Trifasciatosides A-J, steroidal saponins from Sansevieria trifasciata

Article abstract of DOI:10.1248/cpb.c16-00337

Four previously unreported steroidal saponins, trifasciatosides A-D (1-4), three pairs of previously undescribed steroidal saponins, trifasciatosides E-J (5a, b-7a, b) including acetylated ones, together with twelve known compounds were isolated from the n-butanol soluble fraction of the methanol extract of Sansevieria trifasciata. Their structures were elucidated on the basis of detailed spectroscopic analysis, including ¹H-NMR, ¹³C-NMR, ¹H-¹H correlated spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond connectivity (HMBC), total correlated spectroscopy (TOCSY), nuclear Overhauser enhancement and exchange spectroscopy (NOESY), electrospray ionization-time of flight (ESI-TOF)-MS and chemical methods. Compounds 2, 4, and 7a, b exhibited moderate antiproliferative activity against HeLa cells.

Full text of DOI:10.1248/cpb.c16-00337

Vol. 64, No. 9
Chem. Pharm. Bull. 64, 1347–1355 (2016)
1347
Regular Article
Trifasciatosides A–J, Steroidal Saponins from Sansevieria trifasciata
,a,b
Rémy Bertrand Teponno,* Chiaki Tanaka,b Bai Jie,b Léon Azefack Tapondjou,a and
,b
Tomofumi Miyamoto*
a Department of Chemistry, Faculty of Science, University of Dschang; P.O. Box 67, Dschang, Cameroon: and
b Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Kyushu University;
Fukuoka 812–8582, Japan.
Received April 15, 2016; accepted June 7, 2016
Four previously unreported steroidal saponins, trifasciatosides A–D (1–4), three pairs of previously
undescribed steroidal saponins, trifasciatosides E–J (5a, b–7a, b) including acetylated ones, together with
twelve known compounds were isolated from the n-butanol soluble fraction of the methanol extract of San-
sevieria trifasciata. Their structures were elucidated on the basis of detailed spectroscopic analysis, includ-
ing 1H-NMR, 13C-NMR, 1H–1H correlated spectroscopy (COSY), heteronuclear single quantum coherence
(HSQC), heteronuclear multiple bond connectivity (HMBC), total correlated spectroscopy (TOCSY), nuclear
Overhauser enhancement and exchange spectroscopy (NOESY), electrospray ionization-time of flight (ESI-
TOF)-MS and chemical methods. Compounds 2, 4, and 7a, b exhibited moderate antiproliferative activity
against HeLa cells.
Key words Sansevieria trifasciata; Dracaenaceae; steroidal saponin; antiproliferative activity
Sansevieria (Dracaenaceae) is a genus of about 60 species, RP-18 HPLC to afford 22 compounds (Chart 1). The known
native to tropical and subtropical areas from warmer parts ones were identified to 3β-hydroxyspirosta-5,25(27)-dien-1β-
of South Africa to islands east of Africa, north eastern and yl-O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-
northern Africa, the Arabian peninsula, India, Burma, and (1→3)]-α-L-arabinopyranoside (8),15) (25S)-ruscogenin-1-O-α-
Java.1) Members of Sansevieria are of great economic im- L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-
portance as ornamentals, source of fiber and as medicine for arabinopyranoside (9),16) (23S)-3β,23-dihydroxyspirosta-5,25(27)-
curing different ailments. Sansevieria trifasciata commonly dien-1β-yl-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-
known as “snake plant” is an evergreen herbaceous peren- O-[β-D -xylopyranosyl-(1→3)]-α-L-arabinopyranoside
nial plant used for the treatment of inflammatory conditions, (10),7) (23S,24S)-3β,23,24-trihydroxyspirosta-5,25(27)-dien-1β-
snake bite, ear pain, swellings, boils and fever.2,3) Previous yl-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-
chemical studies on this plant led to the isolation of homoiso- xylopyranosyl-(1→3)]-α-L-arabinopyranoside (11),7) (23S,24S)-
flavanones,4) steroidal sapogenins namely 25S-ruscogenin and 3β,23,24-trihydroxyspirosta-5,25(27)-dien-1β-yl-O-(2,3-di-O-
sansevierigenin,5) pregnane glycosides,6) and ten new acety- acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-
lated steroidal saponins.7) Steroidal saponins can be divided (1→3)]-α-L-arabinopyranoside (12),7) (23S,24S)-3β,23-dihydroxy-
into three structural subclasses, spirostane-, furostane- and 1β-[O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-O-[β-
cholestane-type (open chain) glycosides8) and, over the years, D-xylopyranosyl-(1→3)]-α-L-arabinopyranosyl)oxy]spirosta-
have attracted attention for the wide spectrum of their biologi- 5,25(27)-dien-24-yl-β-D-fucopyranoside(13).15) (23S,24S)-3β,23,24-
cal action, particularly cytotoxic, antifungal and in vivo anti- Trihydroxyspirosta-5,25(27)-dien-1β-yl-O-α-L-rhamnopyranosyl)-
tumoral activity.9,10) Acetylated saponins are typical unstable (1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside
compounds, which are extensively distributed in many species (14),7) (23S,24S)-1β-[O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-
of medicinal herbs.11)
O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranosyl)oxy]-
As part of our search for bioactive steroidal saponins from 3β,23-dihydroxyspirosta-5,25(27)-dien-24-yl-β-D-fucopyrano-
Cameroonian medicinal plants,12–14) we have investigated the side (15),7) 1β,2β-dihydroxypregna-5,16-dien-20-one-1-O-α-L-
chemical components of S. trifasciata growing in the western rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-β-D-
highlands of Cameroon. In the present paper, we describe the glucopyranoside (16),6) 1β,2β-dihydroxypregna-5,16-dien-20-
isolation and structure elucidation of four previously unreport- one 1-O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-
ed steroidal saponins, three pairs of previously undescribed (1→3)]-α-L-arabinopyranoside (17),6) and
a
mixture of
steroidal saponins as well as twelve known compounds. Fur- 26-[(β-D-glucopyranosyl)oxy]-3β,22α-dihydroxyfurosta-
thermore, the new isolated compounds were tested for their 5,25(27)-dien-1β-yl-O-α-L-rhamnopyranosyl-(1→2)-α-L-
antiproliferative activity and some of them exhibited moderate arabinopyranoside (18a)17) and 26-[(β-D-glucopyranosyl)-
effect against the HeLa cells.
oxy]-3β-hydroxy-22α-methoxyfurosta-5,25(27)-dien-1β-yl-O-α-
L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside (18b).18)
Compound 1, isolated as a white amorphous solid showed
Results and Discussion
The n-butanol soluble fraction of the methanol extract the pseudo-molecular ion peak at m/z 1071.4953 [M+Na]+
from the aerial part of S. trifasciata was subjected to Se- in the positive electrospray ionization-time of flight (ESI-
phadex LH-20 column chromatography, Medium Pressure TOF)-MS corresponding to the formula C50H80O23Na (Calcd
Liquid Chromatography (MPLC) over silica gel, and then to for C50H80O23Na: 1071.4983). The IR spectrum showed char-
*To whom correspondence should be addressed. e-mail: rteponno@yahoo.fr; miyamoto@phar.kyushu-u.ac.jp
© 2016 The Pharmaceutical Society of Japan

1348
Chem. Pharm. Bull.
Vol. 64, No. 9 (2016)
Chart 1. Structures of the Saponins from S. trifasciata
acteristic absorptions for hydroxy groups (3399cm−1) and C-1 and C-3 positions was revealed by the coupling constants
1
glycosidic linkage bands (1044cm−1). The H-NMR spectrum of H-1 [δ 3.73 (1H, dd, J=11.6, 3.9Hz)] and were supported
displayed signals for three tertiary methyls at δ 0.80 (Me-18), by the nuclear Overhauser enhancement and exchange spec-
1.31 (Me-27) and 1.32 (Me-19), two secondary methyls at troscopy (NOESY) correlations between H-1, H-3 and H-9.18)
δ 1.00 (3H, d, J=6.7Hz, Me-21) and 1.65 (3H, d, J=5.9Hz, Based on the above findings, the structure of the aglycone
Me-6″), an olefinic proton at 5.46 (1H, brs, H-6) along with moiety was then recognized to be (22S,25S) 22,25-epoxy-
four anomeric protons at δ 6.25 (1H, brs, H-1″), 4.84 (1H, d, furost-5-ene-1β,3β,26-triol (1β-hydroxynuatigenin) by inten-
1
J=7.7Hz, H-1‴), 4.80 (1H, d, J=7.7Hz, H-1⁗), and 4.63 (1H, sive H- and 13C-NMR analysis (Table 1) and by comparison
d, J=7.6Hz, H-1′). The 13C-NMR spectrum of 1 consisted of with the literature data.5,20,21) Evaluation of spin–spin cou-
50 carbon signals, including two corresponding to olefinic car- plings and chemical shifts allowed the identification of one
bons at δ 139.0 (C-5) and 125.1 (C-6), one to an acetal carbon 2,3-disubstituted glucopyranosyl unit with the characteristic
at δ 120.6 (C-22), and four to anomeric carbons at 105.0 (C-1‴), C-3 carbon signal at δ 88.5 and terminal rhamnopyranosyl,
104.9 (C-1⁗), 101.4 (C-1″), and 100.1 (C-1′). The acetal carbon xylopyranosyl and glucopyranosyl units. In addition, 1 af-
signal displayed at δ 120.6 and oxygenated carbon signal at δ forded glucose, xylose and rhamnose on acid hydrolysis which
84.0 ascribed to C-22 and C-25, respectively, are characteristic were identified by comparison of retention time with authentic
for the furospirostanols (22,25-epoxyfurostanols).19) Assign- samples by GC-MS. The D-configuration for glucose, xylose
ment of the other proton and carbon signals of the aglycone and L for rhamnose were determined with L-cysteine methyl
was achieved by careful examination of the heteronuclear sin- ester derivatives followed by GC analysis.22) The sequences
gle quantum coherence (HSQC)–heteronuclear multiple bond of the sugar chains and their linkage sites were determined
connectivity (HMBC) and correlated spectroscopy (COSY) by HMBC and NOESY experiments. In the HMBC spectrum,
1
spectra. In the H–1H COSY spectrum of 1, the correlations the anomeric proton signals at δ 6.25 (H-1″), 4.84 (H-1‴), 4.80
from H-1 proton at δ 3.63 (1H, dd, J=11.6, 3.9Hz) via H2-2 at (H-1⁗), and 4.63 (H-1′) exhibited correlations with the carbon
δ 2.34 (1H, q, J=11.7Hz, H-2ax), 2.57 (1H, m, H-2eq), and H-3 signals at δ 76.1 (C-2′), 88.5 (C-3′), 77.4 (C-26) and 84.5 (C-1),
at δ 3.72 (m) to H2-4 at δ 2.63 (1H, t, J=11.6Hz, H-4ax), 2.50 respectively. Accordingly, the structure of 1 was elucidated
(1H, overlapped, H-4eq) indicated the dihydroxylation of the as (22S,25S) 26-O-β-D-glucopyranosyl-22,25-epoxy-furost-5-
A-ring at C-1 and C-3. The β orientation of oxygen atoms at ene-1β,3β,26-triol
1-O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-

Vol. 64, No. 9 (2016)
Chem. Pharm. Bull.
1349
xylopyranosyl-(1→3)]-β-D-glucopyranoside. This compound is Its positive ion mode ESI-TOF-MS showed the pseudo-mo-
a new furospirostanol glycoside to which we gave the trivial lecular ion peak at m/z 891.4452 [M+Na]+ corresponding to
name trifasciatoside A.
the molecular formula C44H68O17Na (Calcd for C44H68O17Na:
Compound 2 was isolated as a white amorphous powder. 891.4349). The IR spectrum displayed the absorptions char-
Table 1. 1H- and 13C-NMR Data of Compounds 1–4 (600, 125MHz, C5D5N–D2O, 20:1)
1
2
3
4
δC
δH
δC
δH
δC
δH
δC
δH
1
2
84.5
37.0
68.0
43.4
139.0
125.1
31.7
32.3
50.2
42.8
24.2
3.73 (dd, 11.6, 3.9)
2.34 (q, 11.7), 2.57 (m)
3.72 (m)
84.8
37.5
68.0
43.4
139.1
125.1
32.2
33.2
50.3
42.7
24.2
3.80*
84.7
37.5
68.0
43.4
139.2
125.0
31.9
33.3
50.4
42.8
24.2
3.75*
84.7
37.3
68.1
43.1
138.9
125.3
32.2
33.1
50.2
42.7
24.2
3.66*
2.40 (q, 12.2), 2.66*
3.79*
2.36 (q, 12.2), 2.56*
3.73 (m)
2.24 (q, 12.0), 2.48*
3.70 (m)
3
4
2.50*, 2.63 (t, 11.6)
—
2.58*, 2.68 (t, 11.2)
—
2.52*, 2.65 (t, 11.4)
—
2.48*, 2.57 (t, 11.6)
—
5
6
5.46*
5.54 (brs)
5.49 (brd, 5.0)
1.57*, 1.79 (m)
1.46*
5.45 (brd, 5.2)
1.40*, 1.74*
1.39*
7
1.38 (m), 1.72*
1.43*
1.5, 1.83 (each m)
nd
8
9
1.52 (brs)
—
1.62 (brs)
1.56 (brs)
—
1.48 (brs)
10
11
—
—
1.50 (brd, 7.5),
2.76 (brd, 10.0)
1.40*, 1.63*
—
1.58 (m)
nd
2.69 (brs)
2.84 (brd, 9.5)
1.49*, 1.69 (m)
—
nd
nd
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
40.3
40.3
56.6
32.6
81.2
62.4
16.7
15.1
38.6
15.1
120.6
33.0
33.6
84.0
77.4
40.3
40.3
56.9
31.7
81.5
62.9
16.9
15.1
41.9
14.9
109.7
33.1
28.9
144.2
65.0
40.7
40.8
56.1
nd
1.41*, 1.62 (m)
—
40.4
40.3
56.8
31.8
81.5
62.5
16.9
15.1
42.5
14.8
110.4
nd
1.38*, 1.59*
—
1.06 (m)
1.22 (m)
1.10 (m)
1.11 (m)
1.29, 1.83 (each m)
4.63 (q, 7.6)
1.82*
1.44, 1.99 (each m)
4.45*
1.46, 1.95 (each m)
4.15 (q, 7.2)
1.56*
1.35, 1.92 (each m)
4.37 (q, 7.6)
1.78*
80.6
62.4
17.3
15.1
42.1
16.4
110.7
29.5
27.9
30.7
69.5
1.83*
0.80 (3H, s)
1.32 (3H, s)
2.13*
0.89 (3H, s)
1.39 (3H, s)
1.97 (m)
0.97 (3H, s)
1.33 (3H, s)
2.27 (m)
0.77 (3H, s)
1.25 (3H, s)
1.83*
1.00 (3H, d, 6.7)
—
1.05 (3H, d, 6.6)
—
0.95 (3H, d, 7.2)
—
1.02 (3H, d, 6.8)
—
1.43*, 1.97 (m)
nd
1.51, 1.79 (each m)
2.27, 2.71 (each m)
—
nd
1.37, 1.79 (each m)
1.31, 2.06 (each m)
1.55 (m)
1.50*, 1.59*
1.57*
26.0
27.4
65.4
—
3.78 (d, 9.9),
4.12 (d, 9.9)
1.31 (3H, s)
4.63 (d, 7.6)
4.04 (t, 8.2)
3.93*
4.04 (d, 12.2), 4.44*
3.63*, 3.73*
3.33 (d, 10.9), 3.97*
27
24.1
100.1
76.1
88.5
70.1
77.5
62.5
101.4
72.1
71.9
73.9
69.5
19.2
105.0
74.5
78.1
70.4
67.0
109.2
100.2
76.0
88.4
70.1
77.4
63.0
101.4
72.1
71.9
73.9
69.5
19.2
105.0
74.5
77.8
70.4
67.0
4.78, 4.83 (each brs)
4.68 (d, 7.7)
4.10*
17.3
100.2
76.0
88.5
70.0
77.5
63.0
101.4
72.2
72.0
73.9
69.5
19.2
105.1
74.5
77.9
70.7
67.0
0.62 (3H, d, 6.4)
4.65 (d, 7.8)
4.05*
16.3
100.0
76.0
88.3
70.2
77.2
63.0
101.2
72.0
71.8
73.4
69.5
15.1
104.8
74.5
77.7
70.4
66.8
0.97 (3H, d, 7.0)
4.55 (d, 7.8)
3.94 (dd, 8.3, 8.4)
3.86 (t, 8.8)
3.60 (dd, 9.1, 9.0)
3.65*
1′
3.99 (dd, 7.8, 8.2)
3.76*
3.94 (dd, 8.3, 8.4)
3.61*
3.70*
3.70*
3.77*
3.72*
4.08*, 4.37 (d, 10.9)
6.25 (brs)
4.67 (brs)
4.49 (brd, 9.6)
4.23 (t, 8.3)
4.73 (m)
4.14*, 4.44*
6.31 (brs)
4.11*, 4.40*
6.28 (brs)
4.31 (d, 10.9), 4.00*
6.11 (brs)
1″
4.72 (brs)
4.68 (brs)
4.60 (brs)
4.55 (brd, 9.1)
4.29 (dd, 9.5, 10.1)
4.80 (m)
4.51 (dd, 9.5, 3.3)
4.23 (t, 9.5)
4.75 (m)
4.40 (dd, 9.6, 3.0)
4.16*
4.64 (m)
1.65 (3H, d, 5.9)
4.84 (d, 7.7)
3.88 (t, 8.3)
4.07*
1.71 (3H, d, 6.0)
4.90 (d, 7.7)
3.93 (t, 7.9)
4.12*
1.66 (d, 6.1)
4.86 (d, 7.7)
3.93 (dd, 7.9, 8.4)
4.06*
1.59 (d, 6.0)
4.79 (d, 7.7)
3.80 (t, 8.3)
4.03 (t, 8.9)
3.98*
1‴
4.05*
4.11*
4.05*
3.65 (t, 10.4), 4.20*
4.80 (d, 7.7)
3.95 (t, 8.3)
4.18*
3.69*, 4.25*
3.65*, 4.20*
3.63*, 4.17*
1⁗ 104.9
75.1
78.2
71.4
4.09*
78.2
3.87*
62.4
4.24*, 4.43 (d, 10.3)
Values in parentheses are coupling constants in Hz. *Overlapped by other signal. nd: not determined.

1350
Chem. Pharm. Bull.
Vol. 64, No. 9 (2016)
acteristic for hydroxy groups (3420cm−1), glycosidic linkages α-L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl(1→3)]-β-D-
(1044 cm−1) and a spirostanol moiety (919 and 836cm−1).12) glucopyranoside (trifasciatoside C).
1
The H-NMR spectrum of 2 showed signals for three steroid
Compound 4 was isolated as white amorphous solid from
methyl groups at δ 0.89 (3H, s, Me-18), 1.05 (3H, d, J=6.6Hz, methanol. It was shown to have the same molecular formula
Me-21) and 1.39 (3H, s, Me-19), an exomethylene group with C44H70O17 as trifasciatoside C (3) from the ESI-TOF-MS which
protons at δ 4.78 (1H, brs) and 4.83 (1H, brs) (H-27), an olefin- exhibited the pseudo-molecular ion peak at m/z 893.4545
ic proton at δ 5.54 (1H, brs), as well as three anomeric proton [M+Na]+ (Calcd for C44H70O17Na: 893.4505). The glycosidic
signals at δ 4.68 (1H, d, J=7.7Hz H-1′), 4.90 (1H, d, J=7.7Hz, nature of 4 was also indicated by the strong absorption bands
H-1‴) and 6.31 (1H, brs, H-1″). These proton signals together at 3406 and 1044cm−1 in the IR spectrum. The 1H-NMR
with the acetalic carbon signal at δ 109.7 (C-22), showing spectrum exhibited two methyl singlets at δ 0.77 (Me-18) and
HMBC correlations with methylene protons at δ 4.04 (1H, d, 1.25 (Me-19), three methyl doublets at δ 0.97 (Me-27), 1.02
J=12.2Hz, H-26a) and 4.44 (H-26b), and other carbon signals (Me-21) and 1.59 (Me-6″), an olefinic proton at δ 5.45 (1H,
at δ 144.2 (C-25), 139.1 (C-5), 125.1 (C-6) and 109.2 (C-27) brd, J=5.2Hz, H-6) as well as three anomeric protons at δ
(Table 2), indicated the presence of a spirosta-5,25(27)-diene 6.11 (1H, brs, H-1″), 4.79 (1H, d, J=7.7Hz, H-1‴) and 4.55
1
type aglycone.16,23,24) The above findings together with carbon (1H, d, J=7.8Hz, H-1′). Comparison of the H- and 13C-NMR
signals observed at δ 84.8 (C-1), 68.0 (C-3), and 81.5 (C-16) spectra of 4 with those of 3 revealed that the structure of the
allowed the identification of the aglycone to spirosta-5,25(27)- rings A–E portion and the sugar moiety attached at C-1 of
diene-1β,3β-diol (neoruscogenin). This was further confirmed the aglycone were identical to those of 3 (Table 1). However,
by correlations observed in the 1H–1H COSY, HSQC and significant differences were recognized in the signals from the
HMBC spectra and was in full agreement with the literature ring F portion mainly the 13C signal of C-26 which was up-
1
data.7,17) The H- and 13C-NMR (Table 1) data and the results field shifted from 69.5ppm in 3 to 65.4ppm in 4. These data
of acid hydrolysis indicated that the sugar chain was consti- indicated that 4 was a steroidal glycoside closely related to 3.
1
tuted of a 2,3-disubstituted glucose, a terminal rhamnose and Extensive analysis of H–1H COSY, HSQC, HMBC, TOCSY
a terminal xylose as in compound 1 (Chart 1). Their linkages and NOESY showed that the only difference was the orienta-
were established by the HMBC correlations observed between tion of the methyl group at C-25. The 25S stereochemistry
H-1′ (δ 4.68) and C-1 (δ 84.8), H-1″ (δ 5.77) and C-2′ (δ 76.0) of the 27-methyl group was deduced based on the difference
and between H-1‴ (δ 4.90) and C-3′ (δ 88.4). Compound 2 was of δ values of the geminal CH2-26 (ΔδH=0.64 ppm)28) and
then elucidated as 3β-hydroxy-spirosta-5,25(27)-dien-1β-yl- the higher field resonance of C-27 (δ 16.3) when compared
O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]- to the 13C-NMR shifts of 25R spirostanes (δ 17.1–17.2).16,29)
β-D-glucopyranoside a new secondary metabolite trivially Although (25S)-ruscogenin-1-O-α-L-rhamnopyranosyl-(1→2)-
named trifasciatoside B.
[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranoside was recently
Compound 3 isolated as white amorphous solid, showed reported as a known compound from Dracaena angustifo-
the [M+Na]+ ion at m/z 893.4564 in the ESI-TOF-MS, cor- lia,30) a systematic search in the literature gave no information
responding to the molecular formula C44H70O17Na (Calcd about its isolation. Consequently, the structure elucidation of 4
for C44H70O17Na: 893.4505) which was also deduced on the trivially named trifasciatoside D is described here for the first
basis of the 13C-NMR spectrum combined with distortionless time.
enhancement by polarization transfer (DEPT) data. Its glyco-
Compounds 5a, b isolated as a white amorphous powder,
sidic nature was shown by strong IR absorptions at 3420 and gave a pink coloration with Ehrlich reagent, indicating that
1
1044 cm−1. The H-NMR spectrum of 3 displayed two methyl it is a furostanol glycoside.31,32) Its ESI-TOF-MS showed two
singlets at δ 0.97 (Me-18) and 1.33 (Me-19), three doublet pseudo-molecular ion peaks at m/z 1073.5100 [M+Na]+ and
methyls at δ 0.62 (Me-27), 0.95 (Me-21) and 1.66 (Me-6″), an 1087.5193 [M+Na]+ corresponding to the molecular formu-
olefinic proton at δ 5.49 (1H, brd, J=5.0Hz, H-6) as well as lae C50H82O23Na and C51H84O23Na, respectively (Calcd for
three anomeric protons at δ 6.28 (1H, brs, H-1″), 4.86 (1H, d, C50H82O23Na: 1073.5139 and C51H84O23Na: 1087.5296). The IR
J=7.7Hz, H-1‴) and 4.65 (1H, d, J=7.8, H-1′). The 13C-NMR spectrum displayed the absorptions characteristic for hydroxy
showed the presence of 27 signals for the aglycone part with groups (3389cm−1), double bonds (1636cm−1) and glycosidic
the remaining 17 carbon signals for the sugar moiety. Compar- linkages (1045cm−1). It appeared to be a mixture of 22-hy-
1
ison of the H- and 13C-NMR data of the aglycone moiety of 3 droxy furostanol saponin 5a and its 22-methoxy derivative
1
1
established by extensive analysis of the H–1H COSY, HSQC, 5b from the NMR analysis. The H- and 13C-NMR spectra
HMBC, total correlated spectroscopy (TOCSY), and NOESY showed a set of duplicated signals corresponding to the com-
spectra (Table 1), with those of (25R)-spirost-5-ene-1β,3β-diol mon parts of the molecules with some significant differences
or [(25R)-ruscogenin] glycosides revealed that they were iden- observed, especially for signals around C-22 (Table 2). The
tical, including the β-orientation of the C-1 and C-3 oxygen 1H-NMR spectrum exhibited signals ascribed to Me-1″ at δ
atoms.25–27) Further inspection of the 13C-NMR data of the 1.68 (3H, d, J=5.1Hz) and Me-19 at δ 1.36 (3H, s), two sig-
sugar moiety in comparison with those reported in the lit- nals due to Me-18 at δ 0.91 (3H, s) and δ 0.85 (3H, s), two
erature showed that xylose and rhamnose, were attached to signals due to Me-21 at δ 1.23 (3H, d, J=6.7Hz) and at δ
the C-3′ and C-2′ positions of the inner glucose, respectively, 1.09 (3H, d, J=6.7Hz), two signals due to Me-27 at δ 0.96
which in turn was attached to the C-1 position of the aglycone (3H, d, J=6.7Hz) and at δ 0.98 (3H, d, J=6.7Hz) as well as
as in compounds 1 and 2.18) This was also supported by the an olefinic proton signals at δ 5.49 (1H, brd, J=6.7Hz, H-6).
results of acid hydrolysis and the downfield shifts observed Other signals of interest were those of four anomeric pro-
for C-1 (δ 84.7), C-3′ (δ 88.5), and C-2′ (δ 76.0). Compound 3 tons at δ 6.30 (1H, brs, H-1″), 4.86 (1H, d, J=7.3Hz, H-1‴),
was then elucidated as (25R)-3β-hydroxyspirost-5-en-1β-yl-O- 4.67 (1H, d, J=7.3Hz, H-1′) and 4.76–4.72 (1H, d, J=7.9Hz,

Vol. 64, No. 9 (2016)
Chem. Pharm. Bull.
1351
Table 2. 13C-NMR Data of Compounds 5a, b–7a, b (125MHz, C5D5N–D2O, 20:1)
No.
5a
5b
6a
6b
7a
7b
1
2
3
4
5
6
7
8
9
84.6
37.6
68.0
43.5
139.1
125.0
31.8
33.1
50.3
42.7
24.1
40.3
40.6
56.8
32.6
81.2
63.7
17.0
15.1
40.7
16.3
110.9
32.3
28.2
34.4
75.3
17.4
100.2
76.1
88.5
70.5
77.5
63.0
101.4
72.2
72.0
74.0
69.5
19.2
105.1
74.5
78.0
70.1
67.0
104.9
75.0
78.2
71.5
78.3
62.6
84.6
37.6
68.0
43.5
139.1
125.0
31.8
33.1
50.3
42.7
24.1
40.5
40.6
56.8
32.6
81.4
64.2
16.8
15.1
40.5
16.2
112.7
31.0
28.1
34.4
75.3
17.5
100.1
76.1
88.5
70.5
77.5
63.0
101.4
72.2
72.0
74.0
69.5
19.2
105.1
74.5
78.0
70.1
67.0
104.9
75.0
78.2
71.5
78.3
62.6
47.4
83.7
37.2
68.0
43.5
139.0
125.2
31.9
32.9
50.3
42.8
23.9
40.5
40.6
56.7
32.2
83.2
61.2
16.8
14.9
37.0
14.6
112.5
69.5
74.3
145.7
60.8
112.9
100.4
73.1
84.1
70.7
66.9
83.7
37.2
68.0
43.4
139.1
125.1
31.9
32.9
50.2
42.8
23.9
40.5
40.6
56.7
32.2
83.2
61.2
16.8
14.8
37.0
14.6
112.5
69.5
74.3
145.7
60.8
112.9
100.4
73.1
84.6
70.7
66.8
82.6
37.3
68.0
43.6
139.2
125.2
31.9
32.9
50.3
42.8
23.9
40.4
40.7
56.6
32.3
83.0
61.3
16.8
15.0
37.4
14.7
111.8
70.0
82.1
143.7
61.5
114.0
100.5
73.2
84.2
708
84.6
37.3
68.0
43.5
139.1
125.0
31.9
32.9
50.2
42.8
23.9
40.4
40.8
56.6
32.3
83.0
61.3
16.8
15.0
37.4
14.7
111.8
70.0
82.1
143.7
61.5
114.0
100.5
73.2
85.0
70.8
66.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1′
66.9
1″
97.5
73.1
67.6
75.8
66.5
18.2
105.8
74.4
77.7
70.7
67.2
100.2
69.3
72.8
72.8
66.4
18.2
106.2
74.4
77.9
70.7
67.1
97.5
73.1
67.7
75.9
66.4
18.2
105.9
74.4
77.7
70.7
67.2
105.9
72.7
75.0
72.6
71.5
17.1
100.3
69.3
73.0
73.0
66.5
18.3
106.4
74.4
78.0
70.7
67.2
105.9
72.7
75.0
72.6
71.5
17.1
1‴
1⁗
OMe
Ac-2″
20.9
20.9
171.2
171.0
Ac-3″
Ac-4″
21.0
171.0
21.1
21.0
170.9
21.1
21.1
21.1
171.3
171.3
171.2
171.2

1352
Chem. Pharm. Bull.
Vol. 64, No. 9 (2016)
H-1⁗) together with a methyl signal at δ 3.21 (OCH3). The the carbon signals at δ 171.3×2, 171.2, 171.0, 21.1×2, 21.0 and
13C-NMR spectrum displayed for the aglycone part signals 20.9 (Table 2). The upfied-shifted carbon signals δ 66.5–66.4
ascribable to C-22 at δ 110.9–112.7, four oxygenated carbon (C-5″) was in favour of the presence of acetyl groups at
signals at δ 84.6 (C-1), 68.0 (C-3), 81.2–81.4 (C-16) and 75.3 C-4″.7,15) Extensive analysis of the 1H–1H COSY, HSQC,
1
(C-27) confirming the furostanol skeleton. Analysis of H–1H HMBC, TOCSY and HMBC spectra showed that the first
COSY, HSQC and HMBC spectra of 5a, b allowed assign- rhamnopyranosyl unit was 2,4-diacetylated and the second
ment of all the signals belonging to the aglycone moiety, 3,4-diacetylated. Compounds 6a, b were then elucidated as
which was identified as furost-5-ene-1β,22α,3β,26-tetrol and a mixture of (23S,24S)-3β,23,24-trihydroxyspirosta-5,25(27)-
22-O-methylfurost-5-ene-1β,22α,3β,26-tetrol, respectively.31,32) dien-1β-yl-O-(2,4-O-diacetyl-α-L-rhamnopyranosyl)-(1→2)-O-
The C-25S configuration was established based on the differ- [β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside 6a (trifasci-
ence of δ values of the geminal CH2-26 (ΔδH=0.57ppm).28) atoside G) and (23S,24S)-3β,23,24-trihydroxyspirosta-5,25(27)-
1
It was evident from the H- and 13C-NMR data and from the dien-1β-yl-O-(3,4-O-diacetyl-α-L-rhamnopyranosyl)-(1→2)-O-
results of acid hydrolysis that the sugar part of 5a, b consisted [β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside 6b (trifas-
of four sugar units. The chemical shifts of all the individual ciatoside H).
protons of each sugar were ascertained from a combination
Compounds 7a, b had a molecular formula of C53H80O24
1
of TOCSY and H–1H COSY spectral analysis, and the 13C determined from the pseudo-molecular ion peak at m/z
chemical shifts of their relative attached carbons were as- 1123.4906 [M+Na]+ (Calcd for C53H80O24Na: 1123.4937) in
signed from the HSQC spectrum (Table 2). This allowed the ESI-TOF-MS. Comparison of the H- and 13C-NMR spectra
1
identification of two glucopyranosyl, one rhamnopyranosyl with those of 6a, b showed similarities, except the presence
and one xylopyranosyl units. The coupling constants observed of one additional deoxypyranosyl moiety in 7a, b. Detailed
for the anomeric protons suggested that the β configuration analysis of the 1H–1H COSY, HSQC, HMBC, TOCSY and
for glucose and xylose, and the α one for rhamnose. An un- HMBC spectra of this compound (Table 2) revealed supple-
ambiguous determination of the sequence and linkage sites mentary signals that were assigned to those of one terminal
was obtained from the HMBC spectrum, which showed key β-fucopyranosyl unit with an anomeric proton at δ 5.00 (1H, d,
correlation peaks between the proton signal at δ 6.30 (H-1″) J=7.6Hz), an anomeric carbon at δ 104.9, oxygenated methine
and the carbon resonance at δ 76.1 (C-2′), the proton δ 4.86 carbons at δ 75.0 (C-3⁗), 72.7 (C-2⁗), 72.6 (C-4⁗) and 71.5
(H-1‴) and the carbon at δ 88.5 (C-3′), the proton at δ 4.67 (C-5⁗), and one methyl carbon signal at δ 17.1 (C-5⁗)33) (Table
(H-1′) and the carbon at δ 84.6 (C-1), and the proton signal at 2). The above information indicated that these two compounds
δ 4.76/4.72 (H-1⁗) and the carbon resonance at δ 75.3 (C-26). have the same aglycone and the same sugar chain at C-1. The
Based on all these evidences, 5a, b was deduced to be a additional fucopyranosyl moiety was shown to be located at
mixture of 26-[(β-D-glucopyranosyl)oxy]-3β,22α-dihydroxy- C-24 as evidenced by the HMBC correlation depicted between
(25S)-furost-5-en-1β-yl-O-α-L-rhamnopyranosyl-(1→2)-O-[β- the anomeric proton at δ 5.00 (H-1′) and the carbon signal
D-xylopyranosyl-(1→3)]-β-D-glucopyranoside (trifasciatoside E at δ 82.1 (C-24). On acid hydrolysis, arabinose, rhamnose,
for 5a) and 26-[(β-D-glucopyranosyl)oxy]-3β-hydroxy-22α- xylose and fucose were obtained. Compounds 7a, b were fi-
methoxy-(25S)-furost-5-en-1β-yl-O-α-L-rhamnopyranosyl- nally shown to be a mixture of, (23S,24S)-3β,23-dihydroxy-1β-
(1→2)-O-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranoside (tri- [O-(2,4-di-O-acetyl-α-L-rhamnopyranosyl-(1→2)-O-
fasciatoside F for 5b).
[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranosyl)oxy]-
Compounds 6a, b were isolated as a white amorphous pow- spirosta-5,25(27)-dien-24-yl-β-D-fucopyranoside and (23S,24S)-
der with a molecular formula of C47H70O20 on the basis of ESI- 3β,23-dihydroxy-1β-[O-(3,4-di-O-acetyl-α-L-rhamnopyranosyl-
TOF-MS data (977.4296 [M+Na]+, Calcd for 977.4358). The IR (1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranosyl)-
spectrum displayed the absorptions characteristic for hydroxy oxy]spirosta-5,25(27)-dien-24-yl-β-D-fucopyranoside, two bides-
groups (3447cm−1), carbonyl groups (1734cm−1), double bond mosidic steroidal saponins trivially named trifasciatosides I
1
(1652 cm−1) and glycosidic linkages (1044cm−1). The H-NMR (7a) and J (7b), respectively.
spectrum showed one signal with regard to Me-21 at δ 1.00
Trifasciatoside A is a furospirostanol saponin having
(3H, d, J=7.0Hz), two signals for Me-18 at δ 0.94 (3H, s) and (22S,25S) 22,25-epoxy-furost-5-ene-1β,3β,26-triol (1β-hydroxy-
0.93 (3H, s), one signal due to Me-19 at δ 1.23 (3H, s) together nuatigenin) as aglycone. Nuatigenin-type steroids are rare
with the olefinic proton signal at δ 5.55 (1H, m, H-6) as well as natural metabolites with important pharmacological potential
the exomethylene proton signals H2-27 at δ 4.94 (1H, brs) and such as anticancer, antagonistic on rheumatoid arthritis, ben-
5.03 (1H, brs). The 13C-NMR spectrum showed signals char- eficial cardiovascular, and antimalarial activities.34) A cyto-
acteristic of 1,3,23,24-tetrahydroxyspirosta-5,25(27)-diene at chrome P450 monooxygenase from Streptomyces virginiae
δ 83.7 (C-1), 68.0 (C-3), 139.0–139.1 (C-5), 125.2–125.1 (C-6), IBL-14 has been recently identified to convert spirostane to
83.2 (C-16), 69.5 (C-23), 74.3 (C-24), 145.7 (C-25) and 112.9 nuatigenin-type steroid. This implies the introduction of a hy-
(C-27) (Table 2). It was evident that the structures of 6a, b droxyl group to the tertiary C-25 atom of the F-ring followed
are closed to (23S,24S)-3β,23,24-trihydroxyspirosta-5,25(27)- by the rearrangement under acidic work-up. It is known that
dien-1β-yl-O-(2,3-di-O-acetyl-α-L-rhamnopyranosyl)-(1→2)- furostanol saponins have two forms of 22-hydroxy and 22-me-
O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside (12) from thoxy in the course of extraction and isolation because of the
the above NMR data and acid hydrolysis which afforded interconversion in the presence of methanol and water.35) This
arabinose, xylose, rhamnose and artifactual aglycones. The phenomenon has already been observed in saponins from As-
1H- and 13C-NMR spectra showed that it was a mixture of paragus cochinchinensis, Asparagus officinalis, Ruscus acu-
two diacetylated steroidal glycosides by the proton signals at leatus, Anemarrhena asphodeloides and Digitalis purpurea.36)
δ 2.13 (3H, s), 2.07 (3H, s), 1.91 (3H, s) and 1.85 (3H, s), and It has recently been suggested that the 22-methoxy furostanol

Vol. 64, No. 9 (2016)
Chem. Pharm. Bull.
1353
saponins are secondary products formed from the correspond- QP2010SE (Shimadzu, Japan) with Inert Cap 5MS/Sil i.d.
ing 22-hydroxy form.37) From the above results, trifasciatoside 0.25×30m (GL Sciences Inc., Japan) [Column temperature:
F could then be the artifact formed from trifasciatoside E. 100–280°C, rate of temperature increase: 10°C/min]. The fol-
Compounds 6a, b–7a, b are acetyl saponins, typical unstable lowing sugar samples were commercially obtained: D-glucose,
compounds which are extensively distributed in many species L-rhamnose, D-fucose (Nacalai Tesque), L-glucose (Aldrich
of medicinal herbs.11,38) Due to the low activation energy of Chem. Co., Japan), D-xylose, D-arabinose, L-arabinose (Kishi-
acetyl migration reaction, it may be difficult to obtain the pure da Chemical Co., Ltd., Japan), L-xylose (Wako Pure Chemical
acetyl saponin. According to Zeng et al., the reaction of acetyl Industries, Ltd., Japan), D-rhamnose (Funakoshi Co., Ltd.,
transfer is faster in water than other solvents, and when com- Japan), L-fucose (Fluka), L-cysteine methyl ester hydrochloride
paring the normal/reverse silica gels, the reaction of acetyl (Kanto Chemical Co., Inc., Japan), N-trimethylsilylimidazole
migration almost did not happen during the process of puri- (TMS-imidazole) (Tokyo Kasei Kogyo Co., Ltd., Japan).
fication by macroporous resin.38) In the future phytochemical
Plant Material The aerial parts of S. trifasciata was col-
investigations on acetyl saponins rich plants, it could then be lected in Dschang (West region of Cameroon) in March 2014
recommended to use a macroporous resin as stationary phase and authentified at the Cameroon National Herbarium, Yaoun-
and avoid the using of water as solvent to be certain if the iso- dé where voucher specimens are deposited (No. HCN 43509).
lated compounds are natural or artifactual.11)
Extraction and Isolation The powdered dried aerial
The ability of the new isolated compounds (1–7a, b) to parts of S. trifasciata (3.3kg) were extracted three times
inhibit the proliferation of HeLa cells was examined. Tri- with MeOH (10L×3) at room temperature and the filtrate
fasciatosides B (2), D (4), and I and J (7a, b) showed weak obtained was evaporated under reduced pressure to yield the
antiproliferative activity with IC50 values of 47.1, 40.7, and crude MeOH extract (497.3g). Part of this extract (450g) was
26.5µM, respectively. The IC50 values of the other newly iso- triturated with EtOAc to yield 102g of the EtOAc-soluble
lated compounds (1, 3, 5a, b, 6a, b) were more than 50µM.
fraction and 340g of the residue. Part of the above residue
In summary, chemical investigation on the aerial parts of S. (70.5g) was suspended in water (1000mL), extracted with
trifasciata collected in Cameroon yielded four previously un- n-butanol (1000mL×3) then concentrated under reduced
reported steroidal saponins namely trifasciatosides A–D (1–4), pressure at 43°C to give 11g of the dried extract. This ex-
three pairs of previously undescribed steroidal saponins, tri- tract was dissolved in MeOH and submitted to a Sephadex
fasciatosides E–J (5a, b–7a, b) and twelve known compounds. LH-20 column eluted with MeOH to yield a crude saponin
The sugar chain residue attached at C-1 of compounds 1–5 rich fraction (6.3g). 5.8g of the crude saponin rich frac-
(α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D- tion were then fractionated by the MPLC on silica gel using
glucopyranosyl) rare in steroidal saponins, is present in CHCl3–MeOH–H2O (40:10:1, 14:6:1, 60:32:7) as eluents,
Ophiopogonin D′ isolated from the tubers of Ophiopogon affording ten main fractions: F-1 (188mg), F-2 (122mg),
japonicus var genuinus,39) and Brodiosaponins A, B, C, E, F F-3 (379mg), F-4 (290mg), F-5 (628mg), F-6 (879mg), F-7
isolated from Brodiaea californica.40) Compounds 2, 4, and (640mg), F-8 (961mg), F-9 (244mg), and F-10 (344mg). F-4
7a, b exhibited moderate cytotoxicity against HeLa cells.
(eluted with CHCl3–MeOH–H2O (40:10:1)) was first sub-
mitted to MPLC using CHCl3–MeOH–H2O (40:10:1) as the
mobile phase to give two main subfractions: F-41 (89mg)
Experimental
General Experimental Procedures Optical rotations and F-42 (100.5mg). F-41 was then submitted to successive
were measured on a JASCO DIP-370 Digital Polarimeter. IR RP-18 HPLC in isocratic conditions (60% MeOH) to afford
spectra were recorded on a JASCO FT-IR-410 Infrared Spec- 7a, b (25.6mg, tR: 14.0min), 10 (3.0mg, tR: 86.0min), and 11
1
trophotometer (Japan). H- and 13C-NMR were performed in (3.1mg, tR: 27.8min). F-5 was eluted with CHCl3–MeOH–H2O
C5D5N–D2O (20:1) on a Varian INOVA-600 NMR Spectrom- (14:6:1), then purified by successive MPLC on silica gel
1
eter (600MHz for H, 150MHz for 13C). All chemical shifts using CHCl3–MeOH–H2O (40:10:1), yielding two subfrac-
(δ) are given in ppm with reference to the residual solvent tions F-51 (76mg) and F-52 (360mg). Subfraction F-51 was
signal and coupling constants (J) are in Hz. ESI-TOF-MS separated by preparative RP-18 HPLC in isocratic conditions
were recorded on a Bruker Micro TOF II LC/MS Spectrom- (80% MeOH, 2.5mL/min) to afford 2 (2.5mg, tR: 21.4min),
eter. Column chromatography was performed using Sephadex 3 (1.7mg, tR: 15.4min), 4 (12.7mg, tR: 23.36min), 8 (3.8mg,
LH-20 (eluted with MeOH) and silica gel 60 (0.040–0.063mm, tR: 27.4min), and 9 (5.2mg, tR: 28.8min). One hundred mil-
Merck) (eluted with CHCl3–MeOH–H2O (40:10:1, 14:6:1, ligrams of F-6 (also eluted with CHCl3–MeOH–H2O (14:6:1))
60:32:7)). MPLC on silica gel 60 (0.040–0.063mm) with were purified by RP-18 HPLC in isocratic conditions (60%
a KISO Power Tool E5305T pump and an Advantec SF-160 MeOH) to yield compounds 14 (4.7mg, tR: 24.8min), 15
fraction collector. TLC were carried out on precoated Kiesel- (24.2mg, tR: 14.24min), 16 (5.49mg, tR: 69.60min), 17 (3.0mg,
gel 60F254 (Merck) plates developed with CHCl3–MeOH–H2O tR: 42.2min), and 18 (8.25mg, tR: 40.36min). F-8 [100mg,
(40:10:1, 14:6:1, 60:32:7) and on Kieselgel 60 RP-8 F254S eluted with CHCl3–MeOH–H2O (14:6:1)] was repeatedly sub-
(Merck KGaA 64271 Darmstadt, Germany) developed with mitted to RP-18 HPLC in isocratic conditions (55% MeOH) to
MeOH–H2O mixtures. They were visualised by spraying with give compounds 1 (3.0mg, tR: 44.6min) and 5a/b (7.54mg, tR:
5% methanolic H2SO4 followed by heating. Preparative HPLC 39.2min). F-2 [eluted with CHCl3–MeOH–H2O (40:10:1)] was
was performed with a JASCO PU-2080 Plus Intelligent HPLC repeatedly separated by RP-18 HPLC in isocratic conditions
pump connected to a 5C18-AR-II column (Nacalai Tesque (60% MeOH) to give compounds 6a, b (6.2mg, tR: 34.2min),
Inc., Japan) and a JASCO Gulliver Series RI-930 Intelligent 12 (3.5mg, tR: 29.48min), and 13 (15.9mg, tR: 17.68min).
IR detector. The flow rate was 2.5mL/min, 50µL of solution
Acid Hydrolysis and GC-MS Each saponin (ca. 1.0mg)
was injected each time. GC-MS was performed with GCMS- was heated in 1M HCl (0.1mL) at 90°C for 3h. The reac-

1354
Chem. Pharm. Bull.
Vol. 64, No. 9 (2016)
tion mixture was dried in vacuo and dissolved in pyridine 2927 (CH), 1636 (C=C), 1378, 1045 (C–O), 625. 1H-NMR
(0.2mL). TMS-imidazole (50µL) was added to the part of (600MHz, C5D5N–D2O, 20:1) δ: 6.30 (1H, brs, H-1′), 5.49
solution (0.1mL) then heated at 50°C for 30min. The reaction (1H, brd, J=6.7Hz, H-6), 4.86 (1H, d, J=7.3Hz, H-1‴), 4.83
mixture was diluted with H2O (0.2mL) and extracted with (1H, m, H-16), 4.76–4.72 (1H, d, J=7.9Hz, H-1⁗), 4.67 (1H,
n-hexane (0.1mL) then analysed by GC-MS by comparison d, J=7.3Hz, H-1′), 3.96 (1H, t, J=7.4Hz, H-3′), 3.77 (1H, m,
with standard samples. L-Cysteine methyl ester hydrochloride H-1), 3.21 (3H, s, OCH3), 1.68 (3H, d, J=5.1Hz, Me-1″), 1.36
(ca. 1.0mg) was added to the remaining pyridine solution (3H, s, Me-19), 1.23 (3H, d, J=6.7Hz, Me-21a), 1.09 (3H, d,
(0.1mL) and heated at 60°C for 1 h then the TMS derivative J=6.7Hz, Me-21b), 0.98 (3H, d, J=6.7Hz, Me-27b), 0.96 (3H,
was prepared the same manner mentioned above and analysed d, J=6.7Hz, Me-27a), 0.91 (3H, s, Me-18a), 0.85 (3H, s, Me-
by GC-MS.
Standard TMS-Sugars (tR, min)
18b). 13C-NMR (150MHz, C5D5N–D2O, 20:1) spectroscopic
data, see Table 2. ESI-TOF-MS m/z: 1073.5100 [M+Na]+ and
TMS-glucose, tR=14.11, 15.01; TMS-xylose, tR=12.08, 12.68; 1087.5193 [M+Na]+ (Calcd for C50H82O23Na: 1073.5139 and
TMS-rhamnose, tR=11.06, 11.88; TMS-arabinose, tR=10.87, C51H84O23Na: 1087.5296). TMS-glucose, tR=14.11, 15.01; TMS-
10.92, 11.29; TMS-fucose, tR=11.17, 11.59, 12.07.
xylose, tR=12.08, 12.68; TMS-rhamnose, tR=11.05, 11.88. D-
Glucose, tR=19.27; D-xylose, tR=17.37; L-rhamnose, tR=18.10.
Trifasciatosides G and H (6a, b)
Standard TMS-Thiazolidine Derivatives (tR, min)
D-Glucose, tR=19.34, L-glucose, tR=19.47, D-xylose, tR=
17.49, L-xylose, tR=17.74, D-rhamnose, tR=18.25, L-rhamnose,
White amorphous powder; IR (KBr) cm−1: 3447 (OH), 2908
tR=18.13, D-arabinose, tR=17.80, L-arabinose, tR=17.48, D- (CH), 2360, 2342, 1734 (C=O), 1653, 1457, 1374, 1248, 1045
1
fucose, tR=18.26, L-fucose, tR=18.54.
Trifasciatoside A (1)
(C–O), 837, 687. H-NMR (600MHz, C5D5N–D2O 20:1) δ:
6.29 (1H, brs, H-1″a), 6.25 (1H, brs, H-1″b), 5.85 (1H, brs,
White amorphous powder; [α]D24 −39.4 (c=0.32, MeOH). H-2″a), 5.76 (1H, brs, H-3″b), 5.75 (1H, brs, H-4″b), 5.03, 4.94
IR (KBr) cm−1: 3399 (OH), 2928 (CH), 1644 (C=C), 1380, (each 1H, brs, H2-27), 4.84 (1H, d, J=7.6Hz, H-1‴b), 4.80 (1H,
1044 (C–O). 1H-NMR (600MHz, C5D5N–D2O, 20:1) and d, J=7.6Hz, H-1‴a), 4.61 (1H, brs, H-24), 4.54 (1H, H-16),
13C-NMR (150MHz, C5D5N–D2O, 20:1) spectroscopic data, 4.53 (1H, overlapped, H-1′), 3.81 (1H, m, H-3), 3.81 (1H, brs,
see Tables 1 and 2. ESI-TOF-MS m/z: 1071.4953 [M+Na]+ H-23), 3.68 (1H, m, H-1), 2.13 (3H, s, OAc-3″b), 2.07 (3H, s,
(Calcd for C50H80O23Na: 1071.4983). TMS-glucose (2mol), OAc-2″a), 1.91 (3H, s, OAc-4″a), 1.85 (3H, s, OAc-4″b), 1.31
tR=14.11, 15.00; TMS-xylose, tR=12.08, 12.68; TMS-rham- (3H, d, J=5.3Hz, Me-6″), 1.23 (3H, s, Me-19), 1.00 (3H, d,
nose, tR=11.05, 11.88. D-Glucose, tR=19.28; D-xylose, tR=17.36; J=7.0Hz, Me-21), 0.94 (3H, s, Me-18b), 0.93 (3H, s, Me-18a).
L-rhamnose, tR=18.09.
Trifasciatoside B (2)
13C-NMR (150MHz, C5D5N–D2O, 20:1) spectroscopic data,
see Table 2. ESI-TOF-MS m/z: 977.4296 [M+Na]+ (Calcd
White amorphous powder; [α]D24 −23.5 (c=0.24, MeOH). IR for C47H70O20Na: 977.4358). TMS-arabinose, tR=10.86,
(KBr) cm−1: 3420 (OH), 2925 (CH), 1636 (C=C), 1376, 1230, 10.91, 11.28; TMS-xylose, tR=12.08, 12.68; TMS-rhamnose,
1
1044 (C–O), 985, 919, 836, 636. H-NMR (600MHz, C5D5N– tR=11.05, 11.88. D-Arabinose, tR=17.59; D-xylose, tR=17.38; L-
D2O, 20:1) and 13C-NMR (150MHz, C5D5N–D2O, 20:1) spec- rhamnose, tR=18.10.
troscopic data, see Tables 1 and 2. ESI-TOF-MS m/z: 891.4452
Trifasciatosides I and J (7a, b)
[M+Na]+ (Calcd for C44H68O17Na: 891.4349). TMS-glucose,
White amorphous powder; IR (KBr) cm−1: 3420 (OH),
tR=14.11, 15.00; TMS-xylose, tR=12.08, 12.68; TMS-rham- 2909 (CH), 2360, 1731 (C=O), 1639 (C=C), 1376, 1250, 1044
1
nose, tR=11.05, 11.88. D-Glucose, tR=19.30; D-xylose, tR=19.35; (C–O), 624, 419. H-NMR (600MHz, C5D5N–D2O, 20:1) δ:
L-rhamnose, tR=18.09.
Trifasciatoside C (3)
6.33 (1H, brs, H-1″a), 6.28 (1H, brs, H-1″b), 5.89 (1H, brs,
H-2″a), 5.79 (1H, H-4″b), 5.78 (1H, H-3″b), 5.53 (1H, H-4″a),
White amorphous powder; [α]D24 −14.9 (c=0.18, MeOH); IR 5.53 1H, (H-6), 5.17, 5.03 (each 1H, brs, H2-27), 5.00 (1H,
(KBr) cm−1 : 3420 (OH), 2925 (CH), 2360, 2342, 1636 (C=C), d, J=7.6Hz, H-1⁗), 4.85 (1H, d, J=7.8Hz, H-1‴b), 4.82 (1H,
1
1541, 1376, 1230, 1044 (C–O), 985, 668. H-NMR (600MHz, overlapped, H-1‴a), 4.71 (1H, brs, H-24), 4.55 (1H, overlapped,
C5D5N–D2O, 20:1) and 13C-NMR (150MHz, C5D5N–D2O, H-1′), 4.54 (1H, m, H-16), 3.87 (1H, brs, H-23), 3.82 (1H, m,
20:1) spectroscopic data, see Tables 1 and 2. ESI-TOF-MS H-3), 3.71 (1H, m, H-1), 2.13 (3H, s, OAc-3″b), 2.05 (3H, s,
m/z: 893.4564 [M+Na]+ (Calcd for C44H70O17Na: 893.4505). OAc-2″a), 1.91 (3H, s, OAc-4″a), 1.84 (3H, s, OAc-4″b), 1.37
TMS-glucose, tR=14.11, 15.01; TMS-xylose, tR=12.08, 12.68; (3H, d, J=6.1Hz, Me-6″), 1.26 (3H, s, Me-19), 1.00 (3H, d,
TMS-rhamnose, tR=11.05, 11.88. D-Glucose, tR=19.32; D- J=7.1, Me-21), 0.90 (3H, s, Me-18b), 0.89 (3H, s, Me-18a);
xylose, tR=17.35; L-rhamnose, tR=18.09.
Trifasciatoside D (4)
13C-NMR (150MHz, C5D5N–D2O, 20:1) spectroscopic data,
see Table 2. ESI-TOF-MS m/z: 1123.4906 [M+Na]+ (Calcd
White amorphous powder; [α]D24 −55.1 (c=0.70, MeOH); IR for C53H80O24Na: 1123.4937). TMS-arabinose, tR=10.86,
(KBr) cm−1: 3406 (OH), 2932 (CH), 2360, 2342, 1636 (C=C), 10.91, 11.28; TMS-xylose, tR=12.06, 12.68; TMS-rhamnose,
1
1456, 1376, 1225, 1044 (C–O), 986, 919, 838, 668. H-NMR tR=11.05, 11.88; TMS-fucose, tR=11.56, 12.06. D-Arabinose,
(600MHz, C5D5N–D2O, 20:1) and 13C-NMR (150MHz, tR=17.60; D-xylose, tR=17.38; L-rhamnose, tR=18.10; D-fucose,
C5D5N–D2O, 20:1) spectroscopic data, see Tables 1 and 2. tR=18.16.
ESI-TOF-MS m/z: 893.4545 [M+Na]+ (Calcd for C44H70O17Na:
Cell Culture and Cell Proliferation Assay Human ma-
893.4505). TMS-glucose, tR=14.11, 15.00; TMS-xylose, lignant epithelial cells (HeLa) were cultured in Eagle’s mini-
tR=12.08, 12.68; TMS-rhamnose, tR=11.05, 11.88. D-Glucose, mum essential medium (EMEM) supplemented with 10% fetal
tR=19.26; D-xylose, tR=17.36; L-rhamnose, tR=18.10.
Trifasciatosides E and F (5a, b)
bovine serum (FBS) kept in an incubator at 37°C in a humidi-
fied air containing 5% CO2. FBS was purchased from Nichirei
White amorphous powder; IR (KBr) cm−1: 3389 (OH), Bioscience Inc. (Tokyo, Japan). Cell viability was determined

Vol. 64, No. 9 (2016)
Chem. Pharm. Bull.
Prod., 73, 1524–1528 (2010).
1355
by a Cell-Titer 96 Aqueous Non-Radioactive Cell Proliferation
(MTS) Assay (Promega, WI, U.S.A.) according to the manu-
facturer’s protocol. HeLa cells (1×104 cells/well) were seeded
in 96 well plates and incubated for 24h, subsequently grown
with compounds for additional 48h, and then cell proliferation
assay was performed.
17) Perrone A., Muzashvili T., Napolitano A., Skhirtladze A., Kemer-
telidze E., Pizza C., Piacente S., Phytochemistry, 70, 2078–2088
(2009).
18) Mimaki Y., Watanabe K., Helv. Chim. Acta, 91, 2097–2106 (2008).
19) Agrawal P. K., Jain D. C., Gupta R. K., Thakur R. S., Phytochemis-
try, 24, 2479–2496 (1985).
20) Gonzalez A. G., Freire R., Hernandez R., Salazar J. A., Suarez E.,
Rev. Lat. Quim., 4, 45–51 (1973).
21) Yoshimitsu H., Nishida M., Nohara T., Phytochemistry, 64, 1361–
1366 (2003).
22) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 35, 501–506
(1987).
Acknowledgments Dr. R. B. Teponno is grateful to The
Matsumae International Foundation (MIF) for financing a
postdoctoral research stay (4 months) at the Graduate School
of Pharmaceutical Sciences, Kyushu University, Japan.
23) Mimaki Y., Kuroda M., Takaashi Y., Sashida Y., Phytochemistry,
47, 79–85 (1998).
24) Kougan G. B., Miyamoto T., Tanaka C., Paululat T., Mirjolet J.-F.,
Duchamp O., Sondengam B. L., Lacaille-Dubois M.-A., J. Nat.
Prod., 73, 1266–1270 (2010).
25) Kuroda M., Mimaki Y., Ori K., Sakagami H., Sashida Y., J. Nat.
Prod., 67, 1690–1696 (2004).
26) Mimaki Y., Aoki T., Jitsuno M., Yokosuka A., Kiliç C. S., Coşkun
M., Nat. Prod. Commun., 3, 1671–1678 (2008).
27) Wang K.-W., Zhang H., Shen L.-Q., Wang W., Carbohydr. Res.,
346, 253–258 (2011).
28) Agrawal P. K., Magn. Reson. Chem., 42, 990–993 (2004).
29) Agrawal P. K., Jain D. C., Pathak A. K., Magn. Reson. Chem., 33,
923–953 (1995).
30) Shen H.-Y., Zuo W.-J., Wang H., Zhao Y.-X., Guo Z.-K., Luo Y., Li
X.-N., Dai H.-F., Mei W.-L., Fitoterapia, 94, 94–101 (2014).
31) Honbu T., Ikeda T., Zhu X.-H., Yoshihara O., Okawa M., Nafady A.
M., Nohara T., J. Nat. Prod., 65, 1918–1920 (2002).
32) Yuan L., Ji T. F., Wang A. G., Yang J. B., Su Y. L., Chin. Chem.
Lett., 19, 461–464 (2008).
33) Agrawal P. K., Phytochemistry, 31, 3307–3330 (1992).
34) Wang W., Wang F.-Q., Wei D.-Z., Appl. Environ. Microbiol., 75,
4202–4205 (2009).
35) Li X.-C., Yang C.-R., Ichikawa M., Matsuura H., Kasai R., Yama-
saki K., Phytochemistry, 31, 3559–3563 (1992).
36) Kite G. C., Porter E. A., Simmonds M. S. J., J. Chromatogr. A,
1148, 177–183 (2007).
37) Sobolewska D., Michalska K., Podolak I., Grabowska K., Phyto-
chem. Rev., 15, 1–35 (2016).
38) Zeng L., Zhu M., Zhong J., Yan W., Phytochem. Lett., 11, 368–372
(2015).
39) Watanabe Y., Sanada S., Tada A., Shoji J., Chem. Pharm. Bull., 25,
3049–3055 (1977).
Conflict of Interest The authors declare no conflict of
interest.
References
1) Carlquist S., Schneider E. L., S. Afr. J. Bot., 73, 196–203 (2007).
2) Da Silva Antunes A., Da Silva B. P., Parente J. P., Valente A. P.,
Phytother. Res., 17, 179–182 (2003).
3) Anbu J. S., Jayaraj P., Varatharajan R., John T., Jisha J., Muthappan
M., Afr. J. Trad. CAM, 6, 529–533 (2009).
4) Tchegnitegni B. T., Teponno R. B., Tanaka C., Gabriel A. F., Ta-
pondjou A. L., Miyamoto T., Phytochem. Lett., 12, 262–266 (2015).
5) González A. G., Freire R., García-Estrada M. G., Salazar J. A.,
Suárez E., Tetrahedron, 28, 1289–1297 (1972).
6) Mimaki Y., Inoue T., Kuroda M., Sashida Y., Phytochemistry, 44,
107–111 (1997).
7) Mimaki Y., Inoue T., Kuroda M., Sashida Y., Phytochemistry, 43,
1325–1331 (1996).
8) Challinor V. L., De Voss J. J., Nat. Prod. Rep., 30, 429–454 (2013).
9) Sautour M., Mitaine-Offer A.-C., Lacaille-Dubois M.-A., J. Nat.
Med., 61, 91–101 (2007).
10) Li R., Wang M.-Y., Li X.-B., J. Med. Plants Res., 6, 2704–2713
(2012).
11) Sparg S. G., Light M. E., Van Staden J., J. Ethnopharmacol., 94,
219–243 (2004).
12) Tapondjou L. A., Ponou K. B., Teponno R. B., Mbiantcha M.,
Djoukeng J. D., Nguelefack T. B., Watcho P., Cadenas A. G., Park
H.-J., Arch. Pharm. Res., 31, 653–658 (2008).
13) Tapondjou L. A., Jenett-Siems K., Böttger S., Melzig M. F., Phyto-
chemistry, 95, 341–350 (2013).
14) Fouedjou R. T., Teponno R. B., Quassinti L., Bramucci M., Petrelli
D., Vitali L. A., Fiorini D., Tapondjou L. A., Barboni L., Phyto-
chem. Lett., 7, 62–68 (2014).
15) Tang L., Wang Z., Wu H., Yokosuka A., Mimaki Y., Phytochemis-
try, 107, 102–110 (2014).
16) Xu M., Zhang Y.-J., Li X.-C., Jacob M. R., Yang C.-R., J. Nat.
40) Mimaki Y., Kuroda M., Nakamura O., Sashida Y., J. Nat. Prod., 60,
592–597 (1997).