Journal of Natural Products
Note
from acetone. Fr. 12 (1.56 g) was chromatographed on a Sephadex
LH-20 column (3 × 57 cm) eluting with EtOH to give 16 (93 mg).
Fr. 15 (4.61 g) and Fr. 16 (2.34 g) were combined and then separated
on a silica gel column (2.6 × 20 cm) using 40% EtOAc/n-hexane as
the mobile phase to give subfractions 15A−15C. Fr. 15B (3.93 g) was
subjected to separation over a Sephadex LH-20 column (4.5 × 50
cm), eluting with 5% EtOAc/MeOH, to give Fr. 15B2, which
recrystallized from MeOH to afford 4 (340 mg). Fr.15C (1.06 g) was
subjected to a Sephadex LH-20 column (3 × 54 cm) eluting with
MeOH to give Fr. 15C1−15C4. Compound 22 (1.2 mg) was
obtained from Fr. 15C3 by semipreparative HPLC (C18, eluent: 30%
CH3CN/H2O, flow rate: 4 mL/min). Fr. 15C2 (0.76 g) was
chromatographed by semipreparative HPLC (C18, elution program:
40%−60%−60%−90% CH3CN/H2O during 0−7−21−22 min, flow
rate: 4 mL/min) to afford 4 (50 mg), 23 (22 mg), and Fr. 15C2m. Fr.
15C2m was further separated by preparative TLC (5% MeOH/
CHCl3) to afford 2 (12 mg) and 3 (15 mg). Fr. 17 (6.4 g) was
subjected to silica gel column chromatography (2.6 × 20 cm), eluting
with 80% EtOAc/n-hexane, to give subfractions 17A−17D. Fr. 17B
(1.96 g) was separated on a Sephadex LH-20 (5% EtOAc/methanol)
and semipreparative HPLC column (C18, eluent: 65% MeOH/H2O,
flow rate: 4.5 mL/min) to afforded 5 (4.0 mg), 6 (30 mg), and 7 (35
mg). Fr. 17D (889 mg) was separated on a Sephadex LH-20 (MeOH)
and semipreparative HPLC column (C18, eluent: 50% CH3CN/H2O,
flow rate: 4 mL/min) to give 8 (9.3 mg). The n-butanol extract (14.3
g) was separated by a Sephadex LH-20 column (7.2 × 65 cm) eluting
with EtOH to give seven fractions (Fr. B1−B7). Compounds 18 (1.17
g) and 19 (204 mg) precipitated successively from Fr. B4 and were
recrystallized further from MeOH. The filtrate of Fr. B4 was
chromatographed by semipreparative HPLC (C18, elution program:
25%−25%−90% CH3CN/0.1% formic acid during 0−10−20 min,
flow rate: 4 mL/min) to afford compounds 13 (15 mg), 17 (24 mg),
and 20 (3.2 mg). Fr. B5 was subjected to semipreparative HPLC (RP-
select B, eluent: 10% to 50% of CH3CN/0.1% formic acid over 30
min, flow rate: 4 mL/min) to give 12 (2.4 mg) and 15 (80 mg). Fr.
B6 (1.79 g) was chromatographed on a Sephadex LH-20 (MeOH)
and semipreparative HPLC column (RP-select B, eluent: 20%
CH3CN/0.1% formic acid, flow rate: 4 mL/min) to afford
compounds 9 (67 mg), 10 (20 mg), 11 (23 mg), and 14 (6.4 mg).
substituted, 7β-hydroxy, and 8β-methyl substituents as being
the same as those of sterebin A. Correspondingly, the structure
of 5 was established as shown.
1
Compounds 6 and 7 exhibited similar H and 13C NMR
spectra (Table 1) with signals stemming from a bisnorditer-
pene skeleton, corresponding to the framework of sterebin A
(8).9 HRESIMS analyses suggested that 6 and 7 have the
molecular formulas C23H36O5 and C23H38O5, respectively. In
1
the H NMR spectra of 6 and 7, the characteristic signals of
sterebin A were observed, along with an angeloyl group [δH
6.12 (dq, H-3′), 2.00 (d, H3-4′), and 1.91 (s, H3-5′)] in 6 and
an isovaleryl group [δH 2.17/2.33 (each d, H2-2′), 2.14 (m, H-
3′), and 0.98/0.99 (each d, H3-4′, 5′)] in 7, which were
confirmed by COSY, NOESY, HMQC, and HMBC experi-
ments. HMBC correlations from H-6 to C-1′ indicated the
location of the angeloyl group in 6 and the isovaleryl group in
7 at C(6)-O−, respectively. Thus, the structures of 6 and 7
were determined as 6α-O-angeloylsterebin A and 6α-O-
isovalerylsterebin A, respectively.
The isolated compounds were assessed for their inhibitory
effects on TNF-α expression in LPS-stimulated RAW 264.7
murine macrophages. The ELISA results showed compounds
9, 16, and 23 at 40 μM slightly suppressed the production of
TNF-α. The inhibition rates of compounds 9, 16, and 23 are
15.5 0.7%, 24 2.2%, and 27.5 5.1%, respectively. The
inhibitory effect of the isolated compounds on PD-L1
expression in LPS-activated MDSCs was also evaluated. The
results showed that LPS strongly up-regulated PD-L1
expression, but the test compounds showed no or little effect
on the expression of PD-L1 in granulocytic MDSCs
(gMDSCs).
EXPERIMENTAL SECTION
■
General Experimental Procedures. The instruments for
measuring optical rotations, recording UV, IR, NMR, ESIMS, and
HRESIMS spectra, and HPLC used to isolate components are the
same as reported previously.16 Silica gel (Merck, 63−200 μm; Fuji
Silysia, 40−70 μm) and Sephadex LH-20 were used for column and
gel filtration chromatography, respectively. Precoated silica gel TLC
plates (Merck, F254) were used for analytical or preparative thin-layer
chromatography, and spots were visualized by UV light (254 nm) or
by spraying with 20% H2SO4 in EtOH followed by heating. Cosmosil
5C18-AR-II (10 × 250 mm; particle size: 5 μm; Nacalai Tesque,
Kyoto, Japan) and LiChrospher 60 RP-select B (10 × 250 mm;
particle size: 5 μm; Merck KGaA, Darmstadt, Germany) columns
were used for semipreparative HPLC separation.
6α-O-Isovalerylnidorellol (1): [α]26 +56.6 (c 0.1, MeOH); UV
D
(MeOH) λmax (log ε) 224 (3.44) nm; IR (neat) νmax 3447, 2960,
1
2930, 1722, 1635, 1467, 1393, 1371, 1193, 1005 cm−1; H NMR
(CD3OD) see Table 1; 13C NMR (CD3OD) see Table 3; HRESIMS
m/z 321.2434 [M − C5H9O]+ (calcd for C20H33O3, 321.2424).
(12S)-Blumdane (2): [α]26 +28.0 (c 0.1, MeOH); UV (MeOH)
D
λmax (log ε) 223 (3.80) nm; IR (neat) νmax 3422, 2923, 1635, 1460,
1
1388, 1107, 1055, 1020 cm−1; H NMR (CD3OD) see Table 1; 13C
NMR (CD3OD) see Table 3; ESIMS m/z 361 [M + Na]+; HRESIMS
m/z 361.2359 [M + Na]+ (calcd for C20H34O4Na, 361.2349).
Reaction of 2 with (R)-MTPA.14,15 (R)-MTPA (25 mg) and thionyl
chloride (1 mL) were mixed and heated under reflux for 3 h. Then,
excess thionyl chloride was removed, a solution of 2 (8 mg) in 4 mL
of dry CH2Cl2, triethylamine (0.1 mL), and (dimethylamino)pyridine
(10 mg) were added, and the solution was stirred at room
temperature for 16 h. The reaction mixture was purified by
preparative TLC (1% MeOH/CHCl3) to yield the (R)-MTPA ester
2-RE: 1H NMR (CDCl3, 600 MHz) δ 1.18 (3H, s, H3-17), 5.00 (1H,
d, J = 9.6 Hz, H-12), 5.08 (1H, d, J = 11.4 Hz, H-15a), 5.32 (1H, d, J
= 18.0 Hz, H-15b), 5.13 (1H, s, H-16a), 5.28 (1H, s, H-16b), 6.32
(1H, dd, J = 10.8, 18.0 Hz, H-14).
Plant Material. The whole plant of B. aromatica was collected in
August 2017 in Hsinchu, Taiwan, and identified by Dr. Cheng-Jen
Chou, retired Research Fellow, National Research Institute of
Chinese Medicine, Taipei, Taiwan. A voucher specimen (NRICM-
30-BA-01) is deposited in the Herbarium of National Research
Institute of Chinese Medicine, Taipei, Taiwan.
Extraction and Isolation. The air-dried whole plant of B.
aromatica (1.8 kg) was cut into segments, extracted three times with
30 L of EtOH at 50 °C, and concentrated to afford an EtOH extract
(166.9 g). The EtOH extract was suspended in H2O (0.5 L) and then
extracted sequentially with n-hexane, EtOAc, and n-BuOH to give n-
hexane, EtOAc, n-BuOH, and H2O extracts, respectively. Due to
similar TLC profiles of n-hexane and EtOAc extracts, these extracts
were combined (as A-extract) for the following separation. A-extract
(61 g) was separated by silica gel column chromatography (8.5 × 55
cm, 94.5 g) using a gradient eluent of EtOAc in n-hexane (10% →
100%) to afford 20 fractions (Fr. 1−20). Fr. 6 (1.159 g) was
chromatographed by semipreparative HPLC (C18, eluent: 70%
CH3CN/H2O, flow rate: 4 mL/min) to give 1 (37 mg). Compound
21 (40 mg) was obtained from Fr. 7 and Fr. 8 after recrystallization
Reaction of 2 with (S)-MTPA.14,15 (S)-MTPA (25 mg) and thionyl
chloride (1 mL) were mixed and heated under reflux for 3 h. Then,
excess thionyl chloride was removed, a solution of 2 (8 mg) in 4 mL
of dry CH2Cl2, triethylamine (0.1 mL), and (dimethylamino)pyridine
(10 mg) were added, and the solution was stirred at room
temperature for 16 h. The reaction mixture was purified by
preparative TLC (1% MeOH/CHCl3) to yield the (S)-MTPA ester
2-SE: 1H NMR (CDCl3, 600 MHz) δ 1.16 (3H, s, H3-17), 5.04 (1H,
d, J = 9.6 Hz, H-12), 5.11 (1H, d, J = 12.0 Hz, H-15a), 5.33 (1H, d, J
D
J. Nat. Prod. XXXX, XXX, XXX−XXX