Biochemical Systematics and Ecology 90 (2020) 104046

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Biochemical Systematics and Ecology

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Taxonomic significance and antitumor activity of alkaloids from Clausena T

lansium Lour. Skeels (Rutaceae)
Wen-Wen Penga,∗,1, Xiao-Xiang Fub,1, Zhong-Hua Xiongb, Hong-Liang Wub, Jing-Wen Changb,
Guang-Hua Huoa, Bao-Tong Lic,∗∗
a
Jiangxi Key Laboratory for Conservation and Utilization of Fungal Resources, Jiangxi Agricultural University, Nanchang, 330045, China
b
College of Agriculture, Jiangxi Agricultural University, Nanchang, 330045, China
c
School of Land Resources and Environment, Jiangxi Agricultural University, Nanchang, 330045, China

ARTICLE INFO ABSTRACT

Keywords: Phytochemical analysis of isolates from the aerial parts of Clausena lansium Lour. Skeels (Rutaceae) led to the
Clausena lansium identification of 14 alkaloids, including two indole alkaloids (1 and 2), one quinoline alkaloid (3), two pyridine
Alkaloids alkaloids (4 and 5), four carbazole alkaloids (6–9) and five amides alkaloids (10–14). The phytochemical
Cytotoxic activity structures of the alkaloids were established by means of NMR and MS spectral analyses. Compounds (4, 5, 14)
were three new natural products, while 1–3 and 10 were firstly reported from the genus Clausena and 8 and 9
were isolated from this species for the first time. The chemotaxonomic significance of these isolated alkaloids has
also been discussed. All the isolated alkaloids were tested for their cytotoxic activity against Hela cancer cell line.
Among them, four carbazole alkaloids 6–9 exhibited weak cytotoxicity with IC50 values ranging from 69.31 to
138.32 μM.

1. Subject and source 2. Previous work

The genus Clausena (Rutaceae) contains approximately 30 species in
the world, which are scattered throughout the subtropical and tropical
regions. There are approximately 10 species as well as 2 varieties in
Southern China (Huang, 1997; Arbab et al., 2012; Peng et al., 2019).
Clausena lansium Lour. Skeels (Rutaceae), belonging to the genus
Clausena of the family Rutaceae, is a high-growing, strongly scented
evergreen tree growing in South China and it flowers from April to May
(Huang, 1997). In the locality, the roots, stems, leaves, and seeds of C.
lansium have also been extensively applied in folk medicine or tradi-
tional Chinese medicine for the treatments of abdominal pain, malaria,
cold, dermatopathy, and snake bites (Arbab et al., 2012; Liu et al.,
2019).
The aerial parts of C. lansium were collected in Qingyuan,
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.
The previous phytochemical investigation on C. lansium has resulted
in the isolation and identification of various types of natural products.
Among them, carbazole alkaloids and coumarins are the predominant
metabolites (Peng et al., 2017a,b). Other types of compounds include
aromatic glycosides, sesquiterpene glycosides, dihydrofuranocoumarin
glycosides, adenosine (Peng et al., 2019), and amides (Song et al.,
2014).
3. Present study
3.1. Extraction and isolation
The air-dried powdered aerial parts of C. lansium (11 Kg) were ex-
tracted by refluxing 95% methanol (20 L each) three times. The three
extraction solvents were combined and concentrated under a vacuum at
room temperature to yield a methanol crude extract. The crude extract
was submitted to the liquid-liquid fractionation with petroleum ether
(PE), ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) (3 × 5 L,

∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: pengwenwen123@sina.com (W.-W. Peng), libt66@163.com (B.-T. Li).
1
contributed equally to this study.

https://doi.org/10.1016/j.bse.2020.104046
Received 5 February 2020; Received in revised form 29 March 2020; Accepted 4 April 2020
Available online 14 April 2020
0305-1978/ © 2020 Published by Elsevier Ltd.
W.-W. Peng, et al.
each), respectively. The three extractive solvents (EtOAc) were com-
bined and condensed to obtain the EtOAc extract (890 g). The EtOAc
part (890 g) was subjected to silica gel column chromatography
(100–200 mesh) eluted with a gradient mixture of PE-Acetone (1 : 0 to
0 : 1) to provide six major fractions: Fractions A-F. Fraction A (52 g)
was chromatographed further on MPLC (MCI column) eluted with 30%,
50%, 70% and 100% MeOH to yield five sub-fractions: A1-A5. Sub-
fraction A2 was chromatographed on silica gel column (200–300 mesh)
eluted with a isocratic system of PE–Acetone (5 : 1) to obtain 17 frac-
tions, which were combined into four groups (A2-1 - A2-4) according to
TLC. A2-1 - A2-4 were isolated and purified separately by semi-pre-
paration HPLC (MeOH – H2O 8 : 2) to yield 2 (5 mg), 3 (7 mg), 12
(6 mg) and 13 (9 mg). In the same way as sub-fraction A2, sub-fraction
A3 was treated with silica gel column (200–300 mesh) eluted with a
gradient system of PE–Acetone (5 : 1–3 : 1) to obtain 34 fractions
combined in 6 groups (A3-1 - A3-6) according to TLC. A3-2 was repeated
chromatographed on silica gel column (200–300 mesh) eluted with a
isocratic system of PE-Acetone (4 : 1) to obtain a mixture of compounds
1, 4, and 5. The mixture was isolated and purified by semi-preparation
HPLC (MeOH–H2O 75 : 25) to yield 1 (6 mg), 4 (5 mg) and 5 (4 mg). A3-
3 was separated over a column of Sephadex LH-20 eluted eluted with a
isocratic system of MeOH–CH2Cl2 (1 : 1) to give four combined frac-
tions (A3-3-1-A3-3-4). A3-3-2 and A3-3-3 was separately isolated and pur-
ified by semi-preparation HPLC (MeOH–H2O 78 : 22 and 75 : 25) to
yield 6 (7 mg), 8 (6 mg), 11 (9 mg), and 14 (5 mg). Sub-fraction A4 was
treated with a column of Sephadex LH-20 eluted eluted with a isocratic
system of MeOH–CH2Cl2 (1 : 1) to give five combined fractions (A4-1-A4-
5). A4-2 was repeated chromatographed on silica gel column (200–300
mesh) eluted with a isocratic system of PE-Acetone (4 : 1) to give 7
(7 mg) and 9 (6 mg). A4-2 was repeated isolated and purified by semi-
preparation HPLC (MeOH–H2O 76 : 24) to yield 10 (4 mg).
3.2. Structure elucidation
Compound 4 was obtained as colorless oil. It was identified as 4-
hydroxypyridine, a new natural product, by analysis of ESIMS, 1H and
13
C NMR, and comparison with data previously published in the lit-
erature (Del Giudice et al., 1984). The 1H NMR data of 4 (Table 1)
showed clear signals for four aromatic protons [δH 7.88 (2H, d,
J = 8.5 Hz) and 6.82 (2H, d, J = 8.5)], and the 13C-NMR and DEPT
data of 4 revealed signals corresponding to 5 carbon signals, comprising
four aromatic methines (δC 2 × 131.6, 2 × 114.6) and one aromatic
quaternary carbon (δC 161.7), indicating a 1-substituted pyridine ring
in 4. Detailed comparison of the NMR data of 4 with those of 4-hy-
droxypyridine (Del Giudice et al., 1984), coupled with ESIMS (118 [M
+ Na]+) of 4, compound 4 was deduced as 4-hydroxypyridine. Al-
though 4 has been reported in many literatures, as a natural product, it
Table 1
1
H-NMR and13C-NMR spectrum of 4, 5, 14.
Position 4 5
δH (J in Hz) δC δH (J in Hz)
1 167.8 (s)
2 7.88 (d, 8.5) 131.6 (d) 7.50 (s)
3 6.82 (d, 8.5) 114.6 (d)
4 161.7 (s)
5 6.82 (d, 8.5) 114.6 (d) 6.76 (d, 6.9)
6 7.88 (d, 8.5) 131.6 (d) 7.49 (d, 6.9)
7 7.50 (s)
8 121.1 (s)
OCH3 3.83 (s)
2
Biochemical Systematics and Ecology 90 (2020) 104046
has never been reported before. So, 4 is reported as a new natural
product for the first time in this paper as showed in Fig. 1.
Compound 5 was obtained as a colorless oil. In the 1H NMR, 13C-
NMR and DEPT spectra of 5 (Table 1), three aromatic protons [δH 7.49
(1H, d, J = 6.9 Hz), 6.76 (1H, d, J = 6.9) and 7.50 (1H, s)] and 5
carbon signals attributable to five aromatic carbons [δC 151.0 (s), 147.2
(s), 123.8 (d), 114.4 (d), and 112.4 (d)] indicated the presence of a 3,4-
disubstituted pyridine ring in 5 (Francois et al., 1993). In addition, the
13
C-NMR spectra of 5 also showed the presence of one methoxy [δC 55.0
(q)]. In the HMBC spectrum (Fig. 2), the correlations from OCH3 (δH
3.83) to C-4 (δC 151.0) revealed that the methoxy was located at C-4.
Coupled with ESIMS (248 [M + Na]+) of 5, compound 5 was estab-
lished as 3-hydroxy-4-methoxypyridine. As a natural product, 5 has
never been reported before, so, 5 is also reported as a new natural
product for the first time in this paper as showed in Fig. 1.
Compound 14, a colorless amorphous solid, was identified as a new
natural product. Its 1H NMR data (Table 1) showed that it possessed
three aromatic protons [δH 7.48 (1H, d, J = 8.7 Hz), 6.78 (1H, d,
J = 8.7) and 7.50 (1H, s)] and one methylene [δH 3.34 (2H, s)].
Analysis of the 13C-NMR and DEPT data of 14 revealed signals corre-
sponding to 8 carbon signals, comprising six aromatic carbons [δC
150.1 (s), 146.2 (s), 122.8 (d), 121.1 (s), 113.4 (d) and 111.5 (d)], one
ester carbonyl carbon [δC 167.8 (s)] and one linking to N methylene [δC
54.0 (t)]. Detailed comparison of the NMR data of 14 with those of 6-
hydroxy-2,3-dihydro-isoindol-1-one (Powers et al., 2009), coupled with
ESIMS (172 [M + Na]+) of 14, compound 14 was deduced as 6-hy-
droxy-2,3-dihydro-isoindol-1-one. Just as 4 and 5, compound 14 has
been reported in chemical synthesis, however, as a natural product, it
has never been reported before. So, 14 is also reported as a new natural
product for the first time in this paper as showed in Fig. 1.
Other compounds were identified as two indole alkaloids, namely 3-
oxoindole (1) (Maugard et al., 2001) and indole-3-carboxaldehyde (2)
(Pedra and Sarma-Mamillapalle, 2012), one quinoline alkaloid, dicta-
mine (3) (Tangjitjaroenkun et al., 2012), four carbazole alkaloids, i.e.,
murrayanine (6) (Li et al., 1991), claulansine G (7) (Liu et al., 2012),
clausine I (8) (Wu et al., 1996) and O-demethylmurrayanine (9)
(Ngadjui et al., 1989), and four amides, paprazine (10) (Rahman et al.,
1992; Suthiphasilp et al., 2019), atanine (11) (Jones et al., 2003; Chen
et al., 2004; Huang et al., 2011), 4-methoxy-1H-quinolin-2-one (12)
(Jones et al., 2003; Chen et al., 2004; Huang et al., 2011) and 4-
methoxy-1-methylquinolin-2-one (13) (Jones et al., 2003; Chen et al.,
2004; Huang et al., 2011) (Fig. 1).
3.3. Cytotoxicity assay
All of the isolates were tested for cytotoxic activity against Hela
cancer cell line using the (SRB) assay as described previously (Peng
14
δC δH (J in Hz) δC
114.4 (d) 3.34 (2H, s) 54.0 (t)
147.2 (s) 146.2 (s)
151.0 (s) 7.48 (d, 8.7) 122.8 (d)
112.4 (d) 6.78 (d, 8.7) 113.4 (d)
123.8 (d) 150.1 (s)
111.5 (d)
55.0 (q)
W.-W. Peng, et al.
Fig. 1. Structures of the compounds isolated from the aerial parts of C. lansium.
Fig. 2. The key HMBC correlation of 5.
et al., 2013, 2018). Known anticancer agent taxol was used as the po-
sitive control (IC50 value of 0.02 μmol L−1). The results revealed that
carbazole alkaloids (6–9) showed weak cytotoxicity for HeLa with IC50
values in the range of 69.31–138.32 μM (Table 2).
4. Chemotaxonomic significance
The genus Clausena is characterized by the presence of several dif-
ferent secondary metabolites, mainly coumarins and carbazole alka-
loids (Peng et al., 2019). In the present study, 14 alkaloids were isolated
from the aerial part of C. lansium, which were classified as indole al-
kaloids (1 and 2), quinoline alkaloid (3), pyridine alkaloids (4 and 5),
carbazole alkaloids (6–9) and amides alkaloids (10–14). Although
compounds (4, 5, 14) have been reported as synthetically produced
Table 2
Cytotoxic activity against Hela cancer cell line of compounds 1–14.
Cell line IC50(μM) Compound
1 2 3 4 5 6
HeLa – – – – – –
“-”indicates no cytotoxic activity; Taxol as positive control.
Biochemical Systematics and Ecology 90 (2020) 104046
chemicals, this is the first report of them occurring naturally in plants.
Moreover, this is the first report of compounds (1–3 and 10) from the
genus Clausena and 8 and 9 from C. lansium. Compounds (1 and 10)
were previously reported in other families. For example, compound 1
from Isatis tinctoria (Woad) (Cruciferae) (Maugard et al., 2001) and 10
from Dasymaschalon dasymaschalum (Blume) Turner I.M. (Annonaceae)
(Rahman et al., 1992) and Fumaria indica (Hausskn.) Pugsley (Fumar-
iaceae) (Suthiphasilp et al., 2019). However, more data are needed to
show a relationship between families Cruciferae, Fumariaceae, Anno-
naceae and Rutaceae. Compound 3 was also found from Zanthoxylum
genera of Rutaceae (Tangjitjaroenkun et al., 2012), which may indicate
a relationship between Clausena and Zanthoxylum genera of Rutaceae.
Compound 2 was previously reported as a metabolite of dithiocarba-
mates in the plant pathogenic fungus Leptosphaeria maculans (Desm.)
Ces. & De Not. (Pedra and Sarma-Mamillapalle, 2012), but it has never
been reported as a plant secondary metabolite, which may suggest that
2 is a product of parallel evolution between two organisms (plant C.
lansium and fungus Leptosphaeria maculans) through different metabolic
pathways.
Compound 8 and 9 have been isolated from Clausena excavata
Burm. F (Wu et al., 1996). and Clausena harmandiana (Pierre) Guill.
(Ngadjui et al., 1989), which shows carbazole alkaloids occur in a range
species of Clausena. Overall, alkaloids are the main active metabolites
of Clausena, especially carbazole alkaloids, which possess structural and
biological diversity (Peng et and Liu et al., 2017). They also appear to of
7 8 9 10 11 12 13 14 Taxol
138.12 81.25 69.31 93.42 – – – – 0.02
3
W.-W. Peng, et al.
systematic importance. However, little attention has been paid toward
the other alkaloids of this plant, the current study demonstrates the
presence of indole, quinoline, and pyridine alkaloids. Therefore, it is
suggested that some scarce alkaloids from this plant probably play a
role in differentiating C. lansium from other species in the genera.
CRediT authorship contribution statement
Wen-Wen Peng: Writing - original draft. Xiao-Xiang Fu: Writing -
original draft. Zhong-Hua Xiong: Investigation. Hong-Liang Wu: Data
curation. Jing-Wen Chang: Investigation. Guang-Hua Huo: Formal
analysis. Bao-Tong Li: Writing - review & editing.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
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).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bse.2020.104046.
Biochemical Systematics and Ecology 90 (2020) 104046
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