Therapeutic Potential of Ozonated Ocimum basilicum L. from Saudi Arabia: Phytochemical Characterization and Enhanced Bioactivities
Abstract
1. Introduction
2. Results and Discussion
2.1. Phytochemical Profiling of Non-Ozonated and Ozonated O. basilicum L. Extracts by HPLC
2.2. Antidiabetic Activity: Inhibition of α-Amylase and α-Glucosidase
2.3. Anti-Alzheimer Activity: Inhibition of Butyrylcholinesterase (BChE)
2.4. Anti-Inflammatory Activity: Inhibition of Hypotonic Red Blood Cell Hemolysis
2.5. Anti-Helicobacter pylori Activity
2.6. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
2.7. Cytotoxicity and Cell Viability in Normal Lung Fibroblasts (WI-38 Cell Line)
2.8. Cytotoxicity and Cell Viability in Colon Epithelial Cells (Caco-2 Cell Line)
3. Materials and Methods
3.1. Collection and Extraction of O. basilicum L.
3.2. HPLC Analysis
3.3. In Vitro α-Amylase Inhibition Assay
3.4. In Vitro α-Glucosidase Inhibition Assay
3.5. In Vitro Anti-Alzheimer Activity (Butyrylcholinesterase Inhibition)
3.6. In Vitro Anti-Inflammatory Activity (Hypotonic Hemolysis Method)
3.6.1. Preparation of Red Blood Cell (RBC) Suspension
3.6.2. Hypotonic Hemolysis Assay
3.7. The Determination of Anti-Helicobacter pylori Activity
3.7.1. Qualitative Agar Diffusion Assay
3.7.2. Quantitative Hemolysin Inhibition Assay
3.8. MIC and MBC Determination
3.9. Cytotoxicity Assay
3.10. Ozone Generation
3.11. Preparation of Resazurin Dye
3.12. Statistical Analysis
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Components | Standard | Non-Ozonated Saudi Basil | Ozonated Saudi Basil | |||
---|---|---|---|---|---|---|
Conc. (µg/mL) | Area | Conc. (µg/mL) | Area | Conc. (µg/mL) | Area | |
Gallic acid | 20 | 248.33 | 727.67 a | 58.61 | 723.78 a | 58.29 |
Chlorogenic acid | 50 | 391.71 | 382.63 a | 48.84 | 1207.83 b | 154.17 |
Catechin | 75 | 329.15 | 0.00 a | 0.00 | 63.59 b | 14.49 |
Methyl gallate | 15 | 291.59 | 125.02 a | 6.43 | 101.77 a | 5.24 |
Caffeic acid | 18 | 220.63 | 288.91 a | 23.57 | 203.97 a | 16.64 |
Syringic acid | 17.2 | 289.68 | 141.54 a | 8.40 | 179.96 a | 10.69 |
Rutin | 50 | 410.85 | 65.71 a | 8.00 | 40.53 a | 4.93 |
Ellagic acid | 70 | 568.31 | 115.05 a | 14.17 | 75.46 a | 9.30 |
Coumaric acid | 20 | 600.85 | 122.46 a | 4.08 | 81.18 a | 2.70 |
Vanillin | 12.9 | 350.04 | 769.67 a | 28.36 | 473.89 a | 17.46 |
Ferulic acid | 20 | 357.33 | 625.38 a | 35.00 | 415.99 a | 23.28 |
Naringenin | 30 | 334.36 | 39.51 | 3.55 | 15.32 | 1.38 |
Rosmarinic acid | 50 | 501.19 | 1604.89 a | 160.11 | 1015.34 a | 101.29 |
Daidzein | 20 | 319.64 | 692.21 a | 43.31 | 408.94 a | 25.59 |
Quercetin | 20 | 300.41 | 1590.07 a | 105.86 | 965.95 a | 64.31 |
Cinnamic acid | 10 | 584.22 | 212.01 a | 3.63 | 131.64 a | 2.25 |
Kaempferol | 20 | 279.09 | 810.83 a | 58.10 | 504.15 a | 36.13 |
Hesperetin | 20 | 418.68 | 23.65 a | 1.13 | 15.19 a | 0.73 |
Conc. (µg/mL) | Mean A540 | α-Amylase Inhibition (%) | SD | SE | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Acar | SB | OSB | Acar | SB | OSB | Acar | SB | OSB | Acar | SB | OSB | |
1000 | 0.053 | 0.093 | 0.072 | 95.8 | 92.5 | 94.2 | 0.002 | 0.004 | 0.003 | 0.001 | 0.001 | 0.001 |
500 | 0.092 | 0.136 | 0.101 | 92.6 | 89.1 | 91.9 | 0.004 | 0.003 | 0.002 | 0.001 | 0.001 | 0.001 |
250 | 0.127 | 0.196 | 0.146 | 89.8 | 84.3 | 88.3 | 0.004 | 0.003 | 0.002 | 0.001 | 0.001 | 0.000 |
125 | 0.153 | 0.282 | 0.177 | 87.7 | 77.4 | 85.8 | 0.003 | 0.003 | 0.006 | 0.001 | 0.001 | 0.002 |
62.5 | 0.237 | 0.392 | 0.266 | 81.0 | 68.5 | 78.7 | 0.004 | 0.004 | 0.004 | 0.001 | 0.001 | 0.001 |
31.25 | 0.323 | 0.480 | 0.359 | 74.1 | 61.5 | 71.2 | 0.003 | 0.003 | 0.007 | 0.001 | 0.001 | 0.002 |
15.62 | 0.419 | 0.589 | 0.487 | 66.4 | 52.7 | 60.9 | 0.007 | 0.003 | 0.014 | 0.002 | 0.001 | 0.005 |
7.81 | 0.522 | 0.708 | 0.581 | 58.1 | 43.2 | 53.4 | 0.005 | 0.006 | 0.009 | 0.001 | 0.002 | 0.003 |
3.9 | 0.615 | 0.816 | 0.678 | 50.7 | 34.5 | 45.7 | 0.003 | 0.003 | 0.003 | 0.001 | 0.001 | 0.001 |
1.95 | 0.782 | 0.918 | 0.786 | 37.3 | 26.4 | 36.9 | 0.003 | 0.003 | 0.002 | 0.001 | 0.001 | 0.001 |
100% | 1.247 | 1.247 | 1.247 | 0.00 | 0.00 | 0.00 | 0.003 | 0.003 | 0.003 | 0.001 | 0.001 | 0.001 |
IC50 | 3.47 a | 13.6 b | 5.09 a |
Conc. (µg/mL) | Mean A540 | α-Glucosidase Inhibition (%) | SD | SE | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Acar | SB | OSB | Acar | SB | OSB | Acar | SB | OSB | Acar | SB | OSB | |
1000 | 0.049 | 0.160 | 0.123 | 97.2 | 90.9 | 93.0 | 0.002 | 0.003 | 0.004 | 0.001 | 0.001 | 0.001 |
500.00 | 0.095 | 0.279 | 0.226 | 94.7 | 84.3 | 87.3 | 0.004 | 0.002 | 0.008 | 0.001 | 0.000 | 0.002 |
250.00 | 0.180 | 0.368 | 0.340 | 89.9 | 79.3 | 80.9 | 0.002 | 0.003 | 0.004 | 0.000 | 0.001 | 0.001 |
125.00 | 0.275 | 0.483 | 0.430 | 84.6 | 72.8 | 75.9 | 0.007 | 0.002 | 0.007 | 0.002 | 0.001 | 0.002 |
62.50 | 0.393 | 0.588 | 0.537 | 77.9 | 67.0 | 69.8 | 0.003 | 0.003 | 0.003 | 0.001 | 0.001 | 0.001 |
31.25 | 0.506 | 0.705 | 0.647 | 71.6 | 60.4 | 63.7 | 0.005 | 0.003 | 0.006 | 0.002 | 0.001 | 0.002 |
15.63 | 0.620 | 0.815 | 0.737 | 65.2 | 54.2 | 58.6 | 0.004 | 0.003 | 0.004 | 0.001 | 0.001 | 0.001 |
7.81 | 0.746 | 0.927 | 0.840 | 58.1 | 47.9 | 52.8 | 0.004 | 0.004 | 0.004 | 0.001 | 0.001 | 0.001 |
3.91 | 0.862 | 1.032 | 0.942 | 51.6 | 42.0 | 47.1 | 0.003 | 0.003 | 0.004 | 0.001 | 0.001 | 0.001 |
1.95 | 0.973 | 1.126 | 1.096 | 45.3 | 36.7 | 38.4 | 0.005 | 0.004 | 0.008 | 0.002 | 0.001 | 0.003 |
Control | 1.745 | 1.745 | 1.745 | 0.00 | 0.00 | 0.00 | 0.006 | 0.006 | 0.006 | 0.002 | 0.002 | 0.002 |
IC50 | 2.91 a | 9.42 b | 6.15 b |
Conc. (µg/mL) | Mean A405 | BChE Inhibition (%) | SD | SE | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
RIV | SB | OSB | RIV | SB | OSB | RIV | SB | OSB | RIV | SB | OSB | |
100 | 0.009 | 0.080 | 0.053 | 95.6 | 62.1 | 74.8 | 0.001 | 0.001 | 0.001 | 0.000 | 0.000 | 0.000 |
50 | 0.020 | 0.096 | 0.068 | 90.5 | 54.4 | 67.8 | 0.001 | 0.001 | 0.001 | 0.000 | 0.000 | 0.000 |
25 | 0.028 | 0.107 | 0.101 | 86.7 | 49.4 | 52.0 | 0.001 | 0.001 | 0.001 | 0.000 | 0.000 | 0.000 |
12.5 | 0.044 | 0.118 | 0.111 | 79.2 | 44.3 | 47.6 | 0.002 | 0.002 | 0.002 | 0.000 | 0.000 | 0.000 |
6.25 | 0.054 | 0.145 | 0.124 | 74.4 | 31.4 | 41.2 | 0.002 | 0.002 | 0.002 | 0.000 | 0.000 | 0.000 |
3.125 | 0.070 | 0.158 | 0.138 | 66.9 | 25.2 | 34.8 | 0.002 | 0.002 | 0.002 | 0.001 | 0.001 | 0.001 |
1.56 | 0.081 | 0.181 | 0.159 | 61.7 | 14.5 | 24.8 | 0.004 | 0.004 | 0.004 | 0.001 | 0.001 | 0.001 |
0.78 | 0.093 | 0.193 | 0.175 | 55.8 | 8.7 | 17.2 | 0.002 | 0.002 | 0.002 | 0.001 | 0.001 | 0.001 |
0.39 | 0.108 | 0.200 | 0.188 | 48.9 | 5.3 | 11.0 | 0.003 | 0.003 | 0.003 | 0.001 | 0.001 | 0.001 |
0.195 | 0.127 | 0.201 | 0.197 | 39.9 | 4.9 | 6.6 | 0.003 | 0.003 | 0.003 | 0.001 | 0.001 | 0.001 |
0.000 | 0.2113 | 0.2113 | 0.2113 | 0.00 | 0.00 | 0.00 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
IC50 | 0.46 a | 31.8 a | 13.4 c |
Conc. (µg/mL) | Mean A540 | Hemolysis Inhibition (%) | SD | SE | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Hypotonic Sol. | Isotonic Sol. | ||||||||||||||
IND | SB | OSB | IND | SB | OSB | IND | SB | OSB | IND | SB | OSB | IND | SB | OSB | |
Control | 1.184 | 1.184 | 1.184 | 0.001 | 0.001 | 0.001 | 0.00 | 0.00 | 0.00 | 0.003 | 0.003 | 0.003 | 0.001 | 0.001 | 0.001 |
1000 | 0.035 | 0.062 | 0.055 | 0.021 | 0.021 | 0.021 | 98.8 | 96.5 | 97.0 | 0.003 | 0.005 | 0.005 | 0.001 | 0.001 | 0.002 |
500 | 0.083 | 0.102 | 0.094 | 0.015 | 0.015 | 0.015 | 94.2 | 92.6 | 93.2 | 0.002 | 0.003 | 0.002 | 0.001 | 0.001 | 0.001 |
250 | 0.104 | 0.141 | 0.138 | 0.011 | 0.011 | 0.011 | 92.0 | 88.9 | 89.2 | 0.003 | 0.008 | 0.005 | 0.001 | 0.003 | 0.001 |
125 | 0.181 | 0.215 | 0.207 | 0.009 | 0.009 | 0.009 | 85.4 | 82.4 | 83.1 | 0.005 | 0.003 | 0.005 | 0.002 | 0.001 | 0.001 |
62.5 | 0.262 | 0.292 | 0.288 | 0.005 | 0.005 | 0.005 | 78.2 | 75.6 | 76.0 | 0.006 | 0.003 | 0.003 | 0.002 | 0.001 | 0.001 |
31.25 | 0.345 | 0.396 | 0.391 | 0.003 | 0.003 | 0.003 | 71.0 | 66.8 | 67.2 | 0.003 | 0.004 | 0.003 | 0.001 | 0.001 | 0.001 |
15.62 | 0.449 | 0.507 | 0.502 | 0.002 | 0.002 | 0.002 | 62.2 | 57.2 | 57.7 | 0.007 | 0.003 | 0.005 | 0.002 | 0.001 | 0.001 |
7.8 | 0.562 | 0.626 | 0.621 | 0.001 | 0.001 | 0.001 | 52.6 | 47.1 | 47.6 | 0.003 | 0.003 | 0.002 | 0.001 | 0.001 | 0.001 |
3.9 | 0.623 | 0.737 | 0.728 | 0.001 | 0.001 | 0.001 | 47.4 | 37.8 | 38.5 | 0.004 | 0.004 | 0.001 | 0.001 | 0.001 | 0.000 |
IC50 | 4.41 a | 8.44 b | 8.04 b |
Substance | Anti-Helicobacter pylori Activity (mm) | SD | SE | |||
---|---|---|---|---|---|---|
R 1 | R 2 | R 3 | Mean | |||
Omeclamox-Pak (control) | 24 | 25 | 24 | 24.5 | 0.577 | 0.190 |
Non-ozonated Saudi basil | 19 | 18 | 19 | 18.7 | 0.577 | 0.188 |
Ozonated Saudi basil | 26 | 27 | 27 | 26.7 | 0.577 | 0.188 |
Sample | A540 of Sample with RBCs | A540 of Sample with Isotonic Solution | Hemolysis (%) | SD | SE | |||||
---|---|---|---|---|---|---|---|---|---|---|
SB | OSB | SB | OSB | SB | OSB | SB | OSB | SB | OSB | |
Control | 1.735 | 1.735 | 0.0001 | 0.0001 | 100 | 100 | 0.011 | 0.011 | 0.004 | 0.004 |
(25% MIC) | 0.417 | 0.212 | 0.008 | 0.007 | 23.6 a | 11.8 b | 0.006 | 0.003 | 0.002 | 0.001 |
(50% MIC) | 0.108 | 0.106 | 0.004 | 0.003 | 6.0 a | 6.0 a | 0.002 | 0.006 | 0.001 | 0.002 |
(75% MIC) | 0.059 | 0.042 | 0.000 | 0.000 | 3.4 a | 2.4 a | 0.004 | 0.003 | 0.001 | 0.001 |
Sample | MIC (µg/mL) | MBC (µg/mL) | MBC/MIC Index (µg/mL) |
---|---|---|---|
Omeclamox-Pak (Control) | 31.25 | 62.5 | 2 * |
Non-ozonated Saudi basil | 15.62 | 31.25 | 2 * |
Ozonated Saudi basil | 31.25 | 31.25 | 1 * |
Sample | Conc. (µg/mL) | Mean of A560 | Viability (%) | Toxicity (%) | SD | SE | IC50 (µg/mL) |
---|---|---|---|---|---|---|---|
WI-38 cell line | None | 0.719 | 100 | 0.00 | None | 0.001 | None |
Non-ozonated Saudi basil | 1000 | 0.06 | 8.35 | 91.65 | 4.01 | 0.004 | 191.06 ± 4.01 a |
500 | 0.085 | 11.87 | 88.13 | 4.01 | 0.002 | ||
250 | 0.232 | 32.32 | 67.68 | 4.01 | 0.007 | ||
125 | 0.483 | 67.23 | 32.77 | 4.01 | 0.010 | ||
62.5 | 0.694 | 96.57 | 3.43 | 4.01 | 0.003 | ||
31.25 | 0.719 | 100.0 | 0.00 | 4.01 | 0.001 | ||
Ozonated Saudi basil | 1000 | 0.035 | 4.96 | 95.04 | 4.84 | 0.002 | 437.89 ± 4.84 b |
500 | 0.275 | 38.2 | 61.75 | 4.84 | 0.005 | ||
250 | 0.643 | 89.5 | 10.52 | 4.84 | 0.005 | ||
125 | 0.705 | 98.05 | 1.947 | 4.84 | 0.003 | ||
62.5 | 0.718 | 99.9 | 0.092 | 4.84 | 0.0008 | ||
31.25 | 0.719 | 100 | 0.00 | 4.84 | 0.0005 |
Sample | Conc. (µg/mL) | Mean of A560 | Viability (%) | Toxicity (%) | SD | SE | IC50 µg/mL |
---|---|---|---|---|---|---|---|
Caco-2 cell line | None | 0.665 | 100 | 0.00 | None | 0.003 | None |
Non-ozonated Saudi basil | 1000 | 0.017 | 2.656 | 97.34 | 1.16 | 0.0003 | 103.70 ± 1.16 a |
500 | 0.018 | 2.756 | 97.24 | 1.16 | 0.0003 | ||
250 | 0.026 | 3.959 | 96.04 | 1.16 | 0.0035 | ||
125 | 0.250 | 37.64 | 62.35 | 1.16 | 0.0077 | ||
62.5 | 0.487 | 73.23 | 26.76 | 1.16 | 0.0066 | ||
31.25 | 0.650 | 97.74 | 2.255 | 1.16 | 0.0046 | ||
Ozonated Saudi basil | 1000 | 0.018 | 2.757 | 97.24 | 0.82 | 0.0003 | 149.14 ± 0.8 a |
500 | 0.018 | 2.807 | 97.19 | 0.82 | 0.0008 | ||
250 | 0.020 | 3.057 | 96.94 | 0.82 | 0.0008 | ||
125 | 0.349 | 52.48 | 47.52 | 0.82 | 0.0040 | ||
62.5 | 0.662 | 99.55 | 0.451 | 0.82 | 0.0020 | ||
31.25 | 0.665 | 100 | 0.00 | 0.82 | 0.0015 |
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Qanash, H.; Alsalamah, S.A.; Bazaid, A.S.; Alghonaim, M.I.; Duhduh, A.; Hudani, I. Therapeutic Potential of Ozonated Ocimum basilicum L. from Saudi Arabia: Phytochemical Characterization and Enhanced Bioactivities. Pharmaceuticals 2025, 18, 1223. https://doi.org/10.3390/ph18081223
Qanash H, Alsalamah SA, Bazaid AS, Alghonaim MI, Duhduh A, Hudani I. Therapeutic Potential of Ozonated Ocimum basilicum L. from Saudi Arabia: Phytochemical Characterization and Enhanced Bioactivities. Pharmaceuticals. 2025; 18(8):1223. https://doi.org/10.3390/ph18081223
Chicago/Turabian StyleQanash, Husam, Sulaiman A. Alsalamah, Abdulrahman S. Bazaid, Mohammed Ibrahim Alghonaim, Amro Duhduh, and Ibtisam Hudani. 2025. "Therapeutic Potential of Ozonated Ocimum basilicum L. from Saudi Arabia: Phytochemical Characterization and Enhanced Bioactivities" Pharmaceuticals 18, no. 8: 1223. https://doi.org/10.3390/ph18081223
APA StyleQanash, H., Alsalamah, S. A., Bazaid, A. S., Alghonaim, M. I., Duhduh, A., & Hudani, I. (2025). Therapeutic Potential of Ozonated Ocimum basilicum L. from Saudi Arabia: Phytochemical Characterization and Enhanced Bioactivities. Pharmaceuticals, 18(8), 1223. https://doi.org/10.3390/ph18081223