In vitro antimalarial activity screening and cytotoxicity assay
In vitro antimalarial activity of Curcuma caesia ethyl acetate and methanol extracts were analyzed for both Chloroquine-resistant (K1) and sensitive (3D7) strains of P. falciparum. IC50 values of ethyl acetate extract were 3.37 µg/ml against 3D7, and 1.53 µg/ml against K1 strain. The methanol extract possesses IC50 values of 8.57 µg/ml against 3D7 and 18.29 µg/ml against K1 strains (Table 1). The IC50 value for promising lead compounds was considered equal to or less than 10.0 µg/ml.
Cytotoxicity was carried out against Vero cell line (C1008; Monkey kidney fibroblast cells). IC50 and CC50 values of both extracts against the P. falciparum sensitive and resistant strains with positive and negative control values along with Selectivity index (SI) has been given in Table 1. SI was calculated by dividing the CC50 value with IC50 and the criteria for selection is that SI value should be equal or more than 50. Higher SI value is preferable for a drug to have favourble safety and efficacy [10]. Interestingly, ethyl acetate extract of Curcuma caesia exhibited profound in vitro antimalarial activity against 3D7 and K1 strains in comparision to methanol extract with higher SI index against K1strain.
Table 1: In vitro antimalarial and cytotoxic activity of Curcuma caesia extracts
Test Samples
|
IC50 (µg/ml)
|
CC50 (µg/ml)
|
Selective Index (SI)
|
3D7
|
K1
|
3D7
|
K1
|
C. caesia ethyl acetate extract
|
3.37
|
1.53
|
13.92
|
4.13
|
9.09
|
C. caesia methanol extract
|
8.57
|
18.29
|
91.20
|
10.64
|
4.98
|
QC-diphosphate*
|
0.005
|
0.303
|
175
|
35,000
|
577.5
|
Podophyllotoxin*
|
na
|
na
|
6.4
|
na
|
na
|
*Values are in µM
ADMET prediction
All the compounds from the GCMS study of both extracts were selected for ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) prediction. From the ADMET result only thirty-one compounds were selected which follows Lipinski rule with no violation and having bioavailability value of 0.55 for docking study. Additional file1, Table S1 shows the bioactivity radar for rapid evaluation of drug-likeness of thirty-one metabolites.
Docking analysis of compounds
Molecular docking studies were carried out to reveal the interactions of phosphoethanolamine methyltransferase enzyme (PfPMT) of Plasmodium falciparum with thirty-one compounds identified from Curcuma caesia methanol and ethyl acetate extracts. Binding energy of all thirty-oneligands against the enzyme is depicted in Additional file 2, Table S2. The higher negative value of binding energy corresponds to a more stable interaction of ligand to the enzyme. Among 31 ligands, β-Selinenol (-6.76 Kcal/mol), α-Eudesmol(-6.59 Kcal/mol), α –Acorenol (-5.85 Kcal/mol), Boldione (-5.55 Kcal/mol) and Xanthinin (-5.40 Kcal/mol) showed the highest binding energy (Table 2). The 2D and 3D interactions docking results were attained by introducing our result into the Discovery Studio Visualizer, enabling us to recognize important interactions between the ligands and the binding site of the receptor. Fig. 1 shows the 2D interaction of all top five compounds with PfPMT which helps in identifying the amino acids which are involved in the interaction. There are 2 amino acids involved in conventional hydrogen bond interaction between β-Selinenol and PfPMT,GLU226 and LYS 225, shown in Fig.2(a) and with a bond length of 2.73 and 2.95. Fig. 2(b) shows the surface view of the hydrogen bond.
Table 2: Binding energy of lead five ligand with PfPMT
Name
|
Binding energy (Kcal/mol)
|
No. of
H-bond
|
Amino acid
|
Bond length (Ångstrom)
|
β-Selinenol
|
-6.76
|
2
|
GLU226, LYS 225
|
2.73Å, 2.95Å
|
α-Eudesmol
|
-6.59
|
2
|
ASN101,ASN74
|
2.55Å, 3.23Å
|
α –Acorenol
|
-5.85
|
1
|
GLN212
|
2.91Å
|
Boldione
|
-5.55
|
1
|
LYS165
|
3.09Å
|
Xanthinin
|
-5.40
|
3
|
PHE116,TRP148,GLU143
|
3.32 Å, 3.17Å, 3.18Å
|