Background
Methods
Plant collection
Preparation of extract/fractions
Chemicals
Preliminary phytochemical screening
Estimation of total phenolic content (TPC)
Estimation of total flavonoid content (TFC)
Antioxidant assays
Sample preparation
Antioxidant activity assessment assays
DPPH radical scavenging activity assay
Superoxide anion radical quenching assay
Hydroxyl radical quenching activity
Hydrogen peroxide radical quenching assay
ABTS radical scavenging activity
Iron chelating power
β- carotene bleaching test
Anti-lipid peroxidation analysis
Total antioxidant capacity (TAC) (Phosphomolybdate assay)
Reducing power assay
Anti-hemolytic activity
Cytotoxicity screening
Cell lines and cell culture
MTT assay
Estimation of cytotoxicity and IC50
HPLC-DAD analysis
Statistical analysis
Results and discussion
Extraction yield
Extract/fraction | Extraction yield (%) | TPC (mg gallic acid equivalent/g dry sample) | TFC (mg rutin equivalent/g dry sample) |
---|---|---|---|
AHM | 15.73e
| 87.6 ± 1.23c
| 127 ± 0.52c
|
AHH | 5.46d
| 57.7 ± 1.17e
| 71 ± 0.86a
|
AHE | 27.77b
| 120.3 ± 1.15b
| 129 ± 1.32b
|
AHC | 1.94f
| 36.0 ± 0.95f
| 34.5 ± 1.13d
|
AHB | 41.66a
| 129 ± 2.98a
| 139 ± 1.04a
|
AHA | 8.05c
| 73.3 ± 1.53d
| 95 ± 0.05c
|
Qualitative phytochemical screening
Compound Class | Samples | |||||
---|---|---|---|---|---|---|
AHM | AHH | AHC | AHE | AHB | AHA | |
Tannins | + | + | + | + | + | + |
Steroids | + | + | + | + | + | + |
Saponins | + | + | + | + | + | + |
Alkaloids | + | + | − | + | + | + |
Flavonoids | + | + | + | + | + | + |
Coumarins | + | − | − | + | − | + |
Terpenoids | + | + | + | + | + | + |
Pholobatanins | + | + | − | + | + | − |
Reducing sugars | − | − | − | − | − | + |
Anthraquinones | + | − | − | − | − | − |
Cardiac Glycosides | + | + | + | + | + | + |
Total phenolic and flavonoid content
Antioxidant activity assessment
EC50 values | |||||||||
---|---|---|---|---|---|---|---|---|---|
Sample | DPPH radical | Superoxide radical | Hydroxyl radical | Hydrogen peroxide radical | ABTS radical | Iron chelating power | β-carotene bleaching inhibition | Total antioxidant index at 250 μg/ml | Lipid peroxidation inhibition |
AHM | 19.5 ± 0.43a
| 43.0 ± 0.37b
| 39.0 ± 0.44a
| 40.3 ± 0.26a
| 193 ± 0.26d
| 41.7 ± 0.34a
| 86.4 ± 0.62d
| 1.6 ± 0.69b
| 156.1 ± 0.76d
|
AHH | 46.5 ± 0.55b
| 59.3 ± 0.36c
| 42.3 ± 0.29b
| 80.6 ± 0.43c
| 401 ± 0.23e
| 91.7 ± 0.17b
| 173.5 ± 0.85d
| 1.2 ± 0.35c
| 562.3 ± 0.55g
|
AHE | 18.0 ± 0.45a
| 38.3 ± 0.40a
| 35.7 ± 0.40a
| 37.2 ± 0.36a
| 173 ± 0.11c
| 40.3 ± 0.21a
| 48.4 ± 0.55b
| 1.7 ± 0.61b
| 61.3 ± 0.52c
|
AHC | 65.0 ± 0.36c
| 90.8 ± 0.55d
| 63.0 ± 0.09c
| 115.6 ± 1.51d
| 600 ± 0.26e
| 130.1 ± 0.17d
| 260.6 ± 1.05f
| 0.7 ± 0.73e
| 467.3 ± 0.60f
|
AHB | 16.7 ± 0.43a
| 49.0 ± 0.41b
| 33.0 ± 0.16a
| 42.0 ± 0.11a
| 98.0 ± 0.12a
| 39.0 ± 0.26a
| 66.2 ± 0.72c
| 1.7 ± 0.41a
| 50.0 ± 0.61b
|
AHA | 39.5 ± 0.51b
| 46.3 ± 0.52b
| 47.0 ± 0.12b
| 68.1 ± 0.36b
| 478 ± 0.12e
| 103.0 ± 0.12c
| 131.6 ± 1.15e
| 1.3 ± 0.14e
| 401.1 ± 0.12e
|
AA | 15.0 ± 0.41a
| - | - | 35.0 ± 0.21a
| 61.0 ± 0.21b
| - | - | 1.8 ± 0.24b
| - |
GA | - | 32.3 ± 0.28a
| 29.0 ± 0.16a
| - | - | - | - | - | 43.5 ± 0.86a
|
C | - | - | - | - | - | 37.7 ± 0.3a
| 40.4 ± 1.01a
| - | - |
BHA | - | - | - | - | - | - | - | 59.3 ± 0.98c
| |
BHT | - | - | - | - | - | - | 50.11 ± 0.59b
| - |
DPPH radical scavenging activity
Superoxide radical scavenging activity
Hydroxyl radical scavenging activity
Hydrogen peroxide radical scavenging activity
ABTS radical scavenging assay
Chelating activity on Fe2+
β-carotene bleaching inhibition
Anti-lipid peroxidation potential
Total antioxidant capacity assay
Reducing power
Anti-hemolytic activity
Samples | Positive control (A) | Negative control (B) | Optical densityC at 560 nm concentration (μg/ml) | % Inhibition of hemolysis at 1000 (μg/ml) | |||
---|---|---|---|---|---|---|---|
100 | 250 | 500 | 1000 | ||||
AHM | 1.45 ± 0.02 | 0.11 ± 0.05 | 0.69 ± 0.01 | 0.85 ± 0.05 | 0.96 ± 0.05 | 1.15 ± 0.01 | 79.3c
|
AHH | 1.45 ± 0.03 | 0.11 ± 0.05 | 0.65 ± 0.05 | 0.71 ± 0.05 | 0.88 ± 0.05 | 0.98 ± 0.05 | 67.6d
|
AHC | 1.45 ± 0.05 | 0.11 ± 0.05 | 0.51 ± 0.05 | 0.63 ± 0.03 | 0.71 ± 0.05 | 0.87 ± 0.02 | 60.0e
|
AHE | 1.45 ± 0.06 | 0.11 ± 0.05 | 0.72 ± 0.04 | 0.89 ± 0.09 | 1.14 ± 0.01 | 1.42 ± 0.02 | 97.9a
|
AHB | 1.45 ± 0.04 | 0.11 ± 0.05 | 0.67 ± 0.05 | 0.81 ± 0.05 | 0.92 ± 0.05 | 1.29 ± 0.03 | 88.9b
|
AHA | 1.45 ± 0.07 | 0.11 ± 0.05 | 0.49 ± 0.05 | 0.59 ± 0.05 | 0.64 ± 0.05 | 0.79 ± 0.05 | 54.5f
|
QR | 1.45 ± 0.08 | 0.11 ± 0.05 | 0.72 ± 0.04 | 0.89 ± 0.09 | 1.14 ± 0.01 | 1.43 ± 0.02 | 98.6a
|
Assessment of anticancer potential by MTT assay
Extract/Fraction | MDA361 cell line | SI | HCC38 cell line | SI | Vero cell line |
---|---|---|---|---|---|
IC50 (μg/ml) | IC50V/IC50M | IC50 (μg/ml) | IC50V/IC50H | IC50 (μg/ml) | |
AHM | 46.9 ± 1.31* | 5.42 | 75.9 ± 1.32* | 3.34 | 254 ± 1.81 |
AHE | 29.9 ± 0.91* | 9.83 | 39.5 ± 0.87* | 7.44 | 294 ± 1.55 |
AHB | 37.1 ± 1.01* | 6.97 | 56.1 ± 0.93* | 4.60 | 258 ± 1.68 |
HPLC-DAD analysis
Compound | Signal wavelength | Retention time (min) | Regression analysis | R2
|
---|---|---|---|---|
Gallic acid | 257 | 4.35 | y = 3.0565x + 8.01 | 0.9998 |
Rutin | 257 | 15.91 | y = 1.7773x−48.49 | 0.9958 |
Catechin | 279 | 9.70 | y = 1.1764x + 7.96 | 0.989 |
Caffeic acid | 325 | 325 | y = 12.569x + 22.66 | 0.9905 |
Apigenin | 325 | 23.53 | y = 12.950x−52.12 | 0.9957 |
Myricetin | 368 | 18.72 | y = 2.560x−15.09 | 0.9994 |
Kaempherol | 368 | 23.59 | y = 10.967x + 125.59 | 0.9964 |
Quercetin | 368 | 21.31 | y = 4.8624x + 35.01 | 0.9993 |
Extract/fraction | Compounds | Signal wavelength | Retention time | Quantity (μg/100 mg dry powder) |
---|---|---|---|---|
AHE | Gallic acid | 275 nm | 4.52 | 52.92 |
Catechin | 279 nm | 11.43 | 8648.0 | |
Myricetin | 368 nm | 17.08 | 34.60 | |
AHB | Gallic acid | 257 nm | 4.41 | 144.70 |
Catechin | 279 nm | 10.55 | 3995.21 |