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An improved method to obtain essential oil, flavonols and proanthocyanidins from fresh Cinnamomum japonicum Sieb. leaves using solvent-free microwave-assisted distillation followed by homogenate extraction
⁎Corresponding author. yanglei@nefu.edu.cn (Lei Yang)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
An efficient solvent-free microwave-assisted distillation followed by homogenate extraction method (SFMD-HE) was established to separate the essential oil, non-volatile flavonols and proanthocyanidins from Cinnamomum japonicum Sieb. leaves. In the SFMD-HE technology applied in this work, no excessive water was introduced into the system, which can reduce the loss of flavonols and proanthocyanidins in the subsequent extraction process, avoid the generation of hydrosol and improve essential oil yield. Furthermore, no drying was required, which can reduce the energy consumption. The homogenate process can reduce the production of dust and environmental pollution, meanwhile, avoid the degradation and deterioration of some thermo-sensitive compounds. Through comparison, solvent-free microwave distillation was selected as the optimal method, and the 71% ethanol volume fraction was chosen for homogenate extraction. Based on the yields of essential oil, flavonols and proanthocyanidins, the process parameters (microwave irradiation power and time for essential oil distillation; homogenate time, liquid-solid ratio, and ethanol volume fraction for flavonol and proanthocyanidin extraction) were optimized. Due to the combined effects of these parameters, greater advantages were found for the proposed process compared with traditional methods. Therefore, the proposed technique is a good alternative for distilling essential oil and extracting non-volatile components from edible plants.
Keywords
Solvent-free microwave-assisted distillation followed by homogenate extraction (SFMD-HE)
Essential oil
Flavonols
Proanthocyanidins
Cinnamomum japonicum Sieb.
1 Introduction
Cinnamomum japonicum Sieb. (syn. Cinnamomum pedunculatum Nees) is an evergreen tree that belongs to the Lauraceae family and is distributed in China, Korea and Japan. C. japonicum Sieb. has been planted as a roadside tree, garden tree and florestation tree species in the southern provinces in China. Currently, leaves and barks of C. japonicum Sieb. have been recommended as a kind of edible and medicinal parts, and are widely used in seasonings, foods, perfumery and Chinese traditional medicine industries. Much research has been performed to study the essential oil (essential oil) or extract of C. japonicum Sieb. (Seo et al., 2013; Lin et al., 2011; Ling et al., 2009; Peng et al., 2013) because of its valuable pharmacological activities (Seo et al., 2013). The leaves and barks of C. japonicum Sieb. have been used for a long time in folk medicine in China, Korea and Japan (Seo et al., 2013; Huang et al., 2011; Kin et al., 2013) mainly due to the observed bioactivities of this plant, including antimicrobial (Lin et al., 2011), anti-inflammatory (Kim and Kim, 2017), inhibition of dopachrome formation (Lin et al., 2011), antiangiogenic (Seo et al., 2013), analgesic and antiepileptic activities (Huang et al., 2011). Pharmacological data indicate that the leaves of C. japonicum Sieb. are rich in flavonoids (Ling et al., 2009), proanthocyanidins (Kin et al., 2013) and essential oil (Peng et al., 2013; Huang et al., 2011). In many cases, pharmacological activities are influenced by the presence and content of these active ingredients. Therefore, developing an efficient method to separate and analyze the active ingredients from the leaves of C. japonicum Sieb. is important.
Plant resources contain various types of natural bioactive compounds (e.g., flavonols, tannins, and essential oil) (Sharma and Janmeda, 2017; Wei and Yang, 2017; Rhazi et al., 2019; Abdelwahab et al., 2017; Siddique et al. 2020). Flavonols are secondary metabolites with low molecular weights are one of the most numerous and widespread groups of phenolic constituents in medicinal plants. Flavonols possess many biological and pharmacological activities due to their phenolic structures. Proanthocyanidins are the most common polyphenolic compounds that naturally occur in cinnamon leaves, pine barks, etc. (Liu et al., 2017; Yang et al., 2012). Plant essential oils, which are natural products, are considered to be non-toxic compounds, and they have been used as natural preservation agents either alone or in combination with other preservatives (Muyima et al., 2002). Therefore, more attention should be paid to these natural compounds due to their potential applications and economic value.
The experimental steps of the traditional method for obtaining the essential oil, and non-volatile flavonols and proanthocyanidins are as follows: (1) leaves of C. japonicum Sieb. are mixed with water in a certain proportion; (2) essential oil in the leaves of C. japonicum Sieb. is extracted by hydrodistillation; (3) the remaining suspension of C. japonicum Sieb. leaves is distilled to remove water; (4) the compounds are extracted with ethanol; (5) the organic solvent is separated from the C. japonicum Sieb. leaves; (6) ethanol is removed from the extract by rotary evaporation to obtain an extract containing flavonols and proanthocyanidins. The yields of target analytes obtained using this method are low due to prolonged heating and the complicated separation process. Alternatively, solvent-free microwave-assisted distillation followed by homogenate extraction (SFMD-HE), which combines microwave irradiation heating with distillation, can rapidly obtain essential oils from plant materials. Solvent-free microwave-assisted distillation consists of placing the fresh leaves in a microwave reaction apparatus with no added solvent while distillation is carried out under atmospheric pressure. When the in situ water from the fresh leaves is irradiated with microwaves, the oil cells rupture due to the increase in inner pressure. This results in the essential oil being released and then distilled by the in situ water from the fresh leaves. Solvent-free microwave-assisted distillation has been applied to extract essential oils from fragrant and medicinal plants (Ma et al., 2012; Sui et al., 2012; Boukroufa et al., 2015), and water has not been introduced in the reaction system, which facilitates subsequent processing. Homogenate process is superior to general extraction methods. This method extracts the target analyte from fresh leaves into the extractant by mechanical cutting, blending and comminuting at a particular speed with no heating or pressure. Moreover, when the pulverization and extraction are combined into one process by homogenate extraction, the influence of external dust is also avoided. This technique has been proven to efficiently extract alkaloids (Wei et al., 2015), naphthoquinones (Liu et al., 2013), anthocyanins (Li et al., 2016) and kernel oil (Chen et al., 2016b). However, its application to the extraction of flavonols and proanthocyanidins from the deoiled leaves of C. japonicum Sieb. has not been reported. In the SFMD-HE technology applied in this work, no excessive water was introduced into the system, which can reduce the loss of flavonols and proanthocyanidins in the subsequent extraction process, avoid the generation of hydrosol and improve essential oil yield. Furthermore, no drying was required, which can reduce the energy consumption.
In order to improve the process consisting of the SFMD-HE of flavonols and proanthocyanidins from C. japonicum Sieb. leaves, the parameters that influence the process were systematically optimized. Additionally, the optimized procedure was compared with traditional techniques.
2 Experimental
2.1 Materials and chemicals
Fresh leaves were manually picked from similar 8-m, 9-year-old C. japonicum Sieb. trees in June 2016 from the outskirts of Leshan, Sichuan Province, China. Voucher specimens were certified by Prof. Huiyan Gu of the Forestry College, Northeast Forestry University, China and stored in the herbarium. To keep the material non-metamorphous, the picked green leaves were flown in a cold chain to the test site, chopped into rectangles with 4–7 mm length and width, blended evenly, and stored in plastic bags in a freezer at −18 °C until the experiment began. After measuring, the moisture content of the C. japonicum Sieb. leaves was 56% ± 3%, which was used in every step of the following experiment.
Reference substances of kaempferol, myricetin, quercetin were purchased from Yuanye Biological Technology Co., Ltd. (Shanghai, China) and (+)-catechin was obtained from Merck (Shanghai, China). For high-performance liquid chromatography determination, chromatographic-grade methanol and phosphoric acid were obtained from Thermo Fisher Scientific (Shanghai, China). Ultrapure water used in solutions and dilutions was obtained from a Milli-Q purification system (Bedford, MA, USA). Each sample to be analyzed by chromatography was passed through a 0.45-μm organic nylon membrane.
2.2 Experimental apparatus
The apparatus used for the solvent-free microwave-assisted distillation of essential oil was a laboratory-scale microwave assisted extraction system (XTrust, Shanghai, China), which consisted of a microwave oven (700 W maximum output power and 2.45 GHz irradiation frequency), a multimode reactor (1 L round-bottomed flask connected to a Clevenger-type essential oil receiving tube). The internal size of the oven cavity was 570 mm × 510 mm × 520 mm, and the microwave diffusion of rotation insures a uniform microwave distribution. Operating microwave irradiation power could be continuously adjusted through the power feedback/control depending on actual needs.
The apparatus used for the homogenate-assisted extraction of flavonols and proanthocyanidins was a Waring blender (Nintaus, Model JZM-3001) purchased from Zhongshan, China, which consists of a 500-mL polycarbonate plastic cylinder container (diam, 4.5 cm), a stainless cross-shaped reamer, a 22,000 r/min reamer rating and a 350 W rated power.
2.3 Distillation and extraction procedure
First, 225 g of chopped fresh leaves of C. japonicum Sieb. (56% moisture content) were placed in a reactor, which was a round-bottomed flask connected to a Clevenger device; inserted in the waveguide; and irradiated using a fixed incident power of either 230, 385, or 540 W. The cooling water circulation system external to the microwave oven was used to condense the vapor containing volatile components. To maintain the extraction conditions consistently, the condensation of water was continuously returned to the flask to supplement the moisture lost from the leaves. The extraction process was continued until no further distillate was obtained. The essential oils obtained by distillation were separated from the aqueous phase. To remove traces of moisture, the minimum amount of anhydrous sodium sulfate needed to dry the essential oil was added. The essential oils containing sodium sulfate were kept in the dark at a low temperature (4 ± 2 °C) until they were analyzed.
The extraction process for flavonols and proanthocyanidins was performed in a blender with 10.0 g of deoiled C. japonicum Sieb. leaves. Different volume fractions of ethanol solvent were tested to select the optimal volume fraction for extraction. The mixture was homogenized for different times, and the supernatant was obtained after a 20 min centrifugation at 4000 r/min and 25 °C. The optimal extraction conditions with respect to the ethanol volume fraction, homogenate time, and the ratio of material to solution were determined based on the content of flavonols and proanthocyanidins obtained during these experiments. Each experiment was conducted in triplicate to ensure accuracy of the experimental results.
To evaluate the proposed process and maximize the output of essential oil from the deoiled leaves of C. japonicum Sieb., BBD was applied to further investigate three independent variables, including homogenate time (X1), liquid-solid ratio (X2) and ethanol volume fraction (X3). The main effect, interaction effect and quadratic effect of the three parameters were estimated using a BBD followed by response surface methodology (RSM). Seventeen free combinations with five replicates at the center point were conducted based on the fluctuation ranges of 2–4 min, 10–20 mL/g, and 60%–80% for X1, X2 and X3, respectively, which were standardized to the interval to fit a full second-order polynomial equation model. The general equation is as follows:
Based on the optimum conditions derived for SFMD-HE, the conventional hydrodistillation (HD) process was carried out by adding 225 g of chopped fresh leaves of C. japonicum Sieb. (moisture content, 56%) and 475 mL of deionized water into a 1-L round bottomed flask connected to a Clevenger apparatus. Then, the sample flask was heated using a heating jacket at 540 W.
2.4 GC–MS analysis of essential oils
The volatile components of the obtained essential oils were analyzed and determined using an Agilent GC–MS system (6890N/5973, Santa Clara, CA) consisting of a GC (Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-5MS capillary column (0.25-μm film thickness, 30 mm × 0.25 mm) and a mass selective detector operating in the electron impact ionization mode (70 eV). GC–MS analysis was performed with the conditions based on the reference (Liu et al., 2011), which are as follows: 1 mL/min carrier gas (helium) flow rate, 1:50 flux ratio, injector temperature of 270 °C, and 1 μL sample (diluted with n-hexane 1:100, v/v) injection volume. The oven temperature was held at 50 °C for 5 min, then programmed to increase from 50 °C to 260 °C at a rate of 5 °C/min, held at this temperature for 5 min, increased to 280 °C at a rate of 15 °C/min and held at this temperature for 10 min. The scan range and detector temperature were 15–500 atomic mass units and 280 °C, respectively. Spectra were obtained as total ion chromatograms. The volatile components of the essential oil were identified by comparing their mass spectral fragmentation patterns with those of analogous compounds in a database (Wiley, Mass Finder 2. Library, NIST05) and MS data in the literature (Chen et al., 2017). The relative amount of each compound in each sample was quantified according to the peak area relative to the total peak area for all compounds (Chen et al., 2015).
2.5 HPLC analysis and quantification
An Agilent 1260HPLC system consisting of a UV detector (Model, G1314B), a binary pump (Model, G1311C), an autosampler (Model, 717 plus), and a temperature control box was used to determine the myricetin, quercetin and kaempferol contents. Chromatographic isolation of the target analytes was performed on a Zorbax XDB-C18 column (4.6 × 250 mm, 5-μm).
The flavonol contents of the extracts of C. japonicum Sieb. leaves were determined using the HPLC method (Li et al., 2016) with modification. Briefly, 15 mL of supernatant was mixed with 30 μL concentrated hydrochloric acid for 1 h at 80 °C. The resulting solution was neutralized with sodium hydroxide solution. Then, 1 mL was passed through a 0.45-μm nylon membrane to prepare it for HPLC detection. Each sample was prepared and injected in triplicate.
The proportion of mobile phase was methanol:water (0.5% phosphoric acid) = 50:50 (v/v) with a flow rate of 1.0 mL/min and a 10 μL injection volume. The sample was run at a temperature of 30 °C, and the UV detection wavelength was set at 360 nm. According to the test conditions, standards of myricetin, quercetin and kaempferol were dissolved in methanol and detected with retention times of 6.0, 9.8 and 14.7 min, respectively. The calibration curves for myricetin, quercetin and kaempferol were: YMyricetin = 67.89X – 60.56 (R2 = 0.9990); YQuercetin = 41.03X – 27.26 (R2 = 0.9996) and YKaempferol = 78.96X – 34.42 (R2 = 0.9999).
2.6 Quantitative analysis of proanthocyanidins
A standard stock solution of (+)-catechin was dissolved in methanol to determine proanthocyanidins extracted from C. japonicum Sieb. leaves using the standard vanillin-HCl method (Chen et al., 2016a). Then, we measured the solvent absorbance using a spectrophotometer (UV-5500PC, Shanghai Metash Instrument Co., Ltd, China) at 500 nm. We calculated the proanthocyanidin content using a calibration curve, which was: YProanthocyanidins = 4.56X + 0.02 (R2 = 0.9992).
3 Results and discussion
3.1 Kinetics and microwave irradiation power for obtaining essential oil
In recent years, a surprising essential oil extraction method using microwave energy has been developed by Chemat’s group (Chemat et al., 2015; Filly et al., 2014; Li et al., 2013). Solvent-free microwave-assisted distillation has been used for obtaining essential oils from the leaves of aromatic plants (Swamy et al., 2014; Yuan et al., 2015; Zhang et al., 2014). For the fresh leaves of C. japonicum Sieb., Fig. 1 shows the extraction kinetics for essential oil under different microwave irradiation powers. To investigate the influence of the microwave irradiation power on the yield of essential oil, extraction procedures were carried out at 230, 385 and 540 W. The overall system was operated at ambient pressure. The average essential oil extraction yield was significantly influenced by the microwave irradiation power. Therefore, to maximize extraction yield with the lowest energy consumption, a 540 W microwave irradiation power was selected for further experiments.
The extraction kinetic curves (operating microwave irradiation powers were 230, 385 and 540 W) for essential oil were constructed. The oil distillation yield increased rapidly with the extension of extraction time for the 540 W microwave irradiation power. Most oil distillation reactions required approximately 25 min to reach the equilibrium yield, and further increases in time produced only a slightly positive effect on their yields. Thus 25 min was sufficient to obtain high yields for the extraction of essential oil.
3.2 Optimization of single extraction conditions
The three parameters of ethanol volume fraction, liquid-solid ratio and homogenate time were optimized in all cases with the univariate method.
3.2.1 Effect of homogenate time on the yields of flavonols and proanthocyanidins
To obtain the optimal homogenate extraction for flavonols and proanthocyanidins, experiments were performed at different homogenate times (1, 2, 3, 4, 5 and 6 min). Fig. 2 displays the experimental results, which indicate that the yields of flavonols (myricetin, quercetin and kaempferol) and proanthocyanidins improved rapidly when the homogenate increased from 1 to 3 min. However, the extraction yields of these two analytes were very low during the first 1 min of homogenate process, which indicated that the shearing process needed sufficient time to break the plant cell walls and thus promote the release of the flavonols and proanthocyanidins into the solvent. However, for long homogenate times, e.g., greater than 4 min, there was no apparent further improvement in extraction yield. Hence, 2–4 min was selected as the appropriate homogenate time for the remaining experiments.
3.2.2 Effect of liquid-solid ratio on the yields of flavonols and proanthocyanidins
The effect of the liquid-solid ratio on the extraction yields of flavonols and proanthocyanidins was determined. The extraction process was carried out over a range of liquid-solid ratios (10–30 mL/g) to assess the effect of this parameter. Fig. 3 shows the yields of the target analytes achieved within the parameter range of 10–20 mL/g, which did not significantly increase with higher proportions of solvent (20–30 mL/g). Hence, a liquid-solid ratio range of 10–20 mL/g was used in the later optimization steps.
3.2.3 Effect of ethanol volume fraction on the yields of flavonols and proanthocyanidins
The experiments were performed at a series of ethanol volume fractions (0–96%) to determine the suitable concentration of ethanol in homogenate for the simultaneous extraction of flavonols and proanthocyanidins. From Fig. 4, it was determined that the extraction yields of flavonols and proanthocyanidins were enhanced with an increase in ethanol volume fraction from 0 to 70%. This may be because the solubility and the extraction properties of ethanol were improved with the gradual increase in concentration. However, for ethanol volume fractions over 70%, the extraction yields of the two analytes showed slight decreases. Therefore, 60–80% was chosen as the appropriate ethanol volume fraction range for further optimization.
3.3 Parameter optimization using response surface methodology
3.3.1 Model building and statistical analysis
The interaction effects among the three parameters (homogenate time X1, liquid-solid ratio X2 and ethanol volume fraction X3) were evaluated using seventeen-run experiments, based on the yields of flavonols and proanthocyanidins. The BBD experimental design and results are listed in Table 1, and the variance analysis of the response surface model is given in Table 2. Generally, the F-test and the P-value were applied to determine the statistical significance and regression coefficient. For flavonols and proanthocyanidins, models with F-values of 102.12 and 72.30 and a P-value of less than 0.0001 illustrated that the models for flavonols and proanthocyanidins performed significantly and can reasonably represent the experimental data. The chance for these large model F-values to occur due to statistical noise was <0.01%. In addition, the F-value and P-value of “Lack of fit” were measured to be 2.26 and 2.59, 0.2241 and 0.1901 for flavonols and proanthocyanidins, respectively, which implied a non-significant “lack of fit”. The large “lack of fit” this large occurred with probabilities of 22.41% and 19.01% due to statistical noise, indicating better model fitting results. The coefficient of determination (R2) is an indicator of the proportion of total variation in the response predicted by the model (Swamy et al., 2014). In the two models for flavonols and proanthocyanidins, R2 of 0.9924 and 0.9894, respectively, implied that the models were suitable for covering 99.24% and 98.94% of variations in the experimental data. Due to the significant F-value, non-significant “lack of fit”, and satisfactory R2, the models were adequate and agreed well with the experimental data (Yuan et al., 2015). The following mathematical models were obtained:
| No. | Factors | Flavonols | Proanthocyanidins | ||||
|---|---|---|---|---|---|---|---|
| Homogenate time (min) | Liquid-solid ratio (mL/g) | Ethanol volume fraction (%) | Predicted yield (%) | Actual yield (%) | Predicted yield (%) | Actual yield (%) | |
| 1 | 2 | 10 | 70 | 10.16 | 10.24 | 54.91 | 55.97 |
| 2 | 4 | 10 | 70 | 11.25 | 11.34 | 61.00 | 61.71 |
| 3 | 2 | 20 | 70 | 11.22 | 11.14 | 58.10 | 57.40 |
| 4 | 4 | 20 | 70 | 12.12 | 12.04 | 68.51 | 67.45 |
| 5 | 2 | 15 | 60 | 9.39 | 9.47 | 43.57 | 44.49 |
| 6 | 4 | 15 | 60 | 10.67 | 10.76 | 46.08 | 47.36 |
| 7 | 2 | 15 | 80 | 9.81 | 9.73 | 44.32 | 43.05 |
| 8 | 4 | 15 | 80 | 10.51 | 10.43 | 58.31 | 57.40 |
| 9 | 3 | 10 | 60 | 9.05 | 8.89 | 42.15 | 40.18 |
| 10 | 3 | 20 | 60 | 10.37 | 10.37 | 40.26 | 40.05 |
| 11 | 3 | 10 | 80 | 9.53 | 9.53 | 41.40 | 41.62 |
| 12 | 3 | 20 | 80 | 10.14 | 10.30 | 53.99 | 55.97 |
| 13 | 3 | 15 | 70 | 12.28 | 12.37 | 70.32 | 71.75 |
| 14 | 3 | 15 | 70 | 12.28 | 12.43 | 70.32 | 70.32 |
| 15 | 3 | 15 | 70 | 12.28 | 12.11 | 70.32 | 68.88 |
| 16 | 3 | 15 | 70 | 12.28 | 12.27 | 70.32 | 68.88 |
| 17 | 3 | 15 | 70 | 12.28 | 12.24 | 70.32 | 71.75 |
| Source | Sum of squares | Degree of freedom | Mean square | F-value | P-value | ||||
|---|---|---|---|---|---|---|---|---|---|
| Flavonols | Proanthocyanidins | Flavonols | Proanthocyanidins | Flavonols | Proanthocyanidins | Flavonols | Proanthocyanidins | ||
| Model | 21.67 | 2253.94 | 9 | 2.41 | 250.44 | 102.12 | 72.30 | <0.0001*** | <0.0001*** |
| X1 | 1.99 | 136.17 | 1 | 1.99 | 136.17 | 84.55 | 39.31 | <0.0001*** | 0.0004*** |
| X2 | 1.87 | 57.22 | 1 | 1.87 | 57.22 | 79.18 | 16.52 | <0.0001*** | 0.0048** |
| X3 | 0.03 | 84.24 | 1 | 0.03 | 84.24 | 1.41 | 24.32 | 0.2741 | 0.0017** |
| X1X2 | 0.01 | 4.63 | 1 | 0.01 | 4.63 | 0.40 | 1.34 | 0.5492 | 0.2854 |
| X1X3 | 0.08 | 32.95 | 1 | 0.08 | 32.95 | 3.56 | 9.51 | 0.1010 | 0.0177* |
| X2X3 | 0.13 | 52.42 | 1 | 0.13 | 52.42 | 5.32 | 15.13 | 0.0544 | 0.0060** |
| X12 | 0.62 | 38.74 | 1 | 0.62 | 38.74 | 26.50 | 11.18 | 0.0013** | 0.0123* |
| X22 | 2.11 | 186.38 | 1 | 2.11 | 186.38 | 89.33 | 53.81 | <0.0001*** | 0.0002*** |
| X32 | 13.67 | 1553.68 | 1 | 13.67 | 1553.68 | 580.05 | 448.55 | <0.0001*** | <0.0001*** |
| Residual | 0.17 | 24.25 | 7 | 0.02 | 3.46 | ||||
| Lack of fit | 0.10 | 16.01 | 3 | 0.03 | 5.34 | 2.26 | 2.59 | 0.2241 | 0.1901 |
| Pure error | 0.06 | 8.24 | 4 | 0.02 | 2.06 | ||||
| Cor total | 21.83 | 2278.19 | 16 | ||||||
| Credibility analysis of the regression equations | Index mark | Standard deviation | Mean | CV % | Press | R2 | Adjust R2 | Predicted R2 | Adequacy precision |
| Flavonols | 0.15 | 10.92 | 1.41 | 1.75 | 0.9924 | 0.9827 | 0.9196 | 27.46 | |
| Proanthocyanidins | 1.86 | 56.72 | 3.28 | 269.03 | 0.9894 | 0.9757 | 0.8819 | 21.05 | |
Generally, a model term is significant when its P-value is less than 0.05, and the opposite is not significant. As shown in Table 2, the linear terms of X1 and X2, and quadratic terms of X12, X22 and X32 were significant for flavonols, while the linear terms of X1, X2 and X3, interaction terms of X1X3 and X2X3, and quadratic terms of X12, X22 and X32 were significant for proanthocyanidins. The predicted R2 of flavonols and proanthocyanidins were 0.9196 and 0.8819, respectively, which was reasonably consistent with the adjusted R2 of 0.9827 and 0.9757. The coefficient of variation (CV) is a general indicator for determining the reproducibility of the model (Zhang et al., 2014); the higher the CV, the higher the variation in the mean value. The CV values for the two models for flavonols and proanthocyanidins were 1.41% and 3.28%, respectively, which was relatively low, indicating a good level of precision for the experiments. “Adeq precision” of 27.46 for flavonols and 21.05 for proanthocyanidins indicated an adequate signal.
3.3.2 Analysis of the response contour
Three-dimensional response surfaces were employed to research the influence of independent variables and their interactive results on the yield of flavonols and proanthocyanidins, using the dependent response (Z-axis) versus two independent variables (X- and Y-axis), while the remaining variable was maintained at 0-level. The response surfaces illustrating the influence of homogenate time (X1) and liquid-solid ratio (X2) on the yield of flavonols and proanthocyanidins are presented in Fig. 5a and d, with the ethanol volume fraction being maintained at 0-level. Both flavonols and proanthocyanidins exhibited a remarkable increase in the yield with increasing homogenate time, but the initial rapid improvement in the yield was followed by an almost constant yield when the time further increased. This phenomenon demonstrated that sufficient time is required for the complete extraction of the target analytes. The influence of the liquid-solid ratio on the yield of flavonols and proanthocyanidins first raised and then slightly decreased with increasing liquid-solid ratio. A higher liquid to material ratio is conducive for permeation of the solvent and release of target analytes because of the larger extra- and intra-cellular concentration difference of the target analytes. Fig. 5b and e depict the response surfaces for the yields of flavonols and proanthocyanidins, regarding the homogenate time (X1) and ethanol volume fraction (X3), with a constant liquid-solid ratio (0-level). A similar trend for the yield of flavonols and proanthocyanidins was obtained. Specifically, the initially obvious improvement was followed by a remarkable reduction when the ethanol volume fraction increased. At an appropriate ethanol volume fraction, it contributes to the destruction of cell walls, movement of the extraction solvent and diffusion of inner cellular components. Fig. 5c and f shows the interacting results of the liquid-solid ratio (X2) and ethanol volume fraction (X3) at a constant homogenate time (0-level).
The optimal results predicted by the BBD software were 4 min homogenate time, 16 mL/g liquid–solid ratio, and 71% ethanol volume fraction. Using the optimum process results mentioned above, the theoretical yields of flavonols and proanthocyanidins were 12.50 mg/g and 72.52 mg/g, respectively.
3.3.3 Verification of predictive models
Five repetitions of the experiments were conducted with the derived conditions (4 min homogenate time, 16 mL/g liquid–solid ratio, and 71% ethanol volume fraction) to validate the reliability of the BBD software. Using the optimized results mentioned above, the practical yields of flavonols and proanthocyanidins were 12.48 ± 0.57 mg/g and 71.97 ± 2.71 mg/g, respectively, which was in reasonable agreement with the predicted results, indicating the reliability of the BBD software.
3.4 Comparison with reference techniques
Fig. 6 shows the variation in yields over time during the extraction of essential oil from the leaves of C. japonicum Sieb. via SFMD-HE and HD. SFMD-HE obviously shortened the treatment time, which took only 1.5 min regardless of the microwave irradiation power, while HD took over 12 min to achieve 100 °C. Compared with HD, SFMD-HE was more efficient for the acquisition of essential oil. The SFMD-HE yield of essential oil was 6.50 ± 0.31 mL/kg for 40 min at 540 W microwave irradiation power, whereas the HD yield 5.46 ± 0.22 mL/kg for 200 min at 540 W conventional electric heating. HD is the most conventional isolation method for essential oils. In the HD technique, the aromatic plant materials are soaked in water for some time, after which the mixture is heated to carry the volatile components away by steam. Lastly, the essential oil is separated. However, a small amount of essential oil components often forms a hydrosol with condensate water, which is not easy to separate and collect. We propose that the formation of hydrosol may lead to the loss of some oxy-components dissolved in the condensate water during the distillation process, resulting in decreased extraction efficiency. These results are similar to those reported by Hamdi et al. (2017) and Timung et al. (2016).
3.5 Chemical components of essential oil
The detailed analysis of the components in the essential oils obtained by SFMD-HE and HD using GC–MS is shown in Table 3, which includes the identified compositions, molecular formulas, retention indices, CAS numbers and relative area percentages. The relative area percentage (RA %) was calculated from the percentage of the peak area of individual volatile components relative to the total peak area and used for comparison. The results show that more components were detected in the SFMD-HE essential oil than in HD and that their RA % in the SFMD-HE and HD were also different. In total, 44 types of components were detected, 42 of which were found in SFMD-HE essential oil; the HD essential oil contained 34 types of components, which was attributed to the extraction mechanism, in which some components dissolved in water could form a hydrosol. The detected components of SFMD-HE and HD essential oils represented 90.86% and 83.61% of the total essential oils, respectively. In general, borneol, cinnamyl acetate, eucalyptol, bornyl acetate, caryophyllene, and γ-elemene were the main compounds in both SFMD-HE and HD essential oils, but the RA% of the individual volatile components in the essential oils were significantly different.
| No.a | Components | RIb | Molecular formula | ID | CAS number | RAe (%) | |
|---|---|---|---|---|---|---|---|
| SFMD-HE | HD | ||||||
| 1 | trans-2-Hexenal | 850 | C6H10O | RIc, MSd | 6728-26-3 | 0.06 | 0.09 |
| 2 | α-Pinene | 917 | C10H16 | RI, MS | 80-56-8 | 0.16 | 1.14 |
| 3 | Camphene | 933 | C10H16 | RI, MS | 5794-03-6 | 0.09 | 0.61 |
| 4 | β-Pinene | 974 | C10H16 | RI, MS | 127-91-3 | ndf | 1.08 |
| 5 | α-Phellandrene | 1003 | C10H16 | RI, MS | 2243-33-6 | 0.071 | 0.254 |
| 6 | Cymene | 1021 | C10H14 | RI, MS | 99-87-6 | 0.19 | 0.03 |
| 7 | Eucalyptol | 1033 | C10H18O | RI, MS | 470-82-6 | 4.48 | 10.13 |
| 8 | Salicylaldehyde | 1041 | C7H6O2 | RI, MS | 90-02-8 | 0.06 | 0.06 |
| 9 | β-Ocimene | 1044 | C10H16 | RI, MS | 13877-91-3 | ndf | 0.07 |
| 10 | γ-Terpinene | 1064 | C10H16 | RI, MS | 99-85-4 | 0.14 | 0.47 |
| 11 | trans-Sabinenehydrate | 1101 | C10H18O | RI, MS | 17699-16-0 | 0.02 | ndf |
| 12 | Linalool | 1104 | C10H18O | RI, MS | 78-70-6 | 0.82 | 0.74 |
| 13 | Fenchol | 1121 | C10H18O | RI, MS | 1632-73-1 | 0.04 | 0.05 |
| 14 | Camphore | 1145 | C10H16O | RI, MS | 464-48-2 | 0.46 | 0.36 |
| 15 | Borneol | 1166 | C10H18O | RI, MS | 507-70-0 | 41.91 | 36.15 |
| 16 | Terpinen-4-ol | 1178 | C10H18O | RI, MS | 562-74-3 | 1.44 | 1.88 |
| 17 | p-Cymen-8-ol | 1185 | C10H14O | RI, MS | 1197-01-9 | 0.02 | ndf |
| 18 | α-Terpineol | 1190 | C10H18O | RI, MS | 98-55-5 | 2.52 | 2.39 |
| 19 | Decanal | 1208 | C10H20O | RI, MS | 112-31-2 | 0.11 | ndf |
| 20 | β-Cyclocitral | 1214 | C10H16O | RI, MS | 432-25-7 | 0.02 | ndf |
| 21 | Isobornyl formate | 1233 | C11H18O2 | RI, MS | 1200-67-5 | 0.03 | 0.04 |
| 22 | Neral | 1237 | C10H16O | RI, MS | 106-26-3 | 0.09 | 0.06 |
| 23 | 2-Phenylethyl acetate | 1256 | C10H12O2 | RI, MS | 103-45-7 | 0.66 | 0.25 |
| 24 | Bornyl acetate | 1287 | C12H20O2 | RI, MS | 76-49-3 | 5.21 | 4.89 |
| 25 | Theaspirane | 1302.5 | C13H22O | RI, MS | 36431-72-8 | 0.02 | 0.03 |
| 26 | trans-Methyl Cinnamate | 1352 | C10H10O2 | RI, MS | 1754-62-7 | 0.01 | ndf |
| 27 | 3,7-Dimethyl-2,6-octadien-1-ol,acetate | 1365 | C12H20O2 | RI, MS | 16409-44-2 | 0.28 | ndf |
| 28 | α-Ylangene | 1370 | C15H24 | RI, MS | 14912-44-8 | 0.07 | 0.05 |
| 29 | β-Elemene | 1441 | C15H24 | RI, MS | 515-13-9 | 0.09 | 0.09 |
| 30 | Caryophyllene | 1446 | C15H24 | RI, MS | 87-44-5 | 4.43 | 5.33 |
| 31 | Aromadendrene | 1449 | C15H24 | RI, MS | 109119-91-7 | 0.07 | 0.16 |
| 32 | Cinnamyl acetate | 1455 | C11H12O2 | RI, MS | 103-54-8 | 17.97 | 10.91 |
| 33 | γ-Elemene | 1465 | C15H24 | RI, MS | 29873-99-2 | 3.93 | 3.09 |
| 34 | δ-Cadinene | 1522 | C15H24 | RI, MS | 483-76-1 | 0.05 | 0.10 |
| 35 | Elemol | 1542 | C15H26O | RI, MS | 639-99-6 | 0.40 | 0.32 |
| 36 | trans-Nerolidol | 1551 | C15H26O | RI, MS | 40716-66-3 | 0.85 | 0.62 |
| 37 | (-)-Spathulenol | 1582 | C15H24O | RI, MS | 77171-55-2 | 0.85 | 0.81 |
| 38 | Caryophyllene oxide | 1589 | C15H24O | RI, MS | 1139-30-6 | 0.80 | 0.74 |
| 39 | Guaiol | 1602 | C15H26O | RI, MS | 489-86-1 | 1.30 | ndf |
| 40 | Tetradecanal | 1614 | C14H28O | RI, MS | 124-25-4 | 0.07 | 0.03 |
| 41 | β-Eudesmol | 1645 | C15H26O | RI, MS | 473-15-4 | 0.11 | ndf |
| 42 | α-Eudesmol | 1652 | C15H26O | RI, MS | 473-16-5 | 0.21 | ndf |
| 43 | α-Bulnesene | 1672 | C15H24 | RI, MS | 3691-11-0 | 0.68 | 0.59 |
| 44 | Ethyl hexadecanoate | 1993 | C18H36O2 | RI, MS | 628-97-7 | 0.07 | ndf |
| Total identified compounds | 90.86 | 83.61 | |||||
| Total terpene hydrocarbons | 9.93 | 13.06 | |||||
| Total oxygenated terpenes | 61.67 | 59.24 | |||||
| Total non terpene oxy-aliphatics | 0.46 | 0.00 | |||||
| Total aromatics | 18.7 | 11.22 | |||||
The SFMD process is a distillation process under microwave heating only using the moisture contained in the fresh plant materials, the heating time for materials is shorter and the content of solvent “water” existed in the system is lower; while the HD process is a conventional essential oil distillation process using water as the solvent, the heating time for materials is longer and the content of solvent “water” existed in the system is higher. In terms of the two processes mentioned above, their own characteristics were existed: non-obvious difference was existed in the essential oil composition, but a certain difference of content was existed in the specific components of essential oil. According to the experimental results, the relative percentages of 2-phenylethyl acetate, bornyl acetate, 3,7-dimethyl-2,6-octadien-1-ol,acetate, cinnamyl acetate and guaiol obtained by SFMD process were higher than that of HD, while which of α-pinene, β-pinene, ɑ-phellandrene and eucalyptol of SFMD essential oil were lower than HD.
4 Conclusions
An efficient technique referred to as SFMD-HE was developed to separate the essential oil and non-volatile flavonols and proanthocyanidins from C. japonicum Sieb. leaves. After optimization, 540 W of microwave power and a 40 min irradiation time were selected for the essential oil distillation. Then, we use the RSM to optimize the homogenate extraction process. The highest yields of the essential oil (6.50 ± 0.31 mL/kg), flavonols (12.48 ± 0.57 mg/g) and proanthocyanidins (71.97 ± 2.71 mg/g) were obtained with the optimal process conditions of 16 mL/g liquid–solid ratio, 4 min homogenate time and 71% ethanol volume fraction. Obvious differences in the relative contents of individual components were observed for essential oils obtained by SFMD-HE and HD using GC–MS analysis, which indicated that the SFMD-HE method presents an efficient isolation method that can also be used for the accurate analysis of essential oil, flavonols and proanthocyanidins from natural plants.
Acknowledgement
The authors thank the National Natural Science Foundation of China (Grant no. NSFC31670580) for financial support.
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