Furostane-type steroidal saponins from the roots of Chlorophytum borivilianum

Article abstract of DOI:10.1002/hlca.200890246

Four new furostanol steroid saponins, borivilianosides A-D (1-4, resp.), corresponding to (3β,5α,22R,25R)-26-(β-D-glucopyranosyloxy)-22- hydroxyfurostan-3-yl O-β-D-xylopyranosyl-(1 → 3)-O-β-D- glucopyranosyl-(1 → 4)-O-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-galactopyranoside (1), (3β,5α,22R,25R)-26-(β-D- glucopyranosyloxy)-22-methoxyfurostan-3-yl O-β-D-xylopyranosyl-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-O-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-galactopyranoside (2), (3β,5α,22R,25R)-26-(β-D- glucopyranosyloxy)-22-methoxyfurostan-3-yl O-β-D-xylopyranosyl-(1 → 3)-O-[β-D-glucopyranosyl-(1 → 2)]-O-β-D-glucopyranosyl-(1 → 4)-β-D-galactopyranoside (3), and (3β,5α,25R)-26-(β-D- glucopyranosyloxy)furost-20(22)-en-3-yl O-β-D-xylopyranosyl-(1 → 3)-O-[β-D-glucopyranosyl-(1 → 2)]-O-β-D-glucopyranosyl-(1 → 4)-β-D-galactopyranoside (4), together with the known tribuluside A and (3β,5α,22R,25R)-26-(β-D-glucopyranosyloxy)-22-methoxyfurostan-3- yl O-β-D-xylopyranosyl-(1 → 2)-O-[β-D-xylopyranosyl-(1 → 3)]-O-β-D-glucopyranosyl-(1 → 4)-O-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-galactopyranoside were isolated from the dried roots of Chlorophytum borivilianum SANT and FERN. Their structures were elucidated by 2D-NMR analyses (COSY, TOCSY, NOESY, HSQC, and HMBC) and mass spectrometry.

Full text of DOI:10.1002/hlca.200890246

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Helvetica Chimica Acta – Vol. 91 (2008)
Furostane-Type Steroidal Saponins from the Roots of Chlorophytum
borivilianum
by Debabrata Acharyaa), Anne-Claire Mitaine-Offera), Nutan Kaushikb), Tomofumi Miyamotoc),
Thomas Paululatd), and Marie-Aleth Lacaille-Dubois*a)
a
´
´
´ ˆ
) Laboratoire de Pharmacognosie, Unite de Molecules dꢀInteret Biologique, UMIB UPRES-EA 3660,
´
´
Faculte de Pharmacie, Universite de Bourgogne, F-21079 Dijon Cedex
(phone: þ 33-380-393229; fax: þ 33-380-393300; e-mail: m-a.lacaille-dubois@u-bourgogne.fr)
b) TERI, Habitat Place, Lodhi Road, New Delhi, 110003, India
c) Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
d) Universitꢁt Siegen, FB8, OC-II (AK Ihmels), Adolf-Reichwein-Strasse 2, D-57068 Siegen
Four new furostanol steroid saponins, borivilianosides A – D (1 – 4, resp.), corresponding to
(3b,5a,22R,25R)-26-(b-d-glucopyranosyloxy)-22-hydroxyfurostan-3-yl O-b-d-xylopyranosyl-(1 ! 3)-O-
b-d-glucopyranosyl-(1! 4)-O-[a-l-rhamnopyranosyl-(1 !2)]-b-d-galactopyranoside (1), (3b,5a,22R,25R)-
26-(b-d-glucopyranosyloxy)-22-methoxyfurostan-3-yl O-b-d-xylopyranosyl-(1 ! 3)-O-b-d-glucopyrano-
syl-(1 ! 4)-O-[a-l-rhamnopyranosyl-(1 ! 2)]-b-d-galactopyranoside (2), (3b,5a,22R,25R)-26-(b-d-glu-
copyranosyloxy)-22-methoxyfurostan-3-yl O-b-d-xylopyranosyl-(1 ! 3)-O-[b-d-glucopyranosyl-(1 !
2)]-O-b-d-glucopyranosyl-(1 ! 4)-b-d-galactopyranoside (3), and (3b,5a,25R)-26-(b-d-glucopyranosyl-
oxy)furost-20(22)-en-3-yl O-b-d-xylopyranosyl-(1 ! 3)-O-[b-d-glucopyranosyl-(1 ! 2)]-O-b-d-gluco-
pyranosyl-(1 ! 4)-b-d-galactopyranoside (4), together with the known tribuluside
A
and
(3b,5a,22R,25R)-26-(b-d-glucopyranosyloxy)-22-methoxyfurostan-3-yl O-b-d-xylopyranosyl-(1 ! 2)-
O-[b-d-xylopyranosyl-(1! 3)]-O-b-d-glucopyranosyl-(1! 4)-O-[a-l-rhamnopyranosyl-(1! 2)]-b-d-gal-
actopyranoside were isolated from the dried roots of Chlorophytum borivilianum Sant and Fern. Their
structures were elucidated by 2D-NMR analyses (COSY, TOCSY, NOESY, HSQC, and HMBC) and
mass spectrometry.
Introduction. – The genus Chlorophytum (Liliaceae) comprises ca. 215 species
which grow as perennial rhizomatous herbs in pantropical regions [1]. They are mainly
cultivated for their ornamental flowers. Traditionally, roots of these species are reputed
to possess various pharmacological utilities having saponins as one of the important
phytochemical constituents [2]. Steroid saponins have attracted the attention for their
structural diversity and significant bioactivities such as cytotoxic, immunomodulating,
antifungal, insecticidal, etc., activities [3 – 5]. Previous phytochemical investigations of
various Chlorophytum species have resulted in the isolation of steroidal saponins
[1][6 – 8]. Chlorophytum borivilianum Sant. and Fern. commonly known as ꢂsafed
musliꢀ in India is traditionally used as a general tonic in treating rheumatism and
increasing general body immunity [9]. It is also reported to have spermatogenic and
antidiabetic properties. A previous report reveals antidiabetic activity which was
related to a polysaccharide fraction [9], but no systematic study on the secondary
metabolites of this plant has been reported so far. In the present article, we describe the
isolation and structure elucidation of four new steroid saponins from the roots of C.
ꢃ 2008 Verlag Helvetica Chimica Acta AG, Zꢄrich

Helvetica Chimica Acta – Vol. 91 (2008)
2263
borivilianum, borivilianosides A – D (1 – 4, resp.), together with two known com-
pounds, tribuluside A (¼(3b,5a,22R,25R)-26-(b-d-glucopyranosyloxy)-22-hydroxy-
furostan-3-yl O-b-d-xylopyranosyl-(1 ! 2)-O-[b-d-xylopyranosyl-(1 ! 3)]-O-b-d-glu-
copyranosyl-(1 ! 4)-O-[a-l-rhamnopyranosyl-(1 ! 2)]-b-d-glactopyranoside)
[10],
and (3b,5a,22R,25R)-26-(b-d-glucopyranosyloxy)-22-methoxyfurostan-3-yl O-b-d-xy-
lopyranosyl-(1 ! 2)-O-[b-d-xylopyranosyl-(1 ! 3)]-O-b-d-glucopyranosyl-(1 ! 4)-O-
[a-l-rhamnopyranosyl-(1 ! 2)]-b-d-galactopyranoside [11].
Results and Discussion. – The BuOH fraction of the EtOH extract of the roots of C.
borivilianum was submitted to multiple chromatographic steps involving vacuum-
liquid chromatography (VLC; reversed-phase RP-18 silica gel) and medium-pressure
liquid chromatography (MPLC; silica gel and reversed-phase RP-18 silica gel) yielding
borivilianosides A – D (1 – 4), tribuluside A [10], and (3b,5a,22R,25R)-26-(b-d-
glucopyranosyloxy)-22-methoxyfurostan-3-yl O-b-d-xylopyranosyl-(1 ! 2)-O-[b-d-xy-
lopyranosyl-(1! 3)]-O-b-d-glucopyranosyl-(1! 4)-O-[a-l-rhamnopyranosyl-(1! 2)]-
b-d-galactopyranoside [11]. Their structures were determined mainly by spectroscopic
methods including 1D- and 2D-NMR experiments (COSY, TOCSY, HSQC, NOESY,
and HMBC) in combination with HR-ESI- and FAB-MS.
Compounds 1 – 4 were isolated as amorphous powders. The sugars obtained by
aqueous acid hydrolysis of each compound were identified by comparison on TLC with
authentic samples as xylose, glucose, galactose, and rhamnose (¼6-deoxymannose) in
the cases of 1 and 2, and as glucose, galactose, and xylose in the cases of 3 and 4. The
absolute configurations of the sugars were determined to be d for glucose, galactose,
and xylose and l for rhamnose by GC analysis of their (trimethylsilyl)imidazole
derivatives (see Exper. Part).

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Helvetica Chimica Acta – Vol. 91 (2008)
Borivilianoside A (1) exhibited in the HR-ESI-MS (pos.) the [M þ Na]þ peak at m/z
1221.5825 consistent with the molecular formula C56H94O27. Negative-ion FAB-MS of 1
displayed a pseudomolecular-ion peak at m/z 1197 ([M ꢀ H]ꢀ) in accordance with the
[M þ Na]þ peak. Other fragment-ion peaks were observed at m/z 1065 ([M ꢀ H ꢀ
132]ꢀ), 903 ([M ꢀ H ꢀ 132 ꢀ 162]ꢀ), and 757 ([M ꢀ H ꢀ 132 ꢀ 162 ꢀ 146]ꢀ) suggesting
the successive elimination of two pentosyl, one hexosyl, and one deoxyhexosyl moiety,
respectively. The aglycone of 1 was identified as (3b,5a,22R,25R)-furostane-3,22,26-
triol by comparison of its NMR spectral data based on correlations observed in the
COSY, NOESY, HSQC, and HMBC spectra with those reported in [12 – 14]. On the
1
basis of H- and 13C-NMR (Tables 1 and 2) and 2D-NMR analyses, the structure of 1
was elucidated as (3b,5a,22R,25R)-26-(b-d-glucopyranosyloxy)-22-hydroxyfurostan-3-
yl O-b-d-xylopyranosyl-(1 ! 3)-O-b-d-glucopyranosyl-(1 ! 4)-O-[a-l-rhamnopyrano-
syl-(1 ! 2)]-b-d-galactopyranoside.
The 1H- and 13C-NMR spectra of 1 exhibited four characteristic Me signals at d(H) 0.77 (s), 0.75 (s),
0.90 (d, J ¼ 6.0 Hz), and 1.23 (d, J ¼ 6.5 Hz), and a quaternary-C-atom resonance at d(C) 110.2 (C(22))
indicating the presence of a steroidal skeleton. In addition, the A/B trans fusion was deduced by the
signals at d(C) 44.2 (C(5)), 54.0 (C(9)), and 12.1 (C(19)) suggesting that 1 was a (5a)-furostanol
derivative [12]. This was confirmed by the NOESY correlation d(H) 3.82 (HꢀC(3))/0.81 – 0.86 (m,
HꢀC(5)). The (22a) configuration of 1 was deduced by considering the downfield shift of signals at d(H)
1.23 (d, J ¼ 6.5 Hz, Me(21)) and 4.86 (HꢀC(16)), which is due to the deshielding effect of the cis-oriented
OHꢀC(22) [15]. The (25R) configuration was deduced by considering the chemical shifts at d(C) 33.6
(C(25)), 74.9 (C(26)), and 16.8 (C(27)) [16][17]. The above was supported by the difference between the
chemical shifts of HaꢀC(26) and HbꢀC(26) (da ꢀ db ¼ 0.27) since the difference is usually > 0.57 for
(25S) configurations and < 0.48 for (25R) configurations [16][17]. The 1H-NMR spectrum of 1
displayed signals for five anomeric H-atoms at d(H) 4.70 (d, J ¼ 7.5 Hz), 4.72 (d, J ¼ 7.5 Hz), 5.04 (d, J ¼
7.8 Hz), 4.83 (d, J ¼ 7.5 Hz), and 5.96 (br. s), which showed HSQC cross-peaks with five anomeric C-
atom signals at d(C) 104.2, 104.1, 104.2, 104.5, and 101.2, resp. The evaluation of chemical shifts and
spin – spin couplings obtained from the 2D-NMR data allowed the identification of two b-glucopyranosyl
units (Glc I and Glc II), one b-galactopyranosyl unit (Gal), one b-xylopyranosyl unit (Xyl), and one a-
rhamnopyranosyl unit (Rha) [18]. The relatively large 3J(1,2) values (7.3 – 8.0 Hz) for the Glc, Gal, and
Xyl indicated b-anomeric orientation for these sugars. The multiplicity of the anomeric 1H-NMR signal
of Rha (br. s) indicated a-anomeric orientation. The linkage of Glc I at the C(26) position was indicated
by long-range coupling (3J) in the HMBC spectrum between the anomeric H-atom at d(H) 4.70 (Glc I
HꢀC(1)) and C(26) at d(C) 74.9 (Agly). The HMBC cross-peak d(H) 4.72 (Gal HꢀC(1))/d(C) 77.6
(Agly C(3)) established the linkage of Gal to C(3) of the aglycone. Furthermore, the HMBC cross-peaks
d(H) 4.30 (Gal HꢀC(2))/d(C) 101.2 (Rha C(1)), d(H) 4.83 (Glc II HꢀC(1))/d(C) 81.0 (Gal C(4)), and
d(H) 5.04 (Xyl HꢀC(1)/d(C) 87.1 (Glc II C(3)) established the sugar sequence in 1.
Borivilianoside B (2) exhibited in the HR-ESI-MS (pos.) the [M þ Na]þ peak at m/z
1235.6043, consistent with the molecular formula C57H96O27. Negative-ion FAB-MS of
2 revealed a pseudomolecular-ion peak at m/z 1211 ([M ꢀ H]ꢀ) in accordance with the
[M þ Na]þ peak. Other fragment-ion peaks were observed at m/z 1079 ([M ꢀ H ꢀ
132]ꢀ), 917 ([M ꢀ H ꢀ 132 ꢀ 162]ꢀ), and 771 ([M ꢀ H ꢀ 132 ꢀ 162 ꢀ 146]ꢀ) corre-
sponding to the loss of one pentosyl, one hexosyl, and one desoxyhexosyl moiety,
respectively. The 1H- and 13C-NMR and 2D-NMR data of 2 (Tables 1 and 2) led to the
establishment of its structure as (3b,5a,22R,25R)-26-(b-d-glucopyranosyloxy)-22-
methoxyfurostan-3-yl O-b-d-xylopyranosyl-(1 ! 3)-O-b-d-glucopyranosyl-(1 ! 4)-O-
[a-l-rhamnopyranosyl-(1 ! 2)]-b-d-galactopyranoside. Since 22-hydroxyfurostane de-

Helvetica Chimica Acta – Vol. 91 (2008)
2265
Table 1. 1H- and 13C-NMR Data ((D5)Pyridine) of the Aglycone Part of 1 – 4. d in ppm, J in Hz; n.d. ¼ not
determined.
1
2
3
4
d(C) d(H)
36.8 0.73a),
d(C) d(H)
36.8 0.73a),
d(C) d(H)
d(C) d(H)
CH2(1)
CH2(2)
36.5 0.68a), 1.40a)
36.5 0.71a), 1.43a)
1.45 – 1.50 (m)
1.45 – 1.50 (m)
29.6 1.17a), n.d.
29.2 1.17a), n.d.
29.0 1.58a),
1.98 – 2.02 (m)
77.1 3.83a)
29.0 1.59a),
1.98 – 2.02 (m)
77.1 3.84a)
HꢀC(3)
77.6 3.82a)
77.6 3.82a)
CH2(4)
34.7 1.25a), n.d.
34.7 1.25a), n.d.
34.0 1.30 – 1.34 (m), 34.0 1.30 – 1.34 (m),
1.72 – 1.76 (m)
43.5 0.78 – 0.84 (m)
28.3 1.00a), 1.06a)
31.5 1.26a), 1.38a)
34.2 1.22 – 1.26 (m)
53.5 0.36 – 0.42 (m)
35.2
1.72 – 1.76 (m)
43.5 0.78 – 0.84 (m)
28.3 1.01a), 1.08a)
31.5 1.24a), 1.36a)
34.2 1.20 – 1.26 (m)
53.5 0.36 – 0.42 (m)
35.2
HꢀC(5)
CH2(6)
CH2(7)
HꢀC(8)
HꢀC(9)
C(10)
CH2(11)
CH2(12)
C(13)
44.2 0.81 – 0.86 (m)
28.6 1.11a), 1.13a)
31.9 1.23a), 1.40a)
34.9 1.26 – 1.33 (m)
54.0 0.39 – 0.43 (m)
35.3
44.2 0.82 – 0.85 (m)
28.6 1.11a), 1.13a)
31.9 1.23a), 1.40a)
34.9 1.26 – 1.33 (m)
54.0 0.39 – 0.43 (m)
35.3
21.2 1.31a), n.d.
39.5 0.97a), 1.63a)
40.8
21.2 1.31a), n.d.
39.5 0.97a), 1.63a)
40.2
20.4 1.06a), 1.28a)
39.2 0.92a), 1.56a)
40.6
20.4 1.08a), 1.27a)
39.2 0.93a), 1.58a)
40.6
HꢀC(14)
55.9 0.90a)
55.9 0.90a)
55.5 0.88a)
55.5 0.89a)
CH2(15)
31.8 1.24a), 1.92a)
31.8 1.24a), 1.92a)
31.5 1.26a),
31.5 1.27a),
1.88 – 1.92 (m)
1.88 – 1.92 (m)
HꢀC(16)
HꢀC(17)
Me(18)
81.0 4.86a)
62.9 1.85 – 1.89 (m)
16.3 0.77 (s)
12.1 0.75 (s)
40.2 2.12 – 2.18 (m)
81.0 4.40a)
80.7 4.37a)
83.1 4.70 – 4.74 (m)
63.5 2.34 – 2.37 (m)
15.5 0.70 (s)
64.0 1.66a)
63.2 1.64 – 1.68 (m)
15.5 0.70 (s)
12.9 0.69 (s)
16.2 0.76 (s)
12.1 0.75 (s)
40.2 2.11 – 2.17 (m)
Me(19)
12.9 0.59 (s)
HꢀC(20)
or C(20)
Me(21)
39.8 2.14 – 2.18 (m) 104.0
15.9 1.23, (d, J ¼ 6.5) 15.9 1.10 (d, J ¼ 6.5) 15.5 1.09 (d, J ¼ 6.5) 11.9 1.55 (s)
C(22)
110.2
112.1
112.0
152.0
CH2(23)
CH2(24)
HꢀC(25)
CH2(26)
Me(27)
36.4 1.99a), n.d.
27.8 1.57a), 1.95a)
33.6 1.80 – 1.85 (m)
74.9 3.55a), 3.82a)
30.0 1.98a), 1.66a)
27.6 1.54a), 1.92a)
33.6 1.80 – 1.85 (m)
74.9 3.55a), 3.82a)
30.8 1.95a), n.d.
27.5 n.d., n.d.
33.8 1.78 – 1.84 (m)
74.2 3.54a), 3.89a)
30.8 1.94a), n.d.
27.2 nd, n.d.
33.8 1.78 – 1.84 (m)
74.2 3.53a), 4.08a)
16.8 0.90 (d, J ¼ 6.0) 16.8 0.90 (d, J ¼ 6.0) 15.9 0.92 (d, J ¼ 6.4) 16.0 0.94 (d, J ¼ 6.4)
MeOꢀC(22)
47.4 3.24 (s) 46.5 3.26 (s)
a) Overlapped with other signals.
rivatives are readily converted into 22-methoxyfurostane derivatives in solutions
containing MeOH [21], the presence of 2 as a natural compound was confirmed by
HPTLC after an extraction of 10 g of the roots of C. borivilianum in refluxing H2O [22].
The comparison of the 1H- and 13C-NMR chemical shifts of 2, assigned from 2D-NMR analysis, with
those of 1 showed that 2 differed from 1 only by the presence of a MeO group instead of an OH group at
C(22) of the aglycone (d(C) 47.4 and d(H) 3.24 (MeOꢀC(22)) and d(C) 112.1 (C(22)) in 2 instead of
d(C) 110.2 (C(22)) in 1). The a-orientation of MeOꢀC(22) was deduced by the observed NOESY
correlation d(H) 3.24 (s, MeO)/d(H) 4.40 (HꢀC(16)) [19][20].

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Helvetica Chimica Acta – Vol. 91 (2008)
Table 2. 1H- and 13C-NMR Data ((D5)pyridine) of the Sugar Moieties of 1 – 4. d in ppm, J in Hz.
1
2
3
4
d(C) d(H)
d(C) d(H)
d(C) d(H)
d(C) d(H)
Glc I:
HꢀC(1) 104.2 4.70 (d, J ¼ 7.5) 104.2 4.70 (d, J ¼ 7.6) 103.6 4.72 (d, J ¼ 7.5) 103.5 4.71 (d, J ¼ 7.5)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
Gal:
74.5 3.90a)
74.5 3.90a)
74.0 3.90a)
74.1 3.92a)
77.6 4.14a)
77.5 4.16a)
77.0 4.16a)
76.9 4.15a)
71.0 4.03a)
71.0 4.02a)
69.8 4.02a)
69.9 4.01a)
77.6 3.83a)
77.6 3.84a)
77.0 3.84a)
76.9 3.83a)
62.1 4.17a), 4.36a)
62.0 4.18a), 4.40a)
61.7 4.18a), 4.37a)
61.8 4.18a), 4.36a)
HꢀC(1) 104.1 4.72 (d, J ¼ 7.5) 104.2 4.72 (d, J ¼ 7.5) 101.4 4.82 (d, J ¼ 7.4) 101.3 4.83 (d, J ¼ 7.4)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
77.0 4.30a)
75.5 4.04a)
81.0 4.40a)
76.7 3.82a)
60.1 4.12a),
76.5 4.22a)
75.5 4.04a)
80.5 4.38a)
76.8 3.83a)
60.1 4.12a),
71.8 4.30a)
71.7 4.31a)
76.5 4.08a)
76.6 4.09a)
78.8 4.49a)
76.1 3.70 – 3.74 (m)
60.0 4.14a), 4.48a)
78.9 4.50a)
76.0 3.70 – 3.74 (m)
60.1 4.14a), 4.46a)
4.52 – 4.56 (m)
4.52 – 4.56 (m)
Glc II:
HꢀC(1) 104.5 4.83 (d, J ¼ 7.5) 104.5 4.81 (d, J ¼ 7.5) 103.6 5.02 (d, J ¼ 7.5) 103.5 5.03 (d, J ¼ 7.5)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
Rha:
75.5 4.02a)
75.5 4.03a)
79.7 4.19a)
79.8 4.20a)
87.1 3.95a)
87.1 3.93a)
86.0 3.98a)
86.1 3.99a)
70.0 4.02a)
70.0 4.02a)
71.0 4.30a)
71.1 4.31a)
76.7 3.70a)
76.7 3.70a)
76.9 3.96a)
77.0 3.97a)
61.9 3.91a), 4.40a)
61.9 3.91a), 4.40a)
61.5 4.26a), 4.37a)
61.6 4.26a), 4.36a)
HꢀC(1) 101.2 5.96 (br. s)
101.2 5.96 (br. s)
72.0 4.63 – 4.67 (m)
71.8 4.26 – 4.30 (m)
73.0 4.22a)
69.2 4.72 – 4.78 (m)
17.9 1.64 (d, J ¼ 6.2)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
Me(6)
72.0 4.63 – 4.67 (m)
71.8 4.26 – 4.30 (m)
73.0 4.22a)
69.3 4.72 – 4.78 (m)
17.9 1.62 (d, J ¼ 6.0)
Xyl:
HꢀC(1) 104.2 5.04 (d, J ¼ 7.8) 104.2 5.07 (d, J ¼ 8.0) 103.6 5.05 (d, J ¼ 7.7) 103.5 5.04 (d, J ¼ 7.7)
HꢀC(2)
HꢀC(3)
HꢀC(4)
CH2(5)
74.4 3.83a)
74.4 3.83a)
73.9 3.82a)
76.9 4.00a)
71.8 4.26a)
66.0 3.58 (t, 10.3),
4.11a)
73.8 3.81a)
77.0 4.02a)
71.9 4.25a)
66.1 3.56 (t, 10.3),
4.11a)
77.5 4.02a)
77.5 4.02a)
71.0 4.03a)
66.5 3.54a), 4.14a)
71.0 4.03a)
66.5 3.54a), 4.14a)
Glc III:
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
103.2 5.44 (d, J ¼ 7.7) 103.1 5.45 (d, J ¼ 7.7)
74.7 3.92a)
74.6 3.93a)
76.5 4.08a)
76.6 4.09a)
70.5 4.00a)
70.6 4.02a)
77.0 3.83a)
77.1 3.81a)
61.4 4.29a), 4.50a)
61.5 4.28a), 4.52a)
a) Overlapped with other signals.

Helvetica Chimica Acta – Vol. 91 (2008)
2267
The molecular formula of Borivilianoside C (3) was determined as C57H96O28
according to the [M þ Na]þ peak at m/z 1251.5991 in the HR-ESI-MS. Negative-ion
FAB-MS showed the pseudomolecular-ion peak at m/z 1227 ([M ꢀ H]ꢀ) in accordance
with the [M þ Na]þ peak. Other fragment-ion peaks were observed at m/z 1065 ([M ꢀ
H ꢀ 162])ꢀ, 933 ([M ꢀ H ꢀ 162 ꢀ 132])ꢀ, and 771 ([M ꢀ H ꢀ 162 ꢀ 132 ꢀ 162])ꢀ,
corresponding to the loss of two hexosyl and one pentosyl moiety. On the basis of
1
extensive H- and 13C-NMR and 2D-NMR analyses (Tables 1 and 2), the structure of
borivilianoside C (3) was determined as (3b,5a,22R,25R)-26-(b-d-glucopyranosyloxy)-
22-methoxyfurostan-3-yl O-b-d-xylopyranosyl-(1 ! 3)-O-[b-d-glucopyranosyl-(1 !
2)]-O-b-d-glucopyranosyl-(1 ! 4)-b-d-galactopyranoside.
1
Most of the H- and 13C-NMR signals corresponding to the aglycone part of 3, assigned from 2D-
NMR experiments, were superimposable with those described above for compounds 1 and 2. The
1H-NMR spectrum of 3 displayed signals for five anomeric H-atoms at d(H) 4.72 (d, J ¼ 7.5 Hz), 4.82 (d,
J ¼ 7.4 Hz), 5.02 (d, J ¼ 7.5 Hz), 5.05 (d, J ¼ 7.7 Hz), and 5.44 (d, J ¼ 7.7 Hz) giving cross-peaks in the
HSQC spectrum with five anomeric C-atoms at d(C) 103.6, 101.4, 103.6, 103.6, and 103.2, resp. The
complete assignment of the glycosidic NMR signals was achieved by analyses of COSY, TOCSY,
NOESY, HSQC, and HMBC data. Evaluation of spin – spin couplings and chemical shifts allowed the
identification of three b-glucopyranosyl (Glc I, Glc II, and Glc III), one b-galactopyranosyl (Gal), and
one b-xylopyranosyl (Xyl) unit. The linkage of Glc I at C(26) of the aglycone was deduced by the HMBC
cross-peak d(H) 4.72 (Glc I HꢀC(1))/d(C) 74.2 (Agly C(26)). The HMBC cross-peaks d(H) 5.05 (Xyl
HꢀC(1))/d(C) 86.0 (Glc II C(3)), d(H) 4.19 (Glc II HꢀC(2))/d(C) 103.2 (Glc III C(1)), and d(H) 5.02
(Glc II HꢀC(1))/d(C) 78.8 (Gal C(4)), and the NOESY correlations d(H) 5.44 (Glc III HꢀC(1))/d(H)
4.19 (Glc II HꢀC(2)) and d(H) 4.82 (Gal HꢀC(1))/d(H) 3.83 (Agly HꢀC(3)) established the linkages
within the oligosaccharide chain at C(3).
The positive-ion HR-ESI-MS of borivilianoside D (4) exhibited the [M þ Na]þ
peak at m/z 1219.5730 consistent with the molecular formula C56H92O27. Negative-ion
FAB-MS showed a pseudomolecular-ion peak at m/z 1195 ([M ꢀ H]ꢀ) in accordance
with the [M þ Na]þ peak. Other fragment-ion peaks were observed at m/z 1033 ([M ꢀ
H ꢀ 162]ꢀ), 901 ([M ꢀ H ꢀ 162 ꢀ 132]ꢀ), 739 ([M ꢀ H ꢀ 162 ꢀ 132 ꢀ 162]ꢀ), corre-
sponding to the loss of two hexosyl and one pentosyl moiety. Extensive study of the 1H-
and 13C-NMR (Tables 1 and 2) and 2D-NMR spectra of 4 led to the establishment of its
structure as (3b,5a,25R)-26-(b-d-glucopyranosyloxy)furost-20(22)-en-3-yl O-b-d-
xylopyranosyl-(1 ! 3)-O-[b-d-glucopyranosyl-(1 ! 2)]-O-b-d-glucopyranosyl-(1 ! 4)-
b-d-galactopyranoside.
The structure elucidation of 4 was supported by comparison of its MS and NMR data with those of 3.
The molecular formula C56H92O27 of 4 indicated a loss of 32 amu as compared to 3. The 1H- and
13C-NMR signals of the aglycone of 4, assigned by extensive 2D-NMR analyses, were almost
superimposable with those of 3, except for the signals at d(C) 39.8 (C(20)) and d(C) 112.0 (C(22)) which
were replaced by two quaternary-C-atom signals at d(C) 104.0 and 152.0, resp. This feature is a
characteristic of a tetra-substituted C(20)¼C(22) bond and was supported by signals at d(H) 1.55 (s,
Me(21)) and 2.34 – 2.37 (m, HꢀC(17)) which were downfield shifted in comparison with the
corresponding signals of 3 at d(H) 1.09 (d, J ¼ 6.5 Hz, Me(21)) and d(H) 1.64 – 1.68 (m, HꢀC(17)).
The presence of five monosaccharide units was suggested by the five anomeric H-atom resonances at
d(H) 4.71 (d, J ¼ 7.5 Hz), 4.83 (d, J ¼ 7.4 Hz), 5.03 (d, J ¼ 7.5 Hz), 5.04 (d, J ¼ 7.7 Hz), and 5.45 ( d, J ¼
7.7 Hz), which exhibited HSQC cross-peaks with d(C) 103.5, 101.3, 103.5, 103.5, and 103.1, resp. The 1H-
and 13C-NMR data of the oligosaccharide moiety of 4, assigned from the 2D-NMR analyses, were about
the same as those of 3, suggesting that 4 possessed the same sugar chain at C(3), i.e., an O-b-d-

2268
Helvetica Chimica Acta – Vol. 91 (2008)
xylopyranosyl-(1 ! 3)-O-[b-d-glucopyranosyl-(1 ! 2)]-O-b-d-glucopyranosyl-(1 ! 4)-b-d-galactopyra-
nosyloxy moiety, and the same sugar unit at C(26), i.e., a b-d-glucopyranosyloxy group.
The authors are greatful to IFCPAR-CEFIPRA, New Delhi, India, for financial support.
Experimental Part
General. Medium-pressure liquid chromatography (MPLC): silica gel 60 (SiO2, 15 – 40 mm; Merck),
Gilson M 305 pump; Bꢀchi glass column (460 ꢁ 25 mm, 460 ꢁ 15 mm, and 230 ꢁ 15 mm), Bꢀchi
precolumn (110 ꢁ 15 mm). Vacuum-liquid chromatography (VLC): reversed-phase SiO2 RP-18 (25 –
40 mm; Merck). TLC and HPTLC: SiO2 60 F254 (Merck); solvent systems: for saponins, CHCl3/
MeOH/H2O 13 :7:2, lower phase (A); for sapogenins, CHCl3/MeOH 19 :1 (B); spray reagent: for the
saponins, Komarowsky reagent, a 5 :1 mixture of 2% 4-hydroxybenzaldehyde/MeOH and 50% H2SO4/
EtOH. GC: Thermoquest gas chromatograph with a DB-1701 capillary column (30 m ꢁ 0.25 mm, i.d.;
J & W Scientific), FID detection; initial temp. 808 for 5 min, then temp. increase to 2708 at the rate of 158/
min; carrier gas He. Optical rotation: AA-OR automatic polarimeter. 1D- and 2D-NMR Spectra: Varian-
Inova-600 spectrometer equipped with a Sun-4-L-X computer system; at 600 (1H) and 150 MHz (13C) in
(D5)pyridine; d in ppm, J in Hz; conventional pulse sequences for COSY, HSQC, and HMBC; TOCSY
with the standard MLEV17 spin-locking sequence and a 90-ms mixing time; mixing time in the NOESY
experiment 500 ms; C-atom type (Me, CH2, CH) by DEPT experiments. Fast-atom-bombardment
(FAB) MS (neg.): Jeol-SX-102 spectrometer; glycerol as matrix; in m/z. HR-ESI-MS (pos.): Micromass
Q-TOF-1 apparatus; in m/z.
Plant Material. The roots of C. borivilianum Sant. and Fern. were provided from Jeevan Herbs in
2006 (New Delhi, India) and identified by Dr. M. Ahmedullah, Botanic Garden of Indian Republic,
BGIR (Noida, India). A voucher specimen (No. 6625) was deposited with the herbarium of the
Laboratory of Pharmacognosy, Faculty of Pharmacy, Burgundy University, France.
Extraction and Isolation. Powdered roots (500 g) of C. borivilianum were refluxed in EtOH (3 ꢁ 2 l)
for 50 min. After evaporation, the resulting EtOH extract (19 g) was suspended in H2O (200 ml) and
partitioned successively with CH2Cl2 (3 ꢁ 300 ml) and BuOH (sat. with H2O; 3 ꢁ 200 ml), yielding after
evaporation the corresponding CH2Cl2 (3.2 g ) and BuOH (4.1g) fractions. An aliquot (4 g) of the BuOH
residue was submitted to VLC (RP-18; MeOH/H2O (0 :100, 50 :50, and 100 :0)): Fractions VLC (1),
VLC (2), and VLC (3) after evaporation. VLC (3) (1.1 g) was submitted to MPLC (system C: SiO2 (15 –
40 mm), CHCl3/MeOH/H2O 13 :7:2, lower phase): Fractions 1 – 17. Fr. 10 was subjected to MPLC
(reversed phase RP-18, MeOH/H2O 40 ! 100%): Fr. 10.1 – 10.6. Fr. 10.4 (92.3 mg) was submitted to
MPLC (system C): 1 (4.7 mg) and 2 (4.3 mg). Fr. 10.8 (41.6 mg) and Fr. 10.6 (38.4 mg) of VLC (3) were
purified by MPLC (system C): 3 (3.4 mg) and 4 (3.9 mg), resp.
Ten grams of powdered roots of C. borivilianum were refluxed in H2O (2 ꢁ 100 ml) for 1 h. The
solvent was evaporated yielding an aq. extract which was analyzed by HPTLC (SiO2 60 F254, system A)
showing the presence of compounds 2 and 3, which means they represent true natural products and not
artefacts of isolation.
Borivilianoside A (¼(3b,5a,22R,25R)-26-(b-d-Glucopyranosyloxy)-22-hydroxyfurostan-3-yl O-b-d-
xylopyranosyl-(1 ! 3)-O-b-d-glucopyranosyl-(1 ! 4)-O-[6-deoxy-a-l-mannopyranosyl-(1 ! 2)]-b-d-
1
galactopyranoside; (1): White amorphous powder. [a]2D0 ¼ ꢀ46.0 (c ¼ 0.10, MeOH). H- and 13C-NMR:
Tables 1 and 2. FAB-MS (neg.): 1197 ([M ꢀ H]ꢀ), 1065 ([M ꢀ H ꢀ 132]ꢀ), 903 ([M ꢀ H ꢀ 132 ꢀ 162]ꢀ),
757 ([M ꢀ H ꢀ 132 ꢀ 162 ꢀ 146]ꢀ). HR-ESI-MS (pos.): 1221.5825 ([M þ Na]þ, C56H94O27Naþ; calc.
1221.5880).
Borivilianoside B (¼(3b,5a,22R,25R)-26-(b-d-Glucopyranosyloxy)-22-methoxyfurostan-3-yl O-b-
d-xylopyranosyl-(1 ! 3)-O-b-d-glucopyranosyl-(1 ! 4)-O-[6-deoxy-a-l-mannopyranosyl-(1 ! 2)]-b-d-
1
galactopyranoside; (2): White amorphous powder. [a]2D0 ¼ ꢀ43.0 (c ¼ 0.10, MeOH). H- and 13C-NMR:
Tables 1 and 2. FAB-MS (neg.): 1211 ([M ꢀ H]ꢀ), 1079 ([M ꢀ H ꢀ 132]ꢀ), 917 ([M ꢀ H ꢀ 132 ꢀ 162]ꢀ),
771 ([M ꢀ H ꢀ 132 ꢀ 162 ꢀ 146]ꢀ). HR-ESI-MS (pos.): 1235.6043 ([M þ Na]þ, C57H96O27Naþ; calc.
1235.6037).

Helvetica Chimica Acta – Vol. 91 (2008)
2269
Borivilianoside C (¼(3b,5a,22R,25R)-26-(b-d-Glucopyranosyloxy)-22-methoxyfurostan-3-yl O-b-
d-xylopyranosyl-(1 ! 3)-O-[b-d-glucopyranosyl-(1 ! 2)]-O-b-d-glucopyranosyl-(1 ! 4)-b-d-galacto-
pyranoside (3): White amorphous powder. [a]2D0 ¼ ꢀ45.6 (c ¼ 0.08, MeOH). 1H- and 13C-NMR: Tables 1
and 2. FAB-MS (neg.): 1227 ([M ꢀ H]ꢀ), 1065 ([Mꢀ H ꢀ 162]ꢀ), 933 ([M ꢀ H ꢀ 162 ꢀ 132]ꢀ), 771 ([M ꢀ
H ꢀ 162 ꢀ 132 ꢀ 162]ꢀ). HR-ESI-MS (pos.): 1251.5991 ([M þ Na]þ, C57H96O28Naþ; calc. 1251.5986).
Borivilianoside D (¼(3b,5a,25R)-26-(b-d-Glucopyranosyloxy)furost-20(22)-en-3-yl O-b-d-xylopyr-
anosyl-(1 ! 3)-O-[b-d-glucopyranosyl-(1 ! 2)]-O-b-d-glucopyranosyl-(1 ! 4)-b-d-galactopyranoside
(4): White amorphous powder. [a]2D0 ¼ ꢀ32.4 (c ¼ 0.10, MeOH). 1H- and 13C-NMR: Tables 1 and 2. FAB-
MS (neg.): 1195 ([M ꢀ H]ꢀ), 1033 ([M ꢀ H ꢀ 162]ꢀ), 901 ([M ꢀ H ꢀ 162 ꢀ 132]ꢀ), 739 ([M ꢀ H ꢀ 162 ꢀ
132 ꢀ 162]ꢀ). HR-ESI-MS (pos.): 1219.5730 ([M þ Na]þ, C56H92O27Naþ; calc. 1219.5724).
Acid Hydrolysis. A soln. of each saponin 1 – 4 (2 mg) in H2O (2 ml) was treated with 2n aq.
CF3COOH (5 ml) and refluxed for 3 h. After extraction with CH2Cl2 (3 ꢁ 5 ml), the aq. layer was
repeatedly concentrated with MeOH until neutral, and then analyzed by TLC (SiO2, CHCl3/MeOH/H2O
8 :5 :1) with authentic samples: galactose (Rf 0.21), glucose (Rf 0.23), xylose (Rf 0.47), and rhamnose
(Rf 0.51) for 1 and 2, and galactose (Rf 0.21), glucose (Rf 0.23), and xylose (Rf 0.47) for 3 and 4.
Furthermore, a silylated derivative of the sugars was prepared according to the procedure described
previously [23]. l-Cysteine methyl ester hydrochloride (Sigma, U.S.A.; 0.06 mol/l) and HMDS/TMCS
(¼ hexamethyldisilazane/trimethylchlorosilane) 3 :1 (Fluka, U.S.A.; 150 ml) were added to the aq.
residue. After centrifugation of the precipitate, the supernatant was concentrated and partitioned
between hexane and H2O (0.1 ml each), and the hexane layer (1 ml) was analyzed by GC. d-Glucose, d-
galactose, d-xylose, and l-rhamnose were detected for 1 and 2, and d-glucose, d-galactose, and d-xylose
for 3 and 4.
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Received May 14, 2008