Background
Methods
Plant materials
Extraction
Fractionation
Qualitative phytochemical evaluation
Determination of total phenolic content
Determination of flavonoid content
GC-MS analysis
Antioxidant activity: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay
The multi-well plate AChE inhibition assay
MTT (3-(4, 5-dimethylthazol-2-yl)-2, 5-diphenyl tetrazonium bromide) cytotoxicity assay
Statistical analysis
Results and discussion
Extraction yield and phytochemical screening
Family of compound | Type of test | interference | ||||
---|---|---|---|---|---|---|
n-hexane | CHCl3
| EtOAc | n-BuOH | H2O | ||
Phenols | FeCl3
| +v | +v | +v | +v | +v |
Tannins | FeCl3
| -v | +v | +v | +v | +v |
Flavonoids | KOH | +v | +v | +v | +v | +v |
Alkaline | +v | +v | +v | +v | +v | |
Lead acetate | +v | +v | +v | +v | +v | |
Quinones | H2SO4
| -v | +v | +v | +v | +v |
Alkaloids | Dragendorff’s | +v | +v | +v | +v | +v |
Wagner’s | +v | +v | +v | +v | +v | |
Triterpenes | Salkowski | +v | +v | +v | +v | +v |
Diterpenes | Copper acetate | -v | -v | -v | -v | -v |
Steroids | Salkowski | +v | +v | +v | +v | +v |
Saponins | Forth | -v | +v | +v | -v | +v |
Total phenolic and flavonoid contents
Sample | Extraction yield (w/w% of dry weight) | Phenolic content (mg /g GAE) | Total flavonoid (mg/g QE) |
---|---|---|---|
Crude | 47.33 | 78.21 ± 2.4 | 91.36 ± 0.84 |
Hexane | 6.18 | 98.78 ± 2.1 | 81.01 ± 0.012 |
CHCl3
| 1.64 | 71.49 ± 0.5 | 118.29 ± 0.21 |
EtOAc | 3.09 | 86.12 ± 0.7 | 80.23 ± 0.03 |
BuOH | 19.09 | 56.20 ± 1.23 | 94.32 ± 0.71 |
H2O | 70.91 | 83.20 ± 3.7 | 40.22 ± 0.28 |
Antioxidant and AChE inhibitory activities
Sample | % RSA | IC50 (mg/ml) |
---|---|---|
Hexane | 76 ± 0.02 | 0.098 ± 0.08 |
CHCl3
| 86 ± 0.03 | 0.099 ± 0.029 |
EtOAc | 84 ± 0.03 | 0.148 ± 0.33 |
BuOH | 83 ± 0.02 | 0.104 ± 0.19 |
H2O | 79 ± 0.10 | 2.015 ± 0.3 |
GCMS analysis
Peak no. | R. Time | Area % | Compunde name | Molecular Formula | Mass |
---|---|---|---|---|---|
1 | 13.736 | 1.68 | 5,5-Dimethyl-1,5-oxasilonan-9-one | C9H18O2Si | 186 |
2 | 15.467 | 5.67 | Quinic acid | C7 H12 O6
| 192 |
3 | 19.188 | 0.29 | Pentadecanoic acid, methyl ester | C16 H32 O2
| 256 |
4 | 19.425 | 0.67 | Ethyl (2E)-3-(4-hydroxy-3-methoxyphenyl)-2-propenoate | C12H14O4
| 222 |
5 | 19.805 | 6.89 | Pentadecanoic acid | C15H30O2
| 242 |
6 | 20.147 | 2.20 | Palmitic acid ethyl ester | C18 H36 O2
| 284 |
7 | 20.761 | 0.53 | 2-Cyclopropylideneadamantane | C13H18
| 174 |
8 | 22.455 | 16.49 | Oleic Acid | C18H34O2
| 282 |
9 | 22.694 | 3.23 | Methyl linoleate | C19 H34 O2
| 294 |
10 | 22.765 | 1.64 | Ethyl octadec-9-enoate | C20H38O2
| 310 |
11 | 22.814 | 0.48 | Linolenic acid methyl ester | C19 H32 O2
| 292 |
12 | 23.122 | 3.91 | Heptadecanoic acid, ethyl ester | C19 H38 O2
| 298 |
13 | 25.920 | 14.21 | Oelic acid amide | C18 H35 N O | 281 |
14 | 25.994 | 0.23 | Oelic acid amide | C18 H35 N O | 281 |
15 | 26.187 | 0.44 | Palmitic acid ethyl ester | C18 H36 O2
| 284 |
16 | 26.265 | 0.60 | Stearic acid amide | C18H37NO | 283 |
17 | 26.509 | 0.43 | 2-Pentyl-2-nonenal | C14H26O | 210 |
18 | 26.761 | 0.43 | Oxirane, hexadecyl- | C18H36O | 268 |
19 | 28.299 | 0.48 | Octadecanal | C18H36O | 268 |
20 | 28.665 | 9.23 | Phthalic acid, mono-(2-ethylhexyl) ester | C16H22O4
| 278 |
21 | 29.192 | 0.97 | Ethyl palmitate | C18 H36 O2
| 284 |
22 | 29.881 | 19.50 | Cinchol | C29 H50 O | 414 |
23 | 32.086 | 0.57 | Ethyl docosanoate | C24H48O2
| 368 |
24 | 32.642 | 1.64 | Spinacene | C30H50
| 410 |
25 | 33.176 | 0.59 | 3-[(Trimethylsilyl)oxy]lanosta-8,24-diene | C33H58OSi | 498 |
26 | 33.343 | 1.44 | Lupenyl acetate | C32H52O2
| 468 |
27 | 35.907 | 3.46 | Stigmast-4-en-3-one | C29H48O | 412 |
28 | 38.248 | 1.09 | Cholesteryl bromide | C27 H45 Br | 448 |
29 | 38.974 | 1.01 | Vitamin E | C29H50O2
| 430 |
Number | R. Time | Area % | Compound name | Molecular Formula | Mass |
---|---|---|---|---|---|
1 | 13.735 | 2.97 | 5,5-Dimethyl-1-oxa-5-silacyclononanone-9 | C9H18O2Si | 186 |
2 | 14.635 | 0.29 | 9-Eicosene, (E)- | C20H40
| 280 |
3 | 15.989 | 1.99 | 2H–Pyran-2-one, 5-ethylidenetetrahydro-4-(2-hydroxyethyl)- | C9H14O3
| 170 |
4 | 17.029 | 0.85 | 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol | C10H12O3
| 180 |
5 | 17.334 | 0.35 | 1-Heptadecene | C17H34
| 238 |
6 | 18.302 | 0.84 | p-Hydroxycinnamic acid, ethyl ester | C11H12O3
| 192 |
7 | 19.187 | 0.35 | 9-Octadecenoic acid, 12-(acetyloxy)-, methyl ester, [R-(Z)]- | C21H38O4
| 354 |
8 | 19.361 | 0.18 | 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione | C17H24O3
| 276 |
9 | 19.422 | 0.15 | alpha.-D-Xylofuranose, 1,2-O-isopropylidene-5-(t-butyldimethylsilyl)- | C14H28O5Si | 304 |
10 | 19.800 | 5.89 | Pentadecanoic acid | C15 H30 O2
| 242 |
11 | 20.115 | 0.30 | 9-Tricosene, (Z)- | C23H46
| 322 |
12 | 20.360 | 7.84 | 2H-1-Benzopyran-2-one, 7-hydroxy-6-methoxy- | C10 H8 O4
| 192 |
13 | 20.761 | 0.62 | Bicylo[4.1.0]heptane, 7-bicyclo[4.1.0]hept-7-ylidene- | C14H20
| 188 |
14 | 21.139 | 0.22 | Trimethylsilyl 3-methoxy-4-(trimethylsilyloxy)cinnamate | C16H26O4Si2
| 338 |
15 | 21.769 | 0.31 | 9-Octadecenoic acid (Z)-, methyl ester | C19H36O2
| 296 |
16 | 21.996 | 25.46 | 2-Propenoic acid, 3-(3,4-dihydroxyphenyl)- | C9 H8 O4
| 180 |
17 | 22.450 | 9.78 | Oleic Acid | C18H34O2
| 282 |
18 | 22.762 | 3.28 | Octadecanoic acid | C18H36O2
| 284 |
19 | 23.104 | 1.19 | 1-Hexadecene | C16 H32
| 224 |
20 | 23.740 | 0.61 | 9,12-Octadecadienoic acid (Z,Z)- | C18H32O2
| 280 |
21 | 25.816 | 0.39 | O O′-BIPHENOL, 4,4’,6,6’-TETRA-T-BUTYL- | C28 H42 O2
| 410 |
22 | 25.909 | 1.08 | 9-Octadecenamide, (Z)- | C18 H35 N O | 281 |
23 | 26.155 | 0.93 | 1-Nonadecene | C19H38
| 266 |
24 | 28.301 | 0.19 | Oxirane, heptadecyl- | C19H38O | 282 |
25 | 28.662 | 1.17 | 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester | C16H22O4
| 278 |
26 | 29.163 | 1.28 | 1-Nonadecene | C19H38
| 266 |
27 | 29.732 | 1.15 | gamma.-Sitosterol | C29H50O | 414 |
28 | 32.053 | 1.83 | 1-Triacontanol | C24H50O | 354 |
29 | 35.350 | 1.02 | 17-Pentatriacontene | C35H70
| 490 |
30 | 35.848 | 5.09 | Methyl commate C | C31 H50 O4
| 486 |
31 | 38.123 | 22.40 | Lup-20 (29)-en-3-ol, acetate, (3.beta.)- | C32H52O2
| 468 |
Cytotoxicity study
Code of extract | Concentration (μg/ml) | IC50 (μg/ml) | IC50
| ||
---|---|---|---|---|---|
Inhibition % ± SD | |||||
500 | 250 | 125 | |||
Hexane | 55.3 ± 0.04 | 50.4 ± 0.02 | 40.9 ± 0.03 | 277.8 | >100 |
Chloroform | 67.6 ± 0.08 | 55.0 ± 0.01 | 35.9 ± 0.05 | 224.9 | >100 |
Control | 95.3 ± 0.00 | <30 |