Isolation of chemical constituents with anti-inflammatory activity from Reineckia carnea herbs

Article abstract of DOI:10.1080/10286020.2019.1575818

Three new saponins (1–3), a new natural product (4) and six other known compounds (5–10) were isolated from the whole Reineckia carnea plant. Their structures were established by comparison of their NMR spectra and MS data with literature data. In addition, all the isolated compounds were evaluated in vitro for anti-inflammatory activities against LPS-stimulated nitric oxide (NO) production in RAW 264.7 macrophages. Compounds 1–4 exhibited anti-inflammatory activities with IC₅₀ values of 37.5 μM, 31.4 μM, 34.6 μM, and 56.1 μM, respectively. Furthermore, compounds 5–10 showed anti-inflammatory activities with IC₅₀ values ranging from 20.3 to 42.9 μM.

Full text of DOI:10.1080/10286020.2019.1575818

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Isolation of chemical constituents with anti-
inflammatory activity from Reineckia carnea herbs
Xu Xu, Ting Tan, Jing Zhang, Zhi-Feng Li, Shi-Lin Yang, Quan Wen & Yu-Lin
Feng
To cite this article: Xu Xu, Ting Tan, Jing Zhang, Zhi-Feng Li, Shi-Lin Yang, Quan Wen & Yu-
Lin Feng (2019): Isolation of chemical constituents with anti-inflammatory activity from Reineckia
carnea herbs, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2019.1575818
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2019.1575818
Isolation of chemical constituents with anti-inflammatory
activity from Reineckia carnea herbs
a,
ꢀ
b,a,
ꢀ
a
a,b
a,b
a
Xu Xu , Ting Tan
, Jing Zhang , Zhi-Feng Li , Shi-Lin Yang , Quan Wen
a,b
and Yu-Lin Feng
a
The National Pharmaceutical Engineering Center (NPEC) for Solid Preparation in Chinese Herbal
b
Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China; State Key
Laboratory of Innovative Drug and Efficient Energy-Saving Pharmaceutical Equipment,
Nanchang 330006, China
ABSTRACT
ARTICLE HISTORY
Three new saponins (1–3), a new natural product (4) and six
other known compounds (5–10) were isolated from the whole
Reineckia carnea plant. Their structures were established by com-
parison of their NMR spectra and MS data with literature data. In
addition, all the isolated compounds were evaluated in vitro for
anti-inflammatory activities against LPS-stimulated nitric oxide
Received 7 November 2018
Accepted 20 January 2019
KEYWORDS
Reineckia carnea; structure
identification; anti-
inflammatory activity
(
NO) production in RAW 264.7 macrophages. Compounds 1–4
exhibited anti-inflammatory activities with IC50 values of 37.5 mM,
1.4 mM, 34.6 mM, and 56.1 mM, respectively. Furthermore, com-
3
pounds 5–10 showed anti-inflammatory activities with IC50 values
ranging from 20.3 to 42.9 mM.
1. Introduction
Reineckia carnea (Andr.) Kunth (referred to herein as R. carnea), an evergreen medi-
cinal herb known in Chinese as “Jixiangcao,” is widely used for medical treatment in
China and Japan [1, 2]. In China, it has been commonly used by the folk people of
CONTACT Quan Wen
Xu Xu and Ting Tan are the co-first authors.
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2019.1575818.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
qwen12@fudan.edu.cn; Yu-Lin Feng
fengyulin2003@126.com
*

2
X. XU ET AL.
the Miao minority as an antitussive, antidote, anti-arthritic, antipyretic, and hemostatic
medicine [3–5]. Extensive phytochemical investigation of R. carnea revealed the presence
of saponins, flavonoids, volatile oils, alkaloids, and others [6, 7]. Among these com-
pounds, saponins have received the greatest attention because of their pharmacological
properties, including anti-inflammatory and antioxidant activities. R. carnea is also used
as a major ingredient in many herbal composition formulae for the treatment of cough
and bronchitis, such as the Chinese medicine “Kesuting syrup” and “compound R. car-
nea buccal tablets.” In this paper, we describe the isolation from R. carnea and structural
elucidation of compounds 1–10 and their inhibitory activity on LPS-stimulated nitric
oxide (NO) production in RAW 264.7 macrophages (Figure 1).
2. Results and discussion
Compound 1 was obtained as a white amorphous powder. The HR-ESI-MS showed a
ꢁ
negative molecular ion peak at m/z 769.4155 [M-H] , corresponding to a molecular
ꢁ
formula of C H O (calcd 769.4089 [M-H] ), and neutral loss (NL) fragments of
3
9
62 15
1
D-fucose and L-rhamnose (146 Da). The H NMR spectrum of 1 (Table 1) showed the
presence of an olefinic proton at dH 6.61 (1H, dd, J ¼ 1.8, 3.1 Hz, H-16), six methyl
group signals at dH 0.96 (3H, s, H-18), 1.29 (3H, s, H-19), 2.24 (3H, s, H-21), 1.47
0
000
(
3H, d, J ¼ 6.4 Hz, H-Fuc-6 ), 1.67 (3H, d, J ¼ 6.2 Hz, H-Rha-6 ), and 1.75 (3H, d,
00
J ¼ 6.2 Hz, H-Rha-6 ), and three anomeric protons at d 5.01 (1H, d, J ¼ 7.7 Hz,
H
Figure 1. Structures of new compounds 1–4.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
1
13
a
Table 1. H NMR and C NMR spectral data for compound 1.
Aglycone of compound 1
Sugar moieties of compound 1
No.
dc
d
H
No.
dc
d
H
0
1
2
78.2
30.0
4.03 (1H)
2.26 (1H)
Fuc-1
99.7
77.2
75.1
74.4
71.4
17.1
101.6
72.5
72.5
74.2
70.0
19.0
99.6
72.4
72.8
73.5
69.2
18.8
5.01 (1H, d, J ¼ 7.7 Hz)
0
2
3
4
5
4.21 (1H)
0
2
.39 (1H)
4.81 (1H)
4.51 (1H)
3.84 (1H)
0
3
4
70.4
31.1
4.23 (1H)
1.54 (1H)
0
0
1
.88 (1H)
6
1.47 (3H, d, J ¼ 6.4 Hz)
6.44 (1H, br.s)
4.89 (1H)
0
0
5
6
34.8
26.8
2.18 (1H)
1.29 (1H)
Rha-1
0
0
0
0
0
0
0
0
0
0
2
3
4
5
6
1
.70 (1H)
1.03 (1H)
.35 (1H)
4.21 (1H)
4.27 (1H)
4.22 (1H)
7
26.8
1
8
9
1
1
33.6
47.0
39.8
22.2
1.50 (1H)
1.34 (1H)
–
1.75 (3H, d, J ¼ 6.2 Hz)
5.45 (1H, br.s)
4.69 (1H)
000
Rha-1
0
0
0
0
0
00
00
00
00
00
0
1
2
3
4
5
6
1.48 (1H)
4.26 (1H)
4.70 (1H)
4.81 (1H)
2
.16 (1H)
1
2
35.6
1.60 (1H)
2
.48 (1H)
1.67 (3H, d, J ¼ 6.2 Hz)
1
1
1
3
4
5
46.5
56.9
32.4
–
1.38 (1H)
1.90 (1H)
2
.15 (1H)
1
1
1
1
2
2
6
7
8
9
0
1
144.6
155.7
16.3
16.8
196.3
27.1
13
6.61 ( 1H, dd, J ¼ 1.8, 3.1 Hz)
–
0.96 (3H, s)
1.29 (3H, s)
–
2.24 (3H, s)
a1
H NMR and C NMR were measured at 600 MHz and 150 MHz in C
5 5
D N, and the experimental temperature was
2
98 K, and only the multiplicities of not overlapping resonances are given.
0
000
00
13
H-Fuc-1 ), 5.45 (1H, br. s, H-Rha-1 ), and 6.44 (1H, br. s, H-Rha-1 ). The C NMR
spectrum (Table 1) showed 39 carbon signals, including one carbonyl carbon at dC
1
96.3 (C-20) and two olefinic carbons at d 144.6 (C-16) and 155.7 (C-17) that indi-
C
cated the presence of an a,b-unsaturated carbonyl group. In addition, six methyls at
0
00
d 16.3 (C-18), 16.8 (C-19), 27.1 (C-21), 17.1 (C-Fuc-6 ), 18.8 (C-Rha-6 ), and 19.0
C
000 000 0
C-Rha-6 ), and three anomeric carbons at d 99.6 (C-Rha-1 ), 99.7 (C-Fuc-1 ), and
C
01.6 (C-Rha-1 ) also appeared in the C NMR spectrum. When comparing to lit-
(
00
13
1
erature NMR data [8], the resonances of 1 were almost identical to those of (17,20-S-
trans)-5b-pregn-16-en-1b,3b-diol-20-one 1-O-b-D-xylopyranosyl-(1!2)-[a-L-rhamno-
pyranosyl]-3-O-a-L-rhamnopyranoside, with the exception of the identity of the sugar:
according to the second-level HR-ESI-MS, compound 1 has three deoxyhexose sugars.
Moreover, acid hydrolysis (0.1 M HCl) of 1 resulted in the production of D-fucose
and L-rhamnose, as identified by HPLC analysis using an optical rotation detector
[
5
9]. HMBC correlations (Figure 2) of H-18/C-12, C-13, C-14 and C-17, H-19/C-1, C-
, C-9 and C-10, H-1/C-3, C-5, C-10 and C-19, revealed the planar structure of the
aglycone moiety to be pregn-16-en-1,3-diol-20-one. Moreover, HMBC correlations of
0
00
0
000
H-Fuc-1 /C-1, H-Rha-1 /C-Fuc-2 , and H-Rha-1 /C-3 showed that the terminal
rhamnose unit was linked to C-2 of the inner fucose unit, that the fucose unit was
linked at C-1 of the aglycone, and that another rhamnose unit was linked at C-3 of
the aglycone. Additionally, the NOESY spectrum (Figure 2) showed correlations of

4
X. XU ET AL.
1
1
Figure 2. Key H- H COSY, HMBC, and NOESY correlations of compound 1.
H-8b/H-5 and H-19, and H-9a/H-1 and H-3, indicating a-axial configurations of H-1
and H-3, and b-orientations of H-5, H-19, 1-OH, and 3-OH, confirming an A/B cis
ring junction pattern. Furthermore, NOE correlations of H-7a/H-9 and H-7b/H-8,
and H-8/H-18 and H-19, signified a B/C trans ring junction pattern; NOE correla-
tions of H-15b/H-8, and H-14/H-15a and H-16, indicated a C/D trans ring junction
pattern; and NOESY correlations of H-21/H-16 signified that the 16-en-20-one was
in an S-trans configuration. Therefore, the structure of 1 was characterized as (17,20-
S-trans)-5b-pregn-16-en-1b,3b-diol-20-one 1-O-a-L-rhamno pyranosyl-(1!2)-b-D-
fucopyranosyl-3-O-a-L-rhamnopyranoside.
Compound 2 was obtained as a white amorphous powder. The HR-ESI-MS
ꢁ
showed a pseudomolecular ion peak at m/z 791.4679 [M-H] , consistent with the
ꢁ
molecular formula C H O (calcd 791.4660 [M-H] ), and NL fragments of D-
4
3
68 13
fucose (146 Da) and D-glucose (162 Da). The NMR data (Table 2) of 2 revealed eight
proton signals arising from methyl at d 0.74 (3H, s, H-24), 0.76 (3H, s, H-26), 0.88
H
(
3H, s, H-29), 0.98 (3H, s, H-30), 1.00 (3H, s, H-25), 1.28 (3H, s, H-27), 1.31 (3H, d,
0
J ¼ 6.4 Hz, H-Fuc-6 ), and 1.95 (3H, s, 28-CH ), with corresponding carbon signals at
3
d 13.1 (C-24), 17.9 (C-26), 25.6 (C-29), 33.2 (C-30), 19.4 (C-25), 22.4 (C-27), 17.2
C
0
(
C-Fuc-6 ), and 24.8 (28-CH ), respectively (HSQC spectrum). Two vinyl protons at
3
d 6.49 (1H, dd, J ¼ 10.8, 2.9 Hz, H-11) and 5.63 (1H, d, J ¼ 10.8 Hz, H-12), together
H
with the two olefinic groups producing signals at d 127.0 (C-11), 127.3 (C-12), 137.5
C
(
C-13), and 133.0 (C-18), showed that the two double bonds could only be located at
1
1(12)
13(18)
1
1
the D
and D
positions. This was further confirmed by the H- H COSY
cross-peaks between dH 2.05 (H-9) and dH 6.49 (H-11)/5.63 (H-12) and the HMBC
correlations (Figure 3) between the proton signals at dH 1.74, 2.52 (H -19), and dH
2
1
.28 (H-27), and the olefinic quaternary carbon at d 137.5 (C-13). In addition, two
C
0
anomeric protons at d 4.44 (1H, d, J ¼ 7.7 Hz, H-Fuc-1 ) and 4.58 (1H, d, J ¼ 7.7 Hz,
H
00
0
00
H-Glc-1 ) that connected to carbons at d 105.9 (C-Fuc-1 ), 106.0 (C-Glc-1 ) were
C
also present in the NMR spectra. These resonances of 2 were consistent with those of
saikosaponin b2 [10] with the only key exceptions being the absence of a carbon sig-
nal at d 65.3 (C-28), and the presence of two new carbon signals, at d 182.1 and
C
C
2
4.2, and a methyl proton signal at d 1.95. Moreover, the HMBC correlation (Figure
H
3
) between dH 1.95 (CH ) and d 182.1 (C-28) revealed the existence of a -COCH
3
3
C

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
1
13
a
Table 2. H NMR and C NMR spectral data for compound 2.
Aglycone of compound 2
Sugar moieties of compound 2
No.
1
dc
d
H
No.
dc
d
H
0
39.4
1.02 (1H)
.90 (1H)
1.80 (1H)
.97 (1H)
Fuc-1
105.9
71.7
85.5
72.2
71.5
17.2
106.0
75.7
78.2
72.7
78.0
62.7
4.44 (1H, d, J ¼ 7.7 Hz)
0
1
2
3
4
5
3.67 (1H)
0
2
26.5
3.62 (1H)
3.65 (1H)
3.35 (1H)
0
0
1
3
4
5
6
83.7
44.3
48.5
19.2
3.66 (1H)
0
–
6
1.31 (3H, d, J ¼ 6.4 Hz)
4.58 (1H, d, J ¼ 7.7 Hz)
3.30 (1H)
00
1.27 (1H)
1.02 (1H)
.49 (1H)
1.33 (1H)
.48 (1H)
Glc-1
0
0
0
0
0
0
0
0
0
0
2
3
4
5
6
1
3.38 (1H)
3.87 (1H)
3.29 (1H)
3.69 (1H)
7
33.2
1
8
9
42.2
55.1
37.6
127.0
127.3
137.5
42.7
–
2.05 (1H, d , J ¼ 4.2 Hz)
3.85 (1H)
10
11
12
13
14
15
–
6.49 (1H, dd, J ¼ 10.8, 2.9Hz )
5.63 (1H, d, J ¼ 10.8 Hz)
–
–
32.3
1.44 (1H)
1
.95 (1H)
1
1
1
1
6
7
8
9
69.3
45.7
133.0
39.7
4.07 (1H)
–
–
1.74 ( 1H, d, J ¼ 13.5 Hz)
2
.52 ( 1H, d, J ¼ 13.5 Hz)
2
2
0
1
33.5
36.1
–
1.32 (1H)
1
.56 (1H)
1.59 (1H)
.04 (1H)
3.30 (1H)
.68 (1H)
2
2
2
3
24.2
65.3
2
3
2
2
2
2
2
2
3
2
4
5
6
7
8
9
0
8-CH
13.1
19.4
17.9
22.4
182.1
25.6
33.2
24.8
13
0.74 (3H, s)
1.00 (3H, s)
0.76 (3H, s)
1.28 (3H, s)
–
0.88 (3H, s)
0.98 (3H, s)
1.95 (3H, s)
3
a1
H NMR and C NMR were measured at 600 MHz and 150 MHz in CD
3
OD, the experimental temperature was 298
K, and the only the multiplicities of not overlapping resonances are given.
group, which connected to C-17. Meanwhile, NOESY correlations (Figure 3) of H3-
4/H -25b, H-5a/H-3, and H-15b/H-16 indicated that 16-OH was in an a-axial con-
2
3
figuration and that H-24 and 3-OH were in b-orientations. Acid hydrolysis (0.1 M
HCl) of 2 afforded D-glucose and D-fucose, which were identified by HPLC equipped
with an optical rotation detector [9]. Thus, compound 2 was characterized as
3
b,16a,23-trihydroxy-11,13(18)-dien-28-methylketone-oleane 3-O-b-D-glucopyranosyl-
(
1!3)-b-D-fucopyranoside.
Compound 3 was obtained as a white amorphous solid. The HR-ESI-MS showed a
ꢁ
pseudomolecular ion peak at m/z 871.4838 [M-H] , consistent with the molecular
formula C H O (calcd 871.4770 [M-H] ), and NL fragments of L-rhamnose
ꢁ
4
4
72 17
1
(146 Da) and D-xylose (132 Da). The H NMR spectrum (Table 3) of 3 showed six
methyl groups, of which two were singlet signals (dH 0.71 and 1.35) and four were

6
X. XU ET AL.
1
1
Figure 3. Key H- H COSY, HMBC, and NOESY correlations of compound 2.
doublet signals (d 1.02, 1.15, 1.72, and 1.80), and three anomeric protons at d 6.53
H
H
0
0
000
(
1
1H, br. s, H-Rha-1 ), 5.47 (1H, br. s, H-Rha-1 ), and 5.09 (1H, d, J ¼ 6.3 Hz, H-Xyl-
0
13
). The C NMR spectrum (Table 3) showed 44 carbon signals, including two car-
bonyl carbons at d 213.3 and 217.9, and three anomeric carbons at d 99.9 (C-Rha-
C
C
000
0
0
0
1
), 100.2 (C-Xyl-1 ), and 101.7 (C-Rha-1 ). HMBC correlations (Figure 4) were
observed from dH 0.71 (H-18) to d 39.3 (C-12)/41.8 (C-13) and 66.6 (C-17), from
C
dH 1.02 (H-21) to d 43.8 (C-20), 66.6 (C-17) and 213.3 (C-22), and from d 2.76
C
H
(
H-17) to d 13.1 (C-18)/213.3 and 217.9. Moreover, the DEPT-135 spectrum showed
C
that the peak at d 67.5 was downfield, implying that the carbonyl signal at d 213.3
C
C
arose from C-22 and the one at d 217.9 from C-16. In addition, correlations in the
C
0
00
0
HMBC spectrum (Figure 4) of H-Xyl-1 /C-1 and H-Rha-1 /C-Xyl-2 showed that the
0
terminal rhamnose unit was linked at C-2 of the inner xylose unit, which was linked
at C-1 of the aglycone. Furthermore, the correlation between H-Rha-1 and C-3 indi-
cated that another rhamnose unit was linked at C-3 of the aglycone. Acid hydrolysis
000
(0.1 M HCl) of 3 released L-rhamnose and D-xylose, which were identified by HPLC
analysis using an optical rotation detector [9]. NOESY correlations of H-8b/H-5 and
H-19, and H-9a/H-1 and H-3, indicated a-configurations for H-1 and H-3, and b-ori-
entations for H-5, H-19, 1-OH and 3-OH, confirming an A/B cis ring junction pat-
tern. Moreover, correlations of H-17/H-21 and H-14a suggested a-configurations for
H-17 and H-21. Therefore, the structure of compound 3 was characterized as
1
b,3b,26-trihydroxy-16,22-dioxo-cholestane
1-O-a-L-rhamnopyranosyl-(1!2)-b-D-
xylopyranosyl-3-O-a-L-rhamnopyranoside.
Compound 4 was obtained as a white amorphous powder. The molecular formula
was deduced as C H O from the HR-ESI-MS result, which showed a positive
2
1
32 3
þ
1
molecular ion peak at m/z 333.2434 [M þ H] . The H NMR spectrum (Table 4) of 4
showed three methyls at d 0.95 (3H, s), 1.33 (3H, s) and dH 2.26 (3H, s) and the
H
presence of an olefinic proton at d 6.63 (1H, dd, J ¼ 1.6, 3.0 Hz, H-16) . The DEPT
H
1
3
and C NMR (Table 4) spectrum displayed 21 carbon signals, including 3 methyls, 7
methylenes, 7 methines, and 4 quaternary carbons. Among them, three methyl carbon

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
1
13
a
Table 3. H NMR and C NMR spectral data for compound 3.
Aglycone of compound 3
Sugar moieties of compound 3
No.
dc
d
H
No.
dc
d
H
0
1
2
79.2
31.5
4.08 (1H)
2.33 (1H)
Xyl-1
100.2
77.0
79.6
71.4
67.1
5.09 (1H, d, J ¼ 6.3 Hz)
0
2
3
4
4.29 (1H)
0
2
.42 (1H)
4.28 (1H)
4.27 (1H)
3.67 (1H)
0
3
4
71.0
31.8
4.22 (1H)
1.63 (1H)
0
5
1
.87 (1H)
4.37 (1H)
6.53 (1H, br.s)
4.85 (1H)
4.67 (1H)
4.28 (1H)
0
0
5
6
35.7
26.2
1.98 (1H)
1.31 (1H)
Rha-1
101.7
72.6
72.8
74.3
69.5
19.1
99.9
72.5
72.5
74.1
70.1
18.7
0
0
0
0
0
0
0
0
0
0
2
3
4
5
6
1
.54 (1H)
1.78 (1H)
.23 (1H)
7
27.3
2
4.85 (1H)
8
9
1
1
33.4
46.7
39.6
22.6
1.39 (1H)
1.44 (1H)
–
1.49 (1H)
2
1.80 (3H, d, J ¼ 6.1 Hz)
5.47 (1H, br.s)
4.85 (1H)
000
Rha-1
0
0
0
0
0
00
00
00
00
00
0
1
2
3
4
5
6
4.67 (1H)
4.35 (1H)
4.42 (1H)
.21 (1H)
1
2
39.3
1.49 (1H)
1
.89 (1H)
–
1.72 (3H, d, J ¼ 6.1 Hz)
1
1
1
3
4
5
41.8
51.6
40.3
1.38 (1H)
2.90 (1H)
3
.00 (1H)
16
17
18
19
20
21
22
23
217.9
66.6
13.1
16.7
43.8
15.7
213.3
37.5
–
2.76 (1H, d, J ¼ 10.6 Hz)
0.71 (3H, s)
1.35 (3H, s)
2.72 (1H)
1.02 (3H, d, J ¼ 6.5 Hz)
–
1.71 (1H)
2
.08 (1H)
1.78 (1H)
.23 (1H)
1.98 (1H)
3.74 (1H)
2
4
27.7
2
2
2
5
6
36.2
67.5
3
.81 (1H)
2
a1
7
17.3
1.15 (3H, d, J ¼ 6.7 Hz)
13
H NMR and C NMR were measured at 600 MHz and 150 MHz in C
5
D
5
N, the experimental temperature was 298 K,
and only the multiplicities of not overlapping resonances are given.
signals at d 16.1, 19.2, and 26.6 were ascribed to C-18, C-19, and C-21, and one car-
C
bonyl carbon at dC 196.2 (C-20) and two olefinic carbons at dC 144.6 (C-16) and
1
55.3 (C-17) indicated the presence of an a,b-unsaturated carbonyl moiety [11].
HMBC correlations (Figure 5) of H-19/C-1, C-5, C-9 and C-10, H-18/C-12, C-13, C-
4 and C-17, H-1/C-3, C-5, C-10 and C-19, H-3/C-2 and C-4, H-6/C-4,C-7, C-8 and
1
C-10, H-8/C-9, C-11 and C-14, H-16/C-13, C-14, C-15, C-17 and C-20, H-21/C-17
and C-20, revealed the planar structure of compound 4 as pregn-16-en-1,3-diol-20-
one. Meanwhile, the NOESY correlations (Figure 5) of H-8b/H-5 and H-19, H-8b
and H-18, H-9a/H-1 and H-3, indicated a-axial configurations of H-1 and H-3, and
b-orientation of H-5, H-8, H-18, H-19, 1-OH and 3-OH, which supported the A/B
cis ring junction pattern. Therefore, the structure of 4 was characterized as 5b-pregn-
16-ene-1b,3b-diol-20-one, as a natural product [12].
Additionally, two known triterpenoid saponins and four flavones were identified,
by comparing their spectra (NMR and MS) and physicochemical data with those

8
X. XU ET AL.
1
1
Figure 4. Key H- H COSY, HMBC, and NOESY correlations of compound 3.
1
13
a
Table 4. H NMR and C NMR spectral data for compound 4.
Compound 4
No.
dc
d
H
1
2
73.1
34.4
4.00 (1H)
2.07 (1H)
2
.10 (1H)
3
4
68.1
34.4
4.39 (1H)
1.40 (1H)
1
.61 (1H)
5
6
31.3
26.2
1.90 (1H)
1.38 (1H)
1
.53 (1H)
1.18 (1H)
.29 (1H)
7
27.0
1
8
9
1
1
32.8
42.5
40.4
20.9
1.76 (1H)
1.33 (1H)
–
0
1
1.53 (1H)
2
.24 (1H)
12
35.4
1.41 (1H)
2
.60 (1H)
1
1
1
3
4
5
46.3
56.2
32.2
–
1.39 (1H)
1.89 (1H)
.16 (1H)
2
1
1
1
1
2
2
6
7
8
9
0
1
144.6
155.3
16.1
19.2
196.2
26.6
6.63 (1H, dd, J ¼ 1.6, 3.0 Hz)
–
0.95 (3H, s)
1.33 (3H, s)
–
2.26 (3H, s)
a1
13
5 5
H NMR and C NMR were measured at 600 MHz and 150 MHz in C D N, the experimental tem-
perature was 298 K, and only the multiplicities of not overlapping resonances are given.
reported in the literature, as prosaikogenin D (5) [13], saikosaponin b2 (6) [10], iso-
rhamnetin-3-O-rutinoside (7) [14], farrerol (8) [15], luteolin (9) [16], and 1,2,8-trihy-
droxy-5,6-dimethoxyxanthone (10) [17] (Figure 1).
All isolated compounds were evaluated for their inhibitory activity on LPS-induced
NO production in RAW 264.7 macrophages. Compounds 1–4 showed IC50 values of

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
1
1
Figure 5. Key H- H COSY, HMBC, and NOESY correlations of compound 4.
3
7.5 mM, 31.4 mM, 34.6 mM, and 56.1 mM, respectively. Compounds 5–10 also
showed inhibitory activities with IC50 values ranging from 20.3 to 42.9 mM (Table 5,
Figure 6). These results showed that the steroids, saponins, and flavones isolated
from R. carnea all had anti-inflammatory activities consistent with the literature
data [18–21].
3
. Experimental
.1. General experimental procedures
The IR spectra were recorded on a TENSOR-27 instrument (Bruker, Karlsruhe,
3
TM
þ
Germany). The HR-ESI-MS was measured on a Triple TOF
5600 system with a
Duo Spray source (AB SCIEX, Foster City, CA, USA). 1D and 2D NMR spectra were
recorded on a AVANCE600 instrument (Bruker, Karlsruhe, Germany) with TMS as
an internal standard. Optical rotations were recorded on a Model 343 polarimeter
(PerkinElmer, Massachusetts, USA). The analytical HPLC was performed on a HPLC
system (Shimadzu, Kyoto, Japan), and separation was performed on a Cosmosil C18
column (4.6 ꢂ 250 mm, 5.0lm). Semipreparative HPLC (Waters, Massachusetts, USA)
was performed on a system comprising a Waters 550 pump equipped with a Waters
2
487 detector and a Cosmosil C18 column (10 mm ꢂ 250 mm, 5.0 lm, Kyoto, Japan).
Sephadex LH-20 gel was purchased from GE Healthcare Bio-Sciences AB (GE,
Uppsala, Sweden). HP20 macroporous resin ODS gel (MeOH as mobile phases) was
purchased from Mitsubishi Chemical Corporation (Mitsubishi, Tokyo, Japan). Silica
gel was purchased from Qingdao Haiyang Chemical Group Corporation (Qingdao,
China). D-fucose (140651-201504), L-rhamnose (111683-201502), D-glucose (111833-
201506), and D-xylose (111508-201605) were purchased from National Institute for
Food and Drug Control (Beijing, China).
3
.2. Plant material
Reineckia carnea was collected on March in 2014 from Anshun city of Guizhou
Province, and identified by Vice Director of Pharmacists Bei Wu, Nanchang Institute
for Food and Drug Control. A voucher specimen (No. Z-140310-01) has been depos-
ited at Jiangxi University of Traditional Chinese Medicine, Nanchang, China.

1
0
X. XU ET AL.
Table 5. IC50 values of compounds 1–10 on LPS-stimulated NO production in RAW
264.7 macrophages.
Compounds
IC50 (lM)
Compounds
IC50 (lM)
1
2
3
4
37.5 ± 3.9
31.4 ± 3.1
34.6 ± 4.3
56.1 ± 7.6
42.9 ± 6.3
20.7 ± 2.8
6
7
8
9
10
30.7 ± 3.2
20.3 ± 2.7
27.7 ± 4.6
25.4 ± 4.2
22.9 ± 3.4
5
Quercetina
a
Quercetin was used as the positive control.
Figure 6. The dose-response curve of NO production of compounds 1–10.
3.3. Extraction and isolation
The air-dried whole plant of R. carnea (20 kg) was powdered and extracted with
ꢃ
8
0% EtOH under reflux for three times at 80 C. After removing the solvent, the
concentrated residue was successively partitioned with petroleum ether, EtOAc
and n-BuOH. The EtOAc extract (305 g) was subjected to column chromatography
(
CC) on silica gel, eluting with gradient solvent system (CH Cl -MeOH, 1:0–0:1)
2 2
to give seven fractions (E-1ꢄE-7). E-6 was subjected to CC on MCI gel, and fur-
ther separated by semipreparative HPLC (MeCN/H O ¼ 23%/77%; 2.0 ml/min;
2
2
2
03 nm) to obtain compound 1 (10.0 mg; t ¼ 22 min), compound 2 (5.3 mg; t ¼
R
R
5 min), compound 5 (2.7 mg; t ¼ 36 min), compound 6 (6.4 mg; t ¼ 20 min).
R
R
The n-BuOH extract (812 g) was subjected to HP-20 macroporous resin column,
and the 60% EtOH eluate (241 g) was separated by silica gel CC, eluting with gra-
dient solvent system (CH Cl -MeOH, 20:1–0:1) to obtain five fractions (S-1ꢄS-5),
2
2
and fraction S4 was further separated by semipreparative HPLC (MeOH/H O ¼
2
3
2%/68%; 2.0 ml/min, 254 nm) to yield compound 7 (4.8 mg; t ¼ 32 min),
R
compound 3 (5.3 mg; t ¼ 27 min), compound 4 (4.6 mg; t ¼ 34 min). The 95%
R
R
EtOH eluate (79 g) was separated by ODS gel CC to yield four fractions (O-1ꢄO-
4
2
), and fraction O3 was further separated by semipreparative HPLC (2.0 ml/min,
54 nm) with MeCN-H O (13:87) as mobile phase to give compound 9 (13.4 mg;
2
t ¼ 26 min), compound
8
(2.8 mg; tR ¼ 28 min), compound 10 (3.7 mg;
R
t ¼32 min).
R

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
3
(
.3.1. (17,20-S-trans)-5b-Pregn-16-en-1b,3b-diol-20-one 1-O-a-L-rhamnopyranosyl-
1!2)-b-D-fucopyranosyl-3-O-a-L-rhamnopyranoside (1)
A white amorphous powder; [a] -46.3 (c 0.15, MeOH); IR (KBr) vmax: 3413, 2918,
2
0
D
ꢁ
1
1
13
1665, 1459, 1074 cm ; H NMR (600 MHz, in C D N) and C NMR (150 MHz, in
5 5
ꢁ
C D N) spectral data, see Table 1; HRESIMS: m/z 769.4155 [M-H] (calcd for
5
5
C H O , 769.4089).
3
9
61 15
3
.3.2. 3b,16a,23-trihydroxy-11,13(18)-dien-28-methylketone-oleane 3-O-b-D-gluco-
pyranosyl-(1!3)-b-D-fucopyranoside (2)
A white amorphous powder; [a] -29.5 (c 0.12, MeOH); IR (KBr) vmax: 3389, 2921,
2
0
D
ꢁ
1
1
13
2
883, 1728, 1072 cm ; H NMR (600 MHz, in CD OD) and C NMR (150 MHz, in
3
ꢁ
CD OD) spectral data, see Table 2; HRESIMS: m/z 791.4679 [M-H] (calcd for
3
C H O , 791.4660).
4
3
67 13
3
(
.3.3.
1!2)-b-D-xylopyranosyl-3-O-a-L-rhamnopyranoside (3)
A colorless amorphous solid; [a] -36.6 (c 0.12, MeOH); IR (KBr) vmax: 3410, 2918,
1b,3b,26-Trihydroxy-16,22-dioxo-cholestane
1-O-a-L-rhamnopyranosyl-
2
0
D
ꢁ
1
1
13
1667, 1652, 1070 cm ; H NMR (600 MHz, in C D N) and C NMR (150 MHz, in
5 5
ꢁ
C D N) spectral data, see Table 3; HRESIMS: m/z 871.4838 [M-H] (calcd for
5
5
C H O , 871.4770).
4
4
71 17
3
.3.4. 5b-Pregn-16-ene-1b,3b-diol-20-one (4)
2
0
A white amorphous powder; [a] þ27.4 (c 0.13, MeOH); IR (KBr) vmax: 3413, 2922,
D
ꢁ
1
1
13
1664, 1456, 1114 cm ; H NMR (600 MHz, in C D N) and C NMR (150 MHz, in
5 5
þ
C D N) spectral data, see Table 4; HRESIMS: m/z 333.2434 [M þ H] (calcd for
5
5
C H O , 333.2385).
2
1
33 3
3
.4. Acid hydrolysis of compounds (1–3)
A solution of each of 1–3 (each 3.0 mg) in 2 ml HCl (0.1 M HCl) was refluxed at
ꢃ
8
0 C for 3 h. After cooling, the reaction mixture was extracted with EtOAc (2 ml ꢂ
3
) to remove the aglycone. The aqueous layer was purified by CC over silica gel
eluted with EtOAc–MeOH–H O (6:1:1) to yield sugar, which was then compared
2
with an authentic sugar sample by co-TLC (EtOAc–MeOH–H O, 6:1:1, R 0.12 for
2
f
xylose, R 0.16 for rhamnose, R 0.28 for glucose, R 0.42 for fucose). On the basis of
f
f
f
the acid hydrolysate of 1–3, D-xylose, L-rhamnose, D-glucose, and D-fucose were con-
firmed by comparison of optical rotation of the liberated sugar with that of an
authentic sample [D-xylose, [a]20 D þ28.8 (c 0.02, H O); L-rhamnose, [a]20 D
2
þ12.4(c 0.15, H O); D-glucose, [a]20 D þ55.2 (c 0.05, H O); D-fucose, [a]20 D þ75.6
2
2
(
c 0.08, H O)].
2
3.5. Analysis of NO production
5
RAW 264.7 macrophages were seeded into 96-well plates with 2 ꢂ 10 cells/well for
4 h and then pre-incubated with different concentrations of compounds for 1 h
2

1
2
X. XU ET AL.
before stimulation with or without LPS (1 lg/ml) for 24 h. The NO concentration in
culture medium was determined by Griess reagent kit. The absorbance at 545 nm was
measured using a multifunction microplate reader (Molecular Devices, Flex Station 3)
[
22]. Sodium nitrite was used as a standard to calculate the nitrite concentration.
Inhibition (%) ¼ (1ꢁ(A
ꢁ Auntreated)/(ALPSꢁA
)) ꢂ 100. The experi-
LPS þ sample
untreated
ments were performed in triplicates, and the data were expressed as mean ± SD.
Quercetin was used as a positive control [23].
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by the National Natural Science Foundations of China
[
grant No. 81860684]; Jiangxi Province key R&D Projects [grant No. 20165BCB19009]; The
Health and Family Planning Commission of Jiangxi Province [grant Nos.
017A291, 20183009].
2
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