what essential oils are high in phenols

With the aim to develop essential oil (EO) multi-antioxidant systems, combinatorial interactions of selected phenol and terpene-rich EOs (from Pimento Berry, Ceylon Cinnamon, Clove, Sage, White thyme; Oregano) enriched with individual polyphenols, crude plant extracts, and mixtures of their major polyphenols were investigated using single electron transfer (SET)-based DPPH and hydrogen atom transfer (HAT)-based ORAC assays. Polyphenols that enriched Eos the most favorably were rosmarinic acid (IC50 of 0.0891–0.1448 mg enriched EO/mg DPPH; 5772–17,879 µmol TE/g enriched EO) and quercetin (IC50 of 0.0682–0.1060 mg enriched EO/mg DPPH; Trolox Equivalents (TE) of 9776–14,567µmol /g enriched EO), whereas p-coumaric acid (IC50 of 0.1865–1.1424 mg enriched EO/mg DPPH; 7451.00–11,588 µmol TE/g enriched EO) and rutin hydrate (IC50 of 0.1140–0.3112 mg enriched EO/mg DPPH; 2298–6227 µmol TE/g enriched EO) were the least favorable. Enrichments with polyphenol mixes and crude extracts exhibited synergistic and additive effects in the SET-based DPPH assay. In the HAT-based ORAC assay, EO enrichments with crude extracts exhibited more additive effects, as well as less antagonistic effects, than enrichments with their major polyphenol mixes, revealing the significant contributions of minor compounds. EOs enriched with crude green tea and apple extracts exhibited synergistic or additive effects, whereas EOs enriched with grape seed and rosemary extracts exhibited equal antagonistic effects. Predictive models were developed to explain the variability between the observed and predicted antioxidant activities of enriched EOs.

To limit the oxidative degradation of lipids during storage and distribution, the food industry has employed the use of synthetic antioxidants, which have since fallen under scrutiny. Indeed, synthetic antioxidants, such as butylated hydroxyanisole, butylated hydroxytoluene, tert-butylhydroquinone, and propyl gallate, are suspected to have carcinogenic, mutagenic, and teratogenic effects after chronic use [1]. With increasing concerns regarding the safety of these preservatives as well as increasing consumer demands for more natural products, the industry is now considering essential oils (EOs) and plant extracts as potential natural replacements. These alternatives are naturally occurring phytochemicals that have shown a wide range of biological activities and physicochemical properties. Indeed, several studies have documented the antioxidant and antimicrobial activities of EOs, plant extracts and their components [1,2].

EOs are composed of volatile compounds that contribute to their antimicrobial and antioxidant properties such as phenols, allylic alcohols, and selected monoterpenes [3,4]. The chemical profile of EOs is often dominated by two or three major compounds, but their antioxidant activity may also rely on synergistic interactions in situ with the EO’s minor components [5]. In our previous study, correlation analysis of the chemical profiles of 38 EOs showed concurrent abundance of some compounds; for instance, monoterpenes and alcohols were concurrently present, and phenol-rich EOs had a higher content of sesquiterpenes [6]. The antioxidant activity of phenols paired with esters and alcohols showed synergistic effects when measured with the ORAC assay, which is a hydrogen atom transfer-based reaction. When phenols were paired with monoterpenes and ketones, antagonistic effects were observed when measuring the antioxidant activity with the DPPH Assay, whose reaction is driven by single electron transfer [6].

Blends of antioxidants can have an additive, synergistic, antagonistic, or indifferent effect [5,7]. In an additive effect, the sum of the individual components will be equal to the combined activity of the blend of antioxidants. When the combined effect of the blend of oils is greater than the sum of their individual components, the effects are synergistic. When antagonism occurs, the combined effect is less than the sum of their individual components. In the present study, combinations of EOs with individual major polyphenols, polyphenols mixes, or crude plant extracts were assessed for their antioxidant activities and combinatorial effects. To the best of our knowledge, no study so far has explored the interactive effects between these combinations. The EOs included pimento berry (HE-PIM-01), clove (HE-CLO-01), oregano (HE-ORI-03), white thyme (HE-THY-03), yellow sage (HE-SAU-01-02), and Ceylon cinnamon (HE-CAN-04), while the extracts included grape seed (EX-RAI-01), green tea (HE-THE-01), apple (EX-POM-04), and rosemary (EX-ROM-04). Two methods utilizing the abilities to jointly transfer electrons or hydrogen atoms were used—the DPPH (2,2′-diphenylpicryl hydrazyl free radical) assay and ORAC (Oxygen Radical Absorbance Capacity) assay. The characterization of interactions between EOs and individual major polyphenols, polyphenols mixes, or crude plant extracts will enable the development of efficient multi-antioxidant systems.

Five essential oils from different botanical sources, of very common use in the food industry as well as in the cosmetic and other industries [5], were chosen as representative prototypes of phenol-rich EOs, namely red thyme (Thymus vulgaris, L.), oregano (Origanum vulgare, L.), savory (Satureja hortensis, L.), which …

3. Antioxidant Capacity of EOs Enriched with Polyphenol Mixes

Aside from rosemary’s polyphenol mix, the IC50 of all unenriched polyphenol mixes were lower than their expected IC50, thus displaying antagonistic effects ( ). This could be due to flavonoid-flavonoid interactions, where H-bonding between flavonoids results in a decrease in the availability of -OH groups [18]. This, in turn, decreases the possibility of interacting with the DPPH radical, lowering the resulting antioxidant capacity. In the ORAC assay, all polyphenol mixes, except those with grape seed, showed synergistic effects. The ORAC value of a pair of flavonoids has been reported to significantly increase when a third flavonoid with a low reduction potential energy is added [19]. Additionally, mixtures of compounds with similar reduction potential energies have shown lower antioxidant activities [20] since the compounds drew electrons away without readily donating them to the AAPH radical. This could account for the antagonism within the grape seed mix. Although the ORAC assay is based on the transfer of a hydrogen atom, an electron is also transferred, which justifies the importance of analyzing the compounds’ reduction potentials.

All EO/grape seed mix enrichments showed synergistic effects in the DPPH assay ( ), except pimento berry (HE-PIM-01, 0.074 mg/mg DPPH), which showed additive effects. Similarly, all enrichments showed synergistic effects in the ORAC assay ( ), except that of oregano (HE-ORI-03, 1219.40 μmol TE/g sample), which exhibited antagonistic effects. All EOs containing eugenol (clove, HE-CLO-01; pimento berry, HE-PIM-01; Ceylon cinnamon, HE-CAN-01) showed similar IC50 and ORAC values, whereas those lacking it (Table S1) exhibited lower ORAC values. This suggests that the mixture’s epicatechin and catechin interacted with the eugenol present in the EOs. As previously mentioned, oregano (HE-ORI-03)/grape seed mix showed antagonistic effects in the ORAC assay, whereas all other enrichments exhibited synergistic effects. Therefore, despite the antagonistic effects within the grape seed polyphenol mix in situ ( ), enriching it with EOs ( ), except oregano (HE-ORI-03), overcomes the antagonism in the ORAC assay. Perhaps this antagonism arises from the large quantity of α-pinene (55%) present in the EO [6]. Therefore, in addition to the antagonism in the grape seed mix, the co-oxidizing activity of α-pinene further decreases the antioxidant activity.

EOs enriched with the green tea mix ( ) showed the lowest IC50 values among all enrichments and moderate to high ORAC values (6208.5–13,681.0 μmol TE/g sample). Pimento berry (HE-PIM-01)/green tea mix showed the highest overall antioxidant capacity (IC50 = 0.009 mg/mg DPPH; ORAC Value = 13,681.0 μmol TE/g sample). This pair’s synergistic effects and low IC50 value could be due to the contributions of eugenol and the EO’s minor components such as methyleugenol. Since clove oil (HE-CLO-01) contains more eugenol (93.5% versus 86.4%), its lower activity confirms that other compounds are influencing pimento berry’s (HE-PIM-01) activity [6]. The green tea mix showed low, antagonistic ORAC values when enriching oregano (HE-ORI-03) and yellow sage (HE-SAU-01-02), due to the lack of phenols in their chemical profiles and/or the presence of pro-oxidants, which can generate reactive species (hydrogen peroxide) that quench the fluorescein and decrease the antioxidant capacity [21].

EOs enriched with the apple polyphenol mix ( ) did not show antagonistic effects in the DPPH assay, however, there was antagonism in the ORAC assay when enriching oregano (HE-ORI-03, 1151.6 μmol TE/g sample), white thyme (HE-THY-03, 1156.4 μmol TE/g sample), and yellow sage (HE-SAU-01-02, 601.6 μmol TE/g sample), with the last enrichment exhibiting the poorest activity. This antagonism could have arisen from the oxidation of the primary antioxidant(s) by the synergist(s) or the regeneration of the latter by the former [7]. Since yellow sage (HE-SAU-01-02) exhibited low, antagonistic ORAC values when paired with chlorogenic acid and rutin ( ), perhaps both enrichments resulted in the oxidation/regeneration, and by combining them, any trace of effective antioxidant was practically lost. Additionally, the possible presence of pro-oxidant monoterpenes in yellow sage could have contributed to the antagonism. The enrichment of clove (HE-CLO-01) with the apple mix showed synergistic effects in both assays (IC50 = 0.013 mg/mg DPPH; ORAC Value = 12,720.5 μmol TE/g sample), whereas enrichments of pimento berry (HE-PIM-01) (IC50 = 0.104 mg/mg DPPH; ORAC value = 11,740.0 μmol TE/g sample) and Ceylon cinnamon (HE-CAN-04) (IC50 = 0.116 mg/mg DPPH; ORAC Value = 11,338.5 μmol TE/g sample) showed additive/synergistic and synergistic/additive effects in the DPPH/ORAC assays, respectively. When paired with the apple mix, these EOs exhibited ORAC values more than ten times greater than the other EOs, due to synergistic interactions with their eugenol. Indeed, the ORAC values increase with the EOs’ increasing eugenol contents (clove, HE-CLO-01, 93.5% > pimento berry, HE-PIM-01, 86.4% > Ceylon cinnamon, HE-CAN-04, 84.6%) [6].

All EOs paired with the rosemary polyphenol mix ( ) did not exhibit any antagonism in the DPPH assay. However, this enrichment yielded some of the highest IC50 values, although all were synergistic. The high absolute values of the IC50 of these enrichments could be attributed to the presence of p-coumaric acid (80% of the mix), which was shown to have a poor antioxidant activity on its own ( ), due to the absence of a catechol group in its structure. In the ORAC assay, enrichments of all EOs exhibited synergistic and additive effects, except those with yellow sage (HE-SAU-01-02, 8620.0 μmol TE/g sample). Pimento berry (HE-PIM-01) and Ceylon cinnamon (HE-CAN-04) EOs exhibited greater ORAC values (16,148.0 μmol TE/g sample, 18,477.00 μmol TE/g sample, respectively) than EOs lacking eugenol. However, the enrichment of clove EO (ORAC value = 12,482.5 μmol TE/g sample) resulted in additive effects, despite having a greater amount of eugenol (93.5% versus 86.4%, 84.6%, respectively), suggesting that interactions with minor oxygenated compounds, such as alcohols, contributed to their synergistic effects.

3. 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Assay

The DPPH assay was carried out according to the method reported by Brand-William et al. [8] with modifications. Varying concentrations of antioxidants were added to Tris-HCl buffer (450 µL, pH 7.4). DPPH (0.1 mM) was then added to all samples and incubated in the dark for 30 min at room temperature. In preliminary trials, 30 min was identified as the appropriate time at which the steady state of scavenging was achieved. Then, the absorbance was read at 517 nm using a Beckman spectrophotometer. Water was used as the blank. As the negative control, 10% methanol in Tris-HCl buffer was used. α-tocopherol in ethanol (2 mg/mL) was used as the positive control. The inhibition ratio (%) was calculated as follows: Inhibition ratio (%) = [(Ac − As)/Ac] × 100(1)

where Ac is the absorbance of the control; As is the absorbance of the sample.

The concentrations of antioxidants were plotted against the estimated inhibition ratio. The IC50 was defined as the concentration required to reduce DPPH by 50%. The IC50 was expressed as mg sample/mg DPPH.

Phenols Chemistry Functional Group | Essential Oil Chemistry

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