The Essential Oil Composition and Antimicrobial Activity of Liquidambar formosana Oleoresin

The oleoresin essential oils of Liquidambar formosana have potential therapeutic benefits. However, current research on L. formosana oleoresin essential oil is still in its early stages, and its chemotypic characterization is undefined. For better leveraging of plant resources and application of the essential oil, we collected 25 L. formosana oleoresin essential oil samples of individual trees from different geographical areas of Southern China. The essential oils were obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry (GC–MS) and gas chromatography–flame ionization detection (GC–FID). The major components of the essential oils were (E)-caryophyllene (3.3%-64.4%), α-pinene (0.6%-34.5%), β-pinene (0.6%-26.0%), camphene (0.3%-17.3%), and limonene (0.2%-7.9%). A chiral GC–MS analysis was carried out on the essential oil samples and (–)-α-Pinene, (–)-β-pinene, (–)-camphene, and (–)-limonene were the dominant enantiomers in L. formosana essential oil. The chemical categories of L. formosana oleoresin essential oils were clarified by agglomerative hierarchical cluster analysis (AHC) and principal component analysis (PCA). The multivariate analyses demonstrated that a total of four chemical groups can be delineated for L. formosana. The L. formosana essential oils were screened for antimicrobial activity against a panel of potentially pathogenic bacteria and fungi and showed promising antimicrobial activities with minimum inhibitory concentration (MIC) ≤ 625 μg/mL. These results highlight the economic value of L. formosana oleoresin essential oil, the importance of L. formosana sustainability, and the potential therapeutic benefits of its oleoresin essential oils.


Introduction
In recent years, essential oils or resins from aromatic plants have been widely applied in the food, cosmetic, and medicinal industries. Research and the related applications of aromatic plants play a more and more important role in preserving biodiversity, encouraging agroecology, and helping social and environmental development. The genus Liquidambar L. is one category of aromatic plants; it includes five species, in which two species and one variety are found in China. Liquidambar formosana Hance is one of the species in the Hamamelidaceae family [1,2]. Known for its bright orange autumn leaves, L. formosana is a large, flowering, deciduous tree.
The fruit of L. formosana (Chinese name LuLuTong) has been used as a traditional Chinese medicine for thousands of years [3]; the pharmacology and phytochemistry of L. formosana has been reviewed [4].
Agglomerative hierarchical cluster (AHC) analysis revealed four clearly defined groups ( Figure 1, Table 2). Group #1 was dominated by α-pinene  There is no obvious correlation between the oleoresin essential oil chemistry from the cluster analysis and the geographical location of the collections, but there are some trends. Most of the trees from the Wangmo collections were dominated by (E)-caryophyllene (groups #2 and #4) and most of the trees from the Leye collection sites were dominated by pinenes (groups #1 and #3). Interestingly, however, adjacent trees (Leye 8 and Leye 9) fell into different clusters (#1 and #2, respectively). Likewise, adjacent trees Leye 6 and Leye 7 were also in different groups (#1 and #3), although both of these groups were dominated by pinenes. Three trees, Wangmo 23, 24, and 25, were collected from the same general area and all showed different chemistries; Wangmo 23 fell into group #2, Wangmo 24 into group #3, and Wangmo 25 into group #4. Furthermore, there does not seem to be a correlation between the time of year (March vs. August) in the observed oleoresin essential oil composition. Thus, for example, samples RE190401D and RE190401E, both collected from Wangmo in March 2019, fell into group #2, along with four Wangmo samples collected in August 2019 (LD190910O, LD190910P, LD190910U, and LD190910V). Similarly, there is little correlation between tree size and oleoresin essential oil composition. Oleoresin essential oils from the largest trees (i.e., LD190910D, LD190910K, and LD190910N) were either in group #1 or group #3 (pinene-rich groups), but one of There is no obvious correlation between the oleoresin essential oil chemistry from the cluster analysis and the geographical location of the collections, but there are some trends. Most of the trees from the Wangmo collections were dominated by (E)-caryophyllene (groups #2 and #4) and most of the trees from the Leye collection sites were dominated by pinenes (groups #1 and #3). Interestingly, however, adjacent trees (Leye 8 and Leye 9) fell into different clusters (#1 and #2, respectively). Likewise, adjacent trees Leye 6 and Leye 7 were also in different groups (#1 and #3), although both of these groups were dominated by pinenes. Three trees, Wangmo 23, 24, and 25, were collected from the same general area and all showed different chemistries; Wangmo 23 fell into group #2, Wangmo 24 into group #3, and Wangmo 25 into group #4. Furthermore, there does not seem to be a correlation between the time of year (March vs. August) in the observed oleoresin essential oil composition. Thus, for example, samples RE190401D and RE190401E, both collected from Wangmo in March 2019, fell into group #2, along with four Wangmo samples collected in August 2019 (LD190910O, LD190910P, LD190910U, and LD190910V). Similarly, there is little correlation between tree size and oleoresin essential oil composition. Oleoresin essential oils from the largest trees (i.e., LD190910D, LD190910K, and LD190910N) were either in group #1 or group #3 (pinene-rich groups), but one of the smallest trees (LD190910W) also yielded a pinene-rich essential oil (group #3). This suggests that there are no major differences in regard to collection site, size of tree, or collection time of year.

Antibacterial and Antifungal Activity
The L. formosana essential oils were screened for antimicrobial activity against a panel of potential dermal and pulmonary pathogenic bacteria (Bacillus cereus, Cutibacterium acnes, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Pseudomonas aeruginosa, and Serratia marcescens) ( Table 3) and fungi (Aspergillus fumigatus, Aspergillus niger, Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, and Candida albicans) ( Table 4). All of the tested essential oil samples demonstrated similar antibacterial and antifungal profiles, which is not surprising considering the similarity of the essential oil compositions. Sartoratto and co-workers have suggested that essential oils showing minimum inhibitory concentration (MIC) values < 500 µg/mL are strong inhibitors, while those with MIC values of 600-1500 µg/mL are moderate inhibitors [24]. Based upon these criteria, the L. formosana oleoresin essential oils showed strong antimicrobial activity against all organisms except for S. marcescens (MIC = 625 µg/mL). In particular, the oil of L. formosana showed excellent antibacterial activity against S. epidermidis (MIC = 78 µg/mL) and strong antifungal activity against A. niger (MIC = 78-313 µg/mL). The mechanisms for antimicrobial activities of essential oils are not completely understood. However, it has been suggested that the lipophilic essential oil components serve to disrupt and penetrate the lipid structure of the cell wall, increasing membrane fluidity and causing the leakage of H 3 O + and K + ions, ultimately leading to cell lysis and death [25,26].
In general, the oleoresin essential oils showed better antibacterial activity against Gram-positive organisms over Gram-negative organisms. Except for S. epidermidis (MIC = 78 µg/mL), the antibacterial activity of L. formosana oleoresin essential oils against Gram-positive organisms was 156 µg/mL, whereas the MIC values against Gram-negative P. aeruginosa were 313 µg/mL and 625 µg/mL against S. marcescens. It has often been observed that Gram-negative bacteria are less susceptible to the inhibitory effects of essential oils than Gram-positive bacteria [27][28][29]. This phenomenon has been attributed to the presence of cell wall lipopolysaccharides in the Gram-negative organisms, which can inhibit the lipophilic essential oil components from diffusing into the cells [30].
The antibacterial activity observed for the L. formosana oleoresin essential oils against S. epidermidis cannot be attributed solely to the activities of the individual major components (Table 3). Thus, for example, (E)-caryophyllene, α-pinene, β-pinene, camphene, and (+)-limonene have MIC values of 313 µg/mL against S. epidermidis, and only (-)-limonene has an MIC value of 78 µg/mL. Similarly, the MIC value for L. formosana oleoresin essential oils against S. pyogenes was 156 µg/mL, but the MIC values for the major components ranged from 313 µg/mL to 625 µg/mL. It is likely that the synergistic effects of the major components, possibly involving minor components, are responsible for the antimicrobial activity [31,32]. Crevelin and co-workers observed synergistic antimicrobial effects in a combination of α-pinene, β-pinene, (E)-caryophyllene, and caryophyllene oxide [33]. Both α-pinene and β-pinene showed good antifungal activity against A. niger, and these components may be responsible for the activity of L. formosana essential oil against this fungal organism. (E)-Caryophyllene, on the other hand, was inactive against A. niger.

Oleoresin Collection
Liquidambar formosana oleoresins were collected from 25 individual trees from several locations in Southern China ( Table 1). The oleoresin tapping practice that was utilized in this work not only increases resin tapping efficiency but also ensures the sustainability of the trees and the ecosystem. It was invented by Zeng and co-workers and named the "downward tapping method of V-shaped" [22]. More specifically, mature L. formosana trees with DBH (diameter at breast height) ≥ 60 cm, without pest infection, qualified for oleoresin tapping. The oleoresin tapping was carried out by local farmers at average temperatures above 15 • C. The tapping surface was on the sun-facing side of the trunk. An area of the trunk was shaved of bark, a medial groove was cut in the center of the shaved area, and a V-shaped ditch was cut from top to bottom along the vertical direction of the trunk. The V-shaped angle (β) was between 60 • and 80 • (Figure 3) and was cut to the first or second annual ring of the tree. A minimum 10-cm space was preserved between each V-shaped ditch to ensure the health of the trees. The oleoresin aggregations were located at the bottom of the V-shaped ditch, which guided the oleoresin into a container. Immediately after the resin was collected, it was transferred to amber-colored glass bottles and stored at 4 • C until distillation.

Oleoresin Collection
Liquidambar formosana oleoresins were collected from 25 individual trees from several locations in Southern China ( Table 1). The oleoresin tapping practice that was utilized in this work not only increases resin tapping efficiency but also ensures the sustainability of the trees and the ecosystem. It was invented by Zeng and co-workers and named the "downward tapping method of V-shaped" [22]. More specifically, mature L. formosana trees with DBH (diameter at breast height) ≥ 60 cm, without pest infection, qualified for oleoresin tapping. The oleoresin tapping was carried out by local farmers at average temperatures above 15 °C. The tapping surface was on the sun-facing side of the trunk. An area of the trunk was shaved of bark, a medial groove was cut in the center of the shaved area, and a V-shaped ditch was cut from top to bottom along the vertical direction of the trunk. The V-shaped angle (β) was between 60° and 80° ( Figure 3) and was cut to the first or second annual ring of the tree. A minimum 10-cm space was preserved between each V-shaped ditch to ensure the health of the trees. The oleoresin aggregations were located at the bottom of the V-shaped ditch, which guided the oleoresin into a container. Immediately after the resin was collected, it was transferred to amber-colored glass bottles and stored at 4 °C until distillation.

Oleoresin Hydrodistillation
The hydrodistillation of the samples of L. formosana oleoresin was performed with an all-glass Clevenger apparatus for 7 h. The water and resin were mixed in a ratio of 6:1 and the hydrodistillation was carried out with constant stirring of the mixture. The rate of hydrodistillation was around 2 mL/min. The isolated oil had a strong resinous aroma with floral, pine, and spicy notes. Oleoresin masses and essential oil yields are summarized in Table 1. The L. formosana oleoresin essential oils were stored in sealed amber vials at 4 °C until chromatographic analysis and bioactivity screening.

Gas Chromatographic-Mass Spectral Analysis
The oleoresin essential oils from L. formosana were subjected to gas chromatographic-mass spectral (GC-MS) analysis, as previously reported [34]:

Oleoresin Hydrodistillation
The hydrodistillation of the samples of L. formosana oleoresin was performed with an all-glass Clevenger apparatus for 7 h. The water and resin were mixed in a ratio of 6:1 and the hydrodistillation was carried out with constant stirring of the mixture. The rate of hydrodistillation was around 2 mL/min. The isolated oil had a strong resinous aroma with floral, pine, and spicy notes. Oleoresin masses and essential oil yields are summarized in Table 1. The L. formosana oleoresin essential oils were stored in sealed amber vials at 4 • C until chromatographic analysis and bioactivity screening.

Gas Chromatographic-Mass Spectral Analysis
The oleoresin essential oils from L. formosana were subjected to gas chromatographic-mass spectral (GC-MS) analysis, as previously reported [34]: Shimadzu GCMS-QP2010 Ultra, electron impact (EI) mode with electron energy = 70 eV, scan range = 40-400 atomic mass units, scan rate = 3.0 scans/s, and Shimadzu GC-MS solution software v. 4.45 (Shimadzu Scientific Instruments, Columbia, MD, USA); ZB-5ms fused silica capillary GC column Phenomenex, Torrance, CA, USA; (5% phenyl)-polymethylsiloxane stationary phase, 0.25 µm film thickness; helium carrier gas, column head pressure = 552 kPa, flow rate = 1.37 mL/min; injector temperature = 260 • C, ion source temperature = 260 • C; GC oven temperature program: initial temperature = 50 • C, temperature increased 2 • C/min to 260 • C. For each sample, a 5% w/v solution in CH 2 Cl 2 was prepared, and 0.1 µL was injected using a split ratio of 30:1. Identification of the individual components of the essential oils was determined by comparison of the Kovats retention indices, determined using a series of n-alkanes, in addition to comparison of the mass spectral fragmentation patterns with those found in the MS databases [35][36][37][38], using the LabSolutions GCMS solution software version 4.45 (Shimadzu Scientific Instruments, Columbia, MD, USA) and with matching factors >90%.

Gas Chromatographic-Flame Ionization Detection
Quantitative analysis of the L. formosana essential oils was carried out by GC-FID, as previously reported [34]: Shimadzu GC 2010 equipped with FID, a split/splitless injector, and Shimadzu autosampler AOC-20i (Shimadzu Scientific Instruments, Columbia, MD, USA), with a ZB-5 capillary column (Phenomenex, Torrance, CA, USA). The GC-FID measurements were carried out using the same oven temperature program as that for GC-MS. Injector temperature = 250 • C, detector temperature = 280 • C, and nitrogen was the carrier gas, with a flow rate of 1.0 mL/min. The concentrations of the oleoresin essential oil components were calculated from raw peak areas, normalized to 100%, without standardization.

Chiral Gas Chromatography-Mass Spectrometry
Chiral GC-MS of the L. formosana oleoresin essential oils was carried out, as reported previously [34]: Shimadzu GCMS-QP2010S (Shimadzu Scientific Instruments, Columbia, MD, USA), electron impact (EI) mode, electron energy = 70 eV; scan range = 40-400 amu, scan rate = 3.0 scans/s; Restek B-Dex 325 chiral capillary GC column (Restek Corp., Bellefonte, PA, USA) (30 m × 0.25 mm ID × 0.25 µm film thickness). Oven temperature program: starting temperature = 50 • C, temperature increased 1.5 • C/min to 120 • C, then 2 • C/min to 200 • C, and kept at 200 • C for an additional 5 min; carrier gas was helium, flow rate = 1.8 mL/min. For each essential oil sample, a 3% w/v solution in CH 2 Cl 2 was prepared, and 0.1 µL was injected using a split ratio of 1:45. The enantiomers of the monoterpenoids were identified by comparison of retention times with authentic samples obtained from Sigma-Aldrich (Milwaukee, WI, USA). The enantiomer percentages were determined from peak areas.
All bacteria were cultured on tryptic soy agar, except for C. acnes, which was grown on tryptic soy agar supplemented with 7% (v/v) defibrinated whole sheep blood (Cleveland Scientific, Bath, OH, USA), under micro-aerophilic conditions [43]. All fungi were cultured on yeast malt agar (Sigma-Aldrich, St. Louis, MO). For the bacteria and fungi, 50 µL of 1% (w/v) solution of the samples in dimethyl sulfoxide (DMSO) was diluted in 50 µL of cation-adjusted Mueller Hinton broth (CAMHB) (Sigma-Aldrich, St. Louis, MO). The sample solutions were then serially diluted (1:1) in fresh CAMHB to obtain concentrations of 2500, 1250, 625, 313, 156, 78, 39, and 20 µg/mL. The microbes were harvested from a fresh culture and added to each well at a concentration of approximately 1.5 × 10 8 CFU/mL for bacteria and 7.5 × 10 7 CFU/mL for fungi, and the 96-well microdilution plates for bacteria were incubated at 37 • C and the fungi were incubated at 35 • C for 24 h. The minimum inhibitory concentration (MIC) was determined as the lowest concentration with no turbidity. Gentamicin (Sigma-Aldrich, St. Louis, MO) was used as a positive antibiotic control and DMSO was used as the negative control (50 µL DMSO diluted in 50 µL broth medium and then serially diluted, as above). For fungi, the above-mentioned method was implemented using a yeast-nitrogen base growth medium (Sigma-Aldrich, St. Louis, MO, USA) and amphotericin B (Sigma-Aldrich, St. Louis, MO, USA) as a positive antifungal control.

Statistical Analysis
Multivariate analyses were carried out on the essential oil compositions of the L. formosana oleoresins. The chemical compositions of the 25 essential oils were treated as operational taxonomic units (OTUs), and the percentage compositions of 25 major components were used to ascertain the associations between the oleoresin essential oil compositions using agglomerative hierarchical cluster (AHC) analysis, using XLSTAT Premium, version 2018.5.53172 (Addinsoft, Paris, France). The dissimilarity of the samples was evaluated using Euclidean distance, and the clusters were defined using Ward's method [44]. The principal component analysis (PCA) was carried out using the 25 major chemical components as variables, with a Pearson correlation matrix, using XLSTAT Premium, version 2018.1.1.60987 (Addinsoft, Paris, France). In all cases, 625 data (25 samples × 25 variables) were utilized for the principal component analysis.

Conclusions
L. formosana trees and their essential oils are important non-timber forest product (NTFP) resources. Due to the lack of research on L. formosana, the resources are under-utilized or even being destroyed. The oleoresin essential oils collected from 25 different L. formosana trees from different regions of Southern China showed very little variation in either chemical composition or enantiomeric distribution. The essential oil yields ranged from 7.7% to 30.2%. The oleoresin essential oils showed promising antibacterial efficacy against Gram-positive bacteria and antifungal activity. The biological potency, coupled with improved tree-tapping methods, promoted L. formosana oleoresin essential oil in terms of its economic potential as well as its therapeutic benefits. This is an innovative research work that extends our understanding of the phytochemistry of this tree as well as providing scientific and practical support for the development and utilization of L. formosana tree resources. As a new NTFP product, the oleoresin essential oils also improve awareness of ecosystem protection.