Optimization of microwave-assisted hydro-distillation essential oil extracted from Rumex Crispus leaves using definitive screening design

2.1 Materials

The Rumex Crispus leaves were collected from different parts of Jimma, South Ethiopia and transported to the laboratory for analysis.

The standard materials utilized for the quantitative and qualitative analysis of the components of the essential oil were methyl eugenol, γ-terpinene, β-myrcene, α-pinene, isopulegol, p- cymene (Fluky, US).

All solvents and reagents (including Gallic acid, anhydrous sodium carbonate, sodium hydroxide, Acetone, n-hexane methanol, NaOH, and Ethanol) analytical grades were used in this experiment were purchased from chemical suppliers (Addis Ababa; Ethiopia).

Instruments such as Oven, Filter paper, hammer mill, Micro Sieve, Measuring cylinder, Digital balance, microwave oven, hydro-distillation, Gas chromatograph-Mass spectrometry (Model: GC-2014-ME83B, Samsung, RI Chi Minh town, Vietnams) were used.

2.2 Methodology

The collected samples were pretreated was carried out (washing, drying and size reduction).

The collected samples were washed and sundried for 4 days to get a fixed weight. The dried Rumex Crispus leaves were grounded into fine form by using a laboratory hammer mill and pestle to increase the surface area and increase the efficiency of oils. Then after the sieve, analysis was conducted through a 45-mesh carbon steel sieve to get a similar particle size. The fine particles were embraced in a glass bottle and kept for the next analyses at 4 °C (Idris et al., 2020).

• Moisture content determination

Moisture content and ash Content of the Rumex crispus leaves were determined according to the American Society for Testing and Materials methods (ASTMD). The plant leaves were washed (to remove impurities such as dust and mineral) and dried and size reduction was carried out. An empty crucible was balanced and its mass was recorded ( $m_1$ ). Three grams (5 g) of the leaf powered was measured ( $m_2$ ) in the crucible and dried at 103 ℃ for a day to obtain constant mass. Then crucible containing the dried powder was allowed to cool in a desiccator and the mass three ( $m_3$ ) was recorded. The moisture content of the leaf was determined as:

Where: $m_1$  = initial mass of the leaf before drying; $m_2$ = mass of the leaf after drying and $m_3$ mass of the crucible and the sample after drying.

2.2.1

2.2.1 Microwave-assisted hydro-distillation (MAHD) essential oil extraction process

Microwave-Assisted Hydro-Distillation (MAHD) with an XH-100A microwave laboratory oven (ltd. German), acting as the heating source to extract the oils and Clevenger distillation equipment (Ranch Chao ltd., Paris; France) was performed. The supplied power level of the microwave oven was determined by applying the LPML-5 test Buffler 1996. The microwave-assisted hydrodistillation experimental set-up is shown in Fig. 1.

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Fig. 1

Schematic diagram of the microwave-assisted hydro-distillation in lab scale for essential oils extraction process.

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The inside space of the microwave oven was 30 × 35 × 46 cm. The apartment bottom flask having the amount of 1L was placed inside the space of the MAHD experiment. The flask was linked to Clevenger equipment via the hole at the upper part of the oven. Fifty grams (50 g) of dried samples and 200 mL of distilled water solution were placed in the bottom flask connected to Clevenger equipment. The mixtures were heated by microwave radiation, the moisture evaporated, the generation of tremendous pressure on the plant cell wall, swelling of the plant cell, rupture of the cell, and leaching of the oils and the oils fractions were collected after distilling according to the designed time. The experiments were conducted beginning from the start of each time to collect the essential oils in the range of extraction parameters (microwave power (400 $-$ 800 W), extraction time (10 $-$ 40 min) and water volume-to-plant mass ratio (6 $-$ 20 mL/g)). Then the essential oils were collected in amber and dehydrated with anhydrous sodium sulfate and stored for the next process at 4 °C. The yield (%) of Rumex Crispus leaves oils are calculated by the following Eqn (2):

(2)

$$\mathrm{Y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}\mathrm{s}\mathrm{o}\mathrm{f}\mathrm{o}\mathrm{i}\mathrm{l}\left(\%\right)=\frac{\mathrm{q}\mathrm{u}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{e}\mathrm{s}\mathrm{s}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{o}\mathrm{i}\mathrm{l}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{e}\mathrm{d}(\mathrm{g})}{\mathrm{q}\mathrm{u}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{R}\mathrm{u}\mathrm{m}\mathrm{e}\mathrm{x}\mathrm{C}\mathrm{r}\mathrm{i}\mathrm{s}\mathrm{p}\mathrm{u}\mathrm{s}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{l}\mathrm{y}\mathrm{u}\mathrm{s}\mathrm{e}\mathrm{d}(\mathrm{g})}$$

2.3 Fourier transforms infrared (FTIR) spectroscopy analyses

All tests were kept at room temperature (25 ℃) for 25 min earlier to the FTIR investigations. Automatic target recognition (single-bounce) was utilized in all spectrum accomplishments Spectrum estimation parameters of resolution n and collection were chosen as 4 cm⁻¹ and 20 scans, separately. OPSU program with a version of 8.3 (Bruker Gnch) was utilized for instrument control and information procurement. Each test was put on a diamond Automatic target recognition crystal with the assistance of a Pasteur pipette. Then automatic target recognition crystal was cleaned using 95 % ethyl alcohol before each spectral accomplishment. Then after, the downplay air spectrum was scanned before each procurement.

2.4 Chemical composition of essential oils

The chemical compositions of the essential oils were identified using gas chromatography-mass spectrometry (GC–MS). Gas Chromatography-Mass Spectrometry (Model: GC-2014-ME83B, Samsung, RI Chi Minh town, Vietnams) was applied to test the constituents that exist in the essential oils of Rumex Crispus leaves. In the beginning, 20 μL of essential oils were added to 2 mL of n-hexane and then dehydrated using Na₂SO₄. For the HP10-MS column, head the column pressure was 7.5 psi. Helium (He) gas was used as carrier gas with a flow rate of 0.5 mL/min and a split ratio of 1:100, injection temperature of 260 °C and injection volume of 1.5 μL.

3.1 Moisture content determination

The moisture content of Rumex crispus leaves was measured and the result was recorded based on the average (mean value) value of the experimental runs. In the present work, the number of moisture contents of Rumex crispus leaf was evaluated as 6.09 ± 0.31 %. The number of moisture contents obtained in this study was closer to the amount of moisture content reported by (Idris et al., 2019) for Rumex crispus leaf 7.57 ± 0.40 %) with a small difference which may be due to plant maturation and environmental condition (Jungová et al., 2022).

3.2 Experimental variables and yields of essential oil

In this study, microwave-assisted hydrodistillation was used during the extraction of essential oils. In addition, the influence of extraction variables microwave power, extraction time and water volume to plant mass ratio on the yield of essential oil was conducted. The experimental design was conducted using a definitive screening design (DSD) with three contributing parameters generated as shown in Table 1. Thirteen (13) numbers of runs were conducted based on the definitive screening design matrix (Table 2). Table 2 shows the experimental and predicted results yields of essential oils. The experimental result indicated that the yield of essential oils extracted from Rumex Crispus leaves ranged from 1.512 - to 4.351 % (w/w). The higher yield of essential oils was found to be 4.351 % corresponding to the predicted values of 4.383 % at microwave power (600 W), extraction time (25 min) and water to plant mass ratio (4.5 mL/g). In contrast, the lowest yield of essential oils extracted was achieved at higher microwave power and extraction time (Table 2 run number 10). The extracted essential oils from Rumex Crispus leaves in the present study have a higher yield compared to the yield of essential oil (3.26 ± 0.05 % (w/w) extracted from Cinnamomum camphora Leaf by Shang et al (Shang et al., 2020) at optimum conditions using microwave-assisted hydro distillation.

The effect of extraction variables on the yield of essential oils was validated by the performance index (R-squared) between actual values and predicted values as plotted in Fig. 2. Visually, the distribution of the experimental data point follows up the 45-degree line, showing the validity of actual data with the predicted data and the coefficients of correlation fit an important level (Hundie et al., 2022).

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Fig. 2

Comparison of experimental and predicted yields of essential oils.

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3.3 Analysis of variance (ANOVA)

Analysis of variance was done based on an approach, in which the method utilizes a variance to test whether the means are distinct or not. It is also used to measure the significance of contributing variables by comparing the dependent variable (yield) at different levels (Beyecha and Abdissa, 2022). The output of ANOVA for the model is indicated in Table 3. It was stated that the model is significant with a 95 % level of confidence when the p-value for the model is below 5 % and insignificance when the p-values are greater than 5 % (Hundie, 2022). In the present study, as shown in Table 3, the p-value of the model was less than 5 %, which shows that the model was strongly significant.

In addition, Table 3 represents the coefficient of regressions (R-squared, R-squared adjusted and R-squared predicted), Root Mean Square Error, and mean of the response. The R-squared shows the percentage of changes between the actual and predicted values of the process and thus it determines how well the model is consistent with the experimental values (Akhbari et al., 2018). The regression result in an R-squared value of 0.99, which stands for a 99 % (Table 3) determination of extraction parameters on the yield of essential oil. The RMSE shows the error between the predicted and the experimental values, thus the lower the RMSE, the better the model describes the response of the process (Hemmat Esfe et al., 2018). The regression equation proposed by the definitive screening design was tested by using multiple regression analysis to determine the predicted value by testing the linear, interaction and quadratic effects of the essential oils extraction parameters, (Eqn (4)).

Where, X₁, X₂ and X₃ represent; microwave power (W), extraction time (min) and water volume to plant mass ratio (mL/g) respectively.

The significances of each term of the extraction variables and their effects were elaborated more in Fig. 4, using p-values and Longworth values.

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Fig. 3

Single parameter effect on the yield of essential oil (a) microwave power, (b) extraction time and (c) water to plant mass ratio.

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Fig. 4

Effect summary of the extraction variables on the yields of essential oils.

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3.4 Effect of extraction parameters on the yield of essential oils

Three important extraction conditions (Microwave power, water volume to plant mass ratio and extraction time) were considered to see their effect on the yield of essential oils extracted from Rumex Crispus leaves.

Microwave power is an important variable ranging from 400 $-$ 800 W in the microwave-assisted extraction of essential oil from plant materials. The other parameters were kept on the extraction time of 60 min and water value to plant mass ratio (4.5 mL/g) and the effect of microwave power at different levels (400 $-$ 800 W) on the yield of essential oil was studied as indicated in Fig. 3a. It can be seen from Fig. 3a that increasing the microwave power (from 400 $-$ 600 W) increases the yield of essential oils attaining the highest value at 600 W, and the utilization of the microwave power of more than 600 W ensued in a slightly diminish the yield of the oils. This means, that the higher microwave power generally contributes to a higher temperature of the extraction and the microwave energy activates plant cell perturbation through the local heating, assimilation and transfer from the external to the inside regions of plant molecules (Chai et al., 2021) and breaks the plant cell membranes, penetrating essential oils in the solvent (Mollaei et al., 2019). According to Shang et al (Shang et al., 2020), by increasing the microwave power, the polarization of polar substances induced by microwave is increased, and increase the motion of substances as well as the cell break is promoted, thereby the outcome of essential oils is enhanced. However, It can be observed that the excessive use of microwave power, reduces recovery due to the thermal degradation of thermo-sensitive substances that affect the yield of essential oils (Chen et al., 2019; Idris et al., 2020). Hence, considering the effects of microwave power on the yield of essential oil, 600 W microwave powers is required to achieve the maximum yield of essential oils in the present study.

The range of 10–40 min was selected for the following optimization considering the other parameters at their optimum values, and the result is shown in Fig. 3b. First, microwave irradiation interacts with the dielectric molecules comprised in the plant particles, creating pressure and then a break of the cell wall. Consequently, the constituents of the essential oil are released into the solvent and evaporate allowing their mass and dipolar moment, with enough time being needed to complete the extraction of each constituent. As the extraction time increased from 20 $-$ 25 min, the yield of essential oil increased, and then after, the yield of essential oils start to decrease. This is because, of the longer extraction time, during the extraction processes, degraded oils due to long-time microwave irradiation, leading to a decrease in the oils yield (Liu et al., 2021). Similar results were reported by (Chen et al., 2020); that further increase in extraction time will reduce the yield of the essential oil. Hence, considering the longer effect of the extraction time on the yield of essential oil, an irradiation time of 25 min is required to reach the equilibrium phase in this study.

The extraction efficiency of essential oil was also studied for the water volume to plant mass ratio ( $1.5-4.5\mathrm{m}\mathrm{L}/$ g) under the parameters of microwave power (600 W) and extraction time (25 min). According to Fig. 3c, the yield of the essential oil increases with increasing water volume to plant mass ratio. It can be seen that, because, of the addition of the water to plant mass ratio, the plant materials diffuse more in water, creating it easier for vapour to fulfil the essential oils (Hou et al., 2019) and could push the transfer of constituents to the solution, increasing the solubility, and raising the extraction efficiency of essential oils (Mehalaine and Chenchouni, 2021). However, if the water volume to mass plant mass ratio is too low, the contact between the plant materials and the solvent is not enough throughout the extraction process and creating the combustion of the plant's powder and causing extraction incomplete (Boudraa et al., 2021), contributing to the minimum yield of essential oils (Almasi et al., 2021). Therefore, in the present work, 4.5 mL/g was taken as the optimum water volume to plant mass ratio.

The effects of interaction parameters on the yield of extraction for essential oils are shown in Fig. 4. In addition, the contributions of each variable can be determined using the p-value and Longworth value. The lower the p-value and the higher the Longworth values, the more significant parameters of the process (Beyecha and Abdissa, 2022). Fig. 5 shows the contribution of all factors to the yield of essential oils utilizing the guideline that, if the variables cross the reference line (in this case vertical blue line), those factors are highly significant in the process (Hundie et al., 2022). In this study according to Fig. 4, the quadratic term ( $X_1^2)$ and interaction terms ( $X_1{X}_2$ ) had a higher significant effect on the yield of essential oils respectively. In addition, microwave power had more significant compared to the other variables (linear terms) (Fig. 4).

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Fig. 5

Effect of extraction conditions on the yield of essential oil after optimization, (a) microwave power, (b) extraction time and water volume to plant mass ratio(c).

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3.5 Optimization of essential oil extraction parameters and model validation

The objective of the present work was the optimization of essential oils extraction parameters from Rumex Crispus leaves using microwave-assisted hydro distillation through a definitive screening design. Different microwave powers (X₁: 400, 600 and 800 W), extraction times (X₂: 10, 25 and 40 min) and water volume to the mass of plant ratio (X₃: 1.5, 3 and 4.5 mL/g) were selected as shown in Table 1. The optimum values of the oil extraction yields were obtained by utilizing the desirability maximization process by a definitive screening design. Then to find the optimum extraction conditions for the yield of essential oil, the DSD model was employed to design the experiment to reduce the number of experiments and the extraction time. The initial prediction of the yield was 4.38 % at the mean value of the experimental conditions. After optimization of the experimental conditions, the yield of essential oil was found to be 4.73 %, corresponding to microwave power (534.89 W), extraction time (23.48 min) and water-to-plant mass ratio (4.5 mL/g) which was a significant amount. Taking these predicted values, experimental runs were triplicated and the obtained results were 4.66 %, 4.67 % and 4.68 %, with an average yield of 4.67 $\pm 0.02$  % of essential oil.

The maximum yield (6.50 %) of essential oil was investigated by Mollaei et al.,[2 1] at optimum operating conditions (microwave power of 980 W and plant/ liquid ratio of 2 (g/100 mL) at 72 min), using microwave-assisted hydrodistillation from Ferulago angulata plant. In the present study, the amount of essential oil extracted from Rumex crispus leaves is somewhat lower than essential oils extracted by (Mollaei et al., 2019). However, the difference may have resulted from plant compositions, plants sources, plant properties, climate conditions and geographical zone, (Abdelmajeed et al., 2013).

The predicted optimum extraction conditions for the yields of essential oil were indicated in Fig. 5. Moreover, the model validity was checked; and the experimental value (4.67 $\pm 0.02$  %) approached the predicted value (with an error of 0.013). Hence, the extraction conditions under microwave-assisted hydro distillation achieved by a definitive screening design may be regarded as accurate and reliable. The result of the present study was approached to the yield of essential oils (6.30 %) extracted from Ferulago angulata using microwave-assisted hydro distillation at, a plant/liquid ratio of 2 g/100 mL, 72 min of extraction time and 980 W of microwave power. The difference between the two yields may be, due to, the oil contents, the type of plant used for oil extraction and its compositions (Mohamad et al., 2019).

In conclusion, the minimum extraction time is a clear indication of MAHD's advantage in terms of cost and energy. Since the longer extraction, time takes more amount of energy, which is cost. The energy demand required to perform the extraction techniques also depends on the power consumption of the microwave oven (in MAHD) (Kusuma and Mahfud, 2017; Sharma and Gupta, 2020). According to a previous study (Kusuma and Mahfud, 2017), the relative power consumption to produce 1 mL of essential oil in hydrodistillation and microwave hydrodistillation was 13.8670 and 2.3134 kWh/ml of essential oil, respectively. In addition, Kusuma and Mahfud (Kusuma and Mahfud, 2017), reported that the environmental effects of pollution generated from CO₂ in the atmosphere were greater in the case of hydrodistillation than those of microwave hydrodistillation (Kusuma and Mahfud, 2017).

3.6 Fourier transforms infrared (FTIR) spectra of Rumex Crispus leaves essential oil

The typical FTIR spectrum of Rumex Crispus essential oil is presented in Fig. 6. The FTIR spectrum of R. Rumex Crispus essential oil had substantial vibrational bands at different wavenumbers (Fig. 6). The spectral band at 3347 cm⁻¹ could be attributed to the stretching vibrations of the OH functional group of alcohols (Salah et al., 2020). The banding with a peak point at 3059 cm⁻¹ will be assigned to the C⚌C–C ring vibrations of volatile aromatic compounds (Tijunelyte et al., 2017). Two bands at 2924 and 2852 cm⁻¹ were attributed to the methylene C—H asymmetric and symmetric stretching oscillations, respectively (Jaleh and Fakhri, n.d.). The band on a peak point at 1670 cm⁻¹ conformed to the C⚌C adulterating vibrations (Drinić et al., 2020). The peak of about 1516 cm⁻¹ was generated from the aromatic ring C⚌C skeleton oscillations of aromatic compounds (Cebi et al., 2021). The strong band at 1452 and 1379 cm⁻¹ were attributed to the COH bending vibration and C—H asymmetric plus symmetric bending vibration, respectively (Chen et al., 2020). Bandings at 1259 and 1234 cm⁻¹ were created from the C—C—O stretching oscillations and C—O stretching oscillations of phenolics (Aziz et al., 2019). The substantial band at 1055 and 1006 cm⁻¹ could be assigned to the hydroxyl group and methylene vibrations, respectively (Cebi et al., 2021; Ciko et al., 2016; Yao and Huang, 2022). The banding at 827 cm⁻¹ could be attributed to the C—H stretching vibration (Cebi et al., 2021; Kusuma and Mahfud, 2017).

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Fig. 6

FTIR spectrum of Rumex Crispus essential oil.

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3.7 Chemical compositions of Rumex Crispus leave essential oils

In the present work, the Rumex Crispus leaves essential oils were analyzed using the GC–MS method. Table 4 indicates the volatile aroma compounds detected in Rumex Crispus essential oils via GC–MS analysis. To identify the chemical compositions of the oils, 25 µL of essential oil was taken from the optimized conditions and mixed in 1.0 mL with dehydrated Na₂SO₄ and n-hexane. Then the chemical compositions of the oils were detected using gas chromatography-mass spectrometry (GC–MS) according to the methods described in the methodology. A total number of 32 compounds (about 98.33 % of the total identified constituents obtained from Rumex Crispus leaves by MAHD) were detected in essential oils.

A similar study was reported by Drinić et al., (Drinić et al., 2020) in which a total of 42 compounds have been detected, indicating more than 99.97 % of the essential oils. Additionally, the author investigated that the essential oils obtained by MAHD had a lower content of monoterpenes hydrocarbons and higher content of oxygenated monoterpenes, which is similar to this study.

The volatile compound profile of Rumex Crispus leaves essential oil was composed of oxygenated monoterpenes, oxygenated sesquiterpenes, monoterpenes, hydrocarbons and other oxygenated compounds. Table 4 indicates the name of compounds, molecular formula, Chemical Abstract Service (CAS), and the percentages content of the components identified by gas chromatograph-mass spectrometry. The table shows that the Rumex Crispus leaves essential oils had high amounts of Oxygenated terpenes and sesquiterpenes and, a lower amount of Monoterpenes. α-santol and β-santol were the primary components of oxygenated terpenes in the essential oil extracted from Rumex Crispus leave because these oxygenated terpenes of the essential oils accounted for about 29.63 % and 25.60 % of yield respectively. However, the lowest content of oxygenated terpenes was Teresantalol (1.55 %). In addition, bore α-bergamot (12.12 %), muciferous (8.02 %), γ-element (3.64 %), cis- lanceol (2.63 %) and α-cedrol (2.65 %) were presented in extracted essential oils of Rumex Crispus leaves.

Boudraa et al. (Boudraa et al., 2021) studied that some hydrocarbon components like geraniol, linalool, and terpineol, as well as their hydrocarbon interaction, will be interrupted by heat stress leading to the evocation of volatilization.

Monoterpene hydrocarbons are also present in low amounts; in the Rumex Crispus leaves essential oils. Monoterpene hydrocarbons are less important than oxygenated compounds in their contribution to the perfume of essential oils (Lucchesi et al., 2004; Chen et al., 2013). In contrast, the oxygenated compounds are more odorous and thus, the most valuable (Tan et al., 2017). Hence, the microwave-assisted hydro distillation extraction method gives the possibility for better procreation of the natural aroma of the plant essential oils (Idris et al., 2019) and is a good alternative for the separation of essential oil components from the plant materials (Jin et al., 2019).