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,

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bisnaphthalimides as new development of DNA [2] A.L.P. Hawariah, Understanding Cancer, Universiti Putra

topoisomerase II inhibitors, Bioorganic & Medicinal Malaysia Press, Serdang, 1998.

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