Phytochemical Study of Stem and Leaf of Clausena lansium

Article abstract of DOI:10.3390/molecules24173124

Clausena lansium Lour. Skeels (Rutaceae) is widely distributed in South China and has historically been used as a traditional medicine in local healthcare systems. Although the characteristic components (carbazole alkaloids and coumarins) of C. lansium have been found to possess a wide variety of biological activities, little attention has been paid toward the other components of this plant. In the current study, phytochemical analysis of isolates from a water-soluble stem and leaf extract of C. lansium led to the identification of 12 compounds, including five aromatic glycosides, four sesquiterpene glycosides, two dihydrofuranocoumarin glycosides, and one adenosine. All compounds were isolated for the first time from the genus Clausena, including a new aromatic glycoside (1), a new dihydrofuranocoumarin glycoside (6), and two new sesquiterpene glycosides (8 and 9). The phytochemical structures of the isolates were elucidated using spectroscopic analyses including NMR and MS. The existence of these compounds demonstrates the taxonomic significance of C. lansium in the genus Clausena and suggests that some glycosides from this plant probably play a role in the anticancer activity of C. lansium to some extent.

Full text of DOI:10.3390/molecules24173124

molecules
Article
Phytochemical Study of Stem and Leaf of
Clausena lansium
Wenwen Peng 1,2,*, Xiaoxiang Fu 2, Yuyan Li 2, Zhonghua Xiong 2, Xugen Shi 2, Fang Zhang 2,
Guanghua Huo 1 and Baotong Li 3,
*
1
Jiangxi Key Laboratory for Conservation and Utilization of Fungal Resources,
Jiangxi Agricultural University, Nanchang 330045, China
College of Agriculture, Jiangxi Agricultural University, Nanchang 330045, China
School of Land Resources and Environment, Jiangxi Agricultural University, Nanchang 330045, China
Correspondence: pengwenwen123@sina.com (W.P.); libt66@163.com (B.L.)
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editors: Jianbo Xiao, Yanbo Zhang and Hui Ni
Received: 23 July 2019; Accepted: 26 August 2019; Published: 28 August 2019
Abstract: Clausena lansium Lour. Skeels (Rutaceae) is widely distributed in South China and has
historically been used as a traditional medicine in local healthcare systems. Although the characteristic
components (carbazole alkaloids and coumarins) of C. lansium have been found to possess a wide
variety of biological activities, little attention has been paid toward the other components of this plant.
In the current study, phytochemical analysis of isolates from a water-soluble stem and leaf extract
of C. lansium led to the identification of 12 compounds, including five aromatic glycosides, four
sesquiterpene glycosides, two dihydrofuranocoumarin glycosides, and one adenosine. All compounds
were isolated for the first time from the genus Clausena, including a new aromatic glycoside (
a new dihydrofuranocoumarin glycoside ( ), and two new sesquiterpene glycosides ( and
1
9
),
).
6
8
The phytochemical structures of the isolates were elucidated using spectroscopic analyses including
NMR and MS. The existence of these compounds demonstrates the taxonomic significance of C. lansium
in the genus Clausena and suggests that some glycosides from this plant probably play a role in the
anticancer activity of C. lansium to some extent.
Keywords: Clausena lansium; aromatic glycosides; sesquiterpene glycosides; dihydrofuranocoumarin
glycosides
1. Introduction
The genus Clausena (Rutaceae) is comprised of approximately 30 species that are scattered
throughout the subtropical and tropical regions, including China, Vietnam, Indonesia, Malaysia,
and the Philippines [1,2]. There are approximately 10 species as well as 2 varieties in China, which
appear in Southern China. Clausena lansium Lour. Skeels (Rutaceae), belonging to the genus Clausena
of the family Rutaceae, is a fruit tree and a species of strongly scented evergreen tree growing in
South China [1,2]. C. lansium is famous for their fruits, which are usually very popular tropical,
health-promoting fruits, while their roots, stems, leaves, and seeds have also been extensively
applied in folk medicine or traditional Chinese medicine for the treatments of abdominal pain,
malaria, cold, dermatopathy, and snake bites [
coumarins, and sesquiterpenes from this plant have reported the neuroprotective [4,5], antitumor [6,7],
hepatoprotective [ ], anti-inflammatory [ ], antifungal [10], antioxidant [11], antiobesity [12],
2,3]. Various biological studies, on the alkaloids,
8
9
nematicidal [13], antimicrobial [14], and hypoglycemic [15] effects of the C. lansium. In our previous
studies, some carbazole alkaloids [16] and coumarins [17] were separated from the stem and leaf of
C. lansium. As a part of our ongoing research into the natural products possessing structural and
biological diversity from C. lansium, a systematic phytochemical study on the stem and leaf of C. lansium
Molecules 2019, 24, 3124; doi:10.3390/molecules24173124
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3124
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was accordingly carried out. The investigation resulted in the separation and characterization of five
aromatic glycosides ( ), two dihydrofuranocoumarin glycosides ( ), four sesquiterpene glycosides
11), and one adenosine (12) (Figure 1). All compounds were isolated for the first time from the
genus Clausena, including a new aromatic glycoside (1), a new dihydrofuranocoumarin glycoside (6),
and two new sesquiterpene glycosides ( and ). The molecular structures of new compounds were
1–
5
6,7
(8–
8
9
established using comprehensive spectroscopic studies. The known compounds were determined
by comparing their experimental data with those described in the literature. The existence of these
compounds demonstrates the taxonomic significance of C. lansium in the genus Clausena and suggests
that some glycosides from this plant probably play a role in the anticancer activity of C. lansium to
some extent.
4
5
6
3
2
HO
1
7
O
HO
OCH3
6'
5'
O
OH
O
O
O
O
O
HO
4'
1'
HO
HO
O
O
O
O
2'
O
3'
OH
OH
OH
O
5''O
HO
OH
1''
4''
OH OH
OH
OH
OH
3''
OH
OH
OH
OH
OH
2''
OH OH
2
4
3
1
HO
3'
HO
4
5
2''
4a
8a
6
3
COOCH3
3''
2
1''
2'
O
O
O
O
O
7
O
O
O
O
6'''
O
O
HO
HO
O
OH
OH
OH
O
O
5'''
OH
OH
1'''
4'''
OH
2'''
OH
OH
OH
OH
OH
OH
OH
7
5
6
O
2
2
5
O
O
O
3
1
3
1
4
4
8
8
10
10
NH2
N
7
9
9
6
5 OH
OH
7
OH
HO
HO
N
N
N
O
O
O
O
O
6'
6'
HO
OH
HO
HO
5'
5'
O
O
O
O
OH
4'
1'
4'
1'
OH
OH
OH
2'
2'
HO
3'
3'
HO
HO
HO
OH
OH
HO
12
OH
11
OH
OH
10
8
9
Figure 1. The chemical structures of compounds 1–12 from the stem and leaf of Clausena lansium.
2. Results and Discussion
2.1. Elucidation of Chemical Structures of Four New Compounds 1,6,8,9
Claulanaroside (1) was obtained as a white amorphous powder, and its elemental composition
was determined to be C18H26O10 by HRESIMS m/z: 425.1623 (Calculated for: [C18H26O10 + Na]+,
425.1628) with 6 degrees of unsaturation. The Infrared Radiation (IR) spectrum exhibited absorptions
for hydroxyl groups (3441 and 1062 cm−1) and the aromatic ring (1618, 1547, and 1498 cm−1). The 1H
NMR data of
1 (Table 1) showed clear signals for five aromatic protons (δH 7.31 (2H, d, J = 7.2 Hz),

Molecules 2019, 24, 3124
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7.23 (2H, m), and 7.18 (1H, d, J = 7.3)), indicating a monosubstituted benzene ring in
1
. The 13C-NMR
spectroscopic data (Table 1) showed the presence of a six-carbon sugar (δC 100.8, 77.2, 76.6, 76.5, 70.3,
and 61.4), a five-carbon sugar (δC 109.3, 79.2, 77.6, 73.9, and 64.6), a benzene ring (δC 137.6, 127.8,
127.8, 127.9, 127.9, and 127.3) and an oxymethylene (δC 70.4). Coupled with the above evidence,
comparison of the 1H- and 13C NMR data of
1 and icariside F2 [18] implied that the aglycone of 1
was methylbenzene and the C-7 position was glycosylated. Acid hydrolysis of
1
afforded d-glucose
and d-apiose, which were detected by derivatization and HPLC analysis [19,20]. The anomeric
configurations of monosaccharide units were confirmed to be
according to their JH1-H2 coupling constants (7–8 and 2–3 Hz, respectively) [18
β
for the d-glucose and d-apiose
21 22]. Detailed
with those of icariside F2 revealed that the positions of the
-d-apiofuranosyl linkage were different in and icariside F2. The -d-apiofuranosyl was located at
C-40 of the
-d-glucopyranosyl of from the HMBC spectrum (Figure 2), which showed that H-1” (δH
5.27) was correlated to C-40 (δC 76.6), according to the
-d-glucopyranosylation-induced downfeld
shift on the -carbon [23]. The above deduction was further supported by downfeld shift observed
for C-40 (δC 76.6 ppm) and upfeld shift for C-60 (δC 61.4 ppm) in
. Consequently, compound was
identified as a methylbenzene-7-O- -d-apiofuranosyl-(1 4)-O- -d-glucopyranoside and named as
claulanaroside, as shown in Figure 1.
3
, ,
comparison of the NMR data of
1
β
1
β
β
1
β
α
1
1
β
→
β
Table 1. 1H NMR (400 MHz, MeOD) and 13C NMR (101 MHz, MeOD) spectroscopic data of 1.
Position
δH (J in Hz)
δC
Position
δH (J in Hz)
δC
1
2
3
4
5
6
7
137.6 (s)
127.8 (d)
127.9 (d)
127.3 (d)
127.9 (d)
127.8 (d)
70.4 (t)
40
50
60
3.84 (m)
3.51 (m)
3.78 (m)
3.60 (m)
5.27 (d, 2.1)
3.31 (m)
76.6 (d)
77.2 (d)
61.4 (t)
7.31 (d, 7.2)
7.23 (m)
7.18 (d, 7.3)
7.23 (m)
7.31 (d, 7.2)
4.56 (d,11.6)
4.80 (d,11.6)
4.30 (d,7.5)
3.16 (m)
1”
2”
3”
4”
109.3 (d)
77.6 (d)
79.2 (s)
73.9 (t)
3.81 (m)
3.55 (m)
3.45 (d, 12.2)
3.41 (d, 12.2)
10
20
30
100.8 (d)
76.5 (d)
70.3 (d)
5”
64.6 (t)
3.38 (m)
O
HO
O
HO
O
O
O
H
OH
O
O
O
O
O
HO
HO
OH
O
HO
OH
O
O
OH
OH
OH
HO
OH
OH
OH
OH
9
1
6
1H-1H COSY
HMBC
Figure 2. Some key HMBC and 1H–1H COSY correlations of 1, 6, and 9.
Claulancoumside (6) was obtained as a white powder crystallization with a blue fluorescence under
the ultraviolet lamp (254 nm). The structure of
6
was deduced for one new dihydrofuranocoumarin
glycoside mainly by its blue fluorescence and experimental data. In the 1H NMR spectrum (Table 2), the
resonance characteristics for a cis-double bond (δH 7.91 (1H, d, J = 9.5 Hz) and 6.25 (1H, d, J = 9.5 Hz)) and
a singlet aromatic proton (δH 7.21 (1H, s)), coupled with the blue fluorescence, suggested the presence
of a trisubstituted coumarin skeleton in
6 [24– 6 showed
26]. The 13C-NMR spectrum (Table 2) of

Molecules 2019, 24, 3124
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20 C-atom signals, including a coumarin skeleton (δC 161.3, 150.1, 144.9, 144.1, 129.3, 127.8, 114.3, 113.8,
and 110.7), a six-carbon sugar (δC 97.2, 76.1, 75.6, 73.1, 69.3, and 59.9), two oxymethines (δC 91.5 and
71.1), two methyls (δC 23.3 and 22.2), and an oxygenated quaternary carbon (δC 77.5 (s)). The 1H and
13C NMR data of
to 8-hydroxysmyrindiol and the C-1” position of
by the HMBC correlation (Figure 2) from H-1000 to C-1” and the downfeld shift observed for C-1” (δC
77.5 ppm) in 23]. Acid hydrolysis and HPLC analysis of afforded a d-glucose [19 20]. The anomeric
configuration of the d-glucose was confirmed to be
according to its large 3JH1-H2 coupling constants
(J = 7.5 Hz) [25
26]. In addition, coupled with the 1H-1H COSY (Figure 2) correlations of H-20/H-30,
a large vicinal coupling constant (6.5 Hz) of two doublets at δH 4.57 (H-20) and 5.35 (H-30) supported
6
and 8-hydroxysmyrindiol [24] demonstrated that the aglycone of
6 was equivalent
6
was glycosylated, which was further confirmed
6
[
6
,
β
,
the cis orientation. The absolute configurations of
6
at C-20 and C-30 were established by comparing its
specific rotation with 10-O-
β
-d-glucopyranosyl-(2S,3R)-3-hydroxynodakenetin ([α]2D0−14.0◦ (pyridine; c
0.5)) [25] and 10-O-
β
-d-glucopyranosyl (2R,3S)-3-hydroxynodakenetin ([α]2D1 +15.1◦ (MeOH; c 0.05)) [26].
Withaspecificrotationvalueof+24.3 (MeOH;c1.1), compound
6
wasassigned tothe 20R,30Sconfigurations.
Therefore, compound was deduced as a 1”-O-
6
β
-d-glucopyranosyl (20R,30S)-30,8-dihydroxyarmesin
and named as claulancoumside, as shown in Figure 1.
Table 2. 1H NMR (400 MHz, MeOD) and 13C NMR (101 MHz, MeOD) spectroscopic data of 6.
Position
δH (J in Hz)
δC
Position
δH (J in Hz)
δC
2
3
4
4a
5
6
7
8
8a
20
30
161.3 (s)
110.7 (d)
144.9 (d)
113.8 (s)
114.3 (d)
127.8 (s)
150.1 (s)
129.3 (s)
144.1 (s)
91.5 (d)
71.1 (d)
1”
2”
3”
77.5 (s)
23.3 (q)
22.2 (q)
97.2 (d)
73.1 (d)
76.1 (d)
69.3 (d)
75.6 (d)
59.9 (t)
6.25 (d, 9.5)
7.91 (d, 9.5)
1.64 (3H, s)
1.63 (3H, s)
4.85 (d, 7.5)
3.16 (m)
3.41 (m)
3.40 (m)
3.19 (m)
3.50 (m)
3.17 (m)
1000
2000
3000
4000
5000
6000
7.21 (s)
4.57 (d, 6.5)
5.35 (d, 6.5)
Clausesquiside A (
The UV spectrum showed absorption maxima at 237 nm. The IR spectrum indicated the presence of
carbonyl (1668 cm−1) and hydroxyl (3447 and 1043 cm−1) groups. The 1H NMR spectrum of
(Table 3)
8
) was obtained as an amorphous powder with [α]2D0 113.4◦ (MeOH; c 0.68).
−
8
showed two trans-olefinic protons (δH 6.92 (1H, dd, J = 15.6 Hz) and 5.79 (1H, dd, J = 15.6, 7.4 Hz)), one
olefinic proton singlet (δH 5.85 (1H, s)), and three methyl singlets (δH 1.91 (3H, s), 0.95 (3H, s), and 0.93
(3H, s)). The 13C NMR spectrum of
8 (Table 3) exhibited a ketonic carbonyl at δC 199.5; four olefinic
carbons at δC 165.3, 133.9, 127.0, and 125.3; an oxymethine at δC 77.6; an oxygenated quaternary carbon
at δC 78.4; an oxymethylene at δC 64.1; three methyls at δC 22.9, 21.6, and 17.7; a quaternary carbon
at δC 40.5; a methylene at δC 48.8; and a six-carbon sugar at δC 99.5, 76.2, 76.2, 73.0, 69.6, and 60.8.
The 1H and 13C NMR spectral data of
only in the presence of an oxymethylene at δC 64.1 in
(6R,9R)-roseoside. The absolute configuration at the 6-position in
from the negative and positive Cotton effects at 241 and 322 nm, respectively, in the CD spectrum [27].
The -d-glucopyranosylation-induced shift-trend rule suggested the absolute configuration at the
9-position of to be R [23 28], which was further supported by the remarkable chemical shift difference
between C-7 (δC 133.9) and C-8 (δC 127.0) of 8, as shown in [27]. So, compound 8 was elucidated as a
8
were very similar to those of (6R,9R)-roseoside [27], differing
, instead of a methyl at δC 21.2 that is found in
was determined to be R, judging
8
8
β
8
,
(6R,9R,4Z,7E,)-6,9,10-trihydroxy-4,7-megastigmadien-3-one-9-O-β-d-glucopyranoside and named as
clausesquiside A, as shown in Figure 1.

Molecules 2019, 24, 3124
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Table 3. 1H NMR (400 MHz, MeOD) and 13C NMR (101 MHz, MeOD) spectroscopic data of 8 and 9.
8
9
Position
Position
δH (J in Hz)
δC
δH (J inHz)
δC
1
2
40.5 (s)
48.8 (t)
1
2
36.8 (s)
48.4 (t)
2.61 (d, 16.6)
2.16 (d, 16.6)
2.48 (d, 15.8)
2.11 (d, 15.8)
3
4
5
6
7
8
9
10
199.5 (s)
125.3 (d)
165.3 (s)
78.4 (s)
127.0 (d)
133.9 (d)
77.6 (d)
64.1 (d)
3
4
5
6
7
8
9
10
200.8 (s)
124.9 (d)
164.3 (s)
57.1 (d)
130.7 (d)
132.1 (d)
78.2 (d)
64.6 (t)
5.85 (s)
5.79 (br s)
2.72 (d, 9.8)
6.92 (dd, 15.6, 6.3)
5.79 (dd, 15.6, 7.4)
4.45 (m)
3.60 (dd, 11.8, 4.2)
3.57 (dd, 11.8, 4.2)
0.95 (3H, s),
5.82 (dd, 15.4, 9.8)
5.55 (dd, 15.4, 7.1)
4.30 (m)
3.61 (dd, 11.6, 4.1)
3.57 (dd, 11.6, 4.1)
0.93 (3H, s),
11
12
13
10
20
30
40
50
60
21.6 (q)
22.9 (q)
17.7 (q)
99.5 (d)
73.0 (d)
76.2 (d)
69.6 (d)
76.2 (d)
60.8 (t)
11
12
13
10
20
30
40
50
60
26.7 (q)
26.2 (q)
22.6 (q)
99.9 (d)
73.5 (d)
76.7 (d)
70.2 (d)
76.7 (d)
61.3 (d)
0.93 (3H, s),
1.91 (3H, s),
4.26 (d, 7.6)
3.25 (m)
3.24 (m)
3.23 (m)
3.13 (m)
0.88 (3H, s),
2.04 (3H, s),
4.21 (d, 7.3)
3.32 (m)
3.25 (m)
3.22 (m)
3.14 (m)
3.81 (dd, 11.2, 2.1)
3.64 (dd, 11.2, 2.1)
3.73 (dd, 12.1, 2.2)
3.68 (dd, 12.1, 2.2)
Clausesquiside B (9), a yellowish amorphous powder, possessed the virtually identical NMR
(Table 3) and MS data as opuntiside A [29]. Its plane structure is determined by the HMBC and
1H-1H COSY spectrums (Figure 2). The CD data (321 (∆ε –7.1), 254 (∆ε +186.2) nm) showed a
positive maximum at 254 nm, which was identical to the CD data of opuntiside A, indicating that
the C-6 of
shift-trend rule [23
shift observed for C-9 (δC 77.6 ppm). Based upon the results of the combined spectroscopic
analyses, the structure of this compound was established as (6R,9R,4Z,7E)-9,10-dihydroxy-
4,7-megastigmadiene-3-one-9-O- -d-glucopyranoside and named as clausesquiside B. As a result, the
9
had an absolute R-configuration. According to the
β-d-glucopyranosylation-induced
,28], the absolute configuration of C-9 was deduced for R from the downfeld
9
β
absolute structure of 9 at C-9 was substantiated for the first time in this study.
2.2. Structural Identification and Function of the Known Compounds 2–5,7,10–12
Comparing the experimental data of the known compounds with those described in the literatures,
the phytochemical structures of the eight known compounds were identified as: Icariside F2 (
Icariside D1 ( ) [30], Vanilloloside ( ) [31], methyl benzoate-2-(6-O- -l-rhanmopyranosyl)-O-
d-glucopyranoside ( -d-glucopyranosyl (2S,3R)-3-hydroxyarmesin ( ) [24], (6R,9R,4Z,7E)-9,10-
) [32], 10-O-
dihydroxy-4,7-megastigmadien-3-one-9-O- -d-glucopyranoside (10) [33], (6R,9S)-Roseoside (11) [27],
and adenosine (12) [34]. The eight known compounds were obtained from the genus Clausena for the first
time, but the aglycone analogs of compounds 10 11 have been isolated from Clausena excavata [35 36
2) [18],
3
4
α
β-
5
β
7
β
2–
5
,
,
,
]
in our previous research about the phytochemical constituents of the genus Clausena, which may
exhibit the chemical relationship between C. lansium and C. excavata. In addition, the analogs of
compound
7 and its aglycone have been previously isolated from three different families such as
Angelica archangelica (Apiaceae) [25], Pleurospermum rivulorum (Apiaceae) [26], Ferulago asparagifolia
(Apiaceae) [37], Notopterygium incisum (Apiaceae) [38], Glehnia littoralis (Apiaceae) [39], Streblus indicus
(Moraceae) [40], Dorstenia brasiliensis (Moraceae) [41], and Aegle marmelos (Rutaceae) [24], which may
revealed a genetic relationship between the Apiaceae, Moraceae, and Rutaceae families. To the best of
our knowledge, the phytochemical constituents of the plant are mainly affected by the genetic and

Molecules 2019, 24, 3124
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environmental factors during plant growth, extraction, and isolation. Therefore, further phytochemical
study on the water-soluble extract of C. lansium from different regions should be performed to achieve
a full understanding of the chemical composition of C. lansium. As a predictable result, more and more
chemical constituents will be used as evidence to support the taxonomic significance of C. lansium in
the genus Clausena.
As mentioned in the introduction, as the characteristic components of the genus Clausena, the
carbazole alkaloids and coumarins have been found to possess a variety of structures and biological
activities [42,43]; however, little attention has been paid toward the other components of this plant,
especially the water-soluble components. The current study demonstrates the presence of the aromatic
glycosides, sesquiterpene glycosides, and coumarin glycosides. The carbazole alkaloids are considered
to be the main anti-cancer components of the genus Clausena [6,7,44–46]. However, several reports
demonstrated that the analogs of aromatic glycosides [47 48] and sesquiterpene glycosides [49] also
,
possessed a certain degree of anti-cancer activity. Therefore, it is suggested that some glycosides from
this plant probably play a role in the anticancer activity of C. lansium to some extent. At the same time,
this also provides some clues about testing anticancer activity and expanding the biological scope of
this work for our future research.
2.3. Characterization of Compounds 1–12
Claulanaroside (1): UV (MeOH) λmax (log ε): 218 (6.31), 265 (6.00), 294(5.76) nm; IR (KBr) νmax
3441, 1618, 1547, 1498, 1248, 1062 cm−1; NMR data (SI 1–SI 5 in Supplementary Material) found in
Table 1; positive ESIMS m/z: 425 [M + Na]+; HRESIMS m/z: 425.1623 (Calculated for: [C18H26O10
+
Na]+, 425.1628).
Icariside F2 (
H-3,5), 7.35 (2H, m, H-2,6), 7.30 (1H, m, H-4), 5.08 (1H, d, J = 2.6 Hz, H-1”), 4.93 (1H, d, J = 12.0 Hz,
2 δ 7.44 (2H, d, J = 7.2 Hz,
): ESIMS m/z: 425 [M + Na]+; 1H-NMR (400 MHz, MeOD):
H-7a), 4.66 (1H, d, J = 12.0 Hz, H-7b), and 4.35 (1H, d, J = 7.4 Hz, H-10). 13C NMR (101 MHz, MeOD)
δ:
137.5 (s, C-1), 128.0 (d, C-2,6), 127.9 (d, C-3,5), 127.4 (d, C-4), 70.4 (t, C-7), 101.8 (d, C-10), 73.6 (d, C-20),
76.7 (d, C-30), 70.5 (d, C-40), 75.6 (d, C-50), 67.3 (d, C-60), 109.6 (d, C-1”), 76.6 (d, C-2”), 79.2 (s, C-3”),
73.7 (d, C-4”), and 64.2 (d, C-5”) (NMR spectrogram SI 6–SI 7 in Supplementary Material).
Icariside D1 (
H-2,6), 7.28 (2H, m, H-3,5), 7.18 (1H, m, H-4), 5.04 (1H, d, J = 2.5 Hz, H-1”), 4.62 (1H, d, J = 7.7 Hz, H-10),
3 δ: 7.29 (2H, J = 7.8 Hz,
): ESIMS m/z: 439 [M + Na]+; 1H-NMR (400 MHz, MeOD):
4.31 (1H, m, H-8a), 3.83 (1H, m, H-8b), and 3.00 (2H, t, J = 7.3 Hz, H-7); 13C NMR (101 MHz, MeOD)
δ:
138.2 (s, C-1), 127.5 (d, C-2,6), 128.1 (d, C-3,5), 125.3 (d, C-4), 35.4 (d, C-7), 69.9 (d, C-8), 102.5 (d, C-10),
73.2 (d, C-20), 76.2 (d,C-30), 69.8 (d, C-40), 75.0 (d, C-50), 66.8 (d, C-60), 109.1 (d, C-1”), 76.1 (d, C-2”), 78.6
(s, C-3”), 73.1 (d, C-4”), and 63.7 (d, C-5”) (NMR spectrogram SI 8–SI 9 in Supplementary Material).
Vanilloloside (
H-5), 7.04 (lH, d, J = 2.1 Hz, H-2), 6.89 (lH, dd, J = 2.1, 8.2 Hz; H-6), 4.63 (1H, d, J = 7.4 Hz, H-10), 4.56
4 δ: 7.14 (lH, d, J = 8.2 Hz,
): ESIMS m/z: 339 [M + Na]+; 1H-NMR (400 MHz, MeOD):
(2H, s; H-7), and 3.97 (3H, s, OCH3); 13C NMR (101 MHz, MeOD)
δ: 136.3 (s, C-1), 111.2 (d, C-2), 149.4
(s, C-3), 145.8 (s, C-4), 116.5 (d, C-5), 119.3 (d, C-6), 63.6 (t, C-7), 101.5 (d, C-10), 76.8 (d, C-20), 76.4 (d,
C-30), 73.5 (d, C-40), 69.9 (d, C-50), 61.1, (d, C-60), and 55.3 (Q, OCH3) (NMR spectrogram SI 10–SI 14 in
Supplementary Material).
Methyl benzoate 2-(6-O-
α
-l-rhanmopyranosyl)-O-
: 7.79 (1H, dd, J = 7.8, 1.6 Hz, H-3), 7.59 (1H, ddd, J = 8.4, 7.8, 1.6 Hz,
β-d-glucopyranoside (5
): ESIMS m/z: 483 [M + Na]+;
1H NMR (400 MHz, MeOD)
δ
H-5), 7.36 (1H, d, J = 8.4 Hz, H-6), 7.17 (1H, dd, J = 7.8, 7.8 Hz, H-4), 4.90 (1H, d, J = 7.3 Hz, H-10), 4.67
(1H, d, J = 1.3 Hz, H-1”), 3.92 (1H, d, J = 9.8 Hz, Hb-60), 3.91 (3H, s, H-8), 3.81 (1H, m, H-2”), 3.77 (1H,
dd, J = 9.7, 3.4 Hz, H-3”), 3.70 (2H, m, Ha-60, H-5”), 3.64 (2H, m, H-20, H-40), 3.62 (1H, t, J = 8.9 Hz,
H-30), 3.50 (1H, t, J = 9.1 Hz, H-50), 3.35 (1H, t, J = 9.6 Hz, H-4”), and 1.14 (1H, d, J = 6.2 Hz, H-6”); 13
C
NMR (101 MHz, MeOD) δ: 157.2 (s, C-1), 120.9 (d, C-2), 130.8 (d, C-3), 122.3 (d, C-4), 133.8 (d, C-5),
117.5 (d, C-6), 167.1 (s, C-7), 102.3 (d, C-10), 73.5 (d, C-20), 75.8 (d, C-30), 71.0 (d, C-40), 76.2 (d, C-50),
66.53 (t, C-60), 100.8 (d, C-1”), 68.5 (d, C-2”), 70.8 (d, C-3”), 72.6 (d, C-4”), 70.0 (d, C-5”), 16.53 (q, C-6”),
and 51.43 (q, OCH3) (NMR spectrogram SI 15–SI 16 in Supplementary Material).

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Claulancoumside (
6
): [α]2D1 +24.3 (MeOH; c 1.1), UV (MeOH) λmax (log
ε
): 325 (4.18), 256 (3.53),
223(4.18) nm; IR (KBr, νmax, cm−1): 3341, 2903, 2834, 1702, 1618, 1527, 1471; NMR data (SI 17–SI 21 in
Supplementary Material) found in Table 2; positive ESIMS m/z: 463 [M + Na]+.
β-d-Glucopyranosyl (2S,3R)-3-hydroxyarmesin (7
10-O- ): ESIMS m/z: 447 [M + Na]+; 1H NMR
(400 MHz, MeOD)
δ
: 7.79 (1H, d, J = 9.6 Hz, H-4), 7.45 (1H, s, H-5), 6.70 (1H, s, H-8), 6.03 (1H, d,
J = 9.6 Hz, H-3), 5.02 (1H, d, J = 6.2 Hz, H-30), 4.31 (1H, d, J = 6.2 Hz, H-20), 4.31 (1H, d, J = 7.8 Hz,
H-1000), 3.14 (2H, m, H-6000), 2.94 (1H, d, J = 6.2 Hz, H-3000), 2.83 (1H, br s, H-5000), 2.82 (1H, m, H-4000),
2.66 (1H, m, H-1000), 1.23 (3H, s, H-3”), and 1.22 (3H, s, H-2”); 13C NMR (101 MHz, MeOD)
δ: 160.9 (s,
C-2), 112.2 (d, C-3), 145.4 (d, C-4), 113.3 (s,0C-4a), 126.1 (d, C-5), 129.1 (s, C-6), 162.8 (s, C-7), 98.2 (d, C-8),
156.5 (s, C-8a), 92.3 (d, C-20), 70.6 (d, C-3 ), 78.0 (s, C-1”), 25.1 (q, C-2”), 23.3 (q, C-3”), 97.8 (d, C-1000),
73.9 (d, C-2000), 77.4 (d, C-3000), 70.3(d, C-4000), 77.1 (d, C-5000), and 61.3 (t, C-6000) (NMR spectrogram SI
22–SI 26 in Supplementary Material).
Clausesquiside A (
8
): [α]2D0
−
113.4◦ (MeOH; c 0.68); UV (MeOH) λmax (log
15.6 (241) and +0.8 (322) nm; IR (KBr, νmax, cm−1): 3447, 2981, 1668, 1105, 1043,
ε): 237 (3.21); CD (c 0.0042,
MeOH) ∆ε nm):
(
λ
−
961; NMR data (SI 27–SI 28 in Supplementary Material) found in Table 3; positive ESIMS m/z: 425
[M + Na]+, HRESIMS m/z: 425.1933 (Calculated for: [C19H30O9 + Na]+, 425.1928).
Clausesquiside B (
9
): [α]2D0
−
101.3◦ (MeOH; c 0.71); CD (c 0.0036, MeOH) ∆ε
(
λ
nm):
−
7.1 (321),
+186.2 (254) nm; IR (KBr, νmax, cm−1): 3439, 2979, 1675, 1100; NMR data (SI 29–SI 33 in Supplementary
Material) found in Table 3; positive ESIMS m/z: 409 [M + Na]+.
(6R,9R,4Z,7E)-9,10-Dihydroxy-4,7-megastigmadien-3-one-9-O-
β
-d-glucopyranoside (10): [α]D20
5.85 (1H, br s, H-4),
:
−
63.13 (c 0.82, MeOH); ESIMS m/z: 393 [M + Na]+; 1H-NMR (400 MHz, MeOD):
δ
5.83 (1H, dd, J = 15.4, 9.4 Hz, H-7), 5.66 (1H, dd, J = 15.4, 7.4 Hz, H-8), 4.43 (1H, m, H-9), 4.31 (1H, d,
J = 7.1 Hz, H-10), 3.90 (1H, dd, 12.0, 2.1 Hz, H-60a), 3.63 (1H, dd, J = 11.0, 3.9 Hz, H-10a), 3.57 (1H, dd,
J = 11.0, 3.9 Hz, H-10b), 3.54 (1H, m, H-60b), 3.30–3.13 (4H, m, H-20, 30, 40 and 50), 2.71 (1H, d, J = 9.4 Hz,
H-6), 2.43 and 2.00 (1H each, d, J = 16.6 Hz, H-2), 1.95 (3H, s, H-13), 1.01 (3H, s, H-12), and 0.97 (3H, s,
H-11); 13C NMR (101 MHz, MeOD):
δ 35.7 (s, C-1), 48.3 (t, C-2), 200.6 (s, C-3), 124.9 (d, C-4), 164.1 (s,
C-5), 55.6 (d, C-6), 128.9 (d, C-7), 134.1 (d, C-8), 78.3 (d, C-9), 7 (t, C-10), 26.7 (q, C-11), 26.2 (q, C-12),
22.4 (q, C-13), 99.7 (d, C-10), 3.4 (d, C-20), 76.7 (d, C-30), 770.3 (d, C-40),76.7 (d, C-50), and 61.4 (d, C-60)
(NMR spectrogram SI 34–SI 35 in Supplementary Material).
(6R,9S)-Roseoside (11): [α]D20
MeOD):
:
−
75.6 (c 1.00, MeOH); ESIMS m/z: 409 [M + Na]+; 1H-NMR (400 MHz,
δ
: 5.90 (1H, d, J = 15.6 Hz, H-7), 5.78 (1H, br s, H-4), 5.64 (1H, dd, J = 15.6, 7.1 Hz, H-8), 4.44
(1H, m, H-9), 4.18 (1H, d, J = 7.5 Hz, H-10), 3.78 (1H, dd, J = 10.5, 2.2 Hz, H- H-60a), 3.63 (1H, dd,
J = 10.5, 2.2 Hz, H-60b), 3.21–3.17 (3H, m, H-20, 30 and 40), 3.11 (1H, m, H-50), 2.55 (1H, d, J = 16.7 Hz,
H-2a), 2.16 (1H, d, J = 16.7 Hz, H-2b), 1.84 (3H, s, H-13), 1.19 (3H, d, J = 6.4 Hz, H-10), 0.94 (3H, s, H-11),
and 0.92 (3H, s, H-12); 13C NMR (101 MHz, MeOD)
δ: 41.1 (s, C-1), 49.4 (t, C-2), 200.0 (s, C-3), 125.8 (d,
C-4), 165.9 (s, C-5) 78.7 (s, C-6), 132.3 (d, C-7), 132.4 (d, C-8), 73.5 (d, C-9), 23.4 (q, C-10), 20.9 (q, C-11),
22.2 (q, C-12), 18.3 (q, C-13), 99.9 (d, C-10), 73.3 (d, C-20), 76.9 (d, C-30), 70.1 (d, C-40), 76.8 (d, C-50), and
61.4 (t, C-60) (NMR spectrogram SI 36–SI 37 in Supplementary Material).
Adenosine (12): ESIMS m/z: 304 [M + Na]+; 1H-NMR (400 MHz, MeOD):
δ
8.07 (1H, s, H-8), 7.90
(1H, s, H-2), 7.13 (2H, br s, 6-NH2), 5.64 (1H, d, J = 6.1 Hz, H-10), 4.38 (1H, m, H-20), 3.90 (1H, m, H-30),
3.73 (1H, m, H-40), 3.41 (1H, m, H-50a), and 3.35 (1H, m, H-50b); 13C NMR (101 MHz, MeOD)
δ 153.0 (d,
C-2), 149.6 (s, C-4), 119.9 (s, C-5), 156.7 (s, C-6), 140.5 (d, C-8), 88.5 (d, C-10), 74.1 (d, C-20), 71.2 (d, C-30),
86.5 (d, C-40), and 62.2 (t, C-50) (NMR spectrogram SI 38–SI 42 in Supplementary Material).
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations and UV spectra were measured using a Horiba SEPA-300 polarimeter (Horiba,
Tokyo, Japan) and shimadzu UV-2401 A spectrophotometer (Shimadzu, Tokyo, Japan), respectively.
IR spectra were recorded with a Tensor 27 Fourier transform infrared spectroscopy (FT-IR) spectrometer

Molecules 2019, 24, 3124
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with KBr pellets (BioRad, Hercules, CA, USA). Mass Spectrometry (MS) were recorded on an API
QSTAR Pular-1 mass spectrometer (VG, Manchester, UK). High Resolution Electrospray Ionization
mass Spectroscopy (HRESIMS) were obtained with a Bruker Daltonics, Inc. micro-TOF-Q spectrometer.
1
The H and 13C NMR spectra were acquired with a Bruker AV-400 (1H: 400 MHz, 13C: 101 MHz)
spectrometer in CD3OD with tetramethylsilane as the internal standard at room temperature (Bruker,
Bremerhaven, Germany). Semipreparative reversed-phase High Performance Liquid Chromatography
(HPLC) was performed on an Agilent 1260 apparatus equipped with a UV detector and an Agilent
Eclipse (XDB-C18, 5
µ
m, 9.4
×
250 mm) column at a flow rate of 2 mL/min (Agilent, Palo Alto, CA, USA).
Column chromatography (CC) was performed on silica gel (100–200 mesh, 200–300 mesh) and TLC
was carried out on precoated silica gel GF254 glass plates (Qingdao Marine Chemical, Inc., Qingdao,
China). Column chromatography (CC) was performed on sephadex LH-20 (Pharmacia, New Jersey,
NJ, USA).
3.2. Plant Material
The stems and leaves of C. lansium (three-year-old) were collected by pruning and air-dried
in Qingyuan (Latitude N 23◦700; Longitude E 113◦030; altitude 71 m), Guangdong Province, China,
in September 2015, which were identified by Professor Zhang Zhi-Yong (a botanist) of College of
Agriculture, Jiangxi Agricultural University, Nanchang, China. A voucher specimen (no. 2015912) has
been deposited in College of Agriculture, Jiangxi Agricultural University.
3.3. Extraction and Isolation
The air dried and powdered stems and leaves of C. lansium (11 Kg) were extracted by refluxing
95% methanol (20 L each) three times. This process yielded methanol-soluble extracts, which were
suspended in water and subsequently extracted with PE, EtOAc, and n-BuOH (3
×
5 L, each),
respectively. The n-BuOH part (130 g) was subjected to a reversed-phase column (RP-18) eluting with
MeOH-Water (10%–100%) to four sub-fractions (A1–A4). A3 was subjected to normal phase silica
gel CC (200–300 mesh) with a gradient system of CH2Cl2-MeOH (9:1-7:3, v/v) to give six fractions
A3-1–A3-6. A3-2 was further separated by normal phase silica gel CC (200–300 mesh) with a isocratic
system of CH2Cl2-MeOH (9:1) to give four fractions A3-2-1–A3-2-4. A3-2-2 was separated by HPLC
(mobile phase: H2O: MeOH (75:25, v/v)) to give
was separated by HPLC [H2O: MeOH (80:20, v/v)] to give
(4 mg) and (10 mg). A3-3 was separated by repeated CC (200–300 mesh) with an isocratic mixture of
2
(8 mg),
4
(6 mg), and
5
(9 mg). In the same way, A3-2-3
1
(6 mg) and
3 (11 mg). Similarly, A3-2-4 gave
6
7
CH2Cl2-MeOH (9:1, v/v) to produce five fractions A3-3-1–A3-3-5. After HPLC separation, A3-3-2 gave
(6 mg) and 10 (9 mg), A3-3-3 gave 9 (5 mg), and A3-3-4 produced 11 (8 mg) and 12 (13 mg) [35,36,48].
8
3.4. Determination of Absolute Configurations of Sugars
Compounds 1,6,8,9 (each compound 2–3 mg) were dissolved in 1 N HCl (4 mL) and heated at
90 ◦C under condition of reflux for 6 h. The reaction product was dissolved in H2O after evaporation
and partitioned with CH2Cl2. The aqueous layer containing sugars was concentrated, and then was
mixed with L-cysteine methyl ester hydrochloride. Anhydrous pyridine (1 mL) was added to the
mixture and heated at 60 ◦C for 2 h. The product was added to isothiocyanate (3 mg) and heated at
60 ◦C for another 2 h. The final reaction mixture was analyzed by HPLC under the following conditions:
an Agilent 1260 chromatograph equipped an Eclipse XDB-C18 column (5
temperature: 35 ◦C; mobile phase: isocratic elution of 25% CH3CN–H2O (V:V) in 50 mmol/L HCl;
flow rate: 0.8 mL/min; injection volume: 10 L; and UV detection wavelength: 250 nm. The standard
µm, 4.6
×
250 mm); column
µ
d-glucose and d-apiose were subjected under the same conditions. After the comparison of the retention
times of monosaccharide derivatives, the samples were confirmed to comprise of d-glucose (19.25 min)
and d-apiose (30.34 min), respectively [19,20].

Molecules 2019, 24, 3124
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4. Conclusions
In conclusion, 12 compounds were isolated from C. lansium. They were obtained from the
genus Clausena for the first time, including four new glycosides. The existence of these compounds
demonstrates the taxonomic significance of C. lansium in the genus Clausena and suggests that some
glycosides from this plant probably play a role in the anticancer activity of C. lansium to some extent.
Supplementary Materials: The 1D- and 2D-NMR spectroscopic data of compounds 1–12 in the paper are
available online.
Author Contributions: W.P. and B.L. designed and supervised the research. W.P. and X.F. analyzed spectroscopic
data. Y.L., Z.X., and X.S. performed the experiment and W.P., X.F., Y.L., Z.X., X.S., F.Z., G.H. and B.L. wrote,
reviewed, and approved the final version of manuscript.
Funding: This work was financially supported by the Funds of National Natural Science Foundation of China (No.
31660094), NSFC-Jiangxi Province (No. 20181BAB204002) and National Key R&D Program of China (2017YFD0301604).
Acknowledgments: We have a special thanks to Zhang Zhi-Yong (a botanist of College of Agriculture, Jiangxi
Agricultural University, Nanchang, China) for the collection and identification of C. lansium.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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