Background
Methods
Microbial strains
Plant collection and sample preparation
Preparation of plant extracts and fractions for antimicrobial and toxicity assays
Extraction of plant material for phytochemical determination
Antimicrobial screening
Culturing bacterial and fungal strains
In vitro microdilution assay
Phytochemical determination
UPLC/MS detection of active compound(s)
Toxicological assays
In vitro cytotoxicity assay
In vitro genotoxicity assay
Statistical analysis
Results
Antimicrobial activity
Antibacterial activity
Plant species | Extract/fraction | MIC (mg/ml) | ||||||
---|---|---|---|---|---|---|---|---|
Ec
|
Kp
|
Ef
| SD |
Pa
|
Sa
| ST | ||
T. diversifolia
| Ethanol | 1.25 | 0.62 |
0.07*
| 0.31 | 1.25 | 0.31 | 0.62 |
Methanol | 0.62 | 0.62 | 0.62 | 0.62 | 1.25 | 0.62 |
0.15
| |
DCM | 0.31 |
0.15
| 0.31 | 0.31 | 0.31 | 0.62 | 0.62 | |
Acetone |
0.15
|
0.15
|
0.07*
| 0.31 | 0.31 | 0.62 | 0.31 | |
Hot water | 1.25 | 0.62 | 2.5 | 1.25 | 1.25 |
0.15
| 0.62 | |
Hexane | 1.25 | 0.31 | 0.62 | 0.62 |
0.07*
| 0.62 | 0.62 | |
Chloroform | 1.25 | 0.62 | 0.31 | 1.25 |
0.07*
|
0.15
| 1.25 | |
Butanol | > 2.5 | 2.5 | > 2.5 | > 2.5 | > 2.5 | > 2.5 | > 2.5 | |
ETAC | 2.5 | 2.5 | 0.62 | > 2.5 |
0.15
| 0.31 | 2.5 | |
T. rotundifolia
| Ethanol | 0.31 |
0.15
| 0.31 | 0.62 | 0.62 |
0.07*
| 0.62 |
Methanol | 0.62 | 0.31 | 2.5 |
0.15
| 0.62 | 0.62 | 0.31 | |
DCM | 0.62 |
0.15
|
0.15
|
0.15
| 0.31 |
0.07*
|
0.15
| |
Acetone | 0.31 |
0.07*
|
0.03**
| 0.31 | 0.31 |
0.07*
|
0.07*
| |
Hot water |
0.01**
| 0.62 |
0.07*
| 0.62 | 1.25 | 0.31 |
0.01**
| |
Hexane | 2.5 | 0.62 | 1.25 | 1.25 | 0.62 | 1.25 | 1.25 | |
Chloroform | 2.5 | > 2.5 | 1.25 | > 2.5 | 0.62 | > 2.5 | > 2.5 | |
Butanol | > 2.5 | > 2.5 | > 2.5 | > 2.5 | > 2.5 | > 2.5 | > 2.5 | |
ETAC |
0.15
| 1.25 | > 2.5 | 1.25 | 1.25 | 0.62 | 1.25 | |
Gent 2 mg/ml (+ve con.) |
0.016*
| 0.50 | 0.50 |
0.004**
|
0.008**
|
0.008**
|
0.08**
|
Antimycobacterial activity
Plant species | Extract/fraction | MIC (mg/ml | |||
---|---|---|---|---|---|
M. aurum
|
M. bovis
|
M. fortuitum
|
M. smegmatis
| ||
T. diversifolia | Ethanol | 0.62 | 0.31 |
0.15
|
0.15
|
Methanol | 0.31 | 0.62 | 0.31 |
0.15
| |
DCM |
0.15
| 0.31 | 0.31 | 0.31 | |
Acetone |
0.15
|
0.15
| 0.62 | 2.5 | |
Hot water | 0.31 | 0.31 | 0.62 | 0.31 | |
Hexane | 0.62 | 0.31 | 1.25 | 2.5 | |
Chloroform | 0.62 | 0.31 | > 2.5 | > 2.5 | |
Butanol | 0.62 | 2.5 | 2.5 | 2.5 | |
ETAC | 0.62 | 0.62 | 0.31 | 2.5 | |
T. rotundifolia | Ethanol | 0.62 | 0.62 | 0.62 | 0.62 |
Methanol | 1.25 | 0.31 | 1.25 | 0.62 | |
DCM |
0.07*
|
0.03**
| 0.03** | 0.07* | |
Acetone | 0.07* | 0.07* | 0.03** |
0.07*
| |
Hot water | > 2.5 | > 2.5 | 2.5 | 1.25 | |
Hexane |
0.15
|
0.15
| 0.62 | 0.31 | |
Chloroform | 2.5 | 0.31 | 0.31 | 0.31 | |
Butanol | > 2.5 | > 2.5 | > 2.5 | > 2.5 | |
ETAC |
0.15
|
0.07*
|
0.15
|
0.15
| |
Strep (+ve con) | – |
> 0.01**
| 0.063 |
> 1.0
| > 0.01 |
Rif (+ve con) | – |
> 0.01**
| > 1.0 |
0.063
| > 0.01 |
Antifungal activity
Plant species | Extract/fraction | MIC (mg/ml) | |||||
---|---|---|---|---|---|---|---|
A. fumigatus
|
C. neoformans
|
C. albicans
| |||||
48 h | 72 h | 48 h | 72 h | 48 h | 72 h | ||
T. diversifolia | Ethanol | 2.5 | 1.25 |
0.07*
| 2.5 | 2.5 | 2.5 |
Methanol | 0.62 | 0.62 |
0.07*
| 0.62 | 2.5 | 2.5 | |
DCM | 0.62 | 1.25 |
0.15
| 0.31 | 2.5 | 2.5 | |
Acetone |
0.15
| 0.31 |
0.07*
|
0.15
|
0.15
|
0.62
| |
Hot water | 0.62 | 0.62 |
0.15
| 1.25 | 1.25 | 1.25 | |
Hexaane |
0.15
|
0.15
|
0.03**
|
0.07*
| 1.25 | 1.25 | |
Chloroform | 0.31 | 1.25 |
0.15
| 0.31 | 1.25 | 1.25 | |
ETAC | 2.5 | 2.5 | 0.31 | 1.25 |
0.15
|
0.15
| |
T. rotundifolia | Ethanol | 1.25 | 1.25 |
0.15
| 0.62 | 1.25 | 1.25 |
Methanol | 0.62 | 1.25 | 0.31 | 0.62 | 2.5 | 2.5 | |
DCM |
0.15
| 1.25 |
0.07*
|
0.15
|
0.07*
|
0.15
| |
Acetone |
0.07*
| 1.25 |
0.07*
|
0.15
|
0.07*
|
0.15
| |
Hot water | 2.5 | > 2.5 | 0.62 | 2.5 | > 2.5 | > 2.5 | |
Hexane | 0.31 | 1.25 | 0.62 | 1.25 |
0.15
|
0.15
| |
Chloroform | > 2.5 | 0.62 | 1.25 | 1.25 | 0.31 | 0.62 | |
Butanol | > 2.5 | > 2.5 | 0.31 | 0.31 | 1.25 | 1.25 | |
ETAC | > 2.5 | 1.25 |
0.15
| 0.62 |
0.15
| 0.62 | |
Amp B (+ve con) |
0.03**
| 0.62 |
0.008**
| 0.125 |
0.03**
| 0.50 |
Phytochemical determination
Phyochemicals | T. diversifolia | T. rotundifolia |
---|---|---|
Phenolics | +++ | +++ |
Flavonoids | +++ | +++ |
Tannins | +++ | +++ |
Alkaloids | + | + |
Saponins | ++ | ++ |
UPLC/MS analysis of active fractions
Toxicity assay
In vitro cytotoxicity
Plant species | Extract | LC50 values (mg/ml) |
---|---|---|
T. diversifolia
| Ethanol | 0.004 ± 0.004 |
Methanol | 0.002 ± 0.001 | |
DCM | 0.004 ± 0.001 | |
Acetone | 0.012 ± 0.009 | |
Hot water | 0.275 ± 0.04 | |
T. rotundifolia
| Ethanol | 0.533 ± 0.113 |
Methanol | 0.518 ± 0.211 | |
DCM | 0.938 ± 0.000 | |
Acetone | 0.782 ± 0.089 | |
Hot water | 0.841 ± 0.325 | |
Doxorubicin +ve (μg/ml) | 0.2009 ± 0.000 |
Plant species | Extract | Selectivity index (SI) | ||||||
---|---|---|---|---|---|---|---|---|
Ec
|
Kp
|
Ef
| SD |
Pa
|
Sa
| ST | ||
T. diversifolia
| EtOH | 0.0033 | 0.0065 | 0.0571 | 0.0129 | 0.0032 | 0.0129 | 0.0065 |
MeOH | 0.0032 | 0.0032 | 0.0032 | 0.0032 | 0.0016 | 0.0032 | 0.0133 | |
DCM | 0.0129 | 0.0267 | 0.0129 | 0.0129 | 0.0129 | 0.0064 | 0.0065 | |
Acetone | 0.0387 | 0.08 | 0.1714 | 0.0387 | 0.0387 | 0.0194 | 0.0387 | |
Hot water | 0.220 | 0.4436 | 0.110 | 0.220 | 0.220 |
1.8333
| 0.4435 | |
T. rotundifolia
| EtOH |
1.719
|
3.553
|
1.719
|
0.861
|
0.861
|
7.614
|
0.861
|
MeOH |
0.836
|
1.671
| 0.207 |
3.453
|
0.836
|
0.836
|
1.671
| |
DCM |
1.513
|
6.253
|
6.253
|
6.253
|
3.026
|
13.4*
|
6.253
| |
Acetone |
2.523
|
11.171*
|
26.067*
|
2.523
|
2.523
|
11.171*
|
11.171*
| |
Hot water |
84.100*
|
1.357
|
12.014*
|
1.357
|
0.673
|
2.713
|
84.100*
|
Plant species | Extract | Selectivity index (SI) | |||||
---|---|---|---|---|---|---|---|
A. fumigatus | C. neoformans | C. albicans | |||||
48 h | 72 h | 48 h | 72 h | 48 h | 72 h | ||
T. diversifolia
| EtOH | 0.0016 | 0.0032 | 0.0571 | 0.0016 | 0.0016 | 0.0016 |
MeOH | 0.0032 | 0.0032 | 0.0286 | 0.0032 | 0.0008 | 0.0008 | |
DCM | 0.0064 | 0.0032 | 0.0267 | 0.0129 | 0.0016 | 0.0016 | |
Acetone | 0.080 | 0.0387 | 0.1714 | 0.08 | 0.08 | 0.0194 | |
Hot water | 0.4436 | 0.4435 |
1.8333
| 0.22 | 0.22 | 0.22 | |
T. rotundifolia
| EtOH | 0.426 | 0.426 |
3.553
|
1.666
| 0.426 | 0.426 |
MeOH |
0.836
| 0.414 |
1.671
|
0.836
| 0.207 | 0.207 | |
DCM |
6.253
|
0.750
|
13.400*
|
6.253
|
13.400*
|
6.253
| |
Acetone |
11.171*
|
0.626
|
11.171*
|
5.213
|
11.171*
|
5.213
| |
Hot water | 0.336 | NA |
1.357
| 0.336 | NA | NA |
In vitro genotoxicity
Plant species | Extracting solvent | Dose (μg/plate) | His + revertants plate | |
---|---|---|---|---|
TA98 | TA100 | |||
T. diversifolia
| Acetone | 5 | 27.33 ± 1.33 | 125.00 ± 1.2 |
50 | 54.67 ± 1.20 | 116.33 ± 1.45 | ||
500 | 45.00 ± 2.52 | 136.00 ± 2.0 | ||
DCM | 5 | 50.00 ± 1.15 | 104.33 ± 1.20 | |
50 | 51.33 ± 1.20 | 115.00 ± 2.00 | ||
500 | 75.33 ± 0.33 | 117.3 ± 0.3 | ||
Hot water | 5 | 56.00 ± 3.00 | 117.00 ± 0.00 | |
50 | 62.00 ± 1.73 | 121.33 ± 0.58 | ||
500 | 63.67 ± 2.52 | 132.33 ± 2.52 | ||
T. rotundifolia
| Acetone | 5 | 32.00 ± 1.53 | 122.00 ± 1.0 |
50 | 25.00 ± 0.58 | 113.00 ± 1.15 | ||
500 | 43.33 ± 1.67 | 108.67 ± 1.20 | ||
DCM | 5 | 35.33 ± 0.88 | 111.33 ± 1.76 | |
50 | 29.00 ± 2.08 | 119.00 ± 1.2 | ||
500 | 26.67 ± 1.67 | 122.00 ± 1.7 | ||
Hot water | 5 | 45.00 ± 3.06 | 112.33 ± 4.04 | |
50 | 52.00 ± 3.00 | 112.00 ± 1.73 | ||
500 | 59.00 ± 1.15 | 124.33 ± 3.06 | ||
4NQO + ve control | 212.7 ± 1.9 | 538.7 ± 5.900 | ||
H2O –ve control | 61.33 ± 1.45 | 130.00 ± 2.5 | ||
10% DMSO –ve control | 57.00 ± 1.15 | 125.7 ± 1.9 |