Efficient separation of Cinnamomum camphora leaf essential oil and in vitro evaluation of its antifungal activity

2.1 Plant materials and chemicals

Fresh C. camphora leaves were gathered from Jiangxi Normal University Yaohu campus (Nanchang, China) and identified by Professor Ronggen Deng (College of Life Science, Jiangxi Normal University, China). The C. camphora leaves were dried in the shade at 25 ± 2 °C for 7 days, then smashed with a grinder, screened through 40 mesh, and stored in a refrigerator.

F. avenaceum, F. sporotrioides, F. culmorum were purchased from China General Microbiological Culture Collection Center (CGMCC, Beijing, China). F. solani were purchased from China Center for Type Culture Collection (CTMCC, Wuhan, China). F. trichothecioides were obtained from Prof. Fenglan Li (Northeast Agricultural University, China). Potato was bought from the supermarket. Neutral protease (purity ≥ 95 %, enzyme activity ≥ 100 U/mg), cellulase (purity ≥ 95 %, enzyme activity ≥ 50 U/mg), hemicellulase (purity ≥ 95 %, enzyme activity ≥ 20 U/mg), pectinase (purity ≥ 95 %, enzyme activity ≥ 500 U/mg), Tween 80, nystatin, and d-Glucose were obtained from Shanghai Yuanye Biotechnology Co., ltd. (Shanghai, China). Agar Powder was gained from Beijing Aoboxing Bio-Tech Co., ltd (Beijing, China). A Milli-Q water purification system (Millipore, Billerica, MA, USA) was used to provide deionized water and used in the whole study.

2.2 CEO extraction

EUP-MAE consisted of two steps, namely enzymatic-ultrasound pretreatment and microwave assisted extraction procedure for the separation of CEO. C. camphora leaves powder (50 g) was mixed with a certain amount of enzyme solution (individual enzyme or enzyme mixture), and then the suspension was placed into KH-250DE ultrasonic bath (Kunshan Leibo Instrument Equipment Co., ltd., Kunshan, China) for enzymatic-ultrasound pretreatment. After the pretreatment, the suspension was transferred to a microwave oven for the extraction of CEO. The effects of eight factors were pre-tested to ascertain the appropriate ranges for the subsequent process optimization, and the corresponding results are shown on Table 1. The obtained CEO was collected in amber bottles after drying with anhydrous sodium sulphate and then placed in a 4 °C refrigerator for the latter analysis. CEO yields were calculated as the milligram of CEO per gram of raw materials.

U-MAE and HD were conducted to make comparisons with EUP-MAE for CEO extraction. U-MAE process was carried out as the same as EUP-MAE optimal conditions without loading any enzymes. As for HD, samples (50 g) were mixed with 900 mL of deionized water, and then heated 240 min by an electric jacket at a heating power of 1000 W. The essential oil was collected in amber bottles, dried with anhydrous sodium sulphate, and then stored at 4 °C before the subsequent analysis.

2.3 Experimental design

In order to optimize the extraction conditions of EUP-MAE, PBD coupled with BBD of RSM were employed. The independent or interactive effects of factors may determine the extraction efficiency, so multivariate analysis can identify the interaction effects between parameters and provide scientific experiment design. Among the experimental design, PBD technology was firstly adopted to screen out the relatively important factors from eight independent variables (A: enzyme dosage, %; B: pretreatment time, h; C: pH; D: ultrasound power, W; E: pretreatment temperature, °C; F: water to solid ratio, mL/g; G: microwave time, min; H: microwave power, W). The whole PBD design consisted of 12 runs with combinations of eight independent variables at high level (+) or low level (−) as shown in Table 2. Then BBD was used to further optimize the yield of CEO, and the effects of four relatively important independent parameters selected from PBD on the yield were investigated. The BBD tested three predicted responses (yield of CEO) in different combinations of four parameters, namely enzyme dosage (%, A), ultrasound power (W, D), microwave time (min, G) and microwave power (W, H) and three levels (−1, 0 and +1). A total of 29 different combinations, including 5 repetitions of the central point, were performed to estimate experimental errors and demonstrate the applicability of the model. The actual values of coding levels and parameters obtained from the pre-experiment are given in Table 1. Through the application of “Design Expert” software version 8.0 (Stat-Ease, Minneapolis, USA), the experimental design was structured and the actual results were analyzed.

2.4 GC–MS analysis

GC–MS analysis of CEOs was operated on a Thermo Trace-1300 ISQ-mass chromatograph (Thermo Fisher Scientific Inc., Waltham, MA, USA) coupled with a HP-5MS capillary column (30 m × 0.25 mm, 0.25 mm i.d., 0.2 μm film thickness). The carrier gas was helium. A 10 μL of the diluted CEOs was injected with a split ratio of 1:2 at a flow rate of 1 mL/min. The heating process was started at 60 °C and kept for 5 min, followed by a constant rate of 10 °C/min to 120 °C and maintained for 5 min, then increased to 200 °C and held for 5 min, and finally enhanced to 280 °C for 8 min. The temperature presets for the sampler and detector were 230 °C and 280 °C, respectively. The MS was run at a voltage of 70 eV in EI mode with a scanning range of 50–500 amu. The volatile compound content was determined by peak area normalization. The chemical constituents of CEO were recognized by comparing with the mass spectrum and retention index (RI) in NIST02 Mass Spectral Library.

2.5 In vitro antifungal activity assays of the CEO

CEO was tested for its antifungal activities against five pathogenic fungi of F. solani, F. culmorum, F. trichothecioides, F. sporotrioides and F. avenaceum. Antifungal activity of CEO was investigated using in vitro contact assays (Ben Ghnaya et al., 2016). CEO was diluted in 5 % of Tween 80 and then mixed with potato dextrose agar (PDA) at 50 °C to a volume of 10 mL. Round mycelial disks with a 5 mm diameter were cut from the circumference of 7 days old cultures and then inoculated in the center of each PDA plates (90 mm diameter) at 25 ± 2 °C for 7 days. PDA plates loaded with 5 % of Tween 80 solution and nystatin to replace CEO solution were recorded as negative and positive controls, respectively. The mycelium growth inhibition rate was calculated using the formula as Eq. (1) follows,

2.6 Statistical analysis

Design Expert 8.0 software (Stat Ease Inc., Minneapolis, MN, USA) was used to perform process design, analyze regression models, and draw response surface plots. All tests were in triplicate. The CEO yields were shown as the mean value ± SD and the significance of their differences were calculated by the ANOVA test using OriginPro 2018 software (OriginLab Corporation, Northampton, MA, USA).

3.1 The selection of enzyme species

Enzyme type could affect the extraction efficiency in the essential oil separation process involving enzymatic treatment (Hu et al., 2020). Therefore, the effects of four commercial enzymes (neutral protease, cellulase, hemicellulase and pectinase) and their mixtures (hemicellulase/pectinase, cellulase/pectinase, cellulase/hemicellulase, and cellulase/hemicellulase/pectinase) on CEO yield using EUP-MAE were studied. As shown in Fig. 1a, higher essential oil yields were achieved with enzymatic pretreatment compared to the control set (without enzymatic treatment). Cellulase possessed the highest essential oil yield compared to other individual enzymes and the control, which is similar to the previous studies (Jiao et al., 2012; Rashed et al., 2018). As cellulase, hemicellulase, and pectinase has the relatively higher CEO yield than neutral protease and the control group, and therefore these three enzymes were mixed to study the enzyme combinations on the essential oil yield in EUP-MAE process. The results in Fig. 1b indicated that the highest CEO yield was obtained by using the mixture of cellulase/hemicellulase/pectinase (1/1/1, w/w/w). This result was in consistent with previous studies, in which an equal quality ratio of cellulase/hemicellulase/pectinase provided the highest yield for the extraction of Cyperus esculentus seed oil and Lavandula angustifolia essential oil (Hu et al.,2020; Rashed et al., 2017). Plant cellular structures determine the enzyme type in the plant essential oil extraction process. Generally, cellulose, hemicellulase, and pectin are the three dominant components of plant cell cytoderm. Once these components were degraded, plant essential oil can be easily released into the solvent (Gil-Chavez et al., 2013). In the EUP-MAE extraction process, the mixed enzymes of cellulase/hemicellulase/pectinase had the higher essential oil yield thanks to their action on the decomposition of C. camphora leaves cell structures. Additionally, cellulase, hemicellulase, and pectinase have similar pH values and temperature ranges (Hu et al., 2020), which is conducive to the synergistic effect of their mixing and improve enzymatic hydrolysis during the extraction process (Nadar et al., 2018). Therefore, an enzyme mixture of cellulase/hemicellulase/pectinase (1/1/1, w/w/w) was selected for further optimization experiments.

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Fig. 1

The selection of enzyme species on CEO extraction using enzymolysis-ultrasound pretreatment and followed by microwave assisted extraction method. Control set: without enzymatic treatment.

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3.2 Optimization of CEO extraction conditions by EUP-MAE

3.2.1

3.2.1 PBD

For EUP-MAE process, there were numerous variables that affect the final essential oil yield. Whereupon, PBD was firstly used to select the variables that had a significant impact on the essential oil yield from eight factors (A: enzyme dosage, %; B: pretreatment time, h; C: pH; D: ultrasound power, W; E: pretreatment temperature, °C; F: water to solid ratio, mL/g; G: microwave time, min; H: microwave power, W). The first-order model equation developed by PBD for the essential oil yield was expressed as Eq. (2) following,

(2)

$$\mathrm{Y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}(\mathrm{m}\mathrm{g}/\mathrm{g})=5.25+0.69\times A+1.56\times {10}^{-3}\times B+0.35\times C-0.02\times D+0.04\times E+0.02\times F+0.07\times G+7.24\times {10}^{-3}\times H$$

The design and results of PBD were presented on Table 2. The influence of each variable on CEO yield can be illustrated by the magnitude of its T value and P value, and the corresponding positive or negative sign of T value represents the positive or negative influence on oil production, respectively. As represented on Table 2 and Fig. 2, all the factors had positive effects on CEO yield except for ultrasound power, which had a negative effect. Enzyme dosage (A), ultrasound power (D), microwave time (G) and microwave power (H) demonstrated significant effects on CEO separation in EUP-MAE procedure, so they were further investigated by BBD. Pretreatment time (B), pH (C), pretreatment temperature (E), and water to solid ratio (F) had no significant effect on the EUP-MAE process. According to the results of pre-experiment and PBD, these insignificant factors were maintained at 2 h for pretreatment time, 5 for pH, 50 °C for pretreatment temperature, and 16 mL/g for water to solid ratio.

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Fig. 2

Pareto chart of PBD for CEO extraction. (A) enzyme dosage; (B) pretreatment time; (C) pH; (D) ultrasound power; (E) pretreatment temperature; (F) water to solid ratio; (G) microwave time; (H) microwave power.

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3.2.3

3.2.3 Response surfaces analysis

The interaction influences of enzyme dosage, ultrasound power, microwave time, and microwave power on CEO yield were illustrated by using the three-dimension profile of multivariate nonlinear regression model. Fig. 3 emphasized the behavior of essential oil yield under the interactions influences of two parameters. In each figure, the other two variables were fixed at a central value. Fig. 3a described the interactive influences of enzyme dosage (A) and microwave time (G) on CEO yield with an ultrasound power (D) of 200 W and a microwave power (H) of 385 W. It can be concluded that increasing of enzyme dosage and microwave time was helpful to significantly enhance essential oil yield. Additionally, when the essential oil reach to the peak, the optimum value of the two parameters were 3.0 % of enzyme dosage and 34 min as microwave time. The reason for this phenomenon may be owing to that the increases of enzyme dosage and microwave time were beneficial to the deconstruction of plant cell walls, thus improving the essential oil yield (Hou et al., 2019). As there is no change on the substrate concentration, further increase in enzyme dosage did not contribute the enhancement of CEO yield, but improved the cost. Fig. 3b showed strong interactive influences between enzyme dosage (A) and microwave power (H) on CEO yield. On the basis of the literature (Gligor et al., 2019), microwave irradiation at the initial stage may contribute a significant increase in extracted yield by making essential oil more solubilization, whereas a further increase in microwave power may lead to the destruction of enzyme or plant substrate, due to decreased essential oil yield at the later phase. As exhibited in Fig. 3c, the interactive influences were depicted between microwave time (G) and ultrasound power (D) on the CEO yield. It was found that the CEO yield improved with the increases of microwave time and ultrasound power, and then decreased gradually with the further increase of these two variables. Fig. 3d showed the interactive influences of ultrasound power (D) and microwave power (H) on CEO yield. The results suggested that CEO yield increased with the increase of microwave power and ultrasound power, and the maximum CEO yield was obtained when the microwave time is about 34 min and the ultrasound power approximately is 190 W. This result may be due to the fact that the increase in ultrasound power produced many cavitation effects in the extracted matrix which promoted hydrolysis of the cell wall by the mixed enzyme, and the enhancement of microwave power accelerated the thermal motion of molecules in plant materials, resulting in active ingredient dissolves faster and improved essential oil yield (Rashed et al., 2017). Whereas, it worth to note that exorbitant ultrasound power and microwave power can degrade or isomerize some heat-sensitive components of target product (Bachtler and Bart, 2021), resulting in decline of the CEO yield.

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Fig. 3

The three-dimensional and contour surface plots from BBD. (a) The interactive influences of enzyme dosage (A) and microwave time (G) on CEO yield; (b) The interactive influences of enzyme dosage (A) and microwave power (H) on CEO yield; (c)The interactive influences of microwave time (G) and ultrasound power (D) on CEO yield; (d) The interactive influences of ultrasound power (D) and microwave power (H) on CEO yield.

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3.3 Comparison of EUP-MAE with reference methods

As illustrated in Table 5, the comparison of the essential oil yield of the developed EUP-MAE technology with other reference technologies (U-MAE and HD) was carried out under optimal conditions. EUP-MAE (19.23 ± 0.12 mg/g) exhibited a significantly higher CEO yield than those of traditional HD (16.37 ± 0.10 mg/g) and U-MAE (17.18 ± 0.15 mg/g). This phenomenon could be attribute to three reasonable respects. Firstly, according to the characteristics of plant matrix cell walls, the mixed enzymes pretreatment of plant materials can fully degrade plant cell walls and significantly increase the separation efficiency (Chari et al., 2013; Domínguez-Rodríguez et al., 2020; Vishwakarma and Malik, 2022). Secondly, ultrasound waves can propagate through any medium as mechanical waves by generating phases of expansion and compression, and induce the formation, development and implosion of cavitation bubbles in the liquid medium, thus accelerating cell collapse and promoting mass transfer behavior (Singla and Sit, 2021); and it is interesting to note that low-intensity ultrasound has been proved to improve the activity of several enzymes, such as cellulase and pectinase, by increasing the binding of the enzyme active site to the substrate (Khadhraoui et al., 2021), thus enhancing the enzymatic hydrolysis reaction and promoting the release of components in the plant matrix. Thirdly, after the high frequency electromagnetic wave produced by microwave can penetrate the water medium and reach the interior of the material, it can be quickly converted into heat, heating the water in the cells and evaporating, so as to rapidly increase the temperature and pressure inside the cells (Rashed et al., 2017). When the intracellular pressure exceeds the pressure the cell wall can withstand, the cell wall ruptures and the plant intracellular components are leached more quickly into the surrounding solvents (Suktham et al., 2021). In summary, EUP-MAE offers an obvious advantage over U-MAE and HD in enhancing the yield of plant essential oils thanks to the synergistic effects of enzymatic, ultrasound, and microwave radiation. Therefore, EUP-MAE is a promising separation technology of plant essential oils.

3.4 Compositions of CEOs obtained by different methods

Chemical components of CEOs isolated by EUP-MAE, U-MAE and HD were presented on Table 5. Based on the peak area normalization method for GC peaks, the contents of each component are calculated and listed in accordance with GC (HP-5MS) elution order. A total of 26 compounds were detected by GC–MS analysis. 23 compounds accounting for 99.66 % were detected on CEO extracted by EUP-MAE. As these for U-MAE and HD, 21 compounds were detected on the essential oils with the total proportions of 97.46 % and 97.90 %, respectively. The CEOs obtained by the three methods have the same main components but different contents. Three main ingredients were detected in CEO were eucalyptol, camphor and α-terpineol, which content of were approximately to those reported in Sriramavaratharajan et al. (2016). In the present study, it was worthy to note that camphor took the second highest proportion in CEO, which was different from the previous research of our group (Liu et al., 2018). This difference may be due to genetic variation or chemical variety (Cano et al., 2008; Lota et al., 2000). Sriramavaratharajan et al. (2016) have also pointed out that there were differences on camphor content in CEOs distributed in adjacent areas.

Notably, among the identified chemical components of CEO obtained by different methods, the proportion of oxygenated compounds was obviously more than that of sesquiterpene hydrocarbons. The content of oxygenated compounds in the CEO extracted from EUP-MAE was significantly higher than that of the essential oil extracted from U-MAE and HD (83.90 % of EUP-MAE, 79.33 % of U-MAE, 72.04 % of HD), while the proportion of sesquiterpene hydrocarbons was evidently lower than that of U-MAE and HD (15.76 % of EUP-MAE, 18.13 % of U-MAE, 25.86 % of HD). The reason behind the above results may be that oxygenated compounds with high dipole moments interact more strongly with microwave radiation and can be extracted more easily than sesquiterpene hydrocarbons with low dipole moments (Filly et al., 2014). Furthermore, the high proportion of oxygenated compounds detected in CEO by EUP-MAE possibly was attributed to the fact that thermal degradation and hydrolysis are reduced resulting from the shortening of distillation time under the combined influence of enzymolysis - ultrasound pretreatment and microwave radiation (Reis et al., 2020). Studies (Ferhat et al., 2006) suggested that oxygenated compounds intensify the aroma and value of essential oils. Therefore, the CEO extracted by our developed EUP-MAE method are more fragrant than these extracted by U-MAE and HD.

3.5 In vitro antifungal activity

Previous studies have reported that essential oil appears to be safer for controlling fungal strains than pure chemical bacteriostatic agents (Derbalah et al., 2011; Masyita et al., 2022). Therefore, the in vitro antifungal activity of CEO on inhibiting mycelial growth of the five fungal pathogens of potato dry rot were investigated. The minimum inhibitory concentration (MIC, mg/mL) and the effective concentration resulting in 50 % inhibition of mycelial growth (EC₅₀, mg/mL) were calculated graphically by linear regression using OriginPro 2018 software, in which CEO concentration and inhibition rate were taken as vertical axis and horizontal axis, respectively. As given on Table 6, the MIC and EC₅₀ values of CEO against F. solani, F. culmorum, F. trichothecioides, F. sporotrioides, and F. avenaceum varied in the range of 2.5–3 mg/mL and 0.74–1.54 mg/mL, respectively. Fig. 4 exhibited the inhibition patterns of CEO (A2-E2) and nystatin (A3-E3) at EC₅₀ values against five Fusarium strains. Compared with the negative control sets (PDA plates with only 5 % of Tween 80 solvent added) in Fig. 4A1-4E1, CEO could inhibit mycelial growths of all tested pathogen strains and exhibited the potential antifungal activity against these strains. Of particular interest was the fact that CEO had obvious influence on the colony color of the tested strains in comparison with nystatin.

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Fig. 4

The inhibition patterns for the mycelial growths of F. solani (A), F. culmorum (B), F. trichothecioides (C), F. sporotrioides (D), and F. avenaceum (E) after treated with CEO (A2-E2) and nystatin (A3-E3). Negative control sets (A1-E1) showed the PDA plates with only 5% of Tween 80 solvent added.

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Based on the previous study (Stević et al., 2014), CEO could be considered as high antifungal activity against the tested fungi. It is considered that plant essential oils possess an antibacterial property that could be a result of a single compound or of a combination of compounds acting synergistically or antagonistically (Lopes-Lutz et al., 2008; Tariq et al., 2019). Pragadheesh et al. (2013) suggested that the lipophilicity of volatile oils helps the components to penetrate the fungal plasma membrane, resulting in cytoplasmic coagulation and mycelial dissolution. Jeldi et al. (2022) pointed out that in the case of antifungal activity, the mechanism of action of essential oils appears to involve disturbing the cell wall and plasma membrane, leading to cell lysis and leakage of intracellular compounds, and ultimately cell death. Conner and Beuchat (1984) put forward the point of view that inhibitory activity was caused by interaction of essential oils in the synthesis of microbial cellular structural units and in enzyme systems associated with energy production. A metabolomics analysis exhibited that CEO has antibacterial activity by interfering with cellular metabolism, especially alanine, aspartic and glutamic acid metabolic pathways (Chen et al., 2020). Based on Chaturvedi et al. (2018), the efficacy of CEO could also be attributed to activity of oxygenated monoterpenes, eucalyptol, camphor and α-terpineol. Polec et al. (2019) has pointed out that the antibacterial activity of essential oil contained the high proportions of oxygen-containing monoterpenes, such as eucalyptol, a terpene that can influence sterols, a component of fungal cell membrane, to produce antibacterial effects. CEO has been observed on controlling the mycelial growth of Choanephora cucurbitarum thanks to the high content of camphor and eucalyptol, which was speculated to play a synergistic role with minor components (Viljoen et al., 2003). Zhou et al. (2014) have pointed out that the antifungal property of α-terpineol against Geotrichum citri-aurantii was ascribed to the destruction of the fungi cell membrane and thus leading to the subsequent leakage of intracellular compounds. Moreover, α-terpineol has been proved to possess the ability to destroy or penetrate into lipid structures (Kararli et al., 1995).