Nitric oxide inhibitory constituents from the barks of Cinnamomum cassia

Article abstract of DOI:10.1016/j.fitote.2016.05.005

Six new compounds including one γ-butyrolactone, cinncassin A (1), two tetrahydrofuran derivatives, cinncassins B and C (2, 3), two lignans, cinncassins D and E (4, 5), and one phenylpropanol glucoside, cinnacassoside D (6), together with 14 known lignans (7-20) were isolated from the barks of Cinnamomum cassia. The structures of 1-6 were elucidated by extensive 1D and 2D NMR spectroscopic data analysis as well as chemical methods, and the absolute configurations were established by experimental and calculated ECD data. The anti-inflammatory activities of the isolates were evaluated on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced BV-2 microglial cells. Compounds 5, 7, 8, and 15 showed potent inhibition activities with IC₅₀ values of 17.6, 17.7, 18.7, and 17.5 μM, respectively.

Full text of DOI:10.1016/j.fitote.2016.05.005

Nitric oxide inhibitory constituents from the barks of Cinnamomum cassia
Shan He, Ke-Wu Zeng, Yong Jiang, Peng-Fei Tu
PII:
DOI:
Reference:
S0367-326X(16)30113-7
doi: 10.1016/j.fitote.2016.05.005
FITOTE 3409
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
15 March 2016
11 May 2016
16 May 2016
Please cite this article as: Shan He, Ke-Wu Zeng, Yong Jiang, Peng-Fei Tu, Nitric oxide
inhibitory constituents from the barks of Cinnamomum cassia, Fitoterapia (2016), doi:
10.1016/j.fitote.2016.05.005
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ACCEPTED MANUSCRIPT
Nitric oxide inhibitory constituents from the barks of
Cinnamomum cassia
Shan He, Ke-Wu Zeng, Yong Jiang*, Peng-Fei Tu*
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China
*Corresponding author. Tel/Fax: +86 10 8280 2719.
E-mail address: yongjiang@bjmu.edu.cn (Y. Jiang).
*Corresponding author. Tel/Fax: +86 10 8280 2750
E-mail address: pengfeitu@vip.163.com (P.F. Tu).

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ABSTRACT
Six new compounds including one γ-butyrolactone, cinncassin A (1), two
tetrahydrofuran derivatives, cinncassins B and C (2, 3), two lignans, cinncassins D
and E (4, 5), and one phenylpropanol glucoside, cinnacassoside D (6), together with
14 known lignans (720) were isolated from the barks of Cinnamomum cassia. The
structures of 16 were elucidated by extensive 1D and 2D NMR spectroscopic data
analysis as well as chemical methods, and the absolute configurations were
established by experimental and calculated ECD data. The anti-inflammatory
activities of the isolates were evaluated on nitric oxide (NO) production in
lipopolysaccharide (LPS)-induced BV-2 microglial cells. Compounds 5, 7, 8, and 15
showed potent inhibition activities with IC50 values of 17.6, 17.7, 18.7, and 17.5 μM,
respectively.
Keywords:
Cinnamomum cassia
Lignans
γ-Butyrolactone
Tetrahydrofuran derivatives
Nitric oxide

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1. Introduction
The plants Cinnamomum cassia Presl (Lauraceae) have been widely cultivated in
the tropical or subtropical areas, such as Yunnan, Guangdong, Guangxi, Hainan,
Guizhou, and Taiwan in China, as well as India, Vietnam, Indonesia, Laos, Thailand,
and Malaysia. The dried barks of C. cassia, well known as Cinnamomi Cortex, are not
only used as a spice and flavoring agent, but also used as a traditional medicine in the
world for the treatment of amenorrhea, rheumatoid arthritis, cardiac palpitation,
diarrhea, and gastrointestinal neurosis[1]. Previous phytochemical and
pharmacological studies on this plant have resulted in the isolation of several kinds of
bioactive compounds such as trans-cinnamaldehyde and 2-methoxycinnamaldehyde
from the essential oil of C. cassia which have been reported to inhibit NF-κB[2], and
cinnamaldehyde has been shown to possess potential in vitro and in vivo
anti-inflammatory activities[5]. There are some other kinds of bioactive
compounds[3-8] such as flavonoids, diterpenoids, comarins and polyphenols which
exhibited antimicrobial, antifungal, immunosuppressive, cytotoxic , and antioxidant

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activities.
In order to search for more new bioactive components from C. cassia, a
systematic phytochemical investigation was carried out. Six new compounds
including one γ-butyrolactone, cinncassin A (1), two tetrahydrofuran derivatives,
cinncassins B and C (2, 3), two lignans, cinncassins D and E (4, 5), and one
phenylpropanol glucoside, cinnacassoside D (6), together with 14 known lignans
(720) were isolated from the barks of C. cassia. Herein, the isolation and structural
elucidation of the six new compounds and the inhibitory effects of the isolates on
LPS-stimulated NO production in BV-2 microglial cells are reported.
2. Experimental
2.1. General experimental procedures
Optical rotation was measured on an Autopol III automatic polarimeter. UV
spectra were obtained on a Shimadzu UV-2450 spectrophotometer. IR spectra were
achieved on a Nicolet Nexus 470 FT-IR spectrometer by KBr pellets. NMR spectra
were recorded on a Varian INOVA-500 FT NMR spectrometer. HR mass spectra were
obtained from a Bruker APEX IV FT-MS (7.0 T) mass spectrometer fitted with an ESI
source. CD spectra were acquired on a JASCO J-810 spectropolarimeter. Analytical
HPLC was performed on an Agilent 1100 series equipped with an Extend-C18 column
(5 μm, 4.6 × 250 mm) and a DAD detector. Semi-preparative HPLC was performed
on an Agilent 1260 system equipped with an Agilent ZORBAX SB-C18 column (5 μm,
9.4 mm × 250 mm; flow rate 3.0 mL/min ). Column chromatography (CC) was

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performed over silica gel (200–300 mesh, Qingdao Marine Chemistry Co., Ltd.,
China), Sephadex LH-20 (Amersham Biosciences, Sweden), and ODS C18 (40–63 μm;
Merck, Germany). TLC was carried out on glass precoated silica gel (GF254) plates
(Qingdao Marine Chemistry Co., Ltd., China). Spots were visualized under UV light
or by spraying with 2% vanillin-sulfuric acid solution.
2.2. Plant material
Cinnamomum cassia was purchased from Guangdong Province, China, and
authenticated by one of the authors, Prof. P.-F. Tu. The voucher specimen (no.
20130326) has been deposited at the herbarium of Modern Research Center for
Traditional Chinese Medicine, Peking University.
2.3. Extraction and isolation
The dried barks of C. cassia (100 kg) was powered and extracted with 85%
aqueous EtOH for 3 times, each for 3 h. After removal of EtOH under reduced
pressure, the aqueous brownish syrup was suspended in water and partitioned with
petroleum, EtOAc, and n-BuOH, successively.
The EtOAc extract was concentrated in vacuo to yield a black residue (3.8 kg)
which was chromatographed on a silica gel CC eluting with gradient of
CHCl3-MeOH (from 30:1 to 1:1) to yield eight fractions (Frs.1→8). Fr.8 (159 g) was
separated on a silica gel CC (CHCl3-MeOH, 20:1 to 1:1) to give six major
subfractions (Frs. A→F). Fr. A (20 g) was purified by Sephadex LH-20, eluting with
CHCl3-MeOH (1:1), to give Frs. A1, A2, and A3. Fr. A1 (200 mg) was crystallized
from cold MeOH to afford 18 (20 mg). Fr. A2 (50.1 mg) was subjected to

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semi-preparative reverse-phase HPLC with CH3CN-H2O (18:82) to afford 17 (8 mg;
tR 10.3 min) and 1 (5.1 mg; tR 19.7 min ). Fr. B (17 g) was chromatographed on
Sephadex LH-20 and its main fraction was purified by semi-preparative HPLC
eluting with CH3CN-H2O (20:80) to yield 5 (5 mg; tR 15.1 min), 7 (6.1 mg; tR 20.6
min), 8 (5.7 mg; tR 25.9 min), and 14 (5.6 mg; tR 32.3 min). Fr. C (5 g) was subjected
to semi-preparative HPLC eluting with CH3CN-H2O (30:70) to give 11 (4.5 mg; tR
21.5 min), 13 (3.0 mg; tR 28.9 min), and 16 (5.4 mg; tR 36.0 min). Fraction D (25 g)
was separated by Sephadex LH-20 (MeOH-H2O, 70:30) to give Frs.D1→D4.
Compounds 2 (3.1 mg; tR 26.8 min), 3 (3.0 mg; tR 34.1 min), and 15 (5.7 mg; tR 41.2
min) were obtained from Fr. D1 after purification by semi-preparative HPLC eluting
with CH3CN-H2O (18:82). Fr. D2 (800 mg) was first subjected to a silica gel CC
(CHCl3-MeOH, 15:1 to 1:1) and then purified by semi-preparative HPLC eluting
with CH3CN-H2O (18:82) to yield 12 (5.5 mg; tR 17.5 min), 19 (16.3 mg; tR 23.2
min), and 20 (30 mg; tR 38.1 min). Compounds 4 (4.5 mg; tR 18.8 min), 9 (4.2 mg; tR
23.9 min), and 10 (12 mg; tR 35.0 min) were obtained from Fr. D3 (220 mg) after
purification by semi-preparative HPLC eluting with CH3CN-H2O (15:85). Fr. F (2 g)
was chromatographed on Sephadex LH-20 (MeOH-H2O, 50:50) to give Frs. F1→F3.
Frs. F3 (50 mg) was purified by semi-preparative HPLC eluting with CH3CN-H2O
(7:93) to yield 6 (10.2 mg; tR18.9 min)
2.3.1. Cinncassin A (1)
Yellow amorphous powder; [α]20D +42 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
203 (2.46), 224 (2.06), 281 (1.56) nm; IR (KBr) νmax 3367, 1760, 1596, 1517 cm1;

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CD (MeOH, △ε) 204 (–13.95), 230 (–7.53) nm; HRESIMS [MH] m/z 285.0770
(calcd for C16H13O5, 285.0763); for 1H NMR and 13C NMR data, see Table 1.
2.3.2. Cinncassin B (2)
White amorphous powder; [α]20D +61 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
206 (2.76), 273 (1.71), 304 (1.51) nm; IR (KBr) νmax 3370, 1594, 1519, 1464 cm1;
CD (MeOH, △ε) 206 (+61.42), 226 (–2.39), 242 (+2.22) nm; HRESIMS [M+Na]+ m/z
1
13
415.1354 (calcd for C20H24O8Na, 415.1369); for H NMR and C NMR data, see
Table 1.
2.3.3. Cinncassin C (3)
White amorphous powder; [α]20D 27 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
204 (2.63), 225 (2.26), 280 (1.75) nm; IR (KBr) νmax 3397, 1765, 1610, 1515 cm1;
CD (MeOH, △ε) 214 (–13.82), 228 (+5.71), 240 (–1.70), 254 (+3.63) nm; HRESIMS
1
[M+Na]+ m/z 457.1477 (calcd for C22H26O9 Na, 457.1475); for H NMR and 13C
NMR data, see Table 1.
2.3.4. Cinncassin D (4)
White amorphous powder; [α]20D 36 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
207 (2.65), 235 (2.23), 289 (1.98), 307(1.88) nm; IR (KBr) νmax 3467, 1766, 1593,
1463 cm1; CD (MeOH, △ε) 212 (+178.85), 226 (5.27), 240 (+6.40), 256 (0.23),
294 (+9.36) nm; HRESIMS [M+H]+ m/z 509.1801 (calcd for C28H29O9, 509.1812);
for 1H NMR and 13C NMR data, see Table 2.
2.3.5. Cinncassin E (5)
White amorphous powder; [α]20D 27 (c 0.3, MeOH); UV (MeOH) λmax (log ε)

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205 (2.65), 303 (2.05), 337 (2.09) nm; IR (KBr) νmax 3466, 1734, 1596, 1511, 1426
cm1; CD (MeOH, △ε) 230 (0.58) nm; HRESIMS [MH] m/z 403.1391 (calcd for
C21H23O8, 403.1393); for 1H NMR and 13C NMR data, see Table 2.
3.3.6. Cinnacassoside D (6)
White amorphous powder; [α]20D +11.2 (c 0.2, MeOH); UV (MeOH) λmax (log ε)
207 (3.52), 255 (2.78); IR (KBr) νmax 3367, 2927, 1705, 1650, 1601, 1453 cm1;
1
HRESIMS m/z 329.1236 [MH] (calcd for C15H21O8 329.1236); for H and 13C
NMR spectral data, see Table 2.
2.4. Acid hydrolysis and GC analysis of compound 6
Compound 6 (4.9 mg) was stirred at 100 °C for 12 h with 2 M CF3COOH (3 mL).
After being cooled to room temperature, the solution was extracted with CHCl3 for
three times. The CHCl3 extract was concentrated in vacuo to yield a white residue (A),
and the water solution was freeze-dried to give a white residue (B).
Residue B was redissolved in 2 mL pyridine with 0.1 M L-cysteine methyl ester
hydrochloride and stirred at 60 °C for 1.5 h. After being cooled and concentrated, the
mixture was treated with hexamethyl-disilazane (HMDS) and trimethylchlorosilane
(TMSCl) (2:1, v/v, 0.6 mL) inpyridine (2 mL), followed by stirring at 60 °C for 0.5 h.
Then, the solution was concentrated to dryness, and the residue was separated with
H2O and n-hexane (2 × 3 mL) [9,10]. The n-hexane fraction was analyzed by GC
using a HP-5 column (0.25 mm × 30 m). The temperature of injector was 250 °C, and
the temperature of detector was 280 °C. A gradient temperature system was applied to
the oven, starting at 160 °C and increasing up to 280 °C at a rate of 5 °C/min, then

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keeping 280 °C for 10 min. The peak of the sugar derivative in the hydrolysate were
identified by comparison of the retention time with the authentic sample of D-glucose
(24.9 min) after similar treatment with TMSCl/HMDS.
2.5. Cell viability
MTT assay was used for Cell viability detection as previously reported [10‒12].
Briefly, BV-2 cells (mouse microglial cells) were purchased from Peking Union
Medical College, Cell Bank (Beijing, China) and maintained in DMEM medium
supplemented with 10% Fetal Bovine Serum (FBS), penicillin (100 U/mL) and
streptomycin (100 g/mL) at 37°C in a humidified atmosphere with 5% CO2. Then,
BV-2 cells were seeded in 48-well plates (5×104 cells/well) for 24 h incubation and
treated with compounds isolated from C. cassia for 24 h with LPS at the
concentration of 1μg/mL (Escherichia coli 0111:B4, Sigma, MO, USA). After that,
BV-2 cells were incubated with MTT solution (500 μL, 0.5 mg/mL in culture medium)
at 37 °C for 4 h. Then the culture supernatant was removed and the residue was
dissolved in DMSO (500 μL). The absorbance was detected under 540 nm by using a
microplate reader (Tecan Trading AG).
2.5. Inhibition of NO production
The production of NO was determined by detecting cell culture supernatants for
nitrite, a major stable product of NO, by Griess reagent[10]. 5×104 cells/well were
seeded in 48-well culture plates for 24 h. Then the cells were treated with different
concentrations of compounds and stimulated with LPS at the concentration of 1μg/mL
for 24 h. After that, the cell culture supernatant (300 μL) was collected to react with

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Griess reagent (100 μL, 1% sulfanilamide/0.1% naphthylethylene diamine
dihydrochloride/2% phosphoricacid) for 10 min in the darkness at room temperature.
The absorbance was measured at 540 nm, using a microplate reader (Tecan Trading
AG). The experiments were performed in parallel for three times. The IC50 was
generated by GraphPad Prism 5 software (GraphPad Software, Inc., San Diego,
California) and quercetin was used as a positive control (IC50 = 15.6 μM).
2.6. ECD calculations
The 3D structures were subjected to random conformation analysis by using
Sybyl-X 1.1 with the MMFF94s molecular mechanics force field. Then, the minimum
conformers were optimized using DFT at the B3LYP/6-31G(d) level in the gas phase
in the Gaussian 09 program. The B3LYP/6-31G(d) harmonic vibrational frequencies
were further calculated to confirm their stability. ECD calculations were performed
using the time-dependent density functional theory (TDDFT) method at the
B3LYP/6-311++G (2d, 2p) level through the CPCM model (MeOH). ECD spectra of
different conformers were simulated using SpecDis with a half-band width of 0.3 ev,
and the final ECD spectra were obtained according to the Boltzmann-calculated
contribution of each conformer.
3. Results and discussion
Cinncassin A (1) was obtained as a yellow amorphous powder. Its molecular
formula was assigned as C16H14O5 on the basis of a quasimolecular ion at m/z
285.0770 [MH] (calcd for C16H13O5, 285.0763) in its HRESIMS. The IR spectrum

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indicated that 1 possesses hydroxy (3367 cm1), carbonyl (1760 cm1), and phenyl
(1596 and 1517 cm1) functionalities. The 1HNMR data revealed the presence of a set
of ABX coupled phenyl protons at δH 6.71 (1H, d, J = 7.5 Hz), 6.69 (1H, d, J = 1.0 Hz)
6.60 (1H, dd, J = 7.5, 1.0 Hz), a group of A2B2 coupled phenyl protons at δH 7.00 (2H,
d, J = 8.5 Hz), 6.73 (2H, d, J = 8.5 Hz), and four aliphatic proton signals at δH 4.61
(1H, br t, J = 8.5 Hz), 4.26 (1H, br t, J = 9.5 Hz), 3.91 (1H, d, J = 12.0 Hz), 3.68 (1H,
13
td, J = 12.0, 8.5 Hz). The C NMR data (Table 1) showed 16 carbon resonances
comprising 12 phenyl carbons, one carbonyl carbon, and three aliphatic carbons. The
1
HMBC correlations of H-3 and H-5 with C-2 as well as H-1H correlations of
H-3/H-4/H-5 revealed the presence of a furanone moiety. The linkage positions of the
two phenyl groups were deduced to be at C-3 and C-4 of the furanone moiety from
the HMBC correlations of H-3 (δH 3.91) with C-1' (δC 128.1), C-2' (δC 130.8), and C-6'
(δC 130.8), and H-4 (δH 3.68) with C-1" (δC 130.1), C-2" (δC 115.6), and C-6" (δC
119.9). Three hydroxy groups were deduced to be attached to the two phenyl groups
to match the molecular composition and the chemical shifts of those aromatic carbons.
The relative configuration of 1 was deduced based on the NOESY spectrum, in which
H-3 was correlated with H-5β (δH 4.26), and H-4 with H-5α (δH 4.61). The absolute
configuration of 1 was determined by comparing the experimental and calculated
ECD spectra. The experimental ECD spectrum of 1 showed negative Cotton effects at
204 and 230 nm, which are in agreement with the calculated ECD data of (3R,4R)-1
(Fig. 3). Based on the above evidences, the structure of cinncassin A (1) was
elucidated
as

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(+)-(3R,4R)-4-(3",4"-dihydroxyphenyl)-3-(4'-hydroxyphenyl)-dihydrofuran-2-one.
Cinncassin B (2) was obtained as a white amorphous powder. HRESIMS analysis
revealed a molecular formula of C20H24O8 from its pseudomolecular ion at m/z
415.1354 [M+Na]+ (calcd for C20H24O8Na, 415.1369). The IR spectrum revealed the
absorption bands for hydroxy (3370 cm1) and phenyl (1594, 1519 and 1464 cm1)
functionalities. The 1H NMR data showed four aromatic proton signals at δH 6.65 (2H,
s) and δH 6.53 (2H, s), revealing the presence of two tetrasubstituted phenyl groups,
four methoxys at δH 3.82 (6H, s) and δH 3.73 (6H, s), and five aliphatic protons at δH
5.14 (1H, d, J = 10.5 Hz, H-2), 4.47 (1H, t, J = 4.5 Hz, H-4), 4.43 (1H, dd, J = 9.5, 4.5
Hz, H-5), 4.01 (1H, d, J = 9.5 Hz, H-5)，3.13 (1H, dd, J = 10.5, 4.5Hz, H-3). In the
13C NMR spectrum, except the data of two phenyl groups and four methoxyls, there
are also four aliphatic carbons, which were proved to the signals of a tetrahydrofuran
moiety from the 1H-1H COSY and HMBC correlations. The HMBC correlations from
H-2 (δH 5.14) to C-1' (δC 133.1), C-2' (δC 104.6), and C-6' (δC 104.6), and from H-3 (δH
3.13) to C-1" (δC 127.8), C-2" (δC 108.6), and C-6" (δC 108.6) verified that the two
phenyl groups are linked to C-2 and C-3 of tetrahydrofuran, respectively. The large
coupling constant of H-2 and H-3 (J = 10.5 Hz) and no correlation between them in
the NOESY spectrum suggested that H-2 and H-3 are trans-relationship. While H-3
and H-4 are cis-relationship on the basis of their NOE effect and their small coupling
constant of J = 4.5 Hz. The CD spectrum displayed a typical coupled Cotton effect,
positive at 242 nm (Δε +2.22) and negative at 226 nm (Δε 2.39), indicating an
exciton coupling between the π→π* transition of the phenyl chromophores. The

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positive chirality revealed the 2S,3S,4R configuration for 2, which was further
confirmed by the calculated ECD data of 2 (Fig. 4). Thus, the structure of cinncassin
B
(2)
was
determined
as
(+)-(2S,3S,4R)-2,3-bis(4-hydroxy-3,5-dimethoxy)-4-hydroxytetrahydrofuran.
Cinncassin C (3) was obtained as a white amorphous powder. HRESIMS
exhibited a sodium-adduct [M+Na]+ ion at m/z 457.1477 (calcd for C22H26O9Na,
1
457.1475), suggesting a molecular formula of C22H26O9. The H NMR data showed
the signals of two sets of 1,3,4,5-tetrasubstituted phenyl groups like 2 [δH 6.65 (2H, s),
6.63 (2H, s), 3.82 (6H, s), 3.81 (6H, s)], a doublet methine at δH 4.00 (1H, d, J = 7.0
Hz), along with six aliphatic protons at δH 2.235.33, five of which are deduced to
form a 2,4-disubstituted tetrahydrofuran moiety from the 1H-1H COSY correlations of
H-2/H-3/H-4/H-5 (Fig. 2). The 13C NMR data (Table 1) showed 22 carbon resonances,
comprising 12 olefinic carbons, four methoxy groups, six aliphatic carbons and a
carboxylic carbon. In the HMBC spectrum, the correlations from H-5 (δH 3.77, 3.65),
H-4 (δH 2.37), and H-3 (δH 2.33, 2.23) to -COOH (δC 180.7) suggested that -COOH
group was linked at C-4. The HMBC correlations of H-6 (δH 4.00) and C-1', 1" (δC
132.8), C-2', 6' (δC 107.1), C-2", 6" (δC 107.6), C-2 (δC 82.5), and C-3 (δC 30.5)
indicated that two tetrasubstituted phenyl units were both connected at C-6. The
relative configuration of 3 was deduced from the NOESY correlations of H-4 with
H-3α (δH 2.23), and H-2 with H-3β (δH 2.33). The absolute configuration of 3 was
determined by comparing the experimental and calculated ECD spectra. Therefore,
the
structure
of
cinncassin
C
(3)
was
assigned
as
()-(2S,

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4R)-2-[6-bis(4-hydroxy-3,5-dimethoxy)]-4-carboxyl-tetrahydrofuran.
Cinncassin D (4) was obtained as a white amorphous powder. Its HRESIMS
showed a [M + H]+ ion at m/z 509.1801 (calcd for C28H29O9, 509.1812), suggesting a
molecular formula of C28H28O9. Meanwhile, the IR spectrum indicated that 4
possesseshydroxy (3467 cm1), aldehyde (1766 cm1), and phenyl (1593 and 1463
cm1) functionalities. The NMR data of 4 (Table 2) showed the presence of one
aldehyde, one 1,3,4-trisubstituted phenyl, two 1,3,4,5-tetrasubstituted phenyl groups,
three methoxyls, two oxygenated methines, two hydroxymethylenes, and two methine
groups. The NMR data of 4 were similar to those of vitrifol A[13], except for
substitution of the n-propanolunit in vitrifol A by an aldehyde group in 4. The HMBC
correlations of -CHO (δH 9.79) andC-1" (δC 132.8), C-6" (δC 122.2), and C-2" (δC
113.9) provided support that the aldehyde group is linked at C-1". The
trans-configurations between H-7 and H-8, and H-7' and H-8' were deduced from
their coupling constants of J = 6.5 Hz, 6.5 Hz[14,15]. The CD spectrum of 4 showed a
positive Cotton effect at 294 nm, indicating a 7S,8R,7'S,8'R configuration[16] and the
absolute configuration of 4 was also confirmed by the calculated ECD data. Therefore,
the
structure
of
cinncassin
D
(4)
was
determined
to
be
()-(7S,8R,7'S,8'R)-2,2',3,3'-tetrahydro-2'-(4-hydroxy-3-methoxyphenyl)-3,3'-bis(hydr
oxymethyl)-7,7'-dimethoxy-5-aldehyde-2,5'-bibenzofuran.
Cinncassin E (5) was obtained as a white amorphous powder. The HRESIMS
displayed a pseudomolecular ion [MH]at m/z 403.1391 (calcd for C21H23O8,
403.1393), consistent with amolecular formula of C21H24O8. The IR absorption bands

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indicated the presence of hydroxy (3466 cm1), aldehyde (1734 cm1), and phenyl
1
(1596 and 1511cm1) functionalities. The HNMR spectrum revealed the signals for
one aldehyde proton at δH 9.58 (1H, d, J = 8.0 Hz), a set of ABX coupled phenyl
protons at δH 7.22 (1H, d, J = 2.0 Hz), 7.16 (1H, dd, J = 8.5, 2.0 Hz), and 7.00 (1H, d,
J = 8.5 Hz), a pair of symmetric 1,3,4,5-tetrasubstituted aromatic protons at δH 6.72
(2H, s), two trans-olefinic protons at δH 7.57 (1H, d, J = 16.0 Hz) and 6.66 (1H, d, J =
16.0, 8.0 Hz), four aliphatic protons at δH 4.80 (1H, d, J = 6.5 Hz), 4.56 (1H, m), and
3.87 (2H, d, J = 5.0 Hz), and three methoxy groups at δH 3.83 (3H, s) and 3.79 (6H, s).
1
The H-1H COSY correlations of H-7 (δH 4.80)/H-8 (δH 4.56)/H2-9 (δH 3.87) and H-7'
(δH 7.57)/H-8' (δH 6.66)/H2-9' (δH 9.58) in combination with HSQC and HMBC
correlations revealed the presence of 1,2,3-phenylpropanetriol and cinnamaldehyde
groups. The HMBC correlation of H-8 and C-4' verified the 8-O-4' linkage; the
correlations of methoxyls to C-3', C-3, and C-5 revealed the linkage positions of the
methoxyls. The large coupling constant of H-7 and H-8 (J = 6.5 Hz) suggested that
H-7 and H-8 are in the threo-form[17]. Meanwhile, the CD spectrum showed a
negative Cotton effect at 230 nm (△ε ‒0.58) , indicating that 5 has a 8R absolute
configuration[17,18]. Thus, the structure of cinncassin E (5) was determined to be
()-threo-(7R,8R)-7'E-4,7,9-trihydroxy-3,3',5-trimethoxy-8-4'-oxyneolignan-7'-en-9'-a
l.
Cinnacassoside D (6) was obtained as a white amorphous powder. The
molecular formula of 6 was assigned as C15H22O8 based on the HRESIMS peak at m/z
329.1236[MH](calcd for C15H21O8 329.1236). The IR spectrum of 6 showed the

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presence of hydroxy (3367 cm1) and phenyl (1650, 1601, 1453 cm1) functionalities.
The NMR data of 6 (Table 2) showed the presence of one monosubstituted phenyl [δH
7.43 (2H, d, J = 7.5 Hz), 7.34 (2H, t, J = 7.5 Hz) and 7.26 (1H, t, J = 7.0 Hz)], two
oxygenated methines [δH 4.91 (1H, d, J = 4.0 Hz) and 3.95 (1H, m)], one
hydroxymethyl [δH 3.64 (2H, m)], and a set of β-glucopyranosyl moiety [δH 4.39 (1H,
d, J = 7.5 Hz)]. The HMBC correlations from H-7 to C-1, C-2, C-6, C-8 and C-9, as
1
well as H-1H correlations of H-7/H-8/H2-9 suggested that 6 is a phenylpropanetriol
glucoside. The HMBC correlations from H-8 to C-1 and C-1', and from H-1' to C-8
revealed that the glucopyranosyl moiety is attached to C-8. Based on the above
evidences,
the
structure
of
6
was
elucidated
to
be
8-(1-O-β-glucopyranosyl)-phenylpropanetriol. The small coupling constant of H-7
and H-8 (J = 4.0 Hz) and ΔC8-C7 = 11.4 ppm (<12.0 ppm) suggested that H-7 and H-8
were in the erythro-form[19]. Acid hydrolysis of 6 gave glucose and
phenylpropanetriol. The positive [α] value{ [α]20 +13.7 (c 0.1, MeOH)} of
D
phenylpropanetriol indicated that its absolute configuration is 7S,8S[20,21].The
absolute configuration of the glucosyl was determined by analysis of its silane
derivative on GC. Therefore, the structure of cinnacassoside D (6) was determined to
be (+)-(7S,8S)-8-(1-O-β-D-glucopyranosyl)-phenylpropanetriol.
The
known
compounds
were
identified
ether
as
(7)[17],
(8)[17],
(9)[22],
(+)-threo-(7S,8S)-guaiacylglycerol-β-coniferyl
(+)-erythro-(7S,8R)-guaiacylglycerol-β-coniferyl
aldehyde
aldehyde
ether
()-erythro-(7R,8S)-guaiacylglycerol-β-O-4'-sinapoyl
ether

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()-erythro-(7S,8R)-syringylglycerol-8-O-4'-(sinapoyl
(+)-erythro-(7R,8S)-guaiacylglycerol-8-vanillin ether(11)[17,24], (7S,8R)-lawsonicin
(12)[14,25], 5'-methoxylariciresinol(13)[26], (+)-(7'R,8R,8'R)-5,5'-
dimethoxylariciresinol (14)[27,28], (+)-(7'S,8R,8'R)-5,5'-dimethoxylariciresinol (15)
[27,28], picrasmalignan (16)[29], (+)-leptolepisol (17)[19,23],
alcohol)
ether(10)[23],
A
C
()-(7R,8S,7'R,8'S)-syringaresinol (18)[14,30,31], (+)-isolariciresinol (19)[32], and
()-secroisolariciresinol (20)[33,34] on the basis of spectroscopic data analysis and
comparison with the physical data reported in the literature (Fig. 1).
One of the clinical usages of C. cassia is for the treatment of rheumatoid arthritis
and osteoarthritis [1], therefore, LPS-induced NO production in BV-2 microglial cells
was used for the evaluation of the anti-inflammatory activities of the isolates of 15
and 720, and their IC50 values are presented in Table 3. Based on the activity assay
data, it could be observed that compounds 4, 5, 7, 8, 10, 12, and 15 showed potent
inhibitory effects on NO production in LPS-induced BV-2 cells with IC50 values of
24.2, 17.6, 17.7, 18.7, 27.0, 20.5, and 17.5 μM respectively. Among the active
compounds, the majority are 8-O-4'-lignans, so their preliminary structure activity
relationship was summarized as follows: 1) compounds with acrylaldehyde group
showed potent anti-inflammatory activities, such as compounds 5, 7, and 8. But when
the acrylaldehyde was changed to aldehyde, the inhibitory effect became much
weaker, such as compound 11; 2) compounds with acrylaldehyde group showed
stronger activity than those having allyl alcohol group (5, 7, 8 > 9, 10); 3) the
methoxy group in the phenyl did not affect the activities of the compounds with

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acrylaldehyde group, for example, the activity of 5 is similar to those of 7 and 8,
whereas it could increase the activity when the acrylaldehyde group was reduced to
the allyl alcohol group, such as 10>9.
The above results indicate that most of the compounds isolated from C. cassia
have potent anti-inflammatory effects, which makes a partial contribution for the
anti-inflammatory activity of C. cassia. The isolates with significant
anti-neuroinflammatory activity have potential to be used as candidates for the
treatment of various neurodegenerative diseases.
Acknowledgment
This study was supported by National Natural Sciences Foundation of China
(NoS. 81222051 and 81530097), and National Key Technology R&D Program “New
Drug
Innovation”
of
China
(Nos.
2012ZX09301002-002-002
and
2012ZX09304-005).
Supplementary data
Supplementary data associated with this article can be found, inthe online version,
athttp://dx.doi.org...
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Fig. 1. Structures of compounds 120 isolated from C. cassia.

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Fig. 2. Key HMBC and 1H-1H COSY correlations of compounds 16.

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Fig. 3. Comparison of the experimental ECD spectrum of 1 in MeOH with the
calculated ECD spectra of (3R,4R)-1 and (3S,4S)-1.

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Fig. 4. Comparison of the experimental ECD spectrum of 2 in MeOH with the
calculated ECD spectra of (2R,3R,4S)-2 and (2S,3S,4R)-2.

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Fig. 5. The CD exciton chirality rule applied of 2.

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Fig. 6. Comparison of the experimental ECD spectrum of 3 in MeOH with the
calculated ECD spectra of (2S,4R)-3 and (2R,4S)-3.