3.1. Components of Rosemary essential oil
REO is a transparent yellowish liquid with unique aroma characteristics, and have a specific gravity of 0.912 g/mL. Its yield approximately 2.48% ± 0.11% (w/w), which is marginally higher than the average yield of 2.10% reported for most rosemary from various geographical regions [9].
A total of 33 components of REO were detected according to GC–MS analysis, and the chemical components are shown in Table 1. The most abundant compounds of REO were 1.8-Cineole (54.05%), alpha-Pinene (20.67%), borneol (5.73%), 1,7,7-trimethyl-bicyclo[2.2.1]heptane (4.99%), menthol (3.34%), D-camphor (2.04%), D-limonene (1.75%), and pulegone (1.44%). These constituents differed from the essential oil detailed by Pellegrini et al [10], who determined the principal compound of REO as camphor (22%) followed by α-pinene (17%), eucalyptol (16%), and borneol (12%). The components differed slightly from those reported by Jordán et al [11], which camphor was reported as the principal compound (24–36%) followed by eucalyptol (19–23%), and myrcene (9–15%). The most common compounds in REO were D-camphor (63.97%), 1,8-cineole (11.52%) and α-pinene (10.08%) [12]. Agreeably, the composition and amount of REO in different plant species were subject to the variation based on growing conditions, fertilisation practices, harvesting season and phenological factors [13].
Table 1
Components and content of REO
| NOa. | Rt (min) | Compound name | CAS number | Contentb. |
| 1 | 7.372 | γ-Terpinene | 99-85-4 | 0.46% |
| 2 | 8.555 | β-Pinene | 18172-67-3 | 0.54% |
| 3 | 8.684 | Myrcene | 123-35-3 | 0.16% |
| 4 | 9.855 | D-Limonene | 5989-27-5 | 1.75% |
| 5 | 10.057 | D-Camphor | 464-49-3 | 2.04% |
| 6 | 10.878 | p-Cymene | 99-87-6 | 0.15% |
| 7 | 12.307 | 3-Octanol | 589-98-0 | 0.17% |
| 8 | 13.676 | 1-Hexanol, 2-ethyl- | 104-76-7 | 0.25% |
| 9 | 13.938 | 1R-α-Pinene | 7785-70-8 | 20.67% |
| 10 | 14.014 | Benzofuran, 4,5,6,7-tetrahydro-3,6-dimethyl- | 494-90-6 | 0.36% |
| 11 | 14.269 | Borneol | 464-43-7 | 5.73% |
| 12 | 14.406 | Linalool | 78-70-6 | 0.08% |
| 13 | 14.547 | verbenone | 1196-01-6 | 0.14% |
| 14 | 14.626 | Terpinen-4-ol | 562-74-3 | 0.14% |
| 15 | 15.049 | Bicyclo[2.2.1]heptane, 1,7,7-trimethyl- | 464-15-3 | 4.99% |
| 16 | 15.337 | dl-Menthol | 89-78-1 | 3.34% |
| 17 | 15.406 | α-Terpineol | 98-55-5 | 0.10% |
| 18 | 15.471 | Terpinen-4-ol | 562-74-3 | 0.34% |
| 19 | 15.645 | Elemene | 515-13-9 | 0.32% |
| 20 | 16.033 | 1.8-Cineole | 470-82-6 | 54.05% |
| 21 | 16.197 | α-Caryophyllene | 6753-98-6 | 0.18% |
| 22 | 16.277 | Pulegone | 89-82-7 | 1.44% |
| 23 | 16.546 | α-Terpineol | 98-55-5 | 0.28% |
| 24 | 16.619 | β- Caryophyllene | 87-44-5 | 0.07% |
| 25 | 16.695 | 1,3,6-Octatriene, 3,7-dimethyl-, (Z)- | 3338-55-4 | 0.07% |
| 26 | 17.086 | Espatulenol | 6750-60-3 | 0.18% |
| 27 | 17.204 | Piperitone | 89-81-6 | 0.68% |
| 28 | 17.372 | (+)-Cyclosativene | 22469-52-9 | 0.05% |
| 29 | 19.603 | Globulol | 489-41-8 | 0.15% |
| 30 | 19.702 | Caryophyllene oxide | 1139-30-6 | 0.69% |
| 31 | 20.101 | γ-Muurolene | 30021-74-0 | 0.05% |
| 32 | 21.721 | α-ylangene | 14912-44-8 | 0.21% |
| 33 | 22.002 | Pinocarvone | 30460-92-5 | 0.20% |
| a The numbering refers to the elution order; b The values (relative peak area percent) represent averages of three determinations. |
To the best of our knowledge, our study suggested that REO from Yuzhou County with high content of 1.8-cineole (54.05%), which differed from that found in other geographical regions. In accordance with the principal compound of the REO in most cases, which present in varying proportions depending on the vegetative stage and bioclimatic conditions in different region [9].
3.2. REO Scavenged Free Radicals in DPPH, ABTS, OH− and O2− Assay
Since REO has been widely applied in oxidative-related foods and biological systems [9], the determination of antioxidant potential of ERO is of great importance. DPPH comprises stable free radical molecules, presenting as a dark purple powder that becomes colorless upon antioxidant reduction. ABTS solution acquires a bold blue-green tint when potassium persulfate is present, and becomes a paler green upon reduction due to hydrogen-donating antioxidants [14]. Superoxide (O2−) is the primary ROS produced through oxygen (O2) molecule reduction by NADPH oxidase during the electron transport chain in mitochondria. This leads to ROS accumulation, including hydrogen peroxide (H2O2), produced by superoxide dismutase. Excess of H2O2 under oxidative stress, give rise to toxic ROS such as hydroxyl ions (HO−), which are produced through catalytic processes in the presence of reduced metals [15].
In this study, results indicated that REO presented antioxidant activity characterized with high reducing power in free radicals scavenging. REO at 62.5–1000 µg/mL exerted dose-dependent antioxidant activity, with the inhibition percentage of 29.23%, 60.93%, 83.77%, 88.4%, and 91.2% of control. In comparison, the scavenging capacities of positive control VC, BHA and BTH (200 µg/mL) were 89.90%, 60.77% and 29.55%, respectively (Fig. 1A). A similar trend was observed in ABTS free radicals scavenging, where REO exerted a similar antioxidative activity with ascorbic acid, followed by BHA and BTH (Fig. 1B). Regarding the OH− and O2− scavenging data, it was noteworthy that the antioxidant capacity of REO was similar to that of VC, but significantly higher than synthe tic antioxidants BTH and BHA (Fig. 1C-D). Specifically, these results agree with those of Wang et al [16], where Rosmarinus officinalis essential oil, 1,8-cineole, β-pinene and α-pinene exerted DPPH radical scavenging activity with an IC50 value of 2.04%, 4.05%, 2.56% and2.28% (v/v), in comparison to the positive control BHT at an IC50 of 2.25% (v/v). Similarly, the REO assessed DPPH assay with IC50 value of 77.6 µL/mL, but vitamin E as positive control with IC50 value of 25.3 µg/mL [17], and REO against DPPH, ABTS, and FRAP radical showed IC50 values of 15.10 mg/mL, 2.21 mg/mL, and 22.84 mg/mL [6].
Comparing with the published literature, this study evaluated the superoxide and hydroxyl ions radicals scavenging ability of REO for the first time. Meanwhile, we also found there is very few reports on the antioxidant mechanism of REO, thus its antioxidative mechanism were uncovered in A549 cells in further study.
3.3. Effect of REO on A549 cell viability
Lung carcinoma-derived A549 cell line typically used as a model for pulmonary oxidative stress study. The effect of REO on cellular viability was examined to determine non-cytotoxic concentration. Initially, A549 cells were incubated with REO (12.5, 25, 50 µg/mL), and no discernible effect on cell viability was observed when compared to control cells. Significantly, REO at 100 µg/mL resulted in a notable reduction in cell viability, corresponding to its antitumor activity in another reported literature [18]. Thus, the nontoxic range of concentrations 12.5–50 µg/mL were selected for later use (Fig. 2A).
Phenomenologically, the protective activity of REO against pulmonary oxidative stress was examined in H2O2-induced A549 cells. As shown in Fig. 2B, exposure to H2O2 at 100 µM resulted in considerable cytotoxicity, resulting in a 42% decrease when compared to control group. However, cell viability restored remarkably with 11–35% following REO pretreatment compared to H2O2-treated cells, indicating that REO exhibited a dose-dependent cytoprotective activity within the range of 12.5–50 µg/mL. Meanwhile, the present study were agree with the results of Razavi-Azarkhiavi, who have demonstrated that extracts of rosemary treatment inhibited H2O2 damage in human lymphocytes strongly [19].
3.4. Inhibition of ROS production in H2O2-treated A549 cells
ROS, comprising H2O2, HO−, O2, have been found specifically target macromolecules (nucleic acids, lipids, proteins), leading to impaired cellular structure and functions [20]. Excessive ROS production disturbs the oxidative and antioxidative system, leading to harm to healthy tissue cells [21].
To substantiate the antioxidative properties of REO, their ability to scavenge cellular ROS was assessed. The levels of intracellular ROS were assessed following DCFH-DA reaction principles, which resulting in fluorescent DCF corresponding to ROS levels. As shown in Fig. 3A, cells with ROS treatment over the range of 12.5, 25, 50 µg/mL showed no significant alteration in ROS levels when compared to control group. Whereas REO at 100 µg/mL caused a significant induction of ROS in response to the reduction of cell viability. However, upon exposure to H2O2, cells caused a pronounced induction of ROS beyond 2-folds, which was in agreement with previous report [22]. However, pretreatment with different range of REO effectively counteracted the overproduction of ROS induced by H2O2. Specifically, ROS decreased by 48.3% with REO treatment at 50 µg/mL in H2O2-induced A549 cells (Fig. 3B). These findings indicated that REO could alleviate oxidative stress in H2O2-induced A549 cells, whilst the fluorescence images of intracellular ROS effectively validated this observation (Fig. 3C). The aforementioned results highlighted the prospective use of REO against oxidative-related pulmonary disorders.
3.5. Effect of REO on enzymatic and non-enzymatic antioxidant parameters in A549 cells
Oxidative stress occurs when oxidants overwhelm antioxidant system, potentially resulting in DNA damage and lipid peroxidation. The intracellular enzymatic antioxidant system, which is composed of a variety of enzymes including CAT and SOD, execute numerous catalytic reactions, neutralize free radicals and prevent the excessive production of ROS [23]. SOD facilitates the conversion of superoxide anions into hydrogen peroxide (H2O2). CAT, efficiently scavenges H2O2 and catalyses the conversion of H2O2 to H2O and O2 [24]. To assess the protective effects of REO, the spectrophotometric method was employed to determine the antioxidant capacity of SOD and CAT. As shown in Fig. 4A-B, we found that H2O2 exposure reduced cellular enzyme activity with CAT, SOD declination remarkably, while REO treatment reversed this trend dose-dependently, even back to their ordinary levels when compared to control. This can be attributed to the phytochemical composition of REO, including 1.8-cineole (54.05%) as the main ingredient, which is known to scavenge ROS [16].
Oxidative stress is elicited by an imbalance between the generation of non-enzymatic antioxidants and the overproduction of ROS [14]. The effect of REO on non-enzymatic antioxidant markers were evaluated by measuring the content of MDA, GSH, and GSH/GSSG in A549 cells. MDA considered a presumptive biomarker for lipid peroxidation, has been a well-established monitor of lipid peroxidation related to oxidative stress [24]. In present study, the level of MDA increased by nearly 2 fold significantly after H2O2 exposure. Contrariwise, the pretreatment with REO showed statistically significant reduction with H2O2-stimulation in a dose-dependent manner. As for GSH, an abundant cellular thiol, plays a crucial role as antioxidant, enzyme cofactor and modulator in oxidative stress status, accelerats the reduction of hydrogen peroxide and hydroperoxides [25]. As depicted in Fig. 4C-E, cells exposure to H2O2 experienced a significant decrease in survival, along with the declination of GSH and overproduction of GSSG. Corresponding to this alternation, the level of GSH/GSSG were elevated significantly, but no obvious changes with GSH + GSSG level in A549 cells (Fig. 4F).
All these results above, coincided with the existence of REO enhanced cellular enzymes SOD and CAT, along with the antioxidant GSH, are crucial in the conversion of H2O2 into H2O [14]. Meanwhile, the observation of enzymatic and non-enzymatic antioxidants alternations in this study, were in accordance with the anti-oxidative potential of REO which prevent CCl4-induced oxidative stress by regulating MDA, GSH, CAT, GSH levels in previous literature [17].
3.6. Effects of REO on Nrf2 signaling pathway in A549 cells
Nrf2 acts as a transcription factor, governs the cellular defense against toxic and oxidative insults, and responsible for the regulation of detoxifying/antioxidant enzymes including SOD, CAT, GPX, protect cells from oxidative stress eventually [26]. Under normal circumstances, Nrf2 remains quiescent in the cytoplasm by binding to Keap1. Upon exposure to oxidative stress, Nrf2 dissociates from Keap1 and relocates to the nucleus triggering the induction of antioxidant/detoxification enzymes including SOD, CAT, NQO1, and HO-1 [27].
Massive evidences have linked anti-oxidative stress with plant-derived bioactive phytochemicals via Nrf2 signaling pathway, which is the vital antioxidant mediator in normalizing ROS level, inhibiting DNA damage, and telomere shortening [28]. To elucidate the mechanism accountable for the antioxidant action of REO, western blot analysis were conducted to determine the protein levels of Nrf2 and its target genes NQO-1, HO-1. As depicted in Fig. 5, there was a minor increase in the protein expression levels of Nrf2 and NQO1 after H2O2 treatment, possibly due to an adaptive response to the oxidative induction in accordance with the reports [29]. Nevertheless, compared with the H2O2-treated group, an appreciable enhancement in protein expression levels of Nrf2, NQO1 and HO-1 dose-dependently, which indicates the amplified activation of Nrf2 signaling pathway by REO treatment. Therefore, REO's antioxidative effect may be partially due to its activation of Nrf2 signaling pathway, and result in an enhancement of detoxification and antioxidant.
3.7. Molecular Docking
The Nrf2-Keap1 pathway is a pivotal signaling pathway involved in combating oxidative stress, and boosting the cellular antioxidant capacity [26]. Inhibitors of Keap1 have the potential to interrupt the covalent interaction between Nrf2 and Keap1. This disruption unleashes the transcriptional machinery of Nrf2, which coordinate cellular antioxidant/detoxification processes, safeguard cells against oxidative stress-induced disorders [30]. Although the literature extensively demonstrates the antioxidant properties of REO, certain exclusively detected volatile compounds within the REO likely have significantly contributed to its notable antioxidant capacity identified in this study. Considering the antioxidant activity and its chemical composition results described above, it should also be noted that the volatile aroma components in REO, including 1.8-cineole (54.05%) are proverbial to exhibit antioxidant biological activity [16]. Regarding the underlying mechanism of REO as Nrf2 activator, potential attachment between 1.8-cineole monomers and the Kelch domain of KEAP1 (PDB ID: 4IQK) were analyzed using the proposed molecular docking model.
Molecular docking based on virtual screening of 1,8-cineole-binding protein the Kelch domain of KEAP1, determined the binding affinity values of the bioactive ligands against the active sites of protein target by AutoDock Vina software. The lowest Gibbs free binding energy estimated as ∆G = -5.5 kcal/mol (< 0 kcal/mol), further confirmed the stability of 1.8-cineole docked into the Kelch domain of KEAP1. As shown in Fig. 6A, the key amino acids involved in the molecular interactions between 1.8-cineole and the Kelch domain of KEAP1, formed alkyl interactions with ALA366, VAL606 and ILE559 by PYMOL software. As shown in Fig. 6B, with Schrödinger molecular modeling software, 1.8-cineole formed alkyl interactions with KEAP1 protein residues ALA366, VAL512 and VAL606, formed Van der Waal’s with GLY417, VAL465, VAL418, GLY464, LEU365, ALA510, GLY511, ILE559, GLY605, GLY558, LEU557, VAL604, GLY367. All these results indicated 1.8-cineole interacted with the Kelch domain of KEAP1 stably through forming alkyl interactions with ALA366, VAL606. Molecular docking demonstrates that 1.8-cineole showed a promising competitive interaction with the Keap1 protein. The outcomes demonstrated that 1.8-cineole may be contributed to REO's antioxidant ability via the activation of Nrf2 pathway.
Indeed, it is very difficult to attribute the antioxidant effect of a total essential oil to one or a few active principles. With our ongoing study, the cooperating results of major compounds, also minor compounds contribute to the REO’s antioxidant activity will be further explored.