Steroidal Alkaloid Oligoglycosides from Solanum lycocarpum
Journal of Natural Products, 2007, Vol. 70, No. 2 213
Extraction and Isolation. The dried fruits of S. lycocarpum (8.7
kg) were extracted three times with MeOH under reflux. Evaporation
of the solvent under reduced pressure provided the MeOH extract (1203
g, 13.8%). The MeOH extract (380 g) was subjected to Diaion HP-20
column chromatography [3.0 kg, H2O f MeOH f acetone] to give
H2O-, MeOH-, and acetone-eluted fractions (275, 85, and 12 g,
respectively). The MeOH-eluted fraction (70 g) was subjected to
normal-phase silica gel column chromatography [2.1 kg, CHCl3-
MeOH-H2O (40:10:1 f 30:10:1f 6:4:1, v/v/v) f MeOH] to give
10 fractions [1 (3.8 g), 2 (2.2 g), 3 (1.0 g), 4 (0.2 g), 5 (1.6 g), 6 (1.6
g), 7 (13.0 g), 8 (18.0 g), 9 (28.0 g), and 10 (2.0 g)].
1.80 (2H, m, H2-11), 1.85 (1H, m, Ha-2), 1.92 (1H, m, Hb-7), 1.92
(1H, m, Hb-23), 1.98 (1H, m, H-8), 2.06 (1H, m, Hb-2), 2.08 (1H, m,
H-20), 2.13 (1H, m, H-14), 2.13 (1H, m, Hb-15), 2.72 (1H, dd, J )
11.2, 13.8 Hz, Ha-4), 2.79 (1H, dd, J ) 4.1, 13.8 Hz, Hb-4), 2.88 (2H,
m, H2-26), 3.07 (1H, m, H-17), 3.90 (1H, m, H-5′′′), 3.92 (1H, m,
H-5′), 3.96 (1H, m, H-3), 3.96 (1H, m, H-12), 3.96 (1H, m, H-2′′′),
4.12 (1H, dd, J ) 8.9, 9.2 Hz, H-3′′′), 4.16 (1H, dd, J ) 8.6, 9.2 Hz,
H-4′′′), 4.25 (1H, m, Ha-6′), 4.25 (1H, m, H-4′′), 4.25 (1H, m, Ha-6′′′),
4.27 (1H, m, H-3′), 4.33 (1H, dd, J ) 6.6, 11.2, Hb-6′′′), 4.43 (1H, dd,
J ) 2.3, 11.8 Hz, Hb-6′), 4.56 (1H, dd, J ) 3.5, 9.5 Hz, H-3′′), 4.60
(1H, m, H-16), 4.64 (1H, dd, J ) 8.0, 9.5 Hz, H-2′), 4.78 (1H, brd, J
≈ 2.0, H-4′), 4.86 (1H, brs, H-2′′), 4.88 (1H, d, J ) 8.0 Hz, H-1′),
4.88 (1H, m, H-5′′), 5.15 (1H, d, J ) 7.7 Hz, H-1′′′), 5.35 (1H, d, J )
4.9 Hz, H-6), 6.24 (1H, brs, H-1′′); 13C NMR (pyridine-d5, 125 MHz),
see Table 2; positive-ion FABMS m/z 922 [M + Na]+; negative-ion
FABMS m/z 898 [M - H]-, 736 [M - C6H11O5]-; HRFABMS m/z
922.4792 (calcd for C45H73NO17Na [M + Na]+, 922.4776).
Acid Hydrolysis of 2 and 4. Solutions of 2 and 4 (each 5.0 mg) in
5% aqueous H2SO4-1,4-dioxane (1:1, v/v) (1.0 mL) were each heated
under reflux for 3 h. After cooling, each reaction mixture was poured
into ice-water and neutralized with Amberlite IRA-400 (OH- form),
and the resin was removed by filtration. Then, the filtrate was extracted
with EtOAc. The aqueous layer was subjected to HPLC analysis under
the following conditions: HPLC column, Kaseisorb LC NH2-60-5, 4.6
mm i.d. × 250 mm (Tokyo Kasei Co., Ltd., Tokyo, Japan); detection,
optical rotation [Shodex OR-2 (Showa Denko Co., Ltd., Tokyo, Japan)];
mobile phase, CH3CN-H2O (85:15, v/v); flow rate, 0.8 mL/min;
column temperature, room temperature. Identifications of (i) l-rhamnose
from 2 and 4, (ii) D-glucose from 2 and 4, and (iii) D-galactose from 4,
present in the aqueous layer, were carried out by comparison of their
retention times and optical rotations with those of authentic samples.
tR: (i) 9.6 min (negative optical rotation); (ii) 13.5 min (positive optical
rotation); (iii) 14.0 min (positive optical rotation).
Fraction 1 (3.8 g) was subjected to reversed-phase silica gel column
chromatography [100 g, MeOH-H2O (50:50 f 70:30 f 90:10, v/v)
f MeOH] to afford seven fractions [1-1 (300 mg), 1-2 (500 mg), 1-3
(720 mg), 1-4 (γ-linolenic acid, 500 mg, 0.02%), 1-5 (1040 mg), 1-6
(linoleic acid, 500 mg, 0.02%), and 1-7 (220 mg)]. Fraction 7 (2 g)
was subjected to reversed-phase silica gel column chromatography [60
g, MeOH-H2O (50:50, v/v) f MeOH] to afford two fractions [7-1
(980 mg) and 7-2 (solamargine, 1, 1.02 g, 0.29%)]. Fraction 8 (2.0 g)
was subjected to reversed-phase silica gel column chromatography [60
g, MeOH-H2O (30:70 f 50:50 f 70:30, v/v) f MeOH] to afford
seven fractions [8-1 (980 mg), 8-2 (60 mg), 8-3 (10 mg), 8-4
(solasonine, 3, 550 mg, 0.22%), 8-5 (30 mg), 8-6 (solamargine, 1, 330
mg, 0.13%), and 8-7 (20 mg)]. Fraction 8-5 (30 mg) was purified by
HPLC [MeOH-H2O (60:40, v/v)] to furnish solasonine (3, 5 mg,
0.002%) and robeneoside A (2, 5 mg, 0.002%). Fraction 9 (3.0 g) was
subjected to reversed-phase silica gel column chromatography [60 g,
MeOH-H2O (30:70 f 60:40, v/v) f MeOH] to afford seven fractions
[9-1 (1520 mg), 9-2 (220 mg), 9-3 (200 mg), and 9-4 (solasonine, 3,
980 mg, 0.40%)]. Fraction 9-3 (200 mg) was purified by HPLC
[MeOH-H2O (60:40, v/v)] to furnish solasonine (3, 91 mg, 0.04%)
and robeneoside A (2, 15 mg, 0.006%). Fraction 10 (2.0 g) was
subjected to reversed-phase silica gel column chromatography [60 g,
MeOH-H2O (10:90 f 30:70 f 50:50, v/v) f MeOH] to afford
six fractions [10-1 (1100 mg), 10-2 (220 mg), 10-3 (100 mg), 10-4
(130 mg), 10-5 (solasonine, 3, 300 mg, 0.01%), and 10-6 (160 mg)].
Fraction 10-4 (130 mg) was purified by HPLC [MeOH-H2O (30:70,
v/v)] to furnish 12-hydroxysolasonine (5, 20 mg, 0.0009%) and
robeneoside B (4, 12 mg, 0.0005%). The known compounds (1, 3, and
5), γ-linolenic acid, and linoleic acid were identified by comparison
of their 1H NMR, 13C NMR, and MS with reported values or
commercial samples.
Bioassay. Animals. Male Wistar rats weighing about 130-150 g
and male ddY mice weighing about 30-32 g were purchased from
Kiwa Laboratory Animal Co., Ltd., Wakayama, Japan. The animals
were housed at a constant temperature of 23 ( 2 °C and were fed a
standard laboratory chow (MF, Oriental Yeast Co., Ltd., Tokyo, Japan).
The animals were fasted for 20-24 h prior to the beginning of the
experiment, but were allowed free access to tap water. All experiments
were performed with conscious rats or mice unless otherwise noted.
Each test sample was suspended in 5% acacia solution, and the solution
was orally administered at 10 mL/kg in each experiment, while the
vehicle was administered orally at 10 mL/kg in the corresponding
control groups. The experimental protocols were approved by Experi-
mental Animal Research Committee at Kyoto Pharmaceutical Univer-
sity.
Robeneoside A (2): white powder (CHCl3-MeOH); mp 205-
29
208 °C; [R]D -112.2 (c 1.0, pyridine); IR (KBr) νmax 3450, 2941,
1
1626, 1280, 1071, 1046, 980 cms1; H NMR (pyridine-d5, 500 MHz)
δ 0.82 (3Η, d, J ) 5.2 Hz, H3-27), 0.99 (3H, s, H3-18), 1.01 (1H, ddd,
J ) 3.8, 13.8, 13.8 Hz, Ha-1), 1.10 (3H, s, H3-19), 1.18 (3Η, d, J )
7.2 Hz, H3-21), 1.61, (1H, m, Ha-15), 1.64 (2H, m, H2-24), 1.64 (3H,
d, J ) 6.0 Hz, H3-6′′′), 1.67 (1H, m, Ha-7), 1.70 (1H, m, H-9), 1.70
(1H, m, H-25), 1.74 (1H, m, Hb-1), 1.77 (1H, m, Ha-23), 1.77 (3H, d,
J ) 6.3 Hz, H3-6′′), 1.85 (1H, m, Ha-2), 1.85 (2H, m, H2-11), 1.85
(1H, m, Hb-23), 1.94 (1H, m, Hb-7), 1.94 (1H, m. H-8), 2.07 (1H, qd,
J ) 7.2, 7.7 Hz, H-20), 2.08 (1H, m, Hb-2), 2.17 (1H, m, H-14), 2.17
(1H, m, Hb-15), 2.74 (1H, dd, J ) 11.2, 13.8 Hz, Ha-4), 2.81 (1H, dd,
J ) 4.3, 13.8 Hz, Hb-4), 2.83 (2H, m, H2-26), 3.08 (1H, dd, J ) 7.7,
7.8 Hz, H-17), 3.62 (1H, m, H-5′), 3.85 (1H, m, H-3), 3.98 (1H, brs,
H-12), 4.08 (1H, dd, J ) 3.4, 12.3 Hz, Ha-6′), 4.20 (1H, brd, J ≈ 12.0
Hz, Hb-6′), 4.22 (1H, m, H-2′), 4.22 (1H, m, H-3′), 4.34 (1H, dd, J )
9.4, 9.5 Hz, H-4′′′), 4.37 (1H, dd, J ) 9.2, 9.5 Hz, H-4′′), 4.39 (1H,
dd, J ) 8.9, 9.2 Hz, H-4′), 4.54 (1H, dd, J ) 3.4, 9.4 Hz, H-3′′′), 4.58
(1H, brd, J ≈ 8.0 Hz, H-16), 4.63 (1H, dd, J ) 3.5, 9.2 Hz, H-3′′),
4.68 (1H, dd, J ) 1.5, 3.4 Hz, H-2′′′), 4.83 (1H, dd, J ) 1.2, 3.5 Hz,
H-2′′), 4.93 (1H, m, H-5′′′), 4.93 (1H, d, J ) 7.2 Hz, H-1′), 4.96 (1H,
m, H-5′′), 5.34 (1H, d, J ) 4.9 Hz, H-6), 5.86 (1H, brs, H-1′′′), 6.40
(1H, brs, H-1′′); 13C NMR (pyridine-d5, 125 MHz), see Table 2;
positive-ion FABMS m/z 906 [M + Na]+; negative-ion FABMS m/z
882 [M - H]-; HRFABMS m/z 906.4824 (calcd for C45H73NO16Na
[M + Na]+, 906.4828).
Serum Glucose in Sucrose-Loaded Rats. Rats weighing about
130-150 g were fasted for 20-24 h. The test compounds suspended
in 5% acacia solution were given orally, and the vehicle was given
orally in the corresponding control groups. Thirty minutes later, 5 mL/
kg of 20% (w/v) sucrose was administered orally (po). Water was given
orally instead of sucrose in a normal group. Blood samples were
collected at 0.5, 1, and 2 h after sucrose loading. The blood was
centrifuged to obtain serum, and serum glucose levels were determined
enzymatically by the glucose-oxidase method (glucose CII-test Wako,
Wako Pure Chemical Industries).
Gastric Emptying in Mice. A solution of 1.5% carboxymethyl
cellulose sodium salt (CMC-Na) containing 0.05% phenol red as a
marker was given intragastrically (0.5 mL/mouse) to conscious mice.
Thirty minutes later, mice were sacrificed by cervical dislocation. The
abdominal cavity was opened, and the gastroesophageal junction and
the pylorus were clamped, then the stomach was removed, weighed,
placed in 14 mL of 0.1 M NaOH, and homogenized. The suspension
was allowed to settle for 1 h at room temperature, and 5 mL of the
supernatant was added to 0.5 mL of 20% trichloroacetic acid (w/v)
and then centrifuged at 3000 rpm for 20 min. The supernatant was
mixed with 4 mL of 0.5 M NaOH, and the amount of phenol red was
determined from the absorbance at 560 nm. Phenol red recovered from
animals sacrificed immediately after the administration of the CMC-
Na solution was used as standards (0% emptying). Gastric emptying
(%) in the 30 min period was calculated according to the following
equation:
Robeneoside B (4): white powder (CHCl3-MeOH); mp 206-
210 °C; [R]D29 -22.2 (c 1.0, pyridine); IR (KBr) νmax 3450, 2938, 1622,
1300, 1071, 1046, 980 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 0.81
(3H, brs, H3-27), 0.97 (1H, m, Ha-1), 0.97 (3H, s, H3-18), 1.09 (3H, s,
H3-19), 1.20 (3H, brs, H3-21), 1.60 (1H, m, Ha-15), 1.62 (1H, m, Ha-
7), 1.62 (2H, m, H2-24), 1.64 (1H, m, H-9), 1.67 (3H, d, J ) 6.0 Hz,
H3-6′′), 1.72 (1H, m, Hb-1), 1.76 (1H, m, Ha-23), 1.76 (1H, m, H-25),