Chemical Profiling and Antioxidant, Antimicrobial, and Hemolytic Properties of Euphorbia calyptrata (l.) Essential oils: in Vitro and in Silico Analysis

Abstract In this work, we sought to validate the use of Euphorbia calyptrata (L.), a Saharan and Mediterranean medicinal plant, in traditional pharmacopeia. GC‐MS/MS identified volatile compounds of potential therapeutic interest. Antioxidant tests were performed using β‐carotene decolorization, DPPH radical scavenging, FRAP, beta‐carotene bleaching, and TAC. The antimicrobial activity was evaluated on solid and liquid media for bacterial and fungal strains to determine the zone of inhibition and the minimum growth concentration (MIC) of the microbes tested. The hemolytic activity of these essential oils was assessed on red blood cells isolated from rat blood. Phytochemical characterization of the terpenic compounds by GC‐MS/MS revealed 31 compounds, with alpha‐Pinene dominating (35.96 %). The antioxidant power of the essential oils tested revealed an IC50 of 67.28 μg/mL (DPPH), EC50 of 80.25.08±1.42 μg/mL (FRAP), 94.83±2.11 μg/mL (beta carotene) and 985.07±0.70 μg/mL (TAC). Evaluating solid media‘s antibacterial and antifungal properties revealed a zone of inhibition between 10.28 mm and 25.80 mm and 31.48 and 34.21 mm, respectively. On liquid media, the MIC ranged from 10.27 μg/mL to 24.91 μg/mL for bacterial strains and from 9.32 μg/mL to 19.08 μg/mL for fungal strains. In molecular docking analysis, the compounds naphthalene, shogunal, and manol oxide showed the greatest activity against NADPH oxidase, with Glide G scores of −5.294, −5.218 and −5.161 kcal/mol, respectively. For antibacterial activity against E. coli beta‐ketoacyl‐[acyl carrier protein] synthase, the most potent molecules were cis‐Calamenene, alpha.‐Muurolene and Terpineol, with Glide G‐scores of −6.804, −6.424 and −6.313 kcal/mol, respectively. Hemolytic activity revealed a final inhibition of 9.42±0.33 % for a 100 μg/mL concentration. The essential oils tested have good antioxidant, antimicrobial, and hemolytic properties thanks to their rich phytochemical composition, and molecular docking analysis confirmed their biological potency.


Introduction
According to the World Health Organization (WHO), in 2002, almost 80 % of the world's population relies on traditional medicine to access primary health care. [1]This reliance on traditional medicine has led to significant economic benefits, particularly in the development of this medical approach and the use of medicinal plants to treat various ailments. [1,2]As a result, it has become more crucial than ever to pursue research to identify the potential active compounds present in these plants. [3,4][7] This research focuses mainly on plants used in traditional Moroccan medicine.These studies have explained the mechanism of these plants' therapeutic action and confirmed their use in traditional medicine.10] It is important to note that essential oils are renowned for their multiple virtues, notably their antibacterial and antioxidant effects. [11]The emergence of antibiotic resistance in bacteria is now a major public health concern.The problem of antibiotic resistance affects all bacterial strains and continues to worsen.Faced with this situation, it is increasingly crucial to look for alternatives, among which medicinal and aromatic plants are proving promising. [12,13]xidative stress plays a central role in many pathological processes, particularly aging-associated, such as atherosclerosis, cancer, autoimmune diseases, and conditions such as Parkinson's and Alzheimer's. [14]Synthetic antioxidants, once seen as a solution in the agri-food, cosmetics, and pharmaceutical sectors, have recently come in for sharp criticism.Indeed, there are long-term concerns about their teratogenic, mutagenic, and carcinogenic potential. [15,16]Plants offer an interesting alternative to synthetic antioxidants, as they contain natural antioxidants. [15]The search for natural substances with antibacterial and antioxidant properties derived from plants is of great scientific importance. [17,18]In this context, studies were undertaken to analyse the volatile compounds of Euphorbia calyptrata (L.), a plant among the species of the Euphorbia genus used in traditional Moroccan medicine. [19,20]This plant is particularly interesting for medicine and the quest for new molecules due to its widespread use among Saharan-Mediterranean populations to treat various infectious diseases.Examination of the chemical composition by GCMS and theoretical study of the terpenic composition extracted from Euphorbia calyptrata by molecular docking is the first part of the objective of this work.The second part of this work is to evaluate the biological activities (antibacterial, antifungal and antioxidant).The third part of the study evaluated the hemolytic activity to determine the in vitro toxicity of essential oils derived from a plant indigenous to the Mediterranean region, known for its use in traditional medicine.

Extraction and Characterization of Components within EO-EC
The essential oil (EO) from crushed seeds was extracted using hydrodistillation for 4 hours at a temperature of 100 °C using a Clevenger apparatus.The chemical profile of the essential oil of Euphorbia calyptrata (EO-EC) was determined using gas chromatography coupled with a spectrometer.For this purpose, we employed a Varian capillary column (Model: TR5-CPSIL-5CB) measuring 50 meters in length, 0.32 mm in diameter, and featuring a film thickness of 1.25 μm.The column underwent a gradual temperature increase, ranging from 45 to 290 °C, at a consistent rate of 4 °C per minute.The injector was consistently maintained at 280 °C, while the MS-Polaris-Q detector was held at 200 °C.Helium served as the carrier gas, flowing at a 1 mL/ min rate.EO-EC was injected in a volume of 1 μL after being diluted in the organic solvent hexane, employing the splitless injection technique.Electron ionization was carried out with an ionization energy of 70 eV.The ion source and interface temperatures were set to 200 °C and 350 °C, respectively.Mass scanning ranged from 30 to 650 m/z.Identification of phytochemical constituents within EO-EC involved comparing their Kovats indices, which were calculated based on the retention times of a series of linear alkanes (C4-C29), with the reference values from standard compounds found in the Adams library and the NIST-MS database version 2.0.

DPPH Radical Scavenging Assay for EO-EC
The antioxidant properties of EO-EC were assessed using established methodologies.In brief, 800 μL of DPPH (0.2 mM, dissolved in methanol) were combined with 200 μL of various EO-EC serial dilutions, ranging from 0 (utilized as the control) to 1 mg/mL.The resulting blend was incubated in darkness for 30 minutes at room temperature (R.T.).Subsequently, absorbances were measured at 517 nm, relative to a control sample comprising 800 μL of DPPH solution and methanol.Parallel positive controls employing quercetin or BHT and a blank control were prepared under identical conditions.The antioxidant activity was quantified as the absorbance inhibition (P.I.) percentage at 517 nm.The DPPH radical scavenging rate was calculated using the formula (1).The IC50 signifies the concentration, whether of EO-EC or BHT and quercetin, necessary to decrease the presence of free radicals (DPPH) in the reaction mixture by 50 %.A lower concentration indicates a higher effectiveness of the tested products: [21] Inhibition % Where, A1: absorbance of the control; A2: absorbance of the presence of sample.

Β-carotene Bleaching Assay of EO-EC
In this assay, the antioxidant capacity of the EO-EC is assessed by quantifying their ability to inhibit the oxidative degradation (bleaching) of β-carotene due to the oxidation products of linoleic acid.This method follows the procedure outlined by Msaada et al., 2017. [22]The β-carotene/linoleic acid emulsion was prepared as follows: 0.5 mg of β-carotene was initially solubilized in 1 mL of chloroform.Subsequently, 25μL of linoleic acid and 200 mg of Tween 40 were introduced into the mixture.The chloroform was completely evaporated using a rotavapor at 40 °C.Afterward, 100 mL of oxygen-saturated distilled water were added, and the resulting emulsion was vigorously stirred.For each test, 100 or 150 μL of the extract solution or the reference antioxidant (BHT) at a concentration of 1 mg/mL were added to 2.5 mL of the previously prepared emulsion.The test tubes were then placed in a water bath and heated to 50 °C.The negative control included all the reagents except the test sample, for which an equal volume of methanol was substituted.The kinetics of emulsion decolorization were continuously monitored at 490 nm, with readings taken at 15-minute intervals over 2 hours until the color of β-carotene completely vanished.The antioxidant activity of EO-EC is computed using the following formula (2): AE0 represents the absorbance at 490 nm of the samples tested at t = 0 minutes, while AE120 corresponds to the absorbance at 490 nm of the samples tested at t = 120 minutes.AC0 represents the absorbance at 490 nm of the control at t = 0 minutes, and AC120 represents the absorbance at 490 nm at t = 120 minutes.

Ferric Reducing Antioxidant Power (FRAP) Assay of EO-EC
The ferric reduction process relies on using antioxidants to convert ferric iron into ferrous salt, forming a blue solution.
Here is a summary of the procedure: In glass tubes, 200 μL of EO-EC at various concentrations and 500 μL of 0.2 M phosphate buffer (pH 6.6) were combined, followed by the addition of 500 μL of 1 % potassium hexacyanoferrate (K 3 Fe(CN) 6 ) in distilled water.The mixture was heated to 50 °C and incubated for 20 minutes in a water bath.Afterward, 500 μL of 10 % trichloroacetic acid was added, and the solution was centrifuged at 3000 rpm for 10 minutes.A 500 μL aliquot of the supernatant was then transferred to another tube, to which 500 μL of double-distilled water (ddH2O) and 100 μL of freshly prepared 1 % FeCl 3 in ddH2O were introduced.A blank, without including EO-EC, was similarly prepared by substituting EO-EC with methanol.Absorbance was measured at 594 nm against the blank, where EO-EC was replaced with methanol, serving as the instrument calibration on a UV-VIS spectrophotometer.Standard antioxidant solutions, either BHT or quercetin, were utilized for positive controls, and their absorbances were recorded in the same manner as for the test samples. [23]

4. Total Antioxidant Capacity (TAC) of EO-EC
The Total Antioxidant Capacity (TAC) of EO-EC was determined using the phosphomolybdate method, as follows: In brief, 100 μL of EO-EC at various concentrations were combined with 1000 μL of a reagent mixture containing sulfuric acid (H 2 SO 4 ), sodium phosphate (Na 2 HPO 4 ), and ammonium molybdate, ensuring their concentrations fell within the ranges of 0.6 M, 28 mM, and 4 mM, respectively.The tubes were then subjected to a temperature of approximately 95 °C for 90 minutes.After cooling, absorbance was measured at 695 nm.The control consisted of 100 μL of methanol mixed with 1000 μL of the reagent mixture.Both samples and controls were incubated under identical conditions.The results are expressed in milligrams of ascorbic acid equivalents per gram (μg AAE/mg). [24]4.Antimicrobial Activity

Determination of the Zone of Inhibition on Solid Media
The disc diffusion test (diameter: 6 mm) was used to assess the antimicrobial activity of E. calyptrata essential oil.In brief, microbial strains, including Staphylococcus aureus (S. aureus), Escherichia coli (E.coli), Klebsiella pneumoniae (K.pneumoniae), Pseudomonas aeruginosa (P.aeruginosa), Condida albicans (C.albicans) and Saccharomyces cerevisiae (S. cerevisiae), were grown in Mueller-Hinton (MH) and malt extract (ME) media.Fresh cultures previously grown in MH and ME media were then used to make decimal dilutions in sterile saline (NaCl: 0.1 %).The final concentration of inoculum (microbial suspension) was set at 10 6 -10 8 CFU/mL.Bacteria and fungi were inoculated into Petri dishes and treated with 20 μL of EO-EC using six-millimeter diameter discs.Conventional antibiotics, such as Streptomycin (Str) for bacterial strains and the antifungal Fluconazole (Flu) for fungal strains, the antimicrobial potency of EO-EC was determined by calculating the zone of inhibition (mm). [25,26]

Determination of minimum EO-EC Concentration (MIC)
The MICs of EO-EC against four bacterial and two fungal strains were determined using established methods.In brief, a sterile 96-well microplate was prepared in aseptic conditions by dispensing 100 μL of EO-EC into the first column of the plate.Subsequently, 50 μL of MH (for bacteria) or ME (for fungi) was added.Serial dilutions were performed with a twofold dilution technique.MICs were determined via a colorimetric method employing TTC (0.2 %).This assessment took place for bacteria one-day following injection, while for fungal strains, it was conducted two days post-inoculation. [13,27].5.Molecular docking

Protein Selection
For our computational investigation, we focused on NAD(P)H Oxidase to assess antioxidant activity. [28]Additionally, we explored the antibacterial activity on E. coli beta-ketoacyl-[acyl carrier protein] synthase and S. aureus nucleoside diphosphate kinase [29] (3).Furthermore, we investigated the antifungal potential on sterol 14-alpha demethylase (CYP51) obtained from the pathogenic yeast C. albicans. [30]

Protein Preparation
NAD(P)H Oxidase, E. coli beta-ketoacyl-[acyl carrier protein] synthase, S. aureus nucleoside diphosphate kinase, and sterol 14-alpha demethylase (CYP51) obtained from the pathogenic yeast C. albicans were obtained from the Protein Data Bank (PDB) through the RCSB database (http://www.rcsb.org/)with following PDBID: 2CDU, 1FJ4, 3Q8 U, and 5FSA respectively.The structure was then imported into the Maestro interface.We followed the protein preparation protocol to ensure its suitability for computational calculations, which involved several steps.These steps included assigning bond orders, adding hydrogen atoms, creating disulfide bonds, and filling in missing side chains and loops using the Prime tool.We capped the termini to eliminate any potential interference and removed water molecules located beyond 5 Å from the hetero group.
Furthermore, we refined the chain by eliminating various heteroatoms and undesired crystallographic water molecules from the crystal structure.For accurate simulation under experimental conditions, we used the PROPKA tool to determine the protonation states of both ligands and residues.Finally, utilizing the OPLS3e force field, we performed energy minimization on the protonated structure, resulting in a lowerenergy protein structure. [31]

Ligand Preparation
All essential oil compounds identified via GC-MS were obtained from the PubChem database.These ligand structures were optimized using the LigPrep tool.To prepare the ligands at a pH of 7.0 � 2.0, we employed the Epik module to preserve specific chirality, desalting, and generating tautomers as required.The 2D ligand structures were then transformed into 3D structures and subjected to geometry minimization using the OPLS3e force field to attain their lowest energy conformations with necessary adjustments.These optimized ligands were subsequently utilized in the docking calculations. [32]

Binding Site Identification and Molecular Docking
We utilized the receptor grid generation tool within the Glide module to identify the docking sites for the ligands within the protein structure.This grid was created to encompass the region around the co-crystallized ligand.We intentionally excluded the co-crystallized ligand from the minimized protein structure to prevent undue influence on the ligand-receptor docking process.The receptor grid was generated using default parameters for Vander Waal's radius scaling factor (1 Å) and a partial charge cut-off (0.25 Å).Flexible docking employed the Glide module, with the ligands fitting into the receptor grid at the Standard Precision (S.P) docking level.Ligands that exhibited a higher affinity for the receptor's active sites were identified based on their molecular interactions with both the ligand and the target protein and their docking scores (5).

Validation of Docked Poses
To verify the precision of the docking process, we utilized the extra precision (XP) Glide docking approach.This entailed redocking the co-crystallized ligand and previously reported inhibitors into the binding site, employing the Glide XP docking mode.The bonding interactions identified during this redocking procedure and those reported in existing literature were employed to validate the docking outcomes.
Blood collected in heparin tubes from a healthy donor is used to prepare the erythrocyte suspension.It is centrifuged at 2500 rpm for ten minutes and, after removal of the plasma, the pellet is washed three times with PBS, then re-suspended in the same volume of discarded plasma.The resulting erythrocyte suspension is diluted 20-fold with PBS.
The hemolytic effect test is performed according to the method of Guo-Xiang and Zai-Qun. [33]In the hemolysis tubes, 20 μL of EO-EC at various concentrations were introduced into 1980 μL of the previously prepared erythrocyte suspension.Subsequently, these tubes were incubated at 37 °C for 60 minutes.After incubation, 250 μL samples were extracted from each tube and mixed with 750 μL of PBS.These mixtures were gently agitated and promptly transferred to an ice bath to halt the reaction.Subsequently, they were subjected to centrifugation at 2500 rpm for ten minutes.
Absorbance measurements were recorded at 548 nm utilizing a spectrophotometer, with a blank containing PBS as the reference.A total hemolysis tube was prepared in parallel conditions and following identical experimental protocols, consisting of 100 μL of erythrocyte suspension and 1900 μL of distilled water.A negative control tube was also created, comprising 250 μL of erythrocyte suspension and 750 μL of PBS buffer solution.The hemolysis rate for the various extracts is determined as a percentage of total hemolysis following a 60minute incubation period, calculated using the following formula:

Statistical Analysis
The results were expressed as the mean � standard deviation (SD).Statistical analyses were conducted using GraphPad Prism (version 8.0.1).The Shapiro-Wilk test was employed for the variables to assess normality, and Levene's test was used to evaluate the homogeneity of variances.Statistical disparities among means were computed through one-way analysis of variance (one-way ANOVA) followed by Tukey's test for multiple comparisons.Significance levels were set at p < 0.05

GC-MS/MS Analysis
Results from gas chromatography-mass spectrometry (GC-MS/ MS) analysis of compounds present in Euphorbia calyptrata (L.) essential oils (Table 1 and Figure 1).31 compounds are identified in EO-EC using GC-MS/MS.The table 3 include information on retention time (RT), compounds identified, retention indices (KI), chemical formulas, and peak area percentages.The list of identified compounds is diverse, covering a range of chemical classes, including monoterpenes, sesquiterpenes, diterpenes and oxygenates such as alcohols and ketones.Peak area percentages indicate the relative abundance of alpha-Pinene, representing 35.96 % of the total sample composition.
The analysis of the essential oil has revealed a diversity of chemical compounds, each belonging to a specific category based on its structure.Monoterpene hydrocarbons (MTH) constitute the largest portion of the total composition, accounting for 38.86 % of the essential oil.Monoterpene oxygenated compounds (MTO) are also present, albeit to a lesser extent, representing 4.07 % of the composition.Sesquiterpene hydrocarbons (STH) form a substantial part at 29.22 %, while oxygenated sesquiterpenes (STO) make a notable contribution of 16.84 %.Diterpene hydrocarbons (DTH) and oxygenated diterpenes (DTO) are in smaller quantities, accounting for 1.63 % and 3.14 % of the total composition, respectively.Lastly, an Others (O) category encompasses compounds that do not fit clearly into the previous categories, contributing 6.17 % to the overall composition of the essential oil.This distribution of chemical classes provides insight into the essential oil's complexity and its constituents' diversity (Table 1).
The compounds identified may play an important role in the pharmacological properties and chemical characteristics of Euphorbia calyptrata.Some of the compounds identified, such as alpha-pinene, Caryophyllene, and others, are known to have potential pharmacological properties due to their specific chemical structures. [34,35]These compounds could contribute to the pharmacological activities of Euphorbia calyptrata (L.). [19]he distribution of chemical classes within an essential oil is significant in pharmacology because each class of compounds possesses specific pharmacological properties.For instance, monoterpene hydrocarbons may exhibit anti-inflammatory and antimicrobial properties, while oxygenated monoterpenes are known for their antiseptic effects.Sesquiterpene hydrocarbons can assist in reducing inflammation, sesquiterpene oxygenated compounds have immunostimulant properties, and diterpenes can have various effects, including anticancer properties.8][39][40][41]

Antioxidant Activity
The data in Table 2 represent the results of the antioxidant activity of OE-EC at different concentrations, measured as a percentage of DPPH inhibition, with the associated standard deviations.At a concentration of 50 μg/ml OE-EC, the antiox-idant activity was 45.08 % � 0.20 %.This means that at this concentration, OE-EC reduces DPPH inhibition by 45.08 % on average, with some variability in the measurements represented by the standard deviation of 0.20 %.
Antioxidant activity increased with the concentration of EO-EC.At a concentration of 1000 μg/ml, antioxidant activity  2. BHT has the lowest IC 50 of the three substances, the most effective at inhibiting antioxidant activity.A lower concentration of BHT (40.58 μg/mL) is required to reduce antioxidant activity by 50 % compared with the concentrations used in the study.EO-EC has an IC 50 of 67.28 μg/mL, indicating that a slightly higher concentration of EO-EC than BHT is required to achieve the same level of 50 % antioxidant inhibition.Quercetin has the highest IC 50 of 72.81 μg/mL, suggesting it is less effective at inhibiting antioxidant activity than BHT and EO-EC (Table 2).An even higher concentration of quercetin is required to achieve the same inhibitory effect.These data provide a valuable indication of the relative ability of the substances to neutralize free radicals in the DPPH assay.
The choice of the DPPH test to assess antioxidant activity is relevant, as it is widely recognized in scientific research for its sensitivity to the ability of compounds to neutralize free radicals.This measurement method is commonly used to assess the antioxidant capacity of plant extracts. [42,43]In addition to the ability to neutralize free radicals, EO-EC has also shown an effect on free radical chains.This suggests that these essential oils can interrupt free radical chain reactions, which may have beneficial consequences for preventing oxidative stress. [15,44]The presence of powerful antioxidant compounds in EO-EC opens opportunities for developing pharmaceutical and cosmetic products. [14,45]These products could potentially help prevent or treat problems linked to oxidative stress, such as degenerative diseases, dermatological conditions, and signs of premature aging. [46,47]C 50 results for EO-EC, BHT, and quercetin measured using the FRAP method are shown in Table 3.The EC 50 is an important measure of antioxidant activity, indicating the concentration required to achieve a 50 % reduction in ferric ions in the FRAP assay.The EC 50 of EO-EC is 80.25 μg/mL � 1.42 μg/mL (Table 3).BHT requires a higher concentration (120.46 μg/mL � 2.03 μg/ mL) than EO-EC to achieve a 50 % reduction in ferric ions in the FRAP test.Quercetin had the lowest EC 50 of the three substances tested (74.48 μg/mL � 1.40 μg/mL), which was the most effective at reducing ferric ions in the FRAP assay (Table 3).The EC 50 results in the FRAP assay confirm that EO-EC has significant antioxidant capacity.Its EC 50 is lower than that of BHT, indicating that EO-EC is more effective than BHT in reducing ferric ions.This suggests that EO-EC could be a potentially useful antioxidant, making it attractive as a natural alternative to synthetic antioxidants such as BHT.
The results of this study firmly established a correlation between antioxidant activity, measured using the FRAP test, and the presence of antioxidant compounds in Euphorbia calyptrata (L.) essential oils.These antioxidant compounds, such as alpha-Pinene, Caryophyllene, and alpha-Muurolol (as the majority compound), play an essential role in the ability of this plant's essential oils to neutralize free radicals. [42,48]Identifying alpha-Pinene, Caryophyllene, and alpha-Muurolol as the main antioxidant compounds in Euphorbia calyptrata (L.) essential oils is essential.It is important to discuss in detail the ability of these compounds to neutralize free radicals, explaining how they act at the molecular level to protect cells and tissues from oxidative damage. [16,42,49]he results for inhibition (%) of beta-carotene by EO-EC, BHT, and quercetin are shown in Table 3. Inhibition of betacarotene by EO-EC was 94.83 � 2.11 %.BHT showed an even higher inhibition of beta-carotene oxidation of 86.42 � 1.35 %.Quercetin also showed a significant ability to inhibit betacarotene oxidation by 66.55 � 2.08 %.In this beta-carotene test, EO-EC proved to be the most effective antioxidant of the three substances tested, closely followed by BHT and then quercetin.
Essential oils from Euphorbia calyptrata (L.) contain antioxidants and have also been shown to affect free radical chains.It is necessary to discuss how these essential oils interrupt these reaction chains, which can benefit health, notably by preventing premature aging and diseases linked to oxidative stress.The presence of powerful antioxidants in Euphorbia calyptrata (L.) essential oils opens potential applications in medicine and cosmetics.These oils could be exploited to develop pharma- ceutical or skin care products aimed at preventing or treating problems linked to oxidative stress, [15,42] such as cardiovascular disease, [14] cancer or skin aging. [50,51]he results of the TAC (Total Antioxidant Capacity) test measuring the antioxidant activity of EO-EC, BHT, and quercetin at different concentrations (100 μg/mL, 250 μg/mL, 500 μg/mL, 1000 μg/mL) are presented in Table 4.At 100 μg/mL, EO-EC has an antioxidant activity of 80.25 � 1.42.This antioxidant activity increases with concentration, reaching 194.83 � 5.11 μg EAA/ mg at 250 μg/mL, 785.39 � 2.58 μg EAA/mg at 500 μg/mL, and 985.07 � 0.70 μg EAA/mg at 1000 μg/mL.
Antioxidant power by the phosphomolybdate method, at low concentration (100 mg/mL), revealed that BHT was the most effective, followed by EO-EC and quercetin, with antiradical power of the order of 120.46 � 2.03, 80.25.08 � 1.42 and 74.48 � 1.40 μg EAA/mg, respectively.The antioxidant activity of BHT also increased with concentration, reaching 360.44 � 7.12 μg EAA/mg at 250 μg/mL, 805.95 � 0.92 μg EAA/mg at 500 μg/mL, and 992.55 � 0.98 μg EAA/mg at 1000 μg/mL.Quercetin showed lower antioxidant activity than EO-EC and BHT at all concentrations tested.The antioxidant activity of quercetin also increased with concentration, reaching 156.55 � 3.45 μg EAA/mg at 250 μg/mL, 583.66 � 5.81 μg EAA/mg at 500 μg/mL, and 886.98 � 1.55 μg EAA/mg at 1000 μg/mL.The results show that EO-EC, BHT, and quercetin have significant antioxidant activity.EO-EC and BHT show similar antioxidant activities at all concentrations tested, with comparable activity values.EO-EC and BHT show similar antioxidant activities, suggesting that EO-EC has an antioxidant potential comparable to BHT, a commonly used synthetic antioxidant.
These results indicate that EO-EC could be a promising candidate as a natural antioxidant.However, further research is needed to assess its efficacy in various contexts and understand its mechanisms of antioxidant action. [52]The results of these studies provide solid evidence of the antioxidant activity of Euphorbia calyptrata (L.) essential oils and of the correlation between this activity and the presence of antioxidant compounds such as alpha-Pinene, Caryophyllene, and alpha-Muurolol. [46,53]These discoveries have great potential for the pharmaceutical industry, cosmetics, and medical research while raising exciting questions for future research.

Antimicrobial Activity
Table 5 displays the inhibitory zone diameters and MICs of E. calyptrata essential oils against the bacterial and fungal strains tested.E. calyptrata essential oils have been shown to have very significant inhibitory activity against the bacterial strains tested.
The EO of this plant was found to have a greater antibacterial effect against Gram (+) bacteria than against Gram (-) bacteria, such that it was found to have an inhibitory activity of the order of 25. 80 � 0.85 and 15.48 � 0.63 mm on solid medium, and a MIC of the order of 10.27 � 0.94 22.62 � 0.74 μg/ mL on liquid medium, for P. aeruginosa and K. pneumonia, respectively.
Essential oils of E. calyptrata are more antifungal than Fluconazole against S. cerevisiae and C. albican.This activity is due to the chemistry of the essential oils, which are high in alpha-Pinene, which has an antimicrobial effect.This molecule has been shown in studies to have an antibacterial effect against various Gram-positive bacteria, and alpha-Pinene has an inhibitory effect against S. aureus with a MIC of 256 μg /mL. [54]mong the majority of EO (5.08 %) beta-caryophyllene compounds is a bicyclic sesquiterpene with a rare 1,1-dimethylcyclobutane ring found in various plants' essential oils.The antibacterial activity of beta-caryophyllene has been reported for several bacterial strains, enhancing their antimicrobial potential.Previous research has found that beta-caryophyllene has antifungal activity.These findings suggest that the antifungal activity observed in our study may result from a combined effect of these EO sesquiterpenes. [55,56]
The docking analysis of Terpineol within the active site of E. coli beta-ketoacyl-[acyl carrier protein] synthase revealed the creation of a lone hydrogen bond with residue THR 300 (Figure 2 B and 3 B).Conversely, Naphthalene formed a single π-π stacking interaction with PHE 57 residue within the active site of Staphylococcus aureus nucleoside diphosphate kinase (Figure 2 C and 3C).In the case of Cadinol, when docked into the active site of sterol 14-alpha demethylase (CYP51) isolated from the pathogenic yeast Candida albicans, it exhibited the formation of a sole hydrogen bond with residue TYR 132 (Figure 2D and 3D).

Hemolytic Activity
Results relating to the inhibition of hemolytic activity at different concentrations are presented in Table 7. Hemolytic activity generally refers to the ability of a substance to prevent or reduce the destruction of red blood cells (hemolysis).The values shown are the percentage inhibition of hemolytic activity at different concentrations.
These results indicate that, as the concentration of the substance increases, there is a dose-dependent increase in the inhibition of hemolytic activity.In other words, higher concentrations of the substance lead to greater red blood cell destruction inhibition.Values expressed as percent inhibition of hemolytic activity indicate the extent to which the substance tested is capable of counteracting hemolysis. [57]The higher the inhibition percentage, the more effectively the substance protects red blood cells from destruction.This can be crucial in contexts such as drug development or research into the toxic effects of chemicals. [33]emolytic activity and inhibition percentages are important parameters in medical research, toxicology and related fields, as they enable us to assess how substances affect the fragility of red blood cells and may have important implications for human health. [58]In toxicology, hemolytic activity can be used to assess the potential harmful effects of certain substances on red blood cells.Strong inhibition of hemolytic activity may indicate that the substance protects against damage to red blood cells. [5]The measurement of hemolytic activity is often used in medical  research to assess the efficacy of drugs or chemical compounds in treating diseases associated with hemolysis, such as hemolytic anemias.Substances inhibiting hemolysis may have therapeutic potential for these affections. [33]

Conclusions
In conclusion, this study looked at essential oils derived from the plant Euphorbia calyptrata (L.), a species deeply rooted in traditional Moroccan medicine and widespread in the Saharan-Mediterranean region.The main objective was to examine their chemical composition and assess their antibacterial and antioxidant properties.The results of this study provide crucial information on the chemical constituents present in the oils of this botanical specimen, as well as their potential roles as antibacterial and antioxidant agents.These data have significant implications for traditional medicinal practices and the ongoing search for new therapeutic compounds.Consequently, this study enhances our understanding of the plausible medicinal properties inherent in Euphorbia calyptrata (L.), laying the foundation for future research efforts aimed at exploiting these compounds to combat infectious diseases and improve general well-being.Cadinol interactions with the active sites of sterol 14-alpha demethylase (CYP51) obtained from the pathogenic yeast Candida albicans.

)
Do S: Optical density of the sample (EOFP); Do Nc: Negative control optical density; Do P.C.: Positive control optical density.

Figure 2 .
Figure 2. 2D Diagrams of ligand interactions with the active site.(a) and (c) Naphthalene interactions with the active sites of NADPH oxidase and Staphylococcus aureus nucleoside diphosphate kinase; (b) Terpineol interactions with the active sites of E. coli beta-ketoacyl-[acyl carrier protein] synthase; (d):Cadinol interactions with the active sites of sterol 14-alpha demethylase (CYP51) obtained from the pathogenic yeast Candida albicans.

Figure 3 .
Figure 3. 3D Diagrams of ligand interactions with the active site.(a) and (c) Naphthalene interactions with the active sites of NADPH oxidase and Staphylococcus aureus nucleoside diphosphate kinase; (b) Terpineol interactions with the active sites of E. coli beta-ketoacyl-[acyl carrier protein] synthase; (d):Cadinol interactions with the active sites of sterol 14-alpha demethylase (CYP51) obtained from the pathogenic yeast Candida albicans.

Table 1 .
Chemical composition of EO-EC essential oils analyzed by GC-MS/MS.

Table 2 .
Antiradical activity and IC 50 of essential oil derived from EO-EC, BHT, and quercetin by DPPH test.

Table 3 .
Antioxidant activity of EO-EC essential oils by FRAP and Betacarotene tests.

Table 4 .
Antioxidant activity of EO-EC essential oils by TAC assay.

Table 5 .
Inhibitory zone diameters and MICs of E. calyptrata essential oils.

Table 6 .
Docking results of ligands inactive.