Five new cycloartane-type triterpenoid saponins from nervilia fordii

Article abstract of DOI:10.1002/hlca.201200139

Five new cycloartane glycosides, nervisides D-H (1-5), were isolated from the AcOEt- and H₂O-soluble portions of the 90% EtOH extract of the aerial part of the plant Nervilia fordii. The structures of the isolated glycosides were elucidated by

Full text of DOI:10.1002/hlca.201200139

150
Helvetica Chimica Acta – Vol. 96 (2013)
Five New Cycloartane-Type Triterpenoid Saponins from Nervilia fordii
by Liu-Bin Weia), Hong-E Yuana), Jin-Man Chena), Qing-Qing Wanga), Hui Wangb), Wen-Cai Yea),
and Guang-Xiong Zhou*a)
a) Institute of Traditional Chinese Medicine and Natural Products, Guangdong Province Key Laboratory
of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, College of
Pharmacy, Jinan University, Guangzhou 510632, P. R. China
(phone: þ 86-20-85221469; fax: þ 86-20-85221559; e-mail: guangxzh@sina.com)
b) School of Medicine, Jinan University, Guangzhou 510632, P. R. China
Five new cycloartane glycosides, nervisides D – H (1 – 5), were isolated from the AcOEt- and H2O-
soluble portions of the 90% EtOH extract of the aerial part of the plant Nervilia fordii. The structures of
the isolated glycosides were elucidated by extensive spectroscopic analysis including HR-ESI-MS and
NMR data. The isolated nervisides D – H were evaluated for the cytotoxic activity in vitro against human-
tumor cell lines (CNE, Hep-2 and HepG2) with the MTT method.
Introduction. – The plant Nervilia fordii (Hance) Schltr. (Orchidaceae) is an
endemic source of herbal medicine in the south of China. The whole plant was used as
the traditional Chinese medicine ꢀQingtiankuiꢁ for the treatment of various diseases,
such as tuberculosis, bronchitis, and pneumonia [1]. The study about an acetylflavonol
of this plant with potent inhibitory activity against the production of nitric oxide (NO)
in RAW264.7 has been reported [2]. The H2O-soluble portion of the plant has shown
antiviral activity against influenza in vitro [3]. The MeOH extracts of the plant with
antiviral activity on Herpes simplex virus type 1 (HSV-1) was investigated [4]. Flavonol
glycosides, steroides, and triterpenes were reported from the plant [4 – 6]. In this paper,
five new cyclolartane glycosides, named nervisides D – H (1 – 5; Fig. 1), were isolated
from the 90% EtOH extract of this plant. The structures of 1 – 5 were determined by
spectroscopic analysis of their HR-ESI-MS and NMR data. The in vitro cytotoxic
activity against human-tumor cell lines (CNE, Hep-2, and HepG2) of these compounds
were measured by the MTT (¼2-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl-2H-tetrazo-
lium bromide) method.
Fig. 1. Compounds 1 – 5, isolated from Nervilia fordii
ꢂ 2013 Verlag Helvetica Chimica Acta AG, Zꢃrich

Helvetica Chimica Acta – Vol. 96 (2013)
151
Results and Discussion. – The 90% EtOH extract of Nervilia fordii was partitioned
successively with petroleum ether, CHCl3, and AcOEt. The AcOEt phase was
subjected to column chromatography (CC; polyamide, Sephadex LH-20, and octadecyl
silica gel (ODS)), and finally to prep. HPLC to give the two new glycosides 4 and 5. The
H2O-soluble portion was subjected to CC (D101 macroporous resin, SiO2, Sephadex
LH-20, and ODS), and finally to prep. HPLC to yield the three new glycosides 1 – 3.
Nerviside D (1) was obtained as white powder. The molecular formula was
established to be C36H60O11 from a quasimolecular ion at m/z 691.4026 ([M þ Na]þ) in
the HR-ESI-MS. The 1H-NMR spectrum (Table 1) displayed the signals of a CH2 group
at d(H) 0.48 and 0.77 (2d, J ¼ 4.3 Hz, 1 H each), of five tertiary Me groups at d(H) 0.95,
1.00, 1.64, 1.65 and 1.66 (5s), of a secondary Me group at d(H) 0.99 (d, J ¼ 6.4 Hz), and
of an anomeric H-atom at d(H) 5.03 (d, J ¼ 7.0 Hz ) . The 13C-NMR spectrum (Table 2)
exhibited signals of 36 C-atoms, of which 31 accounted for the aglycone moiety. The
remaining signals were in good accordance with the presence of a xylose unit at d(C)
106.6 (d, C(1’)), 75.5 (d, C(2’)), 78.1 (d, C(3’)), 71.1 (d, C(4’)), and 67.0 (t, C(5’)) [7].
The resonances assigned to the aglycone moiety arose from six Me groups, eleven CH2
groups, six CH groups, seven quaternary C-atoms, and one COOH group. The signals at
d(C) 72.3 (d), 81.3 (d), 76.5 (s), 76.7 (s), and 65.6 (t) showed five O-bearing C-atoms.
All of the above evidences suggested that 1 was a highly oxygenated cycloartane
(¼ 9,19-cyclolanostane) triterpene monoglycoside [8 – 10]. The anomeric center of the
3
xylopyranosyl moiety was determined to have b-configuration from the large J(1,2)
value. A long-range correlation between an anomeric H-atom at d(H) 5.03 (d, J ¼
7.0 Hz, HꢀC(1’)) and d(C) 81.3 (d, C(3)) in the HMBC spectrum demonstrated that
the sugar moiety was bound to C(3) (Fig. 2). The b-orientation of the sugar moiety at
C(3) was determined from the coupling pattern and constant of HꢀC(3) (dd, J ¼ 12.1
and 4.3 Hz). Furthermore, the HMBCs HꢀC(1)/C(3) and C(5), HꢀC(3)/C(1), C(28),
and C(29), and Me(29)/C(3), C(4), C(5), and C(28) allowed to place a COOH group
at C(4) and an OH group at C(1) of ring A (Fig. 2). In addition, the HMBCs CH2(31)/
C(23), C(24), and C(25) suggested the location of a CH2OH group at C(24) of the side
1
chain. The structure of the aglycone moiety was confirmed by the H,1H-COSY
connectivities HꢀC(1)/HꢀC(2)/HꢀC(3), HꢀC(5)/CH2(6)/CH2(7)/HꢀC(8), CH2(11)/
CH2(12), CH2(15)/CH2(16)/HꢀC(17)/HꢀC(20)/CH2(22)/CH2(23) and HꢀC(20)/
Me(21) (Fig. 2). The relative configurations a and b of OHꢀ(1) and XylOꢀC(3),
respectively, were confirmed by the key ROESY correlations HꢀC(1)/HaxꢀC(2) and
Fig. 2. Major HMBC and 1H,1H-COSY features of 1

152
Helvetica Chimica Acta – Vol. 96 (2013)

Helvetica Chimica Acta – Vol. 96 (2013)
153
Table 2. 13C-NMR Data (C5D5N, 100 MHz) of Compounds 1 – 5
C-Atom
1
2
3
4
5
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
72.3 (d)
37.5 (t)
81.3 (d)
54.7 (s)
37.9 (d)
23.1 (t)
25.8 (t)
48.2 (d)
20.9 (s)
30.1 (s)
26.2 (t)
33.3 (t)
45.6 (s)
49.1 (s)
35.9 (t)
28.4 (t)
53.1 (d)
18.5 (q)
29.7 (t)
37.6 (d)
18.8 (q)
30.3 (t)
30.7 (t)
76.5 (s)
76.7 (s)
26.0 (q)
26.1 (q)
72.4 (d)
37.6 (t)
81.4 (d)
54.8 (s)
37.9 (d)
23.1 (t)
25.9 (t)
48.3 (d)
21.0 (s)
30.2 (s)
26.2 (t)
33.3 (t)
45.7 (s)
49.2 (s)
35.9 (t)
28.4 (t)
52.9 (d)
18.5 (q)
29.7 (t)
36.4 (d)
18.6 (q)
34.2 (t)
29.0 (t)
79.2 (d)
72.9 (s)
26.0 (q)
26.3 (q)
72.3 (d)
37.4 (t)
81.9 (d)
54.7 (s)
37.9 (d)
23.2 (t)
25.8 (t)
48.2 (d)
20.9 (s)
30.2 (s)
26.0 (t)
33.4 (t)
45.7 (s)
49.2 (s)
35.9 (t)
28.5 (t)
53.0 (d)
18.5 (q)
29.8 (t)
37.7 (d)
18.7 (q)
34.2 (t)
29.0 (t)
79.1 (d)
72.8 (s)
26.3 (q)
26.5 (q)
72.4 (d)
37.6 (t)
81.4 (d)
54.7 (s)
37.9 (d)
23.1 (t)
25.8 (t)
48.2 (d)
20.9 (s)
30.1 (s)
26.1 (t)
33.2 (t)
45.6 (s)
49.2 (s)
35.8 (t)
28.3 (t)
52.6 (d)
18.4 (q)
29.6 (t)
36.2 (d)
18.5 (q)
30.6 (t)
33.5 (t)
72.3 (d)
37.4 (t)
81.9 (d)
54.6 (s)
37.9 (d)
23.1 (t)
25.8 (t)
48.2 (d)
20.9 (s)
30.2 (s)
26.1 (t)
33.2 (t)
45.6 (s)
49.2 (s)
35.8 (t)
28.3 (t)
52.6 (d)
18.4 (q)
29.6 (t)
36.1 (d)
18.3 (q)
30.7 (t)
33.5 (t)
C(9)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
C(17)
C(18)
C(19)
C(20)
C(21)
C(22)
C(23)
C(24)
C(25)
C(26)
C(27)
C(28)
C(29)
C(30)
C(31)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
C(6’)
216.6 (s)
76.9 (s)
27.3 (q)
27.4 (q)
180.0 (s)
10.4 (q)
19.5 (q)
216.6 (s)
76.9 (s)
27.3 (q)
27.4 (q)
180.2 (s)
10.5 (q)
19.5 (q)
180.0 (s)
10.4 (q)
19.5 (q)
65.6 (t)
106.6 (d)
75.5 (d)
78.1 (d)
71.1 (d)
67.0 (t)
180.1 (s)
10.4 (q)
19.6 (q)
180.2 (s)
10.5 (q)
19.6 (q)
106.6 (d)
75.5 (d)
78.1 (d)
71.1 (d)
67.0 (t)
105.9 (d)
75.7 (d)
78.3 (d)
71.5 (d)
78.1 (d)
62.8 (t)
106.6 (d)
75.5 (d)
78.1 (d)
71.1 (d)
67.0 (t)
105.9 (d)
75.7 (d)
78.3 (d)
71.4 (d)
78.1 (d)
62.7 (t)
Fig. 3. Key ROESY correlations (H $ H) of 1

154
Helvetica Chimica Acta – Vol. 96 (2013)
HaꢀC(19), and HꢀC(3)/HꢀC(1’), HꢀC(5), and HeqꢀC(2) (Fig. 3). On acid hydrolysis,
1 afforded d-xylose, the structure of which was confirmed by HPLC analysis according
to the method of Tanaka, Kouno, and co-workers [11]. Therefore, the structure of 1 was
deduced to be (1a,3b,24z)-1,3,24,25-tetrahydroxy-24-(hydroxymethyl)cycloartan-28-
oic acid 3-(b-d-xylopyranoside).
Nerviside E (2) was also obtained as white powder. It had the molecular formula
C35H58O10 on the basis of a quasimolecular ion at m/z 637.3954 ([M ꢀ H]ꢀ) in the HR-
1
ESI-MS (negative-ion mode). The H- and 13C-NMR data (Tables 1 and 2) showed
good coincidence with those of 1, except for the side chain, which suggested that 2 was
also a cycloartane triterpene monoglycoside. The HMBC between an anomeric H-atom
at d(H) 5.02 (d, J ¼ 6.9 Hz, HꢀC(1’)) and d(C) 81.4 (d, C(3)) demonstrated that the
sugar moiety was attached to C(3). Furthermore, the HMBCs HꢀC(24)/C(22), C(26),
and C(27), Me(26)/C(24), C(25), and C(27), Me(27)/C(24), C(25), and C(26))
suggested the absence of a CH2OH group at C(24). Additionally, the other key HMBCs
were similar to those of 1 confirming the same substitutions at the cycloartane ring
system as in 1. The key ROESY correlations were identical to that of 1 showing the
same relative configurations in ring A. The configuration at the OH-substituted C(24)
was deduced to be (24R) by comparison with the chemical-shift values of cyclo-
unifolioside C ((24R); C(24) at d(C) 80.3), cyclocantogenin ((24S); C(24) at d(C)
77.0), oleifoliosides A and B ((24S); C(24) at d(C) 77.1) [12][13]. On acid hydrolysis, 2
afforded d-xylose. Thus, the structure of 2 was elucidated as (1a,3b,24R)-1,3,24,25-
tetrahydroxycycloartan-28-oic acid 3-(b-d-xylopyranoside).
Nerviside F (3) was also obtained as white powder. The molecular formula was
determined to be C36H60O11 from a quasimolecular ion at m/z 667.4063 ([M ꢀ H]ꢀ) in
1
the HR-ESI-MS (negative-ion mode). A comparison of the H- and 13C-NMR data
(Tables 1 and 2) of 3 with those of 2 showed that, structurally, 3 closely resembled 2,
except for the presence of a glucose instead of a xylose moiety. Therefore, 3 was also a
cycloartane triterpene monoglycoside. The signals at d(C) 105.9 (d, C(1’)), 75.7 (d,
C(2’)), 78.3 (d, C(3’)), 71.5 (d, C(4’)), 78.1(d, C(5’)), and 62.8 (t, C(6’)) belonged to the
glucose unit [9]. The HMBC between an anomeric H-atom at d(H) 5.06 (d, J ¼ 7.6 Hz,
HꢀC(1’)) and d(C) 81.9 (d, C(3)) established that the sugar moiety was connected to
C(3). The key ROESY correlations were identical to those of 2, indicating the same
relative configurations in the cycloartane ring system. The configuration at the OH-
substituted at C(24) was deduced to be (24R) by comparison with the chemical-shift
values of 2. On acid hydrolysis, 3 afforded d-glucose. From the above evidences, the
structure of 3 was identified to be (1a,3b,24R)-1,3,24,25-tetrahydroxycycloartan-28-oic
acid 3-(b-d-glucopyranoside).
Nerviside G (4) was isolated as white powder. The molecular formula was
calculated as C35H56O10 from a quasimolecular ion at m/z 659.3762 ([M þ Na]þ) in the
1
HR-ESI-MS. The H- and 13C-NMR data (Tables 1and 2) were similar to those of 2,
except for the side chain, which showed that 4 was a cycloartane triterpene
monoglycoside. In the 13C-NMR spectrum of 4, a highly deshielded signal at d(C)
216.6 (s) was expected to represent an isolated C¼O group. The HMBCs Me(26)/
C(24), C(25), and C(27), and Me(27)/C(24), C(25), and C(26) suggested the location
of the C¼O group at C(24). In addition, the sugar moiety was attached to C(3) as
shown by the HMBC between an anomeric H-atom at d(H) 5.03 (d, J ¼ 7.0 Hz,

Helvetica Chimica Acta – Vol. 96 (2013)
155
HꢀC(1’)) and d(C) 81.4 (d, C(3)). The major ROESY correlations were similar to
those of 2, indicating the same relative configurations the in cycloartane ring system.
On acid hydrolysis, 4 afforded d-xylose. Therefore, the structure of 4 was established as
(1a,3b)-1,3,25-trihydroxy-24-oxocycloartan-28-oic acid 3-(b-d-xylopyranoside).
Nerviside H (5) was also isolated as white powder. The molecular formula was
assigned to be C36H58O11 from a quasimolecular ion at m/z 689.3866 ([M þ Na]þ) in the
1
HR-ESI-MS. The H- and 13C-NMR data (Tables 1 and 2) showed good coincidence
with those of 4, except for the sugar moiety, which suggested that 5 was a cycloartane
triterpene monoglycoside. The signals at d(C) 105.9 (d, C(1’)), 75.7 (d, C(2’)), 78.3 (d,
C(3’)), 71.4 (d, C(4’)), 78.1(d, C(5’)), and 62.7 (t, C(6’)) belonged to the glucose unit
[9]. The HMBC between an anomeric H-atom at d(H) 5.08 (d, J ¼ 7.5 Hz, HꢀC(1’))
and d(C) 81.9 (d, C(3)) indicated that the sugar moiety was bound to C(3). In addition,
the other key HMBCs of 5 were similar to those of 4, confirming the same substitutions
at the cycloartane moiety as in 4. The relative configuration was determined to be a for
OHꢀC(1) and b for the 3-(glucosyloxy) substituent at ring A by the key ROESY
correlations HꢀC(1)/HaxꢀC(2) and HꢀC(19a), and HꢀC(3)/HꢀC(1’) and HꢀC(5). On
acid hydrolysis, 5 afforded d-glucose. Accordingly, the structure of 5 was assigned to be
(1a,3b)-1,3,25-trihydroxy-24-oxocycloartan-28-oic acid 3-(b-d-glucopyranoside).
Nervisides D – H (1 – 5) were all found to be inactive (test concentration 100 mg/ml)
in the evaluation for the cytotoxic activity against CNE, Hep-2 and HepG2 cell lines by
the MTT assay method [14].
This research was financially supported by the Foundation of Guangdong Natural Sciences
Committee (No. 8351063201000003). The authors are grateful to Ms. Chun-Chi Feng and Ms. Xiao-Lin
Feng, for the measurement of all NMR spectra and Ms. Hui-Nan Zhao for the recording of the HR-ESI-
MS (College of Pharmacy, Jinan University, Guangzhou, P. R. China).
Experimental Part
General. Column chromatography (CC): polyamide (80 – 100 mesh; Taizhou Luqiao Plaschem Co.,
Ltd., Taizhou, P. R. China), silica gel (SiO2, 200 – 300 mesh; Qingdao Marine Chemical Co. Ltd.,
Qingdao, P. R. China), D101 macroporous resin (Tianjin Agricultural Pesticide Co., Tianjin, P. R. China),
ODS (50 mm; YMC, Tokyo, Japan); Sephadex LH-20 (Phamacia Co. Sweden). TLC: precoated
aluminium silica gel plates (Yantai Chemical Industry Research Institute, Yantai, P. R. China). Anal.
HPLC: Agilent 1200 instrument, equipped with a quaternary pump, a multiple-wavelength detector, an
autosampler, and an UltimateTM-XB-C18 column (5 mm, 250 ꢁ 4.6 mm; Welch, USA); tR in min. Prep.
HPLC: UV detectors from Varian (Palo Alto, CA, USA); UltimateTM-XB-C18 column (5 mm, 21.2 ꢁ
250 mm; Welch, USA). Optical rotations: Jasco P-1030 automatic digital polarimeter. M.p.: X-5
micro-melting point apparatus; uncorrected. UV Spectra: Jasco V-550 UV/VIS spectrometer; lmax (log e)
in nm. IR Spectra: Jasco FT/IR-480 Plus spectrometer; KBr pellets; ˜n in cmꢀ1. NMR Spectra: Bruker-AV-
400 instrument; chemical shifts d in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Agilent-
6210-LC/MSD-TOF mass spectrometer; in m/z.
Plant Material. The aerial part of the plant N. fordii as the experimental material was purchased at
the Qingping market for Chinese Medicinal Material of Guangzhou, P. R. China, in January 2011. The
material was collected in Guangxi and traded to Guangzhou. The original plant of the aerial material was
authenticated as N. fordii (Hance) Schltr. by Prof. G.-X. Zhou, College of Pharmacy, Jinan University.
A specimen (No. 110113) has been deposited with the Department of Pharmacognosy in the college of
Pharmacy, Jinan University, Guangzhou, P. R. China.
Extraction and Isolation. The aerial part of the plant N. fordii (5.0 kg) was extracted with 90% EtOH
under reflux (3 ꢁ 20 l, each 2 h). The combined extracts were concentrated to ca. 2.5 l and then

156
Helvetica Chimica Acta – Vol. 96 (2013)
partitioned successively with petroleum ether, CHCl3, and AcOEt, to afford 330.5 g, 45.6 g, and 18.6 g of
extracts, resp., and 555.0 g of residue from the H2O layer. The AcOEt (18.6 g) fraction was subjected to
CC (polyamide, MeOH/H2O 1:4 ! 1:0): Fractions 1 – 10. Fr. 3 (2.3 g) was fractionated by CC (Sephadex
LH-20, CHCl3/MeOH 1:1): Frs. H1 – H8. Fr. H5 (1.2 g) was subjected to CC (ODS, MeOH/H2O 1:5 !
1:0): Frs. H5-1 – H5-7. Fr. H5-3 (126 mg) was finally purified by prep. HPLC (MeCN/H2O 1:1 containing
0.05% of CF3COOH): 4 (16 mg) and 5 (9 mg). The H2O-soluble portion (500.0 g) was separated by CC
(D101 macroporous resin, EtOH/H2O 0 :1 ! 95 :5): Frs. D1 – D4. Fr. D2 (15.6 g) was subjected to CC
(SiO2, CHCl3/MeOH/H2O 8 :2 :0.2 ! 6 :4 :0.5): Frs. D2-a – D2-h. Fr. D2-e (1.8 g) was fractionated by CC
(Sephadex LH-20, CHCl3/MeOH 1:1): Frs. D2-e-1 – D2-e-9. Fr. D2-e-4 (860 mg) was subjected to CC
(ODS, MeOH/H2O 6 :4 ! 9 :1): Frs. D2-e-4.1 – D2-e-4.15. Fr. D2-e-4.6 (156 mg) was finally purified by
prep. HPLC (MeCN/H2O 45 :55 containing of CF3COOH): 2 (13 mg) and 3 (10 mg). Fr. D2-e-4.2
(125 mg) was purified by prep. HPLC (MeCN/H2O 4 :6 containing 0.05% of CF3COOH): 1 (18 mg).
Acid Hydrolysis and Identification of the Sugar Moieties of Compounds 1 – 5 and Determination of
Their Absolute Configuration. The absolute configuration of the sugar moieties in the structures was
determined by the method of Tanaka, Kouno, and co-workers [11]. Compound 1 (1 mg) was hydrolyzed
with 1n HCl (2 ml) for 2 h at 908. The mixture was concentrated the residue dissolved in pyridine (1 ml)
containing l-cysteine methyl ester (1 mg; Adamas-beta, China), and the mixture heated at 608 for 1 h.
Then, o-tolyl isothiocyanate (5 ml; Sigma, USA) was added, and the mixture was heated at 608 for 1 h.
Then the mixture was directly analyzed by anal. reversed-phase HPLC (308, isocratic elution with 25%
MeCN containing 0.08% of formic acid for 40 min, then washing of the column with 90% MeCN; flow
rate 0.8 ml/min. UV detection at 250 nm): derivative of 1 at tR 25.442 (d-Xyl). Standard sugars (Sigma,
USA) such as d-glycose, l-glycose, d-xylose, and l-xylose were subjected to the same method and their
derivatives recorded: tR 22.291 (d-Glc), 20.298 (l-Glc), 25.536 (d-Xyl), and 23.828 (l-Xyl). Following the
above procedure, the derivatives of 2 – 5 gave peaks at tR 25.391 (d-Xyl), 22.329 (d-Glc), 25.661 (d-Xyl)
and 22.309 (d-Glc), resp.
Nerviside D (¼(1a,3b,24z)-1,3,24,25-Tetrahydroxy-24-(hydroxymethyl)cycloartan-28-oic Acid 3-(b-
D-Xylopyranoside) ¼ rel-(1a,3b,24z)-1,24,25-Trihydroxy-24-(hydroxymethyl)-3-(b-d-xylopyranosyl-
oxy)-9,19-cyclolanostan-28-oic Acid; 1). White powder. M.p. 267 – 2688. [a]2D5 ¼ þ40.5 (c ¼ 0.60, MeOH).
1
UV (MeOH): 203. IR (KBr): 3421, 2943, 2870, 1703. H- and 13C-NMR: Tables 1 and 2. HR-ESI-MS:
691.4026 ([M þ Na]þ, C36H60NaO1þ1 ; calc. 691.4028).
Nerviside E (¼(1a,3b,24R)-1,3,24,25-Tetrahydroxycycloartan-28-oic Acid 3-(b-d-Xylopyrano-
side) ¼ rel-(1a,3b,24R)-1,24,25-Trihydroxy-3-(b-d-xylopyranosyloxy)-9,19-cyclolanostan-28-oic Acid;
2). White powder. M.p. 245 – 2468. [a]2D5 ¼ þ42.8 (c ¼ 0.69, MeOH). UV (MeOH): 202. IR (KBr):
3403, 2943, 2861, 1714. 1H- and 13C-NMR: Tables 1 and 2. HR-ESI-MS: 637.3954 ([M ꢀ H]ꢀ, C35H57Oꢀ10
;
calc. 637.3957).
Nerviside F (¼(1a,3b,24R)-1,3,24,25-Tetrahydroxycycloartan-28-oic Acid 3-(b-d-Glucopyrano-
side) ¼ rel-(1a,3b,24R)-3-(b-d-Glucopyranosyloxy)-1,24,25-Trihydroxy-9,19-cyclolanostan-28-oic Acid;
3). White powder. M.p. 261 – 2628. [a]2D5 ¼ þ51.9 (c ¼ 0.87, MeOH). UV (MeOH): 203. IR (KBr):
3417, 2943, 2870, 1703. 1H- and 13C-NMR: Tables 1 and 2. HR-ESI-MS: 667.4063 ([M ꢀ H]ꢀ, C36H59Oꢀ11
;
calc. 667.4063).
Nerviside G (¼(1a,3b)-1,3,25-Trihydroxy-24-oxocycloartan-28-oic Acid 3-(b-d-Xylopyranoside) ¼
rel-(1a,3b)-1,25-Dihydroxy-24-oxo-3-(b-d-xylopyranosyloxy)-9,19-cyclolanostan-28-oic Acid; 4). White
powder. M.p. 258 – 2598. [a]2D5 ¼ þ27.8 (c ¼ 0.73, MeOH). UV (MeOH): 203. IR (KBr): 3421, 2942, 2873,
1
1708, 1696. H- and 13C-NMR: Tables 1 and 2. HR-ESI-MS: 659.3762 ([M þ Na]þ, C35H56NaO1þ0 ; calc.
659.3766).
Nerviside H (¼(1a,3b)-1,3,25-Trihydroxy-24-oxocycloartan-28-oic Acid 3-(b-d-Glucopyranoside) ¼
rel-(1a,3b)-3-(b-d-glucopyranosyloxy)-1,25-dihydroxy-24-oxo-9,19-cyclolanostan-28-oic Acid; 5). White
powder. M.p. 250 – 2518. [a]2D5 ¼ þ37.6 (c ¼ 0.73, MeOH). UV (MeOH): 204. IR (KBr): 3418, 2939, 2875,
1
1709, 1682. H- and 13C-NMR: Tables 1 and 2. HR-ESI-MS: 689.3866 ([M þ Na]þ, C36H58NaO1þ1 ; calc.
689.3871).

Helvetica Chimica Acta – Vol. 96 (2013)
157
REFERENCES
[1] H.-H. Xu, J.-M. Liu, ꢀDictionary of Zhong Cao Yao Tu Puꢁ, Guangdong Scientific and Technological
Press, Guangzhou, 2007, p. 264.
[2] G.-X. Zhou, C.-L. Lu, H.-S. Wang, X.-S. Yao, J. Asian Nat. Prod. Res. 2009, 11, 498.
[3] Z.-H. Wang, Q. Du, F.-X. Zhang, Shizhen Guo Yi GuoYao 2007, 18, 2940.
[4] L.-W. Tian, Y. Pei, Y.-J. Zhang, Y.-F. Wang, C.-R. Yang, J. Nat. Prod. 2009, 72, 1057.
[5] J. Y. Zhao, Int. J. Appl. Chem. 2006, 2, 201.
[6] H.-S. Zhen, Y.-Y. Zhou, Y.-F. Yuan, H.-H. Mo, Z.-G. Zhong, C.-Y. Liang, Zhong Yao Cai 2007, 30,
942.
[7] J.-H. Ju, D. Liu, G. Lin, Y.-M. Zhang, J.-S. Yang, Y. Lu, N.-B. Gong, Q.-T. Zhang, J. Nat. Prod. 2002,
65, 147.
[8] A. Yokosuka, K. Sato, Y. Mimaki, Phytochemistry 2010, 71, 2174.
[9] Z. Zhao, K. Matsunami, H. Otsuka, T. Shinzato, Y. Takeda, Chem. Pharm. Bull. 2008, 56, 1153.
[10] R. Kasai, A. Sasaki, T. Hashimoto, T. Kaneko, K. Ohtani, K. Yamasaki, Phytochemistry 1999, 51,
803.
[11] T. Tanaka, T. Nakashima, T. Ueda, K. Tomii, I. Kouno, Chem. Pharm. Bull. 2007, 55, 899.
[12] K. D. Kucherbaev, K. K. Uteniyazov, V. V. Kachala, Z. Saatov, A. S. Shashkov, Chem. Nat. Compd.
2002, 38, 447.
˙
[13] M. ꢄzipek, A. A. Dçnmez, Ihsan ꢅalıs¸, R. Brun, P. Rꢃedi, D. Tasdemir, Phytochemistry 2005, 66,
1168.
[14] T. Mosmann, J. Immunol. Methods 1983, 65, 55.
Received March 21, 2012