Diterpenoid glycosides and monoterpenoid glycosides from the fruits of Xanthium chinense

Article abstract of DOI:10.1080/10286020.2018.1526788

Two new diterpenoid glycosides, fructusnoids D (1) and E (2), and two new monoterpenoid glycosides (3, 4), together with three known diterpenoid glycosides (5-7) and three known monoterpenoid glycosides (8-10), were isolated from the fruits of Xanthium chinense. Their structures were elucidated by spectrometric analyses. (Figure presented.).

Full text of DOI:10.1080/10286020.2018.1526788

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Diterpenoid glycosides and monoterpenoid
glycosides from the fruits of Xanthium chinense
Yan Cheng, Jiang Fu, Long Chen, Lu-Lu Li & Jing Qu
To cite this article: Yan Cheng, Jiang Fu, Long Chen, Lu-Lu Li & Jing Qu (2018): Diterpenoid
glycosides and monoterpenoid glycosides from the fruits of Xanthiumꢀchinense, Journal of Asian
Natural Products Research, DOI: 10.1080/10286020.2018.1526788
To link to this article: https://doi.org/10.1080/10286020.2018.1526788
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2018.1526788
Diterpenoid glycosides and monoterpenoid glycosides
from the fruits of Xanthium chinense
Yan Cheng, Jiang Fu, Long Chen, Lu-Lu Li and Jing Qu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia
Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
ABSTRACT
ARTICLE HISTORY
Received 11 July 2018
Accepted 18 September 2018
Two new diterpenoid glycosides, fructusnoids D (1) and E (2), and
two new monoterpenoid glycosides (3, 4), together with three
known diterpenoid glycosides (5-7) and three known monoterpe-
noid glycosides (8-10), were isolated from the fruits of Xanthium
chinense. Their structures were elucidated by spectromet-
ric analyses.
KEYWORDS
Compositae; Xanthium
chinense; diterpenoid
glycoside; monoterpe-
noid glycoside
1. Introduction
The genus Xanthium in the family Compositae contains approximately 25 species
distributed throughout northern and central America, Europe, Asia, and northern
Africa. Chemical investigations of the genus have resulted in the isolation of sesqui-
terpenoids, diterpenoid glycosides, flavonoids, lignans, thiazinediones, and fatty oils.
The fruits of the genus Xanthium are commonly used in clinical medicine for the
treatment of rheumatism, headache, stuffy nose, runny nose, nasosinusitis, etc. [1].
Xanthium has a certain toxicity, and excessive use or improper processing can lead to
poisoning and even death [2–5]. Studies have shown that the main toxic components
in the genus Xanthium are two water-soluble glycosides, carboxyatractyloside and
atractyloside, which were controlled by limiting in the Chinese Pharmacopoeia [6–8].
CONTACT Jing Qu
qujing@imm.ac.cn
State Key Laboratory of Bioactive Substance and Function of Natural
Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, China.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
Y. CHENG ET AL.
Figure 1. Structures of compounds 1–10.
X. chinense Mill. is a widely distributed species in China. To extend the resources
of Xanthium in clinical medicine and make use of Xanthium more safely, a study on
the n-BuOH extraction of the fruits of X. chinense was carried out. Two new diter-
penoid glycosides, fructusnoids D (1) and E (2), and two new monoterpenoid glyco-
sides (3, 4), together with three known diterpenoid glycosides (5-7) and three known
monoterpenoid glycosides (8-10), were isolated from the fruits of X. chinense
(Figure 1). Herein, we report the isolation and structure elucidation of the new diter-
penoid glycosides and monoterpenoid glycosides.
2. Results and discussion
Compound 1 was obtained as a white powder. The HRESIMS [M-H]- ion at m/z
659.2750 indicated a molecular formula of C31H48O13S, suggesting the presence of a
1
sulfate group. The H NMR spectrum (Table 1) proved the presence of three methyl
groups at dH 1.11 (3H, s, H-20), 1.06 (3H, d, J ¼ 5.6 Hz, H-600), 0.85 (3H, t, J ¼ 6.4 Hz,
H-500) and two olefinic protons at dH 5.47 (1H, s, H-17a) and 5.17 (1H, s, H-17b).
The 13C NMR data (Table 2) indicated two carbonyls (dC 178.1, 173.1), two olefinic
carbons (dC 161.2, 108.2), eight oxygenated carbons (dC 100.7, 82.9, 82.1, 78.2, 73.5,
73.0, 71.3, 62.3), two sp3 quaternary carbons (dC 48.5, 41.3), five sp3 methines (dC
53.6, 49.9, 44.5, 43.1, 32.3), nine sp3 methylenes (dC 48.2, 42.4, 36.9, 36.2, 35.6, 33.1,
29.9, 26.7, 18.8), and three methyls (dC 19.9, 17.3, 11.7). These data implied that the
basic skeleton of compound 1 was a diterpene glycoside with an additional substitu-
1
ent group [9]. The correlations in the H-1H COSY spectrum (Figure 2(a)) suggested
the existence of a sugar and 3-methylpentanoyl [10] in compound 1, as did the
HMBC correlations from H-200 to C-100 (dC 173.1), C-300 (dC 32.3), and C-400 (dC
29.9); from H-600 to C-200 (dC 42.4) and C-400; and from H-500 to C-300 and C-400. The
3-methylpentanoyl group and sulfonic group were attached to C-20 and C-30, respect-
ively, indicated by the HMBC correlations from H-20 to C-100, together with the
1
downfield shifts of H-30 (dH 5.32) and C-30 (dC 82.1). The rest of the H-1H COSY
correlations established two spin systems C (1) H2- C (2) H- C (3) H2- C (4) H- C

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1H NMR spectral data of compounds 1, 2, 5-7 (d in ppm, J in Hz).
dH
5b
No.
1
1a
2b
6b
7a
2.49–2.51, m
1.00–1.02, m
4.82–4.84, m
2.93–2.95, m
1.55, overlap
2.86–2.88, m
1.55, overlap
2.31–2.33, m
1.91, d (12.0)
2.16, overlap
1.81, overlap
1.11, overlap
1.47, overlap (2H)
2.33–2.35, m
0.81, t (12.0)
4.16–4.18, m
2.63–2.65, m
1.30, overlap
2.31, dd (12.6,3.6)
0.76, t (12.0)
4.21–4.23, m
2.38–2.40, m
1.20–1.22, m
2.65–2.67, m
1.42–1.51, overlap
1.93–1.95, m
1.63 overlap
2.23–2.25, m
0.69, t (12.0)
4.44–4.46, m
2.42–2.45, m
1.07–1.09, m
2.46–2.48, m
1.30–1.32, m
1.98–2.00, m
1.69–1.71, m
1.93–1.95, m
1.35–1.37, m
1.00, d (7.8)
1.60–1.62, m
1.47–1.49, m
1.62–1.64, m
1.44–1.46, m
2.68–2.70, m
1.65–1.67, m
1.40–1.42, m
3.75, br s
2.49, d (11.2)
1.00 , overlap
4.81–4.83, m
2.93, d (11.2)
1.51–1.53, m
2.85–2.87, m
1.53–1.55, m
2.31–2.33, m
1.91, d (12.0)
2.16, d (13.6)
1.79–1.81, m
1.11, overlap
2
3
4
5
6
1.80–1.82, m
1.85–1.87, m
1.67, overlap
1.67, overlap
1.44–1.46, m
1.12, d (7.8)
1.64, overlap
1.49, overlap
1.64 , overlap
1.49, overlap
2.70–2.72, m
2.87–2.89, m
1.38–1.40, m
3.78, br s
7
1.67–1.69, m
1.42–1.51, overlap
1.05 d (7.8)
9
11
1.63, overlap
1.42–1.51, overlap
1.63, overlap
1.42–1.51, overlap
2.70–2.72, m
1.87, d (12.0)
1.39, dd (11.4, 4.8)
3.76, s
1.45–1.47, m (2H)
12
1.47, overlap
1.38–1.40, m
2.68–2.70, m
1.81, overlap
1.59–1.61, m
4.10, br s
1.42–1.44, m
1.38–1.40, m
2.68–2.70, m
1.81–1.83, m
1.59–1.61, m
4.10, br s
13
14
15
17
5.46–5.48, s
5.18, s
5.18, s
5.17, s
5.48, s
5.17, s
1.11, s (3H)
5.08, s
1.01, s (3H)
4.69, d (7.8)
4.77, dd (9.0, 7.8)
4.40, t (9.0)
5.07, s
0.99, s (3H)
4.60, d (7.8)
4.68, dd (9.6, 8.4)
3.50, t (9.0)
3.38, t (9.0)
3.33, overlap
3.86, dd (12.0, 2.4)
3.70, dd(12.0,5.4)
2.26, d (2H, 6.6)
5.06, s
5.17, overlap
1.11, s (3H)
20
1’
2’
3’
4’
5’
6’
1.08, s (3H)
4.83, overlap
4.83, overlap
4.56, t (9.6)
4.34, t (9.6)
3.51–3.53, m
3.90, d (2H, 3.0)
5.21, d (7.2)
5.60–5.62, m
5.31–5.33, m
4.33–4.35, m
3.83–3.85, m
4.25–4.37, m
4.20–4.22, m
2.74–2.76, m
2.52–2.54, m
2.16, overlap
1.55, overlap
1.22–1.24, m
0.85, t (3H, 6.4)
1.06, d (3H, 5.6)
5.17, overlap
5.60–5.62, m
5.31–5.33, m
4.29–4.31, m
3.83–3.85, m
4.35, d (10.4)
4.21, dd (12.0, 5.6)
2.59–2.61, m (2H)
3.61, t (9.6)
3.39–3.41, m
3.86, dd (12.0, 2.4)
3.71, dd (12.0, 5.4)
2.37–2.39, m
2.17–2.19, m
1.85–1.87, m
1.40–1.42, m
1.20–1.22, m
0.90, t (3H, 7.2)
0.94, d (3H, 6.6)
2’’
2.27 d (2H, 7.2)
3’’
4’’
2.10, sep
0.98, d (3H, 7.2)
2.09, sep
0.96, d (3H, 6.6)
2.36–2.38, m
1.05, d (3H, 5.6)
5’’
6’’
0.98, d (3H, 67.2)
0.96, d (3H, 6.6)
1.02, d (3H, 5.6)
aData were measured at 800 MHz for 1H and 200 MHz for 13C in C5D5N; bData were measured at 600 MHz for H and
1
150 MHz for 13C in CD3OD.
(5) H- C (6) H2- C (7) H2 and C (9) H- C (11) H2- C (12) H2- C (13) H- C (14) H2
(Figure 2(a)). Then, the ent-kaurane skeleton of the aglycon was confirmed by the
HMBC correlations from H-6, H-11, and H-13 to C-8 (dC 48.5); from H-1, H-4, H-5,
and H-11 to C-10 (dC 41.3); from H-17 to C-13 (dC 43.1) and C-15 (dC 82.9); and
from H-15 to C-9 (dC 53.6), C-13, and C-14 (dC 36.9). C-18 was oxidized to a carb-
oxyl group and C-19 was degraded, as suggested by the downfield shifted carbon of
C-4 (dC 44.5) together with the HMBC correlations from H-3, H-4, and H-5 to C-18
(dC 178.1). One hydroxyl group was attached to C-15, indicated by the downfield
shift of C-15. The HMBC correlation from H-2 to C-10 indicated that the sugar moi-
ety was located at C-2. Then, the planar structure of compound 1 was elucidated.
The configuration of compound 1 was elucidated based on NOE correlations
(Figure 2(b)). The presence of the NOE correlation H-2/H-20 and the absence of H-
4/H-2 and H-4/H-20 correlations indicated the relative configurations at C-2, C-4,
and C-10, and the NOE correlations H-5/H-9, H-9/H-15, H-13/H-14a, and H-13/H-

4
Y. CHENG ET AL.
Table 2. 13C NMR spectral data of compounds 1, 2, 5-7 (d in ppm).
dC
No.
1a
2b
5b
6b
7a
1
2
3
4
5
6
7
8
48.2
73.5
35.6
44.5
49.9
26.7
36.2
48.5
53.6
41.3
18.8
33.1
43.1
36.9
82.9
161.2
108.2
178.1
48.4
73.8
41.2
59.1
52.2
24.1
36.2
49.6
55.0
41.4
19.3
33.6
43.6
37.3
83.4
160.3
109.1
175.2
175.5
17.8
101.0
72.6
82.8
70.9
77.6
62.3
173.8
42.6
33.0
30.5
11.7
19.8
48.6
74.1
35.7
44.6
50.4
26.6
36.2
49.6
54.4
41.8
19.2
33.6
43.7
37.2
83.5
160.3
109.0
178.8
48.9
74.5
37.2
47.6
51.2
26.8
37.2
48.9
54.7
41.9
19.3
33.6
43.8
36.4
83.7
160.6
108.8
182.6
48.4
73.8
35.9
44.7
50.0
26.9
36.4
48.7
53.8
41.5
19.0
33.3
43.3
37.1
83.1
161.5
108.4
178.3
9
10
11
12
13
14
15
16
17
18
19
20
1’
17.3
100.7
73.0
82.1
71.3
78.2
62.3
173.1
42.4
32.3
29.9
11.7
19.9
17.2
101.1
75.1
76.3
71.7
77.9
62.6
173.8
44.4
26.8
22.9
22.9
17.5
100.1
73.4
80.4
75.3
76.1
62.3
174.2
44.5
26.4
23.1
23.0
17.5
101.0
73.2
82.3
71.4
78.5
62.5
173.1
44.5
26.2
23.3
23.3
2’
3’
4’
5’
6’
1’’
2’’
3’’
4’’
5’’
6’’
aData were measured at 800 MHz for 1H and 200 MHz for 13C in C5D5N, bData were measured at 600 MHz for H and
1
150 MHz for 13C in CD3OD.
Figure 2. (a) Key HMBC and COSY correlations of 1. (b) Key NOESY correlations of 1.
14b indicated the relative configurations at C-5, C-9, C-15, C-8, and C-13, as well as
the chair conformation of ring C. The anomeric proton signal (dH 5.21, d, J ¼ 7.2 Hz)
indicated that the sugar of compound 1 was attached via a b-linkage. Basic and acid

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 3. 1H NMR (C5D5N, 800MHz) spectral data of compounds 3, 4, 8-10 (d in ppm, J in Hz).
dH
No
1
3
4
8
9
10
4.50, d (12.0)
4.29, overlap
4.54, overlap
5.40–5.42, m
4.66, dd (11.2, 6.4) 4.63, dd (11.2, 6.4) 2.12–2.14, m
4.47. dd (11.2, 8.0) 4.47 dd (11.2, 8.0)
2
3
4
5.57–5.59, m
5.56–5.58, m
5.65–5.67, m
2.22–2.26,
overlap (2H)
5.42–5.44, m
2.40–2.42, m
5.60–5.62, m
2.27–2.29, m
2.21–2.23, m
2.00–2.02, m (2H)
3.32, overlap
2.32, overlap
2.13, overlap
2.13, overlap (2H)
2.21–2.23, m
1.94–1.96, m
5
6
2.22–2.26,
overlap (2H)
2.06–2.08, m (2H)
5.14–5.16, m
1.65, s (3H)
2.25–2.27, m
5.20–5.22, m
2.34–2.36, m
1.21–1.23, m
7
8
6.43–6.45, m
5.27–5.29, m
5.08, overlap
5.08, overlap (2H)
1.76, s (3H)
6.44–6.46, m
5.37–5.39, m
5.05–5.11, overlap
5.05–5.11, overlap (2H)
1.94, s (3H)
1.59, s (3H)
1.18, s (3H)
9
10
1.68, s (3H)
1.69, s (3H)
1.55, s (3H)
1.71, s (3H)
0.83, s (3H)
3.94–3.96, m
4.13, overlap
4.86, d (8.0)
4.02–4.06, overlap
4.23, overlap
4.02–4.06, overlap
4.02–4.06, overlap
4.58, d (11.2)
1’
2’
3’
4’
5’
6’
4.85, d (8.0)
4.04–4.06, m
4.22–4.24, m
4.02, overlap
4.02, overlap
4.58, d (11.2)
4.13–4.15, m
5.72, s
4.81, d (7.2)
4.00–4.06, overlap
4.23, t (8.8)
4.00–4.06, overlap
4.00–4.06, overlap
4.54, overlap
4.13–4.15, m
5.69, s
5.05–5.11, overlap
4.30 s (2H)
4.19, d (12.0)
4.89, d (8.0)
4.04–4.06, m
4.24, t (8.0)
4.02–4.04, m
4.03–4.05, m
4.59, d (11.2)
4.88, d (7.2)
4.04–4.06, m
4.24, t (8.0)
4.02, overlap
4.02, overlap
4.60, d (11.2)
4.12, dd (11.2, 6.4) 4.12, dd (11.2, 4.8) 4.13, overlap
5.68, s
5.10, s
4.30, s (2H)
4.19, d (12.0)
4.05–4.06, m
1’’
2’’
4’’
5’’
5.70, s
5.10, s
4.29, s (2H)
4.19, d (12.0)
4.05–4.07, m
5.68, s
5.10, s
4.29, s (2H)
4.19, overlap
4.02–4.06, overlap
5.11, s
4.31, s (2H)
4.19, d (11.2)
4.06–4.08, m
4.00–4.06, overlap
hydrolysis of 1 afforded D-glucose, which was identified by GC analysis. Compound
1 had a similar structure to that of the previously known compound, 40- desulfated-
atractyloside (7) [9], in which the 3- methylpentanoyl group takes the place of the 3-
methylbuthanoyl in 7. Thus, compound 1 was assigned as shown and named fructus-
noid D.
Compound 2 was obtained as a white powder. The molecular formula of 2 was
established as C32H48O15S based on HRESIMS data at m/z 703.2644 [M-H]ꢀ in con-
junction with the 13C NMR data, containing one more carbon and two more oxygen
1
atoms than compound 1. The H NMR spectrum (Table 1) proved the presence of
three methyl groups at dH 1.01 (3H, s, H-20), 0.94 (3H, t, J ¼ 6.6 Hz, H-600), 0.90 (3H,
d, J ¼ 7.2 Hz, H-500) and two olefinic protons at dH 5.18 (1H, s, H-17a) and 5.08 (1H,
s, H-17b). The 13C NMR data (Table 2) indicated three carbonyls (dC 175.5, 175.2,
173.8), two olefinic carbons (dC 160.3, 109.1), eight oxygenated carbons (dC 101.0,
83.4, 82.8, 77.6, 73.8, 72.6, 70.9, 62.3), three sp3 quaternary carbons (dC 59.1, 49.6,
41.4), four sp3 methines (dC 55.0, 52.2, 43.6, 33.0), nine sp3 methylenes (dC 48.4, 42.6,
41.2, 37.3, 36.2, 33.6, 30.5, 24.1, 19.3), and three methyls (dC 19.8, 17.8, 11.7). These
data were similar to those of 1, with the exception of an additional carbonyl signal
(C-19, dC 175.5) attached to C-4 (dC 59.1), which was confirmed by the downfield
shift of C-4 and the HMBC correlations from H-3/H-5 to C-18/C-19. Therefore,
compound 2 had the same aglycone as that of 40-desulfated-carboxyatractyloside [9].

6
Y. CHENG ET AL.
Table 4. 13C NMR (C5D5N, 200MHz) spectral data for compounds 3, 4, 8-10 (d in ppm).
dC
No
3
4
8
9
10
1
2
3
4
5
6
7
8
75.4
133.1
128.6
27.2
67.6
133.1
130.0
26.8
65.8
122.9
140.6
32.7
27.4
124.9
132.1
18.1
26.1
23.9
103.7
75.4
78.8
72.1
77.2
68.1
109.1
86.3
92.0
74.3
64.6
65.7
121.6
140.8
40.1
26.9
124.9
131.8
26.1
18.0
16.8
103.3
75.4
78.8
72.2
77.4
68.1
109.1
86.2
92.0
74.3
64.6
43.9
145.7
120.5
31.8
41.4
32.1
38.3
26.6
21.4
72.1
103.5
75.4
78.8
72.1
77.4
68.0
109.1
86.2,
92.0
74.3
64.6
31.7
32.1
147.1
139.7
114.0
116.8
14.7
103.3
75.5
78.8
72.0
77.5
68.2
146.4
139.6
114.1
116.9
22.3
103.5
75.3
78.8
72.1
77.3
67.9
9
10
1’
2’
3’
4’
5’
6’
1’’
2’’
3’’
4’’
5’’
109.4
86.2
92.2
74.6
109.1
86.2
92.0
74.3
64.7
64.7
1
The H-1H COSY, HSQC, and HMBC spectra again allowed unambiguous assign-
ment of the structure of compound 2, which differed from 40-desulfated-carboxyatrac-
tyloside only in having a 3-methylpentanoyl ester moiety attached to C-20 of the
glycoside, and then, the structure of 2 was established as assigned and named fructus-
noid E.
Compounds 3 and 4 were obtained as viscous solids. Their identical molecular for-
mula, C21H34O10, was determined by HRESIMS ions at m/z 445.2075 and m/z
1
445.2073 [M-H]ꢀ, respectively. The H and 13C NMR data (Tables 3 and 4) for com-
pounds 3 and 4 suggested that they possessed similar structures with the same sugar
moiety. The doublet at dH 4.85 (1H, d, J ¼ 8.0 Hz, H-10) and a singlet at dH 5.72 (1H,
1
s, H-100) in the H NMR spectrum revealed the presence of two sugar units, with the
signals assigned as the anomeric protons of D-glucose and D-apiose, respectively,
which was confirmed by further NMR spectral analysis and GC analysis after acid
hydrolysis and derivatization. The downfield shift of C-60 (dC 68.2 and 67.9 for 3 and
4, respectively), and the HMBC correlations from H-100 to C-60, C-300, and C-400; from
H-200 to C-300, C-400, and C-500; from H-500 to C-300 indicated that the apiofuranose was
attached to C-6 of the glucopyranose. The b-linkage of the glucopyranose moiety was
characterized by the coupling constant of the anomeric proton at dH 4.85 (1H, d,
J ¼ 8.0 Hz, H-10). As a branched-chain sugar, apiofuranose can occur in four isomeric
forms [11]. A b-D-apiofuranose moiety was confirmed by the anomeric proton signal
(dH 5.72, s) and the NOE correlation between H-200 and H-500 [12]. Thus, it was
finally determined that compounds 3 and 4 shared the same b-D-apiofuranosyl-
(1!6)-b-D glucopyranosyl disaccharide moiety.
1
In addition to the signals of sugar, the H NMR (Table 3) data for compound 3
showed one methyl proton at dH 1.76 (3H, s, H-10); three methylenes at dH 2.22-2.26
(4H, overlap, H-4, 5), 4.50 (1H, d, J ¼ 12.0 Hz, H-1a), and 4.29 (1H, overlap, H-1b);

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
and six olefinic protons at dH 6.43-6.45 (1H, m, H-7), 5.65-5.67 (1H, m, H-3), 5.27-
5.29 (1H, m, H-8a), 5.08 (1H, overlap, H-8b), and 5.08 (2H, overlap, H-9). The 13C
NMR data (Table 4) indicated six olefinic carbons (dC 147.1, 139.7, 133.1, 128.6,
116.8, 114.0), one oxygenated carbon (dC 75.4), two sp3 methylenes (dC 31.7, 27.2),
and a methyl (dC 14.7). The HMBC correlations from H-1 to C-2, C-3, and C-10;
from H-5 to C-4, C-6, and C-9; from H-7 to C-5; and from H-9 to C-4 and C-7, and
the absence of H-3/H-10 NOESY correlations indicated the E configuration at C-3/C-
4, suggested that (E) -2- methyl-6- methyleneocta-2, 7- dien-1-ol [13] was present as
the aglycone in compound 3.
Compound 4 differed from compound 3 in a C-10 signal at dC 22.3 (Table 4), and
the presence of H-3/H-10 NOESY correlations indicated the Z configuration at C-3/
C-4, suggesting that (Z)-2-methyl-6-methyleneocta-2,7-dien-1-ol [14] was present as
the aglycone in compound 4.
The six known compounds 5-10 were identified as 30,40-desulfated-atractyloside (5)
[15], atractyloside (6) [16], 40-desulfated-atractyloside (7) [9], neryl 6-O-b-D-apiofura-
nosyl-b-D- glucopyranoside (8), geranyl 6-O-b-D-apiofuranosyl-b-D-glucopyranoside
(9) [17], and myrtenol 6-O-b-D-apiofuranosyl-b-D-glucopyranoside (10) [18] by
comparison of their NMR (Tables 1–4) and MS data with reported values.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a P2000 automatic digital polarimeter (JASCO
Corporation, Tokyo, Japan). IR spectra were recorded on a Nicolet 5700 FT-IR spec-
trometer (Thermo Electron Corporation, Waltham, MA, USA). ESIMS were measured
on an Agilent 1100 Series LC/MSD trap mass spectrometer (Agilent Technologies
Inc, Santa Clara, CA, USA). HRESIMS were measured on an Agilent Technologies
6250 Accurate-Mass Q-TOF LC/MS instrument (Agilent Technologies Inc, Santa
Clara, CA, USA). NMR spectra were recorded on Bruker AV-III-500, Bruker AV
IIIHD-600, or Bruker AV IIIHD-800 (Bruker Daltonics Inc, Berlin, German), spec-
1
1
trometers for H, 13C, H-1H COSY, HSQC, HMBC, and NOESY. Column chroma-
tography was performed with polyamide (30–60 mesh, Jiangsu Changfeng Chemical
Co., Jiangsu, Ltd, China), macroporous resin (D101, Tianjin Nankai Hecheng Science
& Technology Co., Ltd, Tianjin, China), Sephadex LH-20 (Amersham Pharmacia
Biotech AB, Uppsala, Sweden), and ODS (50 mm, 120 Å, Silicycle Inc., Quebec,
Canada). Preparative HPLC was carried out on a Shimadzu LC-6AD instrument with
an SPD-10A detector (Shimadzu Scientific Instruments Co. Ltd., Kyoto, Japan) using
YMC-Pack ODS-A columns (250 ꢁ 20 mm, 5 mm, or 250 ꢁ 10 mm, 5 mm, YMC Co.,
Ltd, Kyoto, Japan). Two-dimensional high-throughput chromatography was carried
out on a Sepiatec Sepbox-2D-2000 (Sepiatec GmbH, Berlin, Germany) with two pre-
parative HPLC pumps (a gradient pump and a water pump) and two detectors (a UV
detector and an ELSD detector) using 1 first separation column (150 ꢁ 32 mm),
6 second separation columns (250 ꢁ 16 mm), and 18 trap columns (30 ꢁ 32 mm).

8
Y. CHENG ET AL.
3.2. Plant material
The fruits of Xanthium chinense (110 kg) were collected in Xining, Guangxi Province,
China, in September 2013. The plant was identified by Associate Prof. Lin Ma
(Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College). A voucher specimen (No. ID-S-2522) was deposited in the
herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences
and Peking Union Medical College, China.
3.3. Extraction and isolation
A large amount of material (110 kg) was crushed and extracted with 95% EtOH
(150 L; 2 h ꢁ 3) under reflux. After removal of the solvent under vacuum, the extrac-
tum was suspended in H2O and then extracted with petroleum ether, CH2Cl2,
EtOAc, and n-BuOH in turn. Each extract was evaporated under reduced pressure to
result in petroleum ether-soluble (6 kg), CH2Cl2-soluble (450 g), EtOAc-soluble
(250 g), and n-BuOH-soluble (450 g) extracts. The n-BuOH-soluble extract (250 g)
was fractionated by macroporous resin (D101) column chromatography, eluting with
H2O, 10%, 30%, 50%, 70%, and 95% EtOH, to afford six fractions, A–F.
Fraction C (95.3 g) was fractionated by polyamide (30–60 mesh) column chroma-
tography, eluting with H2O, 50% and 95% EtOH, to afford three fractions, C1-C3.
Fraction C1 (70 g) was separated by chromatography on an MCI column, eluting
with EtOH-H2O to yield six major fractions (C1-a–C1-f). Fraction C1-c (6.0g) was
separated by an ODS column using a gradient of MeOH-H2O as the eluent to obtain
seven subfractions, C1-c-1–C1-c-7. Subsequent separation of C1-c-6 (768 mg) by a
Sephadex LH-20 column (H2O: MeOH, 5: 1) afforded subfraction C1-c-6-2 (142 mg),
and then, p-HPLC (25% CH3CN/H2O, t ¼ 4 ml/min, k ¼ 210 nm) of C1-c-6-2 gave
compound 5 (tR ¼35 min, 10 mg).
Fraction D (35.6 g) resulted in four subfractions (D1–D4) after being chromato-
graphed over a silica gel column. Fraction D2 (2g) was subjected to 2D Sepbox high-
throughput preparation to give 23 subfractions, D2-a–D2-w. Subsequent separation of
D2-j (10 mg) by p-HPLC (30% CH3CN/H2O, t ¼ 4 ml/min, k ¼ 210 nm) gave com-
pounds 1 (tR¼ 20 min, 2 mg) and 7 (tR¼ 17 min, 3 mg). D2-i (20 mg) was purified by
p-HPLC (25% CH3CN/H2O, t ¼ 4 ml/min, k ¼ 210 nm) to afford compounds 8 (tR¼
23 min, 2 mg), 9 (tR¼ 26 min, 2 mg), and 10 (tR¼ 48 min, 2 mg). D2-r (10 mg) was
purified by p-HPLC (23% CH3CN/H2O, t ¼ 4 ml/min, k ¼ 210 nm) to afford com-
pounds 3 (tR¼ 23 min, 2 mg) and 4 (tR¼ 21 min, 2 mg).
Fraction D3 (6 g) was fractionated by an ODS column using a gradient of MeOH-
H2O as the eluent to obtain 5 subfractions, D3-a-D3-e. D3-a was afforded as com-
pound 6 (250 mg). D3-c was purified by p-HPLC (17% CH3CN/H2O, t ¼ 4 ml/min,
k ¼ 210 nm) to afford compound 2 (tR¼ 21 min, 2 mg).
3.3.1. Fructusnoid D (1)
C31H48O13S, white powder; [a]2D0 þ64.0 (c 0.00169, H2O); UV (H2O) kmax (log e) 192
(4.02), 255 (3.14) nm; IR vmax 3410, 2931, 1700, and 1076 cmꢀ1; for 1H NMR

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
(C5D5N, 800MHz) and 13C NMR (C5D5N, 200MHz) spectral data (see Tables 1 and
2); HRESIMS: m/z 659.2750 [M-H]ꢀ (calcd for C31H47O13S, 659.2743).
3.3.2. Fructusnoid E (2)
C32H48O15S, white powder; [a]2D0 þ66.9 (c 0.00171, H2O); UV (H2O) kmax (log e) 192
(3.98), 255 (3.47) nm; IR vmax 3441, 2981, 1718, 1250, 1079, 1453, 1379, 1042, and
1
1004 cmꢀ1; for H NMR (CD3OD, 600MHz) and 13C NMR (CD3OD, 150MHz) spec-
tral data (see Tables 1 and 2); HRESIMS: m/z 703.2644 [M-H]- (calcd for
C32H47O15S, 703.2641).
3.3.3. (E)-2-Methyl-6-methyleneocta-2,7-dien-1-ol b-D-apiofuranosyl-(1!6)-b-D-glu-
copyranoside (3)
C21H34O10, viscous solid; [a]2D0 þ54.8 (c, 0.00153 H2O); UV (H2O) kmax (log e) 192
(3.96), 220 (3.59), 255 (3.38) nm; IR vmax 3394, 2936, 1684, 1436, 1207, 1139, 842,
1
803, and 724 cmꢀ1; for H NMR (C5D5N, 800MHz) and 13C NMR (C5D5N, 200MHz)
spectral data (see Tables 3 and 4); HRESIMS: m/z 445.2075 [M-H]ꢀ (calcd for
C21H33O10, 445.2079).
3.3.4. (Z)-2-Methyl-6-methyleneocta-2,7-dien-1-ol b-D-apiofuranosyl-(1!6)-b-D
glucopyranoside (4)
C21H34O10, viscous solid; [a]2D0 þ76.7 (c 0.00215, H2O); UV (H2O) kmax (log e) 192
(3.98), 220 (3.71), 255 (3.28) nm; IR vmax 3406, 2938, 1682, 1433, 1201, 1137, 840,
1
802, and 723 cmꢀ1; for H NMR (C5D5N, 800MHz) and 13C NMR (C5D5N, 200MHz)
spectral data (see Tables 3 and 4); HRESIMS: m/z 445.2073 [M-H]ꢀ (calcd for
C21H33O10, 445.2079).
3.4. Hydrolysis of compounds 1 and 3, and sugar identification
Compound 1 (1.5 mg) was added in 2 M NaOH (2 ml) and stirred for 5 h under
room temperature. The solution was regulated by 0.5M HCl-H2O to pH 2-3, and was
purified by p-HPLC (25% CH3CN/H2O) to remove the side chain on sugar, then the
compound that was removed the side chain on sugar was added in 2 M HCl (2 ml)
and refluxed for 12 h under 90 ꢂC. The solution was extracted three times with
EtOAc. The water layer was dried to yield a residue. Then the residue was dissolved
in pyridine (1 ml) and the L-cysteine methyl hydrochloride (2 mg) was added. The
mixture was refluxed for 2 h under 60 ꢂC, and dried by nitrogen and heated for 0.5 h
under 80 ꢂC. Then the N-trimethylsilylimidazole (1 ml) was added, and heated for 2 h
under 60 ꢂC. Finally, the mixture was added in H2O (2 ml) and partitioned with
n-hexane (2 ml) three times. The organic layer was combined and concentrated, used
for GC analysis. The conditions of GC experiments: capillary column, HP-5 (60
m ꢁ 0.25 mm, with a 0.25 lm film, Dikma); detector, FID; injection temperature,
300 ꢂC; detection temperature, 300 ꢂC; initial temperature, 200 ꢂC, risen to 280 ꢂC at
the rate of 10 ꢂC/min, and sustained for 35 min, then declined to 200 ꢂC at the rate of
40 ꢂC/min and then sustained for 1 min; carrier, N2. The D-glucose (tR ¼ 29.6 min)
was determined by comparing with the standard sugar.

10
Y. CHENG ET AL.
Compound 3 (1.5 mg) was added in 2 M HCl (2 ml) and conducted as the same
way as compound 1. D-glucose (tR ¼ 29.5 min) and D-apiofuranose (tR ¼ 20.3 min)
were determined by comparing with the standard sugars.
Acknowledgements
We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica,
Chinese Academy of Medical Sciences & Peking Union Medical College for measuring the IR,
NMR, MS, and HRESIMS.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This project was financially supported by the National Natural Science Foundation of China [grant
number 81573321], the CAMS Innovation Fund for Medical Science [grant number 2017-I2M-3-
010], Young Elite Scientists Sponsorship Program by CAST [YESS, grant number 2016QNRC001],
and National Science and Technology Project of China [No. 2014ZX09304306-001].
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