Lignans from the stem bark of Syringa pinnatifolia

Article abstract of DOI:10.1016/j.fitote.2016.08.011

Four new lignans, alashinols A–D (1–4), a new hydrolysis product, alashinols E (5), and seven known analogues were isolated from the stem bark of Syringa pinnatifolia Hemsl. These new lignans were characterized using 1D and 2D NMR and MS data, and their absolute configurations were determined by experimental and calculated electronic circular dichroism and X-ray diffraction analyses. Alashinol B (2) exhibited two conformers that adopted an unusual unit cell packing. Anti-inflammatory evaluation revealed that compounds 1, 4, 6, and 8 showed moderate inhibition against NO production in lipopolysaccharide-induced macrophages RAW 264.7 cells with IC₅₀ values range from 43.3–60.9?μM, and 1 decreased the TNF-α and IL-6 level in a concentration-dependent manner at 40–160?μM and exhibited considerable neuroprotective effect against the glutamate-induced injury in PC12 cell line. Analogues 3 and 9 showed protective effects against oxygen glucose deprivation/reoxygenation in H9c2 cells.

Full text of DOI:10.1016/j.fitote.2016.08.011

Lignans from the stem bark of Syringa pinnatifolia
Guozhu Su, Ruifei Zhang, Xinyao Yang, Ruifeng Bai, Xu Yin, Xiaoli
Gao, Li Li, Pengfei Tu, Xingyun Chai
PII:
DOI:
Reference:
S0367-326X(16)30208-8
doi: 10.1016/j.fitote.2016.08.011
FITOTE 3471
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Fitoterapia
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Accepted date:
13 July 2016
18 August 2016
20 August 2016
Please cite this article as: Guozhu Su, Ruifei Zhang, Xinyao Yang, Ruifeng Bai, Xu Yin,
Xiaoli Gao, Li Li, Pengfei Tu, Xingyun Chai, Lignans from the stem bark of Syringa
pinnatifolia, Fitoterapia (2016), doi: 10.1016/j.fitote.2016.08.011
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ACCEPTED MANUSCRIPT
Lignans from the stem bark of Syringa pinnatifolia
Guozhu Su a,b,1, Ruifei Zhang a,b,1, Xinyao Yang a,b,1, Ruifeng Bai a,b, Xu Yin a,b, Xiaoli Gao a, Li Li c,
Pengfei Tu a, and Xingyun Chai a,
a Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing
100029, People’s Republic of China
b School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, People’s Republic
of China
c Department of Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences &
Peking Union Medical College, Beijing, 100050, People’s Republic of China
 Corresponding author, Tel/fax: +8610 6428 6350.
E-mail address: xingyunchai@yeah.net (X. Chai).
[1] These authors contributed to the paper equally.
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ABSTRACT
Four new lignans, alashinols A–D (1–4), a new hydrolysis product, alashinols E (5), and seven
known analogues were isolated from the stem bark of Syringa pinnatifolia Hemsl. These new
lignans were characterized using 1D and 2D NMR and MS data, and their absolute configurations
were determined by experimental and calculated electronic circular dichroism and X-ray diffraction
analyses. Alashinol B (2) exhibited two conformers that adopted an unusual unit cell packing.
Anti-inflammatory evaluation revealed that compounds 1, 4, 6, and 8 showed moderate inhibition
against NO production in lipopolysaccharide-induced macrophages RAW 264.7 cells with IC50
values range from 43.3–60.9 μM, and 1 decreased the TNF-α and IL-6 level in a
concentration-dependent manner at 40–160 μM and exhibited considerable neuroprotective effect
against the glutamate-induced injury in PC12 cell line. Analogs 3 and 9 showed protective effects
against oxygen glucose deprivation/reoxygenation in H9c2 cells.
Keywords:
Syringa pinnatifolia; Lignan; Anti-inflammatory activity; Neuroprotective effect; Antihypoxia
effect
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1. Introduction
The Chinese endemic species Syringa pinnatifolia Hemsl., syn. S. pinnatifolia var. alashanensis
Y.C. Ma & S.Q. Zhou, is a deciduous shrub with pinnately compound leaves mainly found in the
Helan Mountains (Ningxia) and Alxa League (Inner Mongolia). Its peeled stem and root,
‘Shanchenxiang’ in Chinese, serve as one of a representative Mongolian folk medicines against
heart and lung diseases. Previous studies revealed that S. pinnatifolia contains lignans,
sesquiterpenes, and phenylethanoids [1–3]. The essential oils were demonstrated as bioactive
ingredients of Shanchenxiang with anti-myocardial ischemic activity [4].
As a part of systematic studies on anti-myocardial ischemic effect and pharmacologically active
ingredients of Shanchenxiang [2,5], a phytochemical investigation of its stem bark led to the
isolation of four new lignans namely alashinols A‒D (1‒4), a new hydrolysis product namely
alashinol E (5), along with seven known analogues (6‒12). The isolation and structural elucidation
of these compounds are described herein in addition to in vitro assessments of their
anti-inflammatory in RAW264.7 cells, neuroprotective effect in glutamate-induced PC12 cells, and
antihypoxia effect in H9c2 cells.
2. Experimental
2.1 General procedures
HR-ESI-MS were recorded using a Shimadzu liquid chromatography-mass spectrometry-ion
trap time-of-flight instrument. IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR
spectrophotometer using KBr pellets. NMR spectra were measured using a Varian-500 spectrometer.
Specific rotation (SR) was measured using a Rudolph Autopol IV automatic polarimeter and ECD
spectra were acquired using Jasco J-815 spectrometer. Preparative high-performance liquid
chromatography (HPLC) was performed using a Waters apparatus equipped with a Shim-pack
PREP-ODS (H) (250 × 20 mm, 5 µm) and Shiseido Capcell PAK MG Prep C18 (250 × 10 mm, 5
µm). Column chromatography (CC) separations were conducted using silica gel (200–300 mesh,
Qingdao Haiyang Chem. Co. Ltd., China), Sephadex LH-20 (GE Healthcare, Sweden), and Li
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Chroprep RP-18 (Merck, Germany). Thin layer chromatography (TLC) was performed on
pre-coated silica gel GF254 (Qingdao, Haiyang Chem. Co. Ltd, China) and RP-18F254S plates
(Merck, Germany).
2.2 Plant material
The stem bark of S. pinnatifolia, syn. S. pinnatifolia var. alashanensis was collected in July
2013 from Alxa League and authenticated by Prof. Chen-Su-Yi-Le (Alashan Mongolian
Hospital, Inner Mongolia, China). A voucher specimen (SP201307B) was deposited at the
Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese
Medicine.
2.3 Extraction and isolation
Stem bark (1.3 kg) of S. pinnatifolia was refluxed with 95 and 80% EtOH (2.5 L, 1.5 h each).
After removal of solvent in reduced pressure, the residue (325 g) was subjected to silica gel CC,
by a gradient elution of CH2Cl2-MeOH (20:1–1:1) to give nine fractions (A–I). Fractions
exhibiting characteristic signals within δH 3.60‒3.90 and 7.50‒8.00 ppm in 1H-NMR spectrum
were focused on. Specifically, fraction A (48.0 g) was purified by silica gel CC, eluting with
petroleum ether-acetone (2:1–0:1) to yield 12 subfractions (A1–A12). A7 (1.0 g) was separated
into five fractions (A7a–A7e) by Sephadex LH-20 CC using CH2Cl2-MeOH (1:1). A7b (500 mg)
was purified by semipreparative C18 HPLC (Shim-pack PREP-ODS H 250 × 20 mm, 5 µm; 7
mL/min) using 50% aqueous MeOH as a mobile phase to give 1 (8.7 mg, tR = 27.9 min) and 6
(30.1 mg, tR = 60.1 min). Fraction C (31.2 g) was subjected to silica gel CC, eluting with a
gradient of CH2Cl2-acetone (3:1–1:1) followed by purification on Sephadex LH-20 CC
(CH2Cl2-MeOH, 1:1) to yield ten fractions (C1–C10). C4 (982 mg) was purified by
semipreparative C18 HPLC using 50% MeOH as an eluent to yield 4 (5.2 mg, tR = 17.5 min). C5
(690 mg) was purified by HPLC using 15% aqueous acetonitrile (ACN) to afford 11 (6.0 mg, tR
= 37.3 min), 12 (8.3 mg, tR = 42.5 min), 8 (5.4 mg, tR = 44.5 min), and 7 (9.5 mg, tR = 52 min).
Fractions E (10.6 g) and F (8.2 g) were merged and subjected to silica gel CC, eluting with
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EtOAc-MeOH (10:1–0:1) to give eight fractions (EF1–EF8) and 2 (70.5 mg). Fraction EF3 (2.5 g)
was separated by HPLC using 40% MeOH as an eluent to obtain 10 (6.5 mg, tR = 20.1 min), 9 (6.3
mg, tR = 23.5 min), and a mixture that was further purified by HPLC using 16% ACN to yield 3
(6.3 mg, tR = 26.5 min).
Alashinol A (1): white powder; [α]2D5 ‒54.4 (c 0.1, MeOH). UV (MeOH) λmax (log ε): 283 (0.75),
230 (1.90), 206 (4.41) nm. IR (KBr) vmax: 3421, 2918, 1607, 1511, 1453, 1269, 1030 cm–1. ECD
(MeOH) λmax (Δε): 239 (–3.30), 274 (–0.51), 293 (+0.43) nm; 1H and 13C NMR data: see Tables 1
and 2, respectively; HRESIMS m/z: 343.1549 [M + H]+ (calcd for C20H23O5 343.1540).
Alashinol B (2): needle crystal; M. p. 174.5–175 ºC; [α]2D5 –19.2 (c 0.1, MeOH); UV (MeOH)
λmax (log ɛ): 282 (3.90), 228 (4.20), 206 (4.67) nm; IR (KBr) vmax 3423, 2926, 1630, 1384, 1076,
1037 cm–1; ECD (MeOH) λmax (Δε): 209 (+0.45), 230 (+0.50), 241 (–0.95), 282 (–1.01) nm; 1H and
13C NMR data, see Tables 1 and 2, respectively; HRESIMS m/z: 519.1854 [M – H]– (calcd for
519.1872 C26H31O11).
Alashinol C (3): white powder; [α]D25 –3.1 (c 0.1, MeOH); UV (MeOH) λmax (log ɛ): 280 (3.86),
227 (4.20), 206 (4.59) nm; IR (KBr) vmax: 3405, 2926, 1516, 1277, 1073, 1038 cm–1; ECD (MeOH)
λmax (Δε): 209 (+0.73), 228 (+0.49), 240 (–1.1), 275 (–0.87) nm; 1H and 13C NMR data, see Tables 1
and 2, respectively; HRESIMS m/z: 507.1851 [M – H]– (calcd for 507.1872 C25H31O11).
Alashinol D (4): white powder; IR (KBr) vmax 3440, 1632, 1263, 1031 cm–1. UV (MeOH) λmax
(log ɛ) 331 (3.59), 316 (3.56), 283 (4.04), 242 (4.82), 204 (4.63) nm; IR (KBr) vmax 3440, 1632,
1263, 1031 cm–1; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS m/z: 355.1202
[M ‒ H]– (calcd for C20H19O6 355.1187).
Alashinol E (5): white powder; [α]D25 –3.61 (c 0.1, MeOH); UV (MeOH) λmax (log ɛ) 288 (3.67),
214 (4.05) nm; IR (KBr) vmax 3442, 2921, 1636, 1506, 1384, 1037 cm–1; ECD (MeOH) λmax (Δε)
230 (–1.08), 248 (+0.85), 276 (+1.99), 294 (–3.56) nm; 1H and 13C NMR data, see Tables 1 and 2,
respectively; HRESIMS m/z: 357.1357 [M – H]– (calcd for 357.1344 C20H21O6).
Sanshodiol (6): white needles; M. p. 148–149 ºC; [α]2D5 –14.8 (c 0.1, MeOH); UV (MeOH) λmax
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(log ɛ) 284 (3.22), 231 (4.19), 206 (3.94) nm; IR (KBr) vmax 3483, 3423, 2918, 1634, 1513, 1055,
1035 cm–1; ECD (MeOH) λmax (Δε) 206 (+0.90), 236 (+0.74), 245 (–0.55), 292 (–1.42) nm; 13C
NMR data, see Table 2, HRESIMS m/z 357.1332 [M – H]– (calcd for 357.1344 C20H21O6).
2.4 Anti-inflammatory assay
RAW 264.7 macrophages were seeded in 96-well plates (2 × 105 cells/well). The cells were
pretreated with drugs for 1 h and then incubated with LPS (1 μg/mL) for 24 h. The amount of NO
was assessed by determining the nitrite concentration in the cultured RAW 264.7 macrophages
supernatants with Griess reagent, and quercetin was used as a positive control, respectively. The
absorbance was recorded on a microplate reader at a wavelength of 540 nm. Cells viability were
measured by an MTT assay, the absorbance read at 570 nm with an ELISA analyzer [6,7].
2.5 Neuroprotective assay
Rat adrenal pheochromocytoma (PC12) cell lines were seeded in 96-well plates (2 × 105
cells/well). After 24 h, cells were pretreated with drugs for 1 h and then added 50 µL H2O2 (1200
µM). Then, after 24 h, cells were incubated with 100 µL MTT solution (0.5 mg/mL) at 37 ºC for
another 4 h, the supernatants were removed and residue was dissolved with 150 µL DMSO for each
well and subsequently shaked out for 10 min. The absorbance was detected at 570 nm and 630 nm
using a microplate reader after the formazan crystals were dissolved. All compounds were dissolved
as stock solution in DMSO (final solvent concentration less than 0.2% in any assays) [8].
2.6 TNF-α, IL-6, and quantification by ELISA
RAW264.7 cells were seeded in 96-well plates (2 × 104 cells/well). After incubation for 24 h,
the cells were pretreated with 1 for 1 h and then stimulated with LPS (1 µg/mL) for 24 h. The levels
of TNF-α and IL-6 in the supernatant were measured by ELISA according to the manufacture's
protocols.
2.7 Measurement of OGD/re-oxygenation in H9c2 cells
Rat embryonic ventricular H9c2 cardiomyocytes were maintained in Dulbecco’s modified
eagle’s medium (DMEM), supplemented with a 10% fetal bovine serum (FBS),
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penicillin/streptomycin, and 4 mM L-glutamine, in a 5% CO2 incubator at 37 ºC. H9c2 cells were
placed in an anaerobic chamber (HERA cell 150, partial oxygen pressure was maintained below 2
mmHg). The medium was replaced with a pre-warmed (37 ºC) glucose-free balanced salt solution
(116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 1.0 mM NaH2PO4, 1.8 mM CaCl2, 26.2 mM
NaHCO3, 0.025 mM phenol red, and 20 mM sucrose). The solution was bubbled with an anaerobic
gas mix (95% N2, 5% CO2) for 30 min. Cell cultures subjected to OGD were incubated in the
solution at 37 ºC for 1–4 h to produce oxygen deprivation and then re-oxygenated (returned to the
normal aerobic environment). Experimental parameters were assayed at 3–24 h following
re-oxygenation. Briefly, cells were seeded onto 96-well plates (1.0 × 105 cells/mL) and incubated
for 24 h, MTT tetrazolium salt (0.5 mg/mL) was added to each well and incubated in a CO2
incubator for 2 h at 37 ºC, 100 µL DMSO was then added to dissolve formazan crystals, and the
absorbance was recorded at 570 nm. OD values of the treatment group were always normalized to
that of the untreated control group [9,10].
3. Results and discussion
Alashinol A (1), which was obtained as a white powder, showed a unique m/z peak at 343.1549
([M + H]+, calcd for C20H23O5, 343.1540) by HR-ESI-MS and 13C NMR spectroscopic data,
indicating ten degrees of unsaturation. Its infrared (IR) spectrum displayed absorption bands at 3421
and 1511 cm−1 for hydroxy and aryl rings, respectively.
The 1H NMR data (Table 1) indicated that 1 has an ABX coupling system assignable to one
1,3,4-trisubstituted benzene ring [δH 6.64 (1H, d, J = 2.0 Hz, H-2), 6.66 (1H, d, J = 8.0 Hz, H-5),
6.49 (1H, dd, J = 2.0, 8.0 Hz, H-6)], two terminal olefinic protons [δH 4.79 (1H, s, H-9a), 4.91 (1H,
s, H-9b)], two methoxy protons [δH 3.73 (3H, s), 3.83 (3H, s)], two aromatic protons [δH 6.37 (1H, s,
H-2′), δH 6.69 (1H, s, H-5′)] assignable to a tetra-substituted benzene ring, and two oxygen
connected protons [δH 3.55 (1H, dd, J = 2.5, 10.5 Hz, H-9′a) and 3.70 (1H, overlapped, H-9′b)]. The
13C NMR data (Table 2) displayed 20 carbon resonances, including eight quaternary carbons [δC
138.3 (C-1), 148.6 (C-3), 145.9 (C-4, 4′), 152.4 (C-8), 128.5 (C-1′), 147.8 (C-3′), 132.3 (C-6′)], and
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five tertiary aromatic carbons [δC 113.6 (C-2), 115.7 (C-5), 122.5 (C-6), 112.5 (C-2′), 117.0 (C-5′)].
The above assignment suggested 1 to be an arylnaphthalene-type lignan [11], supported by HMBC
correlations (Figure 1) from H-5′, H-2, and H-6 to C-7, from H-7 to C-9, C-1′, and C-8′, from H-5
to C-1 and C-3, from H-6 to C-2 and C-4, from H-9 to C-8′, from H-2′ to C-4′ and C-6′, from H-5′
to C-1′ and C-3′, and from H-7′ to C-2′, C-6′, C-9′, and C-8, as well as from 3′-OMe and 3-OMe to
C-3′ and C-3, respectively, demonstrating the planar structure of 1.
The absence of NOESY correlation between H-7 and H-8′ implied a large spatial distance
between these two protons, its absolute configuration was defined by comparison of
experimental and calculated ECD data. The calculated ECD spectrum of (7R, 8′R)-1 showed
two negative Cotton effects at 239 and 274 nm and a positive Cotton effect at 293 nm,
consistent with experimental data (Figure 2A). Therefore, the absolute configuration of 1
corresponded to (7R, 8′R), establishing its structure.
The molecular formula of alashinol B (2), which formed needle-like crystals, was identified
as C26H32O11 by HR-ESI-MS and 13C NMR spectroscopic data. Its NMR data (Tables 1 and 2)
displayed signals for a glucosyl moiety, which was confirmed by HPLC analysis with authentic
sample after enzymatic hydrolysis, two aromatic rings presenting ABX coupling system, one
furan ring, one methylenedioxy, one methylene, one oxygen-connected methylene, and one
methoxy group, indicating a 7,9′-epoxylignan structure. A comparison of 1H and 13C NMR data
with those of 6 revealed their structural similarity, expect for the C-4-bound glucosyl moiety in
2 supported by HMBC correlation from glc-H-1 [δH 4.83 (d, J = 7.0 Hz)] to C-4 (δC 147.2). The
ROESY correlations between H-7 to H-9 and H-7′b of 2 determined its relative configuration
[12], which was confirmed as 7S, 8R, 8′R by single-crystal X-ray diffraction analysis (Figure 3).
HR-ESI-MS and 13C NMR spectroscopic data indicated that alashinol C (3) matched the
molecular formula C25H32O11. It exhibited similar UV, IR, and NMR data to alashinol B (2),
suggesting their structural resemblance. However, a comparison of NMR data (Tables 1 and 2)
revealed that 3′,4′-methylenedioxy, 4-glucopyranoside in 2 was replaced by 3′-glucopyranoside,
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4′-hydroxy and 4-hydroxy in 3. This discrepancy were confirmed by HMBC correlation from H-1′′
to C-3′ and NOESY correlation of H-1′′ and H-2′. The glucose was determined with the same
method of 2 after enzymatic hydrolysis. Its relative configuration was assigned by NOESY
correlations between H-7 with H-9 and H-7′b. Comparison of the NMR and ECD spectra of 2 and 3
indicated that 3 adopted a (7S, 8R, 8′R) absolute configuration (SI. S35) [12].
Alashinol D (4) displayed a HR-ESI-MS signal at m/z 355.1202 for [M − H]−, consistent with a
molecular ion of the formula C20H20O6 (calcd for C20H19O6: 355.1187) with 11 degrees of
unsaturation. Its 1H NMR data (Table 1) showed signals at δH 6.88 (d, J = 2.0, H-2), 6.93 (d, J = 8.0,
H-5), and 6.73 (dd, J = 2.0, 8.0, H-6) for an 1,2,4-trisubstituted aromatic ring, δH 7.25 (s, H-2′) and
6.76 (s, H-5′) for aromatic protons, δH 3.98 (s) and 3.84 (s) for two methoxys, δH 7.77 (s, H-7′) for
one olefinic proton, and δH 4.61 (d, J = 11.5, H-9a), 4.57 (d, J = 11.5, H-9b), and 4.93 (2H, s, H-9′)
for four hydroxy methylene protons. Its 13C NMR spectrum (Table 2) exhibited 20 carbon
resonances, including 16 olefinic carbons in the low-field region. These assignments, combined
with HMBC analysis, suggested that 4 presented an arylnaphthalene-type lignan [11]. HMBC
correlations (Figure 1) were observed from H-5′, H-2, H-6, and H-9 to C-7, from H-2′ to C-4′, from
H-5′ to C-3′ and C-1′, from H-7′ to C-2′, C-6′, C-9′, and C-8, and from H-9′ to C-8, as well as from
3′-MeO and 3-MeO to C-3′ and C-3, respectively, establishing the final structure.
Alashinol E (5) was obtained by refluxing 2 (18.8 mg) with 1 N HCl (1 mL) for 1 h at 90 ºC and
purifying the hydrolytic product by a Sephadex LH-20 CC using MeOH as eluent. Its HR-ESI-MS
displayed a signal at m/z 357.1359 for [M − H]− corresponding to C20H22O6 (m/z calcd for C20H21O6:
357.1344). Its 1H and 13C NMR spectra exhibited similarities to that of (+)-isolariciresinol, except
for the replacement of methylenedioxy by MeO-3 and HO-4 in 5 [13]. Its 1H NMR data showed a
large J7,8 coupling constant of 10.5 Hz, suggesting the opposite orientation of H-7 and H-8.
Therefore, C-7 and C-8 adopted a trans configuration. NOESY correlation was also observed
between H-8′ and H-7, indicating that C-7 and C-8′ was a cis configuration [14]. The absolute
configuration of 5 was assigned by comparing ECD data with those of (+)-isolariciresinol (8),
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which displayed opposite Cotton effects (Figure 2B). In addition, its SR data ([α]2D5 −3.61,
MeOH) was similar to that of ent-isolariciresinol ([α]2D5 −53.8, MeOH) [15] but opposite to that
of (+)-isolariciresinol (8) ([α]2D5 +34.0, MeOH) on the positive and negative sign of the
equations [13], suggesting a (7R, 8S, 8'S) absolute configuration.
Alashinol B (2) is of special interest because of its two conformers exhibiting an AABB unit
cell packing by X-ray diffraction. These conformers only differ by the benzene ring connected
to the methylenedioxy substituent, which is a typical of 7,9′-epoxylignans. Acid hydrolysis of 2
unexpectedly afforded alashinol E (5) instead of the aglycone needed for SR data measurement.
A process involving acid-mediated furan ring opening, isomerization, and cyclization was
proposed for formation of 5 (Scheme 1).
Seven known lignans were isolated with alashinols A–E. Comparison of their NMR
spectroscopic, SR or ECD data with previous reports facilitated their structural identification.
These lignans included sanshodiol (6) [16], burselignan (7) [13], (+)-isolariciresinol (8) [13],
conicaoside (9) [17], lariciresinol-4-O-β-D-glucopyranoside (10) [18], (–)-olivil (11) [19], and
dysosmarol (12) [20]. With the 13C NMR data, the configuration of 6 was determined by SR and
ECD data analyses for the first time [16] (SI. S35).
These lignans were tested in vitro for anti-inflammatory, neuroprotective effect, and antihypoxia
effect. The results showed that 1, 4, 6, and 8 inhibited the lipopolysaccharide (LPS)-induced NO
production in RAW 264.7 cells with their IC50 values of 60.9, 43.3, 49.8, and 46.0 μM, respectively,
compared with a positive control quercetin (IC50 = 15.3 μM). Moreover, compound 1 exhibited
inhibition on the production of inflammatory mediators TNF-α and IL-6, especially, it significantly
suppressed the increase of IL-6 level in a concentration dependent manner at 40‒160 μM (Figure 4).
Compounds 3 and 9 at 40 μM significantly increased the cell viability with 80.7 ± 5.6% and
79.8 ± 0.7% of the control value (100%), respectively, compared with the model group with
67.0 ± 4.2% of the control value against oxygen glucose deprivation/re-oxygenation injury in
H9c2 cardiomyocytes.
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The present finding not only enriches the lignan study, but also brings potential reference to
clarifying bioactive ingredient of Shanchenxiang with anti-myocardial ischemic function.
4. X-ray Crystal Data for Alashinol B (2)
Needle crystal, C26H32O11, M = 520.52, monoclinic, crystal size 0.45 × 0.20 × 0.15 mm3, space
group P21 (no. 4), a = 25.7104 (6) Å, b = 10.28363 (18) Å, c = 19.2514 (4) Å, α = 90.00, β =
111.621 (2), γ = 90.00, V = 473.89 (17) Å3, Z = 8, µ(Cu Kα) = 0.9064, D calcd = 1.461 mg/mm3, F
(000) = 2208, 39079 reflections measured, 16696 unique (Rint = 0.0281) which were used in all
calculation, final R indexes for I > 2σ (I), R1 = 0.0377, wR2 = 0.0995, R indexes for all data R1 =
0.0520, wR2 = 0. 1112. Flack parameter = –0.05 (7). Crystallographic data for 2 have been
deposited with the Cambridge Crystallographic Data Centre (deposition number: CCDC 1054351).
5. Acid and enzymatic hydrolysis of 2
In acidic conditions, the phenolic glycoside 2 was hydrolyzed to give the free phenol A.
Further hydrolysis of the tetrahydrofuran ring in A was facilitated by the para-phenol group,
affording the para-quinone methide intermediate B. The reactive intermediate B could be
intercepted by the nearby electron-rich aromatic ring, thus forming the observed podophyllotoxin
type product 5 through intermediate C (Scheme 1).
Compound 2 (1.26 mg) was which dissolved in DMSO treated with 0.3 mg of cellulose (Beijing
Biotech Biochemistry Technical Co.) in citric acid buffer solution (PH 4.5, 60 mL). The mixture
was stirred at 40 ºC for 12 h, and then extracted with an equal amount of EtOAc. The EtOAc layer
was evaporated under reduced pressure to obtain the alashinol 2a which was identified by co-TLC
confirmation with compound 6. The β-D-glucopyranoside was dissolved in anhydrous pyridine (1
mL), and then L-cysteine methyl ester hydrochloride (5 mg) was added to the solution. The mixture
was heated at 60 ºC for 1 h, and O-tolyl isothiocyanate (3 mg) was subsequently added. Followed
by heated at 60 ºC for another 1 h. Finally, the reaction mixture was analyzed by C18 HPLC (Aglient
Eclipse XDB 4.6 × 250 mm, 5 μm) using 25% ACN at 0.8 mL/min, and the detection wavelength
was set at 254 nm. The result indicated the retention time was identified for 16.58 min.
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Acknowledgments
This paper is financially supported by grants from the National Natural Science Foundation
of China (Nos. 81473426, 81303309). The authors express their thanks to Prof. Chen-Su-Yi-Le
(Alashan Mongolian Hospital) for his collection and authentication of plant material.
Appendix A. Supplementary data
The MS, NMR, and ECD spectra of 1‒6 and X-ray crystallographic data of 2 are available
in the online version.
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Figure 1. Structures of 1–6 and key HMBC correlations of 1, 2, and 4.
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Figure 2. Calculated ECD spectra of 1 (7R, 8'R), 1a (7S, 8'S), and 1b (7R, 8'R) (A), and ECD spectra of 5 and 8
(B)
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Figure 3. X-ray ORTEP drawing of compound 2.
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Figure 4. Effects of compound 1 on LPS-induced expression of TNF-α and IL-6. RAW264.7 cells were pretreated
with various concentrations of compound 1 for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. Data was
obtained from three independent experiments. ***P < 0.001 vs LPS group.
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Scheme 1. Plausible sequential reaction that transforms 2 into 5 at 90 ºC in 1 N HCl (1 mL)
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Table 1. 1H NMR (500 MHz in CD3OD) of 1–5, δ in ppm and J in Hz
NO.
2
1
2
3
4
5
6.67, d (1.5)
6.74, d (8.0)
6.60, dd (2.0, 8.0)
3.82, d (10.5)
1.76, m
6.64, d (2.0)
6.66, d (8.0)
6.49, dd (2.0, 8.0)
4.52, s
6.92, br s
6.89, br s
6.88, d (2.0)
6.93, d (8.0)
6.73, dd (2.0, 8.0)
5
7.08, d (8.0)
6.81, d (8.0)
4.75, overlapped
2.27, m
6.75, overlapped
6.75, overlapped
4.74, d (7.0)
2.34, m
6
7
8
9a
9b
2'
5'
4.79, s
4.91, s
6.69, s
6.37, s
3.75, m
3.80, m
4.61, d (11.5)
4.57, d (11.5)
7.25, s
3.64, m
3.59, m
3.61, dd (6.5, 11.5)
7.05, br s
3.38, dd (4.0, 11.0)
6.57, br s
6.64, br s
6.66, d (7.5)
6.76, overlapped
6.76, s
6.13, br s
6'
6.58, d (7.5)
2.82, m
2.42, m
2.60, m
3.92, m
3.64, m
3.79, s
6.76, overlapped
2.89, dd (5.0, 13.5)
2.49, dd (2.0, 13.5)
2.73, m
7'a
2.56, dd (8.5, 15.5)
3.04, dd (5.5, 15.5)
2.65, m
7.77, s
4.93, s
2.76, br s
2.74, br s
1.98, m
3.67, m
3.60, m
3.77, s
7'b
8'
9'a
3.55, dd (2.5, 10.5)
3.70, overlapped
3.73, s
3.99, dd (6.5, 8.5)
3.68, m
9′b
MeO-3
MeO-3'
OCH2O
1''
3.83, s
3. 84, s
3.98, s
3.83, s
5.83, br s
4.83, d (7.0)
3.45, m
5.78, br s
4.73, d (7.0)
3.46, m
2''
3''
3.44, m
3.46, m
4''
3.35, m
3.38, m
5''
3.35, m
3.39, m
6''a
6''b
3.82, d (12.5)
3.64, m
3.71, d (11.5)
3.70, m
aChemical shifts (ppm) referred to CD3OD (δH 3.33)
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Table 2. 13C NMR (125 MHz in CD3OD) of 1–5, δ in ppm.
NO.
1
2
3
4
5
6
1
138.3, C
113.6, CH
148.6, C
145.9, C
115.7, CH
122.5, CH
53.4, CH
152.4, C
108.9, CH2
128.5, C
112.5, CH
147.8, C
145.9, C
117.0, CH
132.3, C
35.0, CH2
41.9, CH
65.0, CH2
56.3, CH3
56.4, CH3
139.4, C
111.3, CH
150.7, C
147.2, C
117.8, CH
119.5, CH
83.8, CH
53.9, CH
60.5, CH2
135.8, C
109.9, CH
147.2, C
149.1, C
109.1, CH
122.6, CH
33.7, CH2
43.7, CH
73.5, CH2
135.8, C
110.6, CH
149.0, C
147.0, C
116.0, CH
119.8, CH
84.1, CH
53.9, CH
60.5, CH2
133.7, C
119.0, CH
146.7, C
146.5, C
117.0, CH
124.9, CH
33.4, CH2
43.7, CH
73.5, CH2
56.4, CH3
132.1, C
115.3, CH
148.8, C
146.8, C
116.0, CH
124.2, CH
140.0, C
133.4, C
60.7, CH2
130.1, C
107.4, CH
150.3, C
147.8, C
110.8, CH
130.3, C
127.2, CH
136.8, C
64.5, CH2
56.4, CH3
56.2, CH3
138.3, C
113.7, CH
149.1, C
146.1, C
116.1, CH
123.1, CH
48.4, CH
47.9, CH
62.3, CH2
131.0, C
108.8, CH
147.1, C
147.1, C
110.3, CH
134.8, C
33.9, CH2
39.9, CH
65.8, CH2
56.4, CH3
135.7, C
2
110.7, CH
149.0, C
3
4
147.3, C
5
116.0, CH
119.8, CH
84.1, CH
54.0, CH
60.4, CH2
135.9, C
6
7
8
9
1'
2'
110.0, CH
147.1, C
3'
4'
149.2, C
5'
109.1, CH
122.7, CH
33.8, CH2
43.8, CH2
73.4, CH2
56.4, CH3
6'
7'
8'
9'
MeO-3
MeO-3'
OCH2O
1''
56.7, CH3
102.1, CH2
102.8, CH
74.8, CH
77.7, CH
71.3, CH
78.1, CH
62.5, CH2
101.8, CH2
102.1, CH2
104.2, CH
74.9, CH
77.6, CH
71.3, CH
78.3, CH
62.4, CH2
2''
3''
4''
5''
6''
aChemical shifts (ppm) referred to CD3OD (δC 49.0)
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Graphical Abstract
Lignans from the stem bark of Syringa pinnatifolia
Guozhu Su a,b,1, Ruifei Zhang a,b,1, Xinyao Yang a,b,1, Xu Yin a,b, Ruifeng Bai a,b, Xiaoli Gao a, Li Li c,
Pengfei Tu a, and Xingyun Chai a,
 Corresponding author, Tel/fax: +8610 6428 6350.
E-mail address: xingyunchai@yeah.net (X. Chai).
[1] These authors contributed to the paper equally.
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