Chemical profile and antibacterial activity of essential oil from ironwood (Eusideroxylon zwageri) sawdust

Abstract

The current study describes for the first time the chemical compositions and antimicrobial activity of essential oils from ironwood sawdust (Eusideroxylon zwageri) obtained from Borneo Island. Fifteen terpene metabolites were identified by GC-MS of the ironwood sawdust essential oil extracted by steam distillation. (E)-β-Caryophyllene was the oil’s major compound (35.81 ± 0.25%), while its oxygenated derivative (caryophyllene oxide, 8.78 ± 0.08%) was the third biggest terpene. α-Copaene (13.05 ± 0.02%) was also observed in a significant amount together with other minor sesquiterpene hydrocarbons and oxygenated sesquiterpenes. The oil also exhibited antibacterial activities against Gram-positive Multi-Drug Resistant Staphylococcus aureus (MRSA) and Gram-negative Escherichia coli (strain ESBL) during a disc diffusion assay with inhibition zones of 6.13 ± 0.03 mm and 3.35 ± 0.05 mm, respectively.

Keywords:

• Eusideroxylon zwageri
• ironwood
• sawdust
• essential oil

PUBLIC INTEREST STATEMENT

Throughout history, essential oils have been used as a fragrance in soaps, cosmetics and perfumes, fragrances, food flavoring, and medical aromatherapy. Leaves, flowers, bark, and roots are the main sources of raw material to produce valuable essential oils by steam- or hydro-distillation. The heartwood of slow-growing trees including agarwood and cedarwood was also producing one of the most expensive yet exotic essential oils. Since there was an issue associated with the supply and demand in the essential oil market, therefore more studies need to be carried out to explore industrial wood waste for its essential oil content. This study pioneering the isolation and characterisation of essential oil from ironwood sawdust collected from Borneo, Indonesia.

1. Introduction

Bornean ironwood (Eusideroxylon zwageri) is one of the most treasured and commercially valuable timber trees endemic to the Asian forest (Malaysia, Indonesia, Brunei, and The Philippines). The tree can reach a height of up to 50 m and may live over 1,000 years. The wood is dense, strong, and durable, making it preferred by the indigenous people of Borneo for building houses. Recently, a large amount of industrial sawdust of ironwood was mainly left untreated. Previous studies suggested that wood waste can actually be transformed into commercially valuable items including charcoals, biochars, and essential oils (de Meira et al., 2021; Mishra et al., 2022; Mizushina, 2012; Wijitkosum, 2022).

The wood-derived essential oils are well known for their pleasant aroma and bioactivities making them the popular main ingredient in perfumery, flavour, fragrance industries, and medical aromatherapy (Sadgrove et al., 2022). Some examples of high-value wood-derived essential oils include sandalwood (Santalum album) and cedarwood (Cedrus deodara). The sandalwood essential oil is composed of α-santalol and β-santalol as major volatile molecules responsible for the pleasant aroma. The essential oils are isolated from the heartwood of the biomass by hydro/steam distillation (Tripathi et al., 2021). Meanwhile, the major constituents of cedarwood essential oil are α-himachalene, β-himachalene, and α-atlantone, which exhibited significant antibacterial activities (Chaudhary et al., 2015).

The high demand for sandalwood and cedarwood essential oils does not meet with the declining number of plants available for processing. Therefore, alternatives sources of biomass including sawdust collected from the sawmill need to be explored for essential oil that has distinctive scent. Hydrodistillation of sawdust from four different species of pine trees resulted in essential oils rich in D-limonene and β-caryophyllene (Karaoğul & Alma, 2019). In addition, essential oils of sawdust from Moroccan Thuya (Tetraclinis articulata) contained 22 volatile compounds that were responsible for the characteristic aroma. The oils were hydrodistilled and exhibited a strong antioxidant activity (Saber et al., 2020). Herein we report for the first time the isolation, chemical profile, and antibacterial properties of essential oil isolated from Borneo ironwood (Eusideroxylon zwageri) sawdust.

2. Materials and methods

2.1 Plant material

The ironwood sawdust was obtained from a local sawmill in Borneo, and the dried sawdust (moisture content 5%) (approx. 80 kg) was transported to the laboratory for the experiment. Wood edge cuts, leaves, and other foreign materials were separated from the sawdust. The homogenous and dried sawdust was submitted into steam distillation.

2.2 Steam distillation

Ironwood sawdust (20 Kg) was placed inside a 115 L barrel equipped with a Liebig condenser for cooling the steam. Underneath the material, separated by a stainless strain, was water (8 L) to feed the steam distiller for 12 hours. This customised large-scale steam distillation was fuelled by liquified petroleum gas. The ironwood sawdust essential oil (ISEO) was separated in an oil-water separator, dried over anhydrous Na₂SO₄, and stored in a sealed vial in the dark at 4°C until use. The resulting pale yellow distinctive aromatic oil was expressed as a percentage of dry weight. The experiment was repeated thrice.

2.3 Analysis of the essential oil

The ISEO was analysed by Gas Chromatography equipped with a flame ionisation detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS). The GC-FID analysis was performed on a Shimadzu GC-2010 plus gas chromatography apparatus equipped with FID and a Rtx-5 capillary column (30 m × 0.25 mm, film thickness 0.25 μm). The injector and detector temperatures were set at 200°C and 220°C, respectively. Next, the column temperature program was held at 50°C for 3 min and increased gradually to 220°C at 15°C/min, then maintained for 10 min. Helium was used as the carrier gas at a flow rate of 1 mL/min, and the sample (1 μL) was injected neat with a split ratio of 20:1. The relative percentage of individual compound was calculated based on peak area without using a correction factor. Retention indexes of each component were calculated based on the retention times of the homologous hydrocarbon series C9–C24 injected in the same column temperature program. In addition, GC-MS analysis was carried out using a Shimadzu QP2010 model equipped with an autosampler (AOC-20s), an auto-injector (AOC-20i), and an RTX-5 MS column (30 m × 0.25 mm ID and 0.25 μm film thickness). The column temperature program was the same as that employed in the GC. Next, the mass spectrometer was operated in the electron impact (EI) mode at 70 eV, and the mass scanning range was varied over 35–500 m/z. The ion source and quadrupole temperatures were set at 230°C and 150°C, respectively. The relative percentage of each component was estimated without correction using the GC peak area (FID response). The individual compounds were identified by combining retention index data and mass fragmentation patterns with those in the mass spectral database Wiley 09 and the Adam’s book (Adams, 2007).

2.4 Disc diffusion assays

Antibacterial properties of the oil and hydrosol were evaluated using a disc-diffusion assay (Imane et al., 2020). The growth inhibition of the oils was tested against Gram-positive Multi-Drug Resistant Staphylococcus aureus (MRSA) and Gram-negative Escherichia coli (strain ESBL). One hundred microliters of an overnight bacterial suspension (Mac Farland 0.5) were added into 5 mL pre-warmed growth media in a sterile petri dish. Each essential oil was dissolved in dimethyl sulfoxide (1 mg/mL) and subsequently loaded (10 µL) into sterile filter paper discs (6 mm Ø Whatman No.3). The discs were incubated overnight at 37°C, and the diameter of the inhibition zones was measured. Chloramphenicol (30 μg/disc; Sigma-Aldrich) and DMSO were used as a positive and negative control, respectively. The inhibition zones were classified according to Elaissi et al. as follows: not sensitive for a diameter less than 8 mm, sensitive for a diameter of 8–14 mm, very sensitive for a diameter of 14–20 mm, and extremely sensitive for a diameter larger than 20 mm (Elaissi et al., 2011). Each test was performed in two replicates.

3. Results and discussions

3.1 Essential oils yield

Despite using a different distillation method, the yield of essential oil obtained in this study (0.01% w/w) was way too small compared to those reported from other sawdust (Table 1). Steam distillation was chosen to maintain the presence of possibly thermally instable components in the oil. Therefore, the oil was expected to provide a distinctive aroma what parfumiers are looking for. An obvious explanation would be the nature of the wood. As implied by the name, ironwood is the most durable wood, which is why it was often used to build outdoor ornaments and traditional harbours. As a result, it is tough to break up the oil compartment in the cell of the wood. Another possible reason was that the sawdust has a tiny quantity of oil compared to other wood species. Up to now, there has been no report in the literature presenting the essential oil from ironwood sawdust-only one paper reporting the bio-oil production from burning the ironwood waste (Junaidi et al., 2020). So, there has been very little interest in ironwood essential oil due to its meagre yield. However, that perspective may change since demand has already emerged from fragrance industries owing to its distinctive scents. Therefore, to meet the market, a large-scale distillation of wood sawdust should be established to obtain the desired quantity of products. Accordingly, various extraction techniques may offer an alternative to boost the oil yield.

Table 1. Yield comparisons of essential oil of sawdust from several species of wood

No	Species	Yield (% w/w)	Geographical origin	Distillation method
1	Tetraclinis articulata	2.19	Khemisset, Marocco	Hydro-distillation (Saber et al., 2020)
2	Tetraclinis articulata	1.66	Essaouira, Marocco	Hydro-distillation (Achmit et al., 2021)
3	Cedrus atlantica	5.98	Senoual, Marocco	Hydro-distillation (Jaouadi et al., 2021)
4	Cedrus atlantica	3.51	Itzer, Marocco	Hydro-distillation (Jaouadi et al., 2021)
5	Cedrus atlantica	3.40	Azrou, Marocco	Hydro-distillation (Fidah et al., 2016)
6	Eusideroxylon zwageri	0.01	Borneo, Indonesia	Steam distillation in this study

3.2 Chemical composition of the oil

The GC chromatogram of the ISEO revealed the presence of 18 peaks, representing 95.11% of the total composition (Figure 1). In total, 15 volatile compounds were identified from the well-resolved peak and listed in order of their retention time (Table 2). The identified metabolites were grouped into hydrocarbon sesquiterpene and oxygenated sesquiterpene. The earlier group eluted from the retention time of 13.16 min to 14.96 min, whereas the oxygenated counterparts were observed soon after the later time. The oxygenated terpenoids were commonly detected after the hydrocarbons due to their polarity, as exemplified in several compounds of well-known essential oils, including patchouli (Jain et al., 2022) and eucalyptus (Ndiaye et al., 2018).

Figure 1. GC chromatogram of ironwood essential oil.

Table 2. Chemical composition of ironwood essential oil

RT	Compound	RIExp	RILit	% Concentration
	Sesquiterpene hydrocarbon
13.166	α-Cubebene	1326	1348	6.44±0.00
13.496	α-Copaene	1358	1374	13.05±0.02
13.609	β-Elemene	1369	1389	3.64±0.01
14.005	(E)-Caryophyllene	1408	1417	35.81±0.25
14.340	α-Humulene	1443	1452	7.99±0.02
14.484	α-Amorphene	1458	1483	1.96±0.01
14.702	α-Muurolene	1481	1500	1.67±0.01
14.964	cis-Calamenene	1508	1528	6.15±0.02
				76.71
	Oxygenated sesquiterpene
15.202	Elemol	1532	1548	0.97±0.03
15.592	Caryophyllenyl alcohol	1572	1570	2.03±0.06
15.733	Caryophyllene oxide	1587	1582	8.78±0.08
16.003	Humulene epoxide II	1614	1608	0.96±0.03
16.111	1-epi-Cubenol	1624	1627	2.44±0.06
16.265	Torreyol	1639	1644	3.21±0.02
				18.40

RI_Exp- Retention index from literature and library.

RI_Lit- Retention index calculated on Rtx-5 column (relative to n-Alkane, C9-C24).

RT- Retention time.

trans-Caryophyllene or (E)-β-caryophyllene, a member of bicyclic hydrocarbon sesquiterpene, was the highest component of the ISEO (35.81%). The presence of its isomers (α -humulene) and its oxidative product (caryophyllene oxide) was also evident from the chromatogram but in smaller amounts. In nature, β-caryophyllene mainly occurs as trans-caryophyllene (E isomer) mixed with small amounts of its isomers, (Z)-β-caryophyllene (iso-caryophyllene) and α-humulene (α-caryophyllene), as well as its oxidation derivative, β-caryophyllene oxide (Figure 2) (Fidyt et al., 2016).

Figure 2. Trans‐caryophyllene, its isomers, and oxidative product.

A significant percentage of both (E)-β-caryophyllene and its oxidative product is often associated with the solid wooden odour and they are used as cosmetic and food additives. Natural bicyclic sesquiterpenes, β-caryophyllene and β-caryophyllene oxide are present in essential oils of several species of plants, including basil (Ocimum spp.), cinnamon (Cinnamomum spp.), black pepper (Piper nigrum L.), cloves (Syzygium aromaticum), cannabis (Cannabis sativa L.), lavender (Lavandula angustifolia), oregano (Origanum vulgare L.), and rosemary (Rosmarinus officinalis) (Fidyt et al., 2016). Furthermore, (E)-β-caryophyllene is reported to exert a plethora of pharmacological effects, including antimicrobial (Astani et al., 2011) and analgesic activity (Chavan et al., 2010), anticancer activities, affecting the growth and proliferation of numerous cancer cells (Fidyt et al., 2016), as well as antibacterial activity (Sabulal et al., 2006).

α-Copaene (13.05%), the second most abundant component of the ISEO, was also documented in the essential oil of the inner bark of the Brazilian wood Kielmeyera coriacea in a comparable concentration (14.9%). The compound is often associated with the wood spicy honey aroma exerted by the plant. A significant proportion of this tricyclic sesquiterpene in the essential oil wood was suspected to be responsible for the satisfactory antibacterial properties against the anaerobic bacteria Prevotella nigrescens (Martins et al., 2015). Since α-Copaene has broad application prospects in many fields, including food, medicine, and agriculture, a first de novo biosynthesis has been successfully achieved using a metabolically engineered Escherichia coli strain (Zhang et al., 2022).

cis-Calamenene was previously identified as the most abundant metabolite (27.9%) of essential oil of the tropical fruit Vitex gardneriana collected from Northeast Brazil. The oil also displayed anticholinesterase and antimicrobial activities against Trichophyton rubrum (Pereira et al., 2018). Recently, cis-Calamenene presented in a significant percentage (14.6%) in the oil of four Polyalthia species obtained by hydrodistillation (Shakri et al., 2020).

The ironwood essential oils were not sensitive against Multi-Drug Resistant Staphylococcus aureus (MRSA) and Escherichia coli (strain ESBL) (Table 3 and Figure 3). Despite possessing an excellent antibacterial activity as in an individual β-caryophyllene, the influence of other minor terpenes in the ironwood oil may explain the relatively low inhibition. A relatively low antibacterial activity of wood-derived essential oil was also displayed by the oil from the burl wood sawdust of Tetraclinis articulata obtained using hydro-distillation (Talbaoui et al., 2016).

Figure 3. Examples of inhibition zones experiment.

Table 3. Zone inhibition of the essential oil

	Zone inhibition diameter (mm)
	MRSA	E.coli (ESBL)
Ironwood EO	6.13±0.03	3.35±0.05
Chloramphenicol	24.18±0.03	13.15±0.05
DMSO	-	-

4. Conclusion

This first study on the chemical composition of the ironwood sawdust essential oil revealed that the oil was mainly constituted of sesquiterpene hydrocarbons (76.71%) and a smaller portion of oxygenated sesquiterpenes (18.40%). (E)-β-Caryophyllene and its oxidative product were the two major metabolites observed in the oil that may contribute to the distinctive scent of the oil. An extremely low yield (0.01%) posed another challenge in up-scale oil production. Therefore, further investigation is required to ferment the sawdust prior distillation to increase the oil yield during the steam distillation.

Acknowledgments

We thank Universitas Pendidikan Ganesha, Indonesia for financial support to I.W.M. under DIPA-KR scheme No. 1048/UN48.16/LT/2022. The assistance of MERO Foundation, Bali for performing disc-diffusion assay on the oil samples was highly appreciated.

Disclosure statement

The authors report no declarations of interest.

Additional information

Funding

The work was supported by the DIPA Universitas Pendidikan Ganesha [1048/UN48.16/LT/2022].

Notes on contributors

I Wayan Mudianta

I Wayan Mudianta, Surrounded by tropical forests, our laboratory has explored the essential oils from various plants and biomass. We are also interested in developing techniques to extract the original aroma of endemic flowers by employing enfleurage methods. Enantioselective gas chromatography and thermogravimetric analysis (TGA) are authentication methods that we are investigating in our newly established laboratory in Bali, Indonesia. In collaboration with local companies in Bali, we have fully engaged in developing essential oils with a distinctive scent and unique chemical composition from plants growing in Indonesian fertile soil.

I Nyoman Gede Suma Artha

I Nyoman Gede Suma Artha, founder of CV. Balizen is a local company in Bali that produce and export various handicrafts, aromatherapy, and essential oil-based products. The essential oils were produced in a sustainable and greener way with a strong commitment to preserving the local Balinese culture.

I Wayan Muderawan

I Wayan Muderawan, a Professor in organic chemistry, have been studying the application of beta-cyclodextrin in drug carriers and essential oil encapsulation. Recently, he studies the physical properties and thermal transition of beta CD encapsulated patchouli essential oil using differential scanning calorimetry (DSC).

Ni Wayan Martiningsih

Ni Wayan Martiningsih is a natural product researcher that mainly studies the chemistry and bioactivity of plant extracts and essential oils. She has also focused on the isolation and structure elucidation of secondary metabolites from marine animals.