Asia Pacific
AsPac
J. Mol.
Journal
Biol. Biotechnol.,
of MolecularVol.
Biology
11 (1),
and2003
Biotechnology, 2003
Vol. 11 (1) : 3-7
Antitumor promoting and actioxidant activities of anthraquinones
3
Antitumor promoting and actioxidant activities of anthraquinones isolated
from the cell suspension culture of Morinda elliptica
Jasril1, Nordin H. Lajis2*, Lim Y. Mooi3, Mohd A. Abdullah3,
Mohd A. Sukari2 and Abdul M. Ali3
1
Department of Chemistry, Faculty of MIPA, Universitas Riau, Pekanbaru, Riau, INDONESIA
2
Department of Chemistry, 3 Department of Biotechnology, Universiti Putra Malaysia,
43400 UPM, Serdang, Selangor D. E., MALAYSIA
Received 29 July 2002 / Accepted 11 January 2003
Abstract. Six anthraquinones (nordamnacanthal, alizarin-1-methyl ether, rubiadin, soranjidiol, lucidin-ω-methyl ether and
morindone) isolated from the cell suspension culture of Morinda elliptica were assayed for antitumor promoting and antioxidant
activities. All compounds exhibited strong antitumor promoting activity at the concentration of 2.0 µg/ml when assayed using
the inhibition test of Epstein Barr Virus (EBV) activation on Raji cells. At the concentration of 0.4 µg/ml, only nordamnacanthal
exhibited strong antitumor promoting activity with the inhibition rate and the cell viability of 75.0% and 75.8%, respectively,
which was stronger than the reference compounds genistein and quercetin. In antioxidant assay using ferric thiocyanate (FTC)
method, nordamnacanthal and morindone showed stronger antioxidant activity than α-tocopherol. However when the compounds were assayed for scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, only morindone
was considered to be active as free radical scavenger with fifty percent inhibition concentration (IC50) of 40.6 µg/ml.
Keywords. antitumor promoting, antioxidant, anthraquinones, cell suspension, Morinda elliptica.
INTRODUCTION
Morinda is a genus of the family Rubiaceae and has long been
known to contain substantial amount of anthraquinones
(Thomson, 1971). The roots of these plants are usually rich
in anthraquinones which most often occur as aglycones but
sometimes may also present in the form of glycosides.
Wijnsma and Verpoorte (1986) in their review of
anthraquinones in Rubiaceae, tabled the anthraquinone
contents of ten Morinda species that has been investigated in
recent years. About 90% of these compounds occur as
derivatives of 9,10-anthracenedione with several hydroxy and
other functional groups, such as methyl, hydroxymethyl and
carboxyl. Hydroxyanthraquinones are the active principles
of many phyto-therapeutic drugs (Westendorf et al., 1990).
M. elliptica or locally known as “Menkudu kecil” is one
of the many medicinal plants used by the people in Malaysia.
The leaves may be added to rice for loss of appetite and
taken for headache, cholera, diarrhoea, and particularly in
fever. The pounded leaves are also applied upon the spleen
and wounds. A lotion of them is used for haemorrhoids
and upon the body after childbirth (Burkill, 1966).
A number of anthraquinones have been isolated from
the roots of M. elliptica (Ismail et al., 1997) and the cell
suspension culture of the plant has been established
(Abdullah et al., 1998) The anthraquinones produced in this
suspension culture have also been isolated and characterized
(Jasril et al., 2000). We now wish to report the anti-tumor
promoting and anti-oxidant activities of the anthraquinones
isolated from this cell suspension culture.
MATERIALS AND METHODS
Materials. The sample of anthraquinones including
nordamnacanthal, alizarin-1-methyl ether, rubiadin,
soranjidiol, lucidin-ω-methyl ether and morindone were
isolated from the cell suspension culture of Morinda elliptica
as previously described (Jasril et al., 2000).
Cell Culture. The Raji (human B-lymphoblastoid) cell line
was provided by Prof. K. Koshimizu of Kinki University
*Author for Correspondence.
Mailing address: Department of Chemistry, Universiti Putra Malaysia
43400 UPM, Serdang, Selangor, Malaysia.
Tel/Fax: 60-3-89468080; E-mail: [email protected]
4
AsPac J. Mol. Biol. Biotechnol., Vol. 11 (1), 2003
and HL-60 cell line was obtained from the RIKEN Cell Bank,
Tsukuba, Japan. Cells were cultured in RPMI-1640 (Sigma,
USA) medium with 10 % v/v foetal calf serum (Sera Lab,
UK), 100 IU/ml penicillin (Sigma, USA) and 100 µg/ml
streptomycin (Sigma, USA) as a complete growth medium
(CGM). Cells were maintained in 25 cm3 flask with 10 ml of
CGM at 37 oC with 5% CO2. Every three days the cells were
subcultured by splitting the culture with fresh CGM at a ratio
of 2:8.
Antitumor Promoting Assay. Antitumor promoting assay
was performed in vitro using the inhibition test of EpsteinBarr virus (EBV) activation in Raji cells induced by phorbol12-myristate-13-acetate (PMA) and sodium n-butyrate. Raji
cells (5 x 105 cells/ml) were maintained in a 1 ml of RPMI
1640 medium supplemented with 10% fetal calf serum (FCS)
containing sodium n-butyrate (3 mM), PMA (0.05 µM) and
the test sample (5 µl) at 370C under 5% CO2 for 48 hours.
Early antigen (EA) expressed in Raji cells was detected by an
indirect immunofluorescence method with EA-positive sera
from nasopharyngeal carcinoma (NPC) patients and FITClabeled anti-human IgG as described by Murakami et al.
(1995). The average EA induction was compared to a control
containing only PMA and sodium n-butyrate, the induction
rate (IR) of the control was less than 40%. The inhibitory
rate (IR) of each test sample towards the EBV activation
was classified into four ranks as: +++ (strongly active; (IR ≥
70%), ++ (moderately active; 70% > IR ≥ 50%), + (weakly
active; 50% > IR ≥ 30%), - (inactive; 30% > IR) (Murakami et
al., 1998 and 2000).
Antioxidant Assay. The antioxidant assay of test samples
was carried out using ferric thyocyanate (FTC) method as
described in the modified method of Kikuzaki and Nakatani
(1993). The sample solution was prepared in a screw-capped
vial (φ38 x 75 mm) by dissolving the test sample (2 mg for
pure compound) in 4 ml 99.5% ethanol, 4.1 ml of 2.5%
linoleic acid in 99.5% ethanol, 8 ml of 0.02 M phosphate
buffer (pH 7.0) and 3.9 ml of distilled water. The solution
was then placed in the dark at 40 0C. After 24 hrs 0.1 ml of
this solution was transferred into a test tube (φ16 x 150 mm)
and to it 9.7 ml of 75% ethanol, 0.1 ml of 30% ammonium
thiocyanate and finally 0.1 ml of 2 x 10-2 M ferrous chloride
in 3.5% hydrochloric acid were added. Precisely three minutes
after addition of ferrous chloride, the absorbance was
measured at 500 nm. The measurement according to the
above procedure was taken for each sample every 24 hours
until the absorbance of the control has reached maximum
and depreciated.
Free Radical Scavenging Assay. This assay is based on
the scavenging activity of the stable 1,1-diphenyl-2picrylhydrazyl (DPPH) free radicals (Lee et al., 1998) and
was performed in a 96-well microtiter plate. Stock solution
of the test sample was prepared at 1 mg/ml concentration
Antitumor promoting and actioxidant activities of anthraquinones
in methanol and diluted to the 100 µg/ml (the substock
solutions). The substock solution was introduced into the
respective microtitre wells in a successively descending
amount of 100, 80, 60, 40, 20, 10 and 5 µl. The stock solution
in each well was then diluted with methanol to make the
total volume 195 µl, and finally add 5 µl of diphenyl-ppicrylhydrazyl (DPPH) solution (prepared at 10 mg/ml in
MeOH). The plate was shaken to ensure thorough mixing
before placing it in the dark. After allowing it to stand for 30
minutes, the optical density of the solution was read using
an ELISA Reader at wavelength 517 nm.
RESULTS AND DISCUSSION
Six anthraquinones isolated from the cell suspension culture
of Morinda elliptica were assayed for antitumor promoting and
antioxidant activities. The structures of all anthraquinones
are presented in Figure 1.
In vitro antitumor promoting activity was investigated to
evaluate inhibitory activity of the anthraquinones towards
EBV activation in Raji cells. In this assay, all compounds
exhibited strongest inhibitory activity at the concentration
of 2.0 and 10 µg/ml (Table 1). At 0.4 µg/ml, only
nordamnacanthal exhibited strong inhibition activity with the
inhibition rate and the cell viability of 75.0% and 75.8%,
respectively. Apparently, the activity of nordamnacanthal at
this concentration was stronger than that of genistein and
quercetin (used as reference compounds) which showed the
inhibition rates and the cell viability of 63.4% and 96.1%,
respectively for the former, and 58.9% and 98.8%,
respectively for the latter at the concentration of 1.6 µg/ml.
At this concentration (0.4 µg/ml), alizarin-1-methyl ether,
morindone and lucidin-ω-methyl ether only exhibited
O
R1
8
1
12
9
7
R2
13
2
3
6
R6
10
11
14
R5
Name of compounds
R3
4
5
R4
O
R1
Nordamnacanthal (A-1)
OH
Alizarin-1-methyl ether (A-2) OCH3
Rubiadin (A-3)
OH
Soranjidiol (A-4)
OH
Lucidin-ω-methyl ether (A-5) OH
Morindone (A-6)
OH
R2
R3
R4 R5 R6
CHO
OH
CH3
CH3
CH2OCH3
CH3
OH
H
OH
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
OH
H
OH
Figure 1. Anthraquinones Isolated from Cell Suspension Culture
of Morinda elliptica
AsPac J. Mol. Biol. Biotechnol., Vol. 11 (1), 2003
Antitumor promoting and actioxidant activities of anthraquinones
100.0
90.0
80.0
1
BHT
Toc.
%Inhibition
Absorbance (500 nm)
Control
0.8
A-1
0.6
A-2
A-5
0.4
5
A-6
70.0
60.0
50.0
40.0
30.0
Ascorbic acid
Quercetin
Kaempferol
Morindone
20.0
10.0
0.0
0.2
0
0
1
2
3
4
5
6
2.5
7
5
10
20
30
40
50
Concentration (ug/ml)
Day
Figure 2. Absorbance Values of Anthraquinones Using the FTC
Method
Figure 3. DPPH Free Radical Scavenging Activity
Table 1. Antitumor Promoting Properties of Anthraquinones
µg/ml)
Concentration (µ
Cell viability(%)
Inhibitory rate(%)
Activity rank
Nordamnacanthal
(A-1)
10.0
2.0
0.4
54.1
72.5
75.8
100.0
82.3
75.0
+++
+++
+++
Alizarin-1-methyl
Ether (A-2)
10.0
2.0
0.4
90.2
88.7
90.4
89.3
80.0
63.2
+++
+++
++
Rubiadin (A-3)
10.0
2.0
0.4
65.3
67.9
91.3
72.5
70.5
18.0
+++
+++
-
Soranjidiol (A-4)
10.0
2.0
0.4
88.9
90.4
81.2
89.4
75.7
49.0
+++
+++
+
Lucidin-ω-methyl
Ether (A-5)
10.0
2.0
0.4
71.8
83.3
91.0
79.0
78.2
62.7
+++
+++
+
Morindone (A-6)
10.0
2.0
0.4
83.5
85.7
84.7
82.9
76.0
51.4
+++
+++
++
Genistein
8.0
1.6
0.032
100.0
96.1
98.8
75.2
63.4
58.9
+++
++
++
Quercetin
8.0
1.6
0.032
97.4
98.8
98.9
67.8
58.9
47.4
+++
++
+
Compound
6
AsPac J. Mol. Biol. Biotechnol., Vol. 11 (1), 2003
moderate activity with inhibition rates and cell viability of
63.2% and 90.4%, respectively for alizarin-1-methyl ether,
51.4% and 84.7%, respectively for morindone, and 62.7%
and 91.2%, respectively for lucidin-ω-methyl ether.
However, at this concentration soranjidiol exhibited weak
activity with inhibition rate and cell viability of 49.0% and
81.2%, respectively while rubiadin was found to be inactive.
Antioxidant activity of the anthraquinones was also
measured using FTC method (Figure 2). Nordamnacanthal
and morindone showed stronger antioxidant activity than αtocopherol although they were less active than butylated
hydroxytoluene (BHT), a potent synthetic antioxidant. The
antioxidant activities of alizarin-1-methyl ether and lucidinω-methyl ether were comparable to α-tocopherol. On the
other hand, rubiadin and soranjidiol were inactive.
The antioxidant activity of the anthraquinones has also
been monitored based on scavenging activity of stable 1,1diphenyl-2-picrylhydrazyl (DPPH) radicals (Figure 3). Only
morindone was considered to be active as free radical
scavenger with IC50 value of 40.6 µg/ml. The scavenging
activity of morindone was comparable to kaempferol with
IC50 value of 32.4 µg/ml although was much lower than
ascorbic acid and quercetin with IC50 values of 7.6 µg/ml
and 10.2 µg/ml, respectively.
Ali et al. (2000) reported that the presence of hydroxyl
group at C-1 and C-3 and/or a formyl group at C-2 in the
anthraquinone skeleton, such as nordamnacanthal, lucidinω-methyl ether and rubiadin exert their cytotoxic activity.
Several studies concerning the relationship between the
phenolic structure of flavonoids, lignans and acid-phenols,
and their antioxidant activity have been conducted (Faure et
al., 1990; Cuvelier et al., 1992). Huang et al. (1995) studied the
effect of anthraquinone derivatives on lipid peroxidation in
rat heart mitochondria. They concluded that two hydroxyl
groups arranged at either the meta- or ortho- positions are
required for an anthraquinone to inhibit lipid peroxidation
in rat heart mitochondrial system. However, anthraquinone
derivatives that have no hydroxyl groups as well as two
hydroxyl groups affixed to different rings did not inhibit lipid
peroxidation.
The results of our experiments indicated that there is a
relationship between antioxidant and antitumor promoting
activities in the cases of anthraquinones tested.
Nordamnacanthal being a strong antitumor promoter was
found to be strong antioxidant. Whereas only morindone
was found to be strong radical scavenger, both morindone
and nordamnacanthal were the most active in FTC assay.
This observation suggested that radical scavenging is less
prominent in nordamnacanthal as compared to morindone
in their antioxidant mechanisms. The differences between
the two compounds are that the formyl at C-2 and hydroxyl
at C-3 in nordamnacanthal are replaced by a methyl and
proton groups, respectively in morindone.
Antitumor promoting and actioxidant activities of anthraquinones
ACKNOWLEDGEMENTS
The authors thank the Ministry of Science, Technology &
the Environment of Malaysia for funding the project under
the IRPA Grant.
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