Anti-oxidant activity: Teixeira et al. compared the anti-oxidant activities of OEO and three types of OE (i.e. hot water, cold water, and ethanolic) using three different assays – ferric reducing anti-oxidant power (FRAP), reducing power of Fe³⁺ to Fe²⁺, and DPPH assay14. The chemical constituents of the OEO were characterized by GC-MS and reported the most abundant compounds being carvacrol (14.5%), β-fenchyl alcohol (12.8%), and thymol (12.6%). However, the chemical compositions of the extracts were not reported. FRAP analysis showed OEO (38.5 µmol Fe²⁺/g) and cold water OE (37.7 µmol Fe²⁺/g) having the highest anti-oxidant power, followed by hot water (27.9 µmol Fe²⁺/g) and ethanolic (12.6 µmol Fe²⁺/g). In terms of reducing power, hot water OE (621.7 µmol ascorbic acid/g) was the highest, followed by ethanolic (232.7 µmol ascorbic acid/g), cold water (203.3 µmol ascorbic acid/g), and OEO (74.5 µmol ascorbic acid/g). DPPH assay followed the same trend as the reducing power analysis with EC₅₀ being 25.1 µg/mL for hot water, 64.1 µg/mL for ethanolic, 144.3 µg/mL for cold water, and 1509.1 µg/mL for OEO. The researchers concluded that hot water OE had the highest anti-oxidant activity, followed by ethanolic, cold water, and OEO, respectively. Interestingly, the results of this study did not correlate total phenolic content with anti-oxidant activity, suggesting that other phytochemicals were contributing to the anti-oxidant activity of oregano.

Anti-microbial activity: In the same study, Teixeira et al. also tested the anti-bacterial activity of hot water, cold water, and ethanolic OE and OEO against Brochothrixthermosphacta, Escherichia coli, L. innocua, Listeria monocytogenes, Pseudomonas putida, Salmonella typhimurium, and Shewanellaputrefaciens14. Anti-bacterial activity was tested by paper disc diffusion test and growth in liquid medium. Neither the hot or cold water extracts had any significant inhibitory properties against the growth of any bacteria. Ethanolic OE demonstrated moderate activity against S. typhimurium, E. coli, L. innocua, and L. monocytogenes, resulting in MICs of 13.9 mg/mL, 6.9 mg/mL, 13.9 mg/mL, and 6.9 mg/mL, respectively. OEO displayed strong activity against all bacterial strains, with the strongest against P. putida at 0.4 mg/mL and the weakest against S. putrefaciens. Ethanolic OE and OEO at the calculated MICs reduced bacterial growth in liquid broth dilution of the species susceptible to treatment in the disc diffusion experiment. Coccimiglio et al. tested the anti-bacterial activity of an ethanolic OE against a panel of various gram-negative and gram-positive bacteria using the agar dilution method30. The researchers calculated MICs for OE against all strains of bacteria, and the study noted that OE had the strongest effect against non-mucoid isolates of Pseudomonas aeruginosa (MIC = 6.3 µg/mL), that are more susceptible to immune responses and drug treatment than mucoid isolates. Hernández-Hernández et al. tested the anti-bacterial activity of both free and microencapsulated OEO by disc diffusion method31. Microencapsulation was performed by adding OEO to a modified starch dispersion and homogenizing by ultra-centrifugation. The oils were tested against Bronchothrixthermosphacta, Lactobacillus plantarum, Pseudomonas fragi, Salmonella sp., and Micrococcus luteus. The study showed that free OEO at 25% w/v possessed activity against all species except for L. plantarum, while microencapsulated OEO showed no activity against L. plantarum and Salmonella sp. and was less effective than free OEO against all other species.

Oregano has been evaluated for its anti-oxidant and anti-microbial properties with special application to food preservation. One application of oregano in food preservation is Cypriot pastrami, a type of sun-dried meat product, also known as samarella32. The more modern way of making samarella is to take the meat of a goat or sheep and dry the meat using the sun. Once dry, the meat is washed and sprinkled with oregano. A second application of OEO as a food preservative includes that use of additive-free meats that have been dried and cured. This approach has been recognized in the European Union where essential oils are considered safe food additives at concentrations <2 mg/kg body weight/day33.

Several studies evaluated the effectiveness of food preservation using OE and OEO. Aqueous OE was shown to significantly reduce microbial growth and TBARS values in raw beef samples over a 15 day period compared to controls, although the strongest effect was observed in both experiments when combined with cinnamon and clove extracts34. In a study of anti-oxidant herbs added to animal fat, methanolic OE was determined to have anti-oxidant capacity of 13.59 ± 0.87 nmol equivalent of Vitamin E per gram of fat at a concentration of 5 µg extract per g fat, which was more than double the control group35. The major phytochemicals detected in this study were rosmarinic acid (30.03 mg/g) and carvacrol (16.29 mg/g). OEO prepared by an oil-in-water nanoemulsion inhibited growth of L. monocytogenes, S. Typhimurium, and E. coli on commercial iceberg and romaine lettuce leaves at concentrations of 0.05% and 0.1%, with the latter being the most effective36.

Another strategy of food preservation is to incorporate a combination of extracts to enhance preservation capabilities. Oregano tested in combination with other natural extracts has been reported to enhance its preservative qualities. Chitosan (1%) was found to be effective with OEO (2-4%) in pork meat at inhibiting bacterial growth, protecting from lipid oxidation, and extending shelf-life with minimal effect on sensory qualities37. OEO was tested with caprylic acid at 0.2% and 0.5%, respectively, and found to have a greater anti-microbial effect than either compound alone in minced beef38. A combination of OEO and REO inhibited growth of naturally occurring fungi in table grapes and preserved the sensory and physical (appearance, firmness) qualities during storage39. In a study using cooked chicken, OEO in combination with sage essential oil and honey reduced the amount of lipid oxidation after 48 and 96h as measured by TBARS40. These studies suggest that oregano can be used alone, however, some evidence suggests a combination of other herbs may enhanced food preservation properties.

Another benefit of phytochemicals in food preservation is that they may also improve gastrointestinal health (Table 2). Both OE and OEO have been shown to exhibit anti-inflammatory effects in vivo by suppression of TNF-α expression15^, 41. In mice with streptozotocin-induced diabetes, an ethyl acetate OE compared to control mice. This study found that OE reduced the number of TH1 and TH17 cells and activity measured by expression of the cytokines IFN-γ and IL-17, and both of these results reached statistical significance (P < 0.05)4. Treated mice also had a lower population of pro-inflammatory macrophage F4/80⁺ cells in the peritoneum while also decreasing the expression of IL-6 and IL-1β (P < 0.05), further suggesting that the ethyl acetate OE has anti-inflammatory properties in vivo.

A study by Bukovská et al. investigated the effects combined oregano and thyme essential oils in a colitis mouse model induced by trinitrobenzene sulphonic acid (TNBS) (120 mg/kg in 50% ethanol)42. The oregano and thyme oils were combined at different concentrations and given in the feed for 6 days prior to TNBS administration. The 0.1% oregano and 0.2% thyme combination treatment also had the highest prevention of macroscopic colonic damage (P < 0.05) and also the most recovery in body weight (not statistically significant). This dose also decreased both the mRNA and protein the expression of pro-inflammatory cytokines IL-6 and IL-1β (P < 0.01). The investigators found that oregano and thyme at 0.1% and 0.2%, respectively, had the highest decrease of mortality rate at 33.3% compared to 53.3% in the TNBS-only group, although this result was not statistically significant. These studies suggest that oregano may decrease inflammatory markers in vivo while improving gastrointestinal health.