D. aromatica is a large tropical rainforest species recognised for its valuable timber, locally known as kapur, Borneo camphor, camphor tree, or Sumatran camphor. It can grow up to 60 m in height, with girth of 9 m [1]. The tree which has been classified as critically endangered species, is found only in Malaysian Peninsular, Borneo and Sumatra [2]. The genus Dryobalanops belongs to the Dipterocarpaceae family and is one of the two species that can be found in Peninsular Malaysia, the other being D. oblongifolia [2].

Although ancient Chinese literature reported that D. aromatica is the premium source of borneol, there are no scientific reports on the isolation of borneol from this plant [3,4]. Researches on D. aromatica have so far focused mainly on the tree bark. Moreover, there is limited information on the complete chemical profiling of the extracts or essential oil isolated from the exudates of D. aromatica.

Borneol is an expensive compound used in Chinese and Western medicine for many years. It is an essential ingredient in 65 traditional Chinese medicines [5]. In recent years, natural borneol has been isolated from the leaves of Cinnamomum glanduliferum [6], Cinnamomum camphora [7] and Cinnamomum burmannii [8]. Borneol can also be synthesized from turpentine oil or camphor as Borneolum syntheticum in the form of DL-borneol and iso-borneol. However, synthetic borneol has been shown to exert some toxic effects that due to the presence of iso-borneol and camphor [9]. This makes natural borneol especially D-borneol the safer ingredient for medicinal purposes. However, due to high demand and high production cost, natural borneol is in short supply [10].

In view of the high distribution of D. aromatica in Malaysian Peninsular and the potential of D. aromatica as an alternative source of natural borneol, this study was conducted to determine the presence of borneol and other chemical composition in the essential oil extracted from the exudates of D. aromatica. Further studies were biological activities of the essential oil and the isolated pure compounds, particularly on anti-cancer properties, namely, cytotoxic and anti-tumor promoting activities.

Plant material

Exudates of D. aromatica, obtained either from natural exudates from the tree or by mechanical incision, were collected from Commonwealth Forest Reserve, Selangor, Malaysia. Plant identification and authentication were done by Professor Dr Ong Hean Chooi from University of Malaya, Malaysia and a voucher specimen (UTAR/LTX01/12) was deposited at herbarium of Universiti of Tunku Abdul Rahman.

Fractional distillation of exudates

Exudates were subjected to fractional distillation in double distilled water for 2 h at 80 – 90 °C. After cooling at room temperature for 2 h, the essential oil and distilled water were separated and this was followed by another cycle of fractional distillation. The process was repeated until the yield of essential oil became minimal. The essential oil was then pooled and stored at 20 °C.

GC-MS analysis

The essential oil was analyzed by Agilent 7890A/5975C GC-MS system equipped with HP5ms stationary phase column (30m x 0.25mm internal diameter x 0.25µm film thickness) composed of 5 % phenylpolysiloxane/95 % dimethylpolysiloxane. Helium was used as carrier gas and programmed at a flow rate of 1.0 ml/min. The sample injection volume was 1 µL, with split ratio of 1:10. The initial oven temperature was programmed at 80 °C, held for 5 min, then increased to 140 °C at 10 °C/min, held for 0.5 min and increased to 300 °C at 30 °C/min with final hold for 2 min. Electron ionization system with ionization energy of 70 eV was used. Injector and MS transfer line temperatures were set at 230 and 280 ºC, respectively. The MS system was operated in scan mode with a mass range of 50-400m/z. Identification of compounds were achieved through the determination of retention indices (RI) with reference to a homologous series of n-alkanes (C₈-C₂₀) and by NIST mass spectrum library matching.

Yield of essential oil

Through fractional distillation, different compounds were extracted based on their volatility. Less volatile compounds were separated first followed by more volatile compounds.  The average essential oil obtained from the exudates of D. aromatica was 7.58 % over 28 h of fractional distillation. The higest yield of essential oil was at the first 2 h of fractional distillation, which was 3.24 %; and the yield decreased with increasing number of cycles of fractional distillation.

Essential oil composition

Thirty compounds, which accounted for 97.56 % of essential oil composition from the exudates of D. aromatica were identified using GC-MS analysis (; ). The essential oil consisted of monoterpenes and sesquiterpenes; and the major compounds identified, in order of abundance, were β-caryophyllene (31.76 %), α-pinene (21.49 %), α-caryophyllene (13.50 %), terpinen-4-ol (8.58 %) and α-terpineol (5.89 %). In addition, 0.74 % of borneol was detected.

Borneol is a bicyclic monoterpene that has been used in traditional Chinese medicine for many years as a premium ingredient for treating heart disease, coma and respiratory problems; indigestion and pain relief [11]. In this study, borneol was detected in the essential oil of D. aromatica at 0.74 %.

The other compounds detected accounted for 81.22 % of the total essential oil. This study reported for the first time on the presence of α-pinene, terpinen-4-ol and α-terpineol as the major components of essential oil from the exudates of D. aromatica. This study has identified some compounds not earlier reported by Huang and Lu [3]. These are d-borneol, α-caryophyllene, β-elemene, β-caryophyllene, asiatic acid, dryobalanone, erythrodiol and hydroxy-dammarenone II. The synthesis of phytochemicals can be affected qualitatively or quantitatively in response to environmental conditions, geographical variation, genetic factors and evolution; or physiological variations such as type of plant material, pollinator activity cycle, organ development and mechanical or chemical injuries [12,13]. The differences observed in the chemical composition of essential oil between the present study and that of Huang and Lu might be due to geographical variation of plant species, which led to differences in environmental conditions.

The compounds detected in this study (d-borneol, terpinen-4-ol, α-terpineol, α-pinene and caryophyllene) are terpenoid compounds well known for their anti-microbial, antiviral, anti-inflammatory and cytotoxic effects [14-20].

Twenty-seven compounds have been identified from the essential oil from the exudates of D. aromatica. These terpenoid compounds have great potential in the pharmaceutical, perfumery, aroma-therapeutic, cosmetic, detergent and food industries.