Physical-Chemical Characteristics and Chemical Composition of the Oil
Based on the extractions process of clove oil from dry flower buds, 11.2 ± 0.9 mL of oil was obtained, representing a mass 11.7 ± 1.0 g. As the oil density was determined at 1.041 g·mL-1, the mass yield was estimated at 5.83 ± 0.5 %. In a similar study, Radünz et al. [26] reported yields ranging from 1.87 % to 8.88 %, using a hydrodistillation process to obtain essential oil of clove. Low yields are associated with factors such collection period of the materials, storage conditions and climate, interfering in the oil constitution.
The quality of an essential oil depends on several parameters such as solubility in different organic solvents, density, refraction index, among others. Table 2 presents the physical-chemical parameters obtained to the essential oil of clove, according USP 41 methodology [25]. Through verification of the parameters, it is possible to conclude that the results are satisfactory since all values of the analysis were within the specification limits provided in the regulatory standard.
Table 2 Physical-chemical parameters obtained for the essential oil of clove
Parameters
|
Result Obtained
|
Especification1
|
Density Relative (g·mL-1)
|
1.041
|
1.038 – 1.060
|
Refraction Index (20°C)
|
1.531
|
1.527 – 1.535
|
Color
|
Light yellow
|
-
|
Appearance
|
Clear liquid
|
-
|
Odor
|
Characteristic
|
-
|
Moisture (wt%)
|
0.15
|
-
|
Solubility in Ethanol 70 vol%
|
1:2
|
1:2
|
1 United States Pharmacopeial Convention, 2018.
Table 3 presents the chemical composition of the clove oil, considering the major components that compose the samples. The mass composition relative to the eugenol, the major component of clove oil, is close to that obtained in similar researches, ranging from 83.6 % to 88.4 % [8].
Table 3 Chemical composition of the clove oil used in the tests
Parameters
|
Mass Composition (wt%)
|
Eugenol
|
86.87
|
β-Caryophyllene
|
11.70
|
Humulene
|
0.93
|
Eugenyl Acetate
|
0.11
|
Eugenyl Acetate Synthesis
Selection of the Lipase
Preliminary tests were carried out in order to define which lipase, among the three available enzymes (lyophilized enzyme produced Penicillium sumatrense microorganism, Lipozyme® TL 100L and Lipozyme® CALB L), would present the best response in terms of raw material conversion. At 50 °C, eugenol to acetic anhydride molar ratio of 1:3, enzyme load of 5.5 wt.%, the lipase Lipozyme® TL 100L presented the higher conversion: 85.40 ± 3.2 % after 2 hours of reaction. In these same reaction conditions, the homemade lyophilized enzyme and Lipozyme® CALB L presented conversion of 73.4 ± 4.2 % and 58.3 ± 6.0 %, respectively. Based on these results, lipase Lipozyme® TL 100L was chosen as catalyst to be utilized in the optimization of the reaction process via statistical design. It is important to note the interesting conversion achieved by the lyophilized enzyme produced in laboratory, superior in comparison to the commercial lipase CALB L. This fact demonstrates that the homemade biocatalyst has potential to be applied in the process.
Influence of the Reaction Parameters in the Eugenyl Acetate Synthesis.
In the evaluation of the EA production using Lipozyme® TL 100L as reaction biocatalyst, a full 23 factorial design with three replications of the central point was employed, investigating the effects of the eugenol to acetic anhydride molar ratio, temperature and enzyme load in the process performance. Table 1 presents the matrix of the statistical design with coded and real values of the independent variables and the experimental reaction conversions to EA. The highest conversion into EA occurred at 55 °C, eugenol to acetic anhydride molar ratio of 1:1 and 10 wt% of Lipozyme® TL 100L (assay 7), achieving 91.80 % of conversion. The lowest conversion obtained occurred at 45 °C, eugenol to acetic anhydride molar ratio of 1:1 and 5 wt% of Lipozyme® TL 100L (38.12 % of conversion, assay 1), indicating that a catalyst load superior than 5 wt% is indicated to the reaction.
Results presented in Table 1 were statistically analyzed via Pareto Diagram, according shown in Figure 1. Significant variables presented p < 0.05. Coefficients with a positive sign indicate a synergic effect whereas negative coefficients indicate an antagonistic effect in the ester formation. The Pareto Diagram of standardized effects confirm that the molar ratio between eugenol and acetic anhydride had no significant effect in the EA synthesis, while reaction temperature and lipase load had a significant positive effect (p < 0.05) on ester production. However, it is possible observe that the interaction between the variables “eugenol to acetic anhydride molar ratio” and “reaction temperature” had a negative effect on ester production. Similar tendency was also observed by Silva et al. [4] that, however, used immobilized lipase to produce EA from clove oil.
The statistical analysis of the results presented on Table 1 allowed the obtaining of a model for EA conversion as a function of eugenol to acetic anhydride molar ratio, temperature and lipase load. The model was statistically validated (p < 0.05) by analysis of variance (ANOVA), according presented on Table 4, where a coefficient of determination (R2) of 99.15, proving that the model obtained, with a level of reliability of 95 %, is statistically valid.
Table 4 ANOVA for validation of the model that describes the production of eugenyl acetate using the lipase Lipozyme TL 100L
Source of Variation
|
Sum of Squares
|
Degrees of Freedom
|
Mean Square
|
FCalculated
|
p-value
|
(1) Eugenol to ac. anhydride molar ratio (mol·mol-1)
|
14.55
|
1
|
14.55
|
5.25
|
0.1491 b
|
(2) Temperature (°C)
|
1,516.10
|
1
|
1,516.10
|
5466
|
0.0018 a
|
(3) Lipase load (wt%)
|
814.67
|
1
|
814.67
|
293.75
|
0.0034 ª
|
1 by 2
|
247.87
|
1
|
247.87
|
89.37
|
0.0110 a
|
1 by 3
|
53.41
|
1
|
53.41
|
19.26
|
0.0482 a
|
2 by 3
|
50.96
|
1
|
50.96
|
18.37
|
0.0503 b
|
Lack of Fit
|
17.61
|
2
|
8.80
|
3.17
|
0.2395
|
Pure Error
|
5.55
|
2
|
2.77
|
|
|
Total
|
2720.68
|
10
|
|
|
|
a Significant at “Prob > F” less than 0.05;
b Insignificant at “Prob > F” more than 0.05.
Results presented in the Table 1 and 4 as well as the Pareto diagram (Figure 1) shows an interactive effect between the considered variables. Thus, an independent analysis of the parameters would not be appropriate. Therefore, contour surfaces between the parameters is necessary to find the region with the best response and thus determine the optimum conditions of the system, where the EA conversion is maximum. These results are presented in the Figure 2. The Figure 2a shows the conversion in EA as function of reaction temperature and lipase load, Figure 2b presents the conversion in esters as a function of eugenol to acetic anhydride molar ratio and temperature and Figure 2c simulates the ester conversion ranging with the eugenol to acetic anhydride molar ratio and lipase load. From these results, it is possible to observe that the EA conversion is benefited with the simultaneous increase of the enzyme load and reaction temperature, especially with temperatures higher than 52 °C. It was also possible to observe that at temperatures below 48 °C, even an increase in the eugenol to acetic anhydride molar ratio was not enough for satisfactory EA conversions to be achieved, requiring higher temperatures to be used in the system. Similar situation was observed when the EA conversions were analyzed ranging the eugenol to acetic anhydride molar ratio and lipase load. With lipase loads inferior than 7 wt%, even using a high eugenol to acetic anhydride molar ratio, it was not possible to obtain satisfactory conversions, corroborating the hypothesis presented previously in the Pareto Diagram (Figure 1) that this variable is not significant for the EA conversion. Such finding allowed us to deduce that a minimum eugenol to acetic anhydride molar ratio of 1: 1 is the ideal to be used in the process. On the other hand, as expected, an increase in the lipase load applied to the process returned satisfactory EA conversion.
Therefore, based on the observed results, the reaction conditions that yielded the highest EA conversion were: 55 °C, eugenol to acetic anhydride molar ratio of 1:1 and 10 wt% of lipase load.
Time Course of the Eugenyl Acetate Synthesis
With the optimization of the reaction parameters considered in the EA production, the behavior of reaction temperature, Lipozyme® TL 100L load and eugenol to acetic anhydride molar ratio were evaluated in a time course to the acetylation process. Samples at 20, 40, 60, 120, 180, 240, 300 and 360 min were collected using the optimized reaction conditions previously obtained (55 °C, eugenol to acetic anhydride molar ratio of 1:1 and 10 wt% of lipase). The results obtained are presented on Figure 3.
From the results obtained, it should be note the interesting enzymatic activity of the lipase Lipozyme® TL 100L after 1 hour of process, reaching more than 60 % of feedstock conversion. As the process advance, the increments in conversion decreased according the reaction equilibrium is achieved. With the use of the optimized reaction conditions, it was possible to achieve more than 90% conversion in 5 h of process. Data about the application of lipases in liquid formulation for EA synthesis from clove oil are rare. Few researches about this topic are published, where those that involve enzymatic process, use immobilized lipases as reaction catalyst. Silva et al. [4] investigated the ability of the commercial immobilized lipase Lipozyme® TL IM to catalyze the acetylation of essential oil of clove with acetic anhydride in a solvent-free system. At 70 °C, eugenol to acetic anhydride molar ratio of 1:5 and 5 wt% of lipase, a conversion of 92.86 % was obtained by these authors after 3 h of reaction. In another similar research, Chiaradia et al. [27] reported data of an eugenyl acetate production by esterification of eugenol and acetic anhydride in a solvent-free system using the immobilized lipase Novozym® 435 as catalyst: at 50 °C, eugenol to acetic anhydride of 1:3 and 5.5 wt% of enzyme, a conversion of 99 % was achieved after 6 h of reaction.
Characterization of the Eugenyl Acetate
The chemical structure of the purified EA obtained at optimized reaction conditions was confirmed by the proton nuclear magnetic resonance (1H-NMR) and infrared spectroscopy (FT-IR) analysis.
The results of the FT-IR analysis and the information related to the identified attributions and functional groups are present in Figure 4. The evaluated spectrum shows bands at 2,939 and 2,841 cm-1 that are attributed, respectively, to the asymmetric and symmetric axial deformation of the CH2 group, while the existing one at 3,005 cm-1 is attributed to the =C-H stretch of the aromatic ring structure. In the spectrum it is possible to observe the characteristic presence of the carbonyl band of the ester bound with the aromatic ring at 1,761 cm-1, suggesting that the molecule is, in fact, EA due to the addition of the acyl group in the eugenol molecule. In addition, the spectra present two axial stretch bands of the ester at 1,184 cm-1 (high intensity) and 1,267 cm-1 (medium intensity). The bond stretches of the C-O between 1,214 and 1,033 cm-1 are assigned to the methoxy group. In 1,419 and 1,368 cm-1 there was a folding of CH2 and CH3 [28]. According Engel et al. [28], the stretch of the double aliphatic/aromatic carbon bond is around 1,680 – 1,600 and 1,600 to 1,475 cm-1, respectively. In Figure 4, such stretches occurred in 1,604 and 1,507 cm-1, originating from C = C (aliphatic) and C = C (aromatic). The aromatic compound of the EA molecule presented characteristic bands around 906, 822 and 747 cm-1, which are attributed to off-plane folding, where these occur at 900 – 690 cm-1 and are used to define aromatic ring replacement pattern [28].
Table 5 presents results of 1H and 13C NMR analysis, demonstrating the molecular structure of EA ester, obtained through acetylation reaction. The ester was submitted to detailed analysis of 1H NMR spectra for 1 and 2 D. Through the NMR spectrum of 1H (Supplementary Information 1), DEPT 135 (Supplementary Information 2) and Table 5, was observed a dubbing at δH 7.00 ppm (1H of position 6 carbon, d, J = 8.0 Hz), a voice over at δH 6.96 ppm (1H of position 3 carbon, d, J = 1.8 Hz) and a double dublet at 6.78 (1H of position 5 carbon, dd, J = 8.0 and 1.8 Hz), indicating a tri-substituted aromatic ring. The position of the substituters in the aromatic ring was performed based on the values of chemical displacements, multiplicity and long-distance correlations. The atoms of the functional group that are closer to the oxygen atom have less shielding (higher value of ppm of chemical displacement), than the atoms closest to a carbon atom (lower value of ppm chemical displacement) [28]. Thus, of the 12 carbon signals expected for EA that are observed in the DEPT 135 spectrum, the carbon signal of position 12 is highlighted, according to Table 5, which corresponds to the carbonyl group of the ester with the highest displacement of 169.0 ppm. Additionally, a dublet was observed at δH 3.38 (2H of position 7 carbon, d, J = 6.8 Hz) and two multiplets in δH 6.00 (1H of carbon of position 8, m) and δH 5.12 (2H carbon of position 9, m) combined with the correlations of the HMBC (Supplementary Information 3). It is possible to assign this behavior to the isoprenic unit (alilic group) as one of the substituents linked to the aromatic ring. The presence of two singlets in δH 3.76 (3H of carbon from position 10, s) and 2.25 (3H of carbon of position 11, s) are attributed respectively to hydrogens of the methoxy group and hydrogens of the acetoxi group, associated with data from DEPT 135. It is allowed to characterize the molecular structure of the EA obtained in the acetylation of eugenol with acetic anhydride using the enzyme Lipozyme® TL 100L as catalyst. Santos et al. [29] analyzed the molecule of EA through NMR, verifying the peaks of the additional acetyl group (C-CH3) with lower chemical displacement value, and -COCH3 with higher ppm value were observed in the spectra.
Table 5 1H and 13C NMR data (DMSO-d6, 600 and 150 MHz) of eugenyl acetate ester (δ in ppm and J in Hz)
Eugenyl Acetate
|
|
Position (Carbon)
|
Hydrogen Displacement (δH)
|
Carbon Displacement (δC)
|
1
|
-
|
138.1
|
2
|
-
|
151.2
|
3
|
6.96 (d 1;1.8)
|
113.2
|
4
|
-
|
139.2
|
5
|
6.78 (dd 2; 1.8; 8.0)
|
120.7
|
6
|
7.00 (d 1; 8.0)
|
123.0
|
7
|
3.38 (d 1; 6.8)
|
39.8
|
8
|
6.00 (m 3)
|
137.9
|
9
|
5.12 (m 3)
|
116.3
|
10
|
3.76 (s 4)
|
20.7
|
11
|
2.25 (s 4)
|
56.0
|
12
|
-
|
169.0
|
1 d: doublet; 2 dd: double doublet; 3 m: multiplet; 4 s: singlet.