Anti-inflammatory and Antiosteoporosis Flavonoids from the Rhizomes of Helminthostachys zeylanica

Article abstract of DOI:10.1021/acs.jnatprod.5b01164

Chemical investigation of the rhizomes of Helminthostachys zeylanica led to the isolation of eight new flavonoids including six cyclized geranylflavonoids, ugonins V-X (1-3), (10R,11S)-ugonin N (4), (10R,11S)-ugonin S (5), and ugonin Y (6), as well as two quercetin glucosides, quercetin-4′-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (7) and quercetin-3-O-β-d-glucopyranosyl-4′-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8). The structures of these compounds were established by spectroscopic analyses and acid hydrolysis of the sugar moiety. Among the isolated compounds, 1, 2, 5, 6, ugonins J-S (9-13), ugonstilbene A (14), and ugonin L (23) were evaluated for their anti-inflammatory activity on lipopolysaccharide-induced nitric oxide (NO) production in microglial cells. Except for 1, 5, and 13, all other compounds inhibited NO production with IC₅₀ values of 6.2-10.1 μM and were more potent than the positive control, pyrrolidine dithiocarbamate. Compounds 1, 2, 5, 6, and 10-13 were tested for antiosteoporotic activities, and ugonin K (10) exhibited the highest inhibitory activity against RANKL-induced osteoclast differentiation in RAW264.7 cells with an IC₅₀ value of 1.8 ± 0.2 μM.

Full text of DOI:10.1021/acs.jnatprod.5b01164

Article
pubs.acs.org/jnp
Anti-inflammatory and Antiosteoporosis Flavonoids from the
Rhizomes of Helminthostachys zeylanica
,
†,‡
†
†,§,⊥
Wen-Fei Chiou,†
Yu-Ling Huang,*
Chien-Chang Shen, Yuh-Chiang Shen,
and Chien-Chih Chen#
†National Research Institute of Chinese Medicine, Ministry of Health and Welfare, No. 155-1, Sec. 2, Li-Nung Street, Peitou, Taipei
1
1221, Taiwan
‡
Department of Cosmetic Science, Chang Gung University of Science and Technology, Taoyuan 33302, Taiwan
Institute of Biomedical Sciences, National Chung-Hsing University, Taichung 40227, Taiwan
§
⊥
National Taipei University of Nursing and Health Science, Taipei, Taiwan
#
Department of Biotechnology, Hungkuang University, Taichung 433, Taiwan
S Supporting Information
*
ABSTRACT: Chemical investigation of the rhizomes of Helminthos-
tachys zeylanica led to the isolation of eight new flavonoids including
six cyclized geranylflavonoids, ugonins V−X (1−3), (10R,11S)-ugonin
N (4), (10R,11S)-ugonin S (5), and ugonin Y (6), as well as two
quercetin glucosides, quercetin-4′-O-β-D-glucopyranosyl-(1→2)-β-D-
glucopyranoside (7) and quercetin-3-O-β-D-glucopyranosyl-4′-O-β-D-
glucopyranosyl-(1→2)-β-D-glucopyranoside (8). The structures of
these compounds were established by spectroscopic analyses and acid
hydrolysis of the sugar moiety. Among the isolated compounds, 1, 2,
5
, 6, ugonins J−S (9−13), ugonstilbene A (14), and ugonin L (23)
were evaluated for their anti-inflammatory activity on lipopolysac-
charide-induced nitric oxide (NO) production in microglial cells.
Except for 1, 5, and 13, all other compounds inhibited NO production
with IC values of 6.2−10.1 μM and were more potent than the positive control, pyrrolidine dithiocarbamate. Compounds 1, 2,
50
5
, 6, and 10−13 were tested for antiosteoporotic activities, and ugonin K (10) exhibited the highest inhibitory activity against
RANKL-induced osteoclast differentiation in RAW264.7 cells with an IC50 value of 1.8 ± 0.2 μM.
he rhizomes of Helminthostachys zeylanica (L.) Hook.
(Ophioglossaceae), a folk herbal medicine named “Daodi-
analogues, ugonins V−X (1−3), (10R,11S)-ugonin N (4),
T
(10R,11S)-ugonin S (5), and ugonin Y (6), as well as two
quercetin glucosides, quercetin-4′-O-β-D-glucopyranosyl-(1→
2)-β-D-glucopyranoside (7) and quercetin-3-O-β-D-glucopyra-
nosyl-4′-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside
(8). In addition, the 14 known compounds ugonins J (9), K
Ugon” in Taiwan, have been used as an antipyretic and
1
antiphlogistic agent or to treat bone fracture and contusion.
The previous studies revealed that this medicinal material is
2
−7
rich in prenylated flavonoids such as ugonins A−U. Most of
4
4,5
9
these compounds showed antioxidative or anti-inflammatory
(10), M (11), O (12), S (13), ugonstilbene A (14),
5
,6
activities.
Ugonins J and K also showed extracellular
quercetin (15), quercetin-3-O-β-D-glucopyranoside (16), quer-
cetin-4′-O-β-D-glucopyranoside (17), quercetin-3,4′-di-O-β-D-
glucopyranoside (18), quercetin-3-O-β-D-glucopyranosyl-(1→
8
melanogenesis inhibitory activity, and ugonin U displayed an
immunomodulatory effect in human neutrophils via stimulation
7
9
12
of phospholipase C. Ugonstilbenes A and B and ugonins G, E,
J, K, and L have been tested for their antiosteoporotic activity.
Ugonin K (0.5−10 μM) concentration-dependently increased
alkaline phosphatase (ALP) activity of MC3T3-E1 cells without
detectable cytotoxicity. Ugonin K also promotes osteoblastic
differentiation and mineralization by activation of p38 MAPK-
4)-β-D-glucopyranoside (19), quercetin-3-O-β-D-glucopyra-
nosyl-(1→4)-β-D-glucopyranosyl-4′-O-β-D-glucopyranoside
13
(
20), quercetin-3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyr-
anosyl-4′-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside
13
(
21), and benzoic acid (22). The structures of the isolated
compounds were elucidated on the basis of spectroscopic data,
and the sugar portion was identified by high performance
anion-exchange chromatography after acid hydrolysis. The4
absolute configurations of compounds 2−6 and ugonin L (23)
10
and ERK-mediated expression of Runx2 and osterix. More-
over, ugonin K-stimulated osteogenesis involves estrogen-
receptor-dependent activation of both the nonclassical Src
11
and classical signaling pathways. In this work, the chemical
constituents of an H. zeylanica extract were investigated, and
eight new flavonoids were isolated including six cyclized geranyl
Received: February 26, 2016
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b01164
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Chart 1
Figure 1. COSY (bold lines) and key HMBC (arrows) correlations of 1, 2, and 6−8.
B
DOI: 10.1021/acs.jnatprod.5b01164
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
were determined by comparison of their NOE, specific rotation,
NMR spectrum of 2. In addition, three methyl proton signals
resonated at δ 0.67, 1.04 (H -16, 17) and 1.40 (H -18). As in
and electronic circular dichroism (ECD) data with those of
H
3
3
1
4,15
synthesized compounds.
2
Compounds 1, 2, 5, 6, 9−14, and
the case of 1, the cyclized geranyl group linked at C-2′ was
assigned on the basis of the presence of HMBC cross-peaks
(Figure 1) of H -9 (δ 3.40) and H-5′ (δ 6.94) with C-1′ (δ
4
3 were evaluated for their anti-inflammatory activity on
lipopolysaccharide (LPS)-induced nitric oxide (NO) produc-
tion in microglial cells. In addition, compounds 1, 2, 5, 6, and
2
H
H
C
108.3) and C-3′ (δ 141.0). An oxygenated aliphatic carbon
C
1
0−13 were also tested for their antiosteoporotic activities.
signal (δC 77.8) corresponding to C-11 was observed,
suggesting the presence of an ether linkage between C-11
and HO-3′ to form a dihydropyran ring. By comparing the
RESULTS AND DISCUSSION
■
An ethanol extract of H. zeylanica rhizomes was repeatedly
chromatographed to yield eight new flavonoids: ugonins V−X
1−3), (10R,11S)-ugonin N (4), (10R,11S)-ugonin S (5),
methyl proton chemical shifts (H -16 and H -17) of the
3 3
cyclized geranyl group in synthesized racemic trans-ugonin L
(
(δH 0.95, 1.02), racemic cis-ugonin L (δ 0.62, 0.96), and
H
17
ugonin Y (6), and two quercetin glucosides, quercetin-4′-O-β-
D-glucopyranosyl-(1→2)-β-D-glucopyranoside (7) and querce-
tin-3-O-β-D-glucopyranosyl-4′-O-β-D-glucopyranosyl-(1→2)-β-
D-glucopyranoside (8).
racemic cis-ugonin S (δH 0.66, 0.97), the cis-fused geranyl
group showed one high-field methyl signal near δ 0.65. For 2,
H
the presence of the signal at δ 0.67 was in accordance with this
H
characteristic, and the 10,11-cis-configuration was deduced for
2, which was also supported by the NOE effect between H-10
Compounds 1−8 were obtained as yellow powders. The
spectroscopic data for 1−6 revealed that they possessed the
characteristics of cyclized geranylflavonoids similar to those
and H -18. The (10R,11S)-absolute configuration of compound
3
2 was established by comparing its negative specific rotation
2
−7
([α]25 −134) with that of synthesized (16R,17S)-ugonstilbene
isolated from the same source.
4
Using HRESIMS (m/z
D
34.1374) and 13C NMR data, the molecular formula of 1 was
determined to be C H O . In the H NMR spectrum of 1,
14
25
D
B (24, [α]
−27). Furthermore, both ECD spectra of 2
1
2
5
22
7
[+1.38 (210), +0.40 (221), +1.06 (227)] and 24 [−0.81 (210),
8.20 (221), −6.37 (227)] displayed negative Cotton effects
(CEs) between 210 and 227 nm. In contrast, (16S,17R)-
signals corresponding to the following were observed: a
cyclized geranyl group comprising three methyl singlets at δH
−
25
14
0
.89, 1.03 (H-16, 17), and 1.60 (H-18), two pairs of aliphatic
ugonstilbene B (25, [α]
+36) displayed positive CEs
D
methylene protons at δ 2.02 (2H, m, H-13) and 1.56 (2H, m,
H
[+4.13 (210), +7.45 (221), +5.46 (227)]. Accordingly, the
structure of 2, ugonin W, was defined as (10R,11S)-5,7,4′-
trihydroxy-(5,5,8a-trimethyl-5,6,7,8,8a,9a-hexahydro)xanthen-
[4,3:4,5]furo[3,2-b]-4H-chromen-4-one.
H-14), a benzylic methylene at δ 3.36 (1H, d, J = 17.4 Hz, H-
H
9
) and 3.69 (1H, d, J = 17.4 Hz, H-9), and an olefinic methine
H
4
at δ 5.44 (1H, br s, H-12). In addition, three aromatic proton
signals resonated at δ 6.23 (1H, d, J = 1.8 Hz, H-6), 6.51 (1H,
25
H
The spectroscopic characteristics of 3 (C H O , [α]
2
5
24
7
D
d, J = 1.8 Hz, H-8), and 6.96 (1H, s, H-5′), and a hydrogen-
4
−
162) closely resembled those of ugonin I, except for the
bonded hydroxy group at δ 12.77 (5-OH). H-6 and H-8 were
1
H
absence of a methoxy group at C-3. In the H NMR spectrum
of 3, three methyl proton signals of the cyclized geranyl group
resonated at δ 0.79, 0.98 (H -16, 17), and 1.35 (H -18). A pair
assigned on the basis of HMBC cross-peaks between 5-OH and
C-5 (δ 162.3), C-4a (δ 103.9), and C-6 (δ 99.1), the latter
C
C
C
H
3
3
also correlating to H-8 (Figure 1). Further, H-5′ was assigned
of o-coupled doublets at δ 6.77 (H-5′, J = 8.4 Hz) and 7.02
H
on the basis of its correlations with quaternary C-1′ (δ 105.0)
C
(
H-6′, J = 8.4 Hz) corresponding to the protons of ring B were
and three oxygenated carbons at δ 147.1, 149.4, and 151.4,
C
also observed. The presence of a cyclized geranyl group linked
which indicated the presence of a pentasubstituted B-ring. Both
to C-2′ and HO-3′ was deduced from HMBC cross-peaks
C-1′ and the carbon at δ 147.1 showed correlations with H -9,
C
2
(
Figure 1) between H -9 (δ 3.00, 3.12) and C-1′ (δ 121.7)
2
H
C
suggesting the cyclized geranyl group to be located at C-2′ (δ
C
25
and C-3′ (δ 142.1). The physical data of 4 (C H O , [α]
C
25 26
7
D
1
17.5) and HO-3′. The carbon resonating at δ 147.1 was
C
25
13
−12) and 5 (C25
H
26
O
, [α]
6
D
−31) closely resembled those of
assigned to C-3′. In the C NMR spectrum, an aliphatic
5
1
ugonins N and S, except that the H NMR spectra of 4 and 5
showed one shielded H -16 (or H -17) signal (4: δ 0.68; 5: δ
H
oxygenated tertiary carbon at δ 94.1 was assigned to C-10 on
C
3
3
H
the basis of the HMBC cross-peaks of H-12, H -14, H -16, H -
2
3
3
0
0
.66) compared to those of ugonins N (δ 0.91) and S (δH
.97), suggesting that 4 and 5 could be stereoisomers of
H
1
7, and H -18 with this carbon. Because C-10, C-3′, and C-3
3
(δ
133.9) were assigned referring to C-3 (δ 135.0) of
C
C
1
6
ugonins N and S. Similar to ugonin W (2), the structures of
compounds 3−5 were deduced to have the (10R,11S)-absolute
configurations on the basis of the NOE effect between H-10
broussofluorenone A, C-6′ (δ 149.4 or 151.4) was
C
oxygenated, and 1 had only 22 hydrogens, the ether linkages
were deduced to be between C-10 and HO-3′ to form a
dihydrofuran ring and between HO-3 and C-6′ to form a furan
ring. Accordingly, the structure of 1, ugonin V, was defined as
and H -18 and negative specific rotations. Thus, the structure of
3
3
5
, ugonin X, was defined as (10R,11S)-3,5,7,4′-tetrahydroxy-
″,5″,8″a-trimethyl-4″a,5″,6″,8″a-tetrahydro-4H-chromen-
5
,7,4′-trihydroxyspiro[2,6,6-trimethylcyclohex-2-ene-1,2-furo-
[
3″,2″:2′,3′]flavone, and compounds 4 and 5 as (10R,11S)-
[
5,4:4,5]benzofuro[3,2-b]-4H-chromen]-4-one. The C-10 ab-
ugonins N and S, respectively. Ugonins N (δ 0.91, 1.30), S
solute configuration of this compound has not been determined
H
5
4
(
δH 0.97, 1.05), and L (δH 0.93, 1.01) showed deshielded
yet.
+
signals for H -16 and H -17, suggesting their 10,11-trans
Compound 2, C H O ([M] 436.1534), differed from
ugonin O in the cyclized geranyl group. The DEPT spectra of
3
3
2
5
24
7
5
17
relative configurations. Furthermore, the (10R,11R) absolute
configurations of trans-ugonins L, N, and S were determined by
2
displayed two aliphatic methylene carbons (δ 39.5, C-12; δC
C
comparing their positive specific rotations ([α]25
+82, +15,
+71, respectively) with that of synthesized 4a-epi-ugonstilbene
4
5
18.6, C-13) instead of two olefinic methine carbons (δ 127.9,
D
C
C-12; δC 130.6, C-13) in ugonin O. These two pairs of
B (26, [α]25
+49). Besides, (10R,11R)-ugonins S (13) and L
15
methylene proton signals resonated at δ 2.13 (1H, m, H-12b),
D
H
1
1
.62 (1H, td, J = 3.5, 13.8 Hz, H-12a) and δ 1.88 (1H, m, H-
3b), 1.45 (2H, m, H-13a, overlapped with H-14b) in the H
(23) showed characteristic positive CEs between 210 and 227
nm in their ECD spectra.
H
1
C
DOI: 10.1021/acs.jnatprod.5b01164
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Journal of Natural Products
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1
Table 1. H NMR Spectroscopic Data (500 and 600 MHz) for Ugonins 1−6 (J in Hz)
b
,
c
a
,
d
3 ,
bd
4 ,
ad
5 ,
bd
6 ,
ad
position
1
2
3
6
8
9
6.30, s
6.23, d (1.8)
6.51, d (1.8)
3.36, d (17.4)
6.29, d (2.0)
6.26, br s
6.40, br s
3.00, d (18.0)
3.12, dd
6.26, d (2.0)
6.40, d (2.0)
2.85, br d (18.0)
3.11, dd
6.60, d (2.0)
6.55, s
6.67, s
2.75, dd
3.40 (2H), d (4.5)
2.76, dd
3.69, d (17.4)
(8.4, 18.0)
(4.0, 13.0)
2.94, dd
(
8.4,18.0)
(8.0, 18.0)
2.84, br d (18.0)
(
12.0, 13.0)
2.29, dd
1
1
0
2
1.73, t (4.5)
1.86, m
1.55, br d (8.0)
1.58, br d (7.8)
(
4.0, 12.0)
5.44, br s
1.62, td
5.78, d (10.2)
1.55, m
2.09, m
1.53, td
(4.2, 13.8)
2.14, m
1.42, m
1.94, m
1.33, m
1.42, m
1.96, m
(
3.5, 13.8)
2.48, ddd
2
.13, m
1.45 m
.88, m
1.38, m
.45, m
(5.5, 13.0, 13.0)
1.49, m
1
1
3
4
2.02 (2H), m
1.56 (2H), m
5.83, ddd
1.39, m
1.85, m
1.31, m
1.39, m
1
(1.8, 5.4, 10.2)
1.86, m
1.59, m
1.26, m
1
2.00, br d (18.0)
1.67, ddd
(
4.0, 13.0, 13.0)
0.93 (3H), s
1.06 (3H), s
4.15, d (1.5)
1
6 (17)
8
0.89 (3H), s
0.67 (3H), s
1.04 (3H), s
1.40 (3H), s
0.79 (3H), s
0.98 (3H), s
1.35 (3H), s
0.68 (3H), s
0.89 (3H), s
1.25 (3H), s
0.66 (3H), s
0.97 (3H), s
1.22 (3H) s
1.03 (3H), s
1
1.60 (3H), s
4
.41, d (1.5)
2′
5′
6′
7.38, d (2.4)
6.95, d (8.4)
7.31, dd
7.10, d (2.0)
6.98, d (8.5)
7.59, dd
6.96, s
6.94, s
6.77, d (8.4)
7.02, d (8.4)
6.76, d (8.5)
7.02, d (8.5)
(
2.4, 8.4)
(2.0, 8.5)
5
3
7
-OH
12.77, s
12.95, s
12.23, br s
12.21, br s
13.06, s
-OCH3
-OCH3
3.84 (3H), s
3.93 (3H), s
a
b
c
d
5
00 MHz. 600 MHz. Measured in DMSO-d . Measured in acetone-d .
6
6
The spectroscopic characteristics of 6 with the molecular
between H-1‴ and C-2″ (Figure 1). Furthermore, the H-1″
4
formula C H O closely resembled those of ugonin K (10)
signal showed an HMBC cross-peak with a carbon signal at δ
27
30
7
C
except for an additional methoxy group in 6. The HMBC
correlation (Figure 1) between the methoxy proton signal at δH
146.5 (2 × C, C-3′, 4′) that also correlated with H-2′ (δ 7.69),
H
H-5′ (δ 7.24), and H-6′ (δ 7.62). Subsequently, on the basis
H
H
3
.84 and C-3 (δ 139.5) placed the methoxy group at C-3. The
of the presence of the NOE effect between H-5′ and H-1″, this
sugar moiety was determined to be attached at C-4′. The acid
hydrolysis of 7 revealed the presence of quercetin and a sugar
moiety. The sugar was identified as D-glucose by high-
C
(10R) absolute configuration of 6 was based upon the negative
CEs between 285 and 320 nm in the ECD spectrum in contrast
to the carbonyl derivative of ugonin K with a 10S configuration
4
,18
19
(
280, 300, 335 nm: Δε 0.00, +0.68, +0.15, respectively).
performance anion-exchange chromatography and the
Therefore, the structure of 6, ugonin Y, was defined as (10R)-
positive specific rotation ([α]25 +30). On the basis of the
D
5
,3′,4′-trihydroxy-3,7-dimethoxy-6-(6,6-dimethyl-2-
above data, the structure of 7 was determined to be quercetin-
4′-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside.
methylenecyclohexylmethyl)flavone.
1
13
Compounds 7 and 8 were obtained as yellow, amorphous
The H and C NMR spectra of 8 with the molecular
1
13
powders, and their H and C NMR spectroscopic features
showed the characteristics of quercetin glucosides. The H
formula C H O displayed an additional set of glucose
33
40 22
1
signals compared to 7. Three anomeric proton signals
NMR spectrum of 7, with the molecular formula C H O ,
resonated at δ 4.60 (J = 8.4 Hz, H-1‴), 5.02 (J = 7.8 Hz,
2
7
30 17
H
displayed a broad singlet at δ 12.39 attributed to 5-OH; a pair
H-1″), and 5.49 (J = 7.2 Hz, H-1⁗) with their HMQC-related
H
of m-coupled doublets (J = 1.8 Hz) at δH 6.18 and 6.44
assigned to H-6 and H-8, respectively; and three signals at δH
carbon signals at δ 99.8 (C-1″), 100.7 (C-1⁗), and 104.0 (C-
C
1‴), respectively. In the HMBC spectrum of 8 (Figure 1),
7
.24 (1H, d, J = 9.0 Hz, H-5′), 7.62 (1H, dd, J = 2.4, 9.0 Hz, H-
cross-peaks between H-1‴ and C-2″ (δ 81.6), as well as
C
6′), and 7.69 (1H, d, J = 2.4 Hz, H-2′) attributed to the protons
between H-1″ and C-4′ (δ 147.2), were observed similar to 7,
C
of the ABX-type B-ring. In addition, signals corresponding to
indicating the presence of a 1→2 linkage between two the
glucosyl moieties at C-4′. In addition, the correlation between
two β-linked six-membered hexosyl derivatives were observed,
and two anomeric proton signals resonated at δ 4.60 (J = 7.8
H-1⁗ and C-3 (δ 133.8) suggested that the third glucosyl unit
H
C
Hz, H-1‴) and 5.01 (J = 7.8 Hz, H-1″) with their HMQC-
was linked at C-3. Like 7, the sugar moiety was deduced as D-
glucose. Therefore, the structure of 8 was determined to be
quercetin-3-O-β-D-glucopyranosyl-4′-O-β-D-glucopyranosyl-
(1→2)-β-D-glucopyranoside.
associated carbon signals observed at δ 99.8 (C-1″) and 104.0
C
(
C-1‴), respectively. The remaining sugar signals were assigned
through analyses of 2D COSY, TOCSY, HMQC, and HMBC
spectroscopic data. The 1→2 linkages of the two hexosyl
moieties were deduced on the basis of the HMBC cross-peaks
Among the isolated compounds, 1, 2, 5, 6, 9−14, and 23
were evaluated for their anti-inflammatory activity on LPS-
D
DOI: 10.1021/acs.jnatprod.5b01164
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 2. 13C NMR Spectroscopic Data (125 and 150 MHz) for Ugonins 1−6
b
,
c
a
,
d
3 ,
bd
4 ,
ad
5 ,
bd
6 ,
ad
position
1
2
2
3
4
4
5
6
7
8
8
9
1
1
1
1
1
1
1
146.1, C
133.9, C
169.2, C
103.9, C
162.3, C
99.1, CH
163.7, C
94.8, CH
157.2, C
35.5, CH2
94.1, C
151.9, C
134.9, C
170.6, C
105.6, C
164.2, C
99.8, CH
164.0, C
95.6, CH
158.6, C
21.8, CH2
44.3, CH
77.8, C
149.3, C
137.5, C
176.8, C
104.4, C
162.5, C
99.4, CH
165.8, C
94.6, CH
158.5, C
20.6, CH2
44.4, CH
74.5, C
149.4, C
137.5, C
176.8, C
104.4, C
162.5, C
99.4, CH
165.8, C
94.6, CH
158.5, C
22.0, CH2
44.7, CH
76.6, C
161.0, C
107.6, CH
176.7, C
108.9, C
156.7, C
107.3, C
160.0, C
94.7, CH
158.3, C
19.0, CH2
43.4, CH
76.8, C
156.6, C
139.5, C
179.6, C
106.2, C
159.2, C
113.1, C
164.8, C
90.4, CH
155.9, C
21.7, CH2
53.6, CH
149.0, C
30.2, CH2
24.0, CH2
35.5, CH2
35.2, C
a
a
0
1
135.8, C
123.1, CH
22.1, CH2
32.4, CH2
36.8, C
2
39.5, CH2
18.6, CH2
42.0, CH2
34.7, C
131.6, CH
128.7, CH
42.1, CH2
33.2, C
39.5, CH2
18.7, CH2
42.1, CH2
34.7, C
39.7, CH2
18.8, CH2
42.1, CH2
34.6, C
3
4
5
6 (17)
22.2, CH3
21.6, CH3
32.4, CH3
26.8, CH3
108.3, C
115.9, C
141.0, C
152.1, C
96.9, CH
152.3, C
21.5, CH3
30.7, CH3
27.5, CH3
121.7, C
122.9, C
142.1, C
148.5, C
112.8, CH
123.0, CH
21.8, CH3
32.6, CH3
27.1, CH3
122.0, C
123.3, C
142.8, C
148.8, C
112.7, CH
123.1, CH
21.7, CH3
32.5, CH3
27.1, CH3
124.5, C
113.7, CH
146.3, C
149.1, C
116.5, CH
119.2, CH
28.3, CH3
28.5, CH3
109.4, CH2
123.1, C
116.3, CH
145.8, C
150.5, C
116.2, CH
122.1, CH
60.1
2
2.7, CH3
18
1′
2′
3′
4′
5′
6′
17.6, CH3
105.0, C
117.5, C
147.1, C
151.4, C
97.8, CH
149.4, C
OCH (3)
3
OCH (7)
56.4
3
a
b
c
d
1
25 MHz. 150 MHz. Measured in DMSO-d . measured in acetone-d .
6
6
2
0
induced NO production in microglial cells, and the results are
shown in Table 3. Except compounds 1, 5, and 13, all the other
differentiation (osteoclastogenesis). The results revealed that
none of them had considerable osteoblastogenetic activity,
except ugonin K (10) at 10 μM (data not shown).
Nevertheless, all tested compounds except 13 displayed the
ability to suppress RANKL-induced osteoclast differentiation in
RAW264.7 cells with IC50 values of 1.8−4.6 μM (Table 4);
moreover, ugonin K (10) exhibited the greatest inhibitory
activity with an IC50 value of 1.8 ± 0.2 μM. In these
experiments, genistein was used as the positive control.
Table 3. Inhibition of LPS-Induced Nitric Oxide (NO)
Production in Microglial Cells by Compounds 1, 2, 5, 6, 9−
a
1
4, and 23
compound
IC50 (μM) in LPS-induced NO production
1
2
5
6
9
18.0 ± 4.8
7.0 ± 0.4
19.7 ± 5.2
6.7 ± 0.9
6.5 ± 0.5
8.1 ± 1.3
10.1 ± 1.1
7.7 ± 0.4
24.1 ± 6.1
6.2 ± 0.1
10.1 ± 0.6
10.8 ± 1.3
5
The present and previous studies show that H. zeylanica
rhizomes contain a rich array of anti-inflammatory constituents,
which may support its traditional use as an antipyretic and
antiphlogistic agent. The considerable antiosteoporotic activ-
ities of the constituents of H. zeylanica rhizomes may offer
10
11
12
13
14
23
Table 4. Effects of Compounds 1, 2, 5, 6, and 10−13 on the
Differentiation of Primary Cultured Osteoclasts
a
compound
IC50 (μM) in TRAP activity
PDTC
1
2
5
6
4.5 ± 0.2
3.5 ± 0.3
4.6 ± 0.4
3.2 ± 0.3
1.8 ± 0.2
2.5 ± 0.2
3.3 ± 0.4
>10
a
Data were calculated as 50% inhibitory concentration (IC ). Values
5
0
are expressed as mean ± SEM (n = 3). Pyrrolidine dithiocarbamate
(
PDTC, a NF-kB inhibitor) was used as a positive control.
1
1
1
1
0
1
2
3
compounds inhibited NO production with IC50 values of 6.2−
0.1 μM and were more potent than the NF-kB inhibitor
1
pyrrolidine dithiocarbamate (PDTC), used as the positive
control, with an IC50 value 10.8 μM. In addition, the
antiosteoporotic activities of compounds 1, 2, 5, 6, and 10−
genistein
11.4 ± 0.2
a
Data were calculated as 50% inhibitory concentration (IC ). Values
are expressed as mean ± SEM (n = 3). Genistein was used as a positive
5
0
1
3 were investigated through the increase in osteoblast
differentiation (osteoblastogenesis) or decrease in osteoclast
control.
E
DOI: 10.1021/acs.jnatprod.5b01164
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
evidence for another use of this herbal drug, i.e., to treat bone
fracture and contusions.
(3.84), 362 (4.31) nm; IR (KBr) νmax 3363 (OH), 2923, 1657, 1634,
−
1 1
1
611, 1460, 1364, 1291, 1221, 1001 cm ; H NMR (DMSO-d , 600
6
13
MHz) see Table 1; C NMR (DMSO-d , 150 MHz) see Table 2;
6
+
+
ESIMS m/z 435.23 [M + H] ; HRESIMS m/z 434.1374 [M] (calcd
for C25 , 434.1366).
Ugonin W (2): yellow powder; mp 145−147 °C; [α] −134 (c
EXPERIMENTAL SECTION
■
H O
22 7
2
5
General Experimental Procedures. Melting points were
determined on a Yanaco MP-I3 micro melting point apparatus, and
the thermometer was used without correction. Optical rotations were
measured on a JASCO P-2000 polarimeter. UV spectra were recorded
on a Hitachi U-3310 UV/vis spectrometer. The ECD spectra were
recorded on a JASCO J-715 spectrometer. IR spectra were recorded
on a Nicolet Avatar 320 FT-IR spectrometer. H NMR and C NMR
spectra were measured with a Varian Unity Inova 500 MHz or Varian
VNMRS 600 MHz FT-NMR spectrometer. EIMS spectra were
obtained using a Finnigan Focus GC & DSQ II GC-MS spectrometer
at 70 eV. ESIMS spectra were acquired on a Finnigan MAT LCQ mass
spectrometer. HRESIMS spectra were recorded on a Finnigan MAT
D
1
.0, MeOH); UV (MeOH) λ (log ε) 203 (4.57), 260 (4.23), 351
max
(
1
4.27) nm; IR (KBr) νmax 3399 (OH), 2929, 1650, 1609, 1454, 1377,
332, 1262, 1213, 1160, 1009, 825 cm ; ECD (c 4.59 × 10 M,
−1
−5
MeOH) Δε (nm) +1.38 (210), +0.40 (221), +1.06 (227), −0.80
1
(264), −0.16 (275), −1.53 (304), −0.22 (332), −0.85 (366); H
1
13
13
NMR (acetone-d
, 500 MHz) see Table 1; C NMR (acetone-d
, 125
6
6
−
MHz) see Table 2; ESIMS m/z 435.25 [M − H] ; HRESIMS m/z
436.1534 [M] (calcd for C25H O , 436.1522).
Ugonin X (3): yellow powder; mp 130−132 °C; [α]
MeOH); UV (MeOH) λmax (log ε) 207 (4.65), 254 (4.33), 299
(3.92), 349 (4.04) nm; IR (KBr) νmax 3440 (OH), 2927, 1654, 1600,
+
24 7
25
−162 (c 0.1,
D
−1 1
9
5S mass spectrometer or a Q Exactive Focus mass spectrometer.
Plant Material. The rhizomes of H. zeylanica (L.) Hook. were
1484, 1457, 1372, 1268, 1237, 1175, 1074 cm ; H NMR (acetone-d ,
6
600 MHz) see Table 1; 13C NMR (acetone-d
, 150 MHz) see Table 2;
6
−
+
purchased in Taipei, Taiwan, in June 2010. The plant was identified by
comparison with the voucher specimen (NRICM-99-003) already
deposited at the herbarium of the National Research Institute of
Chinese Medicine, Republic of China.
ESIMS m/z 435.12 [M − H] ; HRESIMS m/z 436.1530 [M] (calcd
for C25H O , 436.1522).
24 7
2
5
D
(10R,11S)-Ugonin N (4): yellow powder; mp 164−166 °C; [α]
−12 (c 0.9, MeOH); UV (MeOH) λmax (log ε) 207 (5.00), 254 (4.56),
Extraction and Isolation. The rhizomes of H. zeylanica (10.8 kg)
were extracted with EtOH (3 × 80 L) at 50 °C for 24 h. The
concentrated EtOH extract (280.5 g) was partioned between EtOAc
and H O as well as between H O and butanol. The EtOAc extract
295 (4.32), 346 (4.27) nm; IR (KBr) νmax 3408 (OH), 2929, 2868,
−1
1
1638, 1601, 1450, 1373, 1234, 1164, 1038, 833 cm ; H NMR
(acetone-d
, 500 MHz) see Table 1; 13C NMR (acetone-d
, 125 MHz)
6
6
−
see Table 2; ESIMS m/z 437.30 [M − H] ; HRESIMS m/z 437.1609
2
2
−
(
215.5 g) was subjected to a silica gel column (70−230 mesh, 10 × 60
[M − H] (calcd for C25H O , 437.1595).
25 7
2
5
D
cm, Merck) eluting with gradient solvent systems of n-hexane−acetone
(10R,11S)-Ugonin S (5): yellow powder; mp 261−263 °C; [α]
(
5
2
10:1, 5:1, 2:1), CH Cl −acetone (10:1, 5:1), CH Cl −MeOH (10:1,
−31 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 216 (4.54), 272 (4.23),
2
2
2
2
:1), and MeOH to yield 87 fractions (3 L each). From fractions 15−
7, n-hexane−acetone (5:1) eluate, the CH Cl -undissolved precip-
337 (4.31) nm; IR (KBr) νmax 3429 (OH), 2943, 2838, 1634, 1600,
−1
1573, 1461, 1353, 1294, 1264, 1156, 1082, 862 cm ; ECD (c 4.74 ×
2
2
−5
itate was obtained and identified as ugonin K (10, 2.5 g). The filtrate
was repeatedly chromatographed over silica gel columns eluting with
n-hexane−acetone (3:1) and CH Cl −acetone (30:1) and Sephadex
10 M, MeOH) Δε (nm) −2.9 (210), −9.86 (215), −1.85 (231),
1
+3.59 (272), −3.97 (313), +1.57 (353), +0.04 (372); H NMR
(acetone-d
, 600 MHz) see Table 1; 13C NMR (acetone-d
, 150 MHz)
2
2
6
6
+
LH-20 columns eluting with MeOH−H O (3:1) to afford ugonins V
see Table 2; ESIMS m/z 423.00 [M + H] ; HRESIMS m/z 423.1809
2
+
(1, 11.6 mg), W (2, 97.8 mg), and X (3, 2.5 mg), (10R,11S)-ugonin N
(4, 4.2 mg), Y (6, 21.0 mg), and O (12, 74.6 mg), and ugonstilbene A
(14, 126.7 mg). The same treatment as above for fraction 31 gave
[M + H] (calcd for C25
H O , 423.1802).
27 6
25
(10R,11R)-Ugonin S (13): [α]
+71 (c 0.5, MeOH); ECD (c 4.74
D
−
5
× 10 M, MeOH) Δε (nm) +3.71 (206), +7.78 (214), +3.69 (225),
1
ugonins M (11, 114.1 mg) and J (9, 208.4 mg). Fractions 42−55,
+1.11 (239), +2.43 (262), + 0.22 (282), +1.95 (308), −0.32 (346); H
CH Cl −acetone (10:1−5:1) eluate, were repeatedly purified by silica
NMR (acetone-d , 600 MHz) δ 7.44 (1H, d, J = 1.8 Hz, H-2′), 7.34
2
2
6
gel columns eluting with CH Cl −MeOH (40:1−20:1) and Sephadex
(1H, dd, J = 1.8, 8.4 Hz, H-6′), 6.95 (1H, d, J = 8.4 Hz, H-5′), 6.59
(1H, s, H-8), 6.36 (1H, s, H-3), 2.77 (1H, dd, J = 4.8, 16.2 Hz, H-9a),
2.35 (1H, dd, J = 13.8, 16.2 Hz, H-9b), 2.07 (1H, m, H-12a), 1.67
2
2
LH-20 columns eluting with MeOH−H O (3:1) to afford (10R,11S)-
2
ugonin S (5, 20.0 mg) and ugonin S (13, 22.5 mg). The n-BuOH
extract (24.9 g) was subjected to a 75C18 OPN column (4.6 × 60 cm,
(1H, m, H-12b), 1.64 (2H, m, H -13), 1.62 (1H, dd, J = 4.8, 13.8 Hz,
2
Cosmosil) eluting with H O−MeOH (80:20−20:80) to yield 50
H-10), 1.50 (1H, m, H-14a), 1.36 (1H, td, 4.8, 12.6 Hz, H-14b), 1.24
2
1
3
fractions (250 mL each). The combined fractions 1−5, H O−MeOH
(3H, s, CH -18), 1.03 and 0.95 (each 3H, s, CH -16, 17); C NMR
2
3
3
(
80:20−60:40) eluate, was chromatographed through a 75C OPN
(acetone-d , 150 MHz) δ 176.7 (C-4), 161.0 (C-2), 160.4 (C-7), 158.5
18
6
column (H O−MeOH, 60:40) and further purified by a Sephadex LH-
(C-8a), 155.3 (C-5), 149.0 (C-4′), 146.3 (C-3′), 124.4 (C-1′), 119.2
(C-6′), 116.4 (C-5′), 113.6 (C-2′), 108.9 (C-4a), 108.0 (C-6), 107.4
(C-3), 94.7 (C-8), 78.6 (C-11), 47.6 (C-10), 42.1 (C-14), 40.4 (C-
12), 34.0 (C-15), 32.3 (C-17), 20.8 (C-16), 20.4 (C-13), 19.9 (C-18),
18.7 (C-9).
2
2
0 column (2.6 × 36 cm) eluting with MeOH−H O (1:1) to give
2
benzoic acid (22, 3.7 mg) and quercetin-3,4′-di-O-β-D-glucopyranoside
18, 37.7 mg). Using the same separation methods as fractions 1−5,
(
the combined fractions 6−11 gave quercetin (15, 6.7 mg), quercetin-3-
O-β-D-glucopyranoside (16, 18.4 mg), quercetin-4′-O-β-D-glucopyr-
anoside (17, 28.9 mg), quercetin-3-O-β-D-glucopyranosyl-(1→4)-β-D-
glucopyranoside (19, 32.6 mg), and quercetin-4′-O-β-D-glucopyrano-
25
Ugonin Y (6): yellow powder; mp 208−210 °C; [α] +3 (c 0.6,
D
MeOH); UV (MeOH) λ
(log ε) 203 (4.64), 264 (4.33), 292
max
(4.00), 363 (4.66) nm; IR (KBr) νmax 3484 (OH), 2931, 2864, 1719,
−1
syl-(1→2)-β-D-glucopyranoside (7, 22.6 mg). The H O extract (24.3
1648, 1593, 1477, 1434, 1354, 1215, 1163, 1056, 810 cm ; ECD (c
2
−5
g) was concentrated and treated with MeOH to afford MeOH-soluble
4.29 × 10 M, MeOH) Δε (nm) −1.42 (260), −0.16 (280), −2.61
1
(
12.6 g) and MeOH-insoluble (1.75 g) portions. The former was
(300), −0.36 (331), −2.62 (369); H NMR (acetone-d , 500 MHz)
6
see Table 1; 13C NMR (acetone-d , 125 MHz) see Table 2; ESIMS m/
z 489.00 [M + Na] ; HRESIMS m/z 466.2002 [M] (calcd for
repeatedly chromatographed on Sephadex LH-20 columns eluting
6
+
+
with MeOH−H O (3:1) to afford quercetin-3-O-β-D-glucopyranosyl-
2
4
′-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (8, 16.4 mg)
C H O , 466.1992).
27
30
7
and quercetin-3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-4′-
Quercetin-4′-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside
O-β-D-glucopyranoside (20, 16.7 mg). The latter was dissolved in
(7): yellow, amorphous powder; mp 216−218 °C; UV (MeOH) λ
max
MeOH−H O (3:1) and chromatographed over Sephadex LH-20
(log ε) 203 (4.79), 253 (4.43), 336 (4.40) nm; IR (KBr) ν 3412
2
max
columns eluting with MeOH−H O (1:1−3:1) to afford 20 (39.0 mg)
(OH), 2921, 1655, 1618, 1597, 1507, 1462, 1368, 1254, 1209, 1168,
2
−1 1
and quercetin-3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-4′-
1070, 805 cm ; H NMR (DMSO-d , 600 MHz) δ 12.39 (1H, br s, 5-
6
O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (21, 38.7 mg).
OH), 7.69 (1H, d, J = 2.4 Hz, H-2′), 7.62 (1H, dd, J = 2.4, 9.0 Hz, H-
6′), 7.24 (1H, d, J = 9.0 Hz, H-5′), 6.44 (1H, d, J = 1.8 Hz, H-8), 6.18
(1H, d, J = 1.8 Hz, H-6), 5.01 (1H, d, J = 7.8 Hz, H-1″), 4.60 (1H, d, J
25
Ugonin V (1): yellow powder; mp 174−176 °C; [α] +24 (c 0.4,
D
MeOH); UV (MeOH) λ
(log ε) 203 (4.51), 264 (4.15), 292
max
F
DOI: 10.1021/acs.jnatprod.5b01164
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1H, 13C, 2D NMR, and HRESIMS spectra of new
compounds 1−8 along with H and C NMR spectra of
known compounds and the values of mean ± SD of
Tables 3 and 4 (PDF)
=
7.8 Hz, H-1‴), 3.72 (1H, br d, J = 10.2 Hz, H-6″b), 3.60 (1H, t, J =
8
1
5
.4 Hz, H-2″), 3.50 (1H, t, J = 9.0 Hz, H-3″), 3.49 (1H, dd, J = 6.0,
0.2 Hz, H-6″a), 3.42 (2H, m, H-6‴), 3.39 (1H, dd, J = 1.8, 6.0, Hz,H-
″), 3.25 (1H, t, J = 9.6 Hz, H-4″), 3.19 (1H, t, J = 8.4 Hz, H-3‴), 3.17
1
13
(
1H, m, H-5‴), 3.13 (1H, t, J = 9.0 Hz, H-4‴), 3.02 (1H, t, J = 8.4 Hz,
13
H-2‴); C NMR (DMSO-d , 150 MHz) δ 176.0 (C-4), 164.6 (C-7),
1
6
60.7 (C-5), 156.3 (C-9), 146.5 (2 × C, C-3′, 4′), 145.7 (C-2), 136.4
AUTHOR INFORMATION
(
(
2
6
6
C-3), 125.4 (C-1′), 119.4 (C-6′), 115.9 (C-5′), 114.7 (C-2′), 104.0
C-1‴), 102.9 (C-10), 99.8 (C-1″), 98.4 (C-6), 93.6 (C-8), 81.9 (C-
■
*
Corresponding Author
Tel: 886-2-28201999, ext. 6701. Fax: 886-2-28264276. E-mail:
ylhuang@nricm.edu.tw.
″), 77.0 (C-5″), 76.8 (C-5‴), 76.2 (C-3‴), 75.8 (C-3″), 74.4 (C-2‴),
9.6 (C-4‴), 69.3 (C-4″), 60.5 (C-6″), 60.4 (C-6‴); ESIMS m/z
25.00 [M − H] ; HRESIMS m/z 626.1502 [M] (calcd for
−
+
C H O , 626.1483).
ORCID
2
7
30 17
Acid Hydrolysis of 7. Compound 7 (7.5 mg) was treated with 5 mL
Yu-Ling Huang: 0000-0001-9914-1716
of 10% HCl at 100 °C for 8 h and gave quercetin and D-glucose
[α]25 +30, c 0.6, H O).
Notes
(
D
2
Quercetin-3-O-β-D-glucopyranosyl-4′-O-β-D-glucopyranosyl-(1→
The authors declare no competing financial interest.
2
°
)-β-D-glucopyranoside (8): yellow, amorphous powder; mp 210−212
C; UV (MeOH) λmax (log ε) 205 (4.76), 267 (4.48), 347 (4.33) nm;
ACKNOWLEDGMENTS
■
IR (KBr) νmax 3432 (OH), 2913, 1655, 1609, 1512, 1360, 1205, 1168,
−1 1
1
074, 827 cm ; H NMR (DMSO-d , 600 MHz) δ 12.54 (1H, br s, 5-
This work was supported in part by the National Research
Institute of Chinese Medicine, Ministry of Health and Welfare,
Republic of China (MOHW103-NRICM-C-315-122304). The
authors are grateful to the Instrumentation Center at National
Taiwan University for HRESIMS measurements. We also thank
Mr. J.-C. Ou for identification of the studied plant, Dr. M.-J.
Don for offering (16R,17S)- and (16S,17R)-ugonstilbene B,
and Dr. M.-K. Lu for confirmation of glucose by high-
performance anion-exchange chromatography.
6
OH), 7.64 (1H, dd, J = 2.4, 9.0 Hz, H-6′), 7.59 (1H, d, J = 2.4 Hz, H-
′), 7.20 (1H, d, J = 9.0 Hz, H-5′), 6.40 (1H, d, J = 1.8 Hz, H-8), 6.17
1H, d, J = 1.8 Hz, H-6), 5.49 (1H, d, J = 7.2 Hz, H-1⁗), 5.02 (1H, d, J
2
(
=
1
8
6
9
7.8 Hz, H-1″), 4.60 (1H, d, J = 8.4 Hz, H-1‴), 3.73 (1H, br d, J =
1.1 Hz, H-6‴b), 3.61 (1H, t, J = 10.8 Hz, H-2″), 3.58 (1H, br d, J =
.4 Hz, H-6⁗b), 3.50 (1H, t, J = 10.8 Hz, H-3″), 3.47 (2H, m, H-6″b,
‴a), 3.43 (2H, m, H-5″, 6″a), 3.34 (1H, m, H-6⁗a), 3.25 (1H, t, J =
.0 Hz, H-4″), 3.20 (2H, m, H-2⁗, 3⁗), 3.17 (2H, m, H-3‴, 5‴), 3.12
(
1H, t, J = 9.0 Hz, H-4‴), 3.07 (2H, m, H-4⁗, 5⁗), 3.02 (1H, t, J = 8.4
13
Hz, H-2‴); C NMR (DMSO-d , 150 MHz) δ 177.4 (C-4), 165.0 (C-
6
7
1
1
9
(
(
6
), 161.2 (C-5), 156.5 (C-9), 155.2 (C-2), 147.2 (C-4′), 146.3 (C-3′),
33.8 (C-3), 124.7 (C-1′), 121.1 (C-6′), 115.9 (C-2′), 115.5 (C-5′),
04.0 (C-1‴), 103.8 (C-10), 100.7 (C-1⁗), 99.8 (C-1″), 98.9 (C-6),
3.8 (C-8), 81.6 (C-2″), 77.7 (C-5⁗), 77.0 (C-5″), 76.9 (C-5‴), 76.5
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