.) KUNTH belongs to the family culated: 447.3110 and also confirmed by 13C-NMR evidence.
Liliaceae, it is the only species of the genus Reineckia and The IR spectrum of 1 showed absorption bands ascribable to
indigenous to China and Japan.1) As a perennial ever-green hydroxyl (3373 cmꢀ1) and spiroketal groups (977, 948, 918,
herb, it can grow well on soils polluted by heavy metals and 898, 822 cmꢀ1); the absorption at 918 cmꢀ1 was of greater in-
1
can produce a repairing effect on the soil that was polluted tensity than that at 898 cmꢀ1 (25(S)-spiroketal).15,16) The H-
by copper.2) The Chinese name “ji xiang cao” means it can NMR spectrum of 1 showed signals for four typical steroid
bring luck and fortune to people, especially when it was cul- methyls; two appeared as singlets at d 0.98 (H-18) and d
tivated as ornamental plant and flowers. The whole plant has 1.34 (H-19) and the other two as doublets at d 1.24
been used as an antitussive, an antarthritic, a hemostatic, and (Jꢁ7.1 Hz, H-21) and d 1.03 (Jꢁ7.2 Hz, H-27). Two methine
an antitode in traditional Chinese medicine.3) It is one of the protons attached to the carbons bearing hydroxyl groups
most important ingredients of many medical prescriptions of were also noted at d 4.02 (1H, br s, H-1) and d 4.36 (1H,
Miao Minority in China and has been successfully developed br s, H-3). The 13C-NMR and distortionless enhancement by
into a medicine to treat cough and sore throats.4) The genus polarization transfer (DEPT) spectrum of 1 showed a quater-
Reineckia is taxonomically close to Rohdea, Tupistra, Con- nary carbon signal at d 110.6, which is the characteristic C-
vallaria, Aspidistra, Liriope and Ophiopogon, and their 22 of a spirostanol skeleton.17) On comparison between the
chemical constituents are characterised by steroidal saponins 13C-NMR spectrum of 1 with that of rhodeasapogenin
with polyhydroxylated skeletons.5—9) During the year 1955— [25(S)-5b-spirostan-1b,3b-diol],14,18) the signal due to the C-
1994, Japanese scholars reported some steroidal sapogenins 17 carbon, which was observed at d 63.1 (CH) in rhodeasa-
and steroidal saponins from this plant.10—13) The steroidal pogenin, appeared at d 89.6 as a quaternary carbon signal,
constituents from the underground parts of R. carnea exhib- accompanied by downfield shifts of the signals due to C-13
ited inhibitory activity on cAMP phosphodiesterase and a po- (ꢂ5.5 ppm), C-16 (ꢂ9.6 ppm), and C-20 (ꢂ2.7 ppm) and up-
tent inhibitor, showing almost equal IC50 values compared to field shifts of signals due to C-12 (ꢀ10.4 ppm), C-14
papaverine, was isolated.13) Formerly, we reported three (ꢀ3.5 ppm), and C-21 (ꢀ5.9 ppm); in addition, the molecu-
spirostanol steroids from R. carnea: 25(S)-5b-spirostan-
1b,3b-diol (rhodeasapogenin, obtained through acid hydroly-
sis), 25(S)-5b-spirostan-1b,3b-diol 1-O-b-D-xylopyranoside,
25(S)-5b-spirostan-1b,3b-diol 1-O-a-L-rhamnopyranosyl-(1→
2)-b-D-xylopyranoside,14) now, the 17a-OH derivatives of the
three spirostanol steroids, corresponding to 25(S)-5b-
spirostan-1b,3b,17a-triol (1), 25(S)-5b-spirostan-1b,3b,17a-
triol 1-O-b-D-xylopyranoside (2), 25(S)-5b-spirostan-1b,3b,
17a-triol 1-O-a-L-rhamnopyranosyl-(1→2)-b-D-xylopyrano
-
side (3) were isolated from this plant. Compounds 1, 2, and 3
are the first naturally occurring steroids possessing unique
structural feature of 5b-spirostan-1b,3b,17a-trihydroxyl.
This paper describes the isolation and structural elucidation
of these new compounds based on spectroscopic data and
chemical transformation.
Compound 1 was isolated as a white amorphous solid with
the molecular formula C27H44O5 deduced from negative-ion
high-resolution (HR)-FAB-MS m/z 447.3124 [MꢀH]ꢀ, cal-
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: mhchiu@mail.kib.ac.cn

54
Vol. 59, No. 1
lar formula of 1 (C27H44O5) has one more oxygen atom than the appearance of an anomeric proton signal at d 4.83 (d,
rhodeasapogenin (C27H44O4). Thus, the introduction of a hy- Jꢁ7.7 Hz) in the 1H-NMR spectrum, and also by five charac-
droxyl group onto C-17 was evident. The 13C-NMR data of teristic 13C signals at d 102.3, 75.2, 78.9, 71.3, and 67.6.21,22)
the C—F rings of 1 was closely related to those of penno- The point of attachment of the sugar moiety in 2 was at 1b-
genin [25(R)-3b,17a-dihydroxy-spirost-5-ene],19) and the OH rather than the more commonly observed 3b-OH group.
characteristic 13C signals that suggested the 17a-hydroxyl This was supported by the 13C-NMR spectrum of the agly-
group as those for pennogenin on the cis-fused D/E ring sys- cone 2 in which there was only a shift for C-1 downfield
tem were: d 46.2 (C-13), 90.0 (C-16), 89.6 (C-17), and 45.2 (ꢂ6.1 ppm, to 79.5 ppm), and by the HMBC correlations of
(C-20). The 17a-OH configuration was further supported by H-1-xylose (d 4.83) with C-1 (d 79.5), this signal in turn
the rotating frame Overhauser effect spectroscopy (ROESY) coupled with H-19 (d 1.35) (Fig. 3). Therefore, the structure
correlations between 20-H (d 2.24) and 18-CH3 (d 0.98) and of 2 was characterized as 25(S)-5b-spirostan-1b,3b,17a-triol
between 16-H (d 4.49) and 15a-H (d 2.11) on a cis-fused 1-O-b-D-xylopyranoside, namely, reineckiagenoside A.
D/E ring system (Fig. 1). The correlations of H-19 (d 1.34)
Compound 3 crystallized as needles from chloroform/
and H-5 (d 2.24) in the ROESY spectrum of 1 supported the methanol/water (7 : 3 : 0.1), negative-ion FAB-MS m/z: 725
5b-H configuration and the cis-fused A/B ring system; the [MꢀH]ꢀ, 579 [Mꢀ147 (rhamnose)ꢀH]ꢀ, 447 [Mꢀ147
broad singlet signal of H-1 (d 4.02, br s) and H-3 (d 4.36, (rhamnose)ꢀ132 (xylose)ꢀH]ꢀ. Negative-ion HR-FAB-MS
br s) on the cis-fused A/B ring system were consistent with gave a quasi-molecular formula ion at m/z 725.4136
the 1b,3b-OH configuration.
[MꢀH]ꢀ, calculated: 725.4112 m/z, corresponding to an em-
Interpretation of the heteronuclear single quantum coher- pirical molecular formula C38H62O13. The IR spectrum
ence (HSQC), heteronuclear multiple bond connectivity showed absorption bands in agreement of 25(S)-spiroketal
(HMBC), and correlation spectroscopy (COSY) spectrum al- moiety (nmax 980, 910, 894, 860 cmꢀ1, intensity 910ꢃ894).
lowed us to assign all the signals of compound 1 unambigu- On acid hydrolysis with 1 M hydrochloric acid dioxane/water
ously (for the key HMBC correlations, see Fig. 2). Thus, the (1 : 1) at 90 °C, 3 liberated 1, 2, L-rhamnose, and D-xylose, 1
structure of 1 was elucidated to be 25(S)-5b-spirostan- and 2 were identified by TLC comparison and further con-
1b,3b,17a-triol, namely, reineckiagenin A.
firmed by 1H- and 13C-NMR spectrum, while L-rhamnose and
Compound 2 was obtained as a white amorphous powder, D-xylose were determined by GC analysis of their
the negative-ion FAB-MS m/z: 579 [MꢀH]ꢀ, 447 [Mꢀ132 trimethylsilylated L-cysteine adducts.
(xylose)ꢀH]ꢀ. The molecular formula C32H52O9 was deter-
On comparison of the whole 13C-NMR spectrum of 3 with
mined by negative-ion HR-FAB-MS [m/z 579.3542 that of 2, a set of additional signals, corresponding to a ter-
(MꢀH)ꢀ, calculated: 579.3533] and 13C-NMR. The IR spec- minal a-L-rhamnopyranosyl unit, appeared (d 101.9, 72.2,
trum of 2 showed characteristic absorptions due to hydroxyl 72.0, 74.5, 69.9, 18.8); the a-configuration of the L-rham-
(3400 cmꢀ1) and spiroketal groups (985, 916, 899, 863 nose was readily confirmed by the 13C shifts; remarkable dif-
cmꢀ1); the absorption at 916 cmꢀ1 was of greater intensity ferences in the 13C shifts at C-3 and C-5 were recognized be-
than that at 899 cmꢀ1 (25(S)-spiroketal). Acid hydrolysis of tween a- and b-L-rhamnopyranosides.22,23) The signal due to
2 with 1 M hydrochloric acid dioxane/water (1 : 1) gave 1 C-2 of the xylose was displaced downfield by 4 ppm and that
(identified by TLC comparison and further confirmed by 1H- due to C-1 of the xylose moved to upper field by 4.1 ppm, in-
and 13C-NMR spectrum) and D-xylose (determined by GC dicating that the C-2 position of the xylose unit was the gly-
analysis of its trimethylsilylated L-cysteine adducts).20) The cosylated position to which the additional L-rhamnose was
presence of a b-xylopyranosyl moiety was demonstrated by linked. The steroidal saponin with the same sugar sequence
(a-L-rhamnopyranosyl-(1→2)-b-D-xylopyranosyl) has been
isolated from R. carnea by Kiyoshi Iwagoe in 1987.9)
The sequence of the sugars and linkage site to the agly-
cone of 3 were further supported by 2D NMR experiments.
In the HMBC spectrum, correlations between H-1-rhamnose
(d 6.57) and C-2-xylose (d 79.2), between H-1-xylose (d
5.05, 1H, d, Jꢁ7.3 Hz) and C-1 (d 76.0) which in turn cou-
pled with H-19 (d 1.34) were observed (Fig. 3).
On the basis of the above evidence, 3 was formulated
as 25(S)-5b-spirostan-1b,3b,17a-triol 1-O-a-L-rhamnopyra-
nosyl-(1→2)-b-D-xylopyranoside, namely, reineckiagenoside
Fig. 1. Selected ROESY Correlations of 1
Fig. 2. Key HMBC Correlations of 1
Fig. 3. Key HMBC Correlations of 2 and 3

January 2011
55
tector. The column was 30QC2/AC-5 quartz capillary column (30 mꢄ0.32
mm) with the following conditions: column temperature: 180 °C/280 °C;
programmed increase, 3 °C/min; carrier gas: N2 (1 ml/min); injection and de-
tector temperature: 250 °C; injection volume: 4 ml, split ratio: 1/50. Thin-
layer chromatography (TLC) was performed on plates pre-coated with silica
gel F254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Silica gel
(200—300 mesh and 10—40 mm), RP-18 (40—63 mm) and Sephadex LH-
20 were used for column chromatography.
B.
17a-OH steroids have been found from Lilium pardar-
inum24) and Paris axialis LI.25) etc. However, 17a-OH
steroids was isolated for the first time from the genus Rei-
neckia. Since 5b-spirostan with 1b,3b-dihydroxyl groups
were found in very limited genera of plants such as
Rhodea,26) we estimated that 5b-spirostan with 1b,3b,17a-
trihydroxyl groups would be very rare as natural products.
Glycosylation of the two steroidal saponins occurred at 1b-
Plant Material The whole plant of R. carnea was collected from Yu-
long Snow Mountain (Nov. 2004), Lijiang City, Yunnan Province, China,
and a voucher specimen (identified by Prof. Xi-wen Li from the department
OH rather than the more commonly observed 3b-OH group of taxonomy, Kunming Institute of Botany, Chinese Academy of Sciences)
as those in the literature.27) The three compounds are the first
naturally occurring steroids with unique structural feature of
5b-spirostan-1b,3b,17a-trihydroxyl. The isolation of the
new steroidal sapogenin may be of help in the chemotaxon-
omy of the family Liliceae.
was deposited at the State Key Laboratory of Phytochemistry of Kunming
Institute of Botany, Chinese Academy of Sciences (No. 200411011).
Extraction and Isolation The air-dried whole plant of R. carnea
(1.90 kg) was extracted with 90% MeOH/H2O under reflux for 3 h each time
(3ꢄ8 l). The extract was filtrated, the filtrate concentrated in vacuo at 45 °C,
and the resulting extract (557 g) suspended in H2O (4 l) and then partitioned
with AcOEt (3ꢄ3 l). The AcOEt extract (137 g) was subjected to column
chromatography over silica gel (800 g) eluting with CHCl3/MeOH to afford
four fractions. Fraction 2 (elution of CHCl3/MeOH, 1 : 0—10 : 1, v/v, 24 g)
was subjected to column chromatography over silica gel (220 g) eluting with
CHCl3/MeOH afford four fractions. Fraction 2.1 (elution of CHCl3/MeOH,
1 : 0—80 : 1, 4 g) was rechromatographed on silica gel eluting with CHCl3/
Experimental
General Procedure Optical rotations were measured with a Horiba
SEPA-300 polarimeter. IR spectral data were determined on a Bio-Rad FTS-
135 spectrometer with KBr pellets. NMR spectra were obtained on Bruker
DRX-500 (500 MHz for 1H-NMR and 125 MHz for 13C-NMR) instrument
with tetramethylsilane (TMS) as the internal standard. The multiplicity of MeOH (100 : 1) to obtain 1 (11 mg). Fraction 2.3 (elution of CHCl3/MeOH,
13C-NMR was determined as DEPT. MS data were obtained on a VG Au-
tospec-3000 mass spectrometer. GC analysis was run on Agilent Technolo-
20 : 1—10 : 1, 7 g) was chromatographed on silica gel eluting with CHCl3/
MeOH 15 : 1, then further purified by RP-18 (70% MeOH/H2O) and
gies HP5890 gas chromatograph equipped with an H2 flame ionization de- sephadex LH-20 (MeOH) to afford 2 (18 mg). Fraction 3 (elution of
Table 1. 1H- and 13C-NMR Spectral Dataa) of Compound 1 and the Aglycone Parts of Compounds 2 and 3
Rhodeasapogeninb)
1
2
3
Position
C
C
H
C
H
C
H
1
73.4
32.8
73.4
32.7
4.02 (br s)
79.5
32.5
4.06 (br s)
76.0
32.5
4.39 (br s)
2ax
2eq
3
4ax
4eq
5
6ax
6eq
7ax
7eq
8
9
10
11
12ax
12eq
13
1.45—1.47 (m)
2.10—2.12 (m)
4.36 (br s)
1.45—1.48 (m)
2.16—2.18 (m)
4.27 (br s)
1.46—1.49 (m)
2.17—2.19 (m)
4.34 (br s)
68.2
34.4
68.2
34.3
66.5
34.4
67.3
34.0
1.68—1.70 (m)
1.92—1.94 (m)
2.23—2.25 (m)
1.40—1.44 (m)
1.82—1.84 (m)
1.20—1.24 (m)
1.68—1.70 (m)
1.68—1.71 (m)
1.27—1.29 (m)
1.62—1.64 (m)
1.94—1.96 (m)
2.21—2.24 (m)
1.42—1.44 (m)
1.90—1.94 (m)
1.33—1.36 (m)
1.60—1.62 (m)
1.67—1.69 (m)
1.26—1.29 (m)
1.60—1.62 (m)
1.93—1.95 (m)
2.48—2.50 (m)
1.46—1.49 (m)
1.90—1.94 (m)
1.33—1.36 (m)
1.60—1.62 (m)
1.67—1.69 (m)
1.29—1.30 (m)
31.2
26.8
31.5
26.7
31.6
26.5
31.8
26.4
26.7
26.6
26.4
26.2
35.8
42.1
40.2
21.1
40.4
36.3
41.9
40.4
20.9
30.0
36.2
41.6
39.4
21.2
29.2
36.1
41.8
39.4
21.3
30.5
1.30—1.33 (m)
1.47—1.49 (m)
2.16—2.18 (m)
1.35—1.37 (m)
1.48—1.50 (m)
2.17—2.19 (m)
1.36—1.39 (m)
1.46—1.49 (m)
2.17—2.19 (m)
40.7
56.4
32.2
46.2
52.9
31.5
45.3
52.8
30.0
45.3
52.8
31.6
14
2.08—2.10 (m)
2.10—1.12 (m)
1.42—1.44 (m)
4.49 (d, 7.1)
2.21—2.24 (m)
2.13—2.15 (m)
1.44—1.46 (m)
4.44 (d, 7.2)
2.15—2.17 (m)
2.18—2.20 (m)
1.46—1.48 (m)
4.43 (d, 7.1)
15ax
15eq
16
17
18
19
20
21
22
23ax
23eq
24
24
25
26ax
26eq
27
81.3
63.1
16.7
19.3
42.5
14.9
109.8
26.4
90.9
89.6
17.8
19.4
45.2
9.0
90.2
90.0
17.5
19.8
45.4
9.6
90.2
90.0
17.5
19.5
45.4
9.6
0.98 (s)
1.34 (s)
2.23—2.25 (m)
1.24 (d, 7.1)
0.96 (s)
1.35 (s)
2.21—2.23 (m)
1.21 (d, 7.2)
0.95 (s)
1.34 (s)
2.20—2.22 (m)
1.20 (d, 7.1)
110.6
26.7
110.3
26.6
110.3
26.6
1.58—1.60 (m)
1.92—1.94 (m)
1.89—1.90 (m)
2.19—2.20 (m)
2.17—2.19 (m)
3.16 (d, 11.1)
1.58—1.60 (m)
1.92—1.94 (m)
1.90—1.92 (m)
2.18—2.20 (m)
2.17—2.19 (m)
3.22 (d, 10.7)
1.60—1.62 (m)
1.92—1.94 (m)
1.90—1.92 (m)
2.19—2.20 (m)
2.18—2.20 (m)
3.23 (d, 10.8)
26.2
26.6
25.7
25.7
27.6
65.2
27.4
63.9
27.4
64.9
27.4
64.9
3.99 (dd, 2.2, 11.1)
1.03 (d, 7.2)
4.03 (dd, 2.5, 10.7)
1.04 (d, 7.0)
4.03 (dd, 2.1, 10.8)
1.04 (d, 7.0)
16.3
16.9
16.2
16.2
a) Measured in 500 Hz for 1H, and 125 Hz for 13C, in C5D5N; coupling constants (J) in Hz are given in parentheses. b) Data in ref. 14.

56
Vol. 59, No. 1
Table 2. 1H- and 13C-NMR Spectral Dataa) for Sugar Parts of Compounds 2 and 3
2
3
C
H
C
H
xyl-1
102.3
75.2
78.9
71.3
67.6
4.83 (d, 7.7)
xyl-1
98.2
79.2
76.9
71.6
67.4
5.05 (d, 7.3)
2
3
4
5
4.00—4.02 (m)
4.17—4.19 (m)
4.22—4.24 (m)
3.77 (dd, 10.6, 10.7)
4.41—4.43 (m)
2
3
4
5
4.18—20 (m)
4.25—27 (m)
4.12—4.14 (m)
3.66 (dd, 10.1, 11.2)
4.34 (dd, 5.2, 11.2)
6.57 (s)
4.77—4.79 (m)
4.73—4.75 (m)
4.21—4.23 (m)
4.73 (m)
Rha-1
101.9
72.2
72.0
74.5
69.9
18.8
2
3
4
5
6
1.73 (d, 6.1)
a) Measured in 500 Hz for 1H, and 125 Hz for 13C, in C5D5N; coupling constants (J) in Hz are given in parentheses.
CHCl3/MeOH, 10 : 0—6 : 1, 18 g) was subjected to column chromatography
gram of Promoting Development for Guizhou and Xibuzhiguang from CAS,
over silica gel (200 g) eluting with CHCl3/MeOH to afford four fractions. Foundation of Key State Lab. of Phytochemistry and Plant Resources in
Fraction 3.3 (elution of CHCl3/MeOH, 9 : 1—7 : 1, 4 g) was chromatograph-
ed over silica gel eluting with CHCl3/MeOH (8 : 1) and further purified by
RP-18 (60% MeOH/H2O) and sephadex LH-20 (MeOH) to obtain 3 (280 mg).
West China (P2008ZZ05), and Knowledge Innovation Programs of the Chi-
nese Academy of Sciences (CAS) (Grant No. KSCX2-YW-G-038, KSCX2-
YW-R-194, 29KZCX2-XB2-15-03).
Reineckiagenin A (1): C27H44O5, white amorphous solid, [a]D20 ꢂ10.4°
References
KBr
(cꢁ0.11, CHCl3), IR n
(cmꢀ1): 3373, 2925, 1057, 977, 948, 918, 898,
max
1) Tsukamoto Y., “The Grand Dictionary of Horticulture,” Shogakukan,
822, intensity 918ꢃ898. Negative-ion FAB-MS m/z: 447 [MꢀH]ꢀ. Nega-
tive-ion HR-FAB-MS m/z 447.3124 [MꢀH]ꢀ, calculated: 447.3110. For the
1H- and 13C-NMR spectral data (see Table 1).
Tokyo, 1988, p. 36.
2) Zhang P., Lian F. Q., Zhu M. Y., Xiong M. L., Journal of Jiangxi Agri-
cultural University, 26, 941—943 (2004).
3) National Administration of Traditional Chinese Medicine, “Dictionary
of Traditional Chinese Medicine,” Shanghai Science and Technology
Publishing House, Shanghai, 1999, pp. 7204—7205.
4) Lu K. M., “Collection of the Drugs in Miao Minority,” Guizhou Peo-
ple’s Publishing House, Guizhou, 1987, pp. 44.
5) Kimura M., Tohma M., Yoshizawa I., Akiyama H., Chem. Pharm.
Bull., 16, 25—33 (1968).
6) Hirai Y., Konishi T., Sanada S., Chem. Pharm. Bull., 30, 3476—3483
(1982).
7) Konishi T., Kiyosawa S., Shoji J., Chem. Pharm. Bull., 32, 1451—
1466 (1984).
8) Yang Q. X., Zhang Y. J., Li H. Z., Yang C. R., Steroids, 70, 732—737
(2005).
9) Kiyoshi I., Tenji K., Shiu K., Yakugaku Zasshi, 107, 140—149 (1987).
10) Takeda K., Okanishi T., Shimaoka A., Yakugaku Zasshi, 75: 560—562
(1955).
Reineckiagenoside A (2): C32H52O9, white amorphous solid. [a]D20 ꢀ3.7°
KBr
max
(cꢁ0.11, MeOH), IR n
(cmꢀ1): 3400, 2926, 1454, 1046, 985, 916, 899,
863, intensity 916ꢃ899. Negative-ion FAB-MS m/z: 579 [MꢀH]ꢀ, 447
[Mꢀ132ꢀH]ꢀ. Negative-ion HR-FAB-MS m/z: 579.3542 [MꢀH]ꢀ, calcu-
lated: 579.3533. For the 1H- and 13C-NMR spectral data (see Tables 1, 2).
Reineckiagenoside B (3): C38H62O13, colorless needles, crystallized from
chloroform/methanol/water (7 : 3 : 0.1), [a]D20 ꢀ9.9° (cꢁ0.14, MeOH), IR
KBr
max
n
(cmꢀ1): 3400, 2930, 1450, 1040, 980, 910, 894, 860, intensity
910ꢃ894. Negative-ion FAB-MS m/z: 725 [MꢀH]ꢀ, 579 [Mꢀ147ꢀH]ꢀ,
447 [Mꢀ147ꢀ132ꢀH]ꢀ. HR-FAB-MS m/z 725.4136 [MꢀH]ꢀ, calculated:
725.4112. For the 1H- and 13C-NMR spectral data (see Tables 1, 2).
Acid Hydrolysis of
2
A solution of 2 (12 mg) in 1 M HCl
(dioxane/water, 1 : 1, 6 ml) was heated at 90 °C for 2 h, the reaction mixture
was diluted with 2 ml H2O and extracted with CHCl3 (5 mlꢄ3), the CHCl3
phase (8.6 mg) was subjected to CC over silica gel eluting with
CHCl3/MeOH (100 : 1 v/v) to give 1 (3.6 mg, identified by TLC comparison
11) Sasaki K., Chem. Pharm. Bull., 9, 684—692 (1961).
12) Takeda K., Okanishi T., Minato H., Shimaoka A., Tetrahedron, 19,
759—772 (1963).
13) Kanmoto T., Mimaki Y., Sashida Y., Nikadido T., Koike K., Ohmoto
T., Chem. Pharm. Bull., 42, 926—931 (1994).
1
and further confirmed by H- and 13C-NMR spectrum). The aqueous layer
was passed through an Amberlite IRA-401 (OHꢀ form) column and the elu-
ate was concentrated to dryness to give D-xylose, which was detected by di-
rect TLC analysis on a HPTLC silica gel 50000 F254 plate using n-
BuOH–Me2CO–H2O (4 : 5 : 1, homogenous) as development and anisalde-
hyde-H2SO4 as detection, comparing with the authentic sample: xylose (Rf
0.58). The absolute configuration of monosaccharide was further determined
by GC analysis of its derivatives to be D-xylose. A solution of the sugar
residue (2.5 mg) in pyridine (2 ml) was added to L-cysteine methyl ester hy-
drochloride (3.3 mg) and kept at 60 °C for 1 h. Then trimethylsilylimidazole
(0.5 ml) was slowly added to the reaction mixture and kept again at 60 °C for
30 min. The supernatant (4 ml) was analyzed by GC, and the retention time
of D- and L-xylose were 13.35 and 14.01 min, respectively.
14) Zhang Z. Q., Chen J. C., Zhang X. M., Li Z. R., Qiu M. H., Helv.
Chim. Acta, 91, 1494—1499 (2008).
15) Eddy C. R., Wall M. E., Scott M. K., Anal. Chem., 25, 266—271
(1953).
16) Jones R. N., Katzenellenbogen K., Dobriner K., J. Am. Chem. Soc., 75,
158—166 (1953).
17) Agrawal P. K., Jain D. C., Gupta R. K., Thakur R. S., Phytochemistry,
24, 2479—2496 (1985).
18) Tori K., Seo S., Terui Y., Nishikawa J., Yashuda F., Tetrahedron Lett.,
22, 2405—2408 (1981).
Acid Hydrolysis of 3 A solution of 3 (22 mg) in 1 M HCl (dioxane/
water, 1 : 1, 12 ml) was heated at 90 °C for 1 h, the reaction mixture was di-
luted with 5 ml H2O and extracted with AcOEt (10 mlꢄ3), the AcOEt phase
(18 mg) was subjected to CC over silica gel eluting with CHCl3/MeOH
(100 : 1—15 : 1) to give 1 and 2 [1 (5.6 mg), 2 (6.8 mg), identified by TLC
comparison and further confirmed by 1H- and 13C-NMR spectrum]. The
aqueous layer was treated and analyzed as described for 2, in which D-xylose
and L-rhamnose was detected by HPTLC (Development solvent: n-
BuOH/Me2CO/H2O, 4 : 5 : 1, D-xylose Rf 0.58, L-rhamnose Rf 0.67), and GC
analysis (retention times for D- and L-xylose, and L-rhamnose were 13.35,
14.01, and 14.97 min, respectively).
19) Marquardt F. H., Chem. Ind. (London), 1978, 94—95 (1978).
20) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 35, 501—506
(1987).
21) Lemieux R. U., Stevens J. D., Can. J. Chem., 44, 249—262 (1966).
22) Gorin P. A. J., Mazurek M., Can. J. Chem., 53, 1212—1223 (1975).
23) Agrawal P. K., Phytochemistry, 31, 3307—3330 (1966).
24) Shimomura H., Sashoda Y., Mimaki Y., Phytochemistry, 28, 3163—
3170 (1989).
25) Chen C. X., Zhou J., Acta Bot. Yunnanica, 6, 111—117 (1984).
26) Miyahara K., Kudo K., Kawasaki T., Chem. Pharm. Bull., 31, 348—
351 (1983).
27) Partil A. D., Baures P. W., Eggleaton D. S., Faucette L., Hemling M.
E., Westley J. W., Johnson P. K., J. Nat. Prod., 56, 1451—1458 (1993).
Acknowledgements The project was financially supported by the Pro-