Phenolic compounds are able to act as reducing agents, hydrogendonors and scavengers of reactive oxygen species as it classified as a commonsecondary metabolites characterized by the presence of an aromatic ring bearingfree hydroxyl groups or engaged with a carbohydrate (Mason et al.
, 1996; Pandey & Rizvi, 2009; Ivanova et al., 2005). Plants polyphenols areplay important role in defense against different types of stress by offeringprotection against reactive oxygen and nitrogen species, UV light, pathogens,parasites and plant predators (Balasundram etal., 2006). Moreover it also playing important role in prevention of chronic diseases, such as cardiovasculardiseases, neurodegenerative disorders, cancer, type II diabetes andosteoporosis (Alothman et al., 2009;Scalbert et al.
, 2005). There are many method to extract the polyphenols from theplant such as Accelerated Solvent Extraction (ASE), Supercritical FluidExtraction (SFE), Soxhlet Extraction (SE), Ultrasound Assisted Extraction (UAE)and others. However, the extraction processes are a major variability factor inthe antioxidant properties of extracts. Indeed, different antioxidantcapacities depending on the factors and affinities between factors used in theextraction such as solvent types, extraction times, extraction temperature,particle size of samples and the method adopted for the influence forextraction influence significantly the composition of the extract (Ksouri et al., 2009; Hayouni et al.
, 2007) . Extracting polyphenolsfrom biological materials by conventional method such as SE is time consuming.By using new method or modern techniques such as Microwaves Assisted Extraction(MAE) or UAE is intended to overcome this time consuming problem and additionalcan increase extraction efficiency, selectivity and kinetics. UAE using ultrasoundor sonication to break the cell membranes and reducing the extraction time andincreasing the extract yield. This application happened by disrupting the cellwall structure and accelerates the diffusion through membranes thus itfacilitates the release of cell contents (Mason et al., 1996). Optimization technique such as Response surface methodology(RSM) was effective technique to analyzing interaction factors for the complexprocess until the process is optimize. The bioactive compound from MCS can beaffected by many factors such as extraction time, solvent, etc.
By using RSMthe number of experimental number needed to analyze many factors interactioncan be reduce. Since the RSM is an effective technique, it is widely use tooptimize extraction parameters such as tannin content, phenolic compound,protein, etc. In this study, the mainobjective was to optimize the extraction condition for Malaysia Cocoa FreshBean in order to achieve the highest polyphenols levels on the choice of themost suitable extraction solvent ratio, extraction time irradiation andtemperature of extraction. Since optimization of TPC ultrasound assistedextraction technique using RSM design not been reported yet, hence an attemptwas made for the first time to investigate and optimize the UAE technique forthe MCS using 3 variables which are ethanol concentration (70–90 v/v %),temperature (45–65 °C), and ultrasound irradiation time (30–60 min). 2.
Materials andMethods2.1 Materials andchemicalsMCS were collected from Pusat Penyelidikan Dan PembangunanKoko (PPPK), Jengka, Pahang,Malaysia. Folin-Ciocalteu phenol reagent, sodiumcarbonate, gallic acid, and ethanol was analytical grade.2.2 Preparation ofaqueous extract of Malaysia Cocoa Fresh Bean (MCFB).
The MCS was dry using Labconco Freeze Dryer and crushed in amechanical blender. For the Extraction 1g of MCS was mixed with 50mL of variousethanol solution. Then the mixture were extract using sonication bath machine(Wiseclean 40khz) for certain duration and temperature base on the RespondSurface Methodology Design. Then the aqueous extract was filtered, removed thesolvent using rotary evaporator and dried using freeze dry to get the crudeextract. The crude extract were store at -40oC in storage vials forfurther analysis.
2.3 RSM design andstatistical analysisA two level factorial and three independence variable (23)was used to determine the optimal condition for UAE by using central compositedesign (CCD). Ethanol concentration (%, X1), UAE temperature (°C, X2),and UAE radiation time (min, X3) were taken as independent variablestested in a 20-run experiment. Five levels included alpha value were coded forthe independent variables, design using CCD and randomize the experimental runas listed in the table 1. As the responses of the design experiments (Y) thetotal phenolic content was taken as the dependent variables.
The data wereanalyze by multiple regression through the second polynomial equation toexpress the responds as a independent variables as following.Data were analyzed by analysis of variance (ANOVA) todetermine the lack of fit and the effects of linear, quadratic, and interactionvariables on total phenolic content. Data analysis and RSM were performed withMinitab software (Version 14)