Optimization and Characterization of Microwave-Assisted Hydro-Distillation Extraction of Essential Oils from Cinnamomum camphora Leaf and Recovery of Polyphenols from Extract Fluid

In this study, the efficiency of microwave-assisted hydro-distillation (MAHD) to extract essential oil from Cinnamomum camphora leaf, and the recovery of polyphenols from extract fluid were investigated. The effects of microwave power, liquid-to-material ratio, and extraction time on the extraction efficiency were studied by a single factor test as well as the response surface methodology (RSM) based on the central composite design method. The optimal extraction conditions were a microwave power of 786.27 W, liquid-to-material ratio of 7.47:1 mL/g, and extraction time of 35.57 min. The yield of essential oil was 3.26 ± 0.05% (w/w), and the recovery of polyphenols was 4.97 ± 0.02 mg gallic acid equivalent/g dry weight under the optimal conditions. Furthermore, the comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) was used to characterize the essential oils of fresh and fallen leaves, and 159 individual compounds were tentatively identified, accounting for more than 89.68 and 87.88% of the total contents, respectively. The main ingredients include sabinene, l-β-pinene, β-myrcene, α-terpineol, 3-heptanone, and β-thujene, as well as δ-terpineol and 3-heptanone, which were first identified in C. camphora essential oil. In conclusion, the MAHD method could extract essential oil from C. camphora with high efficiency, and the polyphenols could be obtained from the extract fluid at the same time, improving the utilization of C. camphora leaf.


Introduction
The Cinnamomum camphora is known as an evergreen tree of Lauraceae, widely distributed in China and Japan [1]. The C. camphora is not only a high-quality resource of wood but also plays an important role in the utilization of fragrance, medicine, and chemical industry due to its special aromatic and medicinal value [1][2][3][4]. In addition, some studies indicated that the extract of C. camphora leaf and its bioactive components have antioxidant, anti-inflammatory, antialgal, antimicrobial, and insecticidal

Effect of Microwave Power
Microwave power is an important influencing factor in the microwave-assisted extraction process. The other conditions were fixed on the liquid-to-material ratio of 6:1 mL/g and the extraction time of 60 min, and the effects of microwave power at 500, 600, 700, 800, and 900 W on the yield of fresh leaf essential oil and the total phenol content (TPC) in the extract fluid were examined, as shown in Figure 1a. According to the experimental results, the essential oil yield increased slightly when the microwave power was 500-800 W, reached the highest at 800 W, and then decreased significantly at 900 W. With the increase of microwave power, the polarization of polar molecules caused by microwave is enhanced, and the heat motion of molecules as well as the cell rupture is promoted, thereby the output of essential oil is increased [29]. On the other hand, excessive microwave power may lead to the decomposition of some thermo-sensitive metabolites, which affects the essential oil yield [18]. On the other hand, as the microwave power ranged from 600 to 800 W, the TPC gradually increased, reaching a peak at 800 W, and then decreasing at 900 W. The high microwave power could result in the degradation of phenolic compounds [30]. Considering the influence of microwave power on the essential oil yield and the TPC value, 800 W was chosen as the optimal microwave power.
when the microwave power was 500-800 W, reached the highest at 800 W, and then decreased significantly at 900 W. With the increase of microwave power, the polarization of polar molecules caused by microwave is enhanced, and the heat motion of molecules as well as the cell rupture is promoted, thereby the output of essential oil is increased [29]. On the other hand, excessive microwave power may lead to the decomposition of some thermo-sensitive metabolites, which affects the essential oil yield [18]. On the other hand, as the microwave power ranged from 600 to 800 W, the TPC gradually increased, reaching a peak at 800 W, and then decreasing at 900 W. The high microwave power could result in the degradation of phenolic compounds [30]. Considering the influence of microwave power on the essential oil yield and the TPC value, 800 W was chosen as the optimal microwave power. Figure 1. Effects of single factors on the yield of essential oil and content of total polyphenols in extract fluid: microwave power (a), liquid-to-material ratio (b), and extraction time (c). Different letters within a, b, c, and d in black color indicate significant differences (p < 0.05) in the essential oil yield among groups; Different letters within a, b, c, d, and e in red color indicate significant differences (p < 0.05) in the total phenol content (TPC) among groups. The same letter indicates no significant difference (p > 0.05) among groups. The significant differences among groups were analyzed by one-way ANOVA with Duncan post hoc test at p < 0.05.

Effect of Liquid-to-Material Ratio
The ratio of liquid-to-material affects the efficiency of microwave extraction, and an appropriate liquid-to-material ratio can simultaneously ensure the extraction efficiency as well as saving production costs. In this section, the extraction efficiency of essential oil and TPC were investigated when the liquid-to-material ratio was set as 2:1, 4:1, 6:1, 8:1, and 10:1 mL/g, under the condition of 800 W and 60 min. The results (Figure 1b) showed that the yield of essential oil rose up with the increase of the liquid-to-material ratio from 2:1 to 6:1 mL/g, and attained the maximum at the ratio of (a) (b) (c) Figure 1. Effects of single factors on the yield of essential oil and content of total polyphenols in extract fluid: microwave power (a), liquid-to-material ratio (b), and extraction time (c). Different letters within a, b, c, and d in black color indicate significant differences (p < 0.05) in the essential oil yield among groups; Different letters within a, b, c, d, and e in red color indicate significant differences (p < 0.05) in the total phenol content (TPC) among groups. The same letter indicates no significant difference (p > 0.05) among groups. The significant differences among groups were analyzed by one-way ANOVA with Duncan post hoc test at p < 0.05.

Effect of Liquid-to-Material Ratio
The ratio of liquid-to-material affects the efficiency of microwave extraction, and an appropriate liquid-to-material ratio can simultaneously ensure the extraction efficiency as well as saving production costs. In this section, the extraction efficiency of essential oil and TPC were investigated when the liquid-to-material ratio was set as 2:1, 4:1, 6:1, 8:1, and 10:1 mL/g, under the condition of 800 W and 60 min. The results (Figure 1b) showed that the yield of essential oil rose up with the increase of the liquid-to-material ratio from 2:1 to 6:1 mL/g, and attained the maximum at the ratio of 6:1 mL/g, and then gradually descended at 8:1 and 10:1 mL/g. When the liquid-to-material ratio is less, the contact between the solvent and sample is inadequate during the extraction process, leading to the lower output of essential oil. With the increase of the liquid-to-material ratio, the essential oil diffuses more in water, making it easier for vapor to carry out the essential oil, and the yield of essential oil increases. When the ratio continues to increase, the essential oil would be dissolved or emulsified by water, resulting in a decrease in the yield [31]. In addition, the variation trend of the TPC of the extract fluid was similar to that of the essential oil. The TPC value increased rapidly from the liquid-to-material ratio of 2:1 to 6:1 mL/g, and the maximum TPC value appeared at the ratio of 6:1 mL/g. From the liquid-to-material ratio of 6:1 to 10:1 mL/g, the TPC value declined slightly. The increase of solvent volume could promote the transfer of components to solution, improve the solubility, and elevate the extraction efficiency of polyphenols [32]. When the ratio of liquid-to-material is higher, the effect of increasing solvent is no longer significant, because most of the polyphenols have been dissolved, but this causes the consumption of solvent. Therefore, 6:1 mL/g was selected as the best liquid-to-material ratio for further optimization by RSM.

Effect of Extraction Time
Under the condition of 800 W and 6:1 mL/g, the effects of an extraction time of 20, 30, 40, 50, and 60 min on the essential oil yield and TPC were studied, and the results are shown in Figure 1c. As the extraction time extended from 20 to 30 min, the essential oil yield obviously enhanced, and then the yield remained almost unchanged when the extraction time increased from 30 to 60 min. In addition, the TPC value also varied with the microwave time. When the microwave-assisted extraction was conducted for 20 min, the TPC value was the highest. With the extension of the extraction time, the TPC values gradually decreased. This was because the polyphenols might degrade during long-time microwave irradiation, resulting in a decrease of the TPC value [32]. Considering the effects of the extraction time on the essential oil yield and the TPC value, 30 min was set as the optimal value for RSM optimization.

Results of Central Composite Design (CCD)
According to the results of the single-factor experiments, the microwave power of 800 W, liquid-to-material ratio of 6:1 mL/g, and extraction time of 30 min were selected as the middle levels of CCD. The design of 20 runs and the corresponding response values, including the yield of fresh leaf essential oil and the TPC values of extract fluids, are shown in Table 1. The experiment results showed that the yield of essential oil ranged from 1.94% to 3.44% (w/w), and the TPC values of extract fluids ranged from 3.29 to 5.44 mg·GAE/g·DW. The data of the CCD design were analyzed by multiple regression fitting, and the quadratic polynomial regression models for the essential oil yield and TPC value of extract fluid were established. Based on the ANOVA analysis, both models were statistically significant (p ≤ 0.0001) with F-values of 14.74 and 26.26, respectively. The determination coefficients (R 2 ) were 0.9299 and 0.9594, both greater than 0.80, indicating that the model fitted well [33]. The adjusted R 2 (R 2 Adj ) was 0.8668 and 0.9299, which were close to their corresponding R 2 , further indicating that the fitting degree of the models was relatively high, and the predicted values had a high correlation with the actual values. Moreover, the high p-values of the "lack of fit" of the models were 0.2473 and 0.1399, suggesting that the "lack of fit" had no statistical significance, which verified the reliability of the models [34]. The variation coefficients (C.V.) of 5.75 and 3.54%, which were both lower than 10%, also indicated the high accuracy and predictability of the equations [35].

Effects of Independent Variables on Essential Oil Yield
The results of the RSM experiments of C. camphora fresh leaf essential oil yield are shown in Table 1. Multiple regression fitting was carried out on the experimental data, and the ANOVA analysis of the model showed that the model was significant. The relationship between the three independent variables and the essential oil yield is described in the following coded regression equation (excluding insignificant coefficients): where Y refers to the yield of fresh leaf essential oil, X 1 is the microwave power, X 2 is the liquid-to-material ratio, and X 3 is the extraction time.
Based on the results of the equation and ANOVA, the linear effect of the extraction time (X 3 ) was positive and significant on the fresh leaf essential oil yield (p < 0.0001), and the cross-product effect between the microwave power and liquid-to-material ratio (X 1 X 2 ) was also positive and significant (p < 0.01), while all the quadratic terms (X 1 2 , X 2 2 and X 3 2 ) were negative and significant (p < 0.05).
To visually illustrate the interaction of the independent variables on the yield of fresh leaf essential oil, the three-dimensional response surfaces and contour plots are shown in Figure 2a-c. Figure 2a displays the interaction between the microwave power and liquid-to-material ratio on the response values with a fixed extraction time of 30 min. It was observed that the yield of essential oil decreased slightly as the microwave power increased from 700 to 900 W, while the yield increased when the liquid-to-material ratio varied from 4:1 to 6:1 mL/g, and then decreased as the liquid-to-material ratio increased to 8:1. According to Figure 2b, the lower microwave power and longer extraction time were beneficial to elevate the essential oil yield at the liquid-to-material ratio of 6:1 mL/g. The essential oil yield markedly increased when the extraction time varied from 20 to 40 min. At a constant extraction time of 40 min, the influence of the microwave power on the essential oil yield was similar to that in Figure 2a. Figure 2c shows the response surface plot between the liquid-to-material ratio and the extraction time on the essential oil yield. Similar to Figure 2a,b, the extended time led to the increase of the essential oil yield, while with the increase of the liquid-to-material ratio, the yield of essential oil first rose and then declined. Combined with the results of ANOVA and the response surfaces above, the influences of the microwave power, liquid-to-material ratio, and extraction time on the yield of essential oil were mainly in a quadratic manner.

Effects of Independent Variables on TPC Value
The RSM results of the TPC value of the extract fluid were measured by the Folin-Ciocalteu method, and the response values are shown in Table 1. The regression analysis was performed on the experimental data, and the relationship between the variables and TPC could be expressed in a coded equation (excluding insignificant coefficients): Where Y refers to the TPC value of the extract fluid, X1 is the microwave power, X2 is the liquid-to-material ratio, and X3 is the extraction time.

Effects of Independent Variables on TPC Value
The RSM results of the TPC value of the extract fluid were measured by the Folin-Ciocalteu method, and the response values are shown in Table 1. The regression analysis was performed on the experimental data, and the relationship between the variables and TPC could be expressed in a coded equation (excluding insignificant coefficients): where Y refers to the TPC value of the extract fluid, X 1 is the microwave power, X 2 is the liquid-to-material ratio, and X 3 is the extraction time.
According to the ANOVA result, the linear terms of the microwave power (X 1 ) and extraction time (X 3 ) were both positive and significant for the TPC value (p < 0.05), while the effect of the liquid-to-material ratio (X 2 ) was statistically reverse and very significant (p < 0.0001). Additionally, the microwave power and extraction time displayed significant negative quadratic effects (X 1 2 and X 3 2 ) on TPC (p < 0.05).
All interaction terms (X 1 X 2 , X 1 X 3 , and X 2 X 3 ) showed no significant influence (p > 0.05). The three-dimensional response surfaces and contour plots, as shown in Figure 3a-c, were obtained to illustrate the interaction of variables on the TPC value. In Figure 3a, when the extraction time was fixed at 30 min, with the increase of the microwave power, the TPC value first increased and then decreased, and the overall change was relatively stable. With the increase of the liquid-to-material ratio, the TPC value increased significantly. Figure 3b exhibits the interaction between the microwave power and extraction time on the TPC value at a liquid-to-material ratio of 6:1 mL/g. As the liquid-to-material ratio and extraction time increased, the TPC value elevated slightly first and then declined, suggesting the quadratic effect of the two parameters on the TPC value. In Figure 3c, the influences of the liquid-to-material ratio and extraction time on the TPC value at the microwave power of 800 W are similar to those in Figure 3a,b. From the three groups of response surface plots, the liquid-to-material ratio had the most significant effect on the TPC value compared with the microwave power and extraction time. Besides, the microwave power and extraction time had quadratic effects on the TPC value.

Verification of the Predicted Value
The optimal extraction conditions of C. camphora fresh leaf essential oil and polyphenols in extract fluid obtained by RSM were as follows: Microwave power of 786.27 W, liquid-to-material ratio of 7.47:1 mL/g, and extraction time of 35.57 min. Under this optimal condition, the predicted yield of essential oil was 3.28% (w/w), and the predicted TPC value of the extract fluid was 5.04 mg·GAE/g·DW. The reliability of the model was determined by the verification experiment under the optimal conditions, and the yield of fresh leaf essential oil was 3.26 ± 0.05% (w/w) and the TPC value was 4.97 ± 0.02 mg·GAE/g·DW. The actual values were close to the predicted values, which proved the accuracy of the model. In addition, the same optimal extraction conditions were used to extract the essential oil of C. camphora fallen leaf, and the yield was 1.87 ± 0.06% (w/w), and the TPC value of extract fluid was 2.56 ± 0.04 mg GAE/g DW. The yield of essential oil extracted from C. camphora fresh leaf and fallen leaf under the optimal condition was significantly different, which might be due to the different essential oil composition of fresh leaf and fallen leaf.

Chemical Profile of C. camphora Essential Oils
The volatile compositions of essential oils from C. camphora fresh leaf and fallen leaf obtained by optimized microwave-assisted hydrodistillation extraction were analyzed by GC×GC-TOFMS. The qualitative analysis was performed by comparing with the mass spectrometry from the National Institute of Standards and Technology (NIST) mass spectrometry library, and the quantitative analysis was conducted by peak area normalization. A total of 159 compounds with a similarity of more than 800 and a peak area of more than 0.02% were identified, accounting for 89.68% and 87.88% of the total essential oil from C. camphora fresh leaf and fallen leaf, respectively, as shown in Table 2. To our knowledge, 109 of these compounds have not been previously identified in C. camphora leaf essential oil, mainly including δ-terpineol, (3-Methyl-oxiran-2-yl)-methanol, 3-heptanone, 2-[2-(ethenyloxy)ethoxy]-ethanol, formaldehyde, cis-sabinene hydrate, (+)-α-terpineol, 2-azido-2,3,3-trimethylbutane, ethanolamine, and (1-hydroxyethylidene)malonic acid diethyl ester [9,14,16,[36][37][38].     According to Table 2, the chemical composition of essential oils of fresh and fallen leaves was similar, and there were 108 identical components in these two essential oils, but the main compounds were different. The major compounds in fresh leaf were sabinene, β-myrcene, δ-terpineol, 3-heptanone, and γ-terpinene, while the major compounds in fallen leaf were l-β-pinene, α-terpineol, δ-terpineol, β-thujene, and (3-methyl-oxiran-2-yl)-methanol. In addition, the GC×GC total ion chromatogram (TIC) contour plots of the essential oils of two kinds of leaves are shown in Figure 4, and the main compounds of C. camphora essential oil are marked. The composition of C. camphora essential oil might be influenced by variety, such as harvest time and maturity [9].

Plant and Reagents
Fresh and fallen leaves of Cinnamomum camphora were obtained from Sichuan province, China, and the plant was authenticated by Dr. Hong-Yan Liu, Research Center for Plants and Human Health, Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu, Sichuan province, China. The leaves were washed, ventilated, and dried at room temperature (25 °C ) for 1 day to dry the blade surface. The dried leaves were ground by a grinder (RS-FS500B, Royalstar Co., Ltd., Hefei, China), and sifted through a 40-mesh sieve (0.425 mm). The moisture

Plant and Reagents
Fresh and fallen leaves of Cinnamomum camphora were obtained from Sichuan province, China, and the plant was authenticated by Dr. Hong-Yan Liu, Research Center for Plants and Human Health, Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu, Sichuan province, China. The leaves were washed, ventilated, and dried at room temperature (25 • C) for 1 day to dry the blade surface. The dried leaves were ground by a grinder (RS-FS500B, Royalstar Co., Ltd., Hefei, China), and sifted through a 40-mesh sieve (0.425 mm). The moisture content of the fresh leaf particles was 54.88 ± 0.04%, and that of fallen leaf particles was 30.22 ± 0.27%. The treated samples were stored at 4 • C for further experiments.
The chemicals, including gallic acid, anhydrous sodium carbonate, and Folin-Ciocalteu reagent, were of analytical grade and were purchased from Sigma-Aldrich St. Louis, MO, USA. Acetone, methanol, and n-hexane were of chromatographic grade and obtained from Macklin Chemical Factory (Shanghai, China). The deionized water was used in experiments.

Microwave-Assisted Hydro-Distillation (MAHD)
The MAHD device consists of a microwave oven (X-100A, Xianghu Instrumental Company, Beijing, China) and a Clevenger apparatus. The C. camphora leaf sample (20.0 g·DW) was placed in a distillation flask, and a certain amount of deionized water was added. The mixture was heated by microwave radiation, and the oil fraction was collected after distilling for a period of time. The anhydrous sodium sulfate was added for drying, and then the dried essential oil was weighed. The yield of essential oil (Y) was calculated as follows: Y (%, w/w) = (dried essential oil mass/dried leaf sample mass) × 100. (3)

Total Phenolic Content Measured by the Folin-Ciocalteu Method
The total phenolic content was determined spectrophotometrically by the Folin-Ciocalteu method according to the literature with minor modification [39]. In brief, the 0.50-mL diluted sample was added to 2.5 mL of 0.2 mol/L Folin-Ciocalteu reagent, and thoroughly mixed. The mixture was incubated for 4 min, then 2 mL of saturated sodium carbonate solution were added. The absorbance of the mixture was determined at 760 nm after incubation shielded from light at room temperature (25 • C) for 2 h. Gallic acid was used as a reference standard, and the results were expressed as mg gallic acid equivalent (GAE)/g dry weight (DW) of leaf samples.

Single-Factor Experiment Design
The single-factor experiment was used to preliminarily optimize the microwave radiation power, liquid-to-material ratio, and extraction time, which are key factors influencing the extraction efficiency. The experimental design of each factor at each level is shown in Table 3.

Response Surface Methodology (RSM) Design
Based on the results of the single-factor experiment, a three-factor five-level response surface analysis based on the central composite design (CCD) was used to optimize the extraction conditions. The RSM design was conducted by Design Expert V8.0.6, with fewer iterations and higher efficiency. The experimental factors and levels are shown in Table 4. In this CCD, 20 different experiments with 6 repetitions of the center points were carried out to fit the following quadratic equation: where Y is the predicted response value (essential oil yield or TPC value); β 0 is the intercept of the equation; β i , β ii , and β ij indicate the coefficients of linear, quadratic, and interaction terms; and x i and x j represent different independent variables (microwave power, liquid-to-material ratio, and extraction time). The mathematical model of extraction conditions of essential oils and polyphenols was analyzed by analysis of variance (ANOVA) to test the adequacy of the equation, and the regression coefficients of each factor were tested. In addition, the three-dimensional surface plots and contour graphs were used to visualize the independence and interaction of each variable on the response values. The optimal conditions were executed, and the results were compared with the predicted values to determine the accuracy of the model.

GC×GC-TOFMS Analysis
The essential oil samples from fresh leaf and fallen leaf of C. camphora were diluted with n-hexane to obtain a solution of a 1% concentration, respectively. The GC×GC-TOFMS analysis was conducted on an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA), connected to a Pegasus 4D time-of-flight mass spectrometer (LECO Corporation, St. Joseph, MI, USA). The GC×GC system consisted of two chromatographic columns in series, and the parameters of these two columns are shown in Table 5. The initial temperature of the first column was set at 60 • C (5 min hold) and then ramped to 240 • C at a rate of 5 • C/min (5 min hold). The secondary oven was maintained 5 • C offset above the primary oven. The carrier gas was helium with a purity of 99.9995%. The injector temperature was maintained at 250 • C. The injection volume was 1 µL, and was performed in the split mode with a split ratio of 1:5. The mass spectrometer scanned in the range of m/z 28-600. The electron impact ionization energy was set at 70 eV. A 5-min solvent delay was conducted to protect the filament from solvent exposure.
All the operations and analysis of data were processed using LECO ChromaTOF ® software. The compositions of oil were identified by comparing the mass spectra with the National Institute of Standards and Technology (NIST) library data. The qualitative results of all compounds were given, including the compound name, Chemical Abstracts Service (CAS) registry number, similarity, retention time, and peak area. The similarity with the database is 1000 units, with the higher the similarity, the better the match. The relative abundance (%) was obtained by the ratio of the peak area of each compound to the sum of the peak areas of all compounds.

Statistical Analysis
All experiments were carried out in triplicate, and the results were expressed as mean ± standard deviation. The statistical analysis was performed using Design Expert V8.0.6 software (Stat-Ease Inc., Minneapolis, MN, USA), and SPSS 26.0 statistics software (IBM Corp., Armonk, NY, USA). The experimental data were statistically analyzed by one-way ANOVA with Duncan post hoc multiple comparisons test, and the significance level was set at p < 0.05.

Conclusions
A microwave-assisted hydro-distillation (MAHD) method was developed for the extraction of essential oils from C. camphora fresh leaf, and recovery of polyphenols from extract fluid. The response surface methodology (RSM) was used to optimize the experimental variables, including the microwave power, liquid-to-material ratio, and extraction time. The fitting models obtained by RSM were proved reliable. The optimal extraction conditions were a microwave power of 786.27 W, liquid-to-material ratio of 7.47:1 mL/g, and extraction time of 35.57 min. Under the optimal conditions, the essential oil yield and TPC value were 3.26 ± 0.05% (w/w) and 4.97 ± 0.02 mg GAE/g·DW, respectively. The microwave-assisted extraction could significantly shorten the extraction time and improve the extraction efficiency. In addition, GC×GC-TOFMS was used to analyze the volatile compounds of C. camphora essential oils from fresh leaf and fallen leaves. The essential oils of these two leaves differed in their chemical profile, and there were 108 common compounds in a total of 159 ingredients of the two leaves. The different composition of fresh leaf and fallen leaves might be influenced by the maturity of the leaves. The main components of fresh leaf were sabinene, β-myrcene, δ-terpineol, 3-heptanone, and γ-terpinene, while l-β-pinene, α-terpineol, δ-terpineol, β-thujene, and (3-methyl-oxiran-2-yl)-methanol were mainly found in the fallen leaf essential oils. Among them, δ-terpineol, 3-heptanone, and (3-methyl-oxiran-2-yl)-methanol were first identified in C. camphora leaf essential oil. Therefore, MAHD extraction is an interesting technology for the industrial production of C. camphora essential oil.