Background
Methods
Solvents and reagents
Extraction
Preparation of extract solutions
Biological evaluation
Phytochemical analysis
Biological evaluation
Antioxidant assays
Total antioxidant capacity (TAC)
Total reducing power (TRP)
Cytotoxicity assays
Brine shrimp lethality assay
Cytotoxicity against THP-1 human leukemia cell line
Cytotoxicity against Hep G2 cell line
Protein kinase inhibition assay
Antimicrobial assays
Antileishmanial assay
Antibacterial assay
Antifungal assay
α-Amylase inhibition assay
Statistical analysis
Results and discussion
Yield of the extract
Phytochemical analysis
TPC
TFC
RP-HPLC polyphenols profiling
Classification of phenolic compounds | Retention time | Extracts (mg/g extract) | |||
---|---|---|---|---|---|
Ea | Dw | M | |||
Phenols | Hydroquinone | 10.07 | Nd | Nd | Nd |
Pyrocatechol | 12.06 | Nd | 0.071 ± 0.003 | Nd | |
Resorcinol | 17.48 | 0.239 ± 0.01 | 0.196 ± 0.02 | Nd | |
Cinnamic acid derivatives | Caffeic acid | 27.51 | 0.224 ± 0.03 | 0.128 ± 0.01 | 1.496 ± 0.2 |
Chlorogenic acid | 25.80 | 2.032 ± 0.02 | 0.985 ± 0.04 | 3.295 ± 0.4 | |
Ferulic acid | 34.87 | 0.105 ± 0.02 | Nd | Nd | |
p-Coumaric acid | 30.16 | 0.063 ± 0.002 | 0.024 ± 0.002 | 0.121 ± 0.02 | |
Sinapic acid | 50.05 | Nd | 1.059 ± 0.01 | 0.860 ± 0.04 | |
Trans cinnamic acid | 54.44 | Nd | 0.068 ± 0.004 | Nd | |
Benzoic acid derivatives | Gallic acid | 15.24 | 3.495 ± 0.24 | 0.089 ± 0.005 | 1.654 ± 0.2 |
Salicylic acid | 28.21 | Nd | Nd | 0.124 ± 0.01 | |
Vanillic acid | 38.48 | 0.127 ± 0.02 | 0.054 ± 0.003 | Nd | |
Flavonol flavonoid | Kaempferol | 73.06 | 0.103 ± 0.01 | 0.412 ± 0.04 | 0.411 ± 0.03 |
Quercetin | 63.13 | 0.038 ± 0.015 | 0.065 ± 0.005 | 0.220 ± 0.02 | |
flavonol glycoside | Rutin | 58.79 | 0.035 ± 0.003 | 0.076 ± 0.003 | Nd |
Flavan-3-ol flavonoid | Catechin | 23.61 | 0.162 ± 0.02 | 0.203 ± 0.02 | Nd |
Tannin | Ellagic acid | 67.35 | Nd | Nd | Nd |
Miscellaneous | Coumarin | 44.85 | 0.402 ± 0.03 | 0.427 ± 0.03 | 1.689 ± 0.2 |
Biological evaluation
Antioxidant assays
Cytotoxicity assessment
Brine shrimp lethality assay
Extracts | Brine shrimp cytotoxicity | THP-1 cytotoxicity | Hep G2 cytotoxicity | Protein kinase inhibition | |||||
---|---|---|---|---|---|---|---|---|---|
% mortality | LC50 (μg/ml) | % inhibition | IC50(μg/ml) | % inhibition | IC50 (μg/ml) | Diameter (mm) at 100 μg/disc | MIC (μg/disc) | ||
200 μg/ml | 20 μg/ml | 20 μg/ml | Clear zone | Bald zone | |||||
Nh | 63.33 ± 5.77bc
| 91.40 ± 0.23 | 31.5 ± 1.5 | > 20 | 61.14 ± 0.70c
| 8.95 ± 0.20 | 10.00 ± 1.0 | 16.00 ± 1.0e
| 12.5 |
Nh-Ea | 73.33 ± 5.77b
| 112.37 ± 1.20 | 45.57 ± 1.00c
| > 20 | 38.43 ± 0.78 | > 20 | 6.67 ± 0.58 | 9.67 ± 0.58 | 100 |
Nh-E | 53.33 ± 5.77cd
| 153.33 ± 1.12 | 35.87 ± 0.85 | > 20 | 57.37 ± 0.67d
| 19.10 ± 0.50 | 6.67 ± 0.58 | 15.00 ± 1.0e
| 25 |
C | 93.33 ± 5.77a
| 85.40 ± 0.26 | 26.67 ± 0.91 | > 20 | 60.50 ± 0.50c
| 9.50 ± 0.28 | 6.67 ± 0.58 | 10.67 ± 0.58fg
| 50 |
Ea | 43.33 ± 5.77 | > 200 | 14.17 ± 1.35 | > 20 | 61.53 ± 0.35c
| 8.63 ± 0.30 | 6.67 ± 0.58 | 9.33 ± 0.58 | 100 |
C-M | 53.33 ± 5.77cd
| > 200 | 18.27 ± 2.91 | > 20 | 20.60 ± 0.30 | > 20 | 9.00 ± 1.0 | 12.00 ± 1.0f
| 100 |
Ea-M | 63.33 ± 5.77bc
| 137.37 ± 0.55 | 19.63 ± 2.87 | > 20 | 31.45 ± 0.15 | > 20 | 9.00 ± 1.0 | 30.00 ± 1.0b
| 12.5 |
A | 63.33 ± 5.77bc
| 123.83 ± 0.95 | 33.13 ± 1.70 | > 20 | 54.71 ± 0.25e
| 19.80 ± 0.20 | – | 12.00 ± 1.0f
| 100 |
M | 93.33 ± 5.77a
| 110 ± 0.75 | 45.37 ± 1.56c
| > 20 | 56.63 ± 0.20d
| 19.76 ± 0.20 | 28 ± 1.0 | 40.00 ± 1.0a
| 12.5 |
A-M | 63.33 ± 5.77bc
| 123.5 ± 0.92 | 77.63 ± 1.33b
| 4.9 ± 0.20 | 25.90 ± 0.19 | > 20 | 15.00 ± 1.0 | 25.0 ± 1.0c
| 50 |
A-E | 93.33 ± 5.77a
| 123.4 ± 0.89 | 48.27 ± 3.00c
| > 20 | 49.80 ± 0.67 | > 20 | 6.67 ± 0.58 | 12.0 ± 1.0f
| 100 |
E | 96.67 ± 5.77a
| 46.91 ± 0.19 | 20.8 ± 1.90 | > 20 | 37.40 ± 0.30 | > 20 | 6.33 ± 0.58 | 7.67 ± 0.58 | – |
A-Dw | 93.33 ± 5.77a
| 52.63 ± 1.11 | 47.43 ± 2.16c
| > 20 | 29.60 ± 0.80 | > 20 | 7.00 ± 1.0 | 15.00 ± 1.0e
| 50 |
M-Dw | 93.33 ± 5.77a
| 64.93 ± 1.40 | 30.47 ± 1.95 | > 20 | 63.07 ± 0.40b
| 8.68 ± 0.25 | – | 12.00 ± 1.0f
| 100 |
Dw | 33.33 ± 5.77 | > 200 | 36.7 ± 1.15 | > 20 | 46.83 ± 0.25 | > 20 | 7.00 ± 1.0 | – | – |
Doxorubicin | 96.67 ± 5.77a
| 5.63 ± 0.25 | 98.37 ± 0.15a
| 5.30 ± 0.20 | |||||
5-Florouracil | 99.2 ± 0.92a
| 5.13 ± 0.13 | |||||||
Vincristine | 99.33 ± 0.83a
| 8.24 ± 0.12 | |||||||
Surfactin | 20 ± 1.0d
| ||||||||
DMSO | – | – | – | – |
Cytotoxicity against THP-I cell line
Cytotoxicity against Hep G2 hepatoma cell line
Protein kinase inhibition assay
Leishmanicidal activity
Antibacterial activity
Extracts |
*Diameter of growth inhibition zone at 100 μg/disc (mm) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
E. coli
| MIC (μg/ml) |
S. aureus
| MIC (μg/ml) |
B. subtilis
| MIC (μg/ml) |
K. pneumoniae
| MIC (μg/ml) |
P. aeruginosa
| MIC (μg/ml) | |
Nh | 7.33 ± 0.58 | – | 9.33 ± 0.58 | – | 8.33 ± 0.58 | – | 7.33 ± 0.58 | – | 8.33 ± 0.58 | – |
Nh-Ea | 6.67 ± 0.58 | – | 7.33 ± 0.58 | – | 6.67 ± 0.58 | – | 7.33 ± 0.58 | – | 7.67 ± 0.58 | – |
Nh-E | 7.33 ± 0.58 | – | 9.33 ± 0.58 | – | 7.67 ± 0.58 | – | 7.33 ± 0.58 | – | 7.33 ± 0.58 | – |
C | 7.67 ± 0.58 | – | 8.67 ± 0.58 | – | 9.33 ± 0.58 | – | 7.33 ± 0.58 | – | 7.33 ± 0.58 | – |
Ea | 7.00 ± 1.00 | – | 6.67 ± 0.58 | – | 10.67 ± 0.58d
| >100 | 7.33 ± 0.58 | – | 8.00 ± 1.00 | – |
C-M | 6.67 ± 0.58 | – | 6.67 ± 0.58 | – | 8.33 ± 0.58 | – | 7.33 ± 0.58 | – | 8.33 ± 0.58 | – |
Ea-M | 6.67 ± 0.58 | – | 6.67 ± 0.58 | – | 10.33 ± 0.58d
| >100 | 7.33 ± 0.58 | – | 9.33 ± 0.58 | – |
A | 6.67 ± 0.58 | – | 8.33 ± 0.58 | – | 9.33 ± 0.58 | – | 7.33 ± 0.58 | – | 9.33 ± 0.58 | – |
M | 6.33 ± 0.58 | – | 6.67 ± 0.58 | – | 16.33 ± 0.58c
| 100 | 8.33 ± 0.58 | – | 7.33 ± 0.58 | – |
A-M | 7.33 ± 0.58 | – | 7.33 ± 0.58 | – | 8.67 ± 0.58 | – | 7.67 ± 0.58 | – | 8.33 ± 0.58 | – |
A-E | 6.67 ± 0.58 | – | 7.33 ± 0.58 | – | 9.33 ± 0.58 | – | 7.33 ± 0.58 | – | 7.33 ± 0.58 | – |
E | 6.67 ± 0.58 | – | 7.33 ± 0.58 | – | – | – | 7.33 ± 0.58 | – | 8.33 ± 0.58 | – |
A-Dw | 6.67 ± 0.58 | – | 6.67 ± 0.58 | – | 8.66 ± 0.58 | – | 7.33 ± 0.58 | – | 9.33 ± 0.58 | – |
M-Dw | 6.67 ± 0.58 | – | 7.33 ± 0.58 | – | 7.33 ± 0.58 | – | 9.33 ± 0.58 | – | 7.33 ± 0.58 | – |
Dw | 6.67 ± 0.58 | – | 6.33 ± 0.58 | – | 8.33 ± 0.58 | – | 7.67 ± 0.58 | – | 8 ± 1.00 | – |
Roxithromycin | 25.67 ± 0.58a
| 0.334 | 24.33 ± 0.58a
| 0.334 | 22.33 ± 0.58a
| 0.334 | 23.22 ± 0.58a
| 0.334 | 25.67 ± 0.58 | 0.334 |
Cefixime | 18.33 ± 0.58b
| 1.11 | 24.33 ± 0.58a
| 0.334 | 18.33 ± 0.58b
| 1.11 | 20.33 ± 0.58b
| 0.334 | – | – |
DMSO | – | – | – | – | – | – |
Antifungal assay
Extracts |
*Diameter of zone of inhibition (mm) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
A. fumigatus
| MIC (μg/disc) |
F. Solani
| MIC (μg/disc) |
A. niger
| MIC (μg/disc) |
A. flavus
| MIC (μg/disc) |
Mucor sp.
| MIC (μg/disc) | |
Nh | 8.67 ± 0.58 | – | 11.33 ± 0.58cd
| 100 | 7.33 ± 0.58 | – | 7.33 ± 0.58 | – | 7.33 ± 0.58 | – |
Nh-Ea | 6.67 ± 0.58 | – | 8.33 ± 0.58 | – | 7.67 ± 0.58 | – | 6.67 ± 0.58 | – | 7.33 ± 0.58 | – |
Nh-E | 8.33 ± 0.58 | – | 12.33 ± 0.58c
| 100 | 7.33 ± 0.58 | – | 7.67 ± 0.58 | – | 6.67 ± 0.58 | – |
C | 7.33 ± 0.58 | – | 10.33 ± 0.58d
| 100 | 7.67 ± 0.58 | – | 7.67 ± 0.58 | – | 6.33 ± 0.58 | – |
Ea | 7.33 ± 0.58 | – | 8.33 ± 0.58 | – | 6.67 ± 0.58 | – | 8.33 ± 0.58 | – | 7.33 ± 0.58 | – |
C-M | 7.67 ± 0.58 | – | 7.33 ± 0.58 | – | 7.67 ± 0.58 | – | 7.67 ± 0.58 | – | 7.33 ± 0.58 | – |
Ea-M | 9.33 ± 0.58 | – | 7.33 ± 0.58 | – | 7.33 ± 0.58 | – | 7.67 ± 0.58 | – | 7.33 ± 0.58 | – |
A | 7.67 ± 0.58 | – | 7.67 ± 0.58 | – | 7.67 ± 0.58 | – | 7.67 ± 0.58 | – | 7.33 ± 0.58 | – |
M | 11.33 ± 0.58c
| 100 | 9.33 ± 0.58 | – | 8.33 ± 0.58 | – | 7.67 ± 0.58 | – | 6.33 ± 0.58 | – |
A-M | 8.33 ± 0.58 | – | 11.33 ± 0.58cd
| 100 | 8.33 ± 0.58 | – | 9.33 ± 0.58 | – | 6.33 ± 0.58 | – |
A-E | 7.33 ± 0.58 | – | 7.33 ± 0.58 | – | 7.67 ± 0.58 | – | 7.33 ± 0.58 | – | 7.33 ± 0.58 | – |
E | 13.67 ± 0.58b
| 50 | 7.33 ± 0.58 | – | 8.33 ± 0.58 | – | 7.33 ± 0.58 | – | 7.33 ± 0.58 | – |
A-Dw | 7.33 ± 0.58 | – | 14.33 ± 0.58b
| 50 | 7.33 ± 0.58 | – | 8.33 ± 0.58 | – | 7.33 ± 0.58 | – |
M-Dw | 7.33 ± 0.58 | – | 8.0 ± 0.58 | – | 7.67 ± 0.58 | – | 6.67 ± 0.58 | – | 7.33 ± 0.58 | – |
Dw | 8.67 ± 0.58 | – | 7.33 ± 0.58 | – | 8.67 ± 0.58 | – | 10.33 ± 0.58b
| 100 | 7.33 ± 0.58 | – |
Clotrimazole | 32.33 ± 0.58a
| 5 | 29.33 ± 0.58a
| 5 | 31.67 ± 0.58a
| 5 | 23.67 ± 0.58a
| 10 | 41.67 ± 0.58a
| 2.5 |
DMSO | – | – | – | – | – | – | – |