Background
Methods
General
Plant material
Chemicals, drugs and biochemical kits
Plant leaves extraction
Selection of best extraction solvent for phenolic contents
Spectrophotometric determination of total phenolic and flavonoid contents
RP- HPLC analysis
Large scale extraction and fractionation
Isolation of the constituents of the chloroform extract
Isolation of the constituents of the n-butanol extract
Antioxidant activity
DPPH radical scavenging assay
Biological study
Microorganisms
Antimicrobial activity
Experimental animals
Acute oral toxicity study
Evaluation of antihyperglycemic activity
Induction of diabetes in mice
Oral glucose tolerance test
Experimental procedure
Evaluation of antihyperlipidemic activity
Induction of hyperlipidemia in rats
Experimental procedure
In-vitro evaluation of the effect of the ethanolic extract and its isolated compound (2) on pancreatic lipase and HMGCoA reductase activities
Statistical analysis
Results
Spectrophotometric determination of total phenolic and flavonoid contents
Solvent | Extraction yield (%)a
| Total flavonoid content (mg quercetin/g) | Total phenolic content (mg GAE/g) |
---|---|---|---|
Ethanol | 20.0 ± 1.2 | 1.5 ± 0.2 | 1.5 ± 0.6 |
Methanol | 12.3 ± 1.7 | 1.1 ± 0.3 | 1.3 ± 0.7 |
Ethyl acetate | 9.7 ± 0.8 | 0.9 ± 0.0 | 0.6 ± 0.0 |
Acetone | 2.4 ± 0.3 | 0.7 ± 0.1 | 0.1 ± 0.0 |
RP- HPLC analysis
Retention time | Identified component | Relative area % |
---|---|---|
6.81 | Pyrogallol | 0.17 |
6.92 | Gallic acid | 0.24 |
8.235 | Protocatechuic acid | 3.95 |
8.444 | Catechin | 4.82 |
8.593 | Chlorogenic acid | 2.97 |
8.950 | Catechol | 1.90 |
10.040 | Caffeic acid | 0.75 |
11.620 | Ferulic acid | 0.75 |
12.466 | Salicylic acid | 11.91 |
12.943 | Ellagic acid | 9.78 |
14.980 | Cinnamic acid | 0.47 |
Total identified constituents | 37.73 |
Retention time | Identified component | Relative area % |
---|---|---|
3.83 | Quercetin | 0.01 |
11.78 | Rosmarinic acid | 0.89 |
12.44 | Rutin | 6.50 |
14.576 | Narenginin | 0.43 |
14.952 | Hispertin | 0.80 |
16.167 | Apigenin | 0.16 |
18.657 | Chrysin | 0.82 |
Total identified constituents | 9.61 |
Isolation of the constituents of the chloroform and n-butanol extracts
Isolated compounds
NO. C | Compound 1 (CDCl3) | Compound 2 (DMSO) |
---|---|---|
1 | 38.6 | 39.0 |
2 | 28.1 | 28.1 |
3 | 78.5 | 79.8 |
4 | 38.8 | 38.5 |
5 | 55.2 | 55.3 |
6 | 18.3 | 18.4 |
7 | 32.7 | 32.7 |
8 | 39.3 | 39.5 |
9 | 47.6 | 47.4 |
10 | 37.1 | 37.0 |
11 | 23.8 | 23.7 |
12 | 125.6 | 125.8 |
13 | 140.6 | 138.9 |
14 | 41.7 | 42.1 |
15 | 27.7 | 28.0 |
16 | 24.6 | 24.4 |
17 | 47.5 | 47.5 |
18 | 53.6 | 53.0 |
19 | 39.4 | 39.4 |
20 | 39.1 | 39.2 |
21 | 31.8 | 30.7 |
22 | 37.0 | 37.4 |
23 | 28.5 | 29.0 |
24 | 16.0 | 15.6 |
25 | 15.6 | 16.5 |
26 | 17.1 | 17.5 |
27 | 23.9 | 23.3 |
28 | 180.0 | 182.0 |
29 | 17.1 | 17.5 |
30 | 21.2 | 21.5 |
OAc | 22.3 | |
CH3COO | 171.0 |
NO. C | Compound 4 CD3OD | Compound 5 CD3OD | Compound 6 CD3OD |
---|---|---|---|
2 | 155.3 | 157.1 | 162.1 |
3 | 137.3 | 134.2 | 103.0 |
4 | 178.2 | 178.0 | 180.0 |
5 | 161.0 | 161.6 | 164.3 |
6 | 98.2 | 98.5 | 103.1 |
7 | 164.8 | 164.6 | 160.0 |
8 | 93.4 | 94.5 | 98.6 |
9 | 157.4 | 156.6 | 157.0 |
10 | 103.3 | 103.3 | 108.4 |
1’ | 121.3 | 122.2 | 122.4 |
2’ | 115.1 | 116.2 | 129.2 |
3’ | 144.7 | 145.5 | 116.4 |
4’ | 148.4 | 148.4 | 161.6 |
5’ | 115.2 | 116.1 | 116.4 |
6’ | 121.5 | 122.2 | 129.2 |
OCH3
| 58.2 | ||
1” | 101.2 | 103.3 | |
2” | 73.8 | 74.9 | |
3” | 76.8 | 77.3 | |
4” | 72.5 | 72.7 | |
5” | 75.8 | 76.7 | |
6” | 67.1 | 65.9 | |
1″‘ | 102.3 | 102.8 | |
2″‘ | 70.8 | 70.8 | |
3″‘ | 70.0 | 71.2 | |
4″‘ | 71.4 | 71.4 | |
5″‘ | 69.1 | 69.1 | |
6″‘ | 18.1 | 18.2 |
Antioxidant activity
DPPH free radical scavenging activity
Biological studies
Antimicrobial activity
Tested sample |
S. aureus
|
P. aeruginosa
|
E. coli
|
C. albicans
|
---|---|---|---|---|
Ethanolic extract | 9.0 ± 1.1 | 22.0 ± 2.4 | 14.0 ± 1.2 | 5.0 ± 1.2 |
n-hexane | 10.0 ± 1.8 | 24.0 ± 1.6 | 25.0 ± 2.6 | 7.0 ± 0.7 |
Chloroform | 7.0 ± 0.7 | 25.0 ± 3.3 | 22.0 ± 3.0 | - |
n-butanol | - | 20.0 ± 2.1 | 21.0 ± 2.1 | - |
Ursolic acid | 23.0 ± 2.6 | 24.0 ± 2.4 | 22.0 ± 1.9 | 13.0 ± 1.2 |
3-β-O acetyl ursolic acid | 30 ± 3.4 | 28.0 ± 3.1 | 29.0 ± 2.3 | 15.0 ± 0.8 |
Ellagic acid | - | 23.0 ± 3.5 | - | - |
Quercetin 3-O- methyl Ether | 25.0 ± 3.1 | 26.0 ± 2.9 | 25.0 ± 2.8 | 21.0 ± 2.3 |
Rutin | 6.0 ± 2.5 | 20.0 ± 1.8 | - | - |
Apigenin7-O-rutinoside | 6.0 ± 1.9 | 18.0 ± 1.6 | - | - |
Gentamicin | 20.0 ± 2.7 | 18.0 ± 1.9 | 22.0 ± 3.4 | - |
Fluconazole | - | - | - | 25 ± 2.5 |
Acute oral toxicity study
Evaluation of antihyperglycemic activity
Antihyperlipidemic activity
Group | Treatment (p.o.) | Day 0 | Day 15 | Day 30 |
---|---|---|---|---|
I | 1% w/v sodium CMC | 211.3 ± 3.6 | 236.1 ± 4.3 (+11.9 ± 0.5) | 253.2 ± 3.4 (+20.2 ± 0.4) |
II | Cholesterol | 209.3 ± 2.8 | 250.4 ± 3.6 (+19.8 ± 0.5)#
| 273.1 ± 4.6 (+30.5 ± 0.3)#
|
III | Cholesterol + lovastatin | 212.4 ± 3.6 | 252.1 ± 2.4 (+18.7 ± 0.3) | 272.1 ± 3.4 (+28.1 ± 0.6) |
IV | Cholesterol + plant extract 250 mg/kg | 211.8 ± 4.2 | 244.4 ± 5.1 (+15.4 ± 0.3)*
| 250.1 ± 3.5 (+18.1 ± 0.6)**
|
V | Cholesterol + plant extract 500 mg/kg | 210.6 ± 5.3 | 241.1 ± 4.3 (+14.5 ± 0.2)*
| 247.8 ± 5.3 (+17.7 ± 0.8)\**
|
Group | Total cholesterol (mg/dl) | Triglyceride (mg/dl) | HDL-C (mg/dl) | LDL-C (mg/dl) | VLDL-C (mg/dl) | Atherogenic index | LDL - C/HDL - C |
---|---|---|---|---|---|---|---|
I | 61.7 ± 2.4 | 60.5 ± 5.6 | 36.3 ± 1.1 | 22.0 ± 0.7 | 12.4 ± 0.5 | 1.7 ± 0.3 | 0.6 ± 0.02 |
II | 84.5 ± 2.5# | 127.5 ± 2.5# | 26.0 ± 1.1# | 39.1 ± 1.3# | 16.9 ± 0.6# | 3.25 ± 0.7 | 1.5 ± 0.2 |
III | 61.5 ± 0.5** (−27.2%) | 58.0 ± 2.1** (−54.5%) | 33.5 ± 0.5** (+28.8%) | 24.3 ± 1.1** (−37.9%) | 12.3 ± 0.1* (−27.2%) | 1.8 ± 0.2 | 0.7 ± 0.05 |
IV | 78.3 ± 3.4 (−7.3%) | 61.3 ± 6.9** (−51.9%) | 30.2 ± 1.4* (+16.2%) | 26.5 ± 0.6** (−32.2%) | 15.7 ± 0.7 (−7.1%) | 2.6 ± 0.6 | 0.9 ± 0.04 |
V | 61.7 ± 1.8** (−27.0%) | 49.0 ± 2.5** (−61.6%) | 32.5 ± 0.6** (+25.0%) | 25.2 ± 0.7** (−35.6%) | 12.9 ± 0.4* (−23.7%) | 1.9 ± 0.3 | 0.8 ± 0.05 |