Introduction
Herbanol combined the March and April reports because the tests took more than a month to complete. This report covers sections 2 and 3 of the project.
In February, the team began testing essential oil from Zataria multiflora, a thyme in the mint family that grows in southern Iran and has edible and medicinal uses. The team chose fresh flaxseed oil as the edible oil because it is rich in unsaturated fatty acids.
Testing took at least 25 days after adding the essential oil. The team placed 10 flaxseed oil samples in dark glass tubes and kept them at 40 °C for 20 days before refrigerating them. The experimental groups were thyme (A) and the synthetic antioxidant BHT (B), each at 50 ppm and 500 ppm, with two samples per concentration. Two control samples contained no antioxidant.
Peroxide value
The team followed the Lubrizol and Iranian national test methods. The peroxide value measures milliequivalents of active peroxide or oxygen per kilogram of fat or oil. Peroxides form as oils oxidize, so the index indicates oxidative damage. The report gives a value below 6 for healthy oils.
Peroxides and other oxidizing compounds react with saturated potassium iodide added to the sample. In the reaction given in the report, ROO reacts with KI to form ROOK and half a molecule of I₂. The team titrated the released iodine with 0.1 N sodium thiosulfate and used starch as an indicator. The report lists I₂ and Na₂S₂O₃ as the reactants and 2NaI and Na₂S₄O₆ as the products.
| Treatment | April 10, 2022 | April 20, 2022 |
|---|---|---|
| Thyme, 500 ppm | 21.75 | 30.90 |
| Thyme, 50 ppm | 20.32 | 45.19 |
| BHT, 500 ppm | 21.36 | 34.17 |
| BHT, 50 ppm | 21.46 | 32.78 |
| Control | 35.14 | 72.25 |
The report’s bar chart compares the two test dates. The peroxide value rose in every group, and the control had the highest value on both dates. The report concludes that the thyme essential oil had a significant effect on peroxide value compared with the control.
Thiobarbituric acid test
Thiobarbituric acid reactive substances (TBARS) form when lipids oxidize. Thiobarbituric acid (TBA) reacts with malondialdehyde (MDA), one of the low-molecular-weight compounds that form when lipid peroxidation products break down. The team tested 10 samples and measured absorbance with a Bio-Tek Kontron N UV-visible instrument.
| Treatment | April 15, 2022 | April 25, 2022 |
|---|---|---|
| Thyme, 500 ppm | 0.209 | 0.340 |
| Thyme, 50 ppm | 0.298 | 0.478 |
| BHT, 500 ppm | 0.240 | 0.452 |
| BHT, 50 ppm | 0.258 | 0.491 |
| Control | 0.410 | 0.685 |
The report’s bar chart shows that absorbance rose in all groups and was highest for the control. The report concludes that the other samples slowed oxidation.
Effect of antioxidant type and concentration
The report plots estimated marginal means of Y for four essences labeled A through D. The maximum-concentration line stays above the minimum-concentration line. Both lines decline overall and the minimum-concentration line drops most sharply between C and D. The report interprets the two lines as leaving open the possibility that antioxidant type and concentration interact.
| Essence | Maximum | Minimum |
|---|---|---|
| A | 2.38 | 2.15 |
| B | 2.15 | 1.97 |
| C | 2.15 | 1.87 |
| D | 1.91 | 1.43 |
Thyme essential oil composition
Zataria multiflora is used as a flavoring and has traditional uses for gastrointestinal disorders, local wounds, respiratory disorders, and colds. Gas chromatography and mass spectrometry (GC/MS) identified 26 compounds. The report calls out carvacrol (50.22%), thymol (21.85%), p-cymene (6.34%), beta-caryophyllene (1.85%), and linalool (1.08%).
The diagrams show carvacrol and thymol as phenols with methyl and isopropyl groups at different positions. Carvacrol is 2-methyl-5-isopropylphenol and thymol is 2-isopropyl-5-methylphenol. The p-cymene diagram shows a benzene ring with methyl and isopropyl groups opposite each other.
The report shows the GC/MS instrument on a lab bench. It used a 30 m DB-1 column with an inner diameter of 0.25 mm and a stationary phase thickness of 0.25 μm. The oven held at 40 °C for 15 minutes and then rose to 230 °C at 4 °C per minute. Helium flowed at 0.5 mL per minute. The report lists carrier gas pressure as 69481 psi. The analysis took 62.5 minutes and used a split ratio of 1 to 500.
| Compound | Retention index | Percentage |
|---|---|---|
| α-Tujene | 16.85 | 0.2 |
| α-Pinene | 17.30 | 0.3 |
| Camphene | 18.11 | 0.2 |
| β-Pinene | 19.67 | 0.48 |
| 3-Octanone | 20.06 | 0.71 |
| β-Myrcene | 20.13 | 1.00 |
| α-Terpinen | 21.73 | 0.44 |
| p-Cymene | 22.48 | 6.34 |
| Limonene | 22.65 | 0.99 |
| 1,8-Cineol | 22.75 | 1.03 |
| γ-Terpinene | 24.11 | 0.33 |
| Sabinene | 25.82 | 0.27 |
| Linalool | 26.39 | 1.08 |
| Borneol | 29.90 | 0.29 |
| Terpinen-4-ol | 30.42 | 0.80 |
| α-Terpineol | 31.35 | 1.65 |
| Thymol methyl ether | 33.02 | 0.41 |
| Carvacrol methyl ether | 33.36 | 2.85 |
| Thymol | 35.96 | 21.85 |
| Carvacrol | 36.25 | 50.22 |
| β-Caryophyllene | 42.01 | 1.85 |
| Aromadendrene | 42.30 | 1.40 |
| β-Humulene | 45.05 | 1.00 |
| Spathulenole | 48.56 | 0.45 |
| Caryophyllene oxide | 48.81 | 1.20 |
| Aromadendrene | 42.38 | 1.40 |
| Total reported | 98.74 | |
Flaxseed oil analysis
The GC/MS used a 30 m DB-WAX column with an inner diameter of 0.25 mm and a stationary phase thickness of 0.25 μm. The oven held at 65 °C for 12 minutes and then rose to 235 °C. Helium flowed at 0.5 mL per minute at a carrier gas pressure of 0.69481 psi. Analysis took 40 minutes and used a split ratio of 1 to 500.
The report describes a two-stage experiment. In the first stage, the team selected pure flaxseed oil and a sample placed intermittently in the oven for 12 days. The team aimed to identify the major components and track how saturated fatty acids changed under accelerated oxidation. GC/MS identified six major fatty acids. Each measurement was repeated three times. The results table labels the heated sample as oil after 20 days in the oven.
| Fatty acid | Pure oil | After 20 days in the oven |
|---|---|---|
| Palmitic acid (C16:0, saturated) | 3.86 | 3.58 |
| Stearic acid (C18:0, saturated) | 2.48 | 2.32 |
| Arachidic acid (C20:0, saturated) | 0.13 | 0.13 |
| Oleic acid (C18:1, monounsaturated) | 16.03 | 14.00 |
| Linoleic acid (C18:2, polyunsaturated) | 15.94 | 13.99 |
| C18:3 (polyunsaturated, labeled linoleic acid in the report) | 59.05 | 56.50 |
The report attributes the small changes in saturated fatty acids to their lack of double or triple bonds. It interprets the larger drops in unsaturated fatty acids as evidence of oxidation and changes in the structure of their double bonds.
Maryam Sanjari, head of the group, prepared the report with the team on May 3, 2022.