Cytotoxic agents from Terminalia arjuna

Article abstract of DOI:10.1055/s-2007-990258

Although a number of chemicals have been isolated from Terminalia arjuna, only a few have been evaluated for their biological significance. As a part of our drug discovery programme for cytotoxic agents from Indian medicinal plants, four novel cytotoxic agents arjunic acid (1), arjungenin (2), arjunetin (3) and arjunoglucoside I (4) were isolated from the bark of T. arjuna. Out of the four compounds, arjunic acid (1) was significantly active against the human oral (KB), ovarian (PA 1) and liver (HepG-2 & WRL-68) cancer cell lines. Further, the most active compound arjunic acid was converted into seven semi-synthetic ester derivatives 5-11. 2-O-Palmitoyl arjunic acid (6) showed two times more activity, while 2,3-di-O-acetyl-, 2-O-p-anisoyl-, 2,3-di-O-benzoyl- and 2, 3-di-O-p-nitrobenzoyl arjunic acid (7-10) showed 1.7-2.3 times less activity than the cytotoxic drug vinblastine against the liver cancer cell lines HepG-2 and WRL-68 respectively. Georg Thieme Verlag KG Stuttgart.

Full text of DOI:10.1055/s-2007-990258

Mohit Saxena1
Uzma Faridi2
Rupal Mishra1
M. M. Gupta1
M. P. Darokar2
S. K. Srivastava1
Digvijai Singh1
Suaib Luqman2
S. P. S. Khanuja2
Cytotoxic Agents from Terminalia arjuna
Abstract
more activity, while 2, 3-di-O-acetyl-, 2-O-p-anisoyl-, 2, 3-di-O-
benzoyl- and 2, 3-di-O-p-nitrobenzoyl arjunic acid (7±10)
Although a number of chemicals have been isolated from showed 1.7±2.3 times less activity than the cytotoxic drug vin-
Terminalia arjuna, only a few have been evaluated for their biolo- blastine against the liver cancer cell lines HepG-2 and WRL-68
gical significance. As a part of our drug discovery programme for respectively.
cytotoxic agents from Indian medicinal plants, four novel cyto-
toxic agents arjunic acid (1), arjungenin (2), arjunetin (3) and ar- Key words
junoglucoside I (4) were isolated from the bark of T. arjuna. Out Terminalia arjuna ´ Combretaceae ´ arjunic acid ´ cytotoxic ´ cyto-
of the four compounds, arjunic acid (1) was significantly active toxicity assay ´ human oral ´ colon ´ liver cancer cell lines
against the human oral (KB), ovarian (PA 1) and liver (HepG-2 &
WRL-68) cancer cell lines. Further, the most active compound ar- Supporting information available online at
junic acid was converted into seven semi-synthetic ester deriva- http://www.thieme-connect.de/ejournals/toc/plantamedica
tives 5±11. 2-O-Palmitoyl arjunic acid (6) showed two times
1486
Introduction
ents of T. arjuna [15], [16], [17], [18]. In a bioassay-guided separa-
tion of cytotoxic constituents from the bark, stem and leaves of T.
Over the past few years triterpenoids from higher plants have arjuna, gallic acid, ethyl gallate flavone, and leutonolin were
shown a wide range of biological activities [1], [2] such as cyto- found, in part, to be responsible for the rational underling the
toxic [3], [4], antitumour [5], antiviral [6], anti-inflamatory [7], use of T. arjuna in traditional cancer treatment [19]. Further in-
anti-HIV [8] and anti-HSV [9], [10]. The Arjun tree (Terminalia vestigation of T. arjuna resulted in the isolation and identifica-
arjuna; Combretaceae) is a well known medicinal plant whose tion of the antimutagenic agent ellagic acid [20] and the moder-
bark is extensively used in Ayurvedic medicine [11], particularly ately cytotoxic agent ellagitanin [21]. As a part of our drug dis-
as cardiac tonic [12], [13], [14]. Considerable work has been car- covery programme for cytotoxic agents from Indian medicinal
ried out on the chemical investigation of different parts of plants [22], [23], [24], the bark of T. arjuna was taken for detailed
Terminalia arjuna, which revealed the presence of a number of chemical investigation.
tannins, triterpenoid acids and their glycosides among others.
Tannins and oleane triterpene derivatives are the major constitu-
Affiliation
1 Analytical Chemistry Division, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, India
2 Genetic Resource and Biotechnology Division, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP,
Lucknow, India
Correspondence
S. K. Srivastava ´ Central Institute of Medicinal and Aromatic Plants ´ P.O. CIMAP ´ Lucknow ± 226015 ´ India ´
Phone: +91-522-235-8723, +91-522-235-9624´ Fax: +91-522-235-7136 ´
E-mail: santoshkumar_1955@yahoo.com
Received June 4, 2007 ´ Revised September 26, 2007 ´ Accepted October 2, 2007
Bibliography
Planta Med 2007; 73: 1486±1490 ꢀ Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2007-990258 ´ Published online November 16, 2007
ISSN 0032-0943

Materials and Methods
ethyl acetate in the ratio of 98:2, 95:5, 90:10, 85:15, 80:20,
75:25, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and
100. Fractions of 100 mL each were collected and detected by
Cell lines, chemicals and biochemicals
KB (human oral), PA 1 (human ovary), HepG-2 and WRL-68 (hu- TLC (SiO2, chloroform:methanol 9:1; vanillin-sulphuric acid),
man liver) cancer cell lines were procured from the cell reposi- which resulted in the isolation and characterization of arjunic
tory of the National Center for Cell Sciences (NCCS) at Pune, Ma- acid, 0.004%, m.p. 280 8C (decomposed) [25], [26]; arjungenin
harastra, India. Purity of each compound was assessed by HPLC 0.007%, m.p. 293±294 8C [27] and arjunetin 0.002%, m.p. 232±
and NMR and was > 99%. Spots on TLC plates were visualized 234 8C [27] (Fig.1).
with a spray reagent [vanillin-ethanol-sulphuric acid (1 g: 95
mL: 5 mL)] followed by heating for 15 min at 110 8C. Bioassays Isolation of arjunglucoside I (4) by preparative HPLC
were carried out as per known protocols. MTT [3-(4, 5-dimethyl- The column fractions eluted with hexane:ethyl acetate (5:95) on
thiazol-2-yl)-2,5-diphenyltetrazolium bromide] was purchased TLC was found to be a mixture of one major and several minor
from Sigma Aldrich (Bangalore, India). The Spectra Max 190 Mi- constituents. Hence, in order to purify the major compound,
cro plate Elisa reader was purchased from Molecular Devices Inc. these fractions were subjected to preparative HPLC, which yield-
(Sunnyvale, CA, USA). Unless stated otherwise, all other reagents ed a crystalline compound (0.001%, m.p. 232±233 8C) charac-
were purchased from Sigma Aldrich.
terized as arjunglucoside-I (Fig.1) on the basis of its physical and
spectroscopic data [28]. Chromatographic conditions employed
for preparative HPLC were: column: Supelcosil LC-18 (25 cm”
Plant material
The bark of Terminalia arjuna was collected from the medicinal 21.2 mm, 12 mm); mobile phase: methanol:H2O (50:50); flow
plants conservatory of the Central Institute of Medicinal and Aro- rate: 17 mL/min; l = 220 nm (SPD-M10Avp Shimadzu photodiode
matic Plants (CIMAP), Lucknow, Uttar Pradesh, India during the detector); column temperature: 268C, respectively. The HPLC used
month of January, 1999, identified in the Department of Botany consisted of LC-8A Shimadzu semipreparative equipment.
and Pharmacognosy at CIMAP where a voucher specimen (No.
5867) is maintained.
Chemical derivatization
The potential cytotoxic activity of arjunic acid prompted us to
carry out chemical derivatization of arjunic acid (Fig. 2) and the
Extraction and isolation of bioactive compounds
The air-dried T. arjuna bark (4.5 kg) was crushed, powdered and preparation of arjunic acid ester derivatives was carried out. Ar-
extracted with ethanol (3”5 L, 24h each). The dried ethanolic junic acid was dissolved in dry pyridine and then the respective
extract (563.7 g) was dissolved in water and successively extract- acid chlorides were added in 1:1.5 ratios. The reaction mixtures
ed with hexane (3”2 L), diethyl ether (3”2 L), ethyl acetate were kept overnight at room temperature (30±45 8C). After com-
(3”2 L) and methanol (3”2 L) to yield hexane (10.8 g), diethyl pletion of the reaction, ice/water was added (~15 mL) and reac-
ether (152.0 g), ethyl acetate (23.5 g) and methanol (329.2 g) ex- tion solutions were extracted three times with chloroform. The
tracts, respectively.
combined chloroform extract was washed with water (until it
was neutralized). The neutralized chloroform extracts were
1487
Further 100 g of diethyl ether extract were column chromato- dried over anhydrous Na2SO4 and the solvent removed under va-
graphed over silica gel (60±120 mesh, 1.5 kg; 6.5”1400 cm). cuum. Further purification of the ester derivatives on short col-
Gradient elution of the column was carried out with hexane and umns afforded the products 5±11 in 85±95% yields. All the deri-
Fig. 1 Cytotoxic compounds from Termina-
lia arjuna.
Saxena M et al. Cytotoxic Agents from ¼ Planta Med 2007; 73: 1486±1490

Fig. 2 General scheme for
the preparation of arjunic
acid ester derivatives.
vatives were characterized on the basis of their spectroscopic [34] prompted us to undertake a detailed chemical investigation
data (for 1H, 13C NMR, MS and [a]D data, see Supporting Informa- of T. arjuna bark, which resulted in the isolation and characteri-
tion).
zation of two triterpenic acids arjunic acid (1) and arjungenin (2)
and two saponins, arjunetin (3) and arjunglucoside (4), respec-
tively.
Cytotoxicity assay
The in vitro cytotoxicity testing was carried out by the method of
Woerdenbag et al. [29]. 2”103 cells/well were incubated in the Compounds 1±4 were tested against the human oral (KB), liver
5% CO2 incubator for 24h to enable them to adhere properly to
(WRL-68 and HepG-2) and ovarian (PA-1) cancer cell lines,
the 96-well polystyrene micro plates. Test compounds dissolved respectively, and the results are presented in Table 1. All the com-
in 100% DMSO in at least 5 doses were added and left for 6 h after pounds had cytotoxic activity against the various cancer cell
which the compound plus media was replaced with fresh media lines. Although arjunic acid (1) was 5 times less active against
and the cells were incubated for another 48 h in the CO2 incuba- liver (HepG-2) cancer cell line with respect to the cytotoxic drug
tor at 37 8C. The concentration of DMSO used in our experiments vinblastine, it was the most active cytotoxic constituent of T.
never exceeded 1.25%, which was found to be non-toxic to cells. arjuna against all tested cancer cell lines. This encouraged us to
Then, 10 mL MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenylte- prepare derivatives of arjunic acid, and a total of seven deriva-
trazolium bromide, Sigma M 2128] was added, and plates were tives (5±11) of arjunic acid (1) was prepared and their cytotoxic
incubated at 37 8C for 4h. 100 mL dimethyl sulphoxide were add- activities were evaluated (Table 1 and Fig. 3). All compounds (1
ed to all wells and mixed thoroughly to dissolve the dark blue and 5±11) were active against the oral, ovarian and liver cancer
crystals. After few minute at room temperature to ensure that cell lines, but among the seven derivatives (5±11) of arjunic acid,
all crystals were dissolved, the plates were read on a Spectra compound 6 and 7±10 were highly active (7±9 times) against
Max 190 Micro plate Elisa reader (Molecular Devices Inc.), at the liver cancer cell lines (HepG-2) and (WRL-68), respectively,
570 nm. Plates were normally read within 1 h of adding the in comparison to the starting material arjunic acid (1). On the
DMSO. The experiments were done in triplicate and the inhibi- other hand compounds 5 and 6 were significantly (4to 5 times)
tory concentration (IC) values were calculated as follows: % inhi- active against the oral cancer (KB) cell line with respect to arjunic
bition = [1±OD (570 nm) of sample well/ OD (570 nm) of control acid (1). When the activity results, IC90, of derivatives were com-
well]”100. IC90 and IC50 are the concentrations in mg/mL required pared with the reference cytotoxic drug, vinblastine, compound
for 90% and 50% inhibition of cell growth as compared to that of 6 showed two times more activity than vinblastine against the
1488
untreated control.
liver cancer HepG-2, while compounds 7±10 showed 1.7 to 2.3
times less activity against the liver cancer cell line WRL-68. But
it was interesting to note that with respect to the IC50 values,
Supporting information
1H-, 13C-NMR, MS and [a]D data of arjunic acid (1) and its deriva- compounds 7±10 showed 1.5±2.4times more activity than vin-
tives (5±11) are available as Supporting Information.
blastine against the liver cancer cell line WRL-68.
While studying the structure-activity relationship (SAR) of arju-
nic acid and its derivatives it was observed that acetylation of ar-
junic acid, such as to give 2,3-di-O-acetylarjunic acid (7), result-
Result and Discussion
Triterpenoids are an integral part of the human diet and in the ed in a ~7-fold increase (IC90) in activity against the liver cancer
last decade many triterpenoids have been reported to possess a (WRL-68) cell line. Further increasing the chain length from the
wide range of cytotoxic activity. Out of many triterpenoids, urso- 2-carbon diacetate (7) to the 12-carbon 2-O-laurylarjunic acid
lic and oleanolic acids have been studied in detail for their cyto- (11) and the 15-carbon compounds, 5 and 6, decreased the activ-
toxic activities such as inhibition of tumor genesis, tumor pro- ity 7- to 10-fold against the liver cancer cell line WRL-68 with re-
motion, angiogenesis [30], ultraviolet-B (UVB) radiation photo- spect to compound 7. But it was interesting to note that replace-
carcinogenesis [31], induction of tumor cell differentiation, inva- ment of the 2,3-diacetyl function with mono- or diaryl groups as
sion of tumor cells, metastasis [30] and effect on acute myeloid in the case of compounds 8±10 did not show any effect on activ-
leukemia [32]. The recent isolation of cytotoxic triterpenoids ity against the liver cancer cell line WRL-68.
from Akebla trifoliate and Clematis lingustifolia [33] and cytotoxic
triterpenoid saponins from Quillaja saponaria Molina (soap tree)
Saxena M et al. Cytotoxic Agents from ¼ Planta Med 2007; 73: 1486±1490

Table 1 IC50 and IC90 (ꢀg/mL) values for four isolated compounds 1 ± 4 and ester derivatives 5 ± 11 of arjunic acid (1) against human cancers cell
lines*
Compounds
KB
PA-1
IC50
HepG-2
IC50
WRL-68
IC50
IC50
IC90
IC90
IC90
IC90
Arjunic acid (1)
5.00  0.05 35.00  0.06
7.50  0.0910.00  0.09 0.70  0.05 .090  0.04
40.00  0.26 60.00  0.13
7.50  0.13 40.00  0.24
Arjungenin (2)
±
±
±
±
±
±
±
±
±
±
±
±
Arjunetin (3)
±
±
±
15.00  0.03 20.00  .020
Arjunglucoside I (4)
±
30.00  0.16 70.00  0.24
±
±
2,3-di-O-palmitoylarjunic acid (5)
2-O-palmitoylarjunic acid (6)
2,3-di-O-acetylarjunic acid (7)
2-O-p-anisoylarjunic acid (8)
2,3-di-O-benzoylarjunic acid (9)
0.70  0.07
0.95  0.08
7.00  0.08 30.00  0.03 40.00  0.03
8.50  0.05 9.00  0.03 20.00  0.02
0.04  0.03
0.45  0.03
6.00  0.05
8.50  0.0945.00  0.11
1.00  0.03 10.00  0.12 50.00  0.98
1.00  0.12 6.00  0.87
1.00  0.38
0.60  0.43
45.00  0.0975.00  0.08 .090  0.10 20.00  0.10
1.00  0.01 45.00  0.01
4.50  0.07 55.00  0.09
4.00  0.08 50.00  0.03
6.50  0.01
2.50  0.02
9.00  0.09
5.00  0.02
9.00  0.22 60.00  0.37
6.00  0.16 65.00  0.64
6.00  0.42
4.50  0.28
2,3-di-O-p-nitrobenzoyl arjunic
acid (10)
3.00  0.04 15.00  0.01 45.00  0.06 60.00  0.05
1.00  0.84 45.00  0.391.00  0.77
6.00  0.82
2-O-lauroylarjunic acid (11)
3.50  0.01 40.00  0.11
9.00  0.06 20.00  0.05
5.00  0.25 55.00  0.19 12.00  0.01 60.00  0.07
0.07  0.03 2.00  0.03 1.45  0.03 2.61  0.03
vinblastine
0.05  0.02
0.82  0.090.03  0.06 0.45  0.07
* Cancer cell lines with their ATCC No. and source organ in parenthesis: KB; CCL 17 (oral cancer), PA-1, CRL 1572 (ovary cancer), HepG-2; HB-8065 (liver cancer), WRL 68; CL 48 (liver cancer).
(±) = inactive (IC50/IC90 > 100 mg/mL).
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From the above results, it may be concluded that arjunic acid es-
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against the liver cancer cell lines HepG 2 and WRL 68, respective-
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Acknowledgements
The authors are thankful to Council of Scientific & Industrial Re-
search (CSIR) for financial support.
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