1 Introduction
Litsea cubeba Lour. Persoon is a small to medium-sized tree that belongs to the Lauraceae family. Its Indonesian name is kemukus and local names are ki lemo or lemo (West Java), krangean (Central Java), and attarasa (North Sumatera). In China, it is known as may chang [1,2]. Products from the tree have multiple applications [3,4]. Each part of the tree has high economical value, from its wood, bark, branches, leafs, fruit, and flowers to its roots [4,5-7]. It is well known as a potential source of essential oil for industrial products such as cosmetics (aroma therapy), soap, perfume, skin cleaner, and acne medicine. Essential oil from this tree is also the source of an agent that is believed to be a carcinostatic (anti-cancer). Other applications of L. cubeba essential oil are: bioinsecticides and anti-termite agents [8,9], antimicrobe active alkaloid
isoquinoline is used as a traditional medicine [10], antibacterial agent [11,12], and antioxidant [13]. A research in Thailand showed that essential oil from L. cubeba can be used as a repellent for the Aedes aegypti mosquito [13].
On Mount Papandayan, L. cubeba grows in various habitats. It is assumed that the amount and quality of the essential oil produced by L. cubeba varies depending on the substrate on which it grows. To confirm this assumption, it was necessary to conduct research to find out the contents of the essential oils taken from L. cubeba trees growing in different habitats.
This study was exciting to conduct because only a limited number of studies have examined the variation of essential oil from L. cubeba trees from different natural habitats. Several studies have been carried out in different habitats, including Mount Ciremai West Java (6), North Sumatra (15) and China (16), but these did not specifically address habitat variation and its relationship with the contents of the essential oil.
Litsea cubeba has never been cultured on a large scale. It grows mainly in natural forests, especially in highlands. The tree is threatened by human exploitation. Its culture is urgently needed for conservation of the species and for supporting the essential oil industry.
The objective of this research was to determine the production of essential oil from L. cubeba leafs harvested from various habitats on Mount Papandayan, West Java, Indonesia. The results were expected to provide information about the yield and contents of the essential oil taken from the different habitats.
2 Methodology
Two methods were used in this research: field exploration and observation, followed by laboratory testing. The determination of sample plots was conducted by purposive sampling based on: 1. stratum (based on land system map); 2. four types of terrain type; 3. altitude; 4. topography (steep/sloping/ ridge).
Leaf samples of L. cubeba were obtained from 15 different habitats, where every habitat was represented by one plot that contained at least one mature tree with a minimum diameter of 20 cm. The leafs were cut from the top of the twigs along 30 cm in all wind directions and canopy positions (top, middle and bottom). 400 g of mixed leaf samples were distilled to obtain essential oil.
Extraction of the essential oil from the leaf samples was conducted by steam distillation for 2 hours or until the essential oil was fully extracted. The leaf
samples were dried by air-drying before distillation. Each distillation needed 200 g of air-dried leaf samples and the distillation was carried out in duplo. The following step was to determine the contents and composition of the essential oil by GC-MS analysis (Agilent Technologies GC System (GC 7890 and 5975 C XLEI/CI MSD)) at 250°C. The temperature of the MS detector was 28°C [14]. The results indicate the variability of the essential oil and its composition from the samples taken from the different habitats.
3 Results and Discussions
3.1 Habitat Variation
Based on a field survey, L. cubeba trees growing at 15 various habitats with different population sizes were selected (Table 1). All habitats were located between 1000 to 2500 m above sea level, ranging from flat to very steep, and were previously disturbed by illegal logging or forest fires.
| Table 3 Habitats of \(L\). | ıbeba on M | Iount Papa | andayan. |
|---|
| Altitude | |||||
|---|---|---|---|---|---|
| Code | Name of location | (m above sea level) | Topography | Landscape | Land cover |
| H1 | Sorok Teko/Tgl Puspa | 1919-1956 | Sloping- slightly steep | Cliff | Forest fire debris |
| H2 | Gn Walirang | 1617-2058 | Flat-very steep | Cliff-Ridge | Bush |
| Н3 | Puntang/S. Cibeureum | 2040 | Very steep | Cliff river/ravine | Forest fire debris |
| H4 | Bungbrun | 2169 | Flat-sloping | Plateau at the top of the hill | Forest fire debris |
| Н5 | Pada Awas/Lutung- Pondok Serok | 2044 | Steep-very steep | Cliff/ravine | Forest fire debris |
| Н6 | Pada Awas/Tibet- Lutung | 2100 | Steep-very steep | Cliff/ravine | Forest fire debris |
| Н7 | Tegal Bungbrun | 2300 | Sloping | Ridge | Young secondary forest |
| Н8 | Lembah Cibeureum | 2161-2160 | Sloping | Plateau | Young secondary forest |
| Н9 | Lutung | 2040 | Steep | Cliff | Young secondary forest |
| H10 | Tibet | 2100 | Sloping | Plateau | Gap/hiatus |
| H11 | Lereng Curug Angklong | 2100 | Sloping | Plateau | Bush |
| H12 | Batu Kasang | 1882 | Slightly steep | Ridge | Young secondary forest |
| H13 | Batu Kasang 2 | 2030 | Very steep | Cliff/ravine | Gap/hiatus |
| H14 | Lembah Puntang/Supa beureum | 2157 | Flat | Valley | Young secondary forest |
| H15 | Tgl Panjang | 2041 | Slightly steep | Cliff | Gap/hiatus |
Litsea cubeba generally grows within a temperature range of 16-26.3°C (with an average temperature of 19.24°C), a humidity range of 60-95%, and light intensity range of 300-85600 lux. The habitats on Mount Papandayan are colder and more humid compared to those in North Sumatra, another natural distribution area of Litsea cubeba in Indonesia apart from West and Central Java [15].
These observations show that L. cubeba can grow in open spaces with various disturbed habitats and has great potential for use as a pioneer tree [15].
3.2 Yield of Essential Oil
The results of the distillation process showed variation in the essential oil content of the L. cubeba leafs harvested from the 15 different habitats (Table 2). The resulting essential oils were either yellowish or colorless and had a pungent smell like citrus mixed with eucalyptus oil (Figure 1).
Table 4 Distillation results of essential oil of L. cubeba harvested from 15 habitats.
| Sample code | Water content (%) | Weight of essential oil | Yield (%) |
|---|---|---|---|
| H4 | 25 | 18.65 | 9.33 |
| H13 | 71 | 18.50 | 9.25 |
| H3 | 31 | 17.85 | 8.93 |
| H8 | 36 | 16.33 | 8.16 |
| H2 | 25 | 16.13 | 8.06 |
| H5 | 40 | 15.55 | 7.77 |
| H7 | 21 | 15.04 | 7.52 |
| H1 | 9 | 14.30 | 7.15 |
| H6 | 43 | 13.88 | 6.94 |
| H9 | 27 | 13.82 | 6.91 |
| H12 | 72 | 13.62 | 6.81 |
| H11 | 56 | 11.08 | 5.54 |
| H15 | 57 | 10.24 | 5.12 |
| H10 | 60 | 9.84 | 4.92 |
| H14 | 56 | 4.27 | 4.27 |
Table 2 shows that the yield of essential oil ranged from 2.76 to 9.33%. The maximum yield in this research was higher than the extraction result of similar leafs originating from Mount Ciremai (5.4%) [6] and China (4.56%) [16].
Among all the locations, the leafs collected from locations H4 and H13 had the highest yield, followed by locations H3, H2, and H8. Further analysis showed that the essential oil yield was not affected by altitude, topography, or landscape factors but only by land cover conditions.
The resuls showed that burnt bush and forest fire debris land cover types tended to have a higher yield compared to other land cover types. This is probably due to the different level of environmental stress received by L. cubeba. Habitat stress, such as limited nutrition in the soil, can increase the production of flavanoid as secondary metabolite [17]. This implies an increase of the chemical compounds in the plant. Further research on soil characteristics is needed to explain this phenomenon.
Figure 1 Color variations of L. cubeba essential oils originating from Mt. Papandayan–(a) and (d): examples of colorless essential oil; (b) and (c): examples with a yellowish color.
3.3 Chemical Composition of Essential Oil
GC-MS analysis showed that the chemical composition of the essential oil extracted from L. cubeba leafs from different habitats was similar, but was different in abundance of each chemical compound (Table 3).
Overall, 16 chemical substances were identified in the leaf extracts, with 8 chemical substance compounds found in all leaf samples, i.e. α-pinene, (-) sabinene, 2-β-pinene, β-myrcene, D-limonene, eucalyptol and α-terpinenyl acetate. There were specific chemical compounds that were found only in a specific habitat, i.e. linalool found only in the leaf samples from locations H1 and H15, and α terpineol found only in the leaf sample from location H12.
Table 3 Chemical composition of essential oil of L. cubeba growing in different habitats.
| Name of chemical | 7 | Abundance (Peak Area (%)) | e (Peak | Area (%) | _ | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| punodwoo | HI | H2 | H3 | H4 | HS | 9H | H7 | H8 | H9 | H10 | H11 | H12 | H13 | H14 | H15 |
| -phellandrene | 0.48 | 0.51 | 0.48 | 0.23 | 0.51 | 0.46 | 0.56 | 0.54 | 0.44 | 0.50 | 0.50 | 0.48 | |||
| -pinene, (-)- | 4.58 | 4.46 | 4.63 | 2.28 | 4.53 | 4.48 | 5.03 | 4.78 | 4.40 | 4.39 | 4.21 | 4.14 | 4.84 | 4.63 | 4.41 |
| Sabinene | 15.78 | 15.99 | 15.56 | 68.05 | 15.74 | 16.05 | 16.33 | 16.07 | 14.81 | 15.51 | 14.45 | 15.22 | 16.54 | 15.28 | 15.14 |
| 2pinene | 4.00 | 3.89 | 4.02 | 1.70 | 4.00 | 3.97 | 4.31 | 4.02 | 4.04 | 3.83 | 3.75 | 3.81 | 4.05 | 3.81 | 3.69 |
| -myrcene | 1.10 | 1.24 | 1.00 | 0.55 | 1.07 | 1.07 | 0.99 | 1.24 | 1.13 | 66.0 | 0.89 | 1.00 | 1.21 | 1.09 | 1.19 |
| D-limonene | 4.64 | 4.00 | 3.78 | 1.74 | 2.09 | 4.25 | 2.82 | 3.99 | 4.93 | 3.90 | 4.94 | 3.10 | 4.59 | 4.26 | 3.58 |
| Eucalyptol | 52.00 | 46.91 | 51.46 | 16.97 | 52.60 | 52.79 | 51.64 | 49.01 | 51.79 | 54.29 | 54.80 | 55.78 | 51.23 | 48.03 | 47.12 |
| 1,4-cyclohexadiene, 1-methyl-4-(1-methylethyl)- | 0.29 | 0.12 | 0.42 | 0.35 | , | , | ī | 0.37 | |||||||
| Cis-sabinene hydrate | 0.53 | 09.0 | 0.23 | 0.63 | 0.64 | 0.53 | 0.58 | 0.74 | 0.77 | 0.82 | 99.0 | 0.62 | |||
| Linalol | 1.51 | 1 | ı | 0.52 | |||||||||||
| 4-terpineol | 0.41 | 0.16 | 0.50 | 0.47 | ì | · | , | i | |||||||
| terpineol | 2.71 | r | |||||||||||||
| Ocimenyl acetate | 0.95 | 0.54 | 1.12 | 0.97 | 1.00 | 1.22 | 1.10 | 0.88 | 0.93 | 0.74 | 1.02 | 1.31 | 1.45 | ||
| Terpinyl acetate (CAS) | 1.41 | 1.10 | • | ||||||||||||
| -terpinenyl acetate | 14.43 | 19.81 | 17.95 | 7.27 | 17.21 | 15.31 | 17.34 | 17.08 | 15.97 | 14.94 | 16.03 | 12.13 | 15.21 | 20.44 | 20.96 |
| Caryophyllene | 0.51 | 0.17 | 0.52 | 0.58 | 0.53 | 09.0 | 0.46 |
This research found three dominant chemicals in the extracted oil, i.e. eucalyptol (16.97-55.78%), α-terpinenyl acetate (7.27-20.44%), and sabinene (14.45-68.05%).
These dominant chemical compounds are different from those found through research conducted in other areas. For example, the abundance of dominant chemical compounds of the L. cubeba essential oil from: (i) Cikole West Java – sineol (56.61%) and citronellol (12.26%) [4]; (ii) Mount Ciremai West Java – sineol (56.61%), β-pinen (14.54%), and citronellol (12.28%) [7]; (iii) Thailand – sabinene (42.69%), 1.8 cineole (18.32%), and β-pinene (6.15%) [18]; (iv) China – monoterpenes (94.4-98.4%), represented mainly by neral and geranial (78.7–87.4%) [16]; (v) Zhao [19] showed that the dominant chemical compounds were citral, eucalyptol, citronellol, 6-octenal, 3,7-dimethyl-2,6 octadien-1-ol (geraniol alcohol), and so on. Wang & Liu [12] discovered that the dominant chemical compounds were citral B (neral), β-phellandrene, and βterpinene.
This research showed that the variety in chemical composition of the investigated samples of essential oil of L. cubeba was quite high. This condition is presumed to be affected by factors in the different habitats. Therefore, further research is needed to understand the ecological preference and its relation to the chemical composition of L. cubeba, which in turn may support the culture of L. cubeba.
4 Conclusions
Litsea cubeba has a tendency to grow well in open spaces with various land covers that have been disturbed. The yield variation of the essential oil produced by L. cubeba in different habitats tends to be high. The highest yield of essential oil was obtained from locations H4 (a burnt area): 9.33% and H13 (an ex-encroachment area): 9.25%, while the lowest yield was found in a dead forest: 4.27%.
In general, it can be concluded that there is a tendency that burnt areas and exencroachment areas produce the highest yield of essential oil. Higher environmental stress results in a higher yield of essential oil. The three dominant chemical compounds found were, i.e. eucalyptol (16.97-55.7%), terpinenyl acetate (7.2- 20.44%), and sabine (14.45-68.05%).
Further research is needed to learn more about the ecological preference and its relation to the chemical compounds of essential oil from L. Cubeba.
Acknowledgements
We would like to express our sincere thanks to the Indonesian Ministry of Education and Culture for financially supporting this research. We also express our gratitude to the Indonesian Ministry of Forestry for providing the research field on Mount Papandayan Nature Reserve, Faculty of Forestry IPB, Faculty of Agriculture Technology IPB, School of Life Sciences & Technology ITB, and Integrated Laboratory of Bandung Polytechnique of Health for the laboratory equipment and analysis.