Antioxidant Activities of a New Chemotype of Piper cubeba L. Fruit Essential Oil (Methyleugenol/Eugenol): In Silico Molecular Docking and ADMET Studies

Piper cubeba L. fruit is an important species used in folk medicine for different types of pains such as rheumatism, chills, flu, colds, muscular aches, and fever. This study examines the chemical constituents, antioxidant activity, and potential inhibitory effect against human peroxiredoxin 5, a key enzyme of P. cubeba essential oil from fruits. Using gas chromatography coupled with mass spectrometry (GC–MS), the principal components were methyleugenol (41.31%) and eugenol (33.95%), followed by (E)-caryophyllene (5.65%), p-cymene-8-ol (3.50%), 1,8-cineole (2.94%), and α-terpinolene (1.41%). Results showed similar scavenging activity via 2,2-diphenyl-1-picrylhydrazyl DPPH radical scavenging activity (IC50 = 110.00 ± 0.08 μg/mL), as well as very potent antioxidant activity against both ferric reducing/antioxidant power (FRAP) (106.00 ± 0.11 μg/mL) and β-carotene bleaching (IC50 = 315.00 ± 2.08 μg/mL) assays when compared to positive butylated hydroxytoluene and ascorbic acid. The molecular docking approach has also been performed to screen the antioxidant activities of the major and potent compounds against human protein target peroxiredoxin 5. Results showed good binding profiles and attributed the strongest inhibitory activity to β-caryophyllene oxide (–5.8 kcal/mol), followed respectively by isocembrol and α-selinene (–5.4 kcal/mol), and viridiflorol (–5.1 kcal/mol). Furthermore, ADME (absorption, distribution, metabolism and excretion)-related physicochemical and pharmacokinetic properties have been assessed and support our in vitro findings. This work demonstrates the powerful antioxidant potency of cubeba pepper and paves the way for the discovery and development of antioxidant agent with high potency.


Essential Oil Characterization
The light-yellow P. cubeba fruit's essential oil was obtained from the hydrodistillation process, with a yield of 1.0% (v/w). The characterization of the volatile compounds through gas chromatography and mass spectrometry (GC-MS) was identified by comparing the retention time on the HP-5MS column and the molecular weight with reference compounds in the NIST library, which revealed the presence of 24 components, representing 98.13% of the total amount (Table 1).  Adams, 2007 [23]. b RI: experimentally retention index calculated against a C8-C25 n-alkanes mixture on the HP-5MS column.
Phenylpropanoids represented the main characterized class (75.41%), followed by monoterpenes, including both hydrocarbonated (9.71%) and oxygenated (3.06%). Hydrocarbonated sesquiterpenes were one of the identified compounds, with a concentration of 1.69%, from the overall identified compounds, in addition to those that were minor oxygenated (8.26%).

Antioxidant Activity
Three complementary assays (DPPH, ferric reducing/antioxidant power (FRAP), and β-carotene bleaching) were used for the evaluation of the antioxidant potential of P. cubeba fruit's essential oil.

FRAP Test
The results of the FRAP assay, expressed in terms of EC 50 (Table 2), showed that P. cubeba fruit's essential oil has a powerful capacity (p < 0.05) to reduce ferric ions to ferrous ions, with an EC 50 of 106.00 ± 0.11 µg/mL, which is three times higher than ascorbic acid, used as a standard (330.00 ± 0.60 µg/mL).

Molecular Docking Study
In order to corroborate the experimental data and understand the possible mechanism behind the antioxidant activity, molecular docking studies were examined using human peroxiredoxin 5 enzyme (PDB: 1HD2), which has a broader activity against ROS and was mostly involved in stress protection mechanisms and in cell differentiation. The listed binding energies of the formed complex between identified P. cubeba fruit's essential oil constituents and peroxiredoxin 5 (Table 3) were found to be in the range −5.8 to −3.7 kcal/mol. From our molecular docking results, it can be suggested that all ligands interact favorably with the target proteins. Herein, we will focus mainly on both of the major constituents, methyleugenol (−4.3 kcal/mol) and eugenol (−4.7 kcal/mol), representing about 75% of the whole P. cubeba fruit's essential oil composition, as well as on those with the four top docking scores, which are β-caryophyllene oxide (−5.8 kcal/mol) followed respectively by isocembrol and α-selinene (−5.4 kcal/mol), and viridiflorol (−5.1 kcal/mol) ( Figure 1).

Methyleugenol
Eugenol β-caryophyllene oxide Isocembrol α-selinene Viridiflorol Their interactions with the catalytic site of the investigated enzyme were analyzed (Table 4 and Figure 2). Their interactions with the catalytic site of the investigated enzyme were analyzed (Table 4 and Figure 2).

ADME Analysis
As one of the best filters in the virtual screening of bioactive molecules, in order to be an effective drug in early preclinical development, the forecasting of ADME (absorption, distribution, metabolism and excretion) profiles of the selected compounds, including their pharmacokinetic and drug-like properties, have been investigated using Swiss ADME (http://www.swissadme.ch/). The selected phytocompounds (Table 5) were found to correctly meet the Lipinski Rule of five, and also share topological polar surface area TPSA values less than 30 Å 2 , suggesting good brain penetration and good lipophilicity behavior, which is expressed by the consensus Log Po/w in the range 2.25-4.75. Their bioavailability score of 0.55 indicates their more drug-like properties. Noticeably, there is no Pglycoprotein (P-gp) substrate justifying their good intestinal absorption and bioavailability; the compounds exhibited high gastrointestinal absorption (GI) (except α-selinene), and only isocembrol and α-selinene were predicted to not cross the blood-brain barrier (BBB). However, the others easily pass the blood-brain barrier (BBB) permeant, and can bind to specific receptors. The study's From our molecular docking results, it was found that β-caryophyllene oxide was identified as the most potent inhibitor of the enzyme, with human peroxiredoxin 5 interacting favorably with its catalytic site via two H-bonding interactions with Gly46 (2.73) and Arg127 (6.35); five van der Waals interactions with Pro40, Thr44, Cys47, Ile119, and Thr147; and four Alkyl/Pi-Alkyl interactions with Pro45 (3.75), Leu116 (4.31) (4.68), and Phe120 (4.89). Isocembrol compound performed several hydrophobic interactions with the following residues of human peroxiredoxin 5 (1HD2): one H bond with Thr147 (2.61), five van der Waals interactions with Thr44, Gly46, Ile119, Arg127, and Gly148l and five Alkyl/Pi-Alkyl interactions with Pro40 (4.03), Pro45 (3.87) (4.31), Leu116 (4.50), and Phe120 (5.31) amino acid residues. The interactions involved between α-selinene and human peroxiredoxin 5 are van der Waals interactions with Ala42, Thr44, Asn76, Phe120, and Arg124; Pi-Sigma interactions with Phe43 (3.80); and Alkyl/Pi-Alkyl interactions with Phe43 (5. 26 included van der Waals reactions with Gly46, Cys47, Phe120, and Arg12; C-H bonds with Thr147 (3.28); and Alkyl interactions with Pro45 (4.75), Leu116 (4.13) (4.42) (5.43), and Ile119 (5.10) residues.

ADME Analysis
As one of the best filters in the virtual screening of bioactive molecules, in order to be an effective drug in early preclinical development, the forecasting of ADME (absorption, distribution, metabolism and excretion) profiles of the selected compounds, including their pharmacokinetic and drug-like properties, have been investigated using Swiss ADME (http://www.swissadme.ch/). The selected phytocompounds (Table 5) were found to correctly meet the Lipinski Rule of five, and also share topological polar surface area TPSA values less than 30 Å 2 , suggesting good brain penetration and good lipophilicity behavior, which is expressed by the consensus Log P o/w in the range 2.25-4.75. Their bioavailability score of 0.55 indicates their more drug-like properties. Noticeably, there is no P-glycoprotein (P-gp) substrate justifying their good intestinal absorption and bioavailability; the compounds exhibited high gastrointestinal absorption (GI) (except α-selinene), and only isocembrol and α-selinene were predicted to not cross the blood-brain barrier (BBB). However, the others easily pass the blood-brain barrier (BBB) permeant, and can bind to specific receptors. The study's components interacted at most with two isoenzymes of the Cytochrome P (CYP) family, confirming their better effectiveness with insignificant toxicity. Their radar plot ( Figure 3A) indicates that all selected compounds are entirely inside the pink area, indicating their better drug likeness alone with a good bioavailability profile. Additionally, pharmacokinetics were evaluated by the boiled-egg model ( Figure 3B). The compounds methyleugenol, eugenol, β-caryophyllene, and viridiflorol appeared in the yellow region (yolk) with a red point, which confirms their high probability of brain penetration acting as a non-substrate of P-gp (PGP−). Plants 2020, 9,

Chemical Composition Analysis vs. Beneficial Role
Plants used as condiments have been noted for their extremely beneficial therapeutics, and have been described with a wide variety of antioxidant compounds able to prevent oxidative stress via their complex composition and richness in bioactive molecules.
As shown, for this oil, phenylpropanoids form the backbone of the characterized compounds. The richness of P. cubeba fruit's essential oil on phenylpropanoids, which are chemically a group of compounds derived from the carbon skeleton of phenylalanine, implicated in plant defense, structural support, and the survival of plants, have been widely reported for their anticonvulsant, analgesic, antidiabetic, and anti-inflammatory potential [24]. Furthermore, they have shown application in the food, pharmaceuticals, cosmetics, textiles, biofuel, and sensors [24]. Macchia et al. [25] have indicated that methyl eugenol is formed through cyclic acid pathways, with phenylalanine as precursor; however, eugenol will be formed under a few reaction stages. Eugenol is then converted easily to methyl eugenol via a methyl donor from sadenosylmethionine (SAM) and enzymatic activity from O-methyltransferase (OMT) [26]. Binu et al. [27] showed that treatment with eugenol improved the peroxidation of the membrane and restored normal heart rate. Also, histological

Chemical Composition Analysis vs. Beneficial Role
Plants used as condiments have been noted for their extremely beneficial therapeutics, and have been described with a wide variety of antioxidant compounds able to prevent oxidative stress via their complex composition and richness in bioactive molecules.
As shown, for this oil, phenylpropanoids form the backbone of the characterized compounds. The richness of P. cubeba fruit's essential oil on phenylpropanoids, which are chemically a group of compounds derived from the carbon skeleton of phenylalanine, implicated in plant defense, structural support, and the survival of plants, have been widely reported for their anticonvulsant, analgesic, antidiabetic, and anti-inflammatory potential [24]. Furthermore, they have shown application in the food, pharmaceuticals, cosmetics, textiles, biofuel, and sensors [24]. Macchia et al. [25] have indicated that methyl eugenol is formed through cyclic acid pathways, with phenylalanine as precursor; however, eugenol will be formed under a few reaction stages. Eugenol is then converted easily to methyl eugenol via a methyl donor from sadenosylmethionine (SAM) and enzymatic activity from O-methyltransferase (OMT) [26]. Binu et al. [27] showed that treatment with eugenol improved the peroxidation of the membrane and restored normal heart rate. Also, histological examination of the cardiac segments has confirmed the beneficial role of eugenol against arsenic-induced oxidative damage. Eugenol has been reported to possess a large spectrum of activity, including antitumor, anti-inflammatory, analgesic, antibacterial, antifungal, anesthetic, and antipyretic potency [28][29][30][31][32][33][34].

Antioxidant Potential
The results prove that P. cubeba fruit's essential oil is able to act as a donor of hydrogen atoms or electrons to reduce the DPPH radical. The potent DPPH scavenging activity of P. cubeba fruit's essential oil could be attributed to the high amount of eugenol and methyleugenol [39,40]. Additionally, Gülçin in 2011 [41] showed that eugenol had the most powerful antioxidant activity and radical-scavenging activity compared to the standards: butylated hydroxyanisole, butylated hydroxytoluene, α-tocopherol, and Trolox. Recently, Gogoi et al. [39] reported that methyleugenol-rich lemongrass essential oil displayed the strongest antioxidant activity on DPPH assay, with IC 50 = 2.263 µg/mL, which is lower than standard ascorbic acid (IC 50 = 2.58 µg/mL). Also, other compounds in small amounts contribute to the scavenging activity, such as oxygenated sesquiterpenes [42].
Our FRAP results were confirmed by those mentioned previously by Zhang et al. [43], indicating the remarkable potency and contribution of eugenol (representing 33.95% in P. cubeba fruit's essential oil via FRAP assay) to donate electrons to reactive free radicals, transforming them into more stable, non-reactive species, and terminating the free radical chain reaction.
β-carotene bleaching results indicate that this potent antioxidant capacity is probably attributable to the presence of diverse compounds in the tested volatile oil, such as terpinen-4-ol, 1,8-cineole, and p-cymene. In fact, Radonic et al. [44] and Tepe et al. [45] showed that oxygenated monoterpenes exert a strong inhibitory effect on the coupled oxidation of β-carotene/linoleic acid.
Indeed, based on the above results, the higher antioxidant activity of P. cubeba fruit's essential oil could be related to this synergistic effect. The high content of methyleugenol/eugenol (75.26%) associated with other constituents (major and minor components) also could have been responsible for their bioactivities. Eugenol possesses the ability to transfer electron or hydrogen atoms by neutralizing free radicals, which can block the oxidative process [46,47]. The presence of methyleugenol/eugenol as the principal chemotype of this oil gives him better antioxidant potency, and therefore good DNA damage protective effects, which can be manifested via direct trapping of the free radicals or inhibiting the propagation of radical chain reactions through transfers of hydrogen or electrons. Overall, the significant antioxidant potential of P. cubeba fruit's essential oil confirms the uses of cubeba fruit as a source of antioxidants, which may provide health-promoting advantages to consumers.

In Silico Study
Computational methods associated with experimental strategies have been of great value in modern drug design, and in the development and discovery of novel promising compounds. Our docking results were also well-correlated with in vitro antioxidant assays, along with the selected phytocompounds interacting perfectly with the protein target. Indeed, in addition to methyleugenol and eugenol, which are discussed above, the natural bicyclic sesquiterpene β-caryophyllene oxide has been proven to have potent antioxidant activity [48]. Isocembrol, also known as thunbergol, is an oxygenated diterpene (alcohol) detected in soft coral eggs Lobophytum compactum and Lobophytum crissum, and has been reported to have potential antioxidant activity [49,50]. Mayachiew and Devahastin [51], using a β-carotene/linoleic acid assay, reported that the Alpinia galangal extract rich α-selinene (called also α-Eudesma-3,11-diene) possesses potent antioxidant activity. Viridiflorol, as an oxygenated sesquiterpenes, was found to possess antioxidant properties [52]. The synergistic action of other minor components contributes to an increase in antioxidant activity that may be taken into account.
As stated above from the docking results, we found that they are in perfect agreement with those obtained by Declercq et al. [53] when analyzing the crystal structure of human peroxiredoxin 5 (1HD2). The authors demonstrated that at the active site of the enzyme, the residue Cys47 located at the N-terminal part of the kinked helix α2, inside a small cavity, was directly involved in peroxide reductase activity by forming an intramolecular disulfide intermediate in the oxidized enzyme. Also, the amino acid Arg127, interacting with the sulfur atom of Cys47 at a distance of 3.3 Å, seems to be responsible for the positively charged active site pocket. On the other hand, Thr44 is present in the active site cavity of human peroxiredoxin 5, with its oxygen atom clearly interacting with the sulfur atom of the catalytic cysteine residue (Cys47) at a distance of 3.0 Å. In addition, they found that one side of the active site pocket contains several hydrophobic residues, including Leu116, Ile119, and Phe120, whose side-chains are located near the benzoate aromatic ring, which is shown to act as hydroxyl radical scavenger (via its benzoate ion). As shown, numerous interactions between human peroxiredoxin 5-selected phytocompounds are close to the active site of the enzyme. Our finding also corroborates perfectly those of Eze et al. [54], demonstrating that the docked 1-(Phenylsulphonyl)-N-propylpyrrolidine-2-carboxamide with human peroxiredoxin 5 exhibited higher binding energy (−13.86 kcal mol −1 ), creating interactions with Thr44, Pro40, Pro 45, Gly46, Arg127, Thr147, and Cys47 residues. Additionally, with the same therapeutic target, caryophyllene oxide (−7.2 kcal/mol) from Cymbopogon citratus essential oil established non-covalent interactions with Pro 40, Thr 147, Thr 44, Phe 120, Pro 45, Leu 116, and Ile 119, sharing a large number of common residues with the active site of the investigated enzyme.
In silico ADME and drug-likeness prediction of the selected P. cubeba fruit essential oil's bioactive molecules demonstrate good pharmacokinetic properties, which encourages further in vivo and in vitro evaluation study of the proposed essential oil to validate the computational findings.
Cubeba pepper fruits ( Figure 4) were purchased commercially from the Saudi market (Baljurashi city, Albaha, Kingdom Saudi Arabia). Samples were cleaned well with tap water, sterilized, and dried in an oven at 60 • C for 72 h before being pulverized into a fine powder for possible use. Dried powder of the fruits (800 g) was subjected to hydrodistillation for 4 h using a Clevenger-type apparatus [51]. The resulting essential oils were dried using anhydrous sodium sulfate, and stored in sealed vials in the dark, at 4 • C for subsequent analysis.

Characterization of Essential Oils by GC-MS
The chemical composition of P. cubeba fruit's essential oil has been identified using a GC-MS apparatus (Agilent Technologies, Santa Clara, CA, USA), as described by Bakari et al. [55]. The column used is an HP-5MS (5% phenyl) of length 30 m, inside diameter 0.25 mm, and film thickness 0.25 μm. The temperature of the injector and detector was 250 °C. The oven temperature was maintained at 100 °C for 1 min, then increased to 260 °C at a rate of 4 °C/min, and then maintained at 260 °C for 10 min. Helium gas has been used as a carrier gas at a constant rate of 1 mL/min. The injection volume was 1 μL. Ionization conditions by impact electronics (EI) were at an ionic energy of 70 eV and a range of swept mass of 50 to 550 m/z.

In Vitro Antioxidant Activity
All tests were conducted in triplicate, and mean values were taken.

DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging activity
The DPPH test was carried out as described by Felhi et al. [56]. A series of dilutions was prepared from the P. cubeba fruit's essential oil solution, each one supplemented with 1 mL of the ethanolic solution of DPPH. Then, the dilutions were incubated for 30 min in the dark, and the absorbance of the mixtures was measured at 517 nm against a negative control treated under the same conditions. Ascorbic acid (AA) was used in this test as the positive control.

Ferric Reducing/Antioxidant Power (FRAP) Assay
The reducing power of the P. cubeba fruit's essential oil was evaluated using the method described by Bakari et al. [9]. The sample was mixed with 2.5 mL of potassium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%). The mixture was incubated for 20 min at 50 °C in a water bath, and then 2.5 mL of trichloroacetic acid (10%) was added.
After centrifugation at 3000 rpm for 10 min at 25 °C, the supernatant was mixed with 2.5 mL of H2O and 0.5 mL of ferric chloride (0.1% FeCl3). A blank without a sample was prepared under the same conditions. The optical density was subsequently measured at 700 nm, and BHT (Butylated hydroxytoluene) was used as positive control.

β-carotene Bleaching Test
The β-carotene bleaching test was performed based on the experimental protocol done by Kadri et al. [8]. Briefly, a mixture containing 20 μL of linoleic acid and 200 mg of Tween 20% (emulsifier) was added to a previously prepared solution by dissolving 0.2 mg of β-carotene in 1 mL of chloroform. After vacuum evaporation of the chloroform at 40 °C, a volume of 50 mL of oxygen sparged water was added to the obtained emulsion. Then, a volume of 5 mL of this emulsion was

Characterization of Essential Oils by GC-MS
The chemical composition of P. cubeba fruit's essential oil has been identified using a GC-MS apparatus (Agilent Technologies, Santa Clara, CA, USA), as described by Bakari et al. [55]. The column used is an HP-5MS (5% phenyl) of length 30 m, inside diameter 0.25 mm, and film thickness 0.25 µm. The temperature of the injector and detector was 250 • C. The oven temperature was maintained at 100 • C for 1 min, then increased to 260 • C at a rate of 4 • C/min, and then maintained at 260 • C for 10 min. Helium gas has been used as a carrier gas at a constant rate of 1 mL/min. The injection volume was 1 µL. Ionization conditions by impact electronics (EI) were at an ionic energy of 70 eV and a range of swept mass of 50 to 550 m/z.

In Vitro Antioxidant Activity
All tests were conducted in triplicate, and mean values were taken.

DPPH (1,1-Diphenyl-2-picrylhydrazyl) Radical Scavenging activity
The DPPH test was carried out as described by Felhi et al. [56]. A series of dilutions was prepared from the P. cubeba fruit's essential oil solution, each one supplemented with 1 mL of the ethanolic solution of DPPH. Then, the dilutions were incubated for 30 min in the dark, and the absorbance of the mixtures was measured at 517 nm against a negative control treated under the same conditions. Ascorbic acid (AA) was used in this test as the positive control.

Ferric Reducing/Antioxidant Power (FRAP) Assay
The reducing power of the P. cubeba fruit's essential oil was evaluated using the method described by Bakari et al. [9]. The sample was mixed with 2.5 mL of potassium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%). The mixture was incubated for 20 min at 50 • C in a water bath, and then 2.5 mL of trichloroacetic acid (10%) was added.
After centrifugation at 3000 rpm for 10 min at 25 • C, the supernatant was mixed with 2.5 mL of H 2 O and 0.5 mL of ferric chloride (0.1% FeCl 3 ). A blank without a sample was prepared under the same conditions. The optical density was subsequently measured at 700 nm, and BHT (Butylated hydroxytoluene) was used as positive control.

β-Carotene Bleaching Test
The β-carotene bleaching test was performed based on the experimental protocol done by Kadri et al. [8]. Briefly, a mixture containing 20 µL of linoleic acid and 200 mg of Tween 20% (emulsifier) was added to a previously prepared solution by dissolving 0.2 mg of β-carotene in 1 mL of chloroform. After vacuum evaporation of the chloroform at 40 • C, a volume of 50 mL of oxygen sparged water was Plants 2020, 9, 1534 14 of 18 added to the obtained emulsion. Then, a volume of 5 mL of this emulsion was introduced into a tube containing 0.5 mL of the sample or BHT (reference synthetic antioxidant). Control solutions have been prepared in parallel. The antioxidant activity was determined according to the optical density of the sample at 470 nm against a control solution prepared as above, but devoid of β-carotene.

Computational Approach
Interactions between the natural compounds and human peroxiredoxin 5 were assessed by in silico molecular docking, in order to explore the preferred orientation of the ligands in the binding site of receptors. The crystal structure was obtained from the Protein Data Bank: human peroxiredoxin 5 (PDB Code: 1HD2). All water molecules were removed, as was the co-crystallized ligand from the structures. Polar hydrogens and Gasteiger charges were assigned with AutoDockTools1.5.2 (ADT), and the PDBQT file format was prepared [57]. The same software was used to select a docking grid. In human peroxiredoxin 5 enzyme (PDB: 1HD2), the grid box site was established at 7.611, 43.828, and 29.921Å (x, y, and z, respectively) using a grid of 70, 58, and 58 points (x, y, and z, respectively) and including a spacing of 0.375 Å.
The structures of the natural compounds were minimized using a conjugate gradient AMMP incorporated in VEGA ZZ [58]. The conversion of the file from PDB to PDBQT was done using AutoDockTools1.5.2. We applied AutoDock Vina software with an exhaustiveness parameter of 32 to perform the docking simulations [59]. ADT was used for the docking conformation analysis. Receptor ligand interactions were visualized by Discovery Studio Visualizer (Dassault Systemes BIOVIA, 2015) [60].

Statistical Analysis
A one-way analysis of variance (ANOVA) and Tukey's post-hoc test was performed to determine significant differences between the responses using the SPSS 19 statistical package (SPSS Ltd., Woking, UK). Means and standard deviations were calculated. Differences between the mean values of the various treatments were determined by the least significant difference test. A probability level of p < 0.05 was used in testing the statistical significance of all experimental data.

Conclusions
The current study aimed to explore a new P. cubeba fruit essential oil chemical composition and its antioxidant activity, in order to provide more information about its health benefits. The oil exhibited higher antioxidant activity than the tested standards. In silico prediction and molecular docking studies showed good correlation between the observed inhibitory activity and the in silico molecular docking scores, with methyleugenol, eugenol, β-caryophyllene oxide, isocembrol, α-selinene, and viridiflorol, the main contributors of the antioxidant activity, sharing many common residues with the human peroxiredoxin 5 active site. Therefore, they may be potent inhibitors, in synergism effect with minor compounds. Our findings demonstrate for the first time that P. cubeba fruit's essential oil can be a new potential resource for future research to design new and more potent antioxidative agents.