Comparison of Chemical Compositions and Antioxidant Activity of Essential Oils from Litsea Cubeba, Cinnamon, Anise, and Eucalyptus

The purpose of this study was to compare the antioxidant activity of litsea cubeba oil (LCO), cinnamon oil (CO), anise oil (AO), and eucalyptus oil (EUC) in vitro. The chemical compositions of the essential oils (EOs) were analyzed using gas chromatography-mass spectrometry (GC-MS). The antioxidant activity of the four EOs was evaluated through scavenging DPPH free radicals, chelating Fe2+, scavenging hydroxyl free radicals, and inhibiting yolk lipid peroxidation. The results showed that the major compounds found in LCO, CO, AO, and EUC are citral (64.29%), cinnamaldehyde (84.25%), anethole (78.51%), and 1,8-cineole (81.78%), respectively. The four EOs all had certain antioxidant activity. The ability to scavenge DPPH radical was ranked in the order of LCO > CO > AO > EUC. The hydroxyl radical scavenging ability was ranked in the order of EUC > CO > LCO > AO. The chelating Fe2+ capacity was ranked in the order of EUC > AO > CO > LCO. The yolk lipid peroxidation inhibition ability was ranked in the order of CO > AO > EUC > LCO. In different antioxidant activity assays, the antioxidant activity of the EOs was different. It was speculated that the total antioxidant activity of an EO may be the result of the joint action of different antioxidant capacities.


Introduction
In livestock production, intensive farming patterns and harmful environmental factors (e.g., bacteria) can lead to oxidative stress in animals. The overproduction of reactive oxygen species and an imbalance of antioxidant systems can cause oxidative stress [1]. Oxidative stress can destroy a variety of cellular components via DNA hydroxylation, protein denaturation, lipid peroxidation, and membrane rupture, which results in cell apoptosis and other cell death patterns [2,3]. Oxidative stress causes oxidative damage to the digestive tract, which, in turn, induces an inflammatory response in the organism. This ultimately leads to stunted growth, reduced immunity, and even death in the animal, which can seriously affect economic efficiency [4]. Eliminating and slowing down oxidative stress is a key step in protecting animals from injury. Some synthetic antioxidants, such as butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA), may have carcinogenic potential and pose a risk to animal health and product quality. Therefore, it is important to find natural green antioxidants to reduce oxidative stress in animals.
Essential oils (EOs) are aromatic and volatile mixtures composed of dozens to hundreds of compounds, among which monoterpenes, sesquiterpenes, and their oxygencontaining derivatives are the main components [2,3,5]. The antioxidative and antibacterial activities of EOs are derived from these components. The toxicity and side effects of EOs are low, meaning they can be widely used in animal husbandry [6]. As a natural antioxidant, EOs have been used in livestock and poultry production. Previous studies have reported that EOs reduce fat oxidation and improve the shelf-life of meat quality [7]. EOs can alleviate oxidative damage and inhibit the activation of NF-κB signaling pathways, thereby alleviating the damage of immune functions caused by oxidative stress [8,9]. Other studies have reported that EO supplementation increased the activity of antioxidant enzymes in the body and improved the growth performance and immune functions of animals [10]. Therefore, the antioxidant activity of EOs has important biological significance [11]. Some studies have indicated that Litsea cubeba (Lour.) Pers. oil (LCO), Cinnamomum cassia (L.) D. Don oil (CO), Pimpinella anisum L. oil (AO), and Eucalyptus spp. oil (EUC) have antibacterial, anti-inflammatory, and antioxidant activities, and these four EOs are widely used in the pharmaceutical and food industries in China [12][13][14][15]. Thus, the purpose of this study was to evaluate the chemical compositions of LCO, CO, AO, and EUC and compare their antioxidant activity via a DPPH radical scavenging assay, Fe 2+ chelating assay, hydroxyl radical scavenging assay, and yolk lipid peroxidation assay. This study provides a theoretical basis for the application of EOs as a natural antioxidant in animal husbandry.

Effect of EOs on Fe 2+ Chelating Ability
Fe 2+ chelating capacity compared to all EOs at all of the concentrations. At the concentration of 12 mg/mL, the chelation rate of VC to Fe 2+ was 82.59%, and the chelation rates of AO and EUC to Fe 2+ were 70.15% and 75.39%, respectively, reaching 84.94% and 91.28% of VC.

Effect of EOs on Hydroxyl Radical Scavenging Rates
The effects of the EO type and the EO concentration on the scavenging rates of hydroxyl radicals are shown in Figure 3. EO type (E) and EO concentration (C) had significant effects on the hydroxyl radical scavenging rate (PE < 0.01, PC < 0.01), and the interactions between EO type and concentration (E × C) were significant (PE × PC < 0.01). The hydroxyl radical scavenging rate quadratically increased (PQ < 0.01) with an increase in the concentration of LCO, CO, AO, and EUC. In comparing rates among EOs, the hydroxyl radical scavenging rate differed (p < 0.05) at five different EO concentration levels (1,4,8,16, and 32 mg/mL) (EUC > CO > LCO > AO). The VC had a consistently greater (p < 0.05) hydroxyl radical scavenging rate compared to all EOs at all of the concentrations. At the concentration of 32 mg/mL, the hydroxyl radical scavenging rate of VC was 85.66%, and the hydroxyl radical scavenging rate of EUC was 75.05%, which reached more than 80% of the level of VC.

Effect of EOs on Hydroxyl Radical Scavenging Rates
The effects of the EO type and the EO concentration on the scavenging rates of hydroxyl radicals are shown in Figure 3. EO type (E) and EO concentration (C) had significant effects on the hydroxyl radical scavenging rate (p E < 0.01, p C < 0.01), and the interactions between EO type and concentration (E × C) were significant (p E × p C < 0.01). The hydroxyl radical scavenging rate quadratically increased (p Q < 0.01) with an increase in the concentration of LCO, CO, AO, and EUC. In comparing rates among EOs, the hydroxyl radical scavenging rate differed (p < 0.05) at five different EO concentration levels (1,4,8,16, and 32 mg/mL) (EUC > CO > LCO > AO). The VC had a consistently greater (p < 0.05) hydroxyl radical scavenging rate compared to all EOs at all of the concentrations. At the concentration of 32 mg/mL, the hydroxyl radical scavenging rate of VC was 85.66%, and the hydroxyl radical scavenging rate of EUC was 75.05%, which reached more than 80% of the level of VC. Figure 4 shows the effect of the EO type and the EO concentration on the lipid peroxidation inhibition rate of yolk. EO type (E) and EO concentration (C) had significant effects on the lipid peroxidation inhibition rate of yolk (p E < 0.01, p C < 0.01), and the interactions of EO type and concentration (E × C) were significant (p E × p C < 0.01). The lipid peroxidation inhibition rate of yolk quadratically increased (p Q < 0.01) with increasing the concentration of LCO, CO, AO and EUC. In comparing among EOs, the lipid peroxidation inhibition rate of yolk differed (p < 0.05) at a concentration of 1.25, 5, 10, 20 mg/mL (PG > CO > AO > EUC > LCO) and at the concentration of 4 mg/mL (CO > AO, EUC > LCO). At the concentration of 2.5 mg/mL, the inhibition rate of PG on yolk lipid peroxidation was 90.41%, and that of CO was 86.42%, reaching 95.59% of PG.   Figure 4 shows the effect of the EO type and the EO concentration on the lipid peroxidation inhibition rate of yolk. EO type (E) and EO concentration (C) had significant effects on the lipid peroxidation inhibition rate of yolk (PE < 0.01, PC < 0.01), and the interactions of EO type and concentration (E × C) were significant (PE × PC < 0.01). The lipid peroxidation inhibition rate of yolk quadratically increased (PQ < 0.01) with increasing the concentration of LCO, CO, AO and EUC. In comparing among EOs, the lipid peroxidation inhibition rate of yolk differed (p < 0.05) at a concentration of 1.25, 5, 10, 20 mg/mL (PG > CO > AO > EUC > LCO) and at the concentration of 4 mg/mL (CO > AO, EUC > LCO). At the concentration of 2.5 mg/mL, the inhibition rate of PG on yolk lipid peroxidation was 90.41%, and that of CO was 86.42%, reaching 95.59% of PG.

Discussion
A reduction in antioxidant defense mechanisms and the production of free radicals can lead to oxidative stress, which damages proteins, lipids, and enzymes [16]. Free radicals exist in the body widely, and they are the main cause of lipid peroxidation and the root of oxidative damage to macromolecular substances [17]. Some studies have shown

Discussion
A reduction in antioxidant defense mechanisms and the production of free radicals can lead to oxidative stress, which damages proteins, lipids, and enzymes [16]. Free radicals exist in the body widely, and they are the main cause of lipid peroxidation and the root of oxidative damage to macromolecular substances [17]. Some studies have shown that oxidative stress can cause inflammation and other diseases by producing excess free radicals to mediate oxidative damage to cells [18]. Antioxidants can scavenge free radicals and stop free radical reactions. As a result of the carcinogenicity and hepatotoxicity of synthetic antioxidants (such as BHA and BHT), EOs have attracted increasing attention as green natural antioxidants. Some studies have found that EOs can prevent free radical chain reactions and delay the lipid peroxidation process [6,19,20].
EOs are complex mixtures of different volatile and non-volatile components [21]. Different EOs have different chemical compositions. In our study, it was found that the active components of the four studied EOs were very complex and varied greatly. Citral, the main component of LCO, and cinnamic aldehyde, the main component of CO, are aldehydes. Anisole, the main component of AO, and 1, 8-eudinolein, the main component of EUC, are terpenoid compounds. The reasons for the differences in EO active components may be related to variety, heredity, maturity stage, culture conditions, etc. [22].
It has been reported that EOs can reduce or eliminate free radicals, block free radical chain reactions, and delay lipid peroxidation [23]. In our study, the antioxidant activity of four EOs was evaluated through scavenging DPPH free radicals, chelating Fe 2+ , scavenging hydroxyl free radicals, and inhibiting yolk lipid peroxidation. Our study found that the antioxidant activity of EOs is related to the EO concentration and EO type. The DPPH and hydroxyl radical scavenging rate, the Fe 2+ chelating ability, and the inhibition rate of the lipid peroxidation of yolk were dose-dependent, and they linearly or quadratically increased with an increase in EO concentration, which suggests that the antioxidant activity of EOs varies with concentration. Similarly, El amrani et al. [24] found that the DPPH radical inhibition percentages of cinnamon and clove essential oils presented a dose dependent effect. Other studies have found that the scavenging effect of AO was stronger than BHA and BHT and increased with the increase of concentration [25]. In DPPH radical scavenging experiments, the position of different EOs changes at different concentrations (cross effect). Similar results have been observed in our previous study, where it was found that the antioxidant activity of five citrus EOs increased with an increase in concentration. Additionally, the scavenging effect of bergamot EO was higher than that of lemon EO at low concentrations of 16-80 mg/mL, but the results were the opposite at higher concentrations of 112-144 mg/mL [26]. This is due to the interaction between the type of EO and its concentration. In addition, different EOs had different antioxidant activity. Wei et al. [27] measured the antioxidant activity of 25 EOs and found that cinnamon leaf oil, clove leaf oil, and thyme oil had good antioxidant activity, while sandalwood oil showed poor antioxidant activity. Similarly, Ghazghazi et al. [28] found significant differences in the antioxidant activity of two EOs (Eucalyptus marginata L. and Eucalyptus pauciflora L. EOs). Another study analyzed the antioxidant activity of LCO extracted in different months, and the results showed that LCO extracted in July had a better hydroxyl radical scavenging rate, and LCO extracted in August had a better DPPH scavenging activity and ferric reducing antioxidant power [29]. In this study, at the same concentration, EUC and CO had good radical hydroxyl scavenging ability, while AO and LCO had poor scavenging ability. This difference may be due to the different chemical compositions of different EOs, and the compositions and contents of EOs may affect their overall antioxidant activity [30].
In addition, the antioxidant activity of EOs is related to their chemical compositions [31]. It has been reported that phenols contribute greatly to the antioxidant capacity of EOs [32]. The phenolic hydroxyl can release hydrogen ions to bind with free radicals and block the oxidative chain reaction [33], and the antioxidant activity of EOs is significantly positively correlated with the phenol content [34]. Mechergui et al. [35] reported that the antioxidant activity of oregano oil mainly depended on the carvall and thymol contents. In addition, fatty acids, esters, ketones, alcohols, and terpenoids have previously been reported to have antioxidant activity [36]. Adefegha et al. [37] showed that the free radical scavenging ability and metal chelating ability of Masson pine EO may be attributed to phenolic monoterpenes and oxygenated monoterpenes in EOs. Some studies have shown that the antioxidant activity of different antioxidants mainly depends on their stability after absorbing lone pair electrons, and phenols > aldehydes > alcohols [38]. The four EOs showed different antioxidant activity, which may have been caused by the different chemical components of different EOs. The active components of EOs are complex and diverse, and whether the antioxidant activity of different active components are based on synergistic, antagonistic, or additive effects needs to be further studied.
Various antioxidant activity assays have been used to evaluate the antioxidant capacity of EOs in vitro, and EOs showed different antioxidant activity using different assays. In our study, we evaluated the antioxidant activity of EOs using four methods: scavenging DPPH radical, chelating Fe 2+ , scavenging hydroxyl radical, and inhibiting yolk lipid peroxidation. DPPH free radical is a stable radical that is widely used as a tool to evaluate the free radical scavenging activity of EOs [39]. The ability of EOs to scavenge DPPH free radicals may be related to their ability to provide hydrogen atoms. EOs can provide hydrogen atoms to DPPH radicals to form stable end products (DPPH-H), which will not further trigger or aggravate lipid oxidation [40]. It has been found that monoterpenes, such as citral, carvone, laurene, and γ-terpinene, have strong DPPH radical scavenging activity [41]. The scavenging ability of EOs against DPPH free radicals is related to their content of unsaturated hydrocarbon. LCO had the strongest scavenging ability against DPPH free radicals among the four EOs, which may be due to the effects of citral, the main component of LCO. The accumulation of Fe 2+ in cells can induce the production of free radicals and malondialdehyde [37,42]. EOs can chelate with metal ions to form stable complexes that inhibit the formation of metal-induced free radicals. Some phenols in EOs can effectively chelate ferrous ions [43]. Singh et al. [44] found that the chelating activity of EOs of Zanthoxylum armatum DC was significantly higher than that of BHA and BHT, which may be attributed to the phenolic compounds contained in EOs that can chelate metal ions. In our study, EUC showed a good Fe 2+ chelating ability and increased in a dose-dependent manner, which may be due to the large number of compounds in the oil and their possible affinity for Fe 2+ . Hydroxyl free radical is a kind of highly active oxidant that can directly damage various biofilms and lead to many diseases [45]. Hydroxyl free radicals can extract electrons from polyunsaturated fatty acids and produce lipid radicals, which can trigger lipid peroxidation. In addition, hydroxyl free radicals are highly reactive products produced via the decomposition of hydrogen peroxide in the presence of Fe 2+ and other metals, which can aggravate oxidative stress in the body, lead to immune system dysfunction, and affect the digestion, absorption, and transformation of nutrients [46]. Thus, a reduction in hydroxyl radicals can reduce lipid peroxidation and protect the immune system. In our study, all four EOs could remove hydroxyl radicals, among which EUC has the best effect. The scavenging effects of EOs on hydroxyl free radicals can not only act on the non-chain process before the oxidation reaction chain to remove the hydroxyl radicals, starting the chain reaction, but also remove the free radicals in the chain extension process, producing a dual-antioxidant effect. Lipid peroxidation is considered to be an important mechanism of cell damage under oxidative stress and is involved in the pathological processes of many diseases [47]. Previous studies have shown that EOs can effectively inhibit lipid peroxidation. Gargouri et al. [48] found that Henna EO showed very high potential for scavenging free radicals and inhibiting lipid peroxidation, which could be used to combat the oxidative stress generated by Raji cells. Alizadeh et al. [49] speculated that the EO of Zeilan cinnamomum L. had a strong free radical scavenging ability and the potential to inhibit lipid oxidation reactions, which was consistent with the results of our experiment. In our study, CO showed the best inhibition of lipid peroxidation of yolk, which may be attributed to the fact that its main component, 1,8 eucalyptol, is a monoterpenoid. It has been found that monoterpene hydrocarbon, γ-terpinene, retards the peroxidation of linoleic acid because its chaincarrying peroxyls are HOO• radicals, which react rapidly with linoleylperoxyl radicals [50]. In our study, the four EOs showed different antioxidant activity in different antioxidant tests. EUC showed good hydroxyl radical scavenging ability and Fe 2+ chelating ability, but poor DPPH radical scavenging ability and yolk lipid peroxidation inhibition. Similarly, Bi et al. [51] found that the EOs from Nandina domestica fruits exhibited significant scavenging activity against DPPH free radical and ATBS free radical, moderate scavenging activity against superoxide anion free radical, and low activity against metal chelating power. This difference may be due to the fact that the total antioxidant activity of EOs is the result of a combination of one or several pathways. In our study, EUC had the best hydroxyl radical scavenging ability and chelating Fe 2+ ability, and CO had better yolk lipid peroxidation inhibition ability and DPPH and hydroxyl radical scavenging ability. Therefore, EUC and CO have good antioxidant activity and have the potential to be used in practical production as a new green antioxidant.
In summary, our results show that the four EOs possessed significant antioxidant activity, but their antioxidant capacity varied with EO type and concentration. In addition, the different antioxidant abilities of EOs may be due to the different chemical compositions of EOs. Combined with the results of the four antioxidant experiments, it is speculated that the total antioxidant activity of EOs may comprise different activities including scavenging free radicals, chelating Fe 2+ , and inhibiting lipid peroxidation etc.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The EO samples were diluted 1:100 with hexane and then injected into an Rxi-5Sil MS (Restek, Bellefonte, Newcastle, PA, USA, 30 m × 0.25 mm × 0.25 µm) column via the GC-MS injection system. The column temperature was initially set at 50 • C and held for 5 min, then increased up to 320 • C at a rate of 6 • C/min for 5 min and solvent delay for 3 min. Helium was used as a carrier with a shunt ratio of 10:1 and a flow rate of 1 mL/min. Total ion flow chromatography (TIC) was obtained by analyzing the samples. The data were collected using the GC-MS solution 2.6 software and screened and matched with NIST and other dedicated standard spectrum libraries to identify each component of the EOs. Finally, the relative content was calculated using area normalization.

DPPH Radical Scavenging Ability Assay
The DPPH free radical scavenging activity was determined according to a previously described method [52]. LCO, CO, AO, EUC, and BHA were diluted with anhydrous ethanol to 2.5, 10, 20, 40, and 80 mg/mL, respectively. A 2 mL EOs diluent and 2 mL 0.2 mmol/L DPPH ethanol solution were taken to prepare 4 mL of the tested solution in a tube. After shaking, the solution was kept away from light at room temperature for 30 min. Next, 200 µL of the tested solution was added to a 96-well microtiter plate. The absorbance was measured using an automatic microplate reader at 517 nm, and the clearance rate was calculated. There were 3 parallel groups in each group. The DPPH free radical scavenging rate was calculated according to Formula (1). A 1 is the absorbance of the ethanol solution of DPPH mixed with the diluent of plant EOs, A 0 is the absorbance of the DPPH ethanol solution mixed with anhydrous ethanol, and A 2 is the absorbance of the plant EOs diluent mixed with absolute ethanol.

Fe 2+ Chelating Ability Assay
The Fe 2+ chelating ability was determined as described by Oboh et al. [53]. LCO, CO, AO, EUC, and strong antioxidant (VC) were diluted with anhydrous ethanol to 2, 6, 8, 10, and 12 mg/mL, respectively. A total of 1 mL of the diluted sample solution was taken, and 0.1 mL of 2 mmol/L FeCl 2 and 0.3 mL of 5 mmol/L phenanthrazine were successively added to the clean test tube. Then, 2.6 mL of distilled water was added to prepare 4 mL of the tested solution, and the solution was left at room temperature for 20 min. A total of 200 µL of the tested solution was added to a 96-well microtiter plate. The absorbance was measured using an automatic microplate reader at 517 nm, and the clearance rate was calculated. There were 3 parallel groups in each group. The chelating capacity of Fe 2+ was calculated according to Formula (2). A 1 is the absorbance of the tested solution added to EOs, A 0 is the absorbance of absolute ethanol sample solution without EOs, and A 2 is the absorbance of distilled water without phenanthrazine.

Hydroxyl Radical Scavenging Ability Assay
The hydroxyl radical scavenging ability was measured according to the method of Radünz [54] with some modifications. LCO, CO, AO, EUC, and strong antioxidant (VC) were diluted with anhydrous ethanol to 1, 4, 8, 16, and 32 mg/mL, respectively. A total of 1 mL of essential oil diluent was taken from each, and 1 mL of a 2 mmol/L FeSO 4 , 1 mL of a 6 mmol/L salicylic acid, and 1 mL of a 2 mmol/L H 2 O 2 were added to prepare 4 mL of the tested sample solution. The solution was mixed well and left for 30 min at room temperature. A total of 200 µL of the tested solution was added to a 96-well microtiter plate. The absorbance was measured using an automatic microplate reader at 510 nm and the clearance rate was calculated. There were 3 parallel groups in each group. The hydroxyl radical scavenging rate was calculated according to Formula (3). A 1 is the absorbance of the tested solution added to EOs; A 0 is the absorbance of absolute ethanol sample solution without EOs; A 2 is the absorbance of distilled water without H 2 O 2 .
The hydroxyl radical scavenging rate

Yolk Lipid Peroxidation Inhibition Assay
The yolk lipid peroxidation inhibition rate was determined according to a previously described method [55]. The yolk of fresh eggs was put into a 50 mL beaker, the same volume of phosphate buffer was added, the sealing film was fastened, and the solution was stored at −4 • C. Four EOs and PG were diluted with absolute ethanol to 1.25, 2.5, 5, 10, and 20 mg/mL sample dilutions. A total of 0.5 mL of egg yolk stock solution was taken and 12 mL of phosphate buffer was added to prepare a 1:25 egg yolk suspension. A total of 200 µL of the yolk suspension was taken, a 100 µL EO sample diluent was added, and then 200 µL of a 25 mmol/L FeSO 4 was added and supplemented to 1 mL with phosphate buffer. After shaking at 37 • C for 30 min, 250 µL of a 20% trichloroacetic acid was added, 3500 r/min centrifugation was performed for 10 min until clarification, the supernatant was absorbed, and 250 µL of a 0.8% TBA was added. A total of 1.5 mL of the sample solution was prepared to be tested and reacted in a boiling water bath for 15 min. A total of 200 µL of the tested solution was added to a 96-well microtiter plate. The absorbance was measured using an automatic microplate reader at 510 nm, and the clearance rate was calculated. There were 3 parallel groups in each group. The yolk lipid peroxidation inhibition rate was calculated according to Formula (4). A 1 is the absorbance of the tested solution added to EOs; A 0 is the absorbance of absolute ethanol sample solution without EOs; A 2 is the absorbance of the tested solution without yolk.

Statistical Analysis
Data for antioxidant activity were analyzed using the SAS 9.4 mixing procedure with a model including EO type, EO concentration, and their interactions as fixed effects and three replications of the experiment as random effects. The effects of increasing the EO concentration were examined through linear and quadratic orthogonal contrasts using the CONTRAST statement of SAS. Differences were declared significant at p ≤ 0.05. The graphs were drawn using the Origin 2021 software (Northampton, MA, USA).

Conclusions
The major compounds found in LCO, CO, AO, and EUC are citral, cinnamaldehyde, anethole, and 1,8-cineole, respectively. The four EOs effectively scavenged DPPH, chelated Fe 2+ , scavenged hydroxyl radicals, and inhibited lipid peroxidation in a dose-dependent manner. Compared with that of the other EOs, the DPPH radical scavenging ability of LCO was the strongest; EUC had the greatest activity in its Fe 2+ chelating ability and hydroxyl radical scavenging rate; and CO had the highest yolk lipid peroxidation ability. It is speculated that the total antioxidant activity of EOs might be the result of the joint actions of different antioxidant capacities.