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The species Elaeis guineensis was classified by Nicolaas Jacquin in 1763. This monocotyledon is native to West Africa and ecologically suits the habitat between the Tropics of Cancer and Capricorn (Latiff, 2000). It was first introduced in Malaysia (then, Malaya) in the 1870s as ornamental plant by the British. Earliest commercial planting took place in 1917 in Rantau Panjang, Selangor and today, oil palm is one of the major commodities of this country. Increased acreage in the 1950s was due to avoidance of rubber dependence by plantation companies and the confidence of its future market.
Oil palm starts to produce fruit as early as its fourth year (Lynn, 1997). Each fruit contains a single seed (kernel) surrounded by a soft oily pulp. The fruits grow in bunches into oily bright-coloured drupes, with the colours depending on their type. The nigrescens are red and blackish when ripe; the virescens are orange while the albescens yellow blackish when ripe. The surface or the skin is the epicarp, the protecting external layer which gives the oil palm fruit its colour. Inside is mesocarp, the 'flesh'. In mature oil palm fruits, the parenchymatous cells of the mesocarp tissue contain oil droplets (Latiff, 2000). The inner-most layer is the endocarp, the hard shell containing the endosperm (kernel). Figure 1 shows the longitudinal section (A) and transverse section with labels (B) of an oil palm fruit.
Figure 1: A) Longitudinal section B) transverse section of E. guineensis fruits
Palm oil is produced from the mesocarp, and palm kernel oil is produced from the kernel. The palm takes five to six months to flower and be pollinated, to develop fruit and ripen and lastly, harvested. The changes and development of the fruits are often tracked weekly after anthesis, WAA. Fruit development starts at approximately two weeks after anthesis (2 WAA). The synthesis of oil begins around 16 WAA with maximum rate of the activity at 17 WAA (Haniff, 2000). This continues up until 20 WAA. The fruits are harvested between 20 to 22 WAA. For palm oil production, the oil from the mesocarp flesh is pressed or leached out using mechanical or chemical means leaving empty fruit bunches and non-oil residues.
The main products from the oil palm are the oils, which serve the food industry and also for non-edible purposes such as oleochemicals, cosmetics and healthcare products. The lipid properties and oil soluble components of palm oil such as the phosphatides, sterols, pigments, tocopherols, tocotrienols and their development in oil palm fruits has been documented extensively (Sundram et al., 2003). In current years, the interest of researchers and the oil palm industry players towards non-oil products from oil palm is increasing. Oil palm industrial wastes have been used for biomass, feedstuff for livestock and fertilizer (Basri et al., 2004) while recent studies found that water soluble residue known as palm oil mill effluent (POME) is rich in antioxidants (Harrison et al., 2007). Previous and recent researches on this species have always weighted in serving commercial purpose and sustainability issue. It is worth mentioning that amidst the brouhaha of genome sequencing and manipulation of genetic material of the species, data on the chemical constituents of the plant tissue is inadequate.
Compounds involved in the photosynthesis, respiration, growth and the development of the plant are dubbed as primary metabolites, which include phytosterols, organic acids, acyl lipids, amino acids and nucleotides. Secondary metabolites on the other hand, are often originated from common progenitors of a primary metabolism function and synthesized from precursors of basic metabolic pathways. Albeit termed secondary, these compounds play vital roles in the survival of their producers and participate in primary functions. Studies from Buer et al. (2007) and Santelia et al. (2008) found that flavonoids act as versatile modulators of auxin transport. The formation and storage of secondary metabolites is generally cell-, tissue- and development-specific and the profiles of the compounds vary accordingly (Wink, 1999).
In higher plants, metabolism studies revealed stable and dynamic pathways containing enzyme complexes for rapid and powerful regulating mechanism in cellular biochemistry. Examples of primary metabolism pathways are the cysteine biosynthesis and the Calvin cycle while examples of secondary metabolisms include the cyanogenic glucoside and phenylpropanoid pathways. In phenylpropanoid pathways, phenylalanine is transformed into a variety of important secondary products including lignins, sinapate esters, stilbenes, and flavonoids. These compounds are of fundamental importance to the plant cell, functioning as phytoalexins and providing defense against pathogens and herbivores, UV sunscreens, pigments, signaling molecules and regulators and major structural components (Winkel, 2004). In the good faith of gaining better insight into the oil palm fruit, studies on these classes of metabolites need to be undertaken. The profile of metabolites accumulated in the fruit tissue during its development will provide important information for understanding the mechanism that delimit the metabolite composition in the oil palm fruit and reveals the underlying developmental shifts during the fruit ripening.
From preliminary experimentation on the fruits, methanolic extracts of oil palm mesocarp tissue showed varying profile in liquid chromatography-mass spectrometry analysis (LC-MS). Figure 2 shows the LC-MS profiles of oil palm mesocarp extract at different developmental stages, namely 10, 12, 14, 15, 16, 18 and 20 WAA. Figure 2
It is suffice to summarize that the objective of this study is to establish phytochemicals profile in the developing mesocarp tissue of oil palm (E. guineensis) fruits. Expected benefits from the study include the discovery of possible metabolite markers signaling fruit ripeness in oil palm, fundamental information on metabolite changes during oil palm fruit ripening, baseline data for comparison with other oil-seed crop and other oil palm species (e.g. E. oleifera, Jessenia bataua) and genetically modified (GM)-oil palm. This important knowledge will further allow efficient uses of plant resources especially the fruits of oil palm.
Plant tissues are always dried or freezed carefully under controlled conditions avoid chemical changes, if not able to be extracted upon harvest. The method of extraction relies on the texture and water content of the plant material and on the type of substance that is studied (Harborne, 1973). After extraction, fractionation of crude extract is favourable to separate main classes of compounds before further analysis. This can be carried out using cartridges containing suitable adsorbent before being diluted off using solvents.
Plant constituents are analyzed using various methods. Chromatographic techniques such as thin layer chromatography (TLC), paper chromatography (PC), column chromatography (CC), gas chromatography (GC) and liquid chromatography (LC) are commonly used in phytochemical studies. For routine and reliable separation and determination of plant compounds, high performance liquid chromatography (HPLC) is used (Ivanauskas et al., 2008). Measured UV spectrum gives useful information on the nature of compounds in complex profiles, which often indicates the class of the compound rather than its exact identity. For this, HPLC profiling methods depend heavily on comparisons with reference compounds.
With mass spectrometer (MS), a small amount of materials is able to give an accurate molecular weight and its unique fragmentation pattern that provide information on its identity, complementing UV absorbance in HPLC. The source of MS convert separated analytes molecules into ions as they elute from the HPLC column and UV detector. The MS determines the m/z value, which is the mass divided by the charge. Fang et al. (2002) characterized hydroxycinnamic acids such as p-coumaric, caffeic and ferulic acid and phenolics such as chlorogenic acid using liquid chromatography (LC) coupled to mass spectrometer (LC/MS) with electrospray ionization (ESI). In tandem MS (MS/MS) analysis, the ions formed from the ESI source are fragmented by adding extra collision energy for the ions to bump into molecules of a bath gas (usually helium or argon). The resulting fragment ions are informative in obtaining structural information of the compound. Nuclear magnetic resonance (NMR) is a robust technique in phytochemical studies where a particular compound is analysed without having to be derivatized or ionized, thus corroborating the identity of the compound.
This work is to serve foremost objectives that are to acquire better understanding of fundamental aspects of the growth of the oil palm fruits and to study valuable phytochemicals in oil palm fruits. Without a doubt, this will benefit the science community and the industry in the long term. The profile of the phytochemicals from the developing oil palm fruits will also be highly valuable in plant physiology and plant taxonomy studies, as well as providing a baseline data for substantial equivalence study against oil palm of different species and breed, transgenic oil palm and other oil crop.
Refer Figure 3 for diagram of methodology.
Phase1: Sample preparation
Oil palm fruit tagging will be performed on E. guineensis var. Tenera palms. The flowers will be tagged at anthesis to accurately follow fruit ages throughout their development. Fruits will be harvested at 10 weeks after anthesis (WAA) followed by 12, 14, 15, 16, 18 and 20 WAA. These harvesting periods covered the transition from green and blackish to fully ripe red fleshy fruit. The fruits were washed and processed immediately upon harvesting. The fruits were peeled to remove the epicarp and sliced into thin chips with a scalpel blade. The processed tissues were immediately frozen in liquid nitrogen before being kept at -80°C until extraction.
Phase 2: Extraction and chromatography
Different extraction methods will be employed to obtain different classes of compounds from the tissues. Extraction will involve solvent extraction and solid phase extraction (SPE) protocols. High performance liquid chromatography (HPLC) will be performed on Dionex UltiMate 3000 comprised a gradient pump with integrated vacuum degasser and mixing chamber and a photodiode array detector. Separation will be performed on C18 reversed phase column or modified C18 columns.
Phase 3: Mass Spectroscopy (MS) and Nuclear Magnetic Resonance (NMR)
After going trough the HPLC detector, the flow was split to allow only 100 µl/min of eluent into the MS. Electrospray ionization (ESI)-MS analysis will be performed on a Bruker MicrOTOF-Q time-of-flight quadrupole spectrometer (Bruker Daltonik GmbH, Germany). Data cquisitions will be performed in both the positive and negative ESI modes. Data acquisition will be performed by HyStar (Hyphenation Star Application) Version 3.2. Further, HPLC elution will be fractionated and collected for multiple reactions monitoring (MRM) MS/MS and NMR analysis for structure elucidation.
Phase 4: Data analysis, interpretation and report writing
Data processing will be carried out with DataAnalysis Version 3.4 by Bruker Daltonik GmbH. Data from chromatographic separations, mass to charge (m/z) observation in MS and interpretation of MS/MS fragment spectra and NMR data will be catalogued in a metabolome database for oil palm fruit.
Figure 3: Diagram of methodology
Flower tagging and fruit harvest
Comparison between Development Stages (Week after Anthesis, WAA)
Separation (chromatography) and fractionation
Figure 3: Diagram of methodology
Activities and Milestone:
1) Collection of plant tissue: leaf of E. guineensis
2) Extraction of metabolites
3) HPLC separation
4) Mass spectrometry (LC/MS)
5) Tandem MS (MS/MS)
6) Fractionation and isolation
7) Data analysis and interpretation
8) Isolation and purification
10) Data analysis and interpretation
11) Report writing and dissemination of results