The Chemical Profiling of Essential Oils from Different Tissues of Cinnamomum camphora L. and Their Antimicrobial Activities

Cinnamomum camphora L. is grown as an ornamental plant, used as raw material for furniture, as a source of camphor, and its essential oil can be used as an important source for perfume as well as alternative medicine. A comparative investigation of essential oil compositions and antimicrobial activities of different tissues of C. camphora was carried out. The essential oils were extracted by hydrodistillation with a Clevenger apparatus and their compositions were evaluated through gas chromatography-mass spectrometry (GC-MS), enantiomeric composition by chiral GC-MS, and antimicrobial properties were assayed by measuring minimum inhibitory concentrations (MICs). Different plant tissues had different extraction yields, with the leaf having the highest yield. GC-MS analysis revealed the presence of 18, 75, 87, 67, 67, and 74 compounds in leaf, branch, wood, root, leaf/branch, and leaf/branch/wood, respectively. The significance of combining tissues is to enable extraction of commercial quality essential oils without the need to separate them. The oxygenated monoterpene camphor was the major component in all tissues of C. camphora except for safrole in the root. With chiral GC-MS, the enantiomeric distributions of 12, 12, 13, 14, and 14 chiral compounds in branch, wood, root, leaf/branch, and leaf/branch/wood, respectively, were determined. The variation in composition and enantiomeric distribution in the different tissues of C. camphora may be attributed to the different defense requirements of these tissues. The wood essential oil showed effective antibacterial activity against Serratia marcescens with an MIC of 39.1 μg/mL. Similarly, the mixture of leaf/branch/wood essential oils displayed good antifungal activity against Aspergillus niger and Aspergillus fumigatus while the leaf essential oil was notably active against Trichophyton rubrum. C. camphora essential oils showed variable antimicrobial activities against dermal and pulmonary-borne microbes.


Introduction
Essential oils are natural, complex, volatile chemical admixtures of an aromatic odor, extracted as secondary metabolites. Different types of essential oils have their applications in the pharmaceutical and flavoring industries. Consumption of essential oils has risen day by day either directly or indirectly because of consciousness in personal health and hygiene, and as alternative medicines [1]. Bacteria and fungi are encountered everywhere in the biosphere due to their metabolic ability and are easily grown under a wide range of environmental conditions; many are known to be pathogenic [2]. Essential oils have often been used in lieu of synthetic chemicals to counter microbial attacks that cause food spoilage, or infections. The available synthetic flavoring and antimicrobial chemicals can, however, be toxic and detrimental to our health if they exceed the prescribed limit of consumption [3]. Therefore, essential oils could be alternative solutions to ensuring food safety, retention of their nutritional value and quality, as well as eliminating human health risk. Some components from different plant essential oils are legally registered flavoring agents in foodstuffs because oxygenated monoterpenes display promising antimicrobial activity [4][5][6][7].
Different parts (roots, wood, branch, and leaf) of Cinnamomum camphora L. are abundant in essential oils, which have linalool, 1,8-cineole, and camphor as major components. Essential oils from C. camphora have chemotypes such as iso-borneol, camphor, 1,8-cineole, linalool, and borneol types [8][9][10]. Biosynthesis of secondary metabolites and their proportions in essential oils lead to variations in composition due to environmental factors (seasonal variation, geographic variation, light availability, herbivory and microbial infection, pH of the soil) pollution, herbicide and pesticide use, and anthropogenic behavior [11]. Due to its diverse climate, geology, and topographical area, Nepal's biodiversity is a repository of secondary metabolites in essential oils. In Nepal, the chemotype of C. camphora essential oil is camphor, and its diverse bioapplication has been explored [12]. It has very significant prospective nutraceutical and pharmaceutical applications [13]. Oxygenated monoterpene chemotypes of C. camphora essential oil have multifunctional bioapplications such as antibacterial activities [14], antifungal activities [15], insecticidal activities [16], anti-inflammatory activities [17], and so on. These activities are related to the lipophilic nature of secondary metabolites in essential oils [18] and may act either by synergistic or antagonistic effects [12,19].
In this work, the chemical and enantiomeric compositions of the essential oils from different tissues (root, wood, branch, and leaf) of C. camphora were analyzed and compared. In addition, their effectiveness against dermal and pulmonary-borne microbes of the essential oils from different tissues of C. camphora L. was explored.

Isolation and Yields of Essential Oils and Yields
The highest essential oil yield was observed for the leaf (2.67%), whereas the lowest yield was from the wood (1.0%) of C. camphora. A slightly higher extraction yield was observed compared to previous reports of C. camphora leaf essential oil [12]. The yield of extraction depends on different factors such as geographical variation, harvesting time, extraction methods, extraction temperature and extraction time. The essential oil yields from different C. camphora tissues are shown in Table 1.

Enantiomeric Composition of Essential Oil Terpenoids
In total, 12, 12, 13, 14 and 14 chiral terpenoid components were evaluated for their enantiomeric distributions in the branch, wood, root, leaf/branch, and leaf/branch/wood essential oils, respectively. Relative percentages of the levorotatory and dextrorotatory enantiomers for each of the chiral components in the essential oils are listed in Table 3. The biosynthesis of levorotatory and dextrorotatory compounds from any geographical location is almost constant and may be used for authentication of C. camphora essential oils. Camphor is the major oxygenated monoterpene and was (+)-camphor-predominant in our study. Similar enantiomeric distributions have been reported for C. camphora essential oil [15]. α-Thujene, sabinene, α-phellandrene, cis-sabinene hydrate, trans-sabinene hydrate, and β-caryophyllene were detected for the first time in this current study. β-Caryophyllene and sabinene were consistently present as dextrorotatory enantiomers. Interestingly, the wood and root essential oils showed contrasting type enantiomeric distributions for α-pinene and camphene. Likewise, the branch and wood essential oils showed oppositive enantiomeric relationships for α-thujene, borneol, and α-terpineol. Additionally, the branch and root essential oils showed contrasting distributions for α-thujene, α-pinene, camphene, borneol, and α-terpineol.

Antibacterial and Antifungal Activity
The essential oils of C. camphora have demonstrated a broad range of antimicrobial activities against different pathogens [12,20,21]. The minimum inhibitory concentrations (MICs) of the different C. camphora essential oil against a panel of bacteria and fungi are presented in Tables 4 and 5, respectively. The wood essential oil showed good antibacterial activity against Serratia marcescens. In the wood oil, camphor, 1,8-cineole, α-terpineol, and safrole were the major components, and the observed activity of the wood oil against S. marcescens may be due to synergism among these and other constituents. Camphor, 1,8-cineole, α-terpineol, and safrole showed only marginal activity against S. marcescens (MIC = 312.5 µg/mL). A study suggested that a synergistic antimicrobial effect occurs between 1,8-cineole and camphor [22]. The essential oils of C. camphora demonstrated weaker antibacterial activities than those of the positive control, gentamicin (MIC < 19.5 µg/mL).  The essential oil from the mixture of leaf/branch/wood of C. camphora displayed good antifungal activity against Aspergillus niger and Aspergillus fumigatus (MIC = 78.1 µg/mL), while the leaf essential oil was notably active against Trichophyton rubrum with an MIC of 78.1 µg/mL. The essential oils of C. camphora demonstrated weaker antifungal activities than those of the positive control, amphotericin B (MIC < 19.5 µg/mL). Safrole alone was effective against Aspergillus fumigatus. However, the safrole-rich root essential oil showed no such effectiveness, which may be due to antagonistic effects among constituents. The antifungal mechanisms of activity of essential oils are not clearly understood. However, it has been postulated that the hydrophobic constituents either disrupt cytoplasmic membranes leading to cytoplasmic leakage, cell lysis, and ultimate death, or inhibition of sporulation [23].

Plant Material and Isolation of Essential Oils
The different fresh plant tissues of C. camphora, collected in May 2019 from Tribhuwan University, Kirtipur, Kathmandu, were cut into smaller pieces. For each plant tissue sample, a ratio of 1:3 plant sample and water was subjected to hydrodistillation using a Clevenger apparatus for 3 h. The essential oils were dried over anhydrous sodium sulfate and stored in bottles at 5 • C until further use. Essential oil yields from different tissues are summarized in Table 1.

Chemical Composition Analysis by Gas Chromatography/Mass Spectrometry (GC-MS)
The essential oils from C. camphora were analyzed by a Shimadzu GCMS-QP2010 Ultra with electron impact (EI) mode with 70 eV along with ZB-5MS capillary GC column. 40-400 m/z scan ranges with a scan rate of 3.0 scan/s. The column temperature was programmed at 50 • C for 2 min and then increased at 2 • C/min to a temperature of 260 • C. The carrier gas was helium with a column head pressure of 552 kPa and a constant flow rate of 1.37 mL/min. The injector temperature was kept at 260 • C. For each essential oil sample, 1:10 (v/v) solution in dichloromethane (DCM) was prepared and 0.3 µL was injected using a split ratio of 1:30. Identification of the individual components of the essential oils was determined by comparison of the retention indices and comparison of the mass spectral fragmentation patterns (over 80% similarity match) with those found in the MS databases using LabSolutions GCMS solution software version 4.45 (Shimadzu Scientific Instruments, Columbia, MD, USA) [24][25][26]. The relative percentages of the individual components are listed in Table 2.

Enantiomeric Analysis by Chiral Gas Chromatography-Mass Spectrometry (CGC-MS)
Enantiomeric analysis of C. camphora essential oil was carried out using a Shimadzu GCMS-QP2010S with EI mode (70 eV) having a Restek B-Dex 325 chiral capillary GC column, 40-400 m/z scan ranges with a scan rate of 3.0 scan/s. The column temperature was programmed at 50 • C, at first increased by 1.5 • C/min to 120 • C and then 2 • C/min to 200 • C. The final temperature of the column was maintained at 200 • C. The carrier gas was helium with a constant flow rate of 1.8 mL/min. For each essential oil sample, a 3% w/v solution in DCM was prepared, and 0.1 µL was injected using a split ratio of 1:45 [24][25][26]. The enantiomer percentages were determined from peak area. Identification of enantiomers was determined by comparison of retention times with authentic samples obtained from Sigma-Aldrich (Milwaukee, WI, USA). Enantiomeric distribution of chiral terpenoid components in C. camphora essential oils are listed in Table 3.

Antibacterial Screening
All tested bacteria were cultured on tryptic soy agar medium. A 5000-µg/mL solution of each essential oil was prepared in dimethyl sulfoxide (DMSO), and 50 µL diluted in 50 µL of cation-adjusted Mueller Hinton broth (CAMHB) (Sigma-Aldrich, St. Louis, MO, USA) was added to the top well of a 96-well microdilution plate. The prepared stock solution of essential oils was then serially two-fold diluted in fresh CAMHB to obtain final concentrations of 2500, 1250, 625, 312.5, 156.3, 78.1, 39.1 and 19.5 µg/mL. Freshly harvested bacteria with approximately 1.5 × 10 8 CFU/mL final concentration (determined using the McFarland standard) were added to each well of 96-well microdilution plates that were incubated at 37 • C for 24 h. Gentamicin (Sigma-Aldrich, St. Louis, MO, USA) and DMSO were used as positive and negative controls, respectively [25,27].

Conclusions
Different plant tissues of Cinnamomum camphora L. were collected from the Tribhuwan University area. The essential oils from these different tissues showed differences in chemical compositions, enantiomeric distributions, and antimicrobial activities. The yield of extraction varied depending upon the tissue used. The significance of combining tissues was to enable extraction of commercial quality essential oil without the need to separate them. However, combining tissues had low extraction yields compared to individual tissues. The oxygenated monoterpenoid camphor was the dominant component in all parts of C. camphora except for the root essential oil, which was rich in safrole. The data analysis in this study can used to create a benchmark for future C. camphora essential oil assessments, as well as authentication for adulteration or consumer safety. The wood essential oil showed the best antibacterial activity against Serratia marcescens among the tested bacterial strains with an MIC of 39.1 µg/mL. The leaf/branch/wood essential oil showed good antifungal activity against Aspergillus niger and Aspergillus fumigatus, while the leaf essential oil showed good antifungal activity against Trichophyton rubrum with an MIC of 78.1 µg/mL. Cinnamomum camphora L. and its essential oils can be used as important source antibacterial and antifungal agents.
Supplementary Materials: The following are available online. Figure S1: Gas chromatogram of leaf essential oil of Cinnamomum camphora L. Figure S2: Gas chromatogram of wood essential oil of Cinnamomum camphora L. Figure S3: Gas chromatogram of root essential oil of Cinnamomum camphora L. Figure S4: Gas chromatogram of branch essential oil of Cinnamomum camphora L. Figure S5: Gas chromatogram of leaf/branch/wood essential oil of Cinnamomum camphora L. Figure S6: Gas chromatogram of leaf/branch essential oil of Cinnamomum camphora L.  Acknowledgments: The authors are thankful to the APRC and ARC for GCMS, Chiral GCMS analysis, and antimicrobial assay. We acknowledge Sunita Satyal and Ambika Satyal for their constructive suggestions and support.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.