Validation of Broth Macrodilution Volatilization Method for Testing of Essential Oils in Liquid and Vapor Phase: Chemical Composition, Cytotoxicity, and Antibacterial Effect of Indian Medicinal Plants against Pneumonia-Causing Pathogens
Abstract
:1. Introduction
2. Results
2.1. Antibacterial Activity and Cytotoxicity
2.2. Chemical Composition
2.3. Chemical Composition of T. ammi EO Vapors
3. Discussion
4. Materials and Methods
4.1. Chemicals
4.2. Plant Material and Sample Preparation
4.3. Bacterial Strains and Culture Media
4.4. Antimicrobial Assay
4.5. Cell Cultures
4.6. Cytotoxicity Assay
4.7. Chemical Analysis of EOs’ Liquid Phase
4.8. Chemical Analysis of EOs’ Vapor Phase
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
References
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Sample | Bacterium/Growth Medium/Minimum Inhibitory Concentration (µg/mL) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Essential Oil | Haemophilus influenzae | Staphylococcus aureus | Streptococcus pneumoniae | Streptococcus pyogenes | x-MIC | ||||
Broth | Agar | Broth | Agar | Broth | Agar | Broth | Agar | ||
Cymbopogon citratus | 256 | 256 | 512 | 1024 | 512 | 1024 | 512 | 1024 | 448 |
Cyperus scariosus. | 1024 | 1024 | 1024 | >1024 | 1024 | >1024 | 1024 | >1024 | 1024 |
Trachyspermum ammi | 128 | 256 | 512 | 512 | 512 | 1024 | 512 | 1024 | 416 |
Positive antibiotic control | 1 a | n.d. | 0.5 b | n.d. | 0.25 c | n.d. | 0.25 d | n.d. | - |
Sample | IC50 ± SD (µg/mL) | IC80 ± SD (µg/mL) | TI |
---|---|---|---|
Essential oil | |||
Cymbopogon citratus | 19.63 ± 1.02 | 29.54 ± 2.18 | 0.065 |
Cyperus scariosus | >258 | >258 | >0.252 |
Trachyspermum ammi | 82.04 ± 3.39 | 156.57 ± 13.88 | 0.376 |
Positive control | |||
vinorelbine | 0.54 ± 0.26 | >10 | n.a. |
RI a | Compound | Cl. b | Column | ||||
---|---|---|---|---|---|---|---|
Content c (%) | Identification f | ||||||
Obs. | Lit. | HP-5MS | DB-WAX | HP-5MS | DB-WAX | ||
915 | 926 | Tricyclene | MH | 0.26 ± 0.12 | tr. d | RI, MS, Std, | MS |
927 | 939 | α-Pinene | MH | 0.20 ± 0.04 | - e | RI, MS, Std | - |
942 | 953 | Camphene | MH | 2.42 ± 0.79 | 1.62 ± 0.34 | RI, MS, Std | MS |
979 | 985 | Sulcatone | MO | 0.35 ± 0.18 | - | RI, MS | - |
1023 | 1029 | D-Limonene | MH | 0.23 ± 0.03 | - | RI, MS | - |
1029 | 1050 | trans-β-Ocimene | MH | 0.31 ± 0.09 | - | RI, MS | - |
1039 | 1011 | 3-Carene | MH | 0.13 ± 0.02 | - | RI, MS, Std | - |
1063 | 1030 | 4-Nonanone | MO | 1.41 ± 0.38 | 1.55 ± 0.97 | RI, MS | MS |
1098 | 1098 | Linalool | MO | 0.46 ± 0.24 | 0.31 ± 0.07 | RI, MS, Std | MS |
1178 | 1184.7 | Isogeranial | MO | 0.70 ± 0.01 | 0.55 ± 0.05 | RI, MS | MS |
1190 | 1189 | α-Terpineol | MO | 0.39 ± 0.03 | - | RI, MS | - |
1238 | 1240 | β-Citral | MO | 35.8 ± 0.61 | 24.3 ± 8.82 | RI, MS, Std | MS |
1268 | 1270 | α-Citral | MO | 48.9 ± 0.55 | 33.2 ± 11.2 | RI, MS, Std | MS |
1376 | 1381 | Geranyl acetate | MO | 2.02 ± 0.20 | tr. | RI, MS | MS |
1412 | 1418 | Caryophyllene | SH | 0.45 ± 0.31 | 0.75 ± 0.05 | RI, MS | MS |
1510 | 1513 | γ-Cadinene | SH | 1.19 ± 0.33 | 1.20 ± 0.23 | RI, MS | MS |
g | 1797 | Geraniol | MO | - | 0.77 ± 0.07 | - | MS |
1582 | 1581 | Caryophyllene oxide | SH | 3.0 ± 1.34 | 2.88 ± 1.40 | RI, MS, Std | MS |
g | 1430 | α-Cyclocitral | MO | - | 0.45 ± 0.04 | - | MS |
g | NA | Isoneral | MO | - | 0.36 ± 0.02 | - | MS |
g | 1669 | Isoborneol | MO | - | 0.83 ± 0.01 | - | MS |
Total content (%) | 99.62 | 68.77 |
RI a | Compound | Cl. b | Column | ||||
---|---|---|---|---|---|---|---|
Content c (%) | Identification f | ||||||
Obs. | Lit. | HP-5MS | DB-WAX | HP-5MS | DB-WAX | ||
927 | 939 | α-Pinene | MH | 1.34 ± 0.10 | 0.50 ± 0.01 | RI, MS, Std | MS |
970 | 980 | β-Pinene | MH | 1.88 ± 0.33 | 0.06 ± 0.00 | RI, MS, Std | MS |
1025 | 1032 | Eucalyptol | MO | 0.24 ± 0.16 | - d | RI, MS | - |
1137 | 1137 | Pinocarveol | MO | 1.85 ± 0.23 | 0.60 ± 0.01 | RI, MS | MS |
1158 | 1165 | Pinocarvone | MO | 0.37 ± 0.32 | 0.09 ± 0.00 | RI, MS | MS |
1168 | 1193 | Myrtenal | MO | 0.41 ± 0.27 | 0.13 ± 0.30 | RI, MS | MS |
1314 | 1327 | Cyprotene | SH | 0.16 ± 0.05 | tr.e | RI, MS | MS |
1344 | 1349 | α-Terpinyl acetate | MO | 1.65 ± 0.18 | tr. | RI, MS | MS |
1371 | 1376 | Copaene | SH | 1.46 ± 0.47 | tr. | RI, MS | MS |
1394 | 1398 | Cyperene | SH | 9.87 ± 0.59 | 8.5 ± 0.04 | RI, MS | MS |
1446 | 1477 | α-Muurolene | SH | 0.16 ± 0.04 | tr. | RI, MS | MS |
1456 | 1461 | Rotundene | SH | 1.94 ± 0.07 | 1.25 ± 0.01 | RI, MS | MS |
1483 | 1473.7 | γ-Patchoulene | SH | 0.19 ± 0.05 | tr. | RI, MS | MS |
1489 | 1491 | Valencene | SH | 0.63 ± 0.08 | 0.57 ± 0.60 | RI, MS | MS |
1518 | 1518 | β-Cadinene | SH | 0.31 ± 0.18 | 0.08 ± 0.40 | RI, MS | MS |
1528 | 1532 | Cyperene epoxide | SO | 2.65 ± 0.26 | 1.50 ± 0.00 | RI, MS | MS |
1541 | 1542 | α-Calacorene | SH | 0.13 ± 0.05 | - | RI, MS | - |
1565 | 1579 | Isoaromadendrene epoxide | SO | 0.62 ± 0.07 | 1.03 ± 0.00 | RI, MS | MS |
1572 | 1627 | Longiverbenone | SO | 1.33 ± 0.15 | 1.20 ± 0.08 | RI, MS | MS |
1582 | 1581 | Caryophyllene oxide | SH | 19.79 ± 0.58 | 17.54 ± 0.12 | RI, MS, Std | MS |
1591 | NA | β-Santalol | SO | 0.38 ± 0.12 | - | RI, MS | - |
1609 | 1608 | Humulene epoxide 2 | SO | 1.69 ± 0.25 | 2.60 ± 0.10 | RI, MS | MS |
1656 | 1604 | Globulol | SO | 0.23 ± 0.04 | 0.39 ± 0.03 | RI, MS | MS |
1664 | 1663 | Patchouli alcohol | SO | 0.50 ± 0.05 | - | RI, MS | - |
1677 | 1676 | Mustakone | SO | 6.26 ± 0.26 | 3.67 ± 0.30 | RI, MS | MS |
1697 | 1694 | Cyperotundone | SO | 29.1 ± 1.11 | 28.91 ± 0.72 | RI, MS | MS |
1750 | 1752 | Aristolone | SO | 3.17 ± 0.77 | 3.73 ± 0.10 | RI, MS | MS |
1808 | 1807 | Nootkatone | SO | 2.17 ± 0.49 | 2.03 ± 0.40 | RI, MS | MS |
g | NA | β-Pinone | MO | - | tr. | - | MS |
g | 1586 | β-Elemene | SH | - | tr. | - | MS |
g | 1652 | cis-Verbenol | MO | - | tr. | - | MS |
g | NA | Aristolochene | SH | - | tr. | - | MS |
g | 1680 | α-Terpineol | tr. | - | MS | ||
g | NA | α-Maaliene | SH | - | tr. | - | MS |
g | 1784 | Myrtenol | MO | - | tr. | - | MS |
g | 2063 | Cubenol | SO | - | 0.49 ± 0.00 | - | MS |
g | 1978 | α-Cedrene epoxide | SO | - | 0.40 ± 0.10 | - | MS |
g | NA | Aromadendrene oxide-(1) | SO | - | 1.35 ± 0.40 | - | MS |
g | NA | Calarene epoxide | SO | - | 0.52 ± 0.02 | - | MS |
g | NA | Diepicedrene-1-oxide | SO | - | 0.05 ± 0.00 | - | MS |
Total content (%) | 91.48 | 77.17 |
RI a | Compound | Cl. b | Column | ||||
---|---|---|---|---|---|---|---|
Content c (%) | Identification e | ||||||
Obs. | Lit. | HP-5MS | DB-WAX | HP-5MS | DB-WAX | ||
920 | 917 | β-Thujene | MH | 0.30 ± 0.03 | 0.17 ± 0.02 | RI, MS | MS |
964 | 925 | α-Pinene | MH | 0.19 ± 0.01 | 0.42 ± 0.13 | RI, MS, Std | MS |
981 | 971 | β-Pinene | MH | 1.87 ± 0.25 | 2.13 ± 0.62 | RI, MS, Std | MS |
994 | 984 | β-Myrcene | MH | 0.21 ± 0.06 | 0.38 ± 0.03 | RI, MS | MS |
1007 | 974 | 2-Carene | MH | 0.20 ± 0.02 | - d | RI, MS | MS |
1050 | 1031 | β-Cymene | MH | 22.6 ± 0.89 | 17.1 ± 3.99 | RI, MS, Std | MS |
1079 | 1065 | γ-Terpinene | MH | 21.5 ± 0.86 | 17.6 ± 0.99 | RI, MS, Std | MS |
1175 | 1086 | Isoterpinolene | MH | 0.08 ± 0.06 | - | RI, MS | MS |
1315 | 1290 | Thymol | MO | 51.2 ± 1.25 | 45.8 ± 4.41 | RI, MS, Std | MS |
f | 1172 | α-Terpinene | MH | - | 0.12 ± 0.01 | - | MS |
f | 1244 | D-Limonene | MH | - | 0.10 ± 0.01 | - | MS |
f | 1195 | β-Phellandrene | MH | - | 0.08 ± 0.03 | - | MS |
f | NA | trans-2-Caren-4-ol | MO | - | 0.18 ± 0.02 | - | MS |
f | 1680 | Terpineol | MO | - | 0.06 ± 0.01 | - | MS |
f | 1635 | Terpinen-4-ol | MO | - | 0.20 ± 0.04 | - | MS |
Total content (%) | 99.47 | 84.26 |
RI a | Compound | Extraction Method/Time (h)/Content b (%) | Ident. e | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Solid Phase Microextraction | Gas Tight Syringe Extraction | ||||||||||||
Obs. | Lit. | 0 | 3 | 6 | 9 | 12 | 0 | 3 | 6 | 9 | 12 | ||
925 | 939 | α-Pinene | 1.27 ± 0.10 | 1.46 ± 0.00 | 1.42 ± 0.01 | 1.41 ± 0.00 | 1.25 ± 0.01 | tr. c | tr. | tr. | tr. | tr. | RI, MS |
961 | 1011 | 2-Carene | 0.64 ± 0.00 | 0.44 ± 0.00 | 0.62 ± 0.01 | 0.65 ± 0.02 | 0.72. ±0.01 | - d | - | - | - | - | RI, MS |
971 | 980 | β-Pinene | 1.91 ± 0.01 | 2.80 ± 0.05 | 2.36 ± 0.00 | 2.31 ± 0.01 | 1.79 ± 0.20 | 6.57 ± 0.20 | 4.93 ± 0.40 | 3.89 ± 0.10 | 4.11 ± 0.01 | 2.02 ± 0.30 | RI, MS |
1031 | 1030 | β-Cymene | 49.14 ± 1.00 | 48.00 ± 1.10 | 46.57 ± 0.80 | 45.97 ± 1.80 | 43.17 ± 0.90 | 52.00 ± 3.50 | 49.18 ± 2.90 | 48.67 ± 2.50 | 46.32 ± 1.40 | 45.60 ± 0.60 | RI, MS |
1065 | 1062 | γ-Terpinene | 39.36 ± 0.70 | 35.41 ± 0.50 | 35.11 ± 0.90 | 33.26 ± 0.60 | 31.01 ± 0.20 | 35.00 ± 2.50 | 32.23 ± 0.09 | 31.60 ± 0.50 | 32.22 ± 1.30 | 28.20 ± 2.90 | RI, MS |
1306 | 1290 | Thymol | 4.96 ± 0.01 | 5.66 ± 0.40 | 9.82 ± 0.08 | 11.90 ± 0.93 | 12.10 ± 0.80 | tr. | 1.23 ± 0.10 | 2.21 ± 0.20 | 2.07 ± 0.03 | tr. | RI, MS |
Total content (%) | 97.25 | 94.21 | 95.9 | 95.5 | 90.66 | 93.40 | 88.57 | 87.98 | 85.72 | 79.05 |
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Chaure, A.; Houdkova, M.; Antih, J.; Urbanova, K.; Doskocil, I.; Naik, M.L.; Patel, K.S.; Kokoska, L. Validation of Broth Macrodilution Volatilization Method for Testing of Essential Oils in Liquid and Vapor Phase: Chemical Composition, Cytotoxicity, and Antibacterial Effect of Indian Medicinal Plants against Pneumonia-Causing Pathogens. Molecules 2023, 28, 4625. https://doi.org/10.3390/molecules28124625
Chaure A, Houdkova M, Antih J, Urbanova K, Doskocil I, Naik ML, Patel KS, Kokoska L. Validation of Broth Macrodilution Volatilization Method for Testing of Essential Oils in Liquid and Vapor Phase: Chemical Composition, Cytotoxicity, and Antibacterial Effect of Indian Medicinal Plants against Pneumonia-Causing Pathogens. Molecules. 2023; 28(12):4625. https://doi.org/10.3390/molecules28124625
Chicago/Turabian StyleChaure, Aishwarya, Marketa Houdkova, Julien Antih, Klara Urbanova, Ivo Doskocil, Mukund Lal Naik, Khageshwar Singh Patel, and Ladislav Kokoska. 2023. "Validation of Broth Macrodilution Volatilization Method for Testing of Essential Oils in Liquid and Vapor Phase: Chemical Composition, Cytotoxicity, and Antibacterial Effect of Indian Medicinal Plants against Pneumonia-Causing Pathogens" Molecules 28, no. 12: 4625. https://doi.org/10.3390/molecules28124625