In vitro antioxidant and anti-inflammatory activities of green cardamom essential oil and in silico molecular docking of its major bioactives

Essential oil of Elettaria cardamomum (Green cardamom) is effective in the treatment of various disease conditions. Globally, Guatemala is the leading producer of green cardamom fruits. Green-cardamom fruits from Guatemala were purchased from a Saudi Arabian market. The Guatemala cardamom essential oil (GCEO) was extracted using hydro-distillation method, and analyzed using gaschromatography/ mass-spectrometry (GC-MS). The antioxidant and anti-inflammatory activities were studied using 2,2-diphenyl-1-picrylhydrazyl-hydrate, ferric-chloride, bovine serum-albumin and proteinase inhibitory assays. GC-MS results confirmed the presence of α- terpinyl acetate and 1,8-cineole as major components. The antioxidant and anti-inflammatory activities of GCEO were found to be significant compared with those of standard ascorbic acid and ibuprofen, respectively. The molecular-docking study revealed the considerable binding potential of α-terpinyl acetate and 1,8-cineole with human-peroxiredoxin-5, tyrosine-kinases and Human-5-LOX receptors. The in-silico activity prediction analysis confirmed the antioxidant and anti-inflammatory potential, whereas the pharmacokinetics confirmed the safety of most of the GCEO compounds.


Introduction
Elettaria cardamomum Maton (Zingiberaceae), termed as the "queen of spices", light green or green cardamom, and small or lesser cardamom, is well known for its characteristic aroma and taste, and it is traditionally used in medicine and food [1]. Currently, Guatemala is the foremost commercial manufacturer of lesser cardamom, which is often used in the preparation of "strong cardamom coffee" called "Gahwa" in Middle East Asia. Lesser cardamom is commonly used in Ayurveda and Unani medicines for the treatment of intestinal disorders [2]. Studies have suggested that cardamom is effective against hepatotoxicity and gastric or ulcerogenic ailments [3]. Elguindy et al. reported that lesser cardamom could be used against hepatocellular cancer. Gilani et al. suggested that lesser cardamom is beneficial against sedation and epilepsy [3]. It has been scientifically evaluated for its antioxidant, anti-inflammatory, antimicrobial [4], anticancer [5], antidiarrheal [6] effects. Studies have reported its effect on multiple myeloma [7]; blood pressure, vasodilation and collective heart complaints as well as its anticholesterol and anticoagulation properties [8]. Gilani et al. reported that lesser cardamom can lower high blood pressure [3]. Goyal et al. reported its cardioprotective effects [9]. Bhaswant et al. reported that it is effective against hepatotoxicity [10]. Nitasha Bhat et al. (2015) reported its effects on obesity, hypothyroidism, diabetes, hepatomegaly, dyslipidaemia and rapid hyperglycaemia [11]. They also stated that a US-patented antidiabetic formulation contains several plant extracts, including that of cardamom. Among respiratory disorders, it can be used to treat cold and cough, asthma, bronchitis, tuberculosis and sore throat. Nitasha Bhat et al. also reported that lesser cardamom could be used to treat as anti-asthmatic [11]. Khan et al. demonstrated and reported the antihistaminic effects of cardamom extracts on isolated rabbit trachea tissues [12]. Kumar et al. (2016) reported that lesser cardamom helps boost and regain memory and demonstrated its antidepressant properties [13]. E. cardamomum capsules possess a wide array of bioactive components, and the central component is α-terpinyl acetate and 1,8-cineole [6,14]. The composition of essential oil depends on the geographical condition chemotype, growing condition, harvesting time of a plant [15]. International organization for standardization (ISO) developed a code for the cardamom oil (ISO 4733:2004) and data show the variation in the major composition of essential oil in different seeds batches [16] and this may be one of the major regions that reflect precise and accurate doses of cardamom oil. The cardamom volatile oil contains antioxidant components, which primarily contribute to its distinctive odour and role in nutraceutical and medicinal value [17]. Some doses of cardamom essential oil, such as doTERRA [18] and Edens Gardens cardamom essential oil 5 mL [19], are available on the market and are used in traditional medicine to treat inflammatory diseases. The doses 175-280 and 233 L/kg were similarly found to be beneficial for anti-inflammatory and analgesic action in a prior study [20]. Heimesaat et al. (2021) observed that a daily intake of 258 mg per kg bodyweight of cardamom essential oil inhibited mice pro-inflammatory mediator [21].
The oxygenated monoterpenes found in the essential oil of green cardamom, such as α-terpinyl acetate, 1,8-cineole and α-terpineol, among others, exhibit antioxidant, anti-inflammatory, gastroprotective, antiviral and antibacterial properties [22,23]. The smell of the extracted fruit oils is due to the presence of the metabolite α-terpinyl acetate, as well as other substances [24]. Lesser cardamom contains up to 50% αterpinyl acetate and 1,8-cineole as phytoconstituents [25,26]. E. cardamomum essential oil has been shown to have antioxidant and anti-inflammatory properties in a number of studies [22,27].
In the oxidative stress, reactive oxygen species (ROS) accumulates in the cells, excessive accumulation of ROS trigger sever cellular enzymes, and may affects the growth, metabolism, differentiation and motility of the normal cells [28]. Human peroxiredoxin 5 (PRDX5) located mainly in the mitochondria, peroxisomes and cytosol while protein tyrosine kinases (PTKs) located in the cellular membrane, both the enzymes along with several other functions involved in the antioxidant protection [29,30]. Human 5-LOX a lipoxygenase-enzyme, that is involved in the leukotrienes biosynthesis and participates in the inflammatory responses, its inhibition may be a potential target for evaluation of a new anti-inflammatory agents [31]. Molecular docking, in silico absorption, distribution, metabolism, elimination (ADME) and prediction of activity spectra for substances (PASS) testing by computational studies are used to predict drug targets, drug likening and pharmacological potential of the active compounds [32].
Considering the enormity of research reports on the pharmacological effects of E. cardamomum essential oil, we aimed to explore the constituents and in vitro antioxidant and anti-inflammatory spectrum of Guatemala cardamom essential oil (GCEO). Further, in silico druglike prediction of several compounds using ADME, PASS and molecular docking studies of the major chemical constituents using three targets, such as human peroxiredoxin 5 (IHD2), protein tyrosine kinases (3CC6) and human 5-LOX (3V99), are also conducted to account and support for the results of in vitro experimental outcomes.

Sample used and extraction of essential oils
Fruit sample of Guatemala E. cardamomum (lesser cardamom), labelled as a brand name "AlMEHBAJ", was obtained from the local market of Al-Kharj (Kingdom of Saudi Arabia, KSA) in January 2020. The sample was deposited in the Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University (PSAU, Al-Kharj, KSA). The fruit was dried and powdered. The essential oil (lower density than water) was extracted using a Clevenger type apparatus (capacity 10 mL) connected onside with 2 L round bottom flask overheating mantle and other side condenser connected with the chiller (Buchi B-741). 100 g of fruit powder was extracted at 60°C for 3 h [6]. The essential oil obtained was collected and dried using anhydrous Na 2 CO 3 ; after complete drying, it was weighed and kept in an amber-coloured air-tight vial and stored at 4°C for performing in vitro antioxidant and antiinflammatory activity assays. The experiment was performed in triplicate, and data were expressed as percentage ± standard deviation.

Characterization of the essential oil composition using gas chromatography-mass spectrometry
GCEO was dissolved in acetone (10 μL/mL) and analysed using gas chromatography (GC) equipped with mass spectrometer detector (MSD) Agilent HP 6890/5977 (Agilent technologies Inc., CA, Santa Clara, USA); the system was operating at 70 eV. 3 μL solution was injected in a split-mode (ratio, 100:1) in a HP-5MS column (Agilent Technologies Inc.) with the following dimensions: length, 30 m; diameter, 0.25 mm; and film-thickness of stationary phase, 0.25 μm. The column oven temperatures, sample injection, separation and identification of compounds were adopted from the previous works [33,34]. In brief, temperatures for the injector and detector were fixed at 270°C and 300°C, respectively. The oven temperature was programmed as follows: 40°C (storage time of 1 min) with a rate of 10°C/min to 110°C (storage time of 1 min), and continued to 300°C (hold time of 5 min), and finally reached to 325°C at the same rate; the total run time was 10 min. Helium constant flow was set at 3 mL/min. The solvent delay was 3 min, and the scan mass/charge range was between 50 and 550 m/z. Agilent MassHunter (NIST14.L) analysis was used for the data analysis. The documentation of compounds was based on the assessment of retention time and spectra of mass with the reference spectra (National Institute of Standards and Technology; NIST, 14.L). Essential oil composition of GCEO was also identified based on the retention indices (RIs) relative to n-alkanes homologous series (C 8 -C 30 ) and by comparing their mass spectra with those recorded in NIST 05 and Wiley 275 inherent mass spectral libraries and with the literature data [35].

DPPH free radical scavenging activity
The free radical scavenging (FRS) ability of GCEO was analysed using the DPPH assay, and the method followed by Alam and Majumdar was used, with some modifications [36]. Briefly, 10 mL DPPH-cation solution (0.1 mM) was prepared in methanol. Different dilutions (62.5-1000 μg/mL) of essential oil and standard ascorbic acid were separately prepared in methanol. Then, 2 mL of diluted sample and 2 mL of DPPH were vortexed and incubated at 26°C for 30 min. After incubation, the maximum absorbance (Ab) was recorded at 517 nm against a blank (2 mL DPPH + 2 mL methanol) by using an ultraviolet-visible (Genesys TM 10S UV-Vis, Thermo Scientific, USA) spectrophotometer. The experiment was performed in triplicate. The percentage of FRS was determined using the following equation:

Fecl 2 reducing assay
The FeCl 2 reducing assay was performed using a previously reported method [37], with minor modifications. In brief, stock solutions (1000 μg/mL) of oils and ascorbic acid were used for preparing different dilutions (200-25 μg/mL). Approximately 1 mL of different concentrations was separately added to 2.5 mL, each of 0.2 mol/L (pH 6.6) phosphate buffer, and 1% C 6 N 6 FeK 3 solution. These solutions were incubated for 30 min at 50°C. To 2.5 mL of above mixtures, 10% TCA was added. Later, these mixtures were centrifuged for 10 min at 3000 g. Approximately 2.5 mL of supernatant of the centrifuged solution was mixed with double distilled water (2.5 mL) and then, the mixture was shaken with 0.5 mL of freshly prepared 0.1% FeCl 3 solution and kept for 10 min. The value of absorbance was noted at 700 nm against blank (n = 3).

Albumin denaturation assay
In vitro anti-inflammatory potential of GCEO was estimated using the BSA denaturation method, with slight modifications, as described by Alam and Singh [38]. The GCEO and ibuprofen were dissolved in methanol to prepare 1 mg/mL solution. Further, five dilutions of GCEO and ibuprofen ranging from 6.25 to 100 μg/mL were prepared in phosphate buffered saline (PBS; pH = 6.8). Approximately 100 μL of essential oil or standard, 1000 μL of BSA (1%) and 1400 μL of BSA were thoroughly mixed. This mixture was incubated at 37°C for 15 min; then, the reaction mixture was heated at 72°C for 5 min. The reaction mixture was cooled, and the absorbance was determined at 660 nm against a blank (1000 μL of 1% BSA solution and 1500 μL PBS) by using a UV-Vis spectrophotometer (Genesys TM 10S UV-Vis, Thermo Scientific, USA). The percentage of inhibition (protein denaturation) of three repeated experiments was determined using the following equation:

Proteinase inhibitory assay
The proteinase inhibitory activity of GCEO was evaluated using trypsin method with slight modifications [39]. Six dilutions of standard and test samples ranging from 6.25 to 200 μg/mL were prepared in methanol. Approximately 1 mL of test sample, 0.06 mg trypsin and 1 mL Tris-HCl buffer (20 mM) were thoroughly mixed. This mixture was incubated at 37°C for 15 min, and then, 1 mL of casein (0.8% w/v) was added to the mixture. The reaction mixture was further incubated at 37°C for 20 min. Subsequently, 2 mL of per-chloric acid (70%) was added to the mixture. The reaction mixture was centrifuged for 5 min at 3000 rpm. The supernatant was separated, and the absorbance was measured at 210 nm against a blank (reaction mixture without sample and standard solution). The percentage of inhibition (protein denaturation) of three repeated experiments was determined using the following equation:

Molecular docking studies
In the present study, the first and second (α-terpinyl acetate and 1,8-cineole respectively) major compounds were selected for the molecular docking. AutoDock-4.2.6 tool was applied for creating the file of PDBQT from traditional PDB files [40]. Three-dimensional (3D) structures of 1HD2: peroxiredoxin 5 proteins [41], 3CC6: tyrosine kinase 2 beta [42] and 3V99: lipoxygenase [43] were downloaded from the PDB database. For molecular docking, the structures of α-terpinyl acetate and 1,8-cineole were drawn and then converted into the PDB format by using ACD/ChemSketch (Version 21) and an open Babel software [44], respectively. The ligands were first optimized and then docked into targeted proteins by using the "Ligand fit" model (AutoDock 4.2.6). Then, the 3D structures of the 1HD2-, 3CC6-and 3V99-targeted proteins were docked with the ligands by using AutoDock software. The BIOVIA Discovery Studio Visualizer 21 software [45] was used for the analysis of docking results. The binding energy, residues and length between the target protein and target ligand were calculated.

PASS and ADME prediction studies
For activity and drug-likening prediction, seven significant percentage compounds α-terpinyl acetate, 1,8cineole, α-terpineol, terpinen-4-ol, β-pinene, linalyl acetate and β-selinene were selected. The structure of the selected compounds was drawn using Chem-Draw (ChemDraw ® JS) ( Figure 5) and converted into the SMILES format and then the SMILES format was simulated using PASS online web tool [46]. The prediction of activity spectra for biologically active substances (PASS), which predicts the probable activity (Pa) and probable inactivity (Pi), was used to predict the pharmacological activities of major compounds present in GCEO by using PASS online web tool. ADME properties of the major compounds (α-terpinyl acetate, 1,8-cineole, α-terpineol, terpinen-4-ol, β-pinene, linalyl acetate and β-selinene) were also predicted using Swis-sADME tool software [47]. The ADME study predicts the lipophilicity, physicochemical properties, drug-likeness and toxicity of compounds.

Statistical analysis
Data were analysed using GraphPad InStat software and is presented as the mean ± standard deviation. The IC 50 value (the essential oil concentration providing 50% inhibition) was calculated from the graph by plotting the percentage inhibition versus concentration of essential oil. Regression equations, correlation coefficient (r), and level of significance (p ≤ 0.05) were used for analysing the antioxidant and anti-inflammatory results.
The percentage yield of Guatemala cardamom was found to be similar to that reported in a previous study [48]. The aroma of GCEO is primarily due to the combined effects of α-terpinyl acetate and 1,8cineole [49]. Several studies have reported that αterpinyl acetate is a major component of the cardamom essential oil [6,50]. The concentration of αterpinyl acetate metabolite is higher than that of other metabolites in high-quality cardamom essential oil; therefore, it may be used as an indicator for the identification of superior-quality cardamom fruits. In volatile oil of Guatemala cardamom, the total concentration of monoterpenes was found to be 87.05%, of which the concentration of oxygenated monoterpenes and monoterpene hydrocarbons was found to be

Antioxidant activities
The DPPH scavenging method was used to assess the antioxidant properties of GCEO. At high concentrations, GCEO has been discovered to have a substantial antioxidant action. At a concentration of 1000 μg/mL, the DPPH-induced free radical scavenging activity of GCEO was determined to be around 67.04 ± 0.04% (Figure 2(A)). Standard ascorbic acid's antioxidant potential was determined to be 99.1 ± 0.07 percent higher than that of GCEO. The IC50 was 588.6 μg/mL, which was larger than the usual ascorbic acid value of 24.02 μg/mL. GCEO's reducing ability was found to be lower than that of ordinary ascorbic acid (Figure 2(B)). In the FeCl2 reduction model, the absorbance value of GCEO (0.448 ± 0.01) of GCEO in the FeCl 2 reducing model was found to be lower than that of ascorbic acid (1.254 ± 0.02). A dose-dependent, noteworthy (p < 0.001) increment in the antioxidant potential, along with an increase in concentration effects, was observed.
The IC 50 value of GCEO indicated a weaker inhibition of the DPPH free radical compared to the standard compounds. The antioxidant effects of essential oils are mainly due to the presence of α-terpinyl acetate, 1,8-cineole, linalyl acetate, β-pinene, α-terpineol and terpinen-4-ol [53][54][55]. Furthermore, our studies align with a previous study [56]. The FeCl 2 assay is dependent on the formation of potassium ferrous cyanide after the reaction between potassium ferric cyanide and tested compounds. Han et al. (2017) reported that the reducing property increases with an increase in the concentration of essential oils [57]. Several studies have revealed that E. cardamomum has a good antioxidant potential against oxidative stress [48,58].

Anti-inflammatory activity
The BSA-and trypsin-induced inflammation assays were assessed to determine the anti-inflammatory activity, and the outcomes are shown in Figure 3(A and B), respectively. The inhibition of albumin-induced inflammation by GCEO extended from 16.98% ± 0.18% to 54.16% ± 0.12% at a concentration range of 6.25-100 μg/mL. The inhibition of trypsin-induced inflammation increased from 14.64% ± 0.94% to 54.82% ± 0.23% with an increase in the concentration from 6.25 to 200 μg/mL. The value of anti-inflammatory activity of GCEO was found to be comparable (p < 0.001) to the standard ibuprofen. The IC 50 values of GCEO were found to be 83.86 and 159.1 μg/mL in the egg albumin and trypsin assay, respectively, which were higher than those of the standard ibuprofen (25.95 and 32.23 μg/mL in the egg albumin and trypsin assay, respectively). In both methods, a dose-dependent increment in the antiinflammatory potential, along with an increase in concentration effects, was observed.
The anti-inflammatory potential of GCEO at similar concentrations of standard (Ibuprofen) exhibited significant (p < 0.001) inhibition of albumin-and trypsininduced inflammation, which indicated the anti-inflammatory properties of GCEO. Several pharmacological studies have been conducted to determine the activity of cardamom essential oil. The present study indicates that GCEO exhibits moderate anti-inflammatory potential. The anti-inflammatory activity of GCEO may be attributed to the presence of a high percentage of αterpinyl acetate, 1,8-cineole, linalyl acetate, α-terpineol, β-pinene, terpinen-4-ol, β-selinene and several other components [25,54,59].
The major metabolite α-terpinyl acetate demonstrated a highest binding-affinity against the lipoxygenase (LO) receptor (PDB ID: 3V99) with the docking score of −6.68 kcal/mol and the lowest affinity against the peroxiredoxin-5 receptor (PDB ID: 1HD2) with a score of −4.96 kcal/mol. The second major compound 1,8-cineole demonstrated similar binding affinities of −5.20 kcal/mol, −5.17 kcal/mol and −5.11 kcal/mol against the 3V99, 3CC6 and 1HD2 receptors, respectively. Docking (molecular) examinations were employed broadly for the assessment of ligand-target connotation to enhance understanding of the pharmacological properties of natural compounds. It delivers mechanisms of probable action and mode of binding within the protein pockets [60]. Two major components of GCEO (α-terpinyl acetate and 1,8-cineole) were selected for docking studies to support the present in vitro antioxidant and anti-inflammatory activities. The compounds, α-terpinyl acetate and 1,8-cineole, were docked with two antioxidant receptors, peroxiredoxin-5 proteins (PDB: 1HD2) and tyrosine kinase 2 beta (3CC6), and one anti-inflammatory receptor, lipoxygenase LO (PDB: 3V99). The docking scores ranged from −4.96 to −6.68 kcal/mol. α-terpinyl acetate interacted with a few amino acid residues within 3V99, 3CC6 and 1HD2, with docking scores of −6.68, −5.68 and −4.96 kcal/mol, respectively. Similarly, 1,8-cineole interacted with several amino acid residues within 3V99, 3CC6 and 1HD2, with docking scores of −5.20, −5.17 and −5.11 kcal/mol, respectively. These results indicated that both these compounds are responsible for the antioxidant and anti-inflammatory activities of GCEO. Interactions of α-terpinyl acetate with 3V99, 3CC6 and 1HD2 proteins indicated ARG666, THR498, ASP113 and SER115, respectively, whereas the interaction of 1,8-cineole with the 3V99, 3CC6 and 1HD2 proteins indicated ASN554, THR498 and ASP113, respectively. Lipoxygenase LO (PDB: 3V99) is a potential target to predict the anti-inflammatory activity of a compound [61]. Human peroxiredoxin-5 (PDB: 1HD2) and tyrosine kinase 2 beta (3CC6) are a potential target to predict the antioxidant potential of drugs [62].

PASS and ADME prediction studies
Seven major compounds ( Figure 5) of GCEO were selected for the antioxidant (Pa) and anti-inflammatory (Pi) prediction studies using the PASS online tool. Table 3 presents the predicted PASS results. The pharmacokinetic features of all major GCEO compounds were also determined using an online tool (SwissADME). The study outcome revealed that all selected compounds fulfil the Lipinski's rule and strongly support oral bioavailability properties ( Table 4). The bioavailability radar graph, which represents physicochemical, pharmacokinetics and drug-likeness properties, is shown in Figure 6(A), and the gastrointestinal (GIT) absorption and blood-brain penetration properties of selected compounds are illustrated in Figure 6(B).  To assess the possible antioxidant and anti-inflammatory profile of the major compounds of GCEO, was used PASS. For all seven compounds, the possibleactivity (Pa) range of 0.132-0.422 and 0.337-0.751 for antioxidant and anti-inflammatory actions, respectively, support our laboratory investigations suggesting good antioxidant and moderate anti-inflammatory effects.
The in vitro GCEO pharmacological effects observed may be due to the combined effect of many compounds. The antioxidant potential among the identified compounds terpinen-4-ol, β-pinene and linalyl acetate -exhibited acceptable Pa values (0.422, 0.399 and 0.384, respectively). However, other compounds exhibited lower antioxidant effects as per the PASS Figure 5. Chemical structure of the major volatile constituents, found in GCEO. GCEO compounds. Results of the online prediction software ADME obtained from in silico ADME Swiss software studies clearly indicated that all the selected volatile compounds have drug-like properties with no violation of any of the drug-likeness rules [63,64]. High GI absorption was predicted for most of the selected compounds; most compounds can be easily absorbed by the GI tract. According to the present Swiss-prediction, the major compounds found in GCEO cannot be affected by the P-glycoprotein of the central nervous system. Drug-like properties and GI absorption of the major compounds of GCEO were also predicted using the bioavailability radar graph and BOILED-Egg prediction. The pink area of radar graph represents drug-likeness, and the compounds present in the yellow zone (except β-selinene) can permeate through the blood-brain barrier.
Overall, molecular docking study of major compounds (α-terpinyl acetate and 1,8-cineole) and PASS prediction study indicated the strong antioxidant and anti-inflammatory potential of cardamom oil as well as ADME predicts the drug likening properties of majority of the compounds. In the docking, α-terpinyl acetate and 1,8-cineole have shown good binding with the  human peroxiredoxin 5 and protein tyrosine kinases receptors. These receptors play an important role in controlling the abnormal growth, metabolism, differentiations and motility of the normal cells through antioxidant mechanism [29,30]. Further, these molecules are studied in PASS prediction to prove the better antioxidant activity. Our in vitro and prediction antioxidant results were strongly supported by several recent studies reporting potent antioxidant effects of cardamom essential oil [25,52]. It is now emphasized that these molecules could be a better antioxidant by PASS predictions.
In addition, the α-terpinyl acetate and 1,8-cineole have been studied for anti-inflammatory activity through inhibition of 5-lipoxygenase enzymes [65,66]. The in vitro anti-inflammatory activity findings are well correlated with the molecular docking and antiinflammatory activities by PASS. The present findings (in vitro and in silico) are also supported by al-Zuhair et al. [20] and Heimesaat et al. [21]. Based on our findings and correlations with other investigators, we have strongly proven that these molecules might be a better antioxidant and anti-inflammatory properties and a good source of future drugs.

Conclusion
Despite the extensive use of essential oils in perfumes, food and pharmaceutical products, modern science has barely explored this treasure scientifically. In the present study, essential oils from cardamom fruits obtained from Guatemala (a large producer) were assessed. GCEO demonstrated strong antioxidant and moderate anti-inflammatory activities. Molecular docking studies of two major compounds (α-terpinyl acetate and 1,8-cineole) of GCEO with the proteins 3V99, 3CC6 and 1HD2 theoretically exhibited activities which are in agreement with the in vitro antioxidant and anti-inflammatory results, as indicated by their significant protein-ligand interaction energy. The antioxidant receptors, human peroxiredoxin 5 and protein tyrosine kinases, as well as the anti-inflammatory receptor 5-lipoxygenase, may be triggered by the essential oil effects of green cardamom. The essential oil of cardamom may be a natural and alternative treatment for oxidative stress and inflammatory reactions. The major compounds present in GCEO exhibited promising in silico docking results, where the PASS prediction supports the in vitro antioxidant and anti-inflammatory outcomes. SwissADME studies clearly indicated that the compounds present in GCEO have drug-like properties. The presence of volatile chemicals in the essential oil of cardamom fruits, such as α-terpinyl acetate, 1,8cineole, linalyl acetate, α-terpineol, β-pinene, terpinen-4-ol and β-selinene, may be responsible for the antioxidant and anti-inflammatory properties. In the future, animal models will be necessary to validate current in vitro or in silico results.