Result and Discussion
3. Result and Discussion
density (OD) was done by a microplate reader at a wavelength of 550 nm. Medium containing only P388
3.1 Isolation and Identification of Lunacridine murine cells used as a positive control. As a
Isolation using preparative TLC method obtained comparison, we used artonin E. The percentage of cell colorless crystal (± 9 mg). Its molecular structure was death (%) was calculated as: identified by TLC with dragendorf reagent, UV-Vis
and IR spectroscopies. The TLC result showed the
Where C is the mean OD 550 of the control group and same Rf value between spot of lunacridine standard T is that of the treated group. The IC
50 was determined
from the dose-response curve.
2.5 Molecular Docking Simulation Molecular structure of lunacridine was built using
Chem 3D ultra 8.0 software (Molecular Modeling and Analysis; Cambridge Soft Corporation, USA (2004)) and geometry optimized by PM3 semiempiric method. Docking study was carried out based on the NMR
structure of the covalent complex between d (CGCTAGGCG)-(GCGATCCGC) and the
biz-thiazole orange (PDB archive code 108D) using Fig. 1 Identification of lunacridine isolated from ethyl
acetate fraction of Sanrego (Lunasia amara Blanco) using
AutoDock 4.0 software. The crystal structure was
eluent of chloroform:methanol (18:1) after sprayed by
downloaded from the protein data bank website
dragendorf reagent. 1: spot of lunacridine standar as the
(www.rscb.org/pdb). The native ligand structure was
comparison; 2: spot of lunacridine isolated.
In Vitro and In Silico Studies of Lunacridine from Lunasia Amara Blanco as Anticancer
Table 1 The comparison of UV-Vis and IR spectrums of lunacridine isolated and lunacridine reported by Ahmad, et al. [11].
UV, λ max nm
IR, cm -1
Lunacridine isolated
Lunacridine
Lunacridine isolated
Lunacridine
204 - 3774.69 3500-3400 216 -
3410.15 3500-3400 240 240 2854.65; 2924.09; 2956.8 - 285 256 1546.91;1512.19 1589, 1565 298 286 1641.42 1647 312 295 1201.65;1170.79; 1116.78 1240, 1209 324 335 3774.69 3500-3400
and the isolated compound after sprayed by dragendorf reagent. It showed that the isolated compound was lunacridine.
The colorless crystal of lunacridine isolated has UV-Vis spectrum as shown in Fig. 2 with maximum absorbances of 204, 216, 240, 285, 298, 312, and 324 nm. This UV-Vis spectrum indicates that there are substituted groups on quinoline ring [9].
The IR spectrum of lunacridine isolated (Fig. 3) Fig. 2 UV-Vis spectrum of lunacridine isolated. Maximum
absorbances were showed by black color.
shows absorptions of 3774.69, 3410.15, 2956.87, 2854.65, 1737.86, 1641.42, 1546.91, 1512.19, 1463.97,
activity or less active on P388 murine leukemia cells. 1201.65, 1170.79, 1116.78 and 750.31 cm -1 . The
3.3 Molecular Docking
stretching at 3774.69 cm -1
indicates the NH group. The
strong stretching at 3410.15 cm -1 indicates the OH
3.3.1 Geometry Optimation
group, the aliphatic CH group is shown by strong Molecular structure of lunacridine was optimized by stretching at 2956.87-2854.65 cm -1 , the secondary
PM3 semiempiric method. Optimization of this amide group is shown by strong stretching at 1641.42
structure aims to obtain a stable molecular structure
cm 0 , the aromatic system is shown by the stretching at characterized by the lowest of ΔH
f (standart formation 1546.91-1512.19 cm -1 , and the C-O/C-O-C group is
enthalpy) value. Model of stable molecule could be shown by the stretching at 1201.65, 1170.79 and
used for docking simulation.
1116.78 cm -1 [10].
3.3.2 Docking Method Validation Validation of the docking simulation was performed
3.2 MTT Colorimetric Assay to choose appropriate parameters for docking of new
The cytotoxic activity of lunacridine with various compounds. The RMSD (Root Mean Square Deviation) concentrations of 1, 3, 10, 30, and 100 μg/mL on P388
value is used as a validation parameter. As cited in Ref.
[13], if the RMSD value of the best-scored μg/mL. This value was obtained by statistic calculation
murine leukemia cells gave the IC 50 value of 39.52
conformation of native ligand docked is ≤ 2.0 Å from from the last three data because these data were in the
the actual native ligand conformation, the prediction is sigmoid area. As cited in Ref. [12], the pure compound
said to be successful. From the validation result as which has the IC 50 value above 4 μg/mL and less 100
shown in Fig. 6 obtained RMSD value of 1.15 Å. It is μg/mL indicates that the compound has less cytotoxic
clearly noticed that the docked native ligand bis thiazole
In Vitro and In Silico Studies of Lunacridine from Lunasia Amara Blanco as Anticancer
Fig. 3 IR spectrum of lunacridine isolated. The strong stretching at 3410.15 cm -1 indicates the OH group.
was exactly superimposed on the actual native ligand with the high binding free energy (-16.37 kcal/mol) and
high inhibition constant (0.995 × 10 μM).
th
3.3.3 Docking of Lunacridine
s d e ll
Docking study was performed on lunacridine into
DNA model using AutoDock 4.0 software. DNA
model was obtained from protein data bank website (www.rscb.org/pdb) with the archive code 108D
Concentration (ug/ml)
Fig. 4 Cells death percentage of P388 murine leukemia cell
(native ligand bis thiazole). This DNA model was used
exposed lunacridine for 72 hours. IC 50 calculated from the
by Filosa [14] as a receptor model for docking
last three data of 10, 30 and 100 μg/mL. Each data point
bis-naftalamida compound as a new drug class of DNA
represents an average taken from three separate wells.
intercalating topoisomerase II inhibitor [14]. OCH 3 4' CH 3 Docking simulation of lunacridine into intercalation
5 4 sites of DNA with various total of energy evaluations
4a 1'
3'
5' CH 3 gave the results as shown in Table 2. The best model reached at 2.5 × 10 7 energy evaluation level with the
6 3 2'
7 8a OH 2 binding free energy and inhibition constant of -6.63
kcal/mol and 13.90 μM respectively. OCH 3 CH 3 The conformation of lunacridine from docking
Fig. 5 2D-stucture of lunacridine.
simulation at the highest energy evaluation level (2.5 ×
In Vitro and In Silico Studies of Lunacridine from Lunasia Amara Blanco as Anticancer
Fig. 7 3D models of the intercalation mode of lunacridine Fig. 6 The superimposition in DNA-intercalation site of
(showed by stick and colored by element) on DNA (showed native ligand docked conformation (showed by stick and
by molecular surface and colored by element). colored by cyan) and actual native ligand conformation (showed by stick and colored by red) with RMSD value of
Table 2 Binding free energy ( ∆Gb) and inhibition constants (Ki) for lunacridine docked into DNA.
Energy evaluation ∆Gb (kcal/mol)
Ki ( μM)
2.5 × 10 5 -6.22 27.51 2.5 × 10 6 -6.50 17.08 2.5 × 10 7 -6.63 13.90
10 7 ) was in accordance with the ability of lunacridine
to intercalate between base pairs of DNA, such as
Fig. 8 Interaction or binding mode between lunacridine
reported by Prescott (2007) [4]. Each level of energy
(showed by stick and colored by element) and base pairs of
evaluations gave ten intercalation models of DNA (showed by line and colored by element). Dipole-dipole
interactions were showed by yellow dotted lines.
lunacridine. As shown in Fig. 7, lunacridine could intercalate between C-G and T-A base pairs, whereas
with the native ligand bis thiazole showed obviously the side chain of lunacridine was located on the minor
different. Lunacridine showed the higher binding free groove of DNA. Quinoline ring of lunacridine could
energy than native ligand bis thiazole. It meant that the perfectly intercalate between DNA base pairs and
binding affinity of lunacridine into DNA was relatively make л-л interaction with purine and pyrimidine bases
weak. This result correlated with the less cytotoxic of DNA because of the planar structure of quinoline
activity of lunacridine on P388 murine leukemia cells. ring.
The active sites of lunacridine docked into DNA The analysis of binding free energy and inhibition
were located on the subtituent of methoxy group constant of lunacridine docked into DNA, if compared
(OCH 3 ) on C4 of quinoline ring, subtituent of carboxyl
In Vitro and In Silico Studies of Lunacridine from Lunasia Amara Blanco as Anticancer
group (C = O) on C2 of quinoline ring and OH group [3] S. Goodwin, A.F. Smith, A.A. Velasquez, E.C. Horning, Alkaloids of Lunasia amara blanco, isolation studies,
on the side chain of lunacridine. In details, the possible Journal of the American Chemical Society 81 (23) (1959)
interaction between lunacridine and base pairs of DNA
6209-6213.
can be seen in Fig. 8. The dipole-dipole interactions [4] A.K. Prescott, S.K. Maciver, I.H. Sadler, R. Kiapranis, occured between carboxyl group (C=O) on C2 of
Lunacridine from Lunasia amara is a DNA intercalating topoisomerase II inhibitors, Journal of
quinoline ring and N9 of adenine (DA5) with the Ethnopharmacology 109 (2007) 289-294.
[5] T. Yuwono, Molecular Biology, Erlangga Press, Jakarta, quinoline ring and carboxyl group (C=O8) of thymine
distance of 3.55 Å, methoxy group (OCH 3 ) on C4 of
2008, p. 102.
(DT4) with the distance of 3.08 Å, OH group on the [6] K.D. Bromberg, N. Osheroff, DNA cleavage and religation by human topoisomerase II alpha at high
side chain of lunacridine and carboxyl group (C=O8) temperature, Biochemistry 40 (2001) 8410-8418.
of thymine (DT4) with the distance of 2.77 Å. The [7] R. Martinez, L.C. Garcia, The search of DNA-intercalators
as antitumoral drugs: What it worked and what did not of quinoline ring, methyl group (CH ) on N1 of quinoline
other subtituents such as methoxy group (OCH 3 ) on C8
work, Current Medicinal Chemistry 12 (2005) 127-151.
D.C. Young, Computational Drug Design, John Wiley & ring and aliphatic side chain of lunacridine did not
Sons Publication, Hoboken, New Jersey, 1964, p. 133. show dipole-dipole interaction with DNA. Therefore,
D. Noerdin, Structure Elucidation of Organic Compound this study suggests to modify these groups to increase
by UV and IR Spectroscopy, Angkasa Press, Bandung, the number of molecular interaction of lunacridine to 1986. [10] R.M. Silverstein, F.X. Webster, D.J. Kiemle,
DNA and to increase the cytotoxic potency of Spectrometric Identification of Organic Compounds, 3rd
lunacridine. ed., John Wiley & Sons, Inc., New York, 2005. [11] M.U. Ahmad, M.A. Rahman, E. Huq, R. Chowdhury,
4. Conclusions
Alkaloids of Zanthoxylum budrunga, Fitoterapia 74 (2003) 191-193.
The less cytotoxic activity of lunacridine probably is [12] M. Muhtadi, E.H. Hakim, Y.M. Syah, L.D. Juliawaty, S.A. due to low affinity and molecular interaction with DNA.
Achmad, I.M. Said, et al., Three oligostilbenoid Therefore, this study suggests to design and to develop
compounds from stem Dipterocarpus retusus Blume lunacridine as a lead compound for anticancer drug. (Dipterocarpaceae), Journal of Mathematics and
Science10 (4) (2005) 137-143.
Acknowledgment [13]
H.I. Ali, K. Tomita, E. Akaho, M. Kunishima, Y. Kawashima, T. Yamagishi, et al., Antitumor studies-Part 2: The author would like to thank to Hasanuddin
Structure-activity relationship study for flavin analogs University for financial support to carry out this including investigations on their in vitro antitumor assay and docking simulation into protein tyrosine kinase,
research under “BPPS DIKTI” grant for 2008-2010 European Journal of Medicinal Chemistry 43 (2008) academic year.
1376-1389. [14] R. Filosa, A. Peduto, S. In Micco, P. de Caprariis, M. Festa,
References
A Petrella, et al., Molecular modeling studies, synthesis [1]
D.E. Thurston, Chemistry and Pharmacology of Anticancer and biological activity of a series of novels and on their Drugs, CRC Press, Boca Raton, FL, 2007, p. 58.
bisnaphthalimides as new development of DNA [2] A.L.P. Hawariah, Understanding Cancer, Universiti Putra
topoisomerase II inhibitors, Bioorganic & Medicinal Malaysia Press, Serdang, 1998.
Chemistry 17 (2009) 13-24.
Journal of Life Sciences 5 (2011) 646-653