Background
No | Plant species | Family | Plant part | Voucher number | Therapeutic Properties |
---|---|---|---|---|---|
1 |
M. siamensis
| Guttiferae | Flowers | SMD 122006002 | |
2 |
M. ferrea
| Guttiferae | Flowers | SMD 122007001 | |
3 |
D. loureiroi
| Dracaenaceae | Stem | SMD 096001007 | |
4 |
P. cablin
| Lamiaceae | Leaves | SMD 142031002 | |
5 |
K. galanga
| Zingiberaceae | Rhizomes | SMD 288013007 | |
6 |
E. stoechadosmum
| Asteraceae | Leaves | SMD 072036002 | Antibacterial activity [29] |
Methods
Plant material and management
Preparation of formulation and extraction
New formula | Plant composition | Mass of component | Yield (%) | |
---|---|---|---|---|
Ethanolic solvent | Aqueous solvent | |||
Formulation 1 |
M. siamensis
| 10 g | 7.32 | 11.66 |
M. ferrea
| 10 g | |||
D. loureiroi
| 10 g | |||
P. cablin
| 10 g | |||
K. galangal
| 10 g | |||
E. stoechadosmum
| 10 g | |||
Formulation 2 |
M. siamensis
| 12 g | 8.35 | 11.41 |
M. ferrea
| 12 g | |||
D. loureiroi
| 12 g | |||
P. cablin . | 12 g | |||
K. galanga
| 12 g | |||
Formulation 3 |
M. siamensis
| 15 g | 10.21 | 11.72 |
M. ferrea
| 15 g | |||
D. loureiroi
| 15 g | |||
P. cablin
| 15 g | |||
Formulation 4 |
M. siamensis
| 20 g | 11.55 | 12.20 |
M. ferrea
| 20 g | |||
D. loureiroi
| 20 g | |||
Formulation 5 |
M. ferrea
| 12 g | 8.38 | 12.47 |
D. loureiroi
| 12 g | |||
P. cablin
| 12 g | |||
K. galanga
| 12 g | |||
E. stoechadosmum
| 12 g | |||
Formulation 6 |
D. loureiroi
| 15 g | 7.22 | 12.67 |
P. cablin
| 15 g | |||
K. galanga
| 15 g | |||
E. stoechadosmum
| 15 g | |||
Formulation 7 |
P. cablin
| 20 g | 2.92 | 14.60 |
K. galanga
| 20 g | |||
E. stoechadosmum
| 20 g |
In vitro cultivation and maintenance of Plasmodium falciparum K1 strain
In vitro assessment of antimalarial activity
In vitro assessment of cytotoxicity
Selectivity index (SI)
Gas chromatography-mass spectrometry analysis
Animal experiments
Four-day suppressive test (Peter’s test)
Groups (n = 5/group) | Extract/drug | Dose (mg/kg) |
---|---|---|
Negative control | 7% Tween 80 | - |
Positive control | Artesunate | 6 |
Positive control | Chloroquine | 25 |
Experimental group 1 | Ethanolic extract of formulation 1 | 200 |
Experimental group 2 | Ethanolic extract of formulation 1 | 400 |
Experimental group 3 | Ethanolic extract of formulation 1 | 600 |
Acute toxicity test
Biochemical analysis
Histopathological examination
Statistical analysis
Results
Percent yield
In vitro antimalarial activity and cytotoxicity
No | List | Ethanolic extract | Aqueous extract | |||||
---|---|---|---|---|---|---|---|---|
IC
50 (µg/ml) |
CC
50 (µg/ml) |
IC
50 (µg/ml) |
CC
50 (µg/ml) | |||||
P. falciparum
| Vero cell | HepG2 cell |
P. falciparum
| Vero cell | HepG2 cell | |||
1 | Formulation 1 | 1.32 ± 0.66 | 79.34 ± 2.36 60.10
| 68.43 ± 1.55 51.84
| 35.72 ± 2.22 | 136.65 ± 13.15 3.83
| 249.30 ± 34.30 6.98
| |
2 | Formulation 2 | 15.69 ± 1.11 | 58.74 ± 1.04 3.74
| 128.40 ± 0.40 8.18
| 14.96 ± 1.11 | 192.10 ± 0.7 12.84
| 176.15 ± 9.65 11.77
| |
3 | Formulation 3 | 2.61 ± 0.25 | 50.02 ± 0.51 19.16
| 51.38 ± 2.85 19.68
| 25.41 ± 3.27 | 185.90 ± 8.8 7.32
| 131.70 ± 4.50 5.18
| |
4 | Formulation 4 | 1.52 ± 0.28 | 37.01 ± 0.34 24.35
| 58.16 ± 7.76 38.26
| 19.79 ± 2.23 | 268.2 ± 19.8 13.55
| 114.80 ± 0.10 5.80
| |
5 | Formulation 5 | 6.14 ± 1.09 | 83.28 ± 0.74 13.56
| 70.56 ± 0.49 11.49
| 22.92 ± 4.12 | 241.00 ± 19.3 10.51
| 121.05 ± 9.05 5.28
| |
6 | Formulation 6 | 2.48 ± 0.34 | 61.76 ± 1.18 24.90
| 128.95 ± 8.05 52.00
| 49.68 ± 0.45 | 130.65 ± 2.95 2.63
| 224.65 ± 14.05 4.52
| |
7 | Formulation 7 | 13.85 ± 1.83 | 88.22 ± 2.17 6.37
| 95.956 ± 4.25 6.93
| 55.75 ± 3.93 | 202.60 ± 17.6 3.63
| 381.15 ± 17.85 6.84
| |
Artesunate | IC50: 1.25 ± 0.52 ng/ml | |||||||
Doxorubicin | CC50: 1.60 ± 0.23 µg/ml |
GC-MS analysis
No | RT | Name of compound | Molecular formula | MW | Peak area (%) |
---|---|---|---|---|---|
1 | 4.311 | Styrene | C8H8
| 104 | 0.06 |
2 | 6.532 | Eucalyptol | C10H18O | 154 | 0.06 |
3 | 8.669 | endo-Borneol | C10H18O | 154 | 0.08 |
5 | 9.324 | 2,4-Cycloheptadien-1-one, 2,6,6-trimethyl- | C10H14O | 150 | 0.24 |
6 | 10.314 | 4-Hydroxy-3-methylacetophenone | C9H10O2
| 150 | 0.03 |
7 | 10.419 | Carvacrol | C10H14O | 150 | 0.02 |
8 | 10.814 | 3,5-Heptadienal, 2-ethylidene-6-methyl- | C10H14O | 150 | 0.08 |
9 | 10.921 | Thymoquinone | C10H12O2
| 164 | 0.04 |
10 | 11.718 | Copaene | C15H24
| 204 | 0.17 |
11 | 11.844 | β-patchoulene | C15H24
| 204 | 0.09 |
12 | 11.905 | Tetradecane | C14H30
| 198 | 0.17 |
13 | 12.113 | Cyperene | C15H24
| 204 | 0.13 |
14 | 12.264 | Cinnamic acid | C9H8O2
| 148 | 0.15 |
15 | 12.395 | Caryophyllene | C15H24
| 204 | 0.33 |
16 | 12.512 | Ethanone, 1-(2-hydroxyphenyl)- | C8H8O2
| 136 | 0.32 |
17 | 12.686 | Patchoulene | C15H24
| 204 | 0.06 |
18 | 12.788 | Seychellene | C15H24
| 204 | 0.13 |
19 | 12.907 | Humulene | C15H24
| 204 | 0.08 |
20 | 12.978 | 2-Propenoic acid, 3-phenyl-, ethyl ester, (E)- | C11H12O2
| 176 | 2.50 |
21 | 13.027 | Alloaromadendrene | C15H24
| 204 | 1.01 |
22 | 13.220 | Gamma.-Muurolene | C15H24
| 204 | 0.13 |
23 | 13.416 | Pentadecane | C15H32
| 212 | 1.85 |
24 | 13.833 | Flamenol | C7H8O3
| 140 | 0.91 |
25 | 13.949 | Naphthalene, 1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-, (1 S-cis)- | C15H24
| 204 | 0.49 |
26 | 16.153 | Ethyl p-methoxycinnamate | C12H14O3 | 206 | 0.35 |
27 | 16.248 | Isoaromadendrene epoxide | C15H24O | 220 | 0.10 |
28 | 16.376 | Patchouli alcohol | C15H26O | 222 | 1.31 |
29 | 16.640 | Acetic acid, 3-hydroxy-6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-2-yl ester | C17H26O3
| 278 | 0.10 |
30 | 17.940 | Ethyl p-methoxycinnamate | C12H14O3
| 206 | 14.32 |
31 | 20.131 | 4,4,8-Trimethyltricyclo[6.3.1.0(1,5)]dodecane-2,9-diol | C15H26O2
| 238 | 0.14 |
32 | 21.529 | n-Hexadecanoic acid | C16H32O2
| 256 | 1.62 |
33 | 21.780 | Naphthalene, decahydro-1,1,4a-trimethyl-6-methylene-5-(3-methyl-2,4-pentadienyl)-, [4aS-(4a.alpha.,5.alpha.,8a.beta.)]- | C20H32
| 272 | 0.07 |
34 | 22.129 | Hexadecanoic acid, ethyl ester | C18H36O2
| 284 | 0.57 |
35 | 23.144 | Kaur-16-ene | C20H32
| 272 | 0.08 |
36 | 24.575 | 9,12-Octadecadienoic acid (Z,Z)- | C18H32O2
| 280 | 0.90 |
37 | 24.667 | Oleic acid | C18H34O2
| 282 | 0.83 |
38 | 25.053 | Octadecanoic acid | C18H36O2
| 284 | 0.26 |
39 | 25.096 | Linoleic acid ethyl ester | C20H36O2
| 308 | 0.37 |
40 | 25.200 | Ethyl oleate | C20H38O2
| 310 | 0.25 |
41 | 28.403 | Oleic acid amide | C18H35NO | 281 | 1.18 |
Four-day suppressive test
Group | Dose (mg/ml) | % Parasitemia | % Suppression |
---|---|---|---|
Negative control | - | 24.53 ± 1.94 | - |
Artesunate | 6 | 00.00 ± 0.00 | 100.00 a, d, e, f
|
Chloroquine | 25 | 00.00 ± 0.00 | 100.00 a, d, e, f
|
Ethanolic extract of CPF-1 (Treated groups) | 200 | 17.20 ± 1.41 | 29.89 ± 5.76 a, b, c, e, f
|
400 | 12.13 ± 0.97 | 50.54 ± 3.95 a, b, c, d, f
| |
600 | 6.87 ± 0.50 | 72.01 ± 2.04 a, b, c, d, e
|
In vivo acute toxicity test
Effects of ethanolic extract of CPF-1 on liver and kidney functions
Group | Liver function test | |||
---|---|---|---|---|
AST (U/L) | ALT (U/L) | ALP (U/L) | ||
Untreated control | 25.40 ± 7.89 | 20.20 ± 1.93 | 93.20 ± 2.29 b
| |
7% Tween 80 | 32.80 ± 9.62 | 25.40 ± 1.94 | 112.00 ± 4.54 a, c
| |
Ethanolic extract of CPF-1 | 35.60 ± 9.09 | 24.00 ± 2.55 | 81.00 ± 6.27 b
| |
Group
|
Kidney function test
| |||
BUN (mg/dL)
|
Cr (mg/dL)
| |||
Untreated control | 19.60 ± 0.68 | 0.20 ± 0.00 | ||
7% Tween 80 | 19.00 ± 0.00 c
| 0.20 ± 0.01 | ||
Ethanolic extract of CPF-1 | 21.00 ± 0.45 b
| 0.22 ± 0.03 |