ABSTRACT
Pogostemon cablin, Melaleuca leucadendra, and Mentha piperita are three aromatic plants that have been reported to produce a high yield of volatile components with medicinal and therapeutic properties. This present study aimed to perform qualitative and semi-quantitative analysis on the volatile components present in the aforementioned aromatic plants. Essential oils from P. cablin and M. leucadendra were obtained from community-based enterprises in Aceh Province, Indonesia. The essential oils were further purified using vacuum rotary evaporator. In addition, we also investigated the essential oils from M. piperita based on the priorly optimized parameters. The volatile components contained in the essential oils were identified using gas chromatography–mass spectrometry (GC-MS) analysis. The qualitative data were derived from the MS data based on the fragmented components separated by the GC and compared with the database. The abundance of each volatile component was determined based on the area percentage of the chromatographic peak. In P. cablin oil, the relative abundance of α-guaiene and seychellene was higher in heavy fraction (17.11 and 10.29, respectively), while patchouli alcohol in light fraction (69.92%). Eucalyptol was found higher in the light fraction of M. leucadendra oil (MO) than that in the heavy fraction (78.87% vs. 17.34%, respectively). As for the M. piperita oil, menthone was found as the predominant component with relative abundance of 21.6%. Essential oils extracted from P. cablin, M. leucadendra, and M. piperita consist of volatile components with medicinal and therapeutic potentials, in which their compositions are affected by the purification process.
Keywords: Aromatic oil, eucalyptol, chromatography–mass spectrometry, seychellene, α-guaiene
INTRODUCTION
Essential oils have gained a spotlight in drug discovery research owing to their medicinal properties that can be utilized as a single-dose therapy or add-on therapy. During the course of the coronavirus disease 2019 pandemic, researchers have even put their attention on essential oils as potential antivirals and immunomodulators.[1] Among many aromatic plants, Pogostemon cablin, Melaleuca leucadendra, and Mentha piperita have been reported to produce volatile compounds with bioactivities. A review article has highlighted that P. cablin could be used as antimicrobial, anti-inflammatory, antioxidant, antimicrobial, analgesic, antitumor, antihypertension, antidiabetic, and so on.[2] Patchouli alcohol (PA) and β-patchoulene are terpenoids from P. cablin that predominantly contribute to its wide array of bioactivities.[2] A study published in 2022 reported that volatile compounds from P. cablin could ameliorate depressive symptoms in rats.[3,4] The compound, PA, was revealed to attenuate malignant cells through a combination with cisplatin.[5] Antibacterial and anti-inflammatory activities have been witnessed for M. leucadendra-derived essential oils, in which the volatile content is predominated by methyleugenol.[6] Further, the aromatic plant has been acknowledged to exert other bioactivities including antikinetoplastid and antiproliferative.[7] The study also revealed that 1,8-cineole occupies 61% of the total volatile components of M. leucadendra.[7] In a review article, M. piperita or peppermint has been recognized as a ethnomedicinal plant with medical properties including antimicrobial, larvicidal, biopesticidal, antioxidant, anti-inflammatory, anticancer, and antidiabetic.[8] L-menthol is the major volatile compound from M. piperita that plays a role in its bioactivities.[9]
In the case of Indonesia – a country with high biodiversity, among 40 available, there are 12 types of essential oils commercially produced across the country.[10,11] Most of the essential oil productions are community based, utilizing simple equipment and tend to be unstandardized.[12] The commercialization of essential oil has become the center of economic activities in several communities in Indonesia.[13] Unfortunately, simple distillation methods used by the communities tend to leave impurities in the essential oils, hence requiring further purification. In this present study, the essential oils procured from the local community would be further purified through fractionation distillation. Other than obtaining a final product with high purity, this study also aimed to see the impact of the purification process to the volatile contents. It is of important to identify the compounds comprising the essential oils since they contribute to the therapeutical properties.
MATERIALS AND METHODS
Sample preparation
The leaves were harvested from P. cablin and M. leucadendra at the age of 5 months – 2 years old and >5 years old, respectively. After the harvesting, the samples were dried and extracted through distillation to produce the essential oils by the local community. P. cablin oil (PCO) and MO were obtained from the community whose chemical components were separated by vacuum rotary evaporator to obtain PCO light fraction (PCO1), PCO heavy fraction (PCO2), MO light fraction (MO1), and MO heavy fraction (MO2). The temperature was set at ranges of 115°C–160°C and 125°C–160°C, for heavy and light fractions, respectively. The fractionation was carried out under 200 kPa. M. piperita oil (PO) was obtained from the Essentials Research Center (ARC) obtained from a procedure reported previously.[3]
Gas chromatography–mass spectrometry analysis
Gas chromatography–mass spectrometry (GC-MS) analysis of essential oils was performed using a Shimadzu GC-2010 plus gas chromatograph at the Instrumentation Laboratory of the Chemistry Department, FMIPA, USK. The analysis was carried out for 50 min. The type of capillary column used was TG-5MS (30 m length, 0.2 mm inner diameter, 0.25 μm film thickness). A total of 2 μL of essential oil were injected into the GC apparatus. The temperature of the GC equipment was calibrated for 4 min at 60°C, then increased to 150°C for 4 min, and increased to 250°C. Helium carrier gas flow rate was set at 1.35 mL/min. In MS measurements, ionization was performed by electron impact at 70 eV. The peaks obtained were compared with the mass spectrum database using Chromeleon software specialized in the interpretation of mass spectral fragmentation patterns.
RESULTS AND DISCUSSION
Identified volatile compounds from Pogostemon cablin oils
Compounds that have been identified in PCO, PCO1, and PCO2 based on GC-MS analysis are presented in Table 1. Based on the results of GC-MS analysis of PCO, PCO1, and PCO2, guaiene, methanoazulene, seychellene, globulol, and PA are the components found in the three patchouli oils. PA (C15H26O) is a compound of the tricyclic tertiary alcohol sesquiterpene group which is the main component determining the quality of patchouli oil with a variety of pharmacological activities.[3] In addition, patchouli oil also has α-guaiene compounds which are classified as major sesquiterpenes, recently thought to have antimicrobial abilities. The α- and δ-guaiene components have been applied in the fragrance industry because they give a sensation of aroma and refreshment. Therapeutical properties including antidiabetic, antileukemia, antiulcer, anti-inflammatory, antiretroviral, and so on have been witnessed in azulene compounds. In addition, seychellene compounds have functions as antioxidants.[14] Patchouli oil contains a large number of sesquiterpene compounds, namely α-/β-/γ-patchoulene, αbulnusene/∆-guaiene, α-guaiene, and seychellene. Sesquiterpene compounds, especially sesquiterpene alcohols from an essential oil, determine pharmacological activity.[15]
Table 1.
Volatile components in Pogostemon cablin oil, Pogostemon cablin oil light fraction, and Pogostemon cablin oil heavy fraction
| Identified compound | Retention time (min) | Relative area (%) | ||
|---|---|---|---|---|
|
| ||||
| PCO | PCO1 | PCO2 | ||
| β-Patchoulene | 20 | 2.8 | 2.86 | |
| (-)-βete-Elemene | 20.2 | 0.67 | 1.63 | |
| Cycloseychellene | 20.8 | 0.81 | ||
| Caryophyllene | 21 | 3.37 | 3.72 | |
| α-Guaiene | 21.4 | 15.24 | 1.4 | 17.11 |
| Seychellene | 21.6 | 8.95 | 1.03 | 10.29 |
| α-Patchoulene | 21.9 | 7.32 | 1.51 | 2.86 |
| Patchoulene | 22 | 0.34 | ||
| Aciphyllene | 22.9 | 2.69 | 1.56 | |
| α-Guajene | 23.1 | 15.98 | 10.41 | |
| 3,7(11)-Eudesmadiene | 23.4 | 0.29 | ||
| Norpatchoulenol | 24.4 | 0.45 | ||
| cis-Z-α-Bisabolene epoxide | 24.7 | 0.29 | ||
| Caryophyllene oxide | 21 | 3.37 | ||
| Isoaromadendrene epoxide | 25.7 | 0.8 | ||
| Globulol | 25.9 | 0.61 | 0.28 | 0.45 |
| Pogostol | 26.6 | 2.23 | ||
| Patchouli alcohol | 26.7 | 34.24 | 69.92 | 20.88 |
| Isoshyobunone | 27.2 | 0.34 | ||
| 2-Pentene, 4, 4-dimethyl- | 8.41 | 0.07 | ||
| α-Maaliene | 23.4 | 0.35 | ||
| 3-Trifluoroacetoxypentadecane | ||||
| ß-Acorenol | 26.6 | 1.58 | ||
| Cedren-13-ol, 8- | 23.7 | 0.31 | ||
| Isoaromadendrene epoxide | 27.2 | 0.78 | 0.1 | |
| cis-Z-α-Bisabolene epoxide | 27.2 | 0.13 | ||
| δ-Elemene | 18.8 | 0.15 | ||
| β-Patchoulene | 20 | 2.86 | ||
| Cycloseychellene | 20.8 | 0.95 | ||
| Aciphyllene | 23 | 3.89 | ||
| (-)-Nootkatene | 23.8 | 0.2 | ||
| Norpatchoulenol | 24.4 | 0.46 | ||
| Caryophyllene oxide | 25 | 0.88 | ||
| 3-Trifluoroacetoxypentadecane | 8.1 | 0.08 | ||
| Cedren-13-ol, 8- | 23.7 | 0.31 | ||
| 4, 11 (13)- Eudesmadien-12-ol | 25 | 2.43 | ||
| Diepicedrene-1-oxide | 25.7 | 1.77 | ||
| Naphthalenone | 27.8 | 0.69 | ||
P. cablin: Pogostemon cablin, PCO: P. cablin oil, PCO1: PCO light fraction, PCO2: PCO heavy fraction
Identifed volatile compounds from Melaleuca leucadendra oils
Results from the GC-MS analysis are presented in Table 2. GC-MS analysis showed that only ß-pinene and eucalyptol (1,8-sineol) were present in both eucalyptus oils. α-and β-pinene are known to be components that belong to monoterpenes and are commonly found in essential oils. These volatile compounds have been suggested by a systematic review to exert antiviral potentials by acting as antagonists of proteins involved in the viral entry and replication.[1] α-and β-pinene are used as anticancer agents through mitochondria-facilitated apoptosis.[16] Eucalyptol (1,8-sineol) is a monoterpene oxide compound that plays an active role in cytotoxicity.[16]
Table 2.
Volatile components in Melaleuca leucadendra oil light fraction and Melaleuca leucadendra oil heavy fraction
| Identified compound | Retention time (min) | Relative area (%) | ||
|---|---|---|---|---|
|
| ||||
| MO | MO2 | MO1 | ||
| α-Pinene | 7.3 | 2.58 | ||
| ß-Pinene | 8.54 | 2.45 | 3.86 | |
| Eucalyptol | 10.1 | 66.7 | 78.87 | 17.34 |
| Terpinolene | 10.9 | 0.53 | ||
| 4-Terpinyl acetate | 14.4 | 0.42 | ||
| L-α-Terpineol | 14.8 | 7.56 | ||
| α-Terpinyl acetate | 19.1 | 1.89 | ||
| Ylangene | 19.8 | 0.74 | 1.03 | |
| Caryophyllene | 21.1 | 3.37 | 9.4 | |
| Humulene | 21.8 | 0.69 | ||
| Cedrene | 22.1 | 0.47 | ||
| β-Selinene | 22.8 | 1.7 | 3.33 | |
| α-Selinene | 23.04 | 3.03 | 5.86 | |
| (.+/-.)-Cadinene | 23.5 | 0.63 | ||
| (-)-Globulol | 25.1 | 3.89 | 7.34 | |
| Ledol | 25.4 | 0.46 | ||
| Cubenol | 26.08 | 0.52 | 0.44 | |
| ß-Acorenol | 26.6 | 0.54 | ||
| ß-Thujene | 7.1 | 0.28 | ||
| L-α-Pinene | 7.3 | 4.45 | ||
| Ethanone, 1-(3-ethyloxiranyl)- | 7.8 | 0.21 | ||
| Hexane, 3-bromo- | 8.1 | 0.41 | ||
| Pseudolimonene | 9.3 | 0.26 | ||
| α-Terpinene | 9.6 | 0.23 | ||
| o-Cymene | 9.9 | 2.08 | ||
| Terpinene | 10 | 0.51 | 0.2 | |
| Isoterpinolene | 11.7 | 0.22 | ||
| 4-Terpinyl acetate | 14.3 | 0.37 | 1.64 | |
| (R)-α-Terpinyl acetate | 14.7 | 2.33 | 22.67 | |
| 4-Menthen-8-ol | 19.1 | 0.14 | ||
| Caryophyllene | 21 | 0.33 | ||
| α-Guaiene | 21.4 | 0.17 | ||
| Seychellene | 21.6 | 0.16 | ||
| α-Bulnesene | 23.1 | 0.16 | ||
| Methyl palmitate | 32.1 | 0.22 | ||
| Estradiols | 32.8 | 0.26 | ||
| α-Terpinyl formate | 19.2 | 4.59 | ||
| (-)-Guaia-6, 9-diene | 20.04 | 0.17 | ||
| Elemene | 20.3 | 0.64 | ||
| ß-Gurjunene | 21.4 | 0.37 | ||
| Spathulenol | 21.6 | 1.42 | ||
| Caryophyllene | 22.1 | 3.07 | ||
| α-Amorphene | 22.6 | 3.07 | ||
| γ-Muurolene | 23.4 | 0.89 | ||
| (+/-)-Cadinene | 23.6 | 1.55 | ||
| γ-Selinene | 23.9 | 0.37 | ||
| 3,7 (11)-Eudesmadiene | 24.1 | 0.49 | ||
| Germacrene β | 24.5 | 0.72 | ||
| Palustrol | 24.7 | 0.29 | ||
| Ledol | 25.6 | 0.58 | ||
| τ-Cadinol | 26.4 | 0.37 | ||
M. leucadendra: Melaleuca leucadendra, MO: M. leucadendra, MO2: MO oil heavy fraction, MO1: MO oil light fraction
Identified volatile compounds from Mentha piperita oils
Compounds found within the PO according to the results of GC-MS are presented in Table 3. GC-MS PO results are generally inseparable from the presence of menthol and menthone compounds that are found in the oil.[17] Menthone has various biological properties such as antibacterial, antifungal, antibiofilm, and anti-inflammatory activities.[1] PO contains compounds belonging to terpenes, aliphatics, and benzenoids. All of these compounds contribute to its volatility properties. Cyclohexanol and cyclohexanone constituted the largest part of the PO. The content of cyclohexanol and cyclohexanone with their compositions of 39.79% and 22.24%, respectively, can inhibit the evaporation of PO.[18]
Table 3.
Volatile components of Mentha piperita oil
| Identified compound | Retention time (min) | Relative area (%) |
|---|---|---|
| 2-Trifluoroacetoxytridecane | 7.7 | 6.31 |
| 3-Trifluoroacetoxypentadecane | 8 | 0.06 |
| ß-Pinene | 8.5 | 2.01 |
| ß-Myrcene | 8.9 | 0.34 |
| L-α-Pinene | 9 | 0.34 |
| δ-Limonene | 10 | 5.02 |
| Neo-iso-isopulegol | 13.4 | 1.49 |
| Menthone | 13.7 | 21.6 |
| p-Menthan-1-ol | 14.3 | 37.2 |
| Neoisomenthol acetate | 14.5 | 0.87 |
| (R)-α-Terpinyl acetate | 14.7 | 0.86 |
| cis-3-Hexenyl isovalerate | 16 | 0.37 |
| Pulegone oxime | 16.1 | 1.43 |
| Neoisomenthol acetate | 17.6 | 5.12 |
| (-)-ß-Bourbonene | 20.1 | 0.26 |
| Caryophyllene | 21 | 1.13 |
| Caryophyllene oxide | 25 | 0.18 |
CONCLUSION
Essential oils from P. cablin, M. leucadendra, and M. piperita are rich with volatile components that have potential medicinal benefits such as antibacterial and immunomodulator. Main volatile compounds contained in the oils from M. leucadendra and M. piperita after the purification were eucalyptol and menthone, respectively. As for P. cablin, the main volatile compounds included a-guaiene, seychellene, and PA. Quantitatively, the highest relative abundance of PA, the main compound of PCOs, was found in the light fraction (69.92%). In the case of MOs, the major compound – eucalyptol was observed to be contained mostly in the heavy fraction (78.87%). As for PO, its purification process is affected by the presence of cyclohexanol (39.79%) and cyclohexanone (22.24%). Changing in compositions after the purification step is caused by the degradation or reaction occurs during the process.
Financial support and sponsorship
This research was funded by the Directorate of Research and Innovation Funding, National Agency for Research and Innovation (BRIN) and Education Fund Management Agency (LPDP), Ministry of Finance of the Republic of Indonesia with grant number: 48/IV/KS/06/2022.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
The authors would like to appreciate the collaboration between Universitas Syiah Kuala (Banda Aceh, Indonesia) and IPB University (Bogor, Indonesia). The authors also acknowledge the assistance from LPPM Universitas Syiah Kuala.
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