Background
Methods
Plant material
Physico-chemical and proximate studies
Extraction of fatty acids
Fatty acid analysis
Instrument and separation conditions of the fatty acids
Amino acid analysis
Elemental analyses
Reagents, water and standard
Ashing procedure and sample preparation for ICP-MS and AAS
ICP-MS instrument and operating condition
Parameters | Settings |
---|---|
Plasma conditions
| |
RF power | 1.40 kW |
Plasma Ar flow rate. | 18.0 L min−1
|
Auxiliary Ar flow rate | 2.25 L min−1
|
Sheath gas flow | 0.20 L min−1
|
Nebulizer gas flow | 1.0 L min−1
|
Sampling depth | 6.50 mm |
Pump Rate | 5 rpm |
Instrument
| |
Sampler cone: Nikel | 1.0 mm orifice diameter |
Skimmer cone: Nikel | 0.5 mm orifice diameter |
Atomic absorption spectrometry (AAS) analyses
Flame photometry analyses
Thermogravimetric analysis
Extraction of essential oils
GC-MS analysis
Identification of the compounds
Antioxidant activity
Cytotoxic activity of brine shrimp lethality bioassay
Antimicrobial activity test
Diffusion method
Dilution method (MIC and MBC determination)
Antifungal activity test
Statistical analysis
Results
Proximate composition
Characteristics | Percent (%) Composition |
---|---|
Moisture (On fresh weight basis) | 78.46 ± 0.23 |
Dry matter (On fresh weight basis) | 21.54 ± 0.23 |
Organic content | 94.71 ± 0.12 |
Ash (On dry weight basis) | 5.29 ± 0.10 |
Ash (On fresh weight basis) | 1.14 ± 0.01 |
Acid insoluble ash | 0.32 ± 0.02 |
Water soluble ash | 1.87 ± 0.00 |
Nitrogen (Kjeldahl method) | 0.32 ± 0.01 |
Protein | 2.01 ± 0.07 |
Crude fibre | 54.09 ± 0.50 |
Fatty oil (Hot extract) | 1.14 ± 0.01 |
Fatty oil (Cold extract) | 0.60 ± 0.11 |
Essential oil (On dry weight basis) | 0.15 ± 0.09 |
Carbohydrate (Calculated by difference) | 28.53 ± 0.30 |
Fatty acid composition
Fatty acid compounds | Cold extract | Hot extract | |||||
---|---|---|---|---|---|---|---|
R
t
| IA | Relative percentage | R
t
| IA | Relative percentage | ||
1. |
aMyristic acid (C14:0) | 9.74 | 31956 | 5.24 | 9.751 | 407 | 0.17 |
2. |
aPalmitic acid (C16:0) | 13.33 | 205940 |
33.81
| 13.334 | 74176 |
31.58
|
3. |
aStearic acid (C18:0) | 16.17 | 30015 | 4.93 | 16.176 | 9810 | 4.17 |
4. |
cOleic acid (C18:1) | 15.83 | 5392 | 0.89 | 15.830 | 41976 |
17.87
|
5. |
bLinoleic acid (C 18:2) | 15.75 | 182845 |
30.03
| 15.754 | 55875 |
23.79
|
6. |
aArachidic Acid (C20:0) | 18.80 | 15831 | 2.60 | 18.809 | 4541 | 1.93 |
7. |
aBehenic acid (C22:0) | 21.24 | 77302 |
12.70
| 21.242 | 23580 |
10.04
|
8. |
aLignoceric acid (C24:0) (Tetracosanoic acid) | 23.61 | 59715 |
9.80
| 23.612 | 24456 |
10.45
|
Total | 608996 | 100 | 234821 | 100 | |||
aSaturated (%) | 69.08 | 58.34 | |||||
bPoly-unsaturated (%) | 30.03 | 23.79 | |||||
cMono-unsaturated (%) | 0.89 | 17.87 | |||||
PUFA/SFA | 0.43 | 0.40 | |||||
AI | 1.771 | 0.77 | |||||
TI | 2.84 | 1.72 |
Amino acid composition
Name of Amino Acids | Sample Retention Time | Standard Retention Time | Sample Individual Area | Standard Individual Area | Concentration | |
---|---|---|---|---|---|---|
g/100 g DB | g/100 g FB | |||||
Serine (Ser)b,g
| 10.392 | 10.471 | 1361824 | 2701518 | 3.58 | 4.01 |
Glutamic acid (Glu)b,d
|
11.212
|
11.535
|
4493784
|
1649736
|
19.37
| 21.67 |
Glycine (Gly)b,g
|
15.262
|
16.138
|
14738830
|
7210517
|
14.53
| 16.26 |
Alanine (Ala)b,g
| 17.360 | 18.191 | 525832 | 1186996 | 3.15 | 3.52 |
Valine (Val)a,g
| 21.543 | 19.752 | 356860 | 1517660 | 1.67 | 1.87 |
Methionine (Met)a,f,g
| 22.448 | 22.300 | 937009 | 1213589 | 5.49 | 6.14 |
Isoleucine (Ile)a,g
| 24.957 | 23.235 | 310181 | 1877608 | 1.17 | 1.31 |
Leucine (Leu)a,g
| 26.182 | 25.570 | 467152 | 1648575 | 2.01 | 2.25 |
Tyrosine (Tyr)b,c,g
| 27.415 | 26.796 | 390655 | 1877669 | 1.47 | 1.65 |
Histidine (His)a,e
| 28.371 | 28.295 | 7778337 | 8098543 | 6.82 | 7.64 |
Lysine (Lys)a,e
|
32.084
|
32.045
|
3868261
|
1609813
|
17.08
| 19.12 |
Arginine (Arg)a,e
| 33.663 | 33.693 | 473612 | 2266037 | 1.48 | 1.66 |
Total | 35702337 | 32858261 | 77.87 | 87.14 | ||
aEssential amino acids | 35.75 | 40.00 | ||||
bNon Essen. Amino Acids | 42.12 | 47.13 | ||||
cAromatic Amino Acids | 1.47 | 1.65 | ||||
dAcidic Amino Acids | 19.37 | 21.67 | ||||
eBasic Amino Acids | 25.40 | 28.42 | ||||
fSulphur Amino Acids | 5.49 | 6.14 | ||||
gNeutral Amino Acids | 33.10 | 37.04 | ||||
% of essential amino acid in total amino acid | 45.90 | 45.90 |
Elemental composition
Elements |
Anethum sowa L. Root | RSD % | Instrumental Method | ||
---|---|---|---|---|---|
Solution Conc. | DWB (ppb) | FWB (ppb) | |||
Be | 0.7977 | 2.09 | 1.90 | 2.84 | ICP-MS |
Ag | 8.039 | 21.10 | 19.23 | 0.97 | ICP-MS |
Cs | 8.2059 | 21.53 | 19.63 | 1.18 | ICP-MS |
Bi | 8.2364 | 21.61 | 19.70 | 1.03 | ICP-MS |
Ga | 9.6419 | 25.30 | 23.07 | 1.18 | ICP-MS |
Co | 26.1657 | 68.67 | 62.61 | 1.36 | ICP-MS |
Li | 121.093 | 317.84 | 289.76 | 0.08 | ICP-MS |
Se | 348.141 | 913.79 | 833.06 | 2.11 | ICP-MS |
Ni | 452.343 | 1187.30 | 1082.40 | 0.76 | ICP-MS |
Elements |
Anethum sowa L. Root | RSD % | Instrumental Method | ||
---|---|---|---|---|---|
Solution Conc. | DWB (ppm) | FWB (ppm) | |||
Mn | 6.56 | 17.21 | 15.69 | 0.1 | AAS |
Al | 53.08 | 139.32 | 127.01 | 0.1 | AAS |
Fe | 78.82 | 206.88 | 188.60 | 0.1 | AAS |
Na | 110 | 288.72 | 263.21 | 0.1 | FP |
K | 490 | 1286.15 | 1172.51 | 0.1 | FP |
Mg | 2903.21 | 7620.33 | 6947.08 | 0.1 | AAS |
Ca | 8991.18 | 23600.01 | 21514.95 | 0.1 | AAS |
Elements |
Anethum sowa L. Root | RSD % | Instrumental Method | ||
---|---|---|---|---|---|
Solution Conc. | DWB (ppb) | FWB (ppb) | |||
Cr | ND | ND | ND | ND | ICP-MS |
Pb | ND | ND | ND | ND | ICP-MS |
Cd | 14.7679 | 38.76 | 35.33 | 1.54 | ICP-MS |
As | 16.5858 | 43.53 | 39.68 | 2.88 | ICP-MS |
Hg | 48.45 | 965.52 | 880.22 | 0.3 | AAS |
Thermal analysis
Events | TG | DTG | ||
---|---|---|---|---|
Mass loss (%) | Tonset-Toffset (OC) | Mass loss (mg/min) | Tonset-Toffset (OC) | |
First step | 92.7 | Troom-275 | 0.151 | Troom-75.9 |
Second step | 53.3 | 276–327 | 0.373 | 76–257 |
Third step | 29.6 | 328–358 | 1.167 | 258–328 |
% Residue | 25.9 | 359–550 | - | - |
Chemical composition of the essential oil
SL No. | RI Index | Name of Compounds | Essential oil | Water extract part | ||
---|---|---|---|---|---|---|
R
t
| Conc. (%) | R
t
| Conc. (%) | |||
1 | 349 | Ethylene oxide | - | ND | 16.249 | 0.294 |
2 | 653 | Glycidol | - | ND | 7.893 | 0.264 |
3 | 707 | n-Pentanal | 9.441 | 0.053 | - | ND |
4 | 721 | Isobutyl acetate | - | ND | 3.769 | 3.733 |
5 | 766 | β-Butyrolactone | - | ND | 3.228 | 5.131 |
6 | 767 | 2,3,3-Trimethyl hexane | 3.677 | 0.068 | - | ND |
7 | 794 | Toluene | 3.606 | 2.40 | 3.635 | 3.327 |
8 | 806 | n-Hexanal | 4.165 | 0.166 | - | ND |
9 | 831 | Furfural | - | ND | 4.800 | 0.48 |
10 | 905 | n-Heptanal | 6.021 | 0.148 | - | ND |
11 | 907 | p-Xylene | 5.422 | 0.895 | - | ND |
12 | 1005 | n-Octanal | 7.885 | 0.238 | - | ND |
13 | 1081 | 2-Phenyl-acetaldehyde | 8.653 | 0.106 | - | ND |
14 | 1197 | p-Cymen-8-ol | 11.026 | 0.878 | 11.027 | 1.109 |
15 | 1224 | 4-Thujen-2-alpha-yl acetate | 11.323 | 0.082 | - | ND |
16. | 1260 | Cyclohexane, 3-hexyl | - | ND | 17.730 | 0.50 |
17. | 1294 | 2-Aminoundecane | 15.164 | 0.244 | - | ND |
18. | 1304 | m-Diaminobenzene | 18.758 | 10.446 | 18.755 | 8.778 |
19. | 1334 | (+/-)-Norephedrine | 18.440 | 0.103 | - | ND |
20. | 1421 | Thymol acetate | 12.773 | 0.878 | - | ND |
21 | 1516 | Myristicin | 15.900 | 0.868 | 15.900 | 0.716 |
22 | 1550 | Elemicin | 16.251 | 0.426 | - | ND |
23 | 1705 | Apiol | 17.897 |
81.99
| 17.887 |
74.779
|
24 | 2187 | 3,4-Dimethyl-benzoic acid 4-tert-bytyl-phenyl ester | - | ND | 17.614 | 0.881 |
Total | 100 | 100 |
Sl No. | Compound classes | Essential oil part (%) | Water extract part (%) |
---|---|---|---|
1. | Hydrocarbon | 3.363 |
3.827
|
2. | Aldehyde | 0.658 | ND |
3. | Alcohol | ND | 0.264 |
4. | Lactone | ND |
5.131
|
5. | Ester | 0.96 |
4.614
|
6. | Oxide | ND | 0.294 |
7. | Hemiterpenoid Aldehyde | 0.053 | 0.48 |
8. | Monoterpenoid Alcohol | 0.878 | 1.109 |
9. | Phenylpropanoid |
83.284
|
75.495
|
10. | Others |
10.793
|
8.778
|
Total | 100 | 100 |
Antioxidant activity on DPPH radical
Essential oil | Standard Antioxidants | |||||
---|---|---|---|---|---|---|
Dose Concentration (mg/mL) | Log Dose | Average percent of Inhibition ± SD | Dose Concentration (μg/mL) | Log Dose | Average percent of Inhibition ± SD of BHT | Average percent of Inhibition ± SD of ASA |
46.5 | 1.66 | 93.43 ± 0.24 | 200 | 2.301 | 93.02 ± 0.50 | 99.05 ± 0.21 |
23.25 | 1.36 | 89.56 ± 0.22 | 100 | 2 | 91.98 ± 0.19 | 98.35 ± 0.17 |
11.625 | 1.06 | 84.17 ± 0.25 | 50 | 1.698 | 85.51 ± 0.76 | 90.71 ± 0.60 |
5.8125 | 0.764 | 64.68 ± 0.21 | 25 | 1.397 | 69.54 ± 0.60 | 85.31 ± 0.60 |
2.90625 | 0.463 | 41.25 ± 0.12 | 12.5 | 1.096 | 55.87 ± 0.82 | 79.79 ± 0.18 |
1.453125 | 0.162 | 32.41 ± 0.31 | 6.25 | 0.795 | 29.69 ± 0.97 | 69.67 ± 0.68 |
0.72656 | −0.138 | 19.71 ± 0.40 | 3.125 | 0.494 | 25.63 ± 0.51 | 37.26 ± 1.12 |
0.36328 | −0.439 | 17.18 ± 0.05 | 1.5625 | 0.193 | 9.05 ± 0.51 | 27.02 ± 1.19 |
Average IC50 value = 3.07 ± 0.01 mg/mL | IC50 value = 11.84 ± 0.29 μg/mL | IC50 value = 3.74 ± 0.05 μg/mL |
Brine shrimp cytotoxicity assay
Dose (μg/mL) | Log Dose (μg/mL) | % of mortality | Probit | LC50 (μg/mL) | |||
---|---|---|---|---|---|---|---|
Vincristine sulphate | Root essential oil | Vincristine sulphate | Root essential oil | Vincristine sulphate | Root essential oil | ||
10 | 1 | 100 | 100 | 7.0959 | 6.723 | ||
5 | 0.699 | 90 | 80 | 6.2802 | 5.8408 | ||
2.5 | 0.3979 | 80 | 60 | 5.8413 | 5.241 | ||
1.25 | 0.0969 | 60 | 50 | 5.2402 |
4.9977
| ||
0.625 | −0.2041 | 60 | 40 |
4.9992
| 4.7482 |
0.48
|
0.81
|
0.3125 | −0.5051 | 40 | 30 | 4.7467 | 4.4782 | ||
0.156 | −0.8069 | 30 | 20 | 4.4774 | 4.1638 | ||
0.078 | −1.1079 | 20 | 20 | 4.1665 | 4.1753 | ||
0.039 | −1.4089 | 10 | 10 | 3.7206 | 3.7198 | ||
0.0195 | −1.71 | 0 | 10 | 0 | 3.8288 |
Antimicrobial activity of Anethum sowa L. root essential oil
Test organisms | Zone of Inhibition in mm | |||||
---|---|---|---|---|---|---|
400 μg/well | 600 μg/well | 800 μg/well | CP | TE | ||
Gram-positive bacteria |
Bacillus subtilis
| nz | 4 | 7 | 27 | 24 |
Bacillus cereus
| 5 | 7 | 8 | 29 | 21 | |
Staphylococcus aureus
| nz | 3 | 7 | 25 | 26 | |
Enterococcus faecalis
| nz | nz | nz | 20 | 16 | |
Gram -negative bacteria |
Escherichia coli 12079
| nz | 4 | 7 | 25 | 9 |
Escherichia coli 2799
| 11 | 13 | 16 | 23 | 11 | |
Pseudomonas aeruginosa
| 8 | 9 | 11 | 29 | 16 | |
Salmonella enteritidis 1375 | nz | nz | 5 | 39 | 23 | |
Salmonella typhi
| nz | 5 | 9 | 35 | 25 | |
Acetobacter aceti
| nz | nz | 3 | 28 | 23 |
Test organisms | Essential Oil | Std. CP | |||
---|---|---|---|---|---|
MIC (μg/mL) | MBC (μg/mL) | MIC (μg/mL) | MBC (μg/mL) | ||
Gram-positive bacteria |
Bacillus subtilis
| 125 | 250 | 1.55 | 12.5 |
Bacillus cereus
| 125 | 500 | 3.125 | 25 | |
Staphylococcus aureus
| 125 | 250 | 6.25 | 50 | |
Enterococcus faecalis
| 62.5 | 125 | 0.37 | 100 | |
Gram -negative bacteria |
Escherichia coli 12079
| 250 | 500 | 0.75 | 50 |
Escherichia coli 2799
| 62.5 | 125 | 0.37 | 6.25 | |
Pseudomonas aeruginosa, | 62.5 | 125 | 3.125 | 25 | |
Salmonella enteritidis 1375 | 62.5 | 250 | 0.37 | 6.25 | |
Salmonella typhi
| 250 | 500 | 0.19 | 3.125 | |
Acetobacter aceti
| 62.5 | 500 | 0.37 | 50 |
Antifungal activity of Anethum sowa L. root essential oil
Fungi | Zone of Inhibition in mm | |||||
---|---|---|---|---|---|---|
Essential oil | Fluconazole | MIC/MBC (μg/mL) | ||||
400 μg/well | 600 μg/well | 800 μg/ well | 100 μg/well | Essential oil | Fluconazole | |
Candida albicans
| - | 3 | 7 | 23 | 125/500 | 62.5/250 |
Aspergillus niger
| - | 5 | 9 | 16 | 250/500 | 125/500 |
Sacharomyces cerevaceae
| - | 4 | 7 | 10 | 62.5/250 | 62.5/250 |
Trichoderma sp.
| - | - | - | 9 | 250/500 | 125/500 |