Background
Methods
Plant extraction
Gas chromatography–mass spectrometry (GC–MS) analysis
Cell culture
Antiproliferative effect
Apoptosis induction effect
Nuclei morphological study by 4´6-diamidino-2-phenylindole (DAPI) staining assay
Analysis of DNA fragmentation
Enhancement of anticancer effect of P. kesiya extract in vitro
IC50 Ratio in U937 | ||||||||||||||
IC50 of P. kesiya (µg/mL) | 0.02 (6) | 0.05 (15) | 0.1 (30) | 0.2 (60) | 0.5 (150) | 1 (300) | 1.5 (450) | 2 (500a) | 1 (300) | 1 (300) | 1 (300) | 1 (300) | 1 (300) | 1 (300) |
IC50 of melphalan (µg/mL) | 1 (15) | 1 (15) | 1 (15) | 1 (15) | 1 (15) | 1 (15) | 1 (15) | 1 (15) | 0.05 (0.75) | 0.1 (1.5) | 0.2 (3) | 0.5 (7.5) | 1.5 (22.5) | 2 (30) |
IC50 Ratio in HepG2 | ||||||||||||||
IC50 of P. kesiya (µg/mL) | 0.02 (1.1) | 0.05 (2.75) | 0.1 (5.5) | 0.2 (11) | 0.5 (27.5) | 1 (55) | 1.5 (83) | 2 (110) | 1 (55) | 1 (55) | 1 (55) | 1 (55) | 1 (55) | 1 (55) |
IC50 of melphalan (µg/mL) | 1 (40) | 1 (40) | 1 (40) | 1 (40) | 1 (40) | 1 (40) | 1 (40) | 1 (40) | 0.05 (2) | 0.1 (4) | 0.2 (8) | 0.5 (20) | 1.5 (60) | 2 (80) |
Statistical analysis
Results
Antiproliferative effects of P. kesiya extract and melphalan singly versus in combination on U937 and HepG2 cell lines
IC50 Ratio | % Antiproliferation | |||
---|---|---|---|---|
P. kesiya
| Melphalan | U937 | HepG2 | Vero |
0 | 1 | 50 | 50 | 50a
|
1 | 0 | 50 | 50 | Inactiveb
|
1 | 1 | 100 ± 7.0 | 95.7 ± 12.2 | 6.8 ± 1.9 |
0.02 | 1 | 46.0 ± 5.7 | Nd | Nd |
0.05 | 1 | 59.8 ± 5.3 | Nd | Nd |
0.1 | 1 | 76.6 ± 13.5 | Nd | 1.0 ± 3.5 |
0.2 | 1 | 79.3 ± 3.7 | 43.7 ± 2.9 | 4.0 ± 6.1 |
0.5 | 1 | 100 ± 7.0 | 82.1 ± 4.9 | 7.3 ± 1.9 |
1.5 | 1 | 100 ± 2.2 | 93.4 ± 1.6 | 16.4 ± 4.7 |
2 | 1 | 100 ± 11.3 | 100 ± 3.1 | 24.7 ± 9.1 |
1 | 0.05 | 46.8 ± 9.5 | 34.2 ± 4.4 | Nd |
1 | 0.1 | 51.8 ± 5.5 | 46.0 ± 9.1 | Nd |
1 | 0.2 | 73.0 ± 4.2 | 60.7 ± 16.4 | 0 ± 3.3 |
1 | 0.5 | 76.4 ± 3.6 | 65.1 ± 9.1 | 1.8 ± 1.2 |
1 | 1.5 | 100 ± 2.0 | 100 ± 10.5 | 8.6 ± 1.4 |
1 | 2 | 100 ± 3.4 | 100 ± 4.9 | 9.5 ± 1.9 |
Synergistic effect of melphalan and P. kesiya extract combination analyzed in U937 leukemic and HepG2 hepatocellular carcinoma cell lines
% Antiproliferation | Cell lines | Combination treatment | DRI | CI | |
---|---|---|---|---|---|
P. kesiya
| Melphalan | ||||
90 % (IC90) | U937 | Fixed [P. kesiya] = IC50
| 2.3 | 7.8 | 0.56 |
Fixed [melphalan] = IC50
| 6.5 | 5.5 | 0.33 | ||
HepG2 | Fixed [P. kesiya] = IC50
| 2.0 | 3.6 | 0.79 | |
Fixed [melphalan] = IC50
| 2.4 | 3.2 | 0.74 | ||
75 % (IC75) | U937 | Fixed [P. kesiya] = IC50
| 1.8 | 8.7 | 0.66 |
Fixed [melphalan] = IC50
| 9.1 | 3.8 | 0.37 | ||
HepG2 | Fixed [P. kesiya] = IC50
| 1.6 | 3.8 | 0.90 | |
Fixed [melphalan] = IC50
| 2.7 | 2.4 | 0.79 |
Extent of dose reduction in the combination treatment compared with single doses of each treatment
Apoptotic induction effect of single treatments and of P. kesiya extract and melphalan combination treatment
IC50 ratio | % Apoptotic cells | ||
---|---|---|---|
IC50 of P. kesiya
| IC50 of Melphalan | U937 | HepG2 |
0 | 1 | 43.1 ± 16.3 | 53.0 ± 6.1 |
1 | 0 | 42.5 ± 4.8b
| 39.7 ± 2.6a
|
1 | 1 | 100a
| 100a
|
0.1 | 1 | 77.0 ± 1.9a
| 66.7 ± 3.1a
|
0.2 | 1 | 100a
| 74.9 ± 12.3a
|
0.5 | 1 | 100a
| 92.8 ± 1.3a
|
1.5 | 1 | 100a
| 98.3 ± 3.0a
|
2 | 1 | 100a
| 100a
|
1 | 0.05 | 38.6 ± 6.1a
| 62.4 ± 3.2a
|
1 | 0.1 | 48.4 ± 3.8a
| 87.9 ± 3.3a
|
1 | 0.2 | 61.3 ± 3.3a
| 90.3 ± 6.0a
|
1 | 0.5 | 100a
| 100a
|
1 | 1.5 | 100a
| 100a
|
1 | 2 | 100a
| 100a
|
GC–MS analysis of P. kesiya extract
Retention time (min) | % of Total area | Assigned compounds |
---|---|---|
11.074 | 2.48 | Pyrocatecol (1,2-benzenediol or brenzcatechin) |
16.412 | 2.66 | trans-Sobrerol |
16.703 | 1.88 | Vanillin |
29.551 | 1.55 | Palmitic acid |
32.878 | 4.49 | Oleic acid |
35.650 | 4.16 | Benzoyl isocyanate |
36.096 | 4.77 | Pimaric acid |
37.170 | 6.52 | Rosin acid |
37.907 | 20.16 | Podocarpa-8,11,13-trien-15-oic acid, 13-isopropyl |
38.525 | 4.31 | 2,3-dimethoxy-5-[2-(3-hydroxy-4-methoxyphenyl)ethenyl]phenol or Combretastatin A3 |
41.788 | 5.98 | Neopine |