Bioactive lignans from the Trunk of Abies holophylla

Article abstract of DOI:10.1021/np4005322

Six new lignans (1-6) were isolated from the trunk of Abies holophylla MAXIM, together with 11 known lignans (7-17). The structures of 1-7 were elucidated by spectroscopic methods, acid hydrolysis, and use of the modified Mosher's method. The effects of the isolates on nerve growth factor induction in a C6 rat glioma cell line were evaluated. Compounds 6, 7, and 13 showed significant induction of nerve growth factor secretion at concentrations of 10 μM. Compounds 1, 5, 6, and 16 showed moderate inhibitory effects on nitric oxide production in lipopolysaccharide-activated BV-2 cells (IC₅₀ 28.5-36.4 μM).

Full text of DOI:10.1021/np4005322

Note
pubs.acs.org/jnp
Bioactive Lignans from the Trunk of Abies holophylla
Chung Sub Kim,† Oh Wook Kwon,‡,§ Sun Yeou Kim,§,⊥ and Kang Ro Lee*,†
†Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
‡Graduate School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Republic of Korea
§Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 406-799, Republic of Korea
⊥College of Pharmacy, Gachon University, Incheon 406-799, Republic of Korea
S
* Supporting Information
ABSTRACT: Six new lignans (1−6) were isolated from the trunk of
Abies holophylla MAXIM, together with 11 known lignans (7−17). The
structures of 1−7 were elucidated by spectroscopic methods, acid
hydrolysis, and use of the modified Mosher’s method. The effects of the
isolates on nerve growth factor induction in a C6 rat glioma cell line were
evaluated. Compounds 6, 7, and 13 showed significant induction of nerve
growth factor secretion at concentrations of 10 μM. Compounds 1, 5, 6, and 16 showed moderate inhibitory effects on nitric
oxide production in lipopolysaccharide-activated BV-2 cells (IC50 28.5−36.4 μM).
H-2′), 6.75 (1H, d, J = 8.0 Hz, H-5′), and 6.74 (1H, dd, J = 8.0,
1.5 Hz, H-6′)], one acetal methine [δH 4.67 (1H, d, J = 1.0 Hz,
H-9)], one oxymethine [δH 4.52 (1H, d, J = 9.0 Hz, H-7′)],
three OCH3 groups [δH 3.84 (3H, s, 3-OCH3), 3.83 (3H, s, 3′-
OCH3), and 3.19 (3H, s, 9-OCH3)], one oxymethylene [δH
3.71 (1H, t, J = 8.5 Hz, H-9′a) and 3.51 (1H, t, J = 8.5 Hz, H-
9′b)], one methylene [δH 2.75 (1H, dd, J = 13.0, 4.5 Hz, H-7a)
and 2.48 (1H, m, H-7b)], and two methine protons [δH 2.56
(1H, m, H-8) and 2.35 (1H, m, H-8′)]. The 13C NMR
spectrum contained 21 signals, including 12 aromatic carbons
for two aromatic rings, one acetal methine [δC 109.5 (C-9)],
one oxymethine [δC 75.8 (C-7′)], one oxymethylene [δC 68.5
(C-9′)], three OCH3 groups [δC 55.2 (3-OCH3), 55.1 (3′-
OCH3), and 53.5 (9-OCH3)], two methine [δC 51.6 (C-8′) and
50.3 (C-8)], and one methylene [δC 39.2 (C-7)] carbons.
These spectroscopic data suggested that 1 was a 9-O-9′ subtype
tetrahydrofuran lignan,4,5 and these data were similar to those
of iso-α-intermedianol except for the absence of signals of an
OCH3 at C-7′ [δH 3.33 (3H, s); δC 56.3].5 1H−1H correlation
spectroscopy (COSY), heteronuclear multiple quantum
correlation spectroscopy (HMQC), and heteronuclear multiple
bond correlation spectroscopy (HMBC) spectra (Figure S2 of
the Supporting Information) confirmed the planar structure of
1. The relative configuration of 1 was elucidated from the
coupling constants and NOESY correlations. The small J value
between H-8/H-9 (1.0 Hz) showed that H-8 and H-9 were in
the trans form5−7 and the NOESY correlations of H-7/H-9 and
H-8′ and H-8/H-7′ corroborated the relative configurations at
C-8, C-9, C-7′, and C-8′ (Figure S3 of the Supporting
Information). In the CD spectrum, negative Cotton effects at
224 and 284 nm indicated that 1 had 8R and 8′R absolute
configuration.8,9 Through Mosher’s method with (R)- and (S)-
Abies holophylla MAXIM (Pinaceae), also known as Man-
churian Fir or Needle Fir, is an evergreen and coniferous tree
that is widely distributed in Korea, China, and Russia.1,2 Several
Abies species have been used in Korean folk medicine for the
treatment of colds, stomach aches, indigestion, rheumatic
diseases, and vascular and pulmonary diseases.2 Previous
phytochemical investigations on A. holophylla reported lignans,
terpenoids, steroids, and phenolic compounds, and their
cytotoxic activities against several tumor cell lines or inhibitory
effect against lipopolysaccharide (LPS)-induced nitric oxide
(NO) production in RAW264.7 macrophages.1,3
As part of our efforts to discover constituents of Korean
medicinal plants with antineuroinflammatory activity, we found
that an EtOAc phase obtained from a MeOH extract (see
Experimental Section) of the trunk of A. holophylla exhibited
inhibitory effects on NO production in murine microglia BV-2
cells. The EtOAc solubles were successively chromatographed
through silica gel, Sephadex LH-20, and prep high-performance
liquid chromatography (HPLC). The result was the isolation of
six new lignans (1−6) and 11 known lignans (7−17). Their
structures were determined by spectroscopic methods,
including one-dimensional (1D) and two-dimensional (2D)
NMR, HRMS, CD experiments, acid hydrolysis, and the use of
the modified Mosher’s method. Herein, we report the isolation
and structural elucidation of compounds 1−7, along with NGF
secretion and inhibitory effects on NO production of isolates
(1−17). (see Figure S1 of the Supporting Information for
structures of the known compounds).
Compound 1 had a molecular formula of C21H26O7, as
determined from the ion peak [M + H]+ at m/z 391.1757 in
positive ion high-resolution fast-atom bombardment mass
1
spectrometry (HRFABMS). The H NMR spectrum showed
the presence of two 1,3,4-trisubstituted aromatic rings [δH 6.77
(1H, d, J = 2.0 Hz, H-2), 6.71 (1H, d, J = 7.5 Hz, H-5), and
6.62 (1H, dd, J = 7.5, 2.0 Hz, H-6) and 6.87 (1H, d, J = 1.5 Hz,
Received: July 1, 2013
Published: November 13, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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MPA, the absolute configuration at C-7′ was confirmed as S
(Figure S4 of the Supporting Information).10,11 (−)-Koreanol12
was reported without determination of the configuration at C-
7′, and its 1H and 13C NMR spectra were quite similar to those
of 1. The J value of H-7′ in (−)-koreanol (d, J = 5.2 Hz) was
reported to be different from that of 1 (d, J = 9.0 Hz),
indicating that (−)-koreanol could be the C-7′ epimer of 1.
Thus, compound 1 was determined to be (8R,9R,7′S,8′R)-
4,4′,7′-trihydroxy-3,3′,9-trimethoxy-9,9′-epoxylignan, and it was
named holophyllol A.
named holophyllol B, was unequivocally defined as
(8S,9S,7′R,8′R)-4,4′,7′-trihydroxy-3,3′,9-trimethoxy-9,9′-epoxy-
lignan.
Compound 3 also had a molecular formula of C21H26O7.
Comparison of the NMR spectra of 3 with those of 2 showed
they were very similar. However, upfield shifts of C-7 (δC 32.9)
and C-9 (δC 105.4) were observed in 13C NMR spectrum of 3,
suggesting that the relative configuration of 3 was identical with
that of 2, except at C-9. The large J value H-8/H-9 (4.5 Hz)
and NOESY correlations of H-7/H-7′ and H-8/H-8′ and H-9
confirmed the relative configuration of 3.5,6,13 The absolute
configurations at C-8 and C-8′ were determined as S and R,
respectively, from the CD spectrum which was very similar to
that of 2, and the 7′R configuration was determined by the
modified Mosher’s method. Thus, the structure of 3 was
established as (8S,9R,7′R,8′R)-4,4′,7′-trihydroxy-3,3′,9-trime-
thoxy-9,9′-epoxylignan, and it was named holophyllol C.
Compound 4 was obtained as a colorless gum. The
molecular formula was determined to be C25H32O9 from the
1
[M + Na]+ ion in the positive ion HRESIMS. The H and 13
C
NMR spectra of 4 suggested that 4 was a dihydrobenzofuran
neolignan glycoside and were similar to those of 11,14 except
for the absence of signals of an OH group at C-5′ (δC 145.1) of
11. 1H−1H COSY, HMQC, and HMBC correlations confirmed
the planar structure of 4. The HMBC correlation of H-1″ to C-
4 indicated that the rhamnose unit was linked to the oxygen at
C-4, and the J value of anomeric proton (J = 1.5 Hz) confirmed
it as α-rhamnose.15 Acid hydrolysis of 4 afforded the aglycone,
cedrusinin, and L-rhamnose ([α]2D5 +9.0), which was identified
by co-TLC confirmation and gas chromatography (GC)
analysis.16,17 The identification of the aglycone was by
comparison of its 1H NMR and MS data.18,19 The trans-
configuration between H-7 and H-8 was confirmed through the
J value (6.0 Hz),20 and its CD spectrum showed the negative
Cotton effect at 236 nm confirming the absolute configurations
as 7R and 8S.21,22 Cedrusinin-4-O-α-L-rhamnopyranoside was
isolated without determination of the absolute configuration at
C-7 and C-8,15 and its H and 13C NMR spectra were quite
1
similar to those of 4. From the opposite sign of optical rotation
values between cedrusinin-4-O-α-L-rhamnopyranoside ([α]2D3
−50.0) and 4 ([α]2D5 +16.0), cedrusinin-4-O-α-L-rhamnopyr-
anoside should be 7S and 8R forms. Thus, the structure of 4
was determined to be (7R,8S)-cedrusinin 4-O-α-L-rhamnoside.
Compound 5 displayed an HRESIMS ion peak, [M + Na]+ at
m/z 529.2052, consistent with the molecular formula
1
C26H34O10. The H and 13C NMR spectra were very similar
to those of 13,23 except for absence of signals of an OCH3 [δH
3.86 (3H, s); δC 55.6]. The location of the two OCH3 groups in
5 was established by the HMBC correlations from 3-OCH3 (δH
3.85) to C-3 (δC 152.2) and from 3″-OMe (δH 3.53) to C-3″
(δC 82.0). Additionally, the HMBC correlation from H-1′ to C-
4 showed that the sugar unit was located at C-4. Acid hydrolysis
of 5 gave the aglycone, 8, which was identified as cedrusin by
Compound 2 was assigned the molecular formula of
C21H26O7 by high-resolution electrospray ionization mass
1
spectrometry (HRESIMS). The H and 13C NMR spectra of
2 closely resembled those of 1 but with an upfield shift of H-9′
(δH 4.18 and 3.99) and downfield shifts of H-7, 8, 7′, and 8′ (δH
2.40 and 2.04, 1.95, 4.42, and 2.22, respectively), indicating that
2 was a stereoisomer of 1. The small J value between H-8/H-9
(1.0 Hz) and NOESY correlations of H-7/H-9 and H-7′ and
H-8/H-8′ and H-9′-OCH3 confirmed the relative configuration
of 2.5−7 The CD spectrum of 2, which showed negative Cotton
effects at 228 and 286 nm, was quite similar to that of
(8S,8′R,9S)-cubebin,9 thus, the absolute configurations at C-8
and C-8′ were determined as S and R, respectively. Mosher’s
esterification with (R)- and (S)-MPA revealed that the absolute
configuration at C-7′ of 2 was R. Therefore, the structure of 2,
24
1
comparison of its H NMR and MS data, and 3-O-methyl-L-
rhamnose, which was identified by co-TLC confirmation,
25,26
1
optical rotation ([α]2D5 +10.3), H NMR and MS data,
and
GC analysis. The relative configuration between H-7 and H-8
of 5 was determined by the J value (6.0 Hz) as trans,20 and the
negative Cotton effect at 245 nm in the CD spectrum
confirmed the 7R and 8S configuration.21,22 Thus, 5 was
elucidated as (7R,8S)-cedrusin 4-O-(3-O-methyl-α-L-rhamno-
side).
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Compound 6 had a molecular formula of C25H34O10, as
determined by the ion peak [M + Na]+ at m/z 517.2048. The
1H and 13C NMR spectra were similar to those of ligraminol
E,27 except for the presence of signals of a xylose unit [δH 4.80
(1H, d, J = 7.0, H-1″), 3.89 and 3.29 (each 1H, m, H-5″), 3.55
(1H, m, H-4″), 3.46 (1H, m, H-2″), and 3.42 (1H, m, H-3″);
δC 102.2 (C-1″), 75.9 (C-3″), 73.2 (C-2″), 69.6 (C-4″), and
secretion (169 9%, 165 9%, and 159 11%, respectively),
which displayed more activitiy than 6-shogaol (129 11%), a
positive control, at a concentration of 10 μM. The other
compounds were considered moderately active or inactive.
Antineuroinflammatory activities of the isolates (1−17) were
also tested via measurement of NO levels using bacterial
endotoxin, LPS, in the murine microglia BV-2 cell line.
Compounds 1, 5, 6, and 16 moderately inhibited NO
production with IC50 values of 36.4, 32.9, 31.0, and 28.5 μM,
respectively, in LPS-stimulated BV-2 cells without cell toxicity
(Table S2 of the Supporting Information).
1
65.4 (C-5″)]. This was supported by H−1H COSY, HMQC,
and HMBC spectra. The xylose unit was placed at C-4 by the
observation of an HMBC correlation from H-1″ to C-4. Acid
hydrolysis of 6 afforded the aglycone, ligraminol E, and D-xylose
([α]2D5 +21.1), which was identified by co-TLC and GC
analysis.16,17 The aglycone was identified as ligraminol E by
EXPERIMENTAL SECTION
27
1
■
comparison of its H NMR and MS data. The absolute
configuration of 6 at C-8 was confirmed to be 8R by the
negative Cotton effect at 232 nm in the CD spectrum of
ligraminol E.27 On the basis of these results, the structure of 6
was established as ligraminol E 4-O-β-D-xyloside.
General Experimental Procedures. Optical rotations were
measured on a JASCO P-1020 polarimeter. Infrared spectra were
recorded on a Bruker IFS-66/S FT-IR spectrometer. CD spectra were
measured on a JASCO J-810 spectropolarimeter. UV spectra were
recorded using an Agilent 8453 UV−visible spectrophotometer. NMR
spectra were recorded on a Varian UNITY INOVA 500 NMR
spectrometer operating at 500 MHz (1H) and 125 MHz (13C),
respectively. HRFABMS spectra were obtained on a JEOL JMS700
mass spectrometer and HRESIMS spectra were recorded on a
Micromass QTOF2-MS. Preparative HPLC was performed using a
Gilson 306 pump with a Shodex refractive index detector. Silica gel 60
(Merck, 230−400 mesh) and RP-C18 silica gel (Merck, 230−400
mesh) were used for column chromatography (CC). TLC was
performed using Merck precoated silica gel F254 plates and RP-18 F254s
plates. The packing material for molecular sieve CC was Sephadex LH-
20 (Pharmacia Company). Low-pressure liquid chromatography was
performed over Merck Lichroprep Lobar-A (240 × 10 mm) column
with an FMI QSY-0 pump (ISCO).
Compound 7 had the molecular formula of C21H26O7.
Compounds 7 and 1 had very similar NMR spectra, but marked
differences included an upfield shift of C-7 (δC 34.2) and C-9
(δC 106.2) in the 13C NMR spectrum and a small change of
chemical shift of methine and methylene protons at H-7, 8, 9,
1
7′, 8′, and 9′ in the H NMR spectrum. Extensive 1D and 2D
NMR analyses confirmed that the planar structure of 7 was the
same as that of 1 but that the configuration at C-9 was different.
The large J value between H-8/H-9 (4.5 Hz) confirmed that H-
8 and H-9 were in the cis form5,6,13 and NOESY correlations of
H-8/H-9 and H-7′ and H-7/H-8′ confirmed the relative
configurations at C-8, C-9, C-7′, and C-8′. Pseudolarkaemin A
was isolated from Pseudolarix kaempferi without determination
of the absolute configuration.13 1H and 13C NMR, HRMS,
specific optical rotation, UV, and IR data of pseudolarkaemin A
were almost identical with those of 7. The absolute
configurations at C-8 and C-8′ were determined to be R by
CD spectrum, which was quite similar to that of 1. The
absolute configuration at C-7′ was elucidated to be S using the
same method as for 1. The irregular Δδ value of H-8′ (+0.023)
was due to the anisotropic effect of the α-aromatic group.28
Thus, 7 was established as (8R,9S,7′S,8′R)-4,4′,7′-trihydroxy-
3,3′,9-trimethoxy-9,9′-epoxylignan. Although compound 7 was
a known, pseudolarkaemin A, the absolute configuration of it
had not been reported earlier.
Plant Material. A. holophylla trunk material was collected in Seoul,
Korea, in January 2012, and the plant was identified by one of the
authors (K.R.L.). A voucher specimen (SKKU-NPL 1205) has been
deposited in the herbarium of the School of Pharmacy, Sungkyunkwan
University, Suwon, Korea.
Extraction and Isolation. The trunk material of A. holophylla (5.0
kg) was extracted with 80% aq MeOH under reflux and was filtered.
The filtrate was evaporated under reduced pressure to obtain a MeOH
extract (280 g), which was suspended in distilled H2O and successively
partitioned with n-hexane, CHCl3, EtOAc, and n-butanol, yielding 23,
43, 17, and 35 g of residues, respectively. The EtOAc-soluble fraction
(17 g) was separated over a RP-C18 silica gel column with 50% aq
MeOH to yield eight subfractions (E1-E8). Fraction E3 (3.4 g) was
separated on a silica gel column (CHCl3−MeOH−H2O, 4:1:0.1) to
give ten subfractions (E31−E310). Fraction E34 (130 mg) was
separated on a Lichroprep Lobar-A RP-C18 column (25% aq CH3CN)
and then by preparative HPLC (25% aq CH3CN), to yield compound
17 (6 mg). Fraction E35 (50 mg) was separated by preparative HPLC
(25% aq CH3CN), to give compound 5 (4 mg). Fraction E38 (610
mg) was separated on a RP-C18 silica gel column (20% aq CH3CN)
and further by preparative HPLC (25% aq CH3CN), to yield
compounds 10 (7 mg) and 11 (20 mg). Compound 9 (9 mg) was
obtained from E310 (90 mg) by preparative HPLC (20% aq CH3CN).
Fraction E5 (2.1 g) was separated over a Sephadex LH-20 column
(60% aq MeOH) to yield eight subfractions (E51−E58). Fraction E52
(460 mg) was separated on a silica gel column (CHCl3−MeOH, 8:1)
and further by preparative HPLC (30% aq CH3CN), to give
compounds 6 (4 mg), 12, (20 mg), 13 (7 mg), and 15 (2 mg).
Fraction E55 (300 mg) was separated on a silica gel Waters Sep-Pak
Vac 6 cm3 (CHCl3−MeOH, 30:1) and then by preparative HPLC
(40% aq CH3CN) to yield compounds 1 (15 mg), 7 (8 mg), 8 (2 mg),
and 14 (2 mg). Fraction E6 (1.2 g) was separated by silica gel CC
(CH3Cl−MeOH, 7:1) to give eight subfractions (E61−E68). Fraction
E61 (79 mg) was purified by preparative HPLC (30% aq CH3CN) to
yield compounds 2 (5 mg), 3 (3 mg), and 16 (4 mg). Fraction E68
(100 mg) was separated on a Sephadex LH-20 column (CH2Cl2-
The 11 known compounds were identified as
(8R,9S,7′S,8′R)-4,4′,7′-trihydroxy-3,3′,9-trimethoxy-9,9′-epoxy-
lignan (7), cedrusin (8),24 (2R,3S)-2,3-dihydro-7-hydroxy-2-
(4′-hydroxy-3′-methoxyphenyl)-3-hydroxymethyl-5-benzofur-
anpropanol 4′-O-β-D-glucopyranoside (9),29 7R,8S-dihydrode-
hydrodiconiferyl alcohol 4-O-β-D-glucopyranoside (10),22
isomassonianoside B (11),14 icariside E4 (12),30 juniperigiside
(13),23 (−)-secoisolariciresinol (14),31 (2R,3R)-2β-(4″-hy-
droxy-3″-methoxybenzyl)-3α-(4′-hydroxy-3′-methoxybenzyl)-
γ-butyrolactone (15),32 conidendrin (16),33 and tanegool
(17)34 by comparison of their spectroscopic data and specific
optical rotation with the reported data (see the Supporting
Information for structures of the known compounds).
Compounds 1−17 were evaluated for their effects on NGF
secretion using an enzyme-linked immunosorbant assay
(ELISA) development kit from C6 glioma cells to measure
NGF release into the medium and in a cell viability by 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)
assay. As shown in Table S1 of the Supporting Information,
compounds 6, 7, and 13 exhibited significant induction of NGF
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Note
MeOH, 1:1) and further by preparative HPLC (30% aq CH3CN) to
give compound 4 (4 mg).
(7R,8S)-Cedrusin. Colorless gum; CD (MeOH) λmax (Δε) 286
(−8.0), 250 (−5.8), 218 (+3.0) nm; UV (MeOH) λmax (log ε) 280
(1.5), 231 (4.5) nm; 1H NMR (CD3OD, 500 MHz) δ 6.98 (1H, br s,
H-2), 6.84 (1H, br d, J = 8.5 Hz, H-6), 6.76 (1H, d, J = 8.5 Hz, H-5),
6.60 (1H, s, H-6′), 6.57 (1H, s, H-2′), 5.48 (1H, d, J = 6.5 Hz, H-7),
3.85 (1H, m, H-9a), 3.82 (3H, s, 3-OCH3), 3.74 (1H, dd, J = 11.0, 7.5
Hz, H-9b), 3.55 (2H, t, J = 6.5 Hz, H-9′), 3.45 (1H, dd, J = 11.0, 6.5
Hz, H-8), 2.56 (2H, t, J = 8.0 Hz, H-7′), 1.79 (2H, m, H-8′); positive
ESIMS m/z 369 [M + Na]+.
Holophyllol A (1). Colorless needles; mp 175−178 °C; [α]2D5 −50.6
(c 0.75, MeOH); IR (KBr) νmax 3379, 2941, 2834, 1517, 1453, 1274,
1032 cm−1; UV (MeOH) λmax (log ε) 281 (1.5), 230 (3.0) nm; CD
1
(MeOH) λmax (Δε) 284 (−1.2), 224 (−5.2) nm; H (CD3OD, 500
MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table S3; positive
HRFABMS m/z 391.1757 [M + H]+ (calcd for C21H27O7, 391.1757).
Holophyllol B (2). Colorless needles; mp 159−161 °C; [α]2D5 −25.0
(c 0.10, MeOH); IR (KBr) νmax 3382, 2947, 2833, 1453, 1032 cm−1;
UV (MeOH) λmax (log ε) 281 (1.6), 229 (3.0) nm; CD (MeOH) λmax
Ligraminol E. Colorless gum; CD (MeOH) λmax (Δε) 232 (−2.0)
1
nm; UV (MeOH) λmax (log ε) 279 (2.0), 231 (4.8) nm; H NMR
1
(Δε) 286 (−1.5), 228 (−3.9) nm; H (CD3OD, 500 MHz) and 13C
(CD3OD, 500 MHz) δ 6.82 (1H, d, J = 8.5 Hz, H-5′), 6.81 (2H, br s,
H-2 and 2′), 6.70 (1H, br d, J = 8.5 Hz, H-6′), 6.68 (1H, br d, J = 8.5
Hz, H-6), 6.67 (1H, d, J = 8.5 Hz, H-5), 4.35 (1H, m, H-8), 3.81 (3H,
s, 3′-OCH3), 3.79 (3H, s, 3-OCH3), 3.63 (2H, m, H-9), 3.55 (2H, t, J
= 6.5 Hz, H-9′), 2.89 (2H, dd, J = 6.0, 2.0 Hz, H-7), 2.61 (2H, t, J =
8.0 Hz, H-7′), 1.79 (2H, m, H-8′); positive ESIMS m/z 385 [M +
Na]+.
NGF and Cell Viability Assay. C6 Glioma cells were used to
measure NGF release into the medium.35 C6 cells were purchased
from the Korean Cell Line Bank (Seoul, Korea) and maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) in
a humidified incubator with 5% CO2. To measure NGF content in
medium and cell viability, C6 cells were seeded into 24-well plates (1
× 105 cells/well). After 24 h, the cells were treated with DMEM
containing 2% FBS and 1% PS with 20 μM of each sample for one day.
Media supernatant was used for the NGF assay using an ELISA
development kit (R&D System, Minneapolis, MN). Cell viability was
assessed by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium
bromide (MTT) assay.
Measurement of NO Production and Cell Viability. BV-2 cells
were maintained in DMEM supplemented with 5% FBS and 1%
penicillin-streptomycin. To measure NO production, BV-2 cells were
plated onto a 96-well plate (3 × 104 cells/well). After 24 h, the cells
were pretreated with compounds for 30 min and stimulated with 100
ng/mL LPS for 24 h. Nitrite, a soluble oxidation product of NO, was
measured in the culture media using the Griess reaction. The
supernatant was harvested and mixed with an equal volume of Griess
reagent (1% sulphanilamide, 0.1% N-1-naphthylethylenediamine
dihydrochloride in 5% phosphoric acid). After 10 min, the absorbance
at 540 nm was measured using an Emax microplate reader (Molecular
Devices, Sunnyvale, CA). Sodium nitrite was used as a standard to
calculate the nitrite concentration. Cell viability was measured using
the MTT assay. NG-Monomethyl-L-arginine (L-NMMA; Sigma-
Aldrich, St. Louis, MO), a well-known NO synthase inhibitor, was
tested as a positive control.
NMR (CD3OD, 125 MHz) data, see Table S3; positive HRESIMS m/
z 413.1570 [M + Na]+ (calcd for C21H26NaO7, 413.1576).
Holophyllol C (3). Colorless needles; mp 164−166 °C; [α]2D5 +12.0
(c 0.05, MeOH); IR (KBr) νmax 3358, 2945, 2832, 1452, 1033 cm−1;
UV (MeOH) λmax (log ε) 281 (1.6), 229 (3.4) nm; CD (MeOH) λmax
1
(Δε) 284 (−1.0), 228 (−2.4) nm; H (CD3OD, 500 MHz) and 13C
NMR (CD3OD, 125 MHz) data, see Table S3; positive HRESIMS m/
z 413.1570 [M + Na]+ (calcd for C21H26NaO7, 413.1576).
Compound 4. Colorless gum; [α]2D5 +16.0 (c 0.05, MeOH); IR
(KBr) νmax 3358, 2945, 2832, 1453, 1033 cm−1; UV (MeOH) λmax (log
ε) 282 (1.6), 229 (4.0) nm; CD (MeOH) λmax (Δε) 292 (+1.7), 236
1
(−9.8) nm; H (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125
MHz) data, see Table S4; positive HRESIMS m/z 499.1938 [M +
Na]+ (calcd for C25H32NaO9, 499.1944).
Compound 5. Colorless gum; [α]2D5 −16.0 (c 0.05, MeOH); IR
(KBr) νmax 3358, 2945, 2832, 1452, 1033 cm−1; UV (MeOH) λmax (log
ε) 280 (1.3), 231 (4.0) nm; CD (MeOH) λmax (Δε) 286 (−7.8), 250
(−4.8), 218 (+2.5) nm; 1H (CD3OD, 500 MHz) and 13C NMR
(CD3OD, 125 MHz) data, see Table S4; positive HRESIMS m/z
529.2052 [M + Na]+ (calcd for C26H34NaO10, 529.2050).
Compound 6. Colorless gum; [α]2D5 + 20.0 (c 0.05, MeOH); IR
(KBr) νmax 3358, 2945, 2832, 1452, 1031 cm−1; UV (MeOH) λmax (log
ε) 279 (1.5), 231 (4.5) nm; CD (MeOH) λmax (Δε) 232 (−1.9) nm;
1H (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, see
Table S4; positive HRESIMS m/z 517.2048 [M + Na]+ (calcd for
C25H34NaO10, 517.2050).
Compound 7. Colorless needles; mp 168−170 °C; [α]2D5 +20.0 (c
0.20, MeOH); IR (KBr) νmax 3359, 2944, 2832, 1515, 1452, 1272,
1032 cm−1; UV (MeOH) λmax (log ε) 281 (1.6), 228 (2.9) nm; CD
1
(MeOH) λmax (Δε) 286 (−1.5), 225 (−5.5) nm; H (CD3OD, 500
MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table S3; positive
HRFABMS m/z 391.1757 [M + H]+ (calcd for C21H27O7, 391.1757).
Acid Hydrolysis of 4, 5, and 6. Compounds 4, 5, and 6 (each 2.0
mg) were individually hydrolyzed by 1 N HCl (1.0 mL) under reflux
conditions for 2 h. The reaction mixtures were extracted with CHCl3,
and the organic layers evaporated under reduced pressure to yield
cedrusinin,18,19 cedrusin,24 and ligraminol E27 (each 1.0 mg). L-
Rhamose, 3-O-methyl-L-rhamnose, and D-xylose were obtained from
the aqueous layers of 4, 5, and 6, respectively, by neutralization
through an Amberlite IRA-67 column. L-Rhamose and D-xylose were
identified by co-TLC confirmation with the authentic sample16 and by
optical rotation ([α]D25 +9.0 for L-rha and [α]2D5 +21.1 for D-xyl).
Identification of 3-O-methyl-L-rhamnose was performed by comparing
the 1H NMR, HRESIMS, and the optical rotation ([α]2D5 +10.3) in the
literature25,26 and co-TLC confirmation with that of 13 [solvent
system (CHCl3−MeOH, 4:1), Rf 0.25], which was hydrolyzed as
above. The absolute configuration of the sugars were determined by a
GC experiment according to reported procedures.16,17
ASSOCIATED CONTENT
* Supporting Information
■
S
Bioactivities of 1−17, 1H and 13C NMR data of 1−7, structures
of known compounds 8−17, HRMS and 1D and 2D NMR data
of 1−7, CD spectra and ΔδR−S values for the (R)- and (S)-
MPA esters of 1−3 and 7, 1H NMR data of 1r−3r and 7r, 1s−
3s and 7s, 4a−6a, and 3-O-methyl-L-rhamnose. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: krlee@skku.edu. Tel: 82-31-290-7710. Fax: 82-31-290-
7730.
■
(7R,8S)-Cedrusinin. Colorless gum; CD (MeOH) λmax (Δε) 292
(+1.8), 236 (−10.0) nm; UV (MeOH) λmax (log ε) 282 (2.0), 229
(4.1) nm; 1H NMR (CD3OD, 500 MHz) δ 7.10 (1H, br s, H-2′), 7.01
(1H, d, J = 2.0 Hz, H-2), 7.00 (1H, d, J = 8.0 Hz, H-6′), 6.90 (1H, d,
H-6), 6.89 (1H, d, J = 8.0 Hz, H-5), 6.80 (1H, d, J = 8.0 Hz, H-5′),
5.48 (1H, d, J = 6.5 Hz, H-7), 3.85 (1H, m, H-9a), 3.82 (3H, s, 3-
OCH3), 3.76 (1H, m, H-9b), 3.56 (2H, t, J = 6.5 Hz, H-9′), 3.47 (1H,
m, H-8), 2.66 (2H, t, J = 8.0 Hz, H-7′), 1.87 (2H, m, H-8′); positive
ESIMS m/z 353 [M + Na]+.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was supported by the Basic Science Research
Program through the National Research Foundation of Korea
■
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dx.doi.org/10.1021/np4005322 | J. Nat. Prod. 2013, 76, 2131−2135

Journal of Natural Products
Note
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(NRF) funded by the Ministry of Education, Science and
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