Chemical Composition of Leaf and Seed Oils of Dryobalanops aromatica Gaertn. (Dipterocarpaceae)

D. aromatica (Figure 1), commonly known as the Bornean Camphor-Tree, and locally known as kapur peringgi, is a large and lofty tree, reaching up to 65 m in height and 7 m in girth. The trunk is usually a straight, cylindrical and clear bole of 30 m–40 m. This species is a well-known and valuable timber tree. The timber has been described as being moderately hard, heavy and durable (Ashton 1964). It is used as an internal wood and resembles mahogany when given a good polish. It has a camphor odour, and the camphor in the wood was sought after and sold as medicine in the past (Burkill 1966). The camphor produced by the tree is less important today than its timber, but in the earlier days the reverse was the case. The species also produces camphoraceous oleo-resin. Chemical Composition of Leaf and Seed Oils of Dryobalanops aromatica Gaertn. (Dipterocarpaceae)

The uses of the wood and camphor of D. aromatica in both eastern and European medicines have been well documented (Burkill 1966;Perry 1980;Siang 1983;Duke & Ayensu 1985). The camphor has also been used by the Malays and the Sumatran people in the ceremonial purification of dead bodies and their preservation until burial (Burkill 1966). A mixture of the volatile oils of D. aromatica, Piper longum, Santalum album, Asarum sieboldi and Alpinia officinarum is said to be effective in the treatment of acute anginal attack (Guo et al. 1983). The methanol extract of the wood is also shown to have antifungal properties (Hong & Abdul Razak 1983;Kim et al. 2005).
Chemical examination on the oleo-resin of D. aromatica shows that it consists of 35% terpenes (including pinene), 10% alcohols (including borneol), 20% sesquiterpenes and 35% resin (Burkill 1966, p. 881). The resin consists mainly of triterpenes (Cheung & Wong 1972) and the oxygenated derivatives of asiatic acid as minor constituents (Cheung & Tokes 1968). The camphor consists of borneol, camphor, camphene, sesquiterpenes and terpineol (Perry 1980, Duke & Ayensu 1985, while the wood extracts contain largely terpenes and fatty acids (Ali & Koh 1991). However, an earlier distillation attempt on the leaves in 1910 yielded little oil and no details were provided (Burkill 1966, p. 881). A later attempt at distilling the leaves and twigs showed that no oil or borneol was obtained (Eaton 1925). Since then, there has not been any study on the essential oils in the leaves of this plant. In addition, no published work can be traced with reference to the volatile oil of its seed. The lack of information and details on both the leaf and seed oils of D. aromatica prompted us to undertake this investigation. Thus, the present study aims to identify and document fully for the first time the chemical constituents present in the essential oils obtained from the fresh leaves and seed of D. aromatica. This study may allow us to identify potential uses and better utilization of these plant parts which are usually discarded when the tree is harvested for its timber.

Plant Material
Fresh leaves ( Figure 2A) and seed ( Figure 2B) of D. aromatica were collected from the Bukit Sawat forest in the Belait District of Brunei Darussalam. The species was identified by the author, Dr Kamariah Abu Salim and confirmed by Awang Ariffin Abdullah Kalat of the Brunei National Herbarium (BRUN), Sg. Liang. A voucher specimen bearing reference no. SN-B000340 was deposited at BRUN.

Isolation of Essential Oils
Immediately, after collection, the fresh leaves and seed were subjected to hydro-distillation in a Neo-Clevenger apparatus for 4 h. The oils were collected in dark brown glass vials and stored at 4°C until further analysis. The percentage compositions of the oils were calculated based on the fresh weight of the respective plant parts.

Properties of Essential Oils
Oil density was determined by using a piknometer, refractive index by a Shimadzu Bausch and Lomb Abbe refractometer, and optical rotation by an Onel Pol S-2 polarimeter.

Gas Chromatography-mass Spectrometry (GC-MS analysis)
GC-MS analysis of the oils was carried out on a Hewlett Packard GCD system. Separation was performed in an Innowax fused silica capillary (FSC) column (60 m × 0.25 mm id; 0.25 mm film thickness). Helium at a flow rate of 1 ml min -1 was used as the carrier gas. The temperature of the GC oven was initially set at 60°C for 10 min, and then increased at a rate of 4°C min -1 to 220°C. It was held isothermally at 220°C for 10 min, programmed to 240°C at a rate of 1°C min -1 , and finally maintained at 240 ο C for 20 min. Split injection was conducted at a flow rate of 50 ml min -1 with a split ratio of 50:1. The temperature of the injector was set at 250 ο C and the ionization energy was 70 eV. The mass range was from 35 to 425 m/z.

Determination of Essential Oil Composition
Chemical identification of the different components of the oils was based on their retention times and comparison of their mass spectra with those of the Wiley GC-MS Library and a home-made library (Baser Library of Essential Oil Constituents). The relative percentage composition of the volatile compounds was calculated from the Total Ion Chromatogramme (TIC), assuming that the relative response factor was equal to 1.

Yields and General Considerations
The yields and physico-chemical properties of the leaf and seed oils of D. aromatica are shown in Table 1.
Although the density, refractive index and optical rotation of both oils appear to show very little difference, the oil yield of the seed was significantly (twenty-nine times) higher than that of the leaves. Thus, the seeds are a better source of oil than the leaves. However, the higher oil yield from the seed may not necessarily prove to be of significant importance for commercial exploitation because the trees only flower and fruit once in every 3-4 years. In addition, the conservation status of the species must be considered and given a priority in any attempt to exploit the seed for commercialization of the oil. It may be better for the discarded seed (when present) during timber extraction to be collected for propagation and re-planting purposes.
The low percentage of oil yield from the leaves seems to be in agreement with earlier work carried out when negligible (Burkill 1966, p. 881) or no yield at all was obtained (Eaton 1925). Thus, any commercial exploitation of discarded leaves for the oil during timber harvest needs to take into account this low yield, available resources and the cost of production.

Chemical Composition
The identified compounds in the oils of D. aromatica leaves and seed, their relative amounts and their retention indices (Kovat's Indices for Polar Column) are shown in Table 2.
The essential oils from the seed and leaves of D. aromatica showed important similarities because out of the 85 identified compounds, 29 (1-8, 10-14, 23, 26, 29, 31, 32, 34, 36, 39, 40, 43, 49, 72-74, 81 and 83, see Table 2) were common in both leaves and seed although in different quantities. However, some specific compounds allowed for the differentiation of the two essential oils. Indeed, 55 compounds (15-21, 24, 25, 27, 28, 30, 33, 35, 37, 38, 41, 42, 44-48, 50-71, 75-80, 82, and 84-86) including an unknown were found only in the leaves and not in the seed, while 2 compounds (9 and 22) were found only in the seed and not in the leaves. This pattern of findings has been similarly obtained in many studies of plant species involving different organs (Rehder et al. 2006;Ghasempour et al. 2007;Bhuiyan et al. 2009;Chowdhury et al. 2009). Thus, common volatile compounds were found to be non-uniformly distributed in different organs of D. aromatic, whilst the different volatile compounds accumulated could be the result of various metabolic processes in the specific cells or vessels of these organs. Table 3 shows the identified volatile compounds listed by chemical class, which to some degree reflects their biosynthetic origin. Out of the 85 identified compounds, 5 were fatty acids and their derivatives, 79 isoprenoids and 1 benzenoid. The presence of fatty acids and isoprenoids, in particular terpenes and sesquiterpenes, had been recorded in the oleoresin, camphor, resin and wood extract of this species (Burkill 1966;Cheung & Wong 1972;Perry 1980;Duke & Ayensu 1985;Ali & Koh 1991). In this study, the fatty acids and their derivatives were found in the leaf oil only, and were represented by C6 compounds, mainly acids and alcohols, which made up 1.4% of the oil. In plants, fatty acids are synthesized in chloroplasts from acetyl-CoA and malonyl-CoA in repetitive reactions that result in longer molecules (Cseke et al. 2006). The alcohols which give the characteristic `green' note or odour of the leaves are biosynthesised from α-linolenic and linoleic acids via their respective hydroperoxides (Stone et al. 1975;Hatanaka et al. 1987;Hatanaka 1993).

Chemical Classification
T h e i s o p r e n o i d s i n b o t h l e a f a n d seed oils were mainly monoterpenes and sesquiterpenes, and their derivatives. The amount of monoterpenes and their derivatives was higher in the seed oil (87.5%) than in the leaf oil (53.4%). However, the sesquiterpenoid fractions were higher in the leaf oil (33%) than in the seed oil (12%). Previous work on the oleo-resin of this plant recorded the presence of 20% sesquiterpenes, an amount which lies in between the contents of leaf and seed oils in this study. In the leaf oil, oxygenated monocyclic monoterpenes (32%) and oxygenated bicyclic sesquiterpenes (20%) dominated whilst bicyclic monoterpenes (73%) and bicyclic sesquiterpenes (11%) formed the major isoprenoids in the seed. Most isoprenoids can be traced back to geranyl-or farnesyl-pyrophosphates (Croteau & Karp 1991). The isoprenoids are synthesized in cytosol from acetyl-CoA via the mevalonic pathway as well as in plastids from pyruvic acid and glyceraldehydes-3-phosphate via 1-deoxy-D-xyluluose-5-phosphate (DOXP) and 2-C-methyl-D-erythritol-4-phosphate (MEP) (Eisenreich et al. 1998;Kuzuyama 2002;Dubey et al. 2003;Eisenreich et al. 2004).
T h e i r r e g u l a r t e r p e n e , 6 -m e t h y l -5-heptene-2-one, occurred in a negligible amount. Similarly, the only benzenoid, α-pdimethylstyrene, was also present in a very small quantity.

CONCLUSION
This study provides the complete chemical profiles of the essential oils obtained from the fresh leaves and seed of D. aromatica. The essential oils of both leaves and seed of D. aromatica were particularly rich in monoterpenes. Sesquiterpenes were present in both oils in lower amounts whilst fatty acids and benzenoid were present in minute  (15) tr -