Isolation, Structural Elucidation, Optical Resolution, and Antineuroinflammatory Activity of Phenanthrene and 9,10-Dihydrophenanthrene Derivatives from Bletilla striata

Article abstract of DOI:10.1021/acs.jnatprod.9b00291

A phytochemical investigation of the aqueous EtOH extract of Bletilla striata tubers afforded 34 phenanthrene and 9,10-dihydrophenanthrene derivatives, including four new compounds, 1-4. These compounds were identified using physicochemical analyses and various spectroscopic methods. Twelve of these compounds were resolved into their enantiomers, and the absolute configurations were determined by comparison of experimental and calculated ECD spectra. The antineuroinflammatory activities were evaluated by measuring the inhibition of nitric oxide production in lipopolysaccharide-stimulated BV-2 microglial cells. Compounds 7, 32, and 33 displayed inhibitory activities, with IC₅₀ values of 1.9, 5.0, and 1.0 μM, respectively, suggesting that they should be subjected to development as potential inhibitors of neuroinflammation.

Full text of DOI:10.1021/acs.jnatprod.9b00291

Article
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Isolation, Structural Elucidation, Optical Resolution, and
Antineuroinflammatory Activity of Phenanthrene and 9,10-
Dihydrophenanthrene Derivatives from Bletilla striata
Di Zhou,†,§ Gang Chen,† Yue-Ping Ma,† Cun-Gang Wang,† Bin Lin,‡ Yan-Qiu Yang,⊥ Wei Li,†,§
Kazuo Koike,§ Yue Hou,*,⊥ and Ning Li*,†,∥
‡
†School of Traditional Chinese Materia Medica and School of Pharmaceutical Engineering, Shenyang Pharmaceutical University,
Shenyang 110016, People’s Republic of China
§Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
⊥College of Life and Health Sciences, Northeastern University, Shenyang 110004, People’s Republic of China
∥State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin
541004, People’s Republic of China
S
* Supporting Information
ABSTRACT: A phytochemical investigation of the aqueous EtOH extract of Bletilla striata tubers afforded 34 phenanthrene
and 9,10-dihydrophenanthrene derivatives, including four new compounds, 1−4. These compounds were identified using
physicochemical analyses and various spectroscopic methods. Twelve of these compounds were resolved into their enantiomers,
and the absolute configurations were determined by comparison of experimental and calculated ECD spectra. The
antineuroinflammatory activities were evaluated by measuring the inhibition of nitric oxide production in lipopolysaccharide-
stimulated BV-2 microglial cells. Compounds 7, 32, and 33 displayed inhibitory activities, with IC50 values of 1.9, 5.0, and 1.0
μM, respectively, suggesting that they should be subjected to development as potential inhibitors of neuroinflammation.
As part of our ongoing investigations aiming at discovery of
natural inhibitors of neuroinflammation,4,5 it was found that an
extract from the tubers of B. striata exerted significant
antineuroinflammatory activities on overactivated microglial
cells. Thus, a chemical investigation was conducted and resulted
in the isolation and structural elucidation of phenanthrene and
9,10-dihydrophenanthrene derivatives (1−34), including four
new compounds (1−4). Twelve of these compounds were
resolved into their enantiomers, and the absolute configurations
were determined using experimental and calculated ECD data.
The antineuroinflammatory activities of the isolated compounds
were evaluated by measuring the inhibition of nitric oxide (NO)
production in lipopolysaccharide (LPS)-stimulated BV-2 micro-
glial cells. The structure−activity relationship is also discussed.
henanthrenes and 9,10-dihydrophenanthrenes are a class of
Pnatural products with limited distribution in the Orchid-
aceae, Dioscoreaceae, Juncaceae, and Combretaceae families.1
These compounds display diverse and promising biological
activities, including anti-inflammatory, antiproliferative, anti-
microbial, spasmolytic, antiplatelet aggregative, antioxidant, and
antiallergic activities.2
Bletilla striata (Thunb.) Reichb. f. (Orchidaceae) is a
perennial herbaceous plant that is widely distributed in East
Asia. The tubers have been applied in traditional Chinese
medicine to relieve swelling, hematemesis, and traumatic
bleeding, as well as to treat skin diseases, such as ulcers, sores,
and chapped skin.3 According to previous phytochemical
studies, phenanthrenes and 9,10-dihydrophenanthrenes are
characteristic constituents of B. striata that possess anti-
inflammatory, antiproliferative, and antimicrobial activities.2
Received: March 29, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b00291
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Figure 1. Structures of 9,10-dihydrophenanthrene derivatives.
dihydrophenanthrene (13),13 lusianthridin (14),14 erianthridin
(15),15 coelonin (16),16 shancigusin G (17),17 shancidin
(18),18 1-(4-hydroxybenzyl)-4-methoxy-9,10-dihydrophenan-
threne-2,7-diol (19),19 1-(4-hydroxybenzyl)-4,7-dimethoxy-
9,10-dihydrophenanthrene-2,8-diol (20),20 2,7-dihydroxy-l-
(4′-hydroxybenzyl)-9,10-dihydrophenanthrene-4′-O-glucoside
(21),9 blestriarene B (22),21 4,4′-dimethoxy-9,10-dihydro-
(6,10-biphenanthrene)-2,2′,7,7′-tetraol (23),11 gymconopin C
(24),12 blestriarene A (25),21 flavanthrinin (26),22 4-methox-
yphenanthrene-2,3,7-triol (27),23 nudol (28),24 1-(4-hydrox-
ybenzyl)-4,7-dimethoxyphenanthrene-2-ol (29),25 1-(4-hydrox-
ybenzyl)-4-methoxyphenanthrene-2,7-diol (30),22 1-(4-hydrox-
ybenzyl)-4,7-dimethoxyphenanthrene-2,8-diol (31),25
bleformin F (32),26 4,8,4′,8′-tetramethoxy-(1,1′-biphenan-
threne)-2,7,2′,7′-tetrol (33),11 and monbarbatain A (34)13
(Figures 1 and 2). Four new compounds (1−4) were named
RESULTS AND DISCUSSION
■
The tubers of B. striata were extracted by refluxing in 95%
aqueous EtOH. The extract was concentrated and sequentially
leached with petroleum ether, EtOAc, and n-BuOH. The EtOAc
fraction, which was enriched in phenanthrenes and 9,10-
dihydrophenanthrenes, significantly decreased NO accumula-
tion in LPS-stimulated BV-2 microglial cells (IC50 72.7 μg/mL).
Further isolation and purification were performed using multiple
chromatographic methods to afford 34 compounds. The
structures of the following known compounds were identified
using comparison of observed and reported physicochemical
data: shanciol H (5),6 3-(4-hydroxybenzyl)-4-methoxy-9,10-
dihydrophenanthrene-2,7-diol (6),7 phochinenin K (7),8
bletilol C (8),9 bletilol B (9),9 pleionesin C (10),10 (2,3-
trans)-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-10-
methoxy-2,3,4,5-tetrahydrophenanthro[2,1-b]furan-7-ol
(11),11 cyrtonesin B (12),12 1,4,7-trihydroxy-2-methoxy-9,10-
B
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bletillatins A−D, respectively, and their detailed structures were
elucidated as described below.
Bletillatin A (1) was isolated as a brownish oil. The molecular
formula C25H24O6 was established based on the 13C NMR data
and a deprotonated molecular ion [M − H]− at m/z 419.1477
(calcd 419.1500 for C25H23O6) in the HRESIMS data. The 1H
NMR spectrum displayed characteristic resonances for two
methylene groups at δH 2.63 (2H, m, Ha,b-9) and 2.64 (2H, m,
Ha,b-10), which revealed the presence of a 9,10-dihydrophenan-
threne skeleton (Table 1). A pair of meta-coupled aromatic
proton resonances at δH 6.31 (1H, d, J = 2.3 Hz, H-1) and δH
6.40 (1H, d, J = 2.3 Hz, H-3) was assigned to the A-ring based on
the HMBC correlations from H-1 to C-10 and C-4a and from H-
3 to C-4a (Figure 3). A pair of aromatic proton resonances at δH
6.67 (1H, s, H-8) and 8.06 (1H, s, H-5) was assigned to the
tetrasubstituted B-ring based on the HMBC correlations from
H-5 to C-4a and C-8a and from H-8 to C-5a and C-9. In
addition, a set of ABX-type aromatic proton resonances was
Figure 2. Structures of the phenanthrene derivatives.
Table 1. 1H (600 MHz) and 13C (150 MHz, Methanol-d4) Data for Compounds 1−4
1
2
3
4
position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
1
2
3
4
4a
5
5a
6
6.31, d (2.3)
108.3
157.6
99.3
159.0
140.3
118.1 6.52, s
156.8
99.6
158.2
117.5
130.4 8.04, d (9.1)
126.0
113.5 6.64, dd (9.1, 2.8)
111.6
155.6
121.8 6.56, s
158.1
120.3
128.9 8.04, d (8.6)
126.1
116.3
158.9
99.1
154.9
141.4
130.3
140.7
113.5
6.40, d (2.3)
8.06, s
6.46, s
125.8 7.88, d (8.5)
142.0
125.4 6.59, dd (8.5, 2.7)
115.2 6.62, dd (8.6,
2.3)
7
8
8a
9
10
159.1
109.0 6.62, d (2.7)
127.3
31.6
31.8
156.2
114.4 6.63, br s
140.6
30.9
27.2
156.6
114.3 6.58, d (2.3)
140.6
31.2
31.4
156.0
114.7
117.3
28.4
6.67, s
a
a
2.63, m
2.66, m
2.59
2.63
2.63
2.57, m
2.50, m
a
2.79, dd (14.3, 6.5); 2.70, dd
(14.3, 7.1)
31.7
10a
1′
2′
3′
4′
5′
6′
7′
8′
9′
116.9
135.4
110.4
140.3
181.3
139.8
134.5
130.5
117.4
157.1 6.36 d (2.9)
117.4
126.6
144.7
117.3
160.3
98.2
6.94, d (1.9)
7.19, d (8.7)
6.95, d (8.7)
a
a
149.5 2.59; 2.45
147.4 2.45; 2.28 m
116.1 5.36, dd (8.7, 6.0)
119.6
88.7
55.2
65.6
32.3
27.5
53.2
a
6.76, d (8.2)
6.83, dd (8.2, 1.9)
5.49, d (5.5)
3.46, dd (7.7, 5.5)
3.84, dd (11.0, 5.5); 3.76, dd
(11.0, 7.7)
6.95, d (8.7)
7.19, d (8.7)
3.96, s
158.2
109.2
130.5 6.37d (2.9)
27.4
Glc
1″
2″
3″
4″
4.83, d (7.6)
3.41, t (7.6)
3.42
102.5
145.2
116.1
158.2
113.6
75.0
78.0
71.4
6.39 d (2.9)
a
3.36, m
6.49, dd (8.0,
2.0)
5″
6″
3.38, m
3.85, dd (12.0, 2.0); 3.65, dd
(12.0, 5.3)
78.1
62.5
6.93, t (8.0)
6.38, dd (8.0,
2.9)
130.2
120.7
α
2.27, t (6.7)
2.47, t (6.7)
3.63, s
38.1
37.7
55.9
α′
3′-
3.81, s
3.82, s
56.4
OCH3
5′-
3.89, s
56.0
OCH3
4-
55.9
3.79, s
55.8
3.28, s
60.3
OCH3
a
Overlapped resonances.
C
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Figure 4. Key NOESY correlations of compounds 1 and 2.
closely matched the experimental data for compounds 1a and
1b, respectively, particularly the Cotton effects (CEs) in the
region of 205−250 nm, which showed different patterns
compared to their diastereomers. Based on the aforementioned
evidence, bletillatin A (1) was elucidated as an enantiomeric
mixture of (aS,7′S,8′R)- and (aR,7′R,8′S)-2-hydroxy-4-me-
thoxy-7′-(4-hydroxy-3-methoxyphenyl)-8′-hydroxymethyl-
7′,8′,9,10-tetrahydrofuro[2,3-b]phenanthrene.
Bletillatin B (2) was isolated as a brownish oil, and the
molecular formula was determined to be C19H19NO4 by the
protonated molecular ion peak at m/z 326.1375 [M + H]+
observed in the HRESIMS data (calcd 326.1387 for
C19H20NO4) and the 13C NMR data. Compound 2 was also a
9,10-dihydrophenanthrene derivate based on the characteristic
resonances of two methylene groups at δH 2.59 (2H, m, Ha,b-9),
2.70 (1H, dd, J = 14.3, 7.1 Hz, Ha-10), and 2.79 (1H, dd, J = 14.3,
Figure 3. Key HMBC correlations of compounds 1−4.
observed at δH 6.76 (1H, d, J = 8.2 Hz, H-5′), 6.83 (1H, dd, J =
8.2, 1.9 Hz, H-6′), and 6.94 (1H, d, J = 1.9 Hz, H-2″), as well as
the resonances due to a three-carbon unit, including an
oxymethine at δH5.49 (1H, d, J = 5.5 Hz, H-7′), a methine at
δH 3.46 (1H, dd, J = 7.7, 5.5 Hz, H-8′), and an oxymethylene at
δH 3.84 (1H, dd, J = 11.0, 5.5 Hz, Ha-9′) and δH 3.76 (1H, J =
11.0, 7.7 Hz, Hb-9′). The connection of the aromatic ring to C-7′
was deduced from the HMBC correlations from H-7′ to C-2′
and C-6′ and from H-8′ to C-1′. Furthermore, the connectivity
of C-8′ and C-6 was deduced from the HMBC correlations from
H-8′ to C-5 and C-7, and the connectivity of C-7′ and C-7
through an ether linkage was based on the HMBC correlations
from H-7′ to C-7. Finally, two methoxy moieties that resonated
at δH 3.81 (3H, s, 3′-OCH3) and 3.82 (3H, s, 4-OCH3) were
assigned to C-3′ and C-4 by the NOESY correlations between
3′-OCH3/H-2′ and 4-OCH3/H-3, respectively. The HMBC
correlations from 3′-OCH3(δH 3.81) and H-2′ (δH 6.95) to C-3′
(δC 149.5) and from 4-OCH3 (δH 3.82) and H-3 (δH 6.40) to C-
4 (δC 159.0) were used to confirm the assignments. The 7′,8′-
trans relative configuration was assigned via the NOESY
correlations between H-7′/Ha,b-9′, H-8′/H-2′, and H-8′/H-6′
(Figure 4). The coupling constant (JH‑7′,H‑8′ = 5.5 Hz) also
supported the conclusion.24
Compound 1 was a racemic mixture based on the specific
rotation value ([α]20D = 0). Considering the axial chirality of the
biphenyl bond, four enantiomerically pure compounds were
possible, namely, (aS,7′S,8′R)-, (aR,7′R,8′S)-, (aS,7′R,8′S)-,
and (aR,7′S,8′R)-1 (Figure 5A). The resolution of compound 1
was achieved using chiral-phase column chromatography to
afford two well-separated peaks (1a and 1b) in a peak area ratio
of 1:1 (Figure 5B). Compounds 1a and 1b are a pair of
enantiomers since they showed opposite specific rotations and
antipodal ECD curves. The absolute configurations of
compounds 1a and 1b were defined by comparing the
experimental and calculated ECD spectra (Figure 5C). The
calculated ECD spectra of (aS,7′S,8′R)-1 and (aR,7′R,8′S)-1
1
7.1 Hz, Hb-10). In the H NMR data, an aromatic proton
resonance at δH 6.46 (1H, s, H-3) assigned to the A-ring and a
set of ABX-type aromatic proton resonances [δH 6.59 (1H, dd, J
= 8.5, 2.7 Hz, H-6), 6.62 (1H, d, J = 2.7 Hz, H-8), 7.88 (1H, d, J
= 8.5 Hz, H-5)] assigned to the B-ring were deduced from the
HMBC correlations from H-3 to C-4a, H-5 to C-4a and C-8a,
and H-8 to C-5a and C-9. A methoxy moiety was assigned to C-4
by the NOESY correlation between 4-OCH3 and H-3. In
addition, HMBC correlations were observed from 4-OCH3 (δH
3.79) to C-4 (δC 158.2). The presence of a 5′-substituted
pyrrolidone moiety was suggested by the typical 13C NMR
resonances [δC 181.3 (C-2′), 32.3 (C-3′), 27.5 (C-4′), and 53.2
(C-5′)], and it was located at C-1 of the 9,10-dihydrophenan-
threne moiety by the HMBC correlations from Ha,b-4′ to C-1
and from H-5′ to C-2 and C-9a. The specific rotation of
compound 2 was zero, suggesting that it was a racemic mixture.
Chiral-phase column chromatography yielded two well-
separated peaks (2a and 2b) in a ratio of approximately 1:1
(Figure 6A and B). The ECD spectrum of compound 2a showed
typical CEs for (aS)-9,10-dihydrophenanthrenes, namely,
positive CEs in the region of 250−325 nm (Figure 6C).8
Meanwhile, the antipodal ECD curves suggested the aR
configuration for compound 2b. The configuration of C-5′
could not be determined from either the NOESY correlations or
calculated ECD spectra. Thus, the structure of bletillatin B (2)
was elucidated as an enantiomeric mixture of (aS)- and (aR)-2,7-
dihydroxy-4-methoxy-1-(2′-oxopyrrolidin-5′-yl)-9,10-dihydro-
phenanthrene.
Bletillatin C (3) was isolated as a brownish powder, and the
molecular formula was determined to be C28H30O9 by the
ammonium adduct ion peak at m/z 528.2246 [M + NH4]+
observed in the HRESIMS data (calcd 528.2234 for
1
C28H34NO9). In the H and 13C NMR data, compound 3
displayed superimposable resonances assignable to the methyl-
D
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Figure 5. Chiral-phase resolution of compound 1. (A) Four possible enantiomers. (B) Chiral-phase HPLC chromatograph. (C) Experimental and
calculated ECD spectra.
ene groups and B-ring, similar to compound 2, but differences
were observed in the A-ring resonances.6 An aromatic proton
singlet at δH 6.52 (1H, s, H-1) was assigned to H-1 by the
HMBC correlations from H-1 to C-4a and C-10. The presence
of a 4-O-β-D-glucopyranosylbenzyl moiety was deduced from
the resonances for a methylene [δH 3.96 (2H, s, H2-7′), δC 27.4
(C-7′)], an AA′XX′-type aromatic ring [δH 6.95 (2H, d, J = 8.7
Hz, H-3′,5′), 7.19 (2H, d, J = 8.7 Hz, H-2′,6′)], and a β-
glucopyranosyl moiety with the anomeric resonances at δH 4.83
(1H, d, J = 7.6 Hz, H-1″). Upon acid hydrolysis, the compound
produced D-glucose, the absolute configurations of which were
confirmed by an HPLC analysis of its thiocarbamate derivative
and a comparison with the standard D-glucose.28 The assign-
ment of a methoxy group at C-4 was accomplished based on the
HMBC correlations from 4-OCH3 and H-7′ to C-4. Thus, the
structure of bletillatin C (3) was determined as 2,7-dihydroxy-3-
(4-O-β-D-glucopyranosylbenzyl)-4-methoxy-9,10-dihydrophe-
nanthrene.
H]+ in the HRESIMS spectrum (calcd 485.1959 for C30H29O6).
The 1H NMR data for compound 4 revealed resonances of four
methylene groups [δH 2.27 (2H, t, J = 6.7 Hz, H2-α), 2.47 (2H, t,
J = 6.7 Hz, H2-α′), 2.50 (2H, m, Ha,b-10), and 2.57 (2H, m, Ha,b-
9)], suggesting a heterodimeric structure. The assignments of a
proton singlet at δH 6.56 (1H, s, H-3) of the A-ring of the 9,10-
dihydrophenanthrene moiety and a set of AMX-type proton
resonances [δH 6.58 (1H, d, J = 2.3 Hz, H-8), 6.62 (1H, dd, J =
8.6, 2.3 Hz, H-6), and δH 8.04 (1H, d, J = 8.6 Hz, H-5)] of the B-
ring were deduced from the HMBC correlations from H-3 to C-
4a, from Ha,b-10 to C-1, from H-5 to C-4a and C-8a, and from H-
8 to C-5a and C-9. On the other hand, the structure of the
bibenzyl group was established by the presence of a set of meta-
coupled aromatic protons [δH 6.36 (1H, d, J = 2.9 Hz, H-4′),
6.37 (1H, d, J = 2.9 Hz, H-6′)] (C ring) and a set of aromatic
proton resonances [δH 6.38 (1H, dd, J = 8.0, 2.9 Hz, H-6″), 6.39
(1H, d, J = 2.9 Hz, H-2″), 6.49 (1H, dd, J = 8.0, 2.0 Hz, H-4″),
6.93 (1H, t, J = 8.0 Hz, H-5″)] for a meta-substituted phenyl
moiety (D-ring), as well as the key HMBC correlations from H2-
α to C-6′ and C-2′, from H-4′ to C-2′ and C-6′, from H2-α′ to C-
2″,6″, from H-5″ to C-1″, and from H-4″ to C-2″ and C-6″. Two
Bletillatin D (4) was isolated as a brownish powder, and its
molecular formula was established as C30H28O6 based on the
presence of a protonated molecular ion at m/z 485.1957 [M +
E
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Figure 6. Chiral-phase resolution of compound 2. (A) Four possible enantiomers. (B) Chiral-phase HPLC chromatograph. (C) Experimental and
calculated ECD spectra.
methoxy groups were assigned to C-3′ and C-5′ by the HMBC
correlations from H-4′ to C-3′ and C-5′; from H-6′ to C-2′ and
C-5′, from 3′-OCH3 to C-3′, and from 5′-OCH3 to C-5′.
Although HMBC correlations did not occur across the 9,10-
dihydrophenanthrene and bibenzyl moieties, the connection of
two quaternary carbons C-1 and C-2′ was reasonable by
assignment of their positions using the HMBC correlations from
H-3 and Ha,b-10 to C-1 and from H-4, H-6, and H-α′ to C-2′.
Thus, the structure of bletillatin D (4) was determined as 2,4,7-
trihydroxy-1-{6-[2-(3-hydroxyphenyl)]ethyl-2,4-dimethoxy}-
phenyl-9,10-dihydrophenanthrene.
16, 18, 21, 23, and 24 exhibited weak inhibitory activities, with
IC50 values ranging from 12.0 to 30.0 μM. Accounting for the
chiralities of 9,10-dihydrophenanthrenes, enantiopure com-
pounds (33a and 33b, Figure 7) were evaluated for their
In addition to compounds 1 and 2, the chiral-phase resolution
of compounds 5, 8−11, 13, 22, 25, 33, and 34 was also
successfully achieved, and the absolute configurations of
enantiopure compounds were determined using the exper-
imental and calculated ECD spectra (Supporting Information).
The antineuroinflammatory activities of all isolated com-
pounds were evaluated using Griess assays in overactivated BV-2
microglial cells stimulated with LPS. A known inhibitor of
neuroinflammation, minocycline, was used as the positive
control (IC50 27.2 μM). The dimeric phenanthrene compound
33 showed the most potent antineuroinflammatory activity, with
an IC50 value of 1.0 μM, followed by compound 32 (IC50 5.0
μM). However, compound 34 exhibited no activity. The
heterodimeric compound 7 also showed potent activity (IC50
1.9 μM). A comparison of compounds 7 and 4 (IC50 60.7 μM),
the structures of which only differ in the presence of a methoxy
group at C-5′ or C-2, revealed that compound 7 showed more
potent activity than compound 4. In addition, compounds 5, 12,
Figure 7. Chemical structures of 33a and 33b.
antineuroinflammatory activities. Results indicated that com-
pounds 33a and 33b exhibited significant inhibitory effects with
IC50 values of 6.9 and 1.4 μM, respectively (minocycline was also
used as the positive control, with IC50 value at 35.7 1.1 μM),
and (aR)-33 (compound 33b) was more active than (aS)-33
(compound 33a). Based on these results, phenanthrenes and
9,10-dihydrophenanthrenes show potential for further develop-
ment as neuroinflammatory inhibitors. Furthermore, the
chiralities of bioactive isomers have an influence on antineuroin-
flammatory activities.
F
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Bletillatin D (4): brownish powder (MeOH); [α]20 0 (c 0.1,
EXPERIMENTAL SECTION
D
■
MeOH); UV (MeOH) λmax (log ε) 280 (2.60) nm, 211 (3.45) nm; 1H
and 13C NMR data (see Table 1); HRESIMS m/z 485.1957 [M + H]+
(calcd for C30H29O6, 485.1959).
General Experimental Procedures. An MCP 200 polarimeter
from Anton Paar (Graz, Austria) was used to measure optical rotations.
1H and 13C NMR spectra were recorded with a Bruker AVIII HD-600
spectrometer in δ (ppm) referenced to tetramethylsilane. HRESIMS
data were collected using an Agilent HPLC 1290 Infinity and a 6540
UHD Accurate TOF-Q mass spectrometer. ECD spectra were recorded
using a Bio-Logic Science MOS-450 spectrometer. The silica gel (SiO2:
50−74 μm) used for column chromatography was produced by
Qingdao Ocean Chemical Group Co. (Qingdao, People’s Republic of
China). The ODS (50 μm) used for column chromatography was
produced by the YMC Company (Kyoto, Japan). HPLC separations
were done by using a YMC ODS-A C18 column (5 μm, 250 mm × 20
mm) with a Shimadzu SPD-20A UV detector and a Shimadzu LC-
20AR pumping system. Sigma-Aldrich (MO, USA) supplied the
deuterium reagents, DMSO-d6, and methanol-d4. Chiral-phase
separations were performed using the chiral-phase chromatographic
column CHIRALPAK IF (Dacicel Chiral pak IF: CHIRALPAK IF
analytic column, 4.6 × 250 mm, 4.6 μm particles, Daicel Chemical
Industries, Tokyo, Japan). Tianjin DaMao provided all reagents for the
chromatographic or analytical experiments (Tianjin, People’s Republic
of China).
Acid Hydrolysis of Compound 3. Compound 3 (1.0 mg) was
dissolved in 4 M HCl (1 mL) and heated at 90 °C for 2 h. The mixture
was extracted with CH2Cl2 after cooling. The aqueous layer was
evaporated to dryness. The residue was added to 3.0 mg of L-cysteine
methyl ester, dissolved in pyridine (1 mL), and heated at 60 °C for 1 h.
o-Tolylisothiocyanate (5 μL) was added to the solution, and the
mixture heated for an additional 1 h. The retention time of the reaction
mixture (thiocarbamate derivative, 18.25 min) was confirmed by a
comparison with authentic D-glucose using HPLC. The HPLC was
equipped with a Shimadzu SPD-20A UV/visible detector and a
Shimpack ODS (H) kit (4.6 × 250 mm, 5 μm particle size) and
analyzed at 35 °C (detection wavelength: 250 nm; flow rate: 1 mL/
min). The mobile phase was CH3CN−H2O (25:75).27,28
Chiral-Phase Resolution. The resolution was conducted using a
chiral-phase column (Dacicel Chiral Pak IF) at 1 mL/min (detector:
UV detection, λ: 210 nm). Chiral-phase separation of compound 1
using HPLC with n-hexane−EtOH (80:20) afforded compounds 1a
(1.0 mg, tR = 11 min) and 1b (0.9 mg, tR = 13 min).
Plant Material. Plant material was purchased from Shaanxi Tasly
Plant Medicine LLC, collected from Shaanxi Province, People’s
Republic of China, in November 2016, and identified by Professor
Yingni Pan (Shenyang Pharmaceutical University). A voucher
specimen (20160911) is deposited in the herbarium of Shenyang
Pharmaceutical University.
(aS,7′S,8′R)-Bletillatin A (1a): [α]20 −26 (c 0.1, MeOH); ECD
D
(MeOH) λmax (Δε) 214 (−0.76), and 282 (+2.27) nm.
(aR,7′R,8′S)-Bletillatin A (1b): [α]20 +26 (c 0.1, MeOH); ECD
D
Extraction and Isolation. The dried tubers of B. striata (16 kg)
were extracted using 95% aqueous EtOH (×3, for 3 h/extraction) using
reflux and then successively leached with petroleum ether, EtOAc, and
n-BuOH. The EtOAc fraction (400 g) was subjected to silica gel
column chromatography (CC) with a gradient of CH2Cl2−MeOH to
produce 10 fractions (Fr.1−Fr.10). Fr.2 was loaded on the ODS
column, eluted with MeOH−H2O, and purified using HPLC to yield
compounds 28 (12.3 mg), 26 (72.3 mg), 29 (21.6 mg), 15 (23.2 mg),
and 20 (17.2 mg). Fr.3 was separated using silica gel CC with gradient
elution using petroleum ether−EtOAc to yield Fr.3-1 and 3-2. These
subfractions were separated on an ODS column and washed with a
gradient of MeOH−H2O to yield compounds 27 (15.1 mg), 6 (21.2
mg), 2 (16.1 mg), 30 (66.2 mg), and 31 (31.2 mg). Fr.4 was loaded
onto an ODS column, eluted with MeOH−H2O, and further purified
by HPLC equipped with an ODS column to give compounds 10 (13.2
mg) and 5 (22.7 mg). Fr.6 was separated on an ODS column (MeOH−
H2O) and then purified using HPLC to afford compounds 32 (3.2 mg),
4 (22.2 mg), and 7 (31.2 mg) after elution with MeOH−H2O. Fr.7 was
subjected to silica gel chromatography with gradient elution using
CH2Cl2−MeOH and purified by HPLC with MeOH−H2O to give
compounds 1 (12.6 mg), 9 (42.3 mg), 8 (33.2 mg), and 12 (26.5 mg).
Fr.9 was isolated using an ODS column and eluted with MeOH−H2O
to produce five subfractions, which were further purified by RP-C18
HPLC to give compounds 14 (23.2 mg), 13 (61.4 mg), 16 (87.2 mg),
18 (67.3 mg), 19 (47.2 mg), 3 (14.7 mg), 21 (21.2 mg), 17 (29.3 mg),
22 (43.2 mg), and 23 (11.6 mg), with CH3CN−H2O. Fr.10 was
subjected to an ODS column and purified by HPLC with CH3CN−
H2O to give compounds 24 (22.5 mg), 25 (50.2 mg), 33 (31.5 mg), 34
(42.3 mg), and 11 (15.4 mg).
(MeOH) λmax (Δε) 211 (+10.47), and 284 (+1.08) nm.
Similarity, chiral-phase separation of compound 2 using HPLC with
n-hexane−EtOH (75:25) afforded compounds 2a (1.2 mg, tR = 9 min)
and 2b (1.1 mg, tR = 12 min).
(aS)-Bletillatin B (2a): [α]20D −25 (c 0.1, MeOH); ECD (MeOH)
λmax (Δε) 213 (+13.71), 230 (−10.41), and 277 (+5.49) nm.
(aR)-Bletillatin B (2b): [α]20D +25 (c 0.1, MeOH); ECD (MeOH)
λmax (Δε) 205 (−25.19), 231 (+8.72), and 277 (−5.22) nm.
Using the same methods, compounds 5, 8−11, 13, 22, 25, 33, and 34
were optically resolved with HPLC using a chiral-phase chromato-
graphic column. The [α]20 data and ECD data of these compounds
D
were measured (Supporting Information).
Cell Viability and NO Production. Cell viability and NO
production were tested using MTT and Griess assays, respectively, as
described in our previous study.4,5
Table 2. Inhibitory Activities of the Compounds on NO
Production in LPS-Activated BV-2 Microglial Cells (Mean
SEM)
compound
IC50 (μM)
compound
24
IC50 (μM)
4
5
6
7
12
15
16
20
21
22
23
60.7 1.6
24.8 1.9
34.6 1.3
1.9 1.9
18.5 2.1
93.5 1.4
15.2 2.2
49.0 2.2
17.9 1.6
46.8 1.7
22.1 1.8
12.0 1.6
39.4 2.4
30.0 1.7
82.0 2.0
30.2 2.2
5.0 1.8
1.0 1.8
6.9 1.4
1.4 1.2
27.2 1.7
25
27
29
30
32
33
33a
Bletillatin A (1): brownish oil (MeOH); [α]20D 0 (c 0.1, MeOH); UV
(MeOH) λmax (log ε) 282 (3.30) nm, 210 (4.60) nm; 1H and 13C NMR
data (see Table 1); HRESIMS m/z 419.1477 [M−H]− (calcd for
C25H23O6, 419.1500).
a
a
Bletillatin B (2): brownish oil (MeOH); [α]20D 0 (c 0.1, MeOH); UV
(MeOH) λmax (log ε) 280 (2.61) nm, 212 (3.39) nm; 1H and 13C NMR
data (see Table 1); HRESIMS m/z 326.1375 [M + H]+ (calcd for
C19H20NO4, 326.1387).
33b
b
minocycline
a
Bletillatin C (3): brownish powder (MeOH); [α]20 −2 (c 0.1,
The data were afforded by the supplementary experiment according
D
MeOH); UV (MeOH) λmax (log ε) 282 (2.15) nm, 212 (3.60) nm; 1H
and 13C NMR data (see Table 1); HRESIMS m/z 528.2246 [M +
NH4]+ (calcd for C28H34NO9, 528.2234).
to the reviewer’s advice using the method described here. Minocycline
was also used as the positive control with an IC50 value of 35.7 1.1
b
μM. Minocycline was used as the positive control.
G
DOI: 10.1021/acs.jnatprod.9b00291
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(14) Wu, Y. P.; Liu, W. J.; Zhong, W. J.; Chen, Y. J.; Chen, D. N.; He,
F.; Jiang, L. Nat. Prod. Res. 2017, 31, 1518−1522.
(15) Zhang, Y. Y.; Song, X. Q.; Mei, W. L.; Zuo, W. J.; Cai, C. H.;
Cheng, J.; Dai, H. F. J. Trop. Subtrop. Bot. 2015, 23, 317−322.
(16) Leong, Y. W.; Kang, C. C.; Harrison, L. J.; Powell, A. D.
Phytochemistry 1997, 44, 157−165.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.9b00291.
■
S
(17) Dong, H. L.; Liang, H. Q.; Wang, C. L.; Guo, S. X.; Yang, J. S.
Magn. Reson. Chem. 2013, 51, 371−377.
1D and 2D NMR and HRESIMS spectra of compounds
1−4; optical resolution and determination of the
structures of compounds 5, 8−11, 13, 22, 25, 33, and
34 (PDF)
(18) Yang, M. H.; Zhao, H. R.; Guo, J.; Yan, H. G.; Fu, X. M.; Cai, L. J.
Yunnan Univ. 2015, 37, 556−563.
(19) Zhang, F.; Zhao, M. B.; Li, J.; Tu, P. F. Chin. Tradit. Herb. Drugs
2013, 44, 1529−1533.
AUTHOR INFORMATION
Corresponding Authors
*(N. Li) E-mail: liningsypharm@163.com. Tel: +86-24-
43520739.
*(Y. Hou) E-mail: houyue@mail.neu.edu.cn. Tel: +86-24-
83656117.
ORCID
(20) Tuchinda, P.; Udchachon, J.; Khumtaveeporn, K.; Taylor, W. C.
Phytochemistry 1989, 28, 2463−2466.
■
(21) Lin, Y. L.; Chen, W. P.; Macabalang, A. D. Chem. Pharm. Bull.
2005, 53, 1111−1113.
(22) Yamaki, M.; Kato, T.; Bai, L.; Inoue, K.; Takagi, S. Phytochemistry
1993, 34, 535−537.
(23) Leong, Y. W.; Harrison, L. J.; Powell, A. D. Phytochemistry 1999,
50, 1237−1241.
(24) Han, G. X.; Wang, L. X.; Zhang, W. D.; Yang, Z.; Li, T. Z.; Jiang,
T.; Liu, W. Y. J. Second Mil. Med. Univ. 2002, 23, 443−445.
(25) Xiao, S. J.; Yuan, F. M.; Zhang, M. S.; Yu, S. Y.; Li, J. D.; Yi, X. D.;
Xu, D. L. J. Asian Nat. Prod. Res. 2017, 19, 140−144.
(26) Lin, C. W.; Hwang, T. L.; Chen, F. A.; Huang, C. H.; Hung, H. Y.;
Wu, T. S. J. Nat. Prod. 2016, 79, 1911−1921.
(27) Wang, C. J.; Wang, L. B. J. Med. Chem. 2013, 23, 235−242.
(28) Chaturvedula, V. S. P.; Yu, O.; Mao, G. H. Eur. Chem. Bull. 2014,
3, 104−107.
Wei Li: 0000-0003-4143-8597
Ning Li: 0000-0001-7410-7327
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The study was partially supported by the National Natural
Science Foundation of China (Grant Nos. 81673323, 81872768,
81473108, 81628012, 81473330, and U1603125), a project of
the State Key Laboratory for Chemistry and Molecular
Engineering of Medicinal Resources (CMEMR2017-B03), the
Program for Liaoning Excellent Talents in University
(LR2015022), and the Fundamental Research Funds for the
Central Universities of China (N162004003) and LiaoNing
Revitalization Talents Program (XLYC1807118) and Liaoning
BaiQianWan Talents Program.
REFERENCES
■
(1) Toth, B.; Hohmann, J.; Vasas, A. J. Nat. Prod. 2018, 81, 661−678.
(2) He, X. R.; Wang, X. X.; Fang, J. C.; Zhao, Z. F.; Huang, L. H.; Guo,
H.; Zheng, X. H. J. Ethnopharmacol. 2017, 195, 20−38.
(3) State Administration of Traditional Chinese Medicine; “Zhong
Hua BenCao” Editorial Committee. Zhong Hua Ben Cao; Shanghai
Science and Technology Press, 1999; Vol. 8, pp 674−679.
(4) Hou, Y.; Li, G. X.; Wang, J.; Pan, Y. N.; Jiao, K.; Du, J.; Chen, R.;
Wang, B.; Li, N. Sci. Rep. 2017, 7, 1−17.
(5) Hou, Y.; Li, N.; Xie, G. B.; Wang, J.; Yuan, Q.; Jia, C. C.; Liu, X.; Li,
G. X.; Tang, Y. Z.; Wang, B. J. Funct. Foods 2015, 19, 676−687.
(6) Liu, X. Q.; Yuan, Q. Y.; Guo, Y. Q. J. Asian Nat. Prod. Res. 2009, 11,
116−121.
(7) Yamaki, M.; Bai, L.; Inoue, K.; Takagi, S. Phytochemistry 1990, 29,
2285−2287.
(8) Yao, S.; Tang, C. P.; Ye, Y.; Kurtan, T.; Kiss-Szikszai, A.; Antus, S.;
Pescitelli, G.; Salvadori, P.; Krohn, K. Tetrahedron: Asymmetry 2008, 19,
2007−2014.
(9) Yamaki, M.; Li, B.; Kato, T.; Inoue, K.; Takagi, S. Phytochemistry
1993, 32, 427−430.
(10) Dong, H. L.; Wang, C. L.; Li, Y.; Guo, S. X.; Yang, J. S. Magn.
Reson. Chem. 2010, 48, 256−260.
(11) Wang, Y.; Guan, S. H.; Meng, Y. H.; Zhang, Y. B.; Cheng, C. R.;
Shi, Y. Y.; Feng, R. H.; Zeng, F.; Wu, J. X.; Zhang, J. X.; Yang, M.; Liu,
X.; Li, Q.; Chen, X. H.; Bi, K. S.; Guo, D. A. Phytochemistry 2013, 94,
268−276.
(12) Auberon, F.; Olatunji, O. J.; Krisa, S.; Antheaume, C.; Herbette,
G.; Bonte, F.; Merillon, J. M.; Lobstein, A. Molecules 2016, 21, 1−9.
(13) Yang, M. H.; Cai, L.; Li, M. H.; Zeng, X. H.; Yang, Y. B.; Ding, Z.
T. Chin. Chem. Lett. 2010, 21, 325−328.
H
DOI: 10.1021/acs.jnatprod.9b00291
J. Nat. Prod. XXXX, XXX, XXX−XXX