*2.5. Statistical Analysis*

Results data were statistically analyzed using Scheffé's test of the SAS system (version 9.2, Cary, NC, USA) with a 95% confidence interval. Scheffé's test is a post hoc multiple comparison method with stringent error control.

*2.5. Statistical Analysis* 

**3. Results and Discussion** 

#### **3. Results and Discussion** *3.1. Changes in Composition of C. osmophloeum Leaf Oil after Thermal Degradation*

*Molecules* **2021**, *26*, x FOR PEER REVIEW 3 of 8

#### *3.1. Changes in Composition of C. osmophloeum Leaf Oil after Thermal Degradation* Constituents of *C. osmophloeum* leaf essential oil were analyzed by GC–MS; a gas

Results data were statistically analyzed using Scheffé's test of the SAS system (ver-

Constituents of *C. osmophloeum* leaf essential oil were analyzed by GC–MS; a gas chromatogram of leaf essential oil is shown in Figure 1A. The main constituent of leaf essential oil was linalool (93.30%), the other minor constituents were 2-methyl benzofuran (1.99%), α-pinene (0.66%), cinnamyl acetate (0.63%), limonene (0.61%), β-caryophyllene (0.59%), methyl chavicol (0.57%), and *trans*-cinnamaldehyde (0.52%), as listed in Table 1. Due to the high content of linalool, *C. osmophloeum* leaf essential oil was classified into the linalool-chemotype. chromatogram of leaf essential oil is shown in Figure 1A. The main constituent of leaf essential oil was linalool (93.30%), the other minor constituents were 2-methyl benzofuran (1.99%), α-pinene (0.66%), cinnamyl acetate (0.63%), limonene (0.61%), β-caryophyllene (0.59%), methyl chavicol (0.57%), and *trans*-cinnamaldehyde (0.52%), as listed in Table 1. Due to the high content of linalool, *C. osmophloeum* leaf essential oil was classified into the linalool-chemotype.

**Figure 1.** Gas chromatogram of linalool-chemotype *C. osmophloeum* leaf oil after thermal degradation. (**A**) 25 °C; (**B**) 100 °C; (**C**) 150 °C. **Figure 1.** Gas chromatogram of linalool-chemotype *C. osmophloeum* leaf oil after thermal degradation. (**A**) 25 ◦C; (**B**) 100 ◦C; (**C**) 150 ◦C.

As presented in Figure 1B, several significant peaks occurred in the gas chromatogram of leaf essential oil after the heat treatment at 100 °C for 30 min. The content of the main constitute, linalool, was reduced from 93.30% to 64.01% (Table 1). New constituents were observed for β-myrcene (5.56%), α-phellandrene (0.91%), α-terpinene (1.49%), *cis*ocimene (4.70%), γ-terpinene, and terpinolene (2.53%) in the thermally degraded leaf essential oil. Major variations were found for the increasing contents of limonene and *trans*ocimene, which were 0.61% and 0.32% in the raw leaf essential oil and obviously increased As presented in Figure 1B, several significant peaks occurred in the gas chromatogram of leaf essential oil after the heat treatment at 100 ◦C for 30 min. The content of the main constitute, linalool, was reduced from 93.30% to 64.01% (Table 1). New constituents were observed for β-myrcene (5.56%), α-phellandrene (0.91%), α-terpinene (1.49%), *cis*-ocimene (4.70%), γ-terpinene, and terpinolene (2.53%) in the thermally degraded leaf essential oil. Major variations were found for the increasing contents of limonene and *trans*-ocimene, which were 0.61% and 0.32% in the raw leaf essential oil and obviously increased to 7.77% and 7.94% in the thermally degraded specimen.

to 7.77% and 7.94% in the thermally degraded specimen. Similar degradation was observed from the gas chromatogram of leaf essential oil after the heat treatment at 150 ◦C for 30 min (Figure 1C). Linalool had a remarkable decrease in content from 93.30% in the original leaf essential oil to 27.54% in the 150 ◦Cdegraded specimens. The peaks of the new compounds generated under more severe heat treatment became more obvious; *trans*-ocimene was present in the degraded leaf essential oil of 20.08%, β-myrcene 17.89%, *cis*-ocimene 11.72%, limonene 11.40%, terpinolene 3.37%,

and α-terpinene 1.69%, in comparison with the original leaf essential oil, where these contents were much smaller. The increased amount (65.22%) of these compounds was close to the decrease in linalool (65.76%). Figure 2 illustrates the degradation mechanism of linalool and chemical structures of degradation products. After heat treatments at 100 ◦C and 150 ◦C, compounds β-myrcene, *cis*-ocimene and *trans*-ocimene were formed through the dehydroxylation of linalool. Moreover, ene cyclization occurred to linalool and its dehydroxylated products, further formed the compounds limonene, terpinolene and α-terpinene.

**Table 1.** Compositions of linalool-chemotype *Cinnamomum osmophloeum* leaf essential oil after thermal degradation.


RT: retention time (min); KI: Kovats index relative to *n*-alkanes (C9 – C24) on a DB-5MS column; rKI: Kovats index on a DB-5MS column in the reference [34].

**Figure 2.** Schematic illustration of the thermal degradation of linalool. **Figure 2.** Schematic illustration of the thermal degradation of linalool.

*3.2. Optimization of Microencapsulation of Leaf Essential Oil with β-Cyclodextrin*  The preparation method of microencapsulation can influence the property of β-cyclodextrin microcapsules. Kfoury et al. (2016) studied the aroma release effect from the solid inclusion complexes of β-cyclodextrin with trans-anethole by two preparation methods, freeze-drying (FD) and co-precipitation coupled to FD (Cop-FD). Cop-FD microcap-Leiner et al. (2013) investigated the pyrolysis behavior of linalool. Linalool was pyrolyzed in a temperature range of 350–600 ◦C under nitrogen and underwent enetype cyclization reactions leading to plinols, four cyclopentanol compounds [38]. The result varied from this study may be due to the different heating temperatures and the environment (under N2 or under air).

sules retained more efficiently trans-anethole than that of FD microcapsules; it revealed

The specimen to β-cyclodextrin ratio and the solvent ratio is the important factors that influence the yield of microcapsules. Yields of linalool and leaf essential oil microencapsulated with β-cyclodextrin by different reaction conditions are presented in Table 2. β-Cyclodextrin completely dissolved in the solution (ethanol/water, 1:2 *v/v*) by heating at 50 °C for 5 min, then the solution was cooled down to 25 °C without adding the core material, and no powders/crystals formed or precipitated even at 4 °C. The highest yield of microcapsule was 94.2% at the linalool to the β-cyclodextrin ratio of 15:85 (*w/w*), which

 0:100 1:2 0.0 ± 0.0 d\* 5:95 1:2 54.3 ± 3.1 a\* Linalool 10:90 1:2 86.9 ± 0.2 b 15:85 1:2 94.2 ± 0.4 c 20:80 1:2 91.0 ± 0.5 b,c 15:85 1:7 93.3 ± 0.6 B 15:85 1:5 98.1 ± 0.1 C Linalool 15:85 1:3 97.0 ± 0.4 C 15:85 1:2 94.2 ± 0.4 B 15:85 1:1 83.4 ± 1.8 A

Leaf essential oil 15:85 1:5 96.5 ± 0.2 \*: Different letters (a–d and A–C) in Table refer to statistically significant difference at the level of *p* <

**EtOH: H2O** 

**(***v/v***) Yield (%)** 

was quite close to the molar ratio (linalool:β-cyclodextrin) of 1:1.

**Specimen Specimen: β-CD** 

0.05 according to Scheffé's test.

**Table 2.** Yields of linalool and leaf essential oil microencapsulated with β-cyclodextrin.

**(***w/w***)** 
