Cytotoxic spirostane-type saponins from the roots of Chlorophytum borivilianum

Article abstract of DOI:10.1021/np800559z

Four new spirostane-type saponins named borivilianosides E-H (1-4) were isolated from an ethanol extract of the roots of Chlorophytum borivilianum together with two known steroid saponins (5 and 6). The structures of 1-4 were elucidated using mainly 2D NM

Full text of DOI:10.1021/np800559z

J. Nat. Prod. 2009, 72, 177–181
177
Cytotoxic Spirostane-Type Saponins from the Roots of Chlorophytum boriWilianum
Debabrata Acharya,† Anne-Claire Mitaine-Offer,† Nutan Kaushik,‡ Tomofumi Miyamoto,§ Thomas Paululat,
Jean-Franc¸ois Mirjolet,3 Olivier Duchamp,3 and Marie-Aleth Lacaille-Dubois*,†
Laboratoire de Pharmacognosie, Unite´ de Mole´cules d’Inte´reˆt Biologique, UMIB UPRES-EA 3660, Faculte´ de Pharmacie, UniVersite´ de
Bourgogne, 21079 Dijon Cedex, France, The Energy and Resources Institute, Habitat Place, Lodhi Road, New Delhi 110003, India, Graduate
School of Pharmaceutical Sciences, Kyushu UniVersity, Fukuoka 812-8582, Japan, UniVersita¨t Siegen, FB8, OC-II (AK Ihmels),
Adolf-Reichwein-Strasse 2, D-57068 Siegen, Germany, and Oncodesign, 20 Rue Jean Mazen, BP 27627, 21076 Dijon Cedex, France
ReceiVed September 5, 2008
Four new spirostane-type saponins named borivilianosides E-H (1-4) were isolated from an ethanol extract of the
roots of Chlorophytum boriVilianum together with two known steroid saponins (5 and 6). The structures of 1-4 were
elucidated using mainly 2D NMR spectroscopic techniques and mass spectrometry. The cytotoxicity of borivilianosides
F (2), G (3), and H (4) and three known compounds was evaluated using two human colon cancer cell lines (HT-29 and
HCT 116).
Steroidal saponins have attracted much attention due to their
structural diversity and significant bioactivities such as cytotoxic,
immunomodulating, antifungal, and insecticidal effects.1,2 Several
Chlorophytum species have been studied chemically and biologi-
cally and were found to contain steroidal saponins.3 Among them,
chloromaloside A from C. malayense showed broad cytotoxicity
against various human cancer cells.4
The dried roots of Chlorophytum boriVilianum Sant. and Fern.
(Liliaceae) are used under the name of “Safed musli” as an Indian
herb for the treatment of rheumatism and increasing general body
immunity.5 This species is also reported to have antidiabetic and
spermatogenic properties.3 In a search for bioactive steroidal
saponins, a previous phytochemical investigation on C. boriVilianum
roots led to the isolation and structure elucidation of four new
furostane-type steroidal saponins called borivilianosides A-D.6 In
a continuing study on the roots of this species, we describe in this
paper the isolation and structure elucidation of four additional new
spirostane-type steroidal saponins, borivilianosides E-H (1-4),
together with two known saponins. The cytotoxic activity of several
of these compounds was evaluated against the HT-29 and HCT
116 colon cancer cell lines.
The n-BuOH-soluble portion of an ethanol-soluble extract of the
roots of C. boriVilianum was subjected to RP-18 vacuum-liquid
chromatography (VLC), followed by successive medium-pressure
liquid chromatography (MPLC) on normal-phase silica gel 60 and
reversed-phase silica gel RP-18, yielding four new steroidal
saponins, borivilianosides E-H (1-4), as well as two known
saponins isolated for the first time from this genus. The known
compounds were identified by extensive NMR and MS analyses
and comparison with literature data as gitogenin 3-O-ꢀ-D-glucopy-
ranosyl-(1f3)-[ꢀ-D-glucopyranosyl-(1f2)]-ꢀ-D-glucopyranosyl-
(1f4)-ꢀ-D-galactopyranoside,7 and 25(R,S)-5R-spirostan-3ꢀ-ol 3-O-
ꢀ-D-xylopyranosyl-(1f3)-[ꢀ-D-glucopyranosyl-(1f2)]-ꢀ-D-
glucopyranosyl-(1f4)-ꢀ-D-galactopyranoside.8
galactose, and xylose (in the case of 4). The absolute configurations
of the sugars were determined to be D for galactose, glucose, and
xylose and L for arabinose and rhamnose by GC analysis of chiral
derivatives of the sugars in the hydrolysate of each compound (see
Experimental Section). In the 1H NMR spectra of the compounds,
the relatively large 3JH-1,H-2 values of the Glc, Xyl, and Ara (between
7.0 and 8.0 Hz) moieties indicated a ꢀ anomeric proton for Gal,
Glc, and Xyl and an R anomeric proton for Ara. The broad singlet
of the anomeric proton of the Rha unit indicated an R-orientation.9
Borivilianoside E (1) exhibited in the HRESIMS (positive-ion
mode) a pseudomolecular ion peak at m/z 1643.7304 [M + Na]+
(calcd for 1643.7310), consistent with the molecular formula,
C73H120O39. Its FABMS (negative-ion mode) showed a quasimo-
lecular ion peak at m/z 1619 [M - H]-, indicating a molecular
weight of 1620. Further fragment ion peaks were observed at m/z
1457 [(M - H) - 162]-, 1311 [(M - H) - 162 - 146]-, 1149
[(M - H) - 162 - 146 - 162]-, 1017 [(M - H) - 162 - 146 -
162 - 132]-, 885 [(M - H) - 162 - 146 - 162 - 132 - 132]-,
and 723 [(M - H) - 162 - 146 - 162 - 132 - 132 - 162]-,
corresponding to the loss of three hexosyls, two pentosyls, and one
desoxyhexosyl moiety. The 1H and 13C NMR spectra in combination
with DEPT and HSQC spectra of 1 exhibited four characteristic
methyls at δH 0.84 (s), 0.87 (s), 1.15 (d, J ) 6.8 Hz), and 0.71 (d,
J ) 4.9 Hz) and a quaternary C atom resonance at δC 109.4 (C-
22), indicating the presence of a steroidal spirostanol skeleton.
Analysis of NMR data showed 13C NMR signals at δC 44.6 (C-5),
54.4 (C-9), and 12.4 (C-19), characteristic of A/B trans-ring fusion,
indicating that 1 is a 5R-steroidal spirostanol derivative. This was
confirmed by the ROESY correlations observed between δH 3.92
(Agly H-3) and 0.92 (Agly H-5). The aglycon of 1 was identified
as (25R)-3ꢀ,5R-spirostan-3-ol (tigogenin) by comparison of 13C and
1H NMR spectroscopic data of 1 obtained from the 2D-NMR spectra
with those reported in the literature.10 The 25R stereochemistry of
the Me-27 group was deduced from the resonances of protons and
carbons at C-25 (δC 30.6), C-26 (δC 66.8, δH 3.52, 3.59), and C-27
(δC 17.3, δH 0.71, d, J ) 4.9 Hz).11 The lower field resonance of
C-27 (δC 17.3) as compared to the 13C NMR chemical shift of (25S)-
spirostanes at δC 16-16.5 and the ROESY correlations between
H-3/H-5, H-11/H3-19, H-11/H3-18, H-9/H-14, H-16/H-17, and
H-17/H3-21 confirmed the relative configuration of 1 as having A/B
trans, B/C trans, C/D trans, D/E cis, and C-20R stereochemistry.11
Compounds 1-4 were isolated as white, amorphous powders.
The sugars obtained by aqueous acid hydrolysis of each compound
were identified by comparison on TLC with authentic samples as
glucose, galactose, xylose, arabinose, and rhamnose (in the case
of 1 and 2), glucose and galactose (in the case of 3), and glucose,
* Corresponding author. Tel: +33 3 80 39 32 29. Fax: +33 3 80 39 33
00. E-mail: m-a.lacaille-dubois@u-bourgogne.fr.
† Faculte´ de Pharmacie, Universite´ de Bourgogne.
‡ The Energy and Resources Institute.
The 1H NMR spectrum of 1 displayed signals for eight anomeric
proton signals at δH 4.82 (d, J ) 7.8 Hz), 4.94 (d, J ) 7.5 Hz),
5.38 (d, J ) 8.0 Hz), 5.26 (d, J ) 7.4 Hz), 5.42 (brs), 6.14 (brs),
5.06 (d, J ) 7.9 Hz), and 5.09 (d, J ) 7.0 Hz), which gave HSQC
correlations with eight anomeric carbon signals at δC 100.2, 105.4,
§ Graduate School of Pharmaceutical Sciences, Kyushu University.
Universita¨t Siegen.
3 Oncodesign.
10.1021/np800559z CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/07/2009

178 Journal of Natural Products, 2009, Vol. 72, No. 1
Notes
(Gal C-2) and the ROESY cross-peak between δH 4.94 (d, J ) 7.5
Hz, Glc-I H-1) and δH 4.48 (Gal H-2) showed the Glc-I unit to be
linked to Gal at C-2. The linkage of Xyl to the 2-position of Glc-I
was deduced by the HMBC correlation observed between δH 5.06
(Xyl H-1, d, J ) 7.9 Hz) and δC 81.2 (Glc-I C-2) and the reverse
correlation observed between δH 4.21 (Glc-I H-2) and δC 104.6
(Xyl C-1). Thus, the first part of the sugar sequence linked at C-3
was characterized as Xyl-(1f2)-Glc-I (1f 2)-[Rha-I (1f6)]-Gal
(1fAgly C-3). Then, the remaining part was established in the
same way. The linkage of Glc-II to the C-3 position of Ara was
deduced by the HMBC correlation between δH 5.38 (Glc II H-1,
d, J ) 8.0 Hz) and δC 81.2 (Ara C-3). The linkage of Glc-III to the
C-3 position of Glc-II was deduced by the HMBC correlation
between δH 5.26 (Glc III H-1, d, J ) 7.4 Hz) and δC 87.5 (Glc-II,
C-3) and the ROESY correlation between δH 5.26 (Glc III H-1, d,
J ) 7.4 Hz) and δH 4.05 (Glc II H-3). The linkage of Rha-II to the
C-4 position of Glc-II was deduced by the HMBC correlation
between δH 6.14 (Rha-II H-1, brs) and δC 81.1 (Glc-II C-4) and
the ROESY correlation between δH 6.14 (Rha-II, H-1) and δH 4.46
(Glc-II, H-4), proving that Rha-II was bound to Glc-II through a
(1f4) linkage. Thus, the sequence of this second part of the
oligosaccharide was elucidated as Glc-III (1f3)-[Rha II-(1f4) Glc-
II-(1f3)-Ara-. Finally the linkage between Ara and Glc-I was
ascertained to be (1f3) by observation of the HMBC correlation
between δH 5.09 (Ara H-1, d, J ) 7.0 Hz) and δC 87.4 (Glc-I C-3)
and the reverse correlation between δH 4.04 (Glc I H-3) and δC
104.9 (Ara C-1). On the basis of the above observations, the
structure of borivilianoside E (1) was elucidated as 25(R)-5R-
spirostan-3ꢀ-ol 3-O-R-L-rhamnopyranosyl(1f4)-[ꢀ-D-glucopyra-
nosyl-(1f3)]-ꢀ-D-glucopyranosyl-(1f3)-R-L-arabinopyranosyl-
(1f3)-[ꢀ-D-xylopyranosyl-(1f2)]-ꢀ-D-glucopyranosyl-(1f2)-[R-
L-rhamnopyranosyl-(1f6)]-ꢀ-D-galactopyranoside.
Borivilanoside F (2) exhibited in the HRESIMS (positive-ion mode)
a [M + Na]+ peak at m/z 1641.7142 (calcd for 1641.7148), consistent
with a molecular formula of C73H118O39. The negative-ion FABMS
displayed a quasimolecular ion peak at m/z 1617 [M - H]-, two mass
units lower than that of 1 and indicating a molecular weight of 1618.
Further fragment ion peaks were observed at m/z 1455 [(M - H) -
162]-, 1309 [(M - H) - 162 - 146]-, 1147 [(M - H) - 162 - 146
- 162]-, 1015 [(M - H) - 162 - 146 - 162 - 132]-, 883 [(M -
H) - 162 - 146 - 162 - 132 - 132]-, 721 [(M - H) - 162 - 146
- 162 - 132 - 132 - 162]-, corresponding respectively to the
successive loss of four hexosyls, two pentosyls, and two desoxyhexosyl
1
moieties. A detailed comparison of the H NMR and 13C NMR
chemical shifts of 1 and 2 obtained from 2D-NMR data (Tables 1
and 2) showed that most signals were superimposable in both
1
molecules except those of ring F in the aglycon part of 2. The H
NMR spectrum of 2 allowed the assignment of two ethylenic
protons at δH 4.79 brs and 4.82 brs (Ha-27 and Hb-27), with both
giving a correlation with δC 108.8 (C-27) in the HSQC spectrum
and long-range correlations in the HMBC spectrum respectively
with δC 144.0 (C-25), δC 65.0 (C-26), and δC 29.0 (C-24), proving
the presence of an exomethylene at C-25 in 2 instead a methyl
105.4, 104.2, 102.9, 101.7, 104.6, and 104.9, respectively. The
evaluation of chemical shifts and spin-spin couplings allowed the
identification of one ꢀ-galactopyranosyl unit (Gal), three ꢀ-glu-
copyranosyl units (Glc I, Glc II, Glc III), two R-rhamnopyranosyl
units (Rha I, Rha II), one ꢀ-xylopyranosyl unit, and one R-ara-
binopyranosyl unit in the molecule of 1.12 The absolute configu-
ration of Glc, Gal, and Xyl was determined as D and those of Rha
and Ara as L, as described above.
group in 1. The aglycon part of 2 was then identified as ∆25(27)
-
tigogenin.13 Sugar analysis and sequencing was carried out using
the same method as for 1. Therefore, the structure of borivilianoside
F (2) was elucidated as 5R-spirost-25(27)-ene-3ꢀ-ol 3-O-R-L-
rhamnopyranosyl(1f4)-[ꢀ-D-glucopyranosyl-(1f3)]-ꢀ-D-glucopy-
ranosyl-(1f3)-R-L-arabinopyranosyl-(1f3)-[ꢀ-D-xylopyranosyl-
(1f2)]-ꢀ-D-glucopyranosyl-(1f2)-[R-L-rhamnopyranosyl-(1f6)]-
ꢀ-D-galactopyranoside.
The sequence of the oligosaccharide chain of 1 was determined
by HMBC and ROESY experiments. The HMBC correlations
1
between the H NMR signal at δH 4.82 (d, J ) 7.8 Hz, Gal H-1)
and the 13C NMR signal at δC 77.0 (Agly C-3) and in the ROESY
spectrum between δH 4.82 (Gal H-1) and δH 3.92 (m, Agly H-3)
suggested the Gal was linked at C-3 of the aglycon. The correlation
1
The molecular formula of borivilianoside G (3) was deduced as
C51H82O24 on the basis of HRESIMS (positive-ion mode, [M +
Na]+ peak at m/z 1101.5110 (calcd for 1101.5094). The FABMS
(negative-ion mode) showed a quasimolecular ion peak at m/z 1077
[M - H]-, indicating a molecular weight of 1078. The NMR data
of this compound revealed that its aglycon differs from the aglycon
in the HMBC spectrum between the H NMR signal at δH 5.42
(brs, Rha-I H-1) and δC 68.6 (Gal C-6) and the reverse correlation
between δH 3.95 (Gal H-6) and δC 102.9 (Rha-I H-1) showed that
the terminal Rha-I was bound to Gal through a (1f6) linkage.
Furthermore, the correlation in the HMBC spectrum between the
1H NMR signal at δH 4.94 (d, J ) 7.5 Hz, Glc-I H-1) and δC 81.3

Notes
Journal of Natural Products, 2009, Vol. 72, No. 1 179
Table 1. 1H and 13C NMR Data for the Aglycon Moieties of 1-4 in Pyridine-d5 (δ in ppm, J in Hz)a
1
2
3
4
position
δC
δH
δC
δH
δC
δH
δC
δH
1
2
3
4
5
6
7
8
37.2
30.0
77.0
34.2
44.6
28.9
32.2
35.3
54.4
35.5
21.4
40.2
40.8
56.4
32.0
81.1
63.2
16.6
12.4
42.0
15.0
109.4
31.8
29.2
30.6
66.8
17.3
0.82, 1.58
-b, 2.04
3.92 m
37.2
30.0
77.0
34.4
44.6
28.9
32.2
35.3
54.6
35.5
21.4
40.2
40.9
56.5
32.0
81.3
63.2
16.8
12.4
42.0
15.0
109.2
32.0
29.0
144.0
65.0
108.8
0.82, 1.58
-b, 2.04
3.92 m
1.92, -b
0.94
1.18, 2.26
0.82, 1.53
1.44
45.6
70.4
84.2
34.1
44.6
28.9
32.1
34.6
54.4
36.8
21.4
40.0
40.4
56.3
32.1
81.4
63.0
16.5
13.4
41.9
14.9
109.2
31.9
29.2
144.0
65.3
108.6
1.16, -b
45.6
70.4
84.3
34.1
44.6
28.9
32.1
34.6
54.4
37.0
21.4
40.0
40.4
56.3
32.1
81.2
63.0
16.5
13.4
41.9
14.9
109.2
32.1
29.8
144.4
65.3
108.6
1.17, 2.18
3.96
3.96
3.88 m
1.50, 1.86
1.16
3.87 m
1.42, 1.86
1.04
1.92, -b
0.92
1.18, 2.26 m
0.82, 1.54
1.44
1.58, 2.70
1.16, -b
-b, 1.53
-b, 1.53
b
b
-
-
9
0.53
0.54
0.60
0.59
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
-b, 1.42
1.06, 1.66
-b, 1.45
1.06, 1.66
-b, 1.48
1.63, -b
-b, 1.48
1.63, -b
1.04
1.42, 2.02
4.48
1.80 m
0.84 s
0.87 s
1.04
1.42, 2.02
4.55
1.80 m
0.82 s
0.86 s
1.04
1.42, 2.02
4.54
1.03
1.42, 2.02
4.55
1.80
0.82 s
0.72 s
1.97 m
1.09 d (6.9)
1.80
0.82 s
0.73 s
1.96
1.96 m
1.15 d (6.8)
1.97 m
1.10 d (6.9)
1.09 d (6.9)
1.66, 1.71
1.26, 1.54
1.40, 1.66
1.40, -b
-b, 2.25
1.58 m, -b
1.58 m, -b
1.36, -b
b
-
3.52 m, 3.59 m
0.71 d (4.9)
4.04, 4.47
4.79, 4.82
4.04, 4.46
4.79, 4.82
4.04, 4.45
4.79, 4.82
a
Overlapping H NMR signals are reported without designated multiplicity. b Not determined.
1
of compound 2 in the NMR signals of ring A (Table 1). The main
difference was the observation of a downfield 13C NMR chemical
shift at δC 70.4 (C-2), showing a secondary alcoholic function at
this position and the downfield 13C NMR chemical shift of C-3 at
δC 84.2 instead of 77.0 in compound 2. The 2R orientation of the
hydroxyl unit was evident from the ROESY cross-peak between
δH 0.73 (s, ax H3-19) and δH 3.96 (m, H-2), proving the axial
orientation of this proton. The comparison of NMR data of 3 with
literature values allowed the identification of the aglycon as the
Borivilianoside H (4) exhibited in the HRESIMS (positive-ion
mode) a pseudomolecular ion peak at m/z 1071.4992 [M + Na]+
(calcd for 1071.4988), consistent with a molecular formula of
C50H80O23. Its FABMS (negative-ion mode) showed a quasimo-
lecular ion peak at m/z 1047 [M - H]-, indicating a molecular
1
weight of 1048. The H and 13C NMR signals assigned from the
2D NMR spectroscopic data, corresponding to the aglycon part of
4 (Table 1), were superimposable with those described for 3,
showing the presence of the same aglycon characterized as 5R-
spirost-25(27)-ene-2R,3ꢀ-diol (∆25(27)gitogenin).13
previously reported 2R,3ꢀ-5R-spirostene-25(27)-2,3-diol (∆25(27)
-
gitogenin),13 which has ben encountered for the first time in the
The 1H NMR spectrum of 4 showed four anomeric proton signals
at δH 4.91 (d, J ) 7.4 Hz), 5.21 (d, J ) 7.6 Hz), 5.59 (d, J ) 7.6
Hz), and 5.25 (d, J ) 7.7 Hz), which gave correlations in the HSQC
spectrum with four anomeric carbon signals at δC 103.3, 104.8,
104.5, and 104.8, respectively. 2D-NMR experiments indicated that
two ꢀ-D-glucopyranosyls, one ꢀ-D-galactopyranosyl, and one ꢀ-D-
xylopyranosyl unit was present (Table 2). The D configuration was
determined as for previously described compounds (1-3). The
cross-peak in the HMBC spectrum between δH 5.25 (Xyl H-1) and
δC 87.2 (Glc I C-3), between δH 5.59 (Glc II H-1) and δC 81.1
(Glc I C-2), between δH 5.21 (Glc I H-1) and δC 79.5 (Gal C-4),
and between δH 4.91 (Gal H-1) and δC 84.3 (Agly C-3), together
with the reverse correlations between δH 4.14 (Glc I H-3) and δC
104.8 (Xyl C-1), between δH 4.36 (Glc I H-2) and δC 104.5 (Glc
II H-1), between δH 4.61 (Gal H-4) and δC 104.8 (Glc I C-1), and
the ROESY correlations between δH 5.25 (Xyl H-1) and δH 4.14
(Glc I H-3), between δH 5.59 (Glc II H-1) and δH 4.36 (Glc I H-2),
between δH 5.21 (Glc I H-1) and δH 4.61 (Gal H-4), and between
δH 4.91 (Gal H-1) and δH 3.87 (Agly H-3) provided evidence for
the oligosaccharide chain at C-3. Therefore, the structure of
borivilianoside H (4) was established as 5R-spirost-25(27)-ene-
2R,3ꢀ-diol 3-O-ꢀ-D-xylopyranosyl-(1f3)-[ꢀ-D-glucopyranosyl-
(1f2)]-ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D-galactopyranoside.
genus Chlorophytum.
1
The H NMR spectrum of 3 displayed four anomeric proton
signals at δH 4.91(d, J ) 7.6 Hz), 5.17 (d, J ) 7.5 Hz), 5.59 (d, J
) 7.6 Hz), and 5.29 (d, J ) 7.5 Hz), which gave correlations with
four anomeric carbon signals at δC 103.2, 104.6, 104.9, and 104.5,
respectively, in the HSQC spectrum. The complete assignment of
the glycosidic NMR signals was achieved by analysis of COSY,
TOCSY, ROESY, HSQC, and HMBC experiments (Table 2).
Evaluation of spin-spin couplings and chemical shifts allowed the
identification of three ꢀ-glucopyranosyl (Glc I, Glc II, Glc III) and
one ꢀ-galactopyranosyl (Gal) unit. The D configuration was
determined as previously described for compounds 1 and 2. The
cross-peaks in the HMBC spectrum between δH 5.29 (Glc III H-1)
and δC 88.6 (Glc I C-3) and between δH 5.59 (Glc II H-1) and δC
81.2 (Glc I C-2), the reverse correlation between δH 4.32 (Glc I
H-2) and δC 104.9 (Glc II C-1), further HMBC correlations between
δH 5.17 (Glc I H-1) and δC 79.6 (Gal C-4), the reverse correlation
between δH 4.58 (Gal H-4) and 104.6 (Glc I C-1) and between δH
4.91 (Gal H-1) and δC 84.2 (Agly C-3), and the ROESY correlations
between δH 5.29 (Glc III H-1) and δH 4.17 (Glc I H-3), between
δH 5.59 (Glc II H-1) and δH 4.32 (Glc I H-2), between δH 5.17
(Glc I H-1) and δH 4.58 (Gal H-4), and between δH 4.91 (Gal H-1)
and δH 3.88 (Agly H-3) provided evidence for the nature of the
oligosaccharide chain at C-3. Therefore, the structure of borivil-
ianoside C (3) was established as 5R-spirost-25(27)-ene-2R,3ꢀ-diol
3-O-ꢀ-D-glucopyranosyl-(1f3)-[ꢀ-D-glucopyranosyl-(1f2)]-ꢀ-D-
glucopyranosyl-(1f4)-ꢀ-D-galactopyranoside.
Since steroidal saponins are reported to possess, to varying
degrees, cytotoxic activity against various cancer cell lines,1-3 we
have tested borivilianosides F (2), G (3), and H (4), 25(R,S)-5R-
spirostan-3ꢀ-ol 3-O-ꢀ-D-xylopyranosyl-(1f3)-[ꢀ-D-glucopyranosyl-
(1f2)]-ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D-galactopyranoside, and the

180 Journal of Natural Products, 2009, Vol. 72, No. 1
Notes
Table 2. 1H and 13C NMR Data for the Sugar Moities of 1-4 in Pyridine-d5 (δ in ppm, J in Hz)a
1
2
3
4
position
δC
δH
δC
δH
δC
δH
δC
δH
Gal-1
2
3
4
5
6
Glc-I-1
2
3
4
5
6
100.2
81.3
75.8
69.6
75.6
68.6
105.4
81.2
87.4
72.4
77.8
62.9
105.4
74.8
87.5
81.1
78.0
61.8
4.82 d (7.8)
4.48
4.13
4.11
3.95
3.95, 4.62
4.94 d (7.5)
4.21
4.04
4.50
100.2
81.3
75.8
69.6
75.6
68.6
105.4
81.2
87.4
72.5
77.8
62.9
105.4
74.8
87.5
81.1
78.0
61.8
4.83 d (7.8)
4.48
4.13
4.11
3.95
3.96, 4.62
4.94 d (7.5)
4.21
4.04
4.50
103.2
72.5
75.3
79.6
76.0
60.5
104.6
81.2
88.6
70.4
77.5
62.9
104.9
75.4
78.3
70.4
78.2
62.3
4.91 d (7.6)
4.54
4.11
4.58
4.04
4.18, 4.60
5.17 d (7.5)
4.32 t (9.0)
4.17
3.82
3.83
4.02, 4.47
5.59 d (7.6)
4.03
4.13
3.96
103.3
72.5
75.6
79.5
75.5
60.5
104.8
81.1
87.2
70.4
77.5
62.9
104.5
75.5
78.2
70.4
78.2
62.7
4.91 d (7.4)
4.54
4.13
4.61
4.04
4.18, 4.60
5.21 d (7.6)
4.36 t (9.0)
4.14
3.81
3.83
4.06, 4.49
5.59 d (7.6)
4.05
4.13
4.09
3.78
3.78
4.00, 4.49
5.38 d (8.0)
3.95
4.05
4.46
4.00, 4.50
5.38 d (8.0)
3.95
4.05
4.47
Glc-II-1
2
3
4
5
6
3.94
4.36, 4.44
3.95
4.38, 4.44
3.88
4.25, 4.53
3.92
4.40, dd
(11.2, 2.1), 4.54
Glc-III-1
2
3
4
5
6
Rha-I-1
2
3
4
5
6
Rha-II-1
2
3
4
5
6
Xyl-1
2
3
4
104.2
75.0
77.4
70.0
77.6
60.4
102.9
71.8
72.5
73.8
69.8
18.5
101.7
72.4
72.6
74.0
69.5
18.4
104.6
74.8
76.7
71.7
66.4
104.9
74.0
81.2
70.7
63.0
5.26 d (7.4)
3.97
4.14
4.10
3.82
4.20, 4.68
5.42 brs
4.72
4.53
4.19
104.3
74.9
77.6
70.0
77.6
60.4
102.9
71.8
72.5
74.0
69.6
18.5
101.7
72.4
72.6
74.0
69.5
18.4
104.6
74.7
76.7
69.1
66.4
104.9
74.5
81.2
70.7
63.0
5.23 d (7.4)
3.98
4.15
4.10
3.94
4.22, 4.66
5.42 brs
4.72
4.53
4.22
104.5
75.4
78.4
71.5
78.6
62.4
5.29 d (7.5)
4.03
4.18
4.11
4.01
4.25, 4.42
4.89
4.89
1.59 d (6.2)
6.14 brs
4.74
4.52
4.22
1.60 d (6.2)
6.25 brs
4.78
4.52
4.26
4.90
4.89
1.71 d (6.3)
5.06 d (7.9)
4.00
4.02
3.94
3.60 t (9.9), 4.16
5.09 d (7.0)
4.00
1.70 d (6.3)
5.06 d (7.9)
3.99
4.02
4.11
3.60 t (9.8), 4.16
5.09 d (7.0)
4.00
4.15
4.42
104.8
75.2
78.2
70.4
67.2
5.25 d (7.7)
3.97
4.08
4.13
3.66 t (9.5), 4.22
5
Ara-1
2
3
4
5
a
4.15
4.42
4.45, -b
4.45, 4.50
Overlapping H NMR signals are reported without designated multiplicity. b Not determined.
1
previously reported borivilianosides A6 and C6 for cytotoxicity
against the HCT 116 and HT-29 human colon tumor cell lines,
using paclitaxel as a standard anticancer drug. As determined by a
MTT assay,14 the only active substance was compound 4, which
showed the best cytotoxicity (0.38 µM in HCT 116 cells and 2.6
µM in HT-29 cells).
(negative-ion mode, glycerol matrix) was conducted on a JEOL SX
102 spectrometer. GC analysis was carried out on a Thermoquest gas
chromatograph. TLC and HPTLC were achieved on precoated silica
gel plates 60 F254 (Merck).15 Isolations were carried out using vacuum-
liquid chromatography (VLC) on reversed-phase RP-18 (Merck, 25-40
µm) and medium-pressure liquid chromatography (MPLC) on silica
gel 60 (Merck, 15-40 µm) and reversed-phase RP-18 (Merck, 25-
40 µm).15
Plant Material. The roots of C. boriVilianum were provided from
Jeevan Herbs in 2006 (New Delhi, India) and identified by Dr. M.
Ahmedullah (Botanic Garden of Indian Republic, BGIR, New Delhi,
India). A voucher specimen (no. 6628) is deposited in the herbarium
of the Laboratory of Pharmacognosy, Faculty of Pharmacy, University
of Burgundy, France.
Extraction and Isolation. The dried, powdered roots of C. boriV-
ilianum (500 g) were refluxed three times with EtOH (3 × 2 L) for
1 h. After evaporation of the solvent in vacuo, the resulting EtOH extract
(19 g) was suspended in water (200 mL) and partitioned successively
with CH2Cl2 (3 × 300 mL) and n-BuOH (3 × 200 mL), yielding after
evaporation of solvents the corresponding CH2Cl2 (3.2 g) and n-BuOH
(4.1 g) fractions. A 4 g aliquot of the n-BuOH residue was submitted
to VLC on C18 reversed-phase silica gel using H2O (3 × 100 mL),
MeOH-H2O mixtures (5:5, 3 × 100 mL), and finally MeOH (100 mL)
Experimental Section
General Experimental Procedures. Optical rotations were recorded
on an AA-OR automatic polarimeter. NMR experiments were measured
on a Varian VNMR-S 600 MHz spectrometer equipped with 3 mm
triple resonance inverse and 3 mm dual broadband probe-heads. Spectra
were recorded in 150 µL of pyridine-d5. Solvent signals were used as
internal standard (pyridine-d5: δH ) 7.21, δC ) 123.5 ppm), and all
spectra were recorded at T ) 35 °C. Pulse sequences were taken from
the Varian pulse sequence library (gCOSY, gHSQCAD, and gHM-
BCAD with adiabatic pulses). TOCSY spectra are acquired using DIPSI
spin-lock and 150 ms mixing time. Mixing time in ROESY experiments:
300 ms. Carbon type (CH3,CH2, CH): DEPT experiments. The 1D and
2D NMR spectra of certain compounds were recorded in pyridine-d5
on a Varian INOVA-600 (600 MHz).15 HRESIMS (positive-ion mode)
was carried out on a Q-TOF 1-micromass spectrometer. FABMS

Notes
Journal of Natural Products, 2009, Vol. 72, No. 1 181
as eluents. After evaporation of the solvents, three fractions were
obtained: Fr-1 (H2O), Fr-2 (MeOH-H2O, 5:5) (300 mg), and Fr-3
(MeOH) (1.1 g). Fr-3 (1.1 g) was submitted to MPLC [system A: silica
gel (15-40 µm), CHCl3-MeOH-H2O (65:35:10, lower phase)] to give
17 subfractions (1-17). Subfraction 7 (38 mg) was rechromatographed
by MPLC [system B: reversed-phase silica gel RP-18 eluted with
MeOH-H2O (40 f 100%)], giving compounds 1 (4.5 mg) and 2 (4.1
mg). Subfraction 3 (90 mg) was submitted to MPLC (system A), giving
compounds 3 (3.4 mg) and 5 (3.6 mg). Subfraction 2 (97.6 mg) was
submitted to MPLC (System B) yielding 4 (3.9 mg) and 6 (6 mg).
tives prepared in a similar way from standard sugars (Sigma-Aldrich).
This, D-glucose, D-galactose, D-xylose, L-arabinose, and L-rhamnose
were detected for 1 and 2, giving single peaks at 18.5, 21.9, 14.0, 12.0,
and 13.5 min, respectively. In the same manner, D-glucose and
D-galactose were identified for 3 and D-glucose, D-galactose, and
D-xylose for 4.
MTT Cytotoxicity Assay. The bioassay was carried out according
to the method described in ref 14 with two human colorectal cancer
cell lines (HCT 116 and HT-29). Paclitaxel was used as positive control
and exhibited IC50 values of 1.1 nM (HCT 116) and 3.6 nM (HT-29).
Borivilianoside E (1): white, amorphous powder; [R]20 -66.7 (c
D
0.20, MeOH); 1H NMR (pyridine-d5, 600 MHz) and 13C NMR
(pyridine-d5, 150 MHz), see Tables 1 and 2; negative FABMS (glycerol
matrix) m/z 1619 [M - H]-, 1457 [(M - H) - 162]-, 1311 [(M - H)
- 162 - 146]-, 1149 [(M - H) - 162 - 146 - 162]-, 1017 [(M -
H) - 162 - 146 - 162 - 132]-, 885 [(M - H) - 162 - 146 - 162
- 132 - 132]-, 723 [(M - H) - 162 - 146 - 162 - 132 - 132 -
162]-; HRESIMS (positive-ion mode) m/z 1643.7304 [M + Na]+ (calcd
for C73H120O39Na 1643.7310).
Acknowledgment. The authors are thankful to IFCPAR-CEFIPRA,
New Delhi, India, for financial support.
Supporting Information Available: Figure S1 showing FABMS
fragments and main HMBC correlations of 1. This information is
available free of charge via the Internet at http://pubs.acs.org.
References and Notes
Borivilianoside F (2): white, amorphous powder; [R]20 -70.8 (c
D
0.20, MeOH); 1H NMR (pyridine-d5, 600 MHz) and 13C NMR
(pyridine-d5, 150 MHz), see Tables 1 and 2; negative FABMS (glycerol
matrix) m/z 1617 [M - H]-, 1455 [(M - H) - 162]-, 1309 [(M - H)
- 162 - 146]-, 1147 [(M - H) - 162 - 146 - 162]-, 1015 [(M -
H) - 162 - 146 - 162 - 132]-, 883 [(M - H) - 162 - 146 - 162
- 132 - 132]-, 721 [(M - H) - 162 - 146 - 162 - 132 - 132 -
162]-; HRESIMS (positive-ion mode) m/z 1641.7142 [M + Na]+ (calcd
for C73H118O39Na 1641.7148).
(1) Lacaille-Dubois, M. A. In Handbook of Medicinal Plants; Yaniv Z.,
Bachrach, U., Eds.; The Haworth Medical Press (HMP):New York,
2005; Chapter 19, pp 399-428.
(2) Lacaille-Dubois, M. A. In Studies in Natural Products Chemistry
Series; Atta-ur-Rahman, Ed.; Elsevier Sciences: Amsterdam, 2005;
Vol. 32, pp 209-246.
(3) Kaushik, N. Phytochem. ReV. 2005, 4, 191–196.
(4) Qiu, S. X.; Li, X. C.; Xiong, Y.; Dong, Y.; Chai, H.; Farnsworth,
N. R.; Pezzuto, J. M.; Fong, H. H. S. Planta Med. 2000, 66, 587–
590.
(5) Narasimhan, S.; Govindarajan, R.; Madhavan, V.; Thakur, M.; Dixit,
V. K.; Mehrotra, S.; Madhusudanan, K. P. Planta Med. 2006, 72,
1421–1424.
(6) Acharya, D.; Mitaine-Offer, A. C.; Kaushik, N.; Miyamoto, T.;
Paululat, T.; Lacaille-Dubois, M. A. HelV. Chim. Acta 2008, 91, 2262-
2269.
(7) Mimaki, Y.; Kanmoto, T.; Sashida, Y.; Nishino, A.; Satomi, Y.;
Nishino, H. Phytochemistry 1996, 41, 1405–1410.
(8) Ikeda, T.; Tsumagari, H.; Honbu, T.; Nohara, T. Biol. Pharm. Bull.
2003, 26, 1198–1201.
(9) Avunduk, S.; Mitaine-Offer, A. C.; Alankus-Caliskan, O.; Miyamoto,
T.; Senol, S. G.; Lacaille-Dubois, M. A. J. Nat. Prod. 2008, 71, 141–
145.
(10) Mimaki, Y.; Kanmoto, T.; Kuroda, M.; Sashida, Y.; Satomi, Y.;
Nishino, A.; Nishino, H. Phytochemistry 1996, 42, 1065–1070.
(11) Agrawal, P. K.; Jain, D. C.; Pathak, A. K. Magn. Reson. Chem. 1995,
33, 923–953.
Borivilianoside G (3): white, amorphous powder; [R]20 -57.9 (c
D
0.20, MeOH); 1H NMR (pyridine-d5, 600 MHz) and 13C NMR
(pyridine-d5, 150 MHz), see Tables 1 and 2; negative FABMS (glycerol
matrix) m/z 1077 [M - H]-, 915 [(M - H) - 162]-, 753 [(M - H)
- 162 - 162]-, 591 [(M - H) - 162 - 162 - 162]-, 429 [(M - H)
- 162 - 162 - 162 - 162)-; HRESIMS (positive-ion mode) m/z
1101.5110 [M + Na]+ (calcd for C51H82O24Na 1101.5094).
Borivilianoside H (4): white, amorphous powder; [R]20 -65.1 (c
D
0.20, MeOH); 1H NMR (pyridine-d5, 600 MHz) and 13C NMR
(pyridine-d5, 150 MHz), see Tables 1 and 2; negative FABMS (glycerol
matrix) m/z 1047 [M - H]-, 915 [(M - H) - 132]-, 753 [(M - H)
- 132 - 162]-, 591 [(M - H) - 132 - 162 - 162]-; HRESIMS
(positive-ion mode) m/z 1071.4992 [M + Na]+ (calcd for C50H80O24Na
1071.4988).
Acid Hydrolysis and GC Analysis. Each compound (3 mg) was
hydrolyzed with 2 N aqueous CF3COOH (5 mL) for 3 h at 95 °C.
After extraction with CH2Cl2 (3 × 5 mL), the aqueous layer was
repeatedly evaporated to dryness with MeOH until neutral and then
analyzed by TLC over silica gel (CHCl3-MeOH-H2O, 8:5:1) by
comparison with authentic samples: galactose (Rf 0.21), glucose (Rf
0.23), xylose (Rf 0.47), arabinose (Rf 0.44), and rhamnose (Rf 0.51) for
1 and 2 and galactose (Rf 0.21) and glucose (Rf 0.23) for 3, and galactose
(Rf 0.21), glucose (Rf 0.23), and xylose (Rf 0.47) for 4. The trimethylsilyl
thiazolidine derivatives of the sugar residue of each compound were
prepared and analyzed by GC.16 The absolute configurations were
determined by comparing the retention times with thiazolidine deriva-
(12) Agrawal, P. K. Phytochemistry 1992, 31, 3307–3330.
(13) Brunengo, M. C.; Tombesi, O. L.; Doller, D.; Gros, E. G. Phytochem-
istry 1988, 27, 2943–2945.
(14) Carmichael, J.; De Graff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell,
J. B. Cancer Res. 1987, 47, 936–942.
(15) Mitaine-Offer, A. C.; Miyamoto, T.; Semmar, N.; Jay, M.; Lacaille-
Dubois, M. A. Magn. Reson. Chem. 2006, 44, 713–716.
(16) Haddad, M.; Miyamoto, T.; Laurens, V.; Lacaille-Dubois, M. A. J.
Nat. Prod. 2003, 66, 372–377.
NP800559Z