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Biochemical Systematics and Ecology 90 (2020) 104046

Contents lists available at ScienceDirect

Biochemical Systematics and Ecology


journal homepage: www.elsevier.com/locate/biochemsyseco

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 previous phytochemical investigation on C. lansium has resulted
the world, which are scattered throughout the subtropical and tropical in the isolation and identification of various types of natural products.
regions. There are approximately 10 species as well as 2 varieties in Among them, carbazole alkaloids and coumarins are the predominant
Southern China (Huang, 1997; Arbab et al., 2012; Peng et al., 2019). metabolites (Peng et al., 2017a,b). Other types of compounds include
Clausena lansium Lour. Skeels (Rutaceae), belonging to the genus aromatic glycosides, sesquiterpene glycosides, dihydrofuranocoumarin
Clausena of the family Rutaceae, is a high-growing, strongly scented glycosides, adenosine (Peng et al., 2019), and amides (Song et al.,
evergreen tree growing in South China and it flowers from April to May 2014).
(Huang, 1997). In the locality, the roots, stems, leaves, and seeds of C.
lansium have also been extensively applied in folk medicine or tradi- 3. Present study
tional Chinese medicine for the treatments of abdominal pain, malaria,
cold, dermatopathy, and snake bites (Arbab et al., 2012; Liu et al., 3.1. Extraction and isolation
2019).
The aerial parts of C. lansium were collected in Qingyuan, The air-dried powdered aerial parts of C. lansium (11 Kg) were ex-
Guangdong Province, China, in September 2015, which were identified tracted by refluxing 95% methanol (20 L each) three times. The three
by Professor Zhang Zhi-Yong (a botanist) of College of Agriculture, extraction solvents were combined and concentrated under a vacuum at
Jiangxi Agricultural University, Nanchang, China. A voucher specimen room temperature to yield a methanol crude extract. The crude extract
(no. 2015912) has been deposited in College of Agriculture, Jiangxi was submitted to the liquid-liquid fractionation with petroleum ether
Agricultural University. (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. Biochemical Systematics and Ecology 90 (2020) 104046

each), respectively. The three extractive solvents (EtOAc) were com- has never been reported before. So, 4 is reported as a new natural
bined and condensed to obtain the EtOAc extract (890 g). The EtOAc product for the first time in this paper as showed in Fig. 1.
part (890 g) was subjected to silica gel column chromatography Compound 5 was obtained as a colorless oil. In the 1H NMR, 13C-
(100–200 mesh) eluted with a gradient mixture of PE-Acetone (1 : 0 to NMR and DEPT spectra of 5 (Table 1), three aromatic protons [δH 7.49
0 : 1) to provide six major fractions: Fractions A-F. Fraction A (52 g) (1H, d, J = 6.9 Hz), 6.76 (1H, d, J = 6.9) and 7.50 (1H, s)] and 5
was chromatographed further on MPLC (MCI column) eluted with 30%, carbon signals attributable to five aromatic carbons [δC 151.0 (s), 147.2
50%, 70% and 100% MeOH to yield five sub-fractions: A1-A5. Sub- (s), 123.8 (d), 114.4 (d), and 112.4 (d)] indicated the presence of a 3,4-
fraction A2 was chromatographed on silica gel column (200–300 mesh) disubstituted pyridine ring in 5 (Francois et al., 1993). In addition, the
13
eluted with a isocratic system of PE–Acetone (5 : 1) to obtain 17 frac- C-NMR spectra of 5 also showed the presence of one methoxy [δC 55.0
tions, which were combined into four groups (A2-1 - A2-4) according to (q)]. In the HMBC spectrum (Fig. 2), the correlations from OCH3 (δH
TLC. A2-1 - A2-4 were isolated and purified separately by semi-pre- 3.83) to C-4 (δC 151.0) revealed that the methoxy was located at C-4.
paration HPLC (MeOH – H2O 8 : 2) to yield 2 (5 mg), 3 (7 mg), 12 Coupled with ESIMS (248 [M + Na]+) of 5, compound 5 was estab-
(6 mg) and 13 (9 mg). In the same way as sub-fraction A2, sub-fraction lished as 3-hydroxy-4-methoxypyridine. As a natural product, 5 has
A3 was treated with silica gel column (200–300 mesh) eluted with a never been reported before, so, 5 is also reported as a new natural
gradient system of PE–Acetone (5 : 1–3 : 1) to obtain 34 fractions product for the first time in this paper as showed in Fig. 1.
combined in 6 groups (A3-1 - A3-6) according to TLC. A3-2 was repeated Compound 14, a colorless amorphous solid, was identified as a new
chromatographed on silica gel column (200–300 mesh) eluted with a natural product. Its 1H NMR data (Table 1) showed that it possessed
isocratic system of PE-Acetone (4 : 1) to obtain a mixture of compounds three aromatic protons [δH 7.48 (1H, d, J = 8.7 Hz), 6.78 (1H, d,
1, 4, and 5. The mixture was isolated and purified by semi-preparation J = 8.7) and 7.50 (1H, s)] and one methylene [δH 3.34 (2H, s)].
HPLC (MeOH–H2O 75 : 25) to yield 1 (6 mg), 4 (5 mg) and 5 (4 mg). A3- Analysis of the 13C-NMR and DEPT data of 14 revealed signals corre-
3 was separated over a column of Sephadex LH-20 eluted eluted with a sponding to 8 carbon signals, comprising six aromatic carbons [δC
isocratic system of MeOH–CH2Cl2 (1 : 1) to give four combined frac- 150.1 (s), 146.2 (s), 122.8 (d), 121.1 (s), 113.4 (d) and 111.5 (d)], one
tions (A3-3-1-A3-3-4). A3-3-2 and A3-3-3 was separately isolated and pur- ester carbonyl carbon [δC 167.8 (s)] and one linking to N methylene [δC
ified by semi-preparation HPLC (MeOH–H2O 78 : 22 and 75 : 25) to 54.0 (t)]. Detailed comparison of the NMR data of 14 with those of 6-
yield 6 (7 mg), 8 (6 mg), 11 (9 mg), and 14 (5 mg). Sub-fraction A4 was hydroxy-2,3-dihydro-isoindol-1-one (Powers et al., 2009), coupled with
treated with a column of Sephadex LH-20 eluted eluted with a isocratic ESIMS (172 [M + Na]+) of 14, compound 14 was deduced as 6-hy-
system of MeOH–CH2Cl2 (1 : 1) to give five combined fractions (A4-1-A4- droxy-2,3-dihydro-isoindol-1-one. Just as 4 and 5, compound 14 has
5). A4-2 was repeated chromatographed on silica gel column (200–300 been reported in chemical synthesis, however, as a natural product, it
mesh) eluted with a isocratic system of PE-Acetone (4 : 1) to give 7 has never been reported before. So, 14 is also reported as a new natural
(7 mg) and 9 (6 mg). A4-2 was repeated isolated and purified by semi- product for the first time in this paper as showed in Fig. 1.
preparation HPLC (MeOH–H2O 76 : 24) to yield 10 (4 mg). Other compounds were identified as two indole alkaloids, namely 3-
oxoindole (1) (Maugard et al., 2001) and indole-3-carboxaldehyde (2)
3.2. Structure elucidation (Pedra and Sarma-Mamillapalle, 2012), one quinoline alkaloid, dicta-
mine (3) (Tangjitjaroenkun et al., 2012), four carbazole alkaloids, i.e.,
Compound 4 was obtained as colorless oil. It was identified as 4- murrayanine (6) (Li et al., 1991), claulansine G (7) (Liu et al., 2012),
hydroxypyridine, a new natural product, by analysis of ESIMS, 1H and clausine I (8) (Wu et al., 1996) and O-demethylmurrayanine (9)
13
C NMR, and comparison with data previously published in the lit- (Ngadjui et al., 1989), and four amides, paprazine (10) (Rahman et al.,
erature (Del Giudice et al., 1984). The 1H NMR data of 4 (Table 1) 1992; Suthiphasilp et al., 2019), atanine (11) (Jones et al., 2003; Chen
showed clear signals for four aromatic protons [δH 7.88 (2H, d, et al., 2004; Huang et al., 2011), 4-methoxy-1H-quinolin-2-one (12)
J = 8.5 Hz) and 6.82 (2H, d, J = 8.5)], and the 13C-NMR and DEPT (Jones et al., 2003; Chen et al., 2004; Huang et al., 2011) and 4-
data of 4 revealed signals corresponding to 5 carbon signals, comprising methoxy-1-methylquinolin-2-one (13) (Jones et al., 2003; Chen et al.,
four aromatic methines (δC 2 × 131.6, 2 × 114.6) and one aromatic 2004; Huang et al., 2011) (Fig. 1).
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- 3.3. Cytotoxicity assay
droxypyridine (Del Giudice et al., 1984), coupled with ESIMS (118 [M
+ Na]+) of 4, compound 4 was deduced as 4-hydroxypyridine. Al- All of the isolates were tested for cytotoxic activity against Hela
though 4 has been reported in many literatures, as a natural product, it cancer cell line using the (SRB) assay as described previously (Peng

Table 1
1
H-NMR and13C-NMR spectrum of 4, 5, 14.
Position 4 5 14

δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) δC

1 167.8 (s)
2 7.88 (d, 8.5) 131.6 (d) 7.50 (s) 114.4 (d) 3.34 (2H, s) 54.0 (t)
3 6.82 (d, 8.5) 114.6 (d) 147.2 (s) 146.2 (s)
4 161.7 (s) 151.0 (s) 7.48 (d, 8.7) 122.8 (d)
5 6.82 (d, 8.5) 114.6 (d) 6.76 (d, 6.9) 112.4 (d) 6.78 (d, 8.7) 113.4 (d)
6 7.88 (d, 8.5) 131.6 (d) 7.49 (d, 6.9) 123.8 (d) 150.1 (s)
7 7.50 (s) 111.5 (d)
8 121.1 (s)
OCH3 3.83 (s) 55.0 (q)

2
W.-W. Peng, et al. Biochemical Systematics and Ecology 90 (2020) 104046

Fig. 1. Structures of the compounds isolated from the aerial parts of C. lansium.

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
Fig. 2. The key HMBC correlation of 5. 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
et al., 2013, 2018). Known anticancer agent taxol was used as the po- a relationship between Clausena and Zanthoxylum genera of Rutaceae.
sitive control (IC50 value of 0.02 μmol L−1). The results revealed that Compound 2 was previously reported as a metabolite of dithiocarba-
carbazole alkaloids (6–9) showed weak cytotoxicity for HeLa with IC50 mates in the plant pathogenic fungus Leptosphaeria maculans (Desm.)
values in the range of 69.31–138.32 μM (Table 2). Ces. & De Not. (Pedra and Sarma-Mamillapalle, 2012), but it has never
been reported as a plant secondary metabolite, which may suggest that
4. Chemotaxonomic significance 2 is a product of parallel evolution between two organisms (plant C.
lansium and fungus Leptosphaeria maculans) through different metabolic
The genus Clausena is characterized by the presence of several dif- pathways.
ferent secondary metabolites, mainly coumarins and carbazole alka- Compound 8 and 9 have been isolated from Clausena excavata
loids (Peng et al., 2019). In the present study, 14 alkaloids were isolated Burm. F (Wu et al., 1996). and Clausena harmandiana (Pierre) Guill.
from the aerial part of C. lansium, which were classified as indole al- (Ngadjui et al., 1989), which shows carbazole alkaloids occur in a range
kaloids (1 and 2), quinoline alkaloid (3), pyridine alkaloids (4 and 5), species of Clausena. Overall, alkaloids are the main active metabolites
carbazole alkaloids (6–9) and amides alkaloids (10–14). Although of Clausena, especially carbazole alkaloids, which possess structural and
compounds (4, 5, 14) have been reported as synthetically produced biological diversity (Peng et and Liu et al., 2017). They also appear to of

Table 2
Cytotoxic activity against Hela cancer cell line of compounds 1–14.
Cell line IC50(μM) Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Taxol

HeLa – – – – – – 138.12 81.25 69.31 93.42 – – – – 0.02

“-”indicates no cytotoxic activity; Taxol as positive control.

3
W.-W. Peng, et al. Biochemical Systematics and Ecology 90 (2020) 104046

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