Study of essential oil extraction from Moroccan Rosmarinus officinalis L. by distillation based on quantum chemical calculation and molecular dynamics simulation

Abstract In this work, essential oils were extracted from rosemary leaves (Rosmarinus officinalis L.) from the eastern region of Morocco using hydrodistillation (Hd) and steam distillation (Sd). The yield, chemical composition, thermal stability, and antioxidant activity of the essential oils obtained by the two methods were compared. Determining the chemical composition of rosemary essential oil, with GC-MS, showed a significant qualitative and quantitative difference in certain components. The Sd method improved the extraction of sesquiterpene compounds with high molecular weight and low vapour pressure. The findings reveal that sesquiterpene and oxygenated monoterpenes in essential oils improved the antioxidant activity and thermal stability. Determining antioxidant activity using DPPH and the β-carotene bleaching assay showed that the Sd and Hd oils presented good radical scavenging activity (IC50 = 4960 μg/mL for Hd oil and 4570 μg/mL for Sd oil), capable of delaying β-carotene discolouration with 16.3 and 9.71% inhibition for Sd and Hd respectively. Thermal stability analysis of the oil products was performed using TGA/DTG analysis, and the results showed that the decomposition points of Hd oil and Sd oil were 362 and 368 K respectively. Furthermore, to identify intermolecular interactions and develop better knowledge of phenomena occurring during the extraction of the family of sesquiterpene compounds by water, quantum chemical calculation and molecular simulation by molecular dynamics (MD) were performed. The results suggested the relative stability of caryophyllene-water and the development of intermolecular bonding interactions of van der Walls and steric effect, improving extraction of the substance studied under steam distillation conditions.


Introduction
Rosemary (Rosmarinus officinalis L.), Salvia Rosmarinus species, belonging to the Lamiaceae family, has been one of the most widely used plants for centuries, due to its wealth of bioactive compounds and its many medicinal effects.Among the plants in this family, basil is also known for its medicinal properties.In traditional medicine, basil leaves are used to treat various diseases such as diarrhoea, cancer, and convulsion 1 .In addition, basil essential oils, which are rich in linalool, have attracted particular interest due to their use in many of the most popular fragrance formulations on the market 2 .
Experiments conducted with Rosmarinus officinalis L. essential oils have demonstrated significant pharmacological effects such as antioxidant, anti-inflammatory, anti-carcinogenic, anti-diabetic and anti-depressant 5 .They are used as a preservative in the food industry, capable of extending the shelf life of food products and retaining their quality during storage 6 .
Several parameters such as plant organ, genetic variety, environmental growing conditions, and extraction process can influence not only extraction time and yield, but also the chemical composition of the extracted oils 7 .Variations in chemical composition result in different behaviours in terms of biological activity and thermal characteristics, and thus in terms of the quality and applicability of the essential oil 8 .
In the eastern region of Morocco, two methods for extracting essential oils (hydrodistillation (Hd) and steam distillation (Sd)) are still widely used.They have the advantage of giving high essential oil yields and respectable production quality.
In hydrodistillation or water distillation, plant material is directly immersed in distilled water, which is boiled.The volatile aromatic compounds and water form an azeotropic mixture which can be evaporated together at a similar pressure and then condensed and separated due to their immiscibility and difference in density.This technique is easy to use and produces high-quality essential oil.However, with this distillation process, components were separated according to their degree of water solubility rather than their boiling point, so some of the volatile compounds and other constituents such as esters, under the prolonged action of hot water, degrade and undergo partial hydrolysis and rearrangement reactions 9 .
Furthermore, the phenomena that control steam distillation are the transfer of oil from the plant surface and its evaporation 10 .The steam passes through the plant matter that contains the compounds which have been transferred to the surface of the plant matrix for separation.The steam condenses and forms a mixture of steam and plant matter compounds.This mixture gets heated further by passing more steam, which continues to pass through the plant matter, evaporating the mixture.Recovery of the essential oil is facilitated by the distillation of two immiscible liquids, namely, water and essential oil, based on the principle that, at boiling temperature, the combined vapour pressure is equal to the ambient pressure.However, the extraction of the components depended not only on their volatility but also on their polarity 9 .Sd is the most widely used technique due to its simplicity, efficiency and cost-effectiveness, minimising the loss of polar compounds and the degradation of heatsensitive compounds 11 .However, this technique is unable to block the presence of undesirable compounds 12 .
Generally, the amount of essential oil produced depends not only on operating conditions such as the length of distillation time, the temperature, and the operating pressure, but also on the accessibility of plant surface compounds for water and the intermolecular interactions between the different chemical groups of oil compounds and water 13 .The intermolecular interaction can be studied using the density functional theory (DFT) quantum chemistry calculation method and the molecular dynamics (MD) method to investigate the water-essential oil azeotropic system interaction.Density functiоnal theory (DFT) is an important method оf theоretical mоdeling and it was used to accurately predict the structures and physical and chemical properties оf mоlecules 14 .It reveals important details about the stable molecular shape of a molecular system, vibrational frequencies, stabilizing charge transfer interaction and potential local reactive sites 15 .Zhu et al. 16 applied the quantum chemical method to calculate the interaction energy, total charge density, deformed charge density and bond length of ionic liquid with butanol.Recently, the main components of essential oils and their interactions with water using the DFT method have been studied for various applications such as aromatherapy medicine and food chemistry [17][18][19] .
Molecular dynamics (MD) simulation is an approach for modelling molecules and studying their interactions on the atomic scale.Molecular dynamics (MD) simulation provides direct access to the microscopic structures of the target molecules which are not easily obtained in the experiment.Thanks to MD simulation, intermolecular interactions between essential oil components and water can be studied through radial distribution function analysis (RDF).
Shen et al. 20 used MD simulation to investigate the mechanism of extractive distillation and the extraction effect of solvents, based on the RDF calculation of organic solvents and ionic liquids.Recently, some works used these both methods to study the separation of a binary azeotropic system by extractive distillation using ionic liquid 21,22 .They studied the interactions between the solvent molecules and ionic liquids to investigate the separation mechanism at the molecular level.The nature of the interactions (H-bonds, van der Waals forces…) was studied through the reduced density gradient (RDG) function.The methodology and approach used can contribute to deeper understanding of the mechanism of interaction between azeotropes and entrainers for extractive distillation processes.
To our knowledge, no study has been carried out regarding Sd and Hd processes using the quantum chemistry calculation method and the molecular dynamics (MD) method to identify the noncovalent interactions in a mixture of steam or water and plant essential oils.The purpose of this study was to investigate the chemical composition of Moroccan rosemary essential oil obtained by hydrodistillation and steam distillation.The chemical composition of the essential oils obtained using these two techniques was characterized using gas chromatography coupled to mass spectrometry (GC-MS).Thermal properties were characterised by thermal analysis (ATD/ATG).In addition, the ability of essential oils, obtained by both processes, to scavenge free radicals was examined with the β-carotene bleaching test and the DPPH method.The difference in the chemical composition of the oils extracted by the two techniques was discussed based on vapour pressure estimation of essential oils' majority compounds.Finally, the nature of the interactions in operating conditions between a sesquiterpene compound, namely caryophyllene, and water was identified using the quantum chemistry method and molecular dynamics through the DFT, the reduced density gradient (RDG) function and radial distribution function analysis (RDF).

Study area and plant material
The essential oils were extracted from a wild population of rosemary (Rosmarinus officinalis L.) harvested in January 2022 in the rural area of Beni-chebal in the province of Taourirt in the eastern region of Morocco (34.172031,  -2.722161).This area has an arid climate.Annual rainfall varies between 230 and 280 mm, with an average value of 255 mm/year.The heaviest rainfall occurs in December and April, while August and July are the driest months.The average temperature is 289.4K, with minimum values of around 277K recorded in January and maximum values of around 315K in August 23 .The leaves of this plant, which give off an aromatic fragrance, are narrow, linear and opposite, dark green on top and lighter underneath.Its flowers are small and range in colour from pale blue to violet.

Hydrodistillation and steam distillation
Several techniques can be used to extract aromatic oils from plants, however, in the eastern region of Morocco (Taourirt), the classical, Hd and Sd techniques remain the most widespread within local cooperatives for the extraction of essential oils of Rosmarinus officinalis L.
The process for obtaining the essential oil with the Sd and Hd methods was conducted in a Clevenger-type distillation apparatus.For hydrodistillation, 100 g samples of dried leaves were immersed in 1L of distilled water.The extraction was carried out at 373K and atmospheric pressure for 3h from the first drop of distillate until the quantity of essential oils stabilised (Fig. 1-A).The distillation was carried out until there was no more essential oil.The condensation vapour produced an organic phase (essential oil) which was isolated from the hydrosol by decantation.The oils obtained were fig 1 dried with sodium sulphate to remove all traces of water and were stored in a refrigerator at 277K until their analysis and characterisation 24 .
Steam distillation was carried out in similar conditions to hydrodistillation.In this process, the plant material was separated from the water and placed in a separating funnel, the lower part of which was connected directly to a water flask while the upper part was connected to the Clevenger apparatus and a refrigerant.Hot water vapour rises through the plant material, carrying away volatile compounds, including essential oils to the condenser, then the steam is liquefied and the mixture of water and oil is separated (Fig. 1-B).The advantage of this method lies in its ability to extract essential oils, while preserving their quality through the use of moderate temperatures below 373K.
The moisture content of the dried sample was 13.90 ± 0.04% (w/w).All extractions were done in duplicate.The yield of essential oil was calculated using the following equation:

GC-MS compounds identification
The essential oils were analysed with gas chromatography using a Shimadzu GC apparatus (Kyoto, Japan) equipped with a BPX25 capillary column with a 5% diphenyl, 95% dimethylpolysiloxane phase (30 m × 0.25 mm internal diameter × 0.25 µm film thickness), coupled to a QP2010 MS.The mobile phase used was pure helium gas (99.99%) at a constant flow rate of 3 mL/min.The injection, ion source and interface temperatures were set at 523K.The temperature programme used for the column oven was 323K (held for 1min), heated to 523K at 10K/min and held for 1 min.The ionisation of the sample components was carried out in EI mode (70 eV).The mass range analysed was 40-300 m/z. 1 μL of each essential oil, diluted with hexane, was injected in splitless mode (split ratio 90:1).Compounds were identified by comparing their retention times (RT) with those of the standards and fragmenting their mass spectra.thermogravimetric analysis (TG) methods were performed on both raw clay samples using a SHIMADZU instrument (DTG-60 H).The experiment was performed on 5-10 mg of sample using a temperature range from 273 to 673K with a heating rate of 5K/min.

In vitro antioxidant activity
In this study, two in vitro methods, with some modifications, were used to determine the antioxidant properties of essential oils: the free radical reduction method (DPPH) and the β-carotene bleach test.For anti-free radical activity, a mass of 2 mg of the DPPH radical was dissolved in 100 mL of pure methanol, and then 1 mL of the DPPH solution was added to 1.5 mL of the methanol solution from the essential oil at different concentrations.After 30 minutes of incubation in the dark, the absorbance was measured using a spectrophotometer (PerkinElmer) at 517 nm.Ascorbic acid was used as a reference.All assays were performed in triplicate.The percentage inhibition (PI) of DPPH was determined using the following equation 25 : Where PI is the percentage of inhibition, A 0 , the optics density of the free radical (DPPH) solutions in the absence of the extract (negative control) and A, the absorbance of the free radical (DPPH) solution in the presence of the extract.
The anti-free radical power was expressed by the IC 50 value calculated from the regression curves.IC 50 corresponds to the inhibitory concentration required to reduce 50% of free radicals.The lower value of IC 50 indicated greater anti-free radical activity.
The β-carotene bleaching test was based on measuring the ability of essential oils to inhibit or delay the loss of the orange colour of β-carotene due to its reaction with free radicals from the oxidation of linoleic acid.The presence of antioxidant molecules in oils made it possible to neutralise the free radicals of linoleic acid and therefore inhibit the destruction and loss of the double bonds of the β-carotene molecule.The β-carotene/linoleic acid emulsion solution was (2)   prepared by solubilising 0.5 mg of β-carotene in 1.5 mL of chloroform, 30 µL of linoleic acid and 250 mg of Tween 80.The chloroform was removed using an evaporator, then 100 mL of oxygen-saturated distilled water (30%) was added.The resulting mixture was shaken vigorously to homogenise it.Then 175 µL of the essential oil solution at a concentration of 2 mg/mL was added to 1.25 mL of the previous emulsion.The reference solution was butylated hydroxy anisole (BHA).Similarly, the control sample was prepared by replacing the oil solution with methanol.The decolourisation of the emulsion of the negative control and the essential oils or BHA was monitored at 490nm for 120 min at constant intervals.The relative antioxidant activity of the extracts (RAA) was determined according to the formula below: Where A 120 is the absorbance of the solution that contains the extract after 120 min of incubation, C 120 and C 0 are respectively the absorbances of the control after and before 120 min of incubation.The kinetics of decolourisation were obtained by monitoring the decrease in absorbance (in UA arbitrary units) as a function of time.

Computational methods
The vapour pressure of the essential oil compounds was estimated with ASPEN software (AspenPlus 14.0, Aspen Technology, Bedford, MA, USA) using the NRTL model.Quantum chemical calculations were performed using the density functional theory (DFT)/B3LYP method with a 6-31 G (d, p) basis.Molecular structure, HOMO-LUMO energies, electronic properties, reactivity, and molecular electrostatic potential (MEP) were determined using Gaussian 09W and GausView version 6.0.16.Reduced density gradient (RDG) analysis was performed using the theory of non-covalent interactions (NCI) and the programs Multiwfn (multifunctional wave function analyzer) and VMD (visual molecular dynamics).
Molecular dynamics simulations were carried out using Materials Studio 7.0 software supplied by Accelrys ® .The three-dimensional structure of caryophyllene and water was downloaded from PubChem databases and imported into the software.The structures were geometrically optimised via the Forcite module using the COMPASS force field, to produce a more stable molecular geometry.The simulation box containing one caryophyllene molecule and 50 water molecules was constructed using the amorphous cell calculation module.After energy minimisation, the cell was subjected to a dynamic calculation using the Forcite program.The canonical NVT ensemble (a system of a fixed number of (N), in a fixed volume (V) and with a fixed temperature (T)) was performed at different temperatures and the time step for the simulation was 1 fs with a period of 500ps, controlled by the NHL thermostat.Then, the cell was equilibrated in the NVE ensemble for 500ps to obtain the final equilibrium system.The molecular trajectories were analysed to establish RDF plots to determine caryophyllene-water and linalool-water intermolecular binary interactions.

Experimental study
The yields of rosemary essential oils obtained by the Hd and Sd methods were respectively 1.49±0.18%and 1.89±0.13%.The excess found for the bulk oil yield of steam distillation over hydrodistillation confirmed the results obtained in previous work 10 .The average yield values obtained for steam distillation were close to those found for Tunisian rosemary 26 .For hydrodistillation, the average value obtained was of the same magnitude as that obtained by previous work on rosemary from the same region 27 and was very high, comparable to the values obtained for Tunis rosemary 26 .

Chemical composition of the rosemary oil obtained
In discussion of the analytical results, we will consider two approaches for the steam distillation results: (i) the calculation is based on the 14 hydrodistillation compounds (Sd 14 ) and (ii) by counting the total number of compounds detected (Sd 30 ).
Table 1 and Fig. S1 (supplementary data) show the results of GC-MS analyses of oils extracted by hydrodistillation (Hd) and steam distillation (Sd).It can be seen that steam distillation produced oils containing more volatile compounds (30 compounds) than oils obtained by hydrodistillation (14 compounds).The steam distillation made possible the extraction of essential oils containing more volatile compounds according to the results obtained for rosemary from Morocco's eastern region 10 .
In oils obtained by steam distillation, the monoterpene family accounted for only 84.7%, followed by compounds belonging to the sesquiterpene family (15.3%).The predominance classification between the 30 compounds (Sd 30 ) was 1.8-cineole (37.4%) > α-pinene (9.32%) > caryophyllene (7.13%) > β-Pinene (6.08%).Based on 14 compounds (Sd 14 ), the predominance ranking of compounds did not change for steam distillation.It was 1,8-cineole (42.3%) > α-pinene (10.7%) > caryophyllene (8.2%) >  1).New compounds from the sesquiterpene family were detected in Sd oils.This family represented 15.3% in Sd 30 , whereas it represented only 1.10% in Hd oils.These results can be explained by the fact that during hydrodistillation, the essential oils inside the plant tissue must diffuse to the plant's surface before evaporating.Heating activates diffusion and evaporation, with the aromatic molecules forming an azeotropic mixture with water vapour, the boiling temperature of which is lower than the boiling temperatures of pure substances 33 .Internal heating of water in situ in the inner part of the plant breaks up the oily and oleiferous bodies.In the case of steam distillation, the oleiferous cells break up further and release their essential oil content 34 .Diffusion is thus accelerated, making it possible to increase distillation yield and extract aromatic compounds with low vapour pressure, such as sesquiterpenes, which are difficult to obtain by hydrodistillation 7 .Moreover, the presence of a lower number of compounds in Hd oils than in Sd oils can also be attributed to the high solubility of some compounds in water when the plant is boiled in water during hydrodistillation 35 .The extraction of sesquiterpene compounds by steam distillation has been demonstrated for Argentine Rosmarinus officinalis L. 36 .In the presence of excess water, in the case of hydrodistillation, many of the hydrocarbon derivatives become vulnerable to chemical modification and degradation.Hydrodistillation causes potential losses of most polar (oxygenated) terpenes 37 ; as is the case with terpeniol, cis-beta, 4-terpineol, linalool, D-verbenone, p-menth-1-en-8-ol and nerolidyl acetate which only appeared in Sd oil.
Table 1 shows that when switching from hydrodistillation to steam distillation, although the number of oxygenated monoterpene derivatives detected increased from 5 to 10 compared to the number of monoterpene hydrocarbon derivatives which increased from 7 to 10, the extraction ratio (oxygenated/hydrocarbons) decreased slightly from 2.10 for Hd to 2.06 for Sd 30 (to 1.95 for Sd 14 ).The decrease in the rate of hydrocarbon degradation can be attributed to the small number of water molecules that coat the oily compounds in the case of steam distillation.Compared to monoterpenes, sesquiterpenes are difficult to isolate by hydrodistillation.When the plant is boiled in water during hydrodistillation, the high molecular weight and low vapour pressure of these molecules hinder the entrainment phenomenon by the flow of vapour.
Table S1 (Supplementary data) shows the variations in the chemical composition of rosemary essential oils around the world.Essential oils from Tunisia 39 and Algeria 40 stand out for their high content of 1,8-cineole, exceeding 50%, while those from Spain and France are characterized by a high concentration of α-pinene, followed by 1,8-cineole 41 .On the other hand, the majority compound of oils from Italy and Portugal is verbenone with a percentage of more than 20% 29 .The essential oil of India is characterised by the predominance of camphor 29 .The variation in the chemical composition observed in rosemary essential oils from different regions of Morocco and different countries is due to several factors such as climatic and geographical conditions that change from one region to another, the harvest period, the nature of the soil and the extraction method that also influences the quality of the essential oil.

Antioxidant activity
The result obtained for the antioxidant activity of essential oils extracted by the two methods Hd and Sd showed that both types of oil show antifree radical activity.The IC 50 values found for Hd oil and Sd oil were 4960 ± 170 and 4570 ± 40 µg/mL respectively.These values were very low compared with the values found by Oualdi et al. 32 (IC 50 = 13900 µg/mL) for oils extracted by steam distillation from rosemary from the eastern region of Morocco.This difference can be attributed to the abundance of volatile compounds in our oils, which have antioxidant properties, or to their synergistic interaction with other compounds, which can explain the low IC 50 values observed.They are higher than the values for oils extracted from rosemary originating from other regions of Morocco: Er-Rich 2770 µg/ mL, El Jadida (302 µg/mL), Taounate (258 µg/ mL), Beni Mellal (236 µg/mL) and Marrakesh (176 µg/mL) 42 .For Algerian rosemary, the IC 50 was found to be 430 µg/mL 43 .The essential oils richest in hydrocarbon and oxygenated monoterpenes have negligible antioxidant activity (IC 50 = 18780 µg/mL) 44 , which could explain the IC 50 values obtained in the current study.Monoterpenes represented 98.7% of Hd oils and 84.7% of Sd oils.A slight improvement in anti-free radical activity was observed in the Sd oils, due to the increase in sesquiterpene levels.Tunisian Rosmarinus officinalis L. oil was rich in sesquiterpenes (22.1%) in which caryophyllene represented 14.5% of oil compounds, and the IC 50 was found to be close to 110 µg/mL 45 .Caryophyllene, sometimes called β-caryophyllene, from the sesquiterpene family, has significant antioxidant activity 46 .
The β-carotene bleaching test showed a strong presence of molecules inhibiting the coupled oxidation of β-carotene and linoleic acid in oils extracted by Sd steam, RAA=16.30%,compared with oils obtained by Hd hydrodistillation, RAA=9.71%.However, it is very difficult to attribute the antioxidant effect of a total essential oil, containing different chemical compounds, to one or a more active ingredients 5 .The maximum RAA value (16.3%) remains very low compared to the BHA reference with relative antioxidant activity of the extracts close to 36.7%.
The kinetics of emulsion decolourisation for the negative control (methanol), the positive control (BHA) and the essential oils are shown in Fig. 2. Absorbance decreased for all four samples.For the positive control (BHA), the decrease in 120 min represented 50% of the initial absorbance.It exceeded 70% for the other samples (Fig. 2-A).The ratio of the absorbance at time t of each sample to the positive control shows that the absorbance of the sample containing the steam oils is markedly slow compared to the hydrodistillation oils (Fig. 2-B).This result highlights the greater antioxidant efficacy of Sd oils compared to Hd oils.This characteristic paves the way for their use as a source of antioxidants in the food industry to extend the shelf life of products, as well as in pharmaceutical and cosmetic formulations.

Thermal stability
The thermal stability of the essential oils obtained is illustrated by the curves of differential thermal analysis and thermogravimetric analysis in air (Fig. 3).After evaporation of all the volatile compounds, the quantity of solid residue remaining at 673K represents 1.17 and 3.25% respectively for the Hd and Sd oils.On the thermogravimetric analysis curves, both oils show almost total evaporation in a single stage.For Hd oils, the horizontal tray is obtained from 393K, while it is at 413K for Sd oils.The 20-degree increase in bearing temperature can be attributed to the presence of Sd oils with molecules with higher evaporation temperatures.For the maximum heat exchange expressed by the endothermic peaks of the DTA curves, the Sd oils showed a peak at a higher temperature (T = 368K) than the Hd oils (T = 362K).Both temperatures are low compared to the boiling temperatures of the oil constituents (Table 1).When terpenes in oils are exposed to temperatures above 313K, they become unstable and evaporate at lower temperatures 47 .
The presence of sesquiterpene compounds, which have a higher boiling point than monoterpene compounds, in Sd oils, could contribute to enhancing thermal stability, resulting in a higher plateau temperature in ATG and a peak in ATD.Table 1 clearly shows that the boiling temperatures of most compounds in the sesquiterpene family, which appear in Sd oils, are significantly higher than those of compounds in the monoterpene family.

Vapour pressure effect on Sd and Hd oil compositions
Thanks to the simulation using the NRTL model that was available in Aspen Plus software, it was possible to estimate the boiling temperature/ vapour pressure of the main compounds present in rosemary oil and, therefore, to highlight the possibilities for extracting sesquiterpene compounds using the Sd method.The evolution in the vapour pressure of the main compounds of rosemary essential oils, from the three main families: monoterpenes hydrocarbons, oxygenated monoterpenes and sesquiterpene hydrocarbons, as a function of temperature is presented in Fig. 4-A.The different curves can be classified into three groups.The first includes monoterpene hydrocarbon compounds that show an exponential evolution in the curves that accelerate at low temperatures.These terpenes were the first to be distilled in large amounts.
The second group corresponds to the oxygenated monoterpenes family, whose curves are slower and only accelerate from 353K.The third group shows a small evolution in vapour pressure as fig 4 a function of temperature.This group includes compounds of the sesquiterpene hydrocarbon family.The latter was only extracted in a minimal amount.This may be due to intermolecular interactions between different chemical groups and/or the molecule size.
As a result, monoterpenes distil quickly whereas sesquiterpenes require a longer distillation time, better accessibility of plant compounds for water, a higher amount of water or a lower entrainment ratio (R).The latter can be estimated using Raoult and Dalton's law, which gives the total pressure and the composition of vapours as a function of partial pressures 48 .It is expressed with the following equation: Where, O and W denote respectively essential oil and water, P is vapour pressure, and M is molar mass.
Fig. 4 gives the values of the entrainment ratio of the main constituents at 353K.Entrainment selectivity between chemical groups in an essential oil, such as between monoterpene hydrocarbons and sesquiterpene hydrocarbons, or between monoterpene hydrocarbons and monoterpene oxygenated compounds, can be established, according to the entrainment ratio values found for particular constituents.
The relationship between the vapour pressure, entrainment ratio and the yields of the oil compounds obtained by the two methods Hd and Sd is also presented in Fig. 4-B.Concerning the phytochemical composition of rosemary leaves, there was a notable correlation between vapour pressure and the efficiency of the distillation methods.The effectiveness of Hd is remarkable when the compounds in the oil have a high vapour pressure and entrainment ratio.This means that volatile compounds can easily be extracted using this method, resulting in high yields.Hd seems to be particularly suitable for oils rich in highly volatile compounds.Compounds with medium vapour pressure, indicate that they have moderate volatility.The yields obtained are comparable, suggesting that both processes are capable of effectively isolating oils containing moderately volatile compounds.In the case of compounds with a low vapour pressure and entrainment ratio, Sd is the most suitable method.Low-volatility compounds are extracted more by this method, resulting in high yields.Sd appears to be more effective for oils containing lowvolatility compounds.The role played by the entrainment ratio in the process of activating the extraction of molecules, from the sesquiterpene family by steam distillation is well manifested for caryophyllene, which has a low entrainment ratio.The interaction of the caryophyllene molecule with water has been investigated by quantum chemical calculations and molecular simulation using molecular dynamics.

Theoretical study
Electronic properties Fig. 4 shows the HUMO-LUMO analysis of pure caryophyllene and a caryophyllene-water mixture for a 1:1 molar ratio.The electron-donating ability of molecules is related to the HOMO energies.The molecules with higher HOMO orbital energy have stronger electron-donating abilities 15 .Fig. 5-A shows that caryophyllene provided the highest HOMO energy (−5.9534 eV).This clearly shows that caryophyllene has the strongest electron-donating capability.The limiting electron density of the occupied and unoccupied molecular orbitals of caryophyllene (Fig. 5-A) was modified by the introduction of a water molecule (Fig. 5-B).
The ionisation potential (IP) of the caryophyllene-water (6.2629 eV) system is higher than that of caryophyllene (5.9543 eV) (an increase of 5.18%).This implies that the mixture is less likely to take part in oxidation reactions.
Our results are in close agreement with those obtained by Akman et al. 14 .In their study of Phlomis bruguieri essential oil constituents, particularly for pure caryophyllene and caryophyllene-water mixture, using GausView version 5 software.However, they differ from those obtained by another study, including the one conducted by Ali et al. 49 , on the essential oil compounds of Mentha longifolia L. and Citrus reticulata L.
The molecular electrostatic potential maps (MEP) are straightforwardly obtained from the geometrical structures and are shown in distribution is related to the dipole moments, partial charges, electronegativity, and the molecule's site of chemical activity.Different colours represent the different values of the electrostatic potential on the surface: The red region represents the most negative electrostatic potential and corresponds to the electron-dense zone.The blue area represents the most positive electrostatic potential, corresponding to areas of low electron density; green represents the zone with zero electrostatic potential.The MEP map of the caryophyllene molecule is mapped in the spectrum of -0.02271 a.u.(deepest red) and 0.02271 a.u (deepest blue) (Fig. 5-C).The areas of high electron density are located around the double bonds, which means that they exhibit the strongest repulsive forces with other atoms.The low electron density zones are located around the hydrogen atoms of the molecule, so they have the strongest attraction for other atoms and are considered to be electron-poor regions.Low electron density regions can interact with other molecules, namely water.The study conducted by Taha et al. 50on modelling the quantum chemistry of river red gum essential oil compounds using Hyperchem 7.5 software revealed that spathulenol, the main component of essential oil, exhibits the highest electrostatic potential, ranging from -0.174 to +0.552 au, followed by eucalyptol.
The MEP map of the water-caryophyllene binary mixture (Fig. 5-D) is shown on a scale from -0.060035 at (dark red) to 0.06035 at (dark blue).The significant increase in electrostatic potential in the mixture results from the interaction between caryophyllene and water.More specifically, the high electron density zone is located above the oxygen atom of the water and represents the reactive zone for electrophilic attacks.In contrast, the low electron density zone is mainly concentrated around the hydrogen atoms of water.
The results obtained from the theoretical calculations of the various physical parameters for caryophyllene and caryophyllene-water were similar to those found in Akman's work 14 .A comparison of the Egap energy difference between the caryophyllene molecule and the caryophyllene mixture shows an increase of 0.12 eV, or 1.86% (Fig. 5).When the water molecule reacts with the caryophyllene molecule, it causes a decrease in the number of electrons in the LUMO orbital, which are likely to take part in chemical reactions, and consequently increases the chemical stability of the molecule.Similarly, adding the water molecule could lead to intermolecular interactions, such as hydrogen bonds and London dispersion forces, or eventually interactions between the oxygen in the water molecule and the hydrogens in the caryophyllene, and thus further stabilise the molecule.Thus, the molecular cohesive energy density of caryophyllene molecules can be reduced, leading to improved vaporisation.

NCI-RDG analysis
To gain deeper understanding of the different types of interactions present in the system, the use of the non-covalent interactions (NCI) approach to examine and define intramolecular or intermolecular interactions is very useful.It is based on the study of the reduced density gradient (RDG), allowing the visualisation of spatial interactions.The RDG is a unitless quantity composed of the electron density and the first derivative, and is expressed as follows 51 : Plotting the electron density against the peaks of the second eigenvalue of the electron density, expressed as a sign(λ 2 )*ρ, generates the RDG scatter plot.It gives information about the strength and nature of the interactions.For a positive sign(λ 2 )*ρ, coloured red, the interactions are repulsive.For a sign(λ 2 )*ρ close, green in colour, there is a weak interaction of the Van der Waals type.For a negative sign(λ 2 )*ρ, blue in colour, the interaction is attractive or bound 21 .
The RDG function combined with the sign(λ 2 ) was used to distinguish van der Waals interactions from other interactions (hydrogen bond and steric effect).As shown in Fig. 6, the values of the sign(λ 2 ) range from -0.05 to 0.05 a.u.This shows that the hydrogen interactions were between -0.05 and -0.02 a.u, the van der Waals interactions between -0.02 and 0.01 a.u, and the steric effect between 0.01 and 0.05 a.u.The presence of large quantities of spikes in the NCI plots, as well as the large spikes in the RDG plots in the water-caryophyllene system (Fig. 6), showed that the interactions are powerful between the components of the mixture compared with the isolated caryophyllene structure.Particularly, compared to the caryophyllene structure, a significant van der Waals effect can be observed through the green spikes on the left corresponding to the negative sign(λ 2 )r and the steric effect by the red band on the right corresponding to the positive sign(λ 2 )r.This indicates a higher contribution of weak interactions (van der Waals) and of strong repulsion (steric effect) in water-caryophylline intermolecular interactions.Note that a nonsignificant change in the hydrogen bond was observed in the presence of water.

Molecular dynamics simulation
Molecular dynamics modelling was used to determine the radial distribution function (RDF), which describes the probability of finding a molecule/atom at a distance (r) from another molecule/atom using the equation below: Where N f is the number of images, N is the number of particles, V is the apparent volume and r is the limiting distance 52 .
Radial distribution function (RDF) was used to explain the relationship between the extraction efficiency of caryophyllene, molecular structure characteristics, and qualitative accessibility of plant surface compounds for water.RDF was calculated with water considering the compound studied as the central molecule.RDF is used to determine a molecule as the central molecule and observe the distribution of water molecules in space.The height of the peak makes it possible to quantify interaction intensity between water and the central molecule 20 .
We supposed that the greater the peak height, the greater the interaction between the central molecule and the water, and the easier the entrainment of the compound for steam distillation and the solubilisation for hydrodistillation.Therefore, the higher the peak height, the better the extraction/separation effect by steam distillation.On the contrary, the compound could stay in the water-plant leaves mixture, due to high binary interaction, leading to bad extraction in the case of hydrodistillation.To validate this supposition, Fig. 7-A shows the radial distribution function for a mixture of 50 water molecules and one linalool molecule, which belongs to the oxygenated monoterpenes family and has a low vapour pressure.Linalool was only detected in small quantities in oils obtained by steam distillation.Temperature, between 353 and 373K, had no effect on the strength of this molecule's intermolecular interactions with water (Fig. 7-B).The presence of a functional group (OH) in the linalool structure makes the intermolecular interactions between this compound and water, stronger.Linalool-water interactions are relatively more significant than caryophyllene-water interactions (Fig. 7-A).In the development of this strong linalool-water interaction, extraction of this molecule was not possible by hydrodistillation.However, when water vapour passed through the plant, by steam distillation, the water molecules grafted onto the linalool and took it with them.
Fig. 8-A shows the evolution in the radial distribution function at temperatures ranging from 348K to 373K for a mixture of watercaryophyllene.The intermolecular interactions at r=2.75Å changed with the temperature of the mixture.They were weak at 348K and maximum at 353K.At temperatures above 353K, the strength of the intermolecular interactions decreased moderately and stabilised at higher temperatures (Fig. 8-B).This result shows that the interactions between water and caryophyllene were influenced by the change in temperature.They were stronger under the temperature conditions of steam distillation (353K) and of hydrodistillation (373K).In conclusion, these results revealed that the intermolecular interactions (van der Walls forces) developed between caryophyllene and water at high temperatures improved the extraction of the sesquiterpenoids by steam distillation.These interactions negatively impacted its separation by hydrodistillation, in which the sesquiterpene stayed in the water and plant leaves mixture, especially as it possesses a high molecular mass and low vapour pressure.

Conclusion
The essential oils of Rosmarinus officinalis L. offer numerous health benefits.In eastern Morocco, they are extracted by steam distillation (Sd) and hydrodistillation (Hd).This study examined the extraction of essential oils from Rosemary by comparing these two methods, Hd and Sd.GC-MS chemical composition analysis highlighted the efficiency of the Sd method in the extraction of sesquiterpene compounds with high molecular weight and low vapour pressure.Antioxidant activity tests, performed using DPPH and the β-carotene bleaching test, demonstrated that Sd oil has a higher antioxidant capacity (IC 50 = 4570 μg/mL, RAA= 16.3%) than Hd oil (IC 50 = CI 50 = 4960 μg/mL, RAA = 9.71%).In addition, thermal stability analysis by TGA-DTG revealed a higher decomposition temperature for Sd oil (368K) compared to Hd oil (362K).These results highlight that oxygenated sesquiterpenes and monoterpenes present in Sd oil have been identified as contributors to both antioxidant activity and thermal stability.
The theoretical findings from the molecular dynamics simulation and quantum chemical calculations revealed that the development of the van der Walls intermolecular bonding interactions and the steric effect improved the relative stability of the sesquiterpenoid compound with water and its extraction using the steam distillation processes.
The results suggested that the specific identification of active compounds among sesquiterpenes could provide promising avenues to optimize the antioxidant and thermal properties of rosemary essential oils.
A strong correlation has been found between the efficiency of the extraction process and the chemical composition; the latter being directly related to the biological activities of the essential oils.Steam distillation promotes the extraction of oxygenated sesquiterpenes and monoterpenes, compounds known for their antioxidant properties.Thus, the enrichment of the oils obtained by this method leads to a significant increase in their antioxidant activity.This research offers valuable insights into the distinct benefits of extraction methods, highlighting the potential of rosemary essential oils as antioxidant agents and exhibiting remarkable thermal stability.These results contribute to enriching the knowledge base on the application of essential oils in various fields, ranging from pharmacology to the food industry.

A B Figure 1 .
Hydrodistillation (A) and steam distillation (B) of rosemary to obtain the essential oil(1)

Figure 4 . 4 )
Figure 4. (A) Variation in vapour pressure as a function of temperature for the main constituents of essential oils.(B) Histogram of vapour pressures at 353K, entrainment ratio and yields of oil compounds in both processes

Figure 7 . 8 Figure 8 .
Figure 7. Radial distribution function of caryophyllene-water and of the linalool-water (A), temperature effect on the interaction linalool-water (B)

Table 1 .
32emical composition of essential oils extracted by Sd and Hd Pinene (6.99%).It found that, of 13 elements detected in oils extracted by steam distillation from rosemary originating from the same region of Morocco, monoterpenes were predominant32.The predominance classification was: 1,8-cineole > α-pinene > camphor > β-Pinene.The compound caryophyllene, which appeared in third place (7.13%) in the Sd 30 oils represented only 0.97% in the Hd oils.It belongs to the sesquiterpene family (Table (a) from ChemSpider, 2023; (b) from Thegoodscentscompany, 2023; Hd and Sd/14, percentage of 14 compounds extracted by hydrodistillation; Sd/30, percentage of 30 compounds extracted by vapour β-