Steroidal Glycosides from AgaVe utahensis
Journal of Natural Products, 2009, Vol. 72, No. 8 1403
passage through an Amberlite IRA-93ZU column and was chromato-
graphed on silica gel eluted with CHCl -MeOH (9:1) to yield 1a (3.0
3
mg) and a sugar fraction (4.2 mg). The sugar fraction was passed
through a Sep-Pak C18 cartridge and a Toyopak IC-SP M cartridge,
which was then analyzed by HPLC under the following conditions:
column, Capcell Pak NH2 UG80 (4.6 mm i.d. × 250 mm, 5 µm,
2
Shiseido, Tokyo, Japan); solvent, MeCN-H O (17:3); flow rate, 0.9
mL/min; detection, RI and OR. Identification of D-galactose and
D-glucose present in the sugar fraction was carried out by comparison
of their retention times and specific optical rotations with those of
authentic samples.
2
8
Compound 2: amorphous solid; [R]
film) νmax 3363 (OH), 2927 (CH), 1075 cm ; H NMR (500 MHz,
N) δ 4.62 (1H, m, H-16), 4.37 (1H, br s, H-3), 1.16 (3H, d, J )
.9 Hz, Me-21), 0.85 (3H, s, Me-19), 0.83 (3H, s, Me-18), 0.70 (3H,
D
-10.0 (c 0.10; MeOH); IR
-1 1
(
5 5
C D
6
Figure 1. Induction of DNA fragmentation by 1 in HL-60 cells.
HL-60 cells were incubated at 37 °C for 18 h with 0, 10, or 20
µg/mL of 1 or 10 µg/mL of etoposide (E). DNA was then extracted
and applied to agarose gel electrophoresis. M: DNA marker.
1
3
d, J ) 5.3 Hz, Me-27), signals for the sugar moiety, see Table 1;
NMR, see Table 2; HRESITOFMS m/z 741.4417 [M + H] (calcd for
C
+
C
39
H
65
O
13, 741.4425).
Compound 3: amorphous solid; [R]
film) νmax 3376 (OH), 2928 (CH), 1072 cm ; H NMR (500 MHz,
N) δ 4.61 (1H, m, H-16), 4.32 (1H, br s, H-3), 1.15 (3H, d, J )
.9 Hz, Me-21), 0.87 (3H, s, Me-19), 0.82 (3H, s, Me-18), 0.69 (3H,
2
8
D
-44.0 (c 0.10; MeOH); IR
-1 1
(
5 5
C D
6
1
3
d, J ) 5.5 Hz, Me-27), signals for the sugar moiety, see Table 1;
NMR, see Table 2; HRESITOFMS m/z 903.4917 [M + H] (calcd for
C
+
C
45
H
75
O
18, 903.4953).
Compound 4: amorphous solid; [R]
film) νmax 3376 (OH), 2926 (CH), 1707 (CdO), 1074, 1030 cm ; H
NMR (500 MHz, C N) δ 4.55 (1H, m, H-16), 4.27 (1H, br s, H-3),
2
8
D
-10.0 (c 0.10; MeOH); IR
-1 1
(
5
D
5
2
.82 (1H, dd, J ) 8.6, 6.8 Hz, H-17), 2.37 (1H, dd, J ) 14.0, 14.0 Hz,
H-11a), 2.21 (1H, dd, J ) 14.0, 4.9 Hz, H-11b), 1.37 (3H, d, J ) 6.9
Hz, Me-21), 1.09 (3H, s, Me-18), 0.83 (3H, s, Me-19), 0.70 (3H, d, J
13
Figure 2. Caspase-3 activity in 1, 2, 3, or etoposide-treated HL-60
cell lysates. HL-60 cells were incubated at 37 °C for 6 h with 0,
) 5.9 Hz, Me-27), signals for the sugar moiety, see Table 1; C NMR,
see Table 2; HRESITOFMS m/z 777.3985 [M + Na] (calcd for
+
1
0, or 20 µg/mL of 1, 2, or 3, or 10 µg/mL of etoposide (E). Each
39 62
C H O14Na, 777.4037).
2
4
Compound 5: amorphous solid; [R]
film) νmax 3356 (OH), 2927 (CH), 1707 (CdO), 1074 cm ; H NMR
(500 MHz, C N) δ 4.55 (1H, m, H-16), 4.31 (1H, br s, H-3), 2.83
D
-4.0 (c 0.10; MeOH); IR
value represents mean ( SE from triplicate determinations.
-1 1
(
(
Sigma-Aldrich, St. Louis, MO); FBS (Bio-Whittaker, Walkersville,
5 5
D
MO); penicillin G sodium salt and streptomycin sulfate (Gibco, Grand
Island, NY). All other chemicals used were of biochemical reagent
grade.
Plant Material. A. utahensis plants were purchased from a garden
center at Japan Cactus Planning Co. (Fukushima, Japan) in 2000 and
identified by Dr. Yutaka Sashida, emeritus professor of the Tokyo
University of Pharmacy and Life Sciences. A voucher specimen has
been deposited in our laboratory (voucher no. AU-2000-001, Laboratory
of Medicinal Pharmacognosy).
Extraction and Isolation. The plant material (2.5 kg) was extracted
with hot MeOH twice (each 6 L). The MeOH extract was concentrated
under reduced pressure, and the concentrate (190 g) was passed through
a Diaion HP-20 column, successively eluted with 30% MeOH, 50%
MeOH, MeOH, EtOH, and EtOAc (each 4 L). The MeOH eluate was
chromatographed on silica gel and eluted with a stepwise gradient of
(1H, br t, J ) 7.8 Hz, H-17), 2.37 (1H, dd, J ) 13.8, 13.8 Hz, H-11a),
2.20 (1H, dd, J ) 13.8, 4.7 Hz, H-11b), 1.37 (3H, d, J ) 6.9 Hz,
Me-21), 1.09 (3H, s, Me-18), 0.85 (3H, s, Me-19), 0.70 (3H, d, J )
1
3
5.5 Hz, Me-27), signals for the sugar moiety, see Table 1; C NMR,
+
see Table 2; HRESITOFMS m/z 777.3981 [M + Na] (calcd for
39 62
C H O14Na, 777.4037).
2
4
Compound 6: amorphous solid; [R]
(film) νmax 3346 (OH), 2929 (CH), 1055 cm ; H NMR (500 MHz,
N) δ 4.60 (1H, m, H-16), 4.42 (1H, br s, H-3), 3.87 (1H, br d, J
D
-52.0 (c 0.10; MeOH); IR
-1 1
5 5
C D
) 12.7 Hz, H-2), 1.14 (3H, d, J ) 6.9 Hz, Me-21), 0.84 (3H, s, Me-
19), 0.81 (3H, s, Me-18), 0.70 (3H, d, J ) 5.5 Hz, Me-27), signals for
1
3
the sugar moiety, see Table 1; C NMR, see Table 2; HRESITOFMS
+
m/z 757.4426 [M + H] (calcd for C39
H
D
65
5
O
14, 757.4374).
2
Compound 7: amorphous solid; [R]
-34.0 (c 0.10; MeOH); IR
-
1 1
(film) νmax 3348 (OH), 2926 (CH), 1073 cm ; H NMR (500 MHz,
5 5
C D N) δ 4.59 (1H, m, H-16), 4.46 (1H, br s, H-3), 3.88 (1H, m, H-2),
CHCl
yielding fractions (I-V). Fraction II was chromatographed on silica
gel eluted with CHCl -MeOH-H O (60:10:1; 50:10:1) and ODS silica
gel with MeOH-H O (8:3; 4:1) and CH CN-H O (5:7) to give 1 (150
3
-MeOH (9:1; 4:1; 3:1; 2:1; 1:1) and finally with MeOH alone,
1.13 (3H, d, J ) 6.9 Hz, Me-21), 0.90 (3H, s, Me-19), 0.80 (3H, s,
Me-18), 0.69 (3H, d, J ) 5.5 Hz, Me-27), signals for the sugar moiety,
3
2
1
3
2
3
2
see Table 1; C NMR, see Table 2; HRESITOFMS m/z 941.4734 [M
+
mg), 2 (14 mg), 3 (820 mg), 4 (13 mg), 5 (4.5 mg), 6 (78 mg), 7 (12
mg), 8 (8.5 mg), and 9 (50 mg). Fraction IV was subjected to CC on
+ Na] (calcd for C45
Compound 8: amorphous solid; [R]
(film) νmax 3375 (OH), 2928 (CH), 1074 cm ; H NMR (500 MHz,
5 5
C D N) δ 4.61 (1H, m, H-16), 4.34 (1H, br s, H-3), 3.82 (1H, br d, J
74
H O19Na, 941.4722).
2
5
D
-2.0 (c 0.10; MeOH); IR
-1 1
silica gel eluted with CHCl
3
-MeOH-H
2
O (20:10:1; 7:4:1) and ODS
CN-H O (1:2; 1:3) to
silica gel with MeOH-H O (2:1; 4:1) and CH
2
3
2
give 10 (42 mg) and 11 (7.0 mg). The furostanol saponins 9 and 11
were obtained as mixtures of C-22 methoxy and C-22 hydroxy forms.
The C-22 hydroxy form was converted to the C-22 methoxy form by
treatment with hot MeOH, and the structure elucidation was carried
out with the C-22 methoxy form.
) 12.8 Hz, H-2), 1.14 (3H, d, J ) 6.8 Hz, Me-21), 0.99 (3H, s, Me-
19), 0.82 (3H, s, Me-18), 0.70 (3H, d, J ) 4.9 Hz, Me-27), signals for
1
3
the sugar moiety, see Table 1; C NMR, see Table 2; HRESITOFMS
+
m/z 941.4689 [M + Na] (calcd for C45
74
H O19Na, 941.4722).
Acid Hydrolysis of 2-8. Compounds 2 (7.0 mg), 3 (10 mg), 4 (13
mg), 5 (4.6 mg), 6 (10 mg),7 (10 mg), and 8 (6.0 mg) were
independently subjected to acid hydrolysis as described for 1 to give
aglycones (1a: 3.5 and 2.9 mg from 2 and 3, 4a: 7.0 and 1.7 mg from
4 and 5, 6a: 4.0, 4.8, and 2.4 mg from 6, 7, and 8, respectively) and a
sugar fractions (2: 2.6 mg, 3: 4.3 mg, 4: 5.0 mg, 5: 1.6 mg, 6: 3.8 mg,
7: 4.1 mg, 8: 3.3 mg). HPLC analysis of the sugar fractions under the
same conditions as in the case of 1 showed the presence of D-galactose
and D-glucose in those of 2-8.
2
8
Compound 1: amorphous solid; [R]
film) νmax 3347 (OH), 2926 (CH), 1058 cm ; H NMR (500 MHz,
N) δ 4.63 (1H, m, H-16), 4.31 (1H, br s, H-3), 1.15 (3H, d, J )
.9 Hz, Me-21), 0.82 (3H, s, Me-19), 0.82 (3H, s, Me-18), 0.70 (3H,
D
-48.0 (c 0.10; MeOH); IR
-1 1
(
5 5
C D
6
1
3
d, J ) 5.6 Hz, Me-27), signals for the sugar moiety, see Table 1;
NMR, see Table 2; HRESITOFMS m/z 741.4417 [M + H] (calcd for
13, 741.4425).
Acid Hydrolysis of 1. A solution of 1 (9.7 mg) in 1 M HCl
dioxane-H O, 1:1; 2 mL) was heated at 95 °C for 2 h under an Ar
atmosphere. After cooling, the reaction mixture was neutralized by
C
+
C
39
H
65
O
2
3
(
2
Compound 9: amorphous solid; [R]
D
-36.0 (c 0.10; MeOH); IR
-1 1
(film) νmax 3364 (OH), 2929 (CH), 1069 cm ; H NMR (500 MHz,