Two new xanthones, a new lignin, and twenty phenolic compounds from Smilax china and their NO production inhibitory activities

Article abstract of DOI:10.1080/10286020.2019.1598395

Two new xanthones smilone A (1), smilone B (2), and a new lignin smilgnin A (3) were isolated from the rhizomes of Smilax china L., together with three known xanthones (4–6), four lignins (7–10), two flavones (11, 12), two stilbenoids (13, 14), and ten organic phenoloids (15–24). Of them, compounds 4–6 were isolated from the genus Smilax for the first time. The structures of 1–24 were elucidated by the extensive analysis of spectral data and compared with the literature. All compounds were evaluated for their inhibitory effects against LPS-induced NO production in RAW264.7 macrophages. Among them, compound 24 exhibited significant inhibitory activity against NO production (IC₅₀ = 1.26 μM), while compounds 3, 6, and 7 showed weak activities at the concentration of 50 μM. (Figure presented.).

Full text of DOI:10.1080/10286020.2019.1598395

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Two new xanthones, a new lignin, and twenty
phenolic compounds from Smilax china and their
NO production inhibitory activities
Xiang-Wei Zheng, Lei Zhang, Wei-Yue Zhang, Yan Zhang, Jian-Ying Wang, Bao-
Cheng Liu, Xiao-Juan Hu, Yan-Long Hong & Yong-Qing Yang
To cite this article: Xiang-Wei Zheng, Lei Zhang, Wei-Yue Zhang, Yan Zhang, Jian-Ying Wang,
Bao-Cheng Liu, Xiao-Juan Hu, Yan-Long Hong & Yong-Qing Yang (2019): Two new xanthones, a
new lignin, and twenty phenolic compounds from Smilax china and their NO production inhibitory
activities, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2019.1598395
To link to this article: https://doi.org/10.1080/10286020.2019.1598395
Published online: 09 Apr 2019.
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2019.1598395
Two new xanthones, a new lignin, and twenty phenolic
compounds from Smilax china and their NO production
inhibitory activities
a
ꢀ
b
ꢀ
c
c
Xiang-Wei Zheng , Lei Zhang , Wei-Yue Zhang , Yan Zhang , Jian-Ying
b
b
b
b
d
Wang , Bao-Cheng Liu , Xiao-Juan Hu , Yan-Long Hong and Yong-Qing Yang
a
Engineering Research Center of Modern Preparation Technology of TCM, Ministry of Education,
b
Shanghai University of Traditional Chinese Medicine, Shanghai 200000, China; Shanghai Innovation
Center of Traditional Chinese Medicine Health Service, Shanghai University of Traditional Chinese
Medicine, Shanghai 200000, China; School of Pharmacy, Shanghai Jiao Tong University, Shanghai
2
2
c
d
00000, China; Yue Yang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai
00000, China
ABSTRACT
ARTICLE HISTORY
Two new xanthones smilone A (1), smilone B (2), and a new lig-
nin smilgnin A (3) were isolated from the rhizomes of Smilax
china L., together with three known xanthones (4–6), four lignins
Received 19 December 2018
Accepted 18 March 2019
KEYWORDS
(
7–10), two flavones (11, 12), two stilbenoids (13, 14), and ten
Smilax china L.; xyxanthone;
lignin; phenoloid; NO
production inhibi-
tory activity
organic phenoloids (15–24). Of them, compounds 4–6 were iso-
lated from the genus Smilax for the first time. The structures of
1–24 were elucidated by the extensive analysis of spectral data
and compared with the literature. All compounds were evaluated
for their inhibitory effects against LPS-induced NO production in
RAW264.7 macrophages. Among them, compound 24 exhibited
significant inhibitory activity against NO production (IC50 ¼ 1.26
mM), while compounds 3, 6, and 7 showed weak activities at the
concentration of 50 lM.
CONTACT Yan-Long Hong
hfuir@163.com
Shanghai Innovation Center of Traditional Chinese Medicine Health
Service, Shanghai University of Traditional Chinese Medicine, Room 8109, No. 1200 Cai Lun Road, Pudong District,
Shanghai 200000, PR China; Yong-Qing Yang dryqyang@163.com Yue Yang Hospital, Shanghai University of
Traditional Chinese Medicine, Shanghai 200000, China
ꢀ
These authors contributed equally to this work.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
X.-W. ZHENG ET AL.
1. Introduction
Smilax china L., a perennial shrub, is one of the most important traditional Chinese
medicines (TCM) in the genus Smilax, and the rhizome of S. china (SCR) called “Da
Tong Jing” is used for treatment of some diseases, such as bone hyperplasia, inflam-
matory, and so on [1]. Previous phytochemistry studies on SCR showed there were
mainly phenolic compounds including flavonoids, phenylpropanoids, and stilbenoids,
as well as steroidal saponins in it. These compounds showed many activities, such as
anti-inflammatory, immunosuppressive and antioxidation.
In clinic, The Chinese drugs pharmaceutics of SCR are widely used for the treat-
ment of inflammation in bone hyperplasia. Previous pharmacological studies showed
its aqueous extract in the dose of 1000 mg/kg (i.g.) had anti-inflammatory and antino-
ciceptive effects compared to physiological saline [2]. Activity screening studies
showed that two steroidal saponins isolated from SCR exhibited anti-inflammatory
activities [3], while the phenolic compounds, as the main identified ingredients of
SCR, were not found to show the activity. Our further investigations on anti-inflam-
matory phenolic ingredients of SCR led to the isolation of two new xanthones (1-2),
a new lignin (3) and 21 known phenolic compounds (4-24) (Figure 1). All the iso-
lates were evaluated for their potential anti-inflammatory activities in LPS-stimulated
RAW264.7 macrophages. Herein we describe the isolation and structure elucidation
of these phenoloids as well as their potential anti-inflammatory activities.
2. Results and discussion
2.1. Chemistry
The ethanol extract of SCR was separated by column chromatography (CC) over sil-
ica gel, MCI gel CHP 20P, Sephadex LH-20, preparative TLC, and semipreparative
HPLC to obtain three new compounds smilone A (1), smilone B (2), smilgnin A (3),
together with 21 known compounds. The known compounds were identified as 1,3,6-
Figure 1. Chemical structures of compounds 1–3.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
trihydroxy-2,7-dimethoxyxanthone (4) [4], 1,3,6-trihydroxy-7-methoxyxanthone (5)
5], 1,3,6,7-tetrahydroxyxanthone (6) [6], syringaresinol (7) [7], 3-O-p-coumaroyl-6-
[
O-feruloyl-b-D-fructofuranosyl-6-O-acetyl-a-D-glucopyranoside (8) [8], (6-O-feruroyl)-
b-D-fructofuranosyl-(6-O-acetyl)-a-D-glucopyranoside (9) [9], arctigenin (10) [10],
catechin (11) [11], naringenin (12) [12], reveratrol (13) [13], cis-resveratrol (14) [14],
4-hydroxyacetophenone neoastilbin (15) [15], 3-hydroxy-4-methoxybenzoic acid (16)
[
16], protocatechuic acid (17) [17], 3,5-dihydroxybenzoic acid (18) [18], p-hydroxy-
benzoic acid (19) [19], 2,5-dihydroxybenzoic acid (20) [20], caffeic acid (21) [21],
syringic acid (22) [22], benzoic acid (23) [23], catechol (24) [24] by alignment with
spectral data reported in the literature. Of them, compounds 4–6 were isolated from
the genus Smilax for the first time.
Compound 1 was obtained as a pale yellow powder, and its molecular formula was
–
deduced to be C H O from the HR-ESI-MS (m/z 317.0652 ([M-H] ). Its UV spec-
1
6
14 7
trum in MeOH (kmax 254 nm) showed the characteristic absorption of benzene rings.
ꢁ
1
The IR spectrum of 1 showed the presence of OH groups (3419 cm ), H-bonded
ꢁ
1
ꢁ1
1
C ¼ O groups (1651 cm ), and aromatic rings (1614, 1589, and 1485 cm ). The H-
NMR spectrum of 1 displayed two uncoupled aromatic H-atoms at d 6.91, 7.39, a H-
bonded OH group at d 13.22 (s), two methoxy groups at d 3.78 (s) and 3.88 (s), and
1
3
an methyl H-atom at d 2.16 (s). The C NMR and DEPT spectra of 1 (Table 1) indi-
cated signals of 16 carbons, including one methyl group at d 8.2, two oxygen-bearing
methyls at d 56.3, 60.6, two methine groups at d 103.2, 104.9, and 11 quaternary car-
bons, which contained one carbonyl and two benzene rings. These observations sug-
gested that 1 had a xanthone skeleton. Additionally the C NMR spectral data of 1
were comparable to those of the known compound 4 [4], except one more methyl in
13
1
. The methyl was determined to be linked at the C-4, which was confirmed by the
cross peaks between d 2.16 (4-CH -H)/d 150.5 (C-3), d 102.0 (C-4) and d 155.9
H
3
C
C
C
(C-4a) in the HMBC spectrum of 1 (Figure 2). The NOESY correlations between dH
7
.39 (H-8)/d 3.88 (7-OCH ), d 13.22 (1-OH) /d 3.78 (2-OCH ) (Figure 3) clearly
H
3
H
H
3
confirmed that the oxygen-bearing methyl (d 3.88, 3.78) groups were located at C-7
H
and C-2, respectively. From these spectral data, the structure of 1 was elucidated as
1,3,6-trihydroxy-2,7-dimethoxy-4-methylxanthone.
Compound 2 was obtained as a pale yellow powder, and its molecular formula was
–
deduced to be C H O from the HR-ESI-MS (m/z 465.1028 [M-H] ). Its UV spec-
2
1
22 12
trum in MeOH (kmax 254 nm) showed the characteristic absorption of benzene rings.
ꢁ
1
The IR spectrum of 2 showed the presence of OH groups (3419 cm ), H-bonded
ꢁ
1
ꢁ1
1
C ¼ O groups (1651 cm ), and aromatic rings (1614, 1589, and 1485 cm ). The H-
NMR spectrum of 1 displayed three uncoupled aromatic H-atoms at d 6.49, 7.31, and
7
3
.49, a H-bonded OH group at d 13.09 (s), two methoxy groups at d 3.76 (s) and
.89 (s), one anomeric proton signal at d 5.23 (d, 1H, J ¼ 7.2 Hz), corresponding to
H
1
3
the carbon resonance at d 100.1 in the C NMR spectrum, suggesting that 2 was a
C
1
3
monoglycoside. The C NMR and DEPT spectra of 2 (Table 1) indicated signals of
twenty one carbons, including two oxygen-bearing methyls at d 56.4, 60.5, one
methylene at d 68.1, eight methine groups of which four were resonated in the region
70–78 ppm, and one methine signal at d 100.1 also suggested the presence of one glu-
cose, the remaining three methine groups at d 94.4, 103.5, and 105.1, ten quaternary

4
X.-W. ZHENG ET AL.
13
Table 1. C NMR spectral data of compounds 1–3 (100 MHz; in DMSO)
d ppm).
(
No.
1
2
3
1
2
3
4
151.6
130.5
150.5
102.0
155.9
155.0
103.2
152.5
146.3
104.9
111.4
102.1
154.4
131.1
151.9
94.4
158.8
153.1
103.5
153.5
147.1
105.1
113.3
102.5
132.8
113.9
148.9
146.4
4
4
5
6
7
8
8
8
9
1
2
3
4
5
6
a
b
116.1
123.0
35.4
47.8
a
b
181.5
130.8
113.7
150.5
149.2
113.1
122.1
38.9
0
0
0
0
0
0
100.1
75.6
77.1
72.2
76.8
68.1
0
0
0
0
0
0
0
0
0
0
0
0
C-7
C-8
C-9
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
42.5
72.9
0
0
0
0
0
0
0
0
0
145.5
113.2
148.8
134.9
121.8
116.1
32.6
35.7
62.3
2
2
3
3
4
-OCH
-OCH
3
60.6
8.2
60.5
0
0
3
56.4
56.4
56.4
-OCH
-OCH
3
0
3
-CH
3
0
C-4 -OCH
3
56.5
7
-OCH
3
56.3
179.9
56.4
179.7
C ¼ O
carbons, which contained one carbonyl and two benzene rings. These observations
1
13
suggested that 2 had a xanthone skeleton. The H and C NMR data of the sugar
moiety of 2 accorded well with those of glucose. Gentle acidic hydrolysis and derivati-
zation of 2 got only one sugar fragment which was identified as trimethylsilyl ether-
D-glucose by comparison with the GC data of an authentic sample, of which the
retention time was 21.086 min (trimethylsilyl ether-L-glucose: 23.156 min). The cou-
pling constants, 7.2 Hz, of the anomeric proton resonance indicated that the sugar
1
13
linkage was in b orientation. The H and C NMR spectral data of 2 (Table 1) were
comparable to those of compound 4 [4], except one more D-glucopyranose in 2, All
the information mentioned above was in support of 2 being the glycoside of 4. The
glucopyranose was determined to be linked at C-6, which was confirmed by the cross
0
peaks between d 5.23 (H-1 )/d 153.5 (C-6) in the HMBC spectrum of 2 (Figure 2).
H
C
The NOESY correlations between d 7.49 (H-8)/d 3.89 (7-OCH ), d 13.09 (1-OH)/
H
H
3
H
0
dH 3.76 (2-OCH ), and dH 5.23 (H-1 )/dH 7.31 (H-5) (Figure 3) clearly confirmed
3

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 2. Key HMBC correlations of compounds 1–3.
Figure 3. Key NOESY correlations of compounds 1–3.

6
X.-W. ZHENG ET AL.
that the oxygen-bearing methyl groups (dH 3.89, 3.76), aromatic hydrogen (dH 5.23)
were located at 7, 2, and 5, respectively. From these spectral data, the structure of 2
was elucidated as 1,3-dihydroxy-2,7-dimethoxyxanthone-6-O-b-D-glucopyanoside.
Compound 3 was obtained as a pale white powder, and its molecular formula was
–
deduced to be C H O from the HR-ESI-MS (m/z 535.2327 ([M-H] ). Its UV spec-
3
1
36 8
trum in MeOH (kmax 203, 222, 280, 321, 320, and 362 nm) showed the characteristic
absorption of a 2,3-two benzyl lactone. The IR spectrum of 3 showed the presence of
ꢁ
1
ꢁ1
OH groups (3380 cm ), lactone groups (1758 cm ), and aromatic rings (1585,
1
ꢁ
1
1
589 cm ). The H-NMR spectrum of 3 indicated three pairs of ABX coupled system
H-atoms at dH 6.69 (d, 1H, J ¼ 2.0 Hz), 6.71 (d, 1H, J ¼ 8.0 Hz) and 6.59 (dd, 1H,
J ¼ 8.0, 2.0 Hz); d 6.58 (d, 1H, J ¼ 2.0 Hz), 6.82(d, 1H, J ¼ 8.0 Hz) and 6.60 (dd, 1H,
H
J ¼ 8.0, 2.0 Hz); d 6.78 (d, 1H, J ¼ 2.0 Hz), 6.70 (d, 1H, J ¼ 8.0 Hz) and 6.63 (dd, 1H,
H
1
3
J ¼ 8.0, 2.0Hz). The C NMR and DEPT spectrum of 3 (Table 1) indicated signals of
3
5
1 carbons, including 18 aromatic carbons, four oxygen-bearing methylenes at d 56.4,
6.4, 56.4, 56.5, one oxygen-bearing propyl at d 32.6, 35.7, 62.3, one carbonyl of lac-
tonic ring at d 181.5. These observations suggested that 3 had a lignanoid skeleton.
1
3
Additionally, the C NMR spectral data of 3 were similar to those of the known
compound 10 (arctigenin) [10], except one additional methoxyphenylpropyl fragment.
The fragment was determined to be linked to the phenolic hydroxyl group at C-4,
0
0
which was confirmed by the cross peak between d 6.71(H-5)/d 145.5 (C-1 ); the
H
C
0
0
00
oxygen-bearing propyl at the C-4 , by the cross peaks between d 2.60 (H-7 )/d
00 00 00 00 00
34.9 (C-4 ), d 148.8 (C-3 ), d 121.8 (C-5 ), d 35.7 (C-8 ), and d 62.3 (C-9 );
C C C C
the methoxy at C-2 by the cross peaks between d 3.80 (CH -H)/d 145.5 (C-1 ),
d 113.2 (C-2 ) and d 148.8 (C-3 ); the 7-methylene at the C-8 by the cross peaks
between d 2.54 (H-7)/d 46.8 (C-8) and d 181.5 (C-9); the 7 -methylene at C-8 by
the cross peaks between dH 2.55 (H-7 )/dC 42.5 (C-8 ) and d 72.9 (C-9 ) in the
H
C
1
0
0
00
H
3
C
0
0
00
C
C
0
0
H
C
C
0
0
0
C
HMBC spectrum of 3 (Figure 2). The NOESY correlations between d 2.54 (H-7)/d
H
H
0 0
.51 (H-8 ), d 2.55 (H-7 )/d 2.49 (H-8) (Figure 3) clearly confirmed that the config-
H H
2
0
uration of C-8 and C-8 was converse, consistent with arctigenin. And the optical
rotation of compound 3 (–18.71) and arctigenin (–28.70) were both negative. These
0
observations suggested that absolute configurations of C-8 and C-8 were R by com-
parison with those of arctigenin. From these spectral data, the structure of 3 was elu-
0
0
00
cidated as 4-(2 -methoxy- 4 -hydroxypropyl-phenol)-arctigenin.
2
.2. Biological studies
Nitric oxide (NO) was an important media in inflammation. The production of LPS-
induced NO in RAW264.7 macrophages might reflect the degree of inflammation
and offered a measure to evaluate the effects of compounds on the inflammatory pro-
cess. All of the isolates of SCR were assessed for their inhibitory effects against LPS-
induced NO production in RAW264.7 macrophages (Figure 4). Compound 24
showed significant inhibitory activity against NO production (IC50 ¼1.26 lM), and
compounds 3, 6, and 7 exhibited weak activities (Figure 4). The inhibitory effects
were not caused by nonspecific cytotoxicity to the cells because they had no effect on
cell viability, as determined by MTT assay at the tested concentrations. In contrast,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Figure 4. Inhibitory effects of compounds 1–24 against LPS-induced NO production in RAW264.7
macrophages. (a) Inhibitory effects of compounds 1–24 against LPS-induced NO production in
RAW264.7 macrophages. (Compounds concentration: 50lM, 10 lM; P: parthenolide, positive con-
trol, 10 lM). (b) The effects of compounds 1–24 on cell viability by MTT assay in RAW264.7
macrophages.
compound 10 showed a stronger inhibitory effect on NO production in LPS-stimu-
lated RAW 264.7 macrophages, whereas the NO inhibition could not be distinguished
from the cytotoxicity observed in the MTT assay (Figure 5).
2
.3. Conclusion
In this study, we identified 24 phenolic ingredients of SCR including 2 new xan-
thones, a new lignin, and 21 known phenolic compounds. All the compounds were
evaluated for their inhibitory effects on LPS-induced NO production in RAW264.7
macrophages. Among them compound 24 showed significant inhibitory activity, and
compounds 3, 6, and 7 exhibited weak activities at a high concentration. Our research
enriched the diversity of phenolic compounds from SCR, and especially identified
new compounds and xanthones firstly from the genus Smilax, and we also found
three NO production inhibitory active phenolic ingredients. As the main ingredients,
further detailed evaluation on the anti-inflammatory activity of the phenoloids from
SCR remained to be studied.

8
X.-W. ZHENG ET AL.
Figure 5. The dose inhibition curves of nitric oxide produced by compound 24 (The data were
obtained from three independent experiments and expressed as the means ± SEM.).
3. Experiment
3.1. General experimental procedures
Optical rotation was measured with an MCP-500 polarimeter (Anton Paar). The UV
spectrum was conducted on a Shimadzu UV-1800 spectrometer (Shimadzu, Kyoto,
Japan). The FT-IR spectra were obtained on a Bruker Vector 22 spectrometer (Bruker
1
13
Optik GmbH, Ettlingen, Germany) with KBr pellets. H and C NMR spectra were
recorded on a Bruker DRX-400 NMR spectrometer (Bruker BioSpin GmbH,
Rheinstetten, Germany) using TMS as an internal standard. ESI-MS data were
obtained on a Q-TOF micromass spectrometer. HRESI–MS data were recorded on a
Q-TOF micromass spectrometer (Waters, Milford, MA). Gas chromatography was
performed with HP-5892 II FID detector, and an HP-20M (Carbowx 20M) capillary
column (25 m ꢂ 0.32 mm ꢂ 0.3 lm). Materials for column chromatography were sil-
ica gel (200–300 mesh; Jiangyou Silica Gel Development Co. Ltd., Yantai, China), sil-
ica gel H (10–40 mm; Jiangyou Silica Gel Development Co. Ltd., Yantai, China),
Sephadex LH-20 (40–70 mm; Amersham Pharmacia Biotech AB, Uppsala, Sweden),
MCI (75–150 mm; Mitsubishi Chemical Corporation; Tokyo, Japan), and YMC-gel
ODS-A (50 mm; YMC, Allentown, PA). The chromatographic systems also included
semi-preparative HPLC that was performed on a HPLC system equipped with a
Waters 1525 pump and a Waters 2487 Dual Wavelength Detector using a Thermo

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
hypersil C18 column (5 lm, 150 ꢂ 250 mm, i.d.), together with preparative TLC
Jiangyou Silica Gel Development Co. Ltd., Yantai, China).
(
3
.2. Plant material
The rhizomes of Smilax china were gathered in Quzhou city, Fu Jian province,
China, in May 2016, and identified by Professor Bao-Kang Huang (Second Military
Medical University, Shanghai, China). A voucher specimen (Zheng 5598) has been
deposited in the Herbarium of the Shanghai University of Traditional
Chinese Medicine.
3
.3. Extraction and isolation
The rhizomes of S. china (4.0 kg) were smashed and heated to reflux three times with
ꢃ
9
5% ethanol at 75 C for 1 h each time, and filtered. The combined filtrate was con-
centrated to get crude extracts (0.80 kg). The crude extracts were subjected to column
chromatography over silica gel (200–300 mesh, 4 kg) and eluted with gradient
CH Cl /MeOH/H O (15:0.4:1, 10:0.4:1 and 5:0.4:1) to separate it into five fractions:
2
2
2
Frs. A–E (eluted with 2.75 L CH Cl /MeOH/H O (15:0.4:1) to afford fraction A
2
2
2
(
(
(
(
0.21 g); continually with 5.0 L CH Cl /MeOH/H O (15:0.4:1) to afford fraction B
2 2 2
0.25 g); continually with 5.0 LCH Cl /MeOH/H O (10:0.4:1) to afford fraction C
2
2
2
4.08 g); continually with 2.75 LCH Cl /MeOH/H O (10:0.4:1) to afford fraction D
2
2
2
2.0 g); finally with 5.0 L CH Cl /MeOH/H O (5:0.4:1) to afford fraction E (0.9 g).
2
2
2
Fraction A (0.21 g) was chromatographed over MCI gel CHP20P (MeOH/H O,
2
1
:9–4:6), and ODS (MeOH/H O, 2:8–5:5) to afford 23 (8.0 mg), 22 (26 mg), 21
2
(35 mg). Fraction B (0.25 g) was chromatographed over MCI gel CHP20P (MeOH/
H O, 3:7–8:2) to yield four subfractions B1–B4. Subfraction B1 (0.11 g) was subjected
2
to MCI gel CHP 20P (MeOH/H O, 2:8–6:4) to yield 20 (43 mg), 19 (25 mg).
2
Subfraction B2 (0.042g) was chromatographed over ODS (MeOH/H O, 2:8–6:4), MCI
2
gel CHP20P (MeOH/H O, 3:7–7:3), and sephadex LH-20 (100% MeOH) to yield 15
2
(
12.0 mg). Subfraction B4 (0.058g) was purified by preparative TLC (CH Cl /MeOH/
2 2
H O (15:0.4:1)) to yield 1 (10 mg) and 2 (26 mg). Fraction C (4.08 g) was chromato-
2
graphed over MCI gel CHP 20P (MeOH/H O, 3:7–9:1) to yield three subfractions,
2
C1-C3. Subfraction C2 (1.07g) was purified by preparative TLC (CH Cl /MeOH/H O
2
2
2
(10:0.4:1)) to afford 3 (21 mg), Sub-fraction C3 (2.69 g) was subjected to CC over
MCI gel CHP20P (MeOH/H O, 4:6–1:0) to yield three subfractions, C3-1, C3-2, C3-
2
3. Subfraction C3-2 (1.12 g) was purified by semipreparative HPLC (isocratic model;
mobile phase: 35% MeOH; Detection wavelength: 256 nm; flow rate: 5 ml/min) to
afford 9 (81 mg, retention time: 23.6 min), 10 (43 mg, tR:35.7 min), 11 (26 mg, tR:
38.6 min), and 12 (51 mg, tR:45.7 min). Subfractions C3-3 (1.45 g) was purified by
semipreparative HPLC (gradient model; mobile phase: MeOH/H O, 20–80% over
2
3
0 min, MeOH and H O contained 0.1% TFA; Detection wavelength: 256 nm; flow
2
rate: 5 ml/min) to yield 4 (28 mg, tR: 8.5 min), 5 (19 mg, tR: 14.9 min) , 6 (34 mg, tR:
8.5 min), and 7 (16 mg, tR: 26.4 min) . Fraction D (2.0 g) was chromatographed over
MCI gel CHP 20P (MeOH/H O, 4:6–1:0) and Sephadex LH-20 (100% MeOH) to
1
2

1
0
X.-W. ZHENG ET AL.
afford 13 (62 mg). Fraction E (1.0 g) was chromatographed over MCI gel CHP20P
MeOH/H O, 4:6–8:2) to yield 8 (37mg) and 16 (19 mg). Fraction F (0.9 g) was sub-
(
2
jected to CC over MCI gel CHP20P (MeOH/H O, 6:4–1:0), Sephadex LH-20 (100%
2
MeOH), and silica gel (DCM/MeOH, 9:1–8:2) to afford 14 (15 mg), 17 (25 mg), 18
(39 mg), and 24 (32 mg).
3
.3.1. Smilone A (1)
2
2
ꢁ1
Pale yellow amorphous powder; [a] –29.49 (c 0.21, M OH); IR (KBr) ꢀ (cm ):
D
e
3
419, 1651, 1614, 1589, 1485; UV (MeOH) k
(loge): 235 (3.29), 243 (4.12), 260
max
1
(3.58), 320 (4.38), 362 (4.27); H NMR (400 MHz, DMSO-d , d ppm): 2.16 (s, 3H,4-
6
CH ), 3.78 (s, 3H, 2-OCH ), 3.88 (s, 3H,7-OCH ), 6.91 (s, 1H, 5-H), 7.39 (s, 1H, 8-
3
3
3
1
3
H), 13.22 (s, 1H, 1-OH); CNMR spectral data: Table 1; HR-ESI-MS: m/z 317.0652
M-H] (calcd. for C H O , 317.0661).
–
[
16 14 7
3
.3.2. Smilone B (2)
2
2
ꢁ1
Pale yellow amorphous powder; [a] –11.21 (c 0.22, M OH); IR (KBr) ꢀ (cm ):
D
e
3
2
419, 1651, 1614, 1589, 1485; UV (MeOH) kmax (loge): 235 (3.29), 243(4.12),
1
60(3.58), 320(4.38), 362(4.27); HNMR (400 MHz, DMSO-d , d ppm): 3.76 (s, 3H, 2-
6
OCH ), 3.89 (s, 3H, 7-OCH ), 3.23–3.25 (m, 1H, glc-2-H), 3.28–3.30 (m, 1H, glc-4-
3
3
H), 3.29–3.31 (m, 1H, glc-5-H), 3.39 (dd, 1H, J ¼ 12, 1.6 Hz, glc-3-H), 3.90 (dd, 2H,
J ¼ 12.0, 1.6 Hz, glc-6-H), 5.23 (d, 1H, J ¼ 7.2 Hz, glc-1-H), 6.49 (s, 1H, 4-H), 7.31 (s,
1
3
1
H, 5-H), 7.49 (s, 1H, 8-H), 13.09(s, 1H, 1-OH); CNMR spectral data: Table 1. HR-
–
ESI-MS: m/z 465.1028 [M-H] (calcd for C H O , 465.0133).
2
1
22 12
3
.3.3. Smilgnin A (3)
2
2
ꢁ1
Pale white amorphous powder; [a] –18.71 (c 0.22, M OH); IR (KBr) ꢀ (cm ): 3380,
D
e
1
3
3
2
2
758, 1614, 1585, 1589; UV (MeOH) k
20 (4.38), 362 (4.27); H NMR (400 MHz, DMSO-d , d ppm): 3.77 (s, 3H, 3 -OCH ),
(loge): 203 (3.29), 222 (4.12), 280 (3.58),
max
1
0
6
3
0
0
0
.79 (s, 3H, 3-OCH ), 3.80 (s, 3H, 2 -OCH ), 3.83 (s, 3H, 4 -OCH ), 1.80-1.82 (m,
3
3
3
0
0
0
H, 8 -H), 2.48-2.50 (m, 1H, 8-H), 2.50-2.52 (m, 1H, 8 -H), 2.53–2.55 (m, 2H, 7-H),
.54–2.56 (m, 2H, 7 -H), 2.59–2.61 (m, 2H, 7 -H), 3.92 (dd, 1H, J ¼ 9.7, 1.8 Hz, 9 -
0
00
0
0
Ha), 4.17 (dd, 1H, J ¼ 9.7, 1.8 Hz, 9 -He), 6.59 (dd, 1H, J ¼ 8.0, 2.0 Hz, 6-H), 6.59 (d,
0
0
1
6
H, J ¼ 2.0 Hz, 2 -H), 6.60(dd, 1H, J ¼ 8.0, 2.0 Hz, 5 -H), 6.63 (dd, 1H, J ¼ 8.0, 2.0 Hz,
0
0
00
-H), 6.69 (d, 1H, J ¼ 2.0 Hz, 2-H), 6.70 (d, 1H, J ¼ 8.0 Hz, 5 -H), 6.71 (d, 1H,
0
0
0
13
J ¼ 8.0 Hz, 5-H), 6.78 (d, 1H, J ¼ 2.0 Hz, 2 -H), 6.82(d, 1H, J ¼ 8.0 Hz, 6 -H); CNMR
–
spectral data: Table 1; HR-ESI-MS: m/z 535.2327 [M-H]
(calcd for
C H O , 535.2332).
3
1
36 8
3
.4. Acid hydrolysis and GC analysis of 2
The mixture of 2 (5 mg) and 5% HCl-EtOH (1:1, 5 ml) was heated in 2.0 mol/l HCl/
ꢃ
MeOH (1:1, v/v, 10 ml) at 90 C for 4 h. After drying by evaporation, the reaction
mixture was partitioned between EtOAc and H O, and then neutralized by passing
2
through an ion-exchange resin (Amberlite MB-3) column. The H O layer was con-
2
centrated to produce a sugar residue. The residue was dealt with D-cysteine (0.1 mg)

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
ꢃ
in water (0.05 ml) and pyridine (0.03 ml) at 60 C for 1 h with stirring. When the solv-
ent was evaporated and the reaction mixture was dried, it was mixed with pyridine
(
0.03ml), hexamethyldisilazane (0.03ml), and trimethylsilyl chloride (0.03ml). Then it
ꢃ
was heated at 60 C for 30min. Ultimately, the supernatant was applied to GC.
3
.5. GC analysis of acidolysis product of 2
ꢃ
The GC temperature program was initiated at 80 C and held for 1.0 min, increased
ꢃ
ꢃ
to 200 C at 30 C/min and held for 20 min. The Flame ionization detector temperature
ꢃ
was set at 280 C with the flow rate (H :Air:N ¼ 40:400:30 ml/min). And the retention
2
2
time of the hydrolysis and acetylation product of 2 was consistent with that of authen-
tic trimethylsilyl ether-D-glucose (21.086 min) (Sigma-Aldrich, Shanghai, China).
3
.6. Cell culture
The RAW 264.7 mouse macrophage cell line was got from China Cell Line Bank
Beijing, China). The cells were incubated in RPMI-1640 complemented with 10%
(
heat-inactivated fetal bovine serum, 2.05 mM L-glutamine, penicillin (100 U/ml), and
ꢃ
streptomycin (100 U/ml) at 37 C under a humidified atmosphere of 5% CO .
2
3
.7. Determination of nitric oxide (NO) production
The assessment was performed using the MTT method. Cells were planted onto 96-
4
well plates (4 ꢂ 10 cell/well) and treated with 10 or 50 lM of samples for 1 h before
stimulated with 100 ng/ml (for RAW 264.7) of lipopolysaccharide. After incubation
for 24 h, an equal volume (50 ll) of the supernatant was blended with Griess Reagent
in a 96-well plate. The mixture was let to react for 15 min, and the release of NO was
measured at 540 nm on a Flexstation 3 and the positive control is parthenolide.
3
.8. MTT assay for cell viability
4
Cells were seeded in 96-well plates at 1 ꢂ 10 cells/well for 24 h, and the cells were
treated with 100 ll of different concentrations of samples (10 or 50 lM) for 24 h.
Subsequently, 10 ll of 5 mg/ml MTT in double distilled water was put in each well,
and the cells were incubated for 4 h. The media was eliminated and resolved with
100 ll/well DMSO. The optical density was measured at 490 nm on a Flexstation 3
(Molecular Devices, Silicon Valley, CA).
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was financially supported by grant from Project of National Sci-Tech Major Special
Item (2018-ZX09731016), Project of Natural Science Foundation of Shanghai (18ZR1436600),

1
2
X.-W. ZHENG ET AL.
and Project of Innovation Foundation of Shanghai Municipal Health Planning Commission
ZYKC201603003).
(
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