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Ca
Research Article
DOI:10.13179/canchemtrans.2013.01.03.0027
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
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Ca
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
167
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
122.2
H6
111.9
H7
8
1a
4a
5a
8a
1
2
131.0
145.5
118.9
126.4
143.9
23.4
122.6
3CH3
3CH3
1OH
8OH
3CHO
NH
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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COOH
20
HO
MeO
N
H
OMe
17
12
11
OH
13
14
2
O
10
16
15
7
6
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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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