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1 Introduction Plants contain a broad range of bioactive compounds such as lipids, phytochemicals, pharmaceutics, flavors, fragrances and pigments. Plant extracts are widely used in the food, pharmaceutical and cosmetics industries. Extraction techniques have been widely investigated to obtain such valuable natural compounds from plants for commercialization. Various novel techniques including ultrasound-assisted extraction, microwave-assisted extraction, supercritical fluid extraction, and accelerated solvent extraction have been developed for the extraction of nutraceuticals from plants in order to shorten the extraction time, decrease the solvent consumption, increase the extraction yield, and enhance the quality of extracts. However traditional methods, such as soxhlet extraction, which have been used for many decades, are very time consuming and require relatively large quantities of solvents (Luque de Castro & Garcia-Ayuso, 1998). The massive wide scale use of organic solvents by a diverse range of global industries represents a serious threat to the environment. In response, the Montreal Protocol was introduced in 1987 with an initial objective to restrict or eliminate the manufacture and use of particularly damaging ozone depleting solvents such as chlorofluorocarbons (CFCs). Hannay and Hogart (1879) early observations of the dissolution of solutes in supercritical fluid (SCF) media introduced the possibility of a new solvent medium. Consequently, supercritical fluid extraction (SFE) as an environmentally responsible and efficient extraction technique for solid materials was introduced and extensively studied for separation of active compounds from herbs and other plants (Modey et al., 1996).

1.2 Research Background In an article published in 1971 by the Florida Horticultural Society reveals the benefit and nutrients derived from plants B.hispida includes the content, seed and leaves. In addition to medicinal properties at all division plants, the seeds can live for more than ten years before in the seedling. Therefore this research will be conducted to emphasize the investigation on the main bioactive compounds contain from seed by using supercritical carbon dioxide (SCCO2) extraction. Supercritical fluid extraction (SFE) has been documented as an effective method for preparing bioactive products from plant materials (Modey et al., 1996). In addition, to date there is no established research is carried out on the chemical constituents extract from B.hispida seed. Therefore, the result from the research is important to be revealed and document in order to evaluate the therapeutic properties for their uses in pharmaceutical or food industries. However, the addition of small percentage of polar organic solvent and pressure swing technique are expected to enhance the polarity and solvating power of supercritical fluid carbon dioxide to extract bioactive from the solid matrix sample.

1.3 Research Hyphothesis In this work, SC-CO2 extraction of bioactive compounds from B. hispida seed will be to investigate the effect of the operating parameters on the global extraction yield. It is understood, that extraction using pure carbon dioxide at its supercritical condition may not be successful due to lower in polarity (non polar solvent). However, the addition of small percentage of polar organic solvent and pressure swing technique are expected to enhance the polarity and solvating power of supercritical fluid carbon dioxide to extract bioactive from the solid matrix sample. 1.4 Research Problem Generally known that the SC-CO2 extraction process is feasible to obtain quality results as well as selective extraction results. As the seed is usually coated by a hard shell, there were a restriction of the extraction system can be run effectively. As the pressure swing technique approaches will be consider as improvement of SC-CO2 process where it can improve the quantity and quality of extraction, especially of hard grains due to botanical structure of the material used. The swing technique of SC-CO2 is considered an economic process due to the lack of CO2 gas consumed for the extraction process.

2.0 Literature Review 2.1 Extraction Method

Natural materials contain various extractable fractions of bioactive compounds. Among these, essential oils are particularly relevant since they represent the basic material for many foods, cosmetics, and pharmaceutical preparations. Several studies have been devoted to the extraction of essential oils and of related products by several extraction methods. Conventional production methods such as solvent extraction and soxhlet, although effective for extraction, can lead to degradation of heat sensitive compounds as well as leaving traces of toxic solvents in the solute. This is a concern for food and medicinal extracts. Supercritical fuid technology may be a viable alternative to solvent extraction methods. The phenomenon of supercritical fuid technology extraction was observed over 100 years ago but has been slow to find commercial applications, due in part to the sophisticated and expensive high pressure equipment and technology required (Jarvis and Morgan, 1997). Since the end of the 1970s, supercritical fluids have been used to isolate natural products, but for a long time applications relayed only on few products. Now the development of processes and equipment is beginning to pay off and industries are getting more and more interested in supercritical techniques (Brunner, 2005). This interest is also reflected in the high amount of scientific papers dealing with supercritical fluid extraction (SFE) published in recent years. Moreover,

industrial applications of SFE have experienced a strong development since the early 1990s in terms of patents (Schultz, 2007). 2.2 Properties of Supercritical Fluids A phase diagram, as shown in Figure 2, describes the physical stage of a substance of fixed composition. In this pressure-temperature diagram, there are three lines describing the sublimation, melting and boiling processes. These lines also define the regions corresponding to the gas, liquid and solid states. Points along the lines (between the phases) define the equilibrium between two of the phases. The vapor pressure (boiling) starts at the triple point and ends at the critical point (CP). The critical region has its origin at its critical point, so at this point, a fluid heated to above the critical temperature (Tc) and compressed to above the critical pressure (Pc) is known as a supercritical fluid. Frequently the term of compressed liquid, is used to indicate a supercritical fluid, a near-critical fluid, an expanded liquid or a highly compressed gas. The critical temperature is therefore the highest temperature at which a gas can be converted to a liquid by an increase in pressure. Carbon dioxide is generally the most desirable solvent for supercritical fuid extraction (SFE). The critical temperature of carbon dioxide is only 30.9C and 73.8 bar, which makes it attractive for the extraction of heat sensitive compounds its high diffusivity combined with its easily tuneable solvent strength.. In addition it is an inert, non-flammable,non-explosive, inexpensive, odourless, colourless, clean solvent that leaves no solvent residue in the product, it is also non-toxic and is generally recognized as safe by FDA and EFSA accepted as a harmless ingredient in pharmaceuticals and food (Mendiola et al., 2007). 2.3 Optimization of Supercrital Fluid Extraction

The design of processes using supercritical solvents is strongly dependent on the phase equilibrium scenario, which is highly sensitive to changes in operating conditions. Therefore, phase equilibrium engineering plays a key role in the synthesis and design of these processes. For a successful application of SFE technology, several critical variables must be taken into consideration in the SFE process. One of the main aspects that should be considered in SFE is the extraction optimization. The use of the optimum values for the different variables influencing the SFE extractions could significantly enhance the recovery or extraction yield of a target compound. With the aim to effectively optimize these variables (mainly extraction temperature, pressure, time, type and percentage of modifiers, sample size, etc.), different approaches have been applied. Those strategies can be grouped in phase equilibrium strategies and experimental design statistical optimization. Phase equilibrium and mass transfer are not modelling problems; they are limit stages of the process that can justify the results. Therefore, it is very important to organize the experiments based in these

fundamentals, to contribute to the understanding of the extraction problem. Despite of the valuable information obtained by phase equilibrium engineering, it is a common practice to optimize the processes using experimental designs and statistical modelling. The use of experimental designs is one of the most common strategies when it comes to set up robust extraction methods (Bianchi and Careri, 2008). Response surface methodology (RSM) is among the popular possibility used in optimization study; RSM was recently been applied to optimize the process parameters for supercritical carbon dioxide extraction of passion fruit oil (Liu et al., 2009). The use of RSM allowed the simultaneous graphical optimization of the extraction temperature, pressure and extraction time. Besides, RSM using a full factorial composite design involving 3 factors (temperature, pressure and extraction time) and 3 levels, was employed to fit a second order polynomial model for the maximization of lycopene SFE extraction from tomato pomace (Huang et al., 2008; Gelmez et al., 2009) and bioactive flavonoid from Strobilanthes crispus leaves (Liza et al., 2010).

2.4 Pressure Swing Technique

Use of SC-CO2 with the pressure swing, which consists of pressurization and depressurization steps has been proposed for separating cashew nut shell liquid (CNSL) from cashew nut shells (Smith et al., 2003). CNSL is difficult to remove from the shells, due to the hard outer shell of the cashew nut, the intricate honeycombed features of the pericarp and the thermally sensitive nature of the CNSL (Setianto et al., 2001; Smith et al., 2003). Shobha and Ravindranath (1991) reported that 0.7 g of CNSL was obtained for every kilogram of CO2 in their study on continuous extraction of CNSL from cashew nut shell with supercritical CO2 at 25 MPa and 313.2 K. Setianto et al. (2001) examined separation of CNSL with pressure swing steps including temperature effects. From the point of view of these studies, Setianto et al. (2001) and Smith et al. (2003) concluded that applying pressure swing steps greatly increased the possible yields that could be obtained over those from continuous constant pressure extractions and further, reduced the amount of CO2 required in the separation. However, the use of pressure swing extraction with SC-CO2 has not yet been apply to separation of any bioactive compound extraction from plant seed.

2.5 Application of Supercritical Extraction Carbon Dioxide

Based on the interest and research that have been done in the world, a sharp increases of SCCO2 extraction method clearly reflects that this industry promises a discovery which is useful especially in the areas of food and nutraceutical technologies. Thus the findings of this

study coupled with pressure swing technique were will be viewed as a new approach for comparison with the currently SC-CO2 continous extraction process. The founding information that obtained from this study may provide new approaches to disclosure the bioactive compound obtained from B.hispida seed extraction and also will be a reference to the SC-CO2 industry involving extraction of bioactive compounds separation from plant seed.

Objective 1. To investigate the effect SC-CO2 extraction parameter of Benincasa hispida seed 2. To optimize the extraction condition of Benincasa hispida seed 3. To characterize the bioactive compounds from essential oil extraction of Benincasa hispida seed 4. To compare extraction between the SC-CO2 and SC-CO2-pressure swing technique.