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Research Article

DOI:10.13179/canchemtrans.2013.01.03.0027

A New Cytotoxic Carbazole Alkaloid Isolated from the Stem


Bark of Malaysian Clausena excavata
Tian Hai Peh1, Gin Keat Lim2**, Yun Hin Taufiq-Yap1*, Gwendoline Cheng Lian Ee1,
Marwardi Rahman1, and Mohd Aspollah Sukari1
1

Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Malaysia
2
School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia
* Corresponding author, Email: yap@science.upm.edu.my Phone: +603 8946 6809
** Co-author, Email: limgk@usm.my Phone: +604 653 4028 Fax: +604 657 4854
Received: July 22, 2013 Revised: August 25, 2013 Accepted: August 30, 2013 Published: September 2, 2013

Abstract: Malaysia is one of the richest in its biodiversity in the world. There are not less than 12,000
plants species in its rain forest. The aim of this study is a continuous investigation for medicinal plants in
Malaysia, especially the chemical constituents that are poses significant activities, i.e. anti-cancer.
Clausena excavata (Rutaceae) has been known as a very rich in carbazole alkaloids, coumarins and
limonoids species. In the present study, one new carbazole alkaloid, 1,8-dihydroxy-3-formyl-4prenylcarbazole (ClausineTH), and two other known compounds, Clausenarin (coumarin) and
ClausineK (carbazole alkaloid), were isolated from the methanol extract of the stem bark of Clausena
excavata, collected from Kedah, Malaysia. Structures of these compounds were confirmed by various
spectroscopic analyses including GC-MS, NMRs, and FTIR. All pure compounds isolated were tested for
their cytotoxicity against CEM-SS cell lines, with the ClausineTH and ClausineK gave a very strong
activity with an IC50 value of 2.1 g/mL and 5.1 g/mL, respectively.
Keywords: Clausena excavata; carbazole alkaloid; ClausineTH; ClausineK;
cells line

Clausenarin; CEMSS

1. INTRODUCTION
Malaysia has around 12,000 species of flowering plants, of which around 1300 are said to be
medicinal [1] and only about few hundreds have been investigated for their potential. Clausena excavata
from the Rutaceae family is a wild shrub that grows up to 1.5 meters high is extensively distributed
throughout Southeast Asian, India and China. Clausena excavata, locally known as pokok kemantu

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Ca
3'

2'

5
6
7

A
8

OH

1'

CHO

4
5a

4a
1a

8a

N
H
OH

Figure 1: Structure of ClausineTH (1)


(ghostly tree) or pokok cerek (diarrhea tree) is one of the Malaysian species of ulam (salad) that is
known to have high antioxidant properties. Various parts of the plant have been used traditionally,
throughout the Southeast Asia countries for the treatment of cold, malaria, AIDS, dermatopathy,
abdominal pain, and snake-bite, and also as a detoxification agent [2-3].
Clausena excavata is very rich in carbazole alkaloids, coumarins and limonoids [4]. Up to date,
there are approximately sixty carbazole alkaloidal compounds and fifty coumarin compounds isolated
from the leaves, bark or roots of Clausena excavata [2], with the recent (years 2010 and 2012) isolations
of binorponcitrin [2] (coumarin dimer), dictamine [2] (furoquinoline alkaloid), murrayacine [2] and
sansokamine [5] (carbazole alkaloids). Additional to that, a new carbazole alkaloid (excavatineA) and
two new alkaloids (excavatineB and C) were isolated from the Clausena excavata collected from
Yunnan, P. R. China reported by Peng et al. in 2013 [6].
Pharmaceutical and biological agents are derived from two major sources, natural and synthetic.
The later including the biotechnological adaptation of nature compounds. These natural sources have
yielded a wealth of biologically active compounds, many of these have entered the market place, either as
pharmaceutical agents or biological tools for the world [7].
As for the history of Clausena excavata, as early as 1994, ClausineD [8] and clausenaquinoneA
[3] have been reported showed potent inhibitory activity of the rabbit platelet aggregation.
ClausenaquinoneA [3] also gave a potent inhibitory activity of cytotoxic against the HCT-8, RPMI-7951
and TE 671 tumor cells. Several biological activities have also been on the genus of Clausena. From
clausena anisata, carbazole alkaloids [9], clausenol and clausenine isolated demonstrated high antimicrobial and anti-fungi acticities against the microorganism tested. While from the extract of clausena
harmandiana [10], three known compounds isolated, heptaphylline, dentatin and clausarin exhibited antiplasmodial activity against Plasmodium falciparum.
2. MATERIAL AND METHODS
2.1 Plant Material
The stem bark of Clausena excavata used in this study was obtained from Jabi, Kedah, Malaysia.
The sample was identified by Dr. Rusea Go of Biology Department, Universiti Putra Malaysia. Voucher
specimens have been deposited in the Herbaria of this Department.
2.2 Extraction of Clausena excavata (Stem Bark)
The stem bark of Clausena excavata was air dried and ground into fine powder to give 800 g
sample. Extraction was performed by continuously soaking the dried ground plant material with a range
of solvents such as hexane, ethyl acetate (denoted as EA thereafter) and methanol. Hexane was used first
to remove non-polar organic compounds, waxes and fats. The sample was drained out after three days and

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CHO

CHO

CH3
N
H

N
H
OH

OH

OH

m/e 295

OH

m/e 280

CH2CH=C(CH3)2

CH=C(CH3)2

+
CHO

N
H

N
H
OH

CHO

OH

OH

m/e 240

OH

m/e 226
Figure 2: Fragmentation Patterns of (1) in the MS spectra

H3C

CH3

H
H

H
H

CHO

N
H
OH

H
OH

Figure 3: 13C1H Coupling Pattern Observed in HMBC Spectra of (1).


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this procedure was repeated twice. This process was followed by the treatment with EA and lastly
methanol was used to remove more polar compounds. Each extract obtained was filtered and evaporated
to dryness. After removal of the solvent, the crude methanol extract (39.2 g) was obtained.
A sample (10.0 g) of the crude methanol extract was directly chromatographed on a silica gel
column and eluted with mixtures of chloroform (denoted CH3Cl thereafter), EA and acetone to afford 48
fractions. Fractions 5 and 6 were combined (1.2 g) and re-chromatographed in a silica gel mini column to
give 15 fractions. Fractions 2 and 3 were eluted with 100 % chloroform, the solvent was removed and the
solid obtained was recrystallized from EA to give yellow needles crystal (12 mg). This solid gave a single
spot on thin-layer chromatograph (denoted as TLC hereafter) with an Rf = 0.65 (CHCl3 : EA = 9 : 1)
2.3 Cytotoxicity Assay
The CEMSS cell line (T-Lymphoblastic Leukemia) was obtained from the National Institutes
Cancer, Frederick, Maryland, USA. The cells were cultured and maintained in growth medium as
described [11]. Cytotoxicity was determined by performing the micro titration assay [12]. The tests were
performed in 96-well micro- titer plates. Each well was added with 100 L of varying concentrations of
isolated pure compounds prepared from the stock solutions by serial dilution in RPM I1640 medium.
Subsequently, each well was filled with 100 L of the cell suspension in complete growth medium at 12
105 cells/mL. The controls for each sample contain only untreated cells. The assay for each
concentration of isolated compounds was performed in triplicate and the culture plates were incubated at
37 oC with 5 % (v/v) CO2 for three days. The cytotoxicity index used was IC50, the concentration that
yielded 50 % cell inhibition when compared with the untreated control. Isolated compounds gave
cytotoxic index (IC50) 10 g/mL.
3. RESULTS AND DISCUSSION
3.1 Isolation of 1,8-dihydroxy-3-formyl-4-prenylcarbazole (ClausineTH)
ClausineTH (1) (see in figure 1) was obtained as yellowish needles (12 mg) with a melting point
of 194 196 oC. The mass spectrum indicated a molecular ion peak at m/z 295, which is consistent with
the molecular formula C18H17NO3. The peak at m/z 280 was due to the loss of a methyl group from the
molecular ion. One characteristic mass fragment at m/z 226 is due to the loss of a prenyl group [
CH2CH=C(CH3)2]. Meanwhile, the loss of two methyl groups gave a fragment ion at m/z 265 (refer to
Figure 2 for fragmentation patterns).
The IR spectrum indicated the presence of a broad band at 3438 cm-1 due to NH and OH groups.
The absorption observed at 2924 cm-1 was assigned to the CH stretching for CH3 and CH2. The spectrum
also showed a strong carbonyl band at 1655 cm-1. This was further confirmed by the 13CNMR which
indicated the presence of a C=O peak at 197.0 ppm.
The 1H-NMR spectrum of (1) showed a similar signal pattern to that of Clausine-D [13], except
for the absence of a hydroxyl signal at 8.89. The presence of a 3,3-dimethylallyl group in the compound
was suggested by the 1H-NMR signal: two singlet methyl signals at 1.68 and 1.86; a benzylic methylene
doublet at 3.74 (J = 6.8 Hz); a multiplet for vinylic proton at 5.40 and the mass fragmentation ion at
m/e 226 coupled with the appearance of the carbon signals at 25.9, 18.1, 23.4, 122.6 and 133.0 ppm in the
13
C-NMR spectrum which were assigned to 3CH3, 3CH3, C1, C2 and C3. The lack of
substituent in ring A was suggested by the three mutually coupling aromatic protons at 6.91 (dd, J = 7.8,
1.0 Hz), 7.08 (t, J = 8.0, 7.8 Hz) and 7.59 (dd, J = 7.8, 0.8 Hz) which were assigned to H7, H6 and H
5, respectively. The lower field signal at 7.59 is characteristic of H-5 of carbazoles [2-5, 13-17]. All

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Table 1: 1H, 13CNMR Chemical Shift () and Coupling Patterns of the Protons in HETCOR, COSY,
DEPT and HMBC Techniques of ClausineTH (1)
H/C

1H

13C

1
2
3
4
5

8.29 (s)
7.59 (dd, 7.8, 0.8 Hz)

158.5
126.7
116.4
110.6
111.9

H13C
HETCOR
H2
H5

7.08 (t, 8.0, 7.8 Hz)

122.2

H6

6.91 (dd, 7.8, 1.0 Hz)

111.9

H7

8
1a
4a
5a
8a
1
2

3.74 (d, 6.8 Hz, 2H)


5.40 (m)

131.0
145.5
118.9
126.4
143.9
23.4
122.6

3CH3
3CH3
1OH
8OH
3CHO
NH

1.68 (d, 1.2 Hz)


1.86 (s)
11.73 (s)
8.89 (br s)
9.97 (s)
10.23 (br s)

DEPT

H1
H2

H1H
COSY
7.08 (H-6),
6.91 (H-7)
7.59 (H-5),
6.91 (H-7)
7.08 (H-6),
7.59 (H-5)
5.40 (H2)
3.74 (H1)

133.0

25.9
18.1
197.0
-

3CH3
3CH3
-

CH3
CH3
CH
-

C
CH
C
C
CH

HMBC
(Figure 3)
CHO, H2
CHO
CHO, H1
H1, CHO
H7

CH

H5

CH

H5

C
C
C
C
C
CH2
CH

H5
H1, H2
H5
H6, H5
H7
H2
H1, 3CH3,
3CH3
H1, 3CH3,
3CH3
3CH3, H2
3CH3, H2
H2
-

Table 2: Cytotoxicity Activity of Pure Compounds against CEMSS Cells Line (T-Lymphoblastic
Leukemia)
Pure Compounds

IC50 (g/mL)

ClausineTH (1)

2.1

ClausineK (2)

5.1

Clausenarin (3)

Doxorubicin

0.1

Tamoxifen

36

Colchicine

0.02
Reference Compounds [18]

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these assignments were further substantiated by 1H1H COSY spectrum.


The 13CNMR spectrum showed the presence of 18 carbons atoms of which nine were quaternary
carbons at 110.6, 116.4, 118.9, 126.7, 131.0, 133.0, 143.9, 145.5 and 158.5 ppm and assigned to C4, C
3, C4a, C5a, C8, C3, C8a, C1a and C1, respectively. Six methine carbons which were observed
at 111.9, 112.0, 122.2, 122.6, 126.7 and 197.0 ppm were assigned to C7, C5, C6, C2, C2 and CHO,
respectively. The methylene carbon at 25.9 ppm is due to C-1 and two methyl groups resonate at 18.1 and
25.9 ppm. The 13CNMR signals of (1) were assigned using the DEPT experiment. 1H13C connectivity
obtained from 1H13C HETCOR and other correlations are summarized in Table 1. These spectral data
suggested the structure of (1). For clarity, the proposed gross structure was unambiguously confirmed by
the HMBC.
3.2 Cytotoxicity Assay
The pure organic compounds were tested on the viability of CEMSS cells line (T-Lymphoblastic
Leukemia). Table 2 shows the IC50 value of each compound. ClausineTH (1) gave an IC50 value of 2.1
g/mL, which is considered chemotherapeutically significant (IC50 4 g/mL). However, Clausine-K (2)
is considered active against cytotoxic activity with an IC50 value 5.1 g/mL, whereas Clausenarin (3) is
considered not cytotoxic with an IC50 value 30 g/mL.
O
23
21
22

COOH

20

HO
MeO

N
H

OMe

17

12
11

OH

13
14

2
O

10

16
15

7
6

Figure 4: Structure of Clausine-K (2) and Clausenarin (3)


Clausine-K (2). Yellow prisms (hot acetone), m.p. 254-256 oC. MS: C15H13NO4, m/z 271 [M]+.
UV max nm (log ): 244 (2.29), 257 (0.80), 282 (1.91), 303 (0.65), 320 (0.65).
IR max cm-1: 3412,
3317, 2930, 1665, 1618, 1579, 1443, 1346, 1322, 1287, 1239, 1201, 1163, 1116, 1083, 1036, 823, 679,
552. EIMS m/z (rel. int.): 271 ([M]+, 100), 256 (28), 240 (16), 212 (15), 196 (15), 183 (11), 169 (12), 153
(17), 140 (15), 127(14). 1HNMR (DMSOd6): 3.81 (3H, s, 7OMe), 3.87 (3H, s, 2OMe), 6.77 (1H,
dd, J = 8.5, 2.2 Hz, H6), 6.97 (1H, d, J = 2.2 Hz, H8), 7.03 (1H, s, H1), 7.94 (1H, d, J = 8.5, 2.2 Hz,
H5), 8.39 (1H, s, H4), 11.28 (1H, br. s, NH). 13C-NMR (DMSOd6): 55.3 (2OMe), 56.0 (7OMe),
94 (C1), 95.1 (C8), 108.1 (C6), 112.3 (C3), 115.8 (C4a), 116.3 (C5a), 120.5 (C5), 123.1 (C4),
141.6 (C8a), 143.4 (C1a), 157.4 (C2), 158.1 (C7) and 167.5 (C=O).
Clausenarin (3). White needles (hot acetone), m.p. 292-294 oC. Lit.. R UV max nm (log ): 269
(0.05), 243 (0.03), 208 (0.91). IR max cm-1: 3483, 3432, 2950, 1719, 1638, 1398, 1301, 1162, 1117, 1065,
1028, 920, 875, 804, 601, 474. 1HNMR (DMSOd6): 1.05 (3H, s, CH3), 1.37 (3H, s, CH3), 1.52 (3H, s,
CH3), 1.58 (3H, s, CH3), 1.65 (3H, s, CH3), 1.70 (1H, d, J =6.3 Hz, H2), 1.86 (1H, d, J =14.9 Hz, H
2), 2.48 (1H, dd, J =3.9, 3.9 Hz, H12), 2.60 (1H, dd, J =3.9, 4.2 Hz, H5), 2.63 (1H, s, H9), 2.86 (1H,

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s, H6), 3.14 (1H, t, J =14.2 Hz, H12), 3.47 (1H, d, J =15.4 Hz, H6), 3.72 (1H, s, H12), 4.00 (1H,
d, J =2.6 Hz, 1OH), 4.15 (1H, t, J =6.6 Hz, H1), 4.62 (1H, d, J =4.4 Hz,
11OH), 4.76 (1H, t, J
=5.1 Hz, H11), 5.54 (1H, s, H17), 6.48 (1H, d, J =1.0 Hz, H22), 7.57 (1H, t, J =1.7 Hz, H23), 7.59
(1H, t, J =0.7 Hz, H21). 13C-NMR (DMSOd6): 18.4 (CH3), 19.6(CH3), 20.5(CH3), 23.9(CH3),
33.6(CH3), 36.9 (C13), 40.0
(C12), 40.2 (C6), 44.6 (C2), 46.2 (C8), 47.1 (C9), 52.4 (C10),
50.7 (C5), 54.4 (C15), 65.5 (C14), 66.6 (C11), 70.5 (CC1), 79.0 (C17), 84.4 (C4), 111.0 (C22),
121.8 (C20), 142.3 (C21), 144.1 (C23), 168.0 (C16), 170.8 (C3), 208.7 (C7).
4. CONCLUSION
A new carbazole alkaloid (ClausineTH) and two known compounds ClausineK and
Clausenarin were successfully isolated as pure compounds from the methanol crude extract of the stem
back of Clausena excavata collected from Jabi, Kedah, Malaysia. These compounds were isolated from
the conventional column chromatography method and the structure elucidations we done using the
standard spectroscopic analyses (i.e. GC-MS, 1D-NMR, 2D-NMR and FTIR spectroscopy). The pure
compounds isolated were also tested for their cytotoxic activity against the CEM-SS cell lines. The
ClausineTH and ClausineK gave very significant activities, with the IC50 value of 2.1 g/mL and 5.1
g/mL, respectively. This study again proven that there are still lot more natural products in our nature
need further investigation before it could be beneficial to us.
ACKNOWLEDGEMENT
Authors wish to thank Mr. Tan Boon Keat for his help in conducting the cytotoxic testing and Dr.
Rusea Go for identifying the plant materials. This project was financially supported by the Malaysian
Government under the IRPA programme.
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