Variations in the Chemical Composition of Essential Oils in Native Populations of Korean Thyme, Thymus quinquecostatus Celak.

The genus Thymus (Lamiaceae) contains numerous medicinally important species. Among them, Thymus quinquecostatus Celak. has been extensively utilized as a traditional medicine and a food flavoring agent in the Korean peninsula, owing to its unique aroma. In particular, T. quinquecostatus has been used for the treatment of gastroenteritis, inflammation, stomach problems, liver disease, arthritis, arteriosclerosis, and menstrual problems. This study aimed to investigate the chemical diversity of essential oils among 103 Korean native populations of T. quinquecostatus. For this purpose, seedlings of T. quinquecostatus populations were purchased from different regions in the Korean Peninsula, and seedlings were grown in the experimental field under the same environmental conditions. The chemical compositions of steam-distilled essential oils were determined using GC-MS. In total, 212 components were identified from 103 populations of T. quinquecostatus. Furthermore, principal component analysis (PCA) was performed in order to understand variations in the essential oil compositions among 103 Korean native populations of T. quinquecostatus. According to the essential oil compositions, 30 components were selected for PCA. Based on the most abundant essential oil components, four chemotypes were identified in T. quinquecostatus populations. PCA and cluster analyses revealed that 103 individuals of T. quinquecostatus could be classified into four clusters, such as thymol, geraniol, geranyl acetate, and linalool. Furthermore, dendrogram construction demonstrated that geraniol and geranyl acetate, as well as linalool and thymol groups, were closely related. This study suggested the significant chemical polymorphism of essential oils in local populations of T. quinquecostatus in Korea. It could be concluded that the intraspecific variations in the essential oil compositions may be associated with genetic diversity among the individuals.


Introduction
Essential oils exhibit various biological properties, due to the presence of thousands of low-molecular-weight volatile components in them, primarily terpenes and their oxygenated derivatives, such as alcohols, aldehydes, esters, and phenols [1]. Essential oils isolated from Thymus species are well known for their potential use in food industries as flavoring agents, due to their unique fragrances. The genus Thymus (Lamiaceae) contains about 300 species of medicinally important aromatic plants [2]. In traditional systems of medicine, infusions and decoctions obtained from the leaves and flowers of Thymus species have been utilized in the treatments of several diseases, including complications in digestion, in addition to circulatory, genital, nervous, urinary, skin, and respiratory conditions [3]. It has been shown that essential oils and compounds isolated from different Thymus species exhibited strong antioxidant, antimicrobial, anti-tumoral, insecticidal, anti-inflammatory, and neuroprotective properties [2][3][4]. Essential oils obtained from different Thymus species (Lamiaceae) mainly contain 35-55% thymol, followed by carvacrol, geraniol, and linalool.
Thymol is widely used for flavor or as a fragrant material, in the preparation of herbal teas, and as antimicrobial and insecticidal agents [5].
Among the various species of Thymus, T. quinquecostatus Celak. is a medicinally important aromatic plant that is native to the Korean peninsula [5,6]. In Korea, there are two varieties of T. quinquecostatus, such as Bak-ri-hyang and Ulleungdo thyme (T. quinquecostatus var. japonica). In Korea, T. quinquecostatus has been traditionally used to treat diaphoretic, flatulence, and liver disorders, as well as stomachaches, menstrual problems, and coughs, in addition to being a flavoring agent. This plant has a high industrial value, due to its distinctive aroma [7]. Previous studies found that T. quinquecostatus has a lot of therapeutical potential. T. quinquecostatus extracts showed strong antioxidant and free radical scavenging potential [7,8], in addition to antimicrobial [9], anti-diabetic [10], anti-aging [11], hepatoprotective [12], anti-tumoral [13], and anti-inflammatory properties [14]. T. quinquecostatus extract effectively improved mitochondrial function and attenuated oxidative stress in lipopolysaccharide-induced RAW 264.7 macrophages [14]. A recent study reported that the polyphenol-rich extract of T. quinquecostatus ameliorated cerebral ischemia-reperfusion injuries in rats [15].
Essential oil composition is a significant quality parameter for the commercial applications of T. quinquecostatus species. Recently, several studies reported that the same plant species collected from different locations exhibited different essential oil compositions, mostly in the yield and concentrations of major components in the essential oils [16][17][18]. Previously, some studies reported on the chemical composition of T. quinquecostatus essential oils, and their compositions varied significantly according to different geographical origins [19][20][21][22][23]. The essential oil compositions of T. quinquecostatus cultivars that were collected from three mountains in Korea showed three different chemotypes, such as geraniol, thymol, and linalool [5]. The yield, chemical components, and biological activities of essential oils are severely influenced by various factors, including environmental conditions, genotype, growth stage, extraction technique, etc. [24,25]. Principal component analysis (PCA) is extensively employed to understand the relationships and similarities between, as well as within species, according to the major components in the essential oils. PCA analysis provides insight into the distribution of essential oil components in plant populations [4,26,27].
There are no detailed studies on the diversity in the composition of essential oils within Korean populations of T. quinquecostatus. Hence, the present study aimed to investigate the chemical diversity in the essential oils of T. quinquecostatus populations collected from different regions in the Korean peninsula via GC-MS analysis. Furthermore, PCA was performed, in order to identify intraspecific variations in T. quinquecostatus populations.

The Yield and Color of T. quinquecostatus Essential Oils
A total of 107 (4 seedlings decayed due to being frozen) Korean native populations of T. quinquecostatus were selected in the present study (Table 1). Seedlings collected from 103 individuals were planted under the same environmental conditions, cultivated during the flowering stage, and used for the isolation of essential oils. The average yield of essential oils obtained from the aerial parts of T. quinquecostatus populations was 0.40% (v/w). The highest yield was obtained from the sample site T64 (0.8%) (Gwacheon-si, Gyeonggi-do) ( Table 1). The color of extracted essential oil was classified into red, orange, dark yellow, yellow, pale yellow, transparent yellow, and lemon.

Essential Oil Compositions of T. quinquecostatus Populations
Based on the GC-MS analyses, 212 different compounds were detected in the essential oils of 103 T. quinquecostatus individuals. All of the essential oil samples, qualitatively and quantitatively, exhibited different chemical compositions. Thymol, geraniol, geranyl acetate, and linalool were the predominant components in essential oils (Supplementary  Table S1). Among them, 30 components were detected in most of the essential oils, and these components were used for further PCA studies. Table 2 shows the compound names, CAS numbers, chemical formulas, and retention index values, with the total number of essential oil samples. The essential oils of T. quinquecostatus populations mainly contained oxygenated monoterpenes, followed by monoterpene hydrocarbons. Six chemotypes, carvacrol, geraniol, geranyl acetate, linalool, o-cymene, and thymol, were distinguished according to the essential oil compositions (Table 3 and Figure 1). In particular, linalool (80.33%) was the most abundant component in sample T9. The maximum number of samples were grouped under the thymol chemotype (39), followed by the geraniol (30) and geranyl acetate (26)
A dendrogram was constructed on the basis of of cluster analysis results ( Figure 3). The essential oils from 103 T. quinquecostatus populations comprised 4 clusters. Group I contains individuals of T. quinquecostatus essential oils with the highest amount of geraniol. Group II comprises T. quinquecostatus individuals with the highest amount of geranyl acetate; Group III contains T. quinquecostatus individuals with the highest amount of linalool, and Group IV contains T. quinquecostatus individuals with the highest amount of thymol.

Discussion
T. quinquecostatus is a medicinally important aromatic plant in Korea. Traditionally, the essential oil of T. quinquecostatus has been used to treat various disorders, in addition to being used as a flavoring agent. The biological properties of the essential oils were highly correlated with the chemical class of the components. The types of essential oil components and their concentrations offer unique features to each essential oil. Further-

Discussion
T. quinquecostatus is a medicinally important aromatic plant in Korea. Traditionally, the essential oil of T. quinquecostatus has been used to treat various disorders, in addition to being used as a flavoring agent. The biological properties of the essential oils were highly correlated with the chemical class of the components. The types of essential oil components and their concentrations offer unique features to each essential oil. Furthermore, the most significant characteristic of an essential oil is its fragrance. Therefore, this study aimed to evaluate the chemical diversity within Korean native populations of T. quinquecostatus. For this purpose, seedlings of T. quinquecostatus collected from 103 different regions of Korea were grown under identical field conditions. The cultivated plants were harvested during the flowering stage, and the essential oils were isolated using the steam distillation technique. Kim et al. [5] also reported that the yield of essential oils from T. quinquecostatus ranged from 0.12 to 0.43%. In this study, 103 individuals of T. quinquecostatus were divided into six chemotypes, such as carvacrol, geraniol, geranyl acetate, linalool, o-cymene, and thymol, based on the major components found in their essential oils. In our previous study, Wolchul and Odae cultivars of Korean native T. quinquecostatus showed different chemotypes according to their essential oil compositions, even though they were placed into the same cluster on the basis of RAPD analysis results [5]. Previously, Shin and Kim [9], and Kim et al. [5], found that thymol (41.70 and 30.54%), γ-terpinene (16.00 and 23.92%), and p-cymene (13.00 and 11.13%) were major compounds in the essential oils of T. quinquecostatus (Supplementary Table S2).
Our previous study reported that the predominant compounds in the essential oils of T. quinquecostatus that were obtained from Wolchul and Jiri cultivars were different from the Odae cultivar. Geraniol (42.94%) and geranyl acetate (26.49%) were the most abundant components in the Wolchul cultivar, and linalool (47.89%) and thymol (15.98%) were the major compounds found in the Jiri cultivar [5]. Similarly with our report, T. quinquecostatus essential oils that were obtained from Cheongwon and Shandong Yimeng in China exhibited different chemotypes, such as trans-geraniol and p-cymene, respectively [19]. Chiang et al. [28] found that there was a significant variation in the essential oil compositions of T. quinquecostatus that were collected from different places in Korea, such as the high mountains of Jeju Island, the mid-mountainous region of Jeju, Gapyeong, and Ulleungdo. He et al. [21] identified 103 essential oil components from T. quinquecostatus collected from four different regions in China, and reported that 1,8-cineole, linalool, terpinen-4-ol, γ-terpinene, borneol, β-bisabolene, and α-pinene were commonly found in the essential oils that were obtained from all four regions. In particular, thymol and carvacrol were observed to possess identical structures, and that the degree of their transformation to each other may have been influenced by differences in environmental conditions [4]. It was also reported that the enzyme, geraniol dehydrogenase, specifically converts geraniol into geranial, and nerol into neral [29].
These studies revealed that various ecological and physiological factors play a major role in the composition of essential oils obtained from plants. However, the essential oils were isolated from T. quinquecostatus, which were grown under identical environmental conditions with similar edaphic factors. Therefore, the genetic makeup of T. quinquecostatus populations may be associated with their essential oil compositions. Quan et al. [30] demonstrated that T. quinquecostatus wild populations registered higher levels of genetic and clonal variations between patches within species. Rustaiee et al. [31] found genetic diversity and variations in the essential oil compositions of six Thymus species. Previous studies suggested that genetic diversity at the intraspecific level plays a crucial role in the composition of essential oils and their concentrations [32][33][34]. In addition, Choi et al. [35] suggested that harvesting time highly influenced the quality of thyme essential oils, and revealed that the essential oil content was found to be higher during the flowering period. Ghasemi Pirbalouti et al. [36] suggested that hybridization and introgression within species can also influence variations in the composition of essential oils of different Thymus species. PCA can be employed as a valuable tool to understand relationships among the data. Furthermore, PCA is a useful pattern recognition technique to classify and discriminate plant samples, specifically essential oil compositions [16]. In the chemical composition, some of the components were present in only one essential oil, while other components were present in all of the essential oils. In this study, among 30 essential oil components that were selected from 103 T. quinquecostatus populations, the first three principal components accounted for 90.014% of the total variation. Thus, those 30 essential oil components were subsequently selected for PCA analysis. The cluster analysis supported the discrimination of T. quinquecostatus populations that was achieved through the PCA. Thymus species from different places in Iran were categorized into three chemotypes, such as thymol, geraniol/linalool, and carvacrol, according to cluster analysis [4].
A study reported that 11 Thymus species were classified into 3 major groups, according to morphological traits using PCA and cluster analyses [37]. A recent study also used PCA and cluster analyses to investigate differences in the composition of essential oils from five Thymus species, such as T. atticus, T. leucotrichus, T. striatus, T. zygioides, and T. perinicus [6]. Satyal et al. [38] found 20 different chemotypes in the essential oil compositions of 85 T. vulgaris samples, based on cluster analysis. Among them, 39 samples were grouped under the thymol chemotype. In another study, thymol and carvacrol were major essential oil components that were found in four T. vulgaris varieties, but significant variations were observed in the concentrations of major components in essential oils that were extracted before versus after the flowering stage [39].
On the basis of the essential oil composition-based dendrogram, T. quinquecostatus samples were classified into four major clusters. The results of correlations clearly demonstrated a relationship among Korean native populations of T. quinquecostatus and their chemical components (Table 5). Essential oil composition is a major factor used to detect a chemical between and within species. In this study, the statistical analysis results indicated that essential oil showed chemical diversity within the species, even though the collected seedlings were grown under identical field conditions.

Collection and Cultivation of T. quinquecostatus Populations
Seedlings of T. quinquecostatus were purchased from 107 different regions in the Korean peninsula. The collection period was between 2019 and 2020. The seedlings of T. quinquecostatus were separately planted in two rows at 25 cm intervals in the same experimental field, which was a 1650 m 2 area that was located at Chuncheon, Gangwan-do, Republic of Korea. The plants were cultivated during the flowering season, and essential oils were extracted. Four samples were found decayed, due to being frozen. Thus, a total of 103 samples were used for further experimentation.

Essential Oil Extraction
The essential oils from the aerial parts of 103 populations of T. quinquecostatus were separately extracted using a steam distillation extraction technique. The plant samples were steam distilled at the boiling condition for 90 min, using a steam distillation apparatus (EssenLab Plus, Hanil Lab Tech Co., Ltd., Yangju, Korea). The yield (%, v/w) of essential oil was calculated on the basis of the weight of fresh sample. After extraction, the moisture in the isolated essential oil was removed using anhydrous sodium sulfate. The purified essential oil was kept at 4 • C prior to GC-MS analysis.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
For the identification and quantitative analysis of components in the essential oils, a Varian CP3800 gas chromatograph coupled with a Varian 1200 L mass detector (Varian, Palo Alto, CA, USA) were used. The GC-MS was performed using a VF-5MS (Agilent, Santa Clara, CA, USA) polydimethylsiloxane (30 m × 0.25 mm × 0.25 µm) capillary column. The GC oven temperature was programmed from 50 • C (for 5 min) to 250 • C (for 3 min), at a rate of 5 • C/min, then increased to 300 • C at a rate of 20 • C/min; the final temperature was maintained for 5 min. The injector and the ion source temperature were 250 • C and 280 • C, respectively. A volume of 1 µL of the sample was injected, using a split ratio of 1:20. The carrier gas used was helium at a constant flow rate of 1 mL/min. For the mass spectra analysis, the ionization voltage was set to 70 eV, and the mass range was set to 30-500 m/z. The essential oil components of T. quinquecostatus were identified by comparing the mass spectrum data from the National Institute of Standards and Technology (NIST, 3.0) library, and the retention indices (RI) relative to a homologous series of n-alkanes (C 8 -C 20 ) that was reported in the literature [40].

Statistical Analysis
For statistical analysis, the chemical compositions of essential oils of 103 T. quinquecostatus populations were integrated and sorted according to their RI values. Subsequently, the common essential oil components that appeared in 40 or more T. quinquecostatus individuals were selected separately from the raw data. Statistical analysis was performed on the basis of the extracted data, and then cluster analysis was carried out. PCA was carried out to analyze multiple data on the concentration of components in the essential oils, and to determine associations between the essential oil components and the collection sites of the T. quinquecostatus seedlings. All statistical analyses were carried out using IBM SPSS ver. 26 (IBM Corp. Released 2016, Chicago, IL, USA).

Conclusions
The data of the present study demonstrated that a significant chemical variation was observed within Korean native T. quinquecostatus populations based on the collection sites of the seedlings. One hundred and three T. quinquecostatus populations were grouped into six chemotypes, such as carvacrol, geraniol, geranyl acetate, linalool, o-cymene, and thymol. According to the PCA and cluster analysis, T. quinquecostatus populations could be categorized into four groups, such as thymol, geraniol, geranyl acetate, and linalool. These results indicated that the chemical composition of essential oils and their major components are excellent biomarkers to help understand the intraspecific variations among aromatic species. Further studies, in connection with genetic analyses of T. quinquecostatus populations, are necessary, in order to confirm the observed variations within the species.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27217203/s1, Table S1. The area percent of 30 components in the essential oils obtained from 103 T. quinquecostatus individuals. Table S2. Comparison of major essential oil components (area %) of T. quinquecostatus from previous publications

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the essential oils of Thymus quinquecostatus are available from the authors.