Background
Methods
Solvents and reagents
Collection and extraction
Extract recovery
Phytochemical analysis
Determination of total phenolic content (TPC)
Determination of total flavonoid content (TFC)
RP-HPLC quantitative analysis
Biological evaluation
Antioxidant assays
Cytotoxicity assays
Brine shrimp lethality assay
Cytotoxicity against THP-1 human leukemia cell line
Cytotoxicity against Hep G2 cell line
Cytotoxicity against isolated lymphocytes
Protein kinase inhibition assay
Antileishmanial assay
α-Amylase inhibition assay
Antifungal assay
Statistical analysis
Results and discussion
Effect of extraction solvent on the extract yields
Phytochemical analysis
TPC
TFC
RP-HPLC
Standards | Retention time (min) | Samples | |||||
---|---|---|---|---|---|---|---|
Et | CM | MEt | DA | M | D | ||
Hydroquinone | 10.07 | -- | -- | -- | -- | -- | -- |
Pyrocatechol | 12.06 | 0.049 | 0.013 | 0.361 | 0.232 | 0.111 | 0.017 |
Gallic acid | 15.24 | 0.534 | 0.499 | 2.822 | 5.282 | 2.112 | 1.382 |
Resorcinol | 17.48 | -- | -- | -- | -- | -- | -- |
Catechin | 23.61 | -- | 0.150 | 2.291 | 1.380 | 0.808 | 0.095 |
Chlorogenic acid | 25.80 | 1.009 | 2.699 | 9.478 | 15.336 | 7.122 | 5.162 |
Caffeic acid | 27.51 | -- | -- | -- | -- | -- | -- |
Vanillic acid | 28.21 | 1.271 | -- | -- | -- | -- | -- |
p-Coumaric acid | 30.16 | 0.534 | 0.146 | 0.232 | 1.026 | 0.339 | 0.036 |
Ferulic acid | 34.87 | 0.701 | 0.046 | 0.461 | 0.173 | 0.192 | -- |
Sinapinic acid | 38.48 | -- | -- | -- | -- | -- | -- |
Coumarin | 44.85 | 0.105 | 0.105 | 0.368 | -- | 0.149 | -- |
Salicylic acid | 50.05 | -- | -- | -- | -- | -- | -- |
Trans cinnamic acid | 54.44 | -- | -- | -- | -- | -- | -- |
Rutin | 58.79 | 1.181 | 0.094 | -- | 0.245 | 0.407 | -- |
Quercetin | 63.13 | 0.164 | 0.060 | 0.969 | 0.616 | 0.085 | 0.059 |
Ellagic acid | 67.35 | -- | -- | -- | -- | -- | -- |
Kaempferol | 73.06 | 0.291 | 0.069 | 0.284 | -- | 0.129 | -- |
Biological evaluation
Antioxidant assays
Cytotoxicity assessment
Brine shrimp lethality assay
Samples | Brine shrimp cytotoxicity (μg/ml) | THP-1 cytotoxicity (μg/ml) | Hep G2 cytotoxicity (μg/ml) | Isolated lymphocytes | Protein kinase inhibition (μg/disc) | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
% mortality | LC50
| % inhibition | IC50
| % inhibition | IC50
| % inhibition | IC50
| Diameter (mm) at 100 μg/disc | MIC | ||
200 | 20 | 20 | 20 | Clear zone | Bald zone | ||||||
NH | 33.6 ± 2.5
a
| >200 | 22.32 ± 2.41
c
| > 20 | 46.73 ± 0.85
a
| >20 | 8.76 ± 1.12 | >20 | -- | 7 ± 0.15
c
| -- |
C | 80.00 ± 0.49
b
| 96.45 ± 0.98 | 14.24 ± 1.77
a
| > 20 | 43.20 ± 1.56
a
| >20 | 10.43 ± 2.56 | >20 | -- | 25 ± 0.2
a
| 12.5 |
A | 42.5 ± 0.94
b
| >200 | 82.43 ± 1.68 | 3.88 ± 0.53 | 41.20 ± 1.56
a
| >20 | 14.43 ± 1.21 | >20 | -- | 8 ± 0.47 | -- |
EtA | 80.0 ± 0.47
a
| 77.7 ± 1.14 | 38.42 ± 2.13
b
| > 20 | 38.03 ± 2.28
b
| >20 | 12.43 ± 1.43 | >20 | -- | 7 ± 0.39
c
| -- |
Et | 50.0 ± 1.25
c
| >200 | 47.25 ± 2.99 | > 20 | 41.32
a
± 2.56
b
| >20 | 15.21 ± 1.22 | >20 | -- | 27 ± 0.47
a
| 12.5 |
CE | 91.5 ± 0.94
c
| 49.62 ± 0.22 | 19.56 ± 3.22 | > 20 | 9.72 ± 0.87 | >20 | 13.87 ± 2.12 | >20 | -- | 9 ± 0.52
b
| -- |
CM | 100.0 ± 1.70
a
| 34.54 ± 0.16 | 21.56 ± 2.87 | > 20 | 28.27 ± 0.85 | >20 | 6.65 ± 1.54 | >20 | -- | 7 ± 0.21 | -- |
EEt | 100.0 ± 0.94 | 81.4 ± 0.17 | 43.12 ± 3.18 | > 20 | 42.79 ± 0.76
a
| >20 | 5.86 ± 1.01 | >20 | -- | 9 ± 0.15 | -- |
MEt | 80.30 ± 2.36 | 69.9 ± 0.18 | 71.5 ± 4.11 | 5.59 ± 0.25 | 23.51 ± 1.54 | >20 | 14.76 ± 2.22 | >20 | -- | 10 ± 0.41
b
| -- |
E | 100.0 ± 0.94
a
| 86.62 ± 1.35 | 82.52 ± 1.45
b
| 4.95 ± 0.53 | 42.30 ± 1.12
a
| >20 | 12.87 ± 1.12 | >20 | -- | 26 ± 0.42
a
| 12.5 |
DA | 82.40 ± 1.25 | 49.1 ± 1.18 | 62.85 ± 2.45 | 9.24 ± 0.53 | 26.21 ± 1.21 | >20 | 16.34 ± 4.45 | >20 | -- | 8 ± 0.35 | -- |
M | 91.50 ± 1.70 | 71.0 ± 0.74 | 22.53 ± 0.83
a
| > 20 | 45.50 ± 1.43
a
| >20 | 18.45 ± 3.98 | >20 | -- | 28 ± 0.35
a
| 12.5 |
DM | 80.50 ± 1.70 | 92.3 ± 1.70 | 42.54 ± 2.45 | > 20 | 9.64 ± 0.76
c
| >20 | 22.54 ± 5.16 | >20 | -- | 8 ± 0.35 | -- |
D | 90.0 ± 1.70 | 78.4 ± 1.70 | 48.54 ± 0.83
a
| > 20 | -- | >20 | 12.11 ± 1.15 | >20 | -- | 9 ± 0.55 | -- |
Doxorubicin | 100 | 5.93 | 98 ± 0.18 | 5.1 | |||||||
5-Florouracil | 100 | 5 | |||||||||
Vincristine | 100 | 8.1 | 73.45 ± 1.56 | 6.66 ± 0.22 | |||||||
Surfactin | 30 ± 1.02 | ||||||||||
DMSO | -- | -- | -- | ||||||||
1% DMSO in PBS/sea water | -- | -- | -- | -- | -- |
Cytotoxicity against THP-1 cell line
Cytotoxicity against Hep G2 cell line
Cytotoxicity against isolated lymphocytes
Protein kinase inhibition assay
Antileishmanial potential
α-Amylase inhibition assay
Antifungal assay
Samples |
aDiameter of growth inhibition zone at 100 μg/disc | |||
---|---|---|---|---|
A. fumigatus
|
F. Solani
|
A. niger
|
A. flavus
| |
NH | 7 ± 0.24 | 8 ± 1.85 | 7 ± 1.21 | 7.5 ± 0.45 |
C | 7.5 ± 1.0 | 7 ± 0.25 | 7.5 ± 1.2 | 7.5 ± 0.25 |
A | 7.5 ± 0.56 | 7.5 ± 1.21 | 7.5 ± 1.5 | 7.5 ± 0.32 |
EtA | 7 ± 0.45 | 8 ± 0.56 | 7.5 ± 0.78 | 6.5 ± 0.41 |
Et | 8 ± 0.52 | 8 ± 1.40 | 7 ± 0.25 | 8 ± 0.35 |
CE | 7.5 ± 0.45 | 7 ± 0.35 | 8 ± 0.50 | 7.5 ± 0.30 |
CM | 7 ± 0.35 | 8 ± 0.40 | 7 ± 0.57 | 7 ± 0.25 |
EEt | 7.5 ± 1.2 | 7 ± 0.35 | 7.5 ± 0.20 | 7 ± 0.65 |
MEt | 8 ± 0.40 | 7 ± 0.65 | 8 ± 1.10 | 7.5 ± 0.67 |
E | 8 ± 0.45 | 7.5 ± 1.43 | 8 ± 1.0 | 9 ± 0.98 |
DA | 7 ± 0.40 | 7 ± 1.50 | 7 ± 1.67 | 8 ± 0.55 |
M | 7 ± 0.50 | 9 ± 1.54 | 8 ± 0.76 | 7.5 ± 0.69 |
DM | 7.5 ± 0.5 | 8 ± 0.78 | 7.5 ± 0.80 | 6.5 ± 0.55 |
D | 8 ± 0.55 | 7.5 ± 0.45 | 9 ± 0.75 | 7 ± 0.40 |
Clotrimazole | 23 ± 0.12 | 24 ± 0.98 | 22 ± 1.03 | 24 ± 1.01 |
DMSO | -- | -- | -- | -- |