CHAPTER 1

INTRODUCTION

1.1 Research Background

In recent years, there has been an increased interest to identify plant compound especially bioactive compounds that have positive impact to human health. Bioactive compounds are secondary metabolite produced by plants which associated with plant growth and development besides the primary biosynthetic. A lot of studies have been conducted to identify these bioactive compounds which include studies on antioxidant activity and anti-carcinogenicity, their ability to lower cholesterol level and obesity and many more.

Interestingly, the success of the characterization of a plant compound may lead to the development of new foods or supplements with activities that can promote health. During the last decade, these foods are called functional foods, which are generally accepted as those that contain naturally beneficial health qualities (Boeu et al., 2009). 30

Most species of plants for example vegetables and fruits are capable of producing bioactive compounds such as phenolic compounds, flavonoids, tannins, steroid and many more. Bioactive compounds in plants are very useful as the source of modern pharmacology, medical treatment and as natural beneficial compounds in vegetable feed, food and supplements. The benefits of bioactive compounds are also related with their antioxidant activities (Ryan et al., 2002). Antioxidant can be classified as synthetic and natural and plant is one of the sources of natural antioxidant.

Fruits can be found abundantly in many species with variety of genera. Fruits are also rich in bioactive compounds, vitamins, minerals, fibre and high antioxidant activities. Due to the facts that some fruits are seasonal, may not last longer and yet, have high nutritional value, fruits are being processed to make dried products, juices, cake, jams and many more. In food processing industry, edible portion of the fruits are processed into products and the fruits wastes such as the peels, seeds and stones are discarded since they are not being utilised for any commercial purposes. Many by-products from food processing including fruit waste contain high polyphenol with potential application as food antioxidants to prevent against some diseases (Servili, 2002).

The use of fruits wastes such as peels and seeds for commercial purposes especially as antioxidants still remains low due to their lack in popularity and lack of research. Several researches are performed to study the antioxidant levels and activities in fruit wastes and many authors had come out with findings that some fruit waste contains higher content of phenolic compound and antioxidant level than the whole fruit itself (Baydar, 2006). This interesting fact should be used as a stepping stone to perform more researches on the potential of fruit wastes as functional foods and source of antioxidants.

1.2 Problem Statement

The uses of industrial, domestic and agriculture wastes as process feedstock, secondary energy source and many more has recently generated tremendous interest. Food industries especially manufacturing of fruits based products produce huge amount of solid fruits waste such as seeds, peel and stones after the edible part of the fruits has been processed. If these wastes are not properly dealt with, it may lead to problems such as flies and rats at the processing room and create a displeasing smell if being stored for a longer time. Apart from that, it could also possibly lead to environmental pollution if these wastes are being discarded at landfill.

Fruits are being acknowledged for their high content of vitamins, fibres, minerals as well as its high antioxidants levels. Based on these facts, the edible parts of the fruits are being processed for human consumption while the seeds, peel and the stones are being discarded without being acknowledged that they might have high content of bioactive compounds especially phenolic compounds which is useful for human health. These compounds can be used as the food supplements, food ingredients and many more. Apart being used as source of healthy compounds for the development of dietary supplements, the possibility of environmental pollution also can be reduced when these wastes are being fully utilized.

However, the current scenarios shows most of the solid waste from fruits which has high phenolic contents with high antioxidant activities often been discarded during food products processing. Thus, this research attempts to explore the potential of solid fruit waste from A.muricata especially in terms of the phenolic content. A.muricata is famous for its high content of bioactive compounds and some researchers claim that A.muricata can cure cancer. In food industry, the pulp of A.muricata is usually being processed for its juice while the seeds and peels of A.muricata will normally being discarded. This research will compare the phenolic content in pulp, seeds and peel of A.muricata.

1.3 Objectives of Research

The objectives of this research are to: To extract the phenolic compounds from pulp, peel and seeds of A.muricata To compare the concentration and total phenolic acid content found in pulp, peel and seeds of A.muricata To quantify the antioxidant activity from the extracted phenolic compounds

1.4 Scope of Research

In this research, A.muricata was used as the raw material where bioactive compound which is phenolic compound is to be extracted from pulp, seeds and peel of A.muricata. In order to explore the potential of A.muricata solid waste, the scopes of research is divided into three parts which are the preparation of the samples (pulp, seeds and peel of A.muricata), the extraction of phenolic compounds from A.muricata samples and the quantification of total phenolic contents and its antioxidant activities.

The extraction method used is very crucial to ensure a maximum recovery of phenolic compounds from the A.muricata samples for further analysis. Before the extraction process, the preparation of the samples also plays an important role in increasing the efficiency of the extraction process. In this research, the fruits samples are dried by using freeze dryer to turn them into powder form. Freeze drying is a good method for drying process as it can preserve the antioxidant content inside the fruit waste and to ensure that the antioxidants are not ruined, deformed or destroyed during the preparation of the extract. The fine textures of samples powder will improves the kinetics of analytic extraction and also increase the contact of sample surface with the solvent system.

In the extraction process, various method of extraction is available such as solvent extraction, ultrasound-assisted extraction (UAE), supercritical fluid extraction (SFE), microwave-assisted extraction (MAE) and pressurized liquid extraction (PLE). Among all, solvent extraction method is widely used for the extraction of phenolic compounds from plant based material because it is simple and inexpensive. Usually methanol is selected as the solvent and the selection of solvent largely depends on the nature of the desired compound being targeted (Guyot et al., 1998). Extraction conditions such as the extraction time, pH of the solvent, solvent to water ratio and temperature are carefully studied to ensure the maximum recovery of the desired compounds. All these parameter are further discussed in literature review section.

Next, concentration and total phenolic acid content analysis need to be performed on the extracted sample to quantify the total phenolic content. The total phenolic content is usually determine using Follin-Ciocalteu reagent in comparison with standard Gallic acid and the results are expressed in terms of g GAE/g dry sample. Phenolic compounds are well known for its antioxidant properties and the activity of antioxidant of the extracted phenolic compound can be determined by using DPPH assay. DPPH assay is free-radical scavenging assay which can be used to measure how much free radical can be scavenged by the extracted phenolic compound.

In a conclusion, in order to fulfil the objectives of this research project, three important steps which are pre-treatment of samples, extraction process and quantification of total phenolic content and its antioxidant activities need to be carried out. Parameters and conditions for every processes mentioned above need to be carefully studied to ensure a maximum recovery of phenolic compounds for an optimum result for this research project.

CHAPTER 2

LITERATURE REVIEW

2.1 Overview

Malaysia is one of the countries with the largest biodiversity which includes a large number of fruits species. Majority of fruit species contain high phenolic contents and most of researches performed were focusing on the phenolic contents from the edible part of the fruits. In this research, A.muricata is used as the raw material where the phenolic compounds are to be extracted from the pulp, seeds and the peel of A.muricata using solvent extraction. Hence, this chapter will discuss on the backgrounds of A.muricata, the bioactive compounds in the fruits, the extraction parameters involved and the quantification of phenolic contents and its antioxidant activities.

2.2 A.muricata

Annona muricataLinn or soursop is a small tree from theAnnonaceaefamily. This local plant which originated from South America is now widely being cultivated throughout tropical regions of the world. A.muricata is more commonly known as Durian Belanda in Malaysia. It is one of Malaysia tropical fruits with heart-shaped to oval, ranging in size from a few inches to over 1 foot in length. The fully mature A.muricata is green or light greenish yellow. The ripe, mature A.muricata is soft to the touch and people normally detect the ripeness by the softness of the fruits rather by its colour. The outside of the fruit is thorny while the pulp is white and juicy with brownish seeds.

A.muricata with several genera are characterized by the presence of alkaloids, acetogenins, and cyclopeptide metabolites. In recent years, these compounds have attracted interest because different scientific evaluations have shown them to possess significant pharmacological activities for example insecticidal, parasiticidal, antiviral, antifungal, antitumoral and cytotoxic. Most of the previous phytochemical studies on this species include a study done by Gleye on the roots of A.muricata. Through this study, it has shown that A.muricata contains more than 50 compounds of class acetogenins (Gleye, 1999). Acetogenins are chemicals that have shown various interesting biological properties including cytotoxicity. In vitro studies have shown that acetogenins give therapeutic effects toward neoplasic cells in comparison with normal cells which suggesting their potential use as antitumoral agents.

The seed of A.muricata has also been studied on its presence of a phytochemical called Annomuricatin C, a new cyclohexapeptide where Alassane through a study has confirmed the presence of this compound. This compound has been isolated from the methanol extract of the seeds of A.muricata. Annomuricatin C was found to be cytotoxic with potential therapeutic applications where it can inhibit the growth of tumoural KB cells, with an inhibiting concentration, IC50 of 1.0 M (Alassane et al., 2005). Other important components of theA.muricata seeds have also been studied where the composition, physical and chemical properties of the oil extracted from the seeds make it found potentially attractive in the food sector.

Another interesting study on the extract of A.muricatawas its effect on Herpes simplex virus. In this study, researchers have looked at the ability of A.muricata extract to inhibit the cytopathic effect of HSV-1 on vero cells as indicative of anti-HSV-1 potential. It was shown that, the extract was able to inhibit the growth of the virus cell with the minimum inhibitory concentration of 1 mg/ml (Padma et al., 1998). A lot of research done have revealed the bioactive compounds contained in A.muricata which have very interesting therapeutic benefits and this has sparked interest of other researcher to dig more about the benefits of A.muricata. These previous studies have initiate an idea to this research to extract the phenolic compounds from various part of A.muricata (seed, pulp and peel) where phenolic compounds are also found to be very beneficial to human health.

Figure 2.1: A.muricata fruit

2.3 Phenolic Compounds

Plant contains a lot of phenolic compounds that can be found everywhere in parts of plants which consists of an aromatic ring, having one or more hydroxyl substituent and range from simple phenolic molecules to highly polymerised compounds. Phenolic compounds are basically categorised into several classes where phenolic acids, flavonoids and tannins are regarded as the main dietary phenolic compounds (King & Young, 1999; Harborne, 1989; Harborne et al., 1999; Bravo, 1998).

Table 2.1: Examples of phenolic compounds classesClassStructure

Acethophenones, phenylacetic acids

Biflavonoids ()2

Condensed tannins (proanthocyanidins or flavolans ()n

Flavonoids, isoflavonoids

Hydroxycinnamic acids, phenylpropanoids(coumarins, isocoumarins, chromones, chromenes)

Hydroxybenzoic acids

Napthoquinones

Lignins, neolignans()2

Lignins ()n

Simple phenolics, benzoquinones

Stilbenes, anthraquinones

Xanthones

2.3.1 Phenolic Acids

Phenolic acids comprises of two subgroups which are the hydroxybenzoic and hydroxycinnamic acids. Examples of hydroxybenzoic acids are Gallic, p-hydroxybenzoic, protocatechuic, vanillic and syringic acids where all of them have the C6C1 structure. On the other hand, hydroxycinnamic acids are aromatic compounds which have a three-carbon side chain (C6C3). Examples of hydroxycinnamic acids includes caffeic, ferulic, p-coumaric and sinapic acids (Bravo, 1998).

Figure 2.2: Generic structure of hydroxybenzoic acid

Figure 2.3: Generic structure of hydroxycinnamic acid

2.3.2 Flavonoids

Flavonoids is the largest group of plant phenolics in which over half of the eight thousand of them are naturally occurring phenolic compounds (Harborne et al., 1999). Flavonoids are low molecular weight compounds and have fifteen carbon atoms where these carbons are arranged in a C6C3C6 configuration. The structure of flavonoids consists of two aromatic rings A and B, joined by a 3-carbon bridge, usually in the form of a heterocyclic ring, C. The aromatic ring A is derived from the acetate/ malonate pathway while ring B is derived from phenylalanine through the shikimate pathway (Merken & Beecher, 2000). The type of major flavonoid classes will be determined by the substitution pattern to ring C which later can be either flavonols, flavones, flavanones, flavanols, isoflavones, flavanonols and anthocyanidins (Hollman & Katan, 1999).

Figure 2.4: Generic structure of flavonoids

2.3.3 Tannins

Tannins are the third important group of phenolic acid and are relatively high molecular weight compounds. Tannins may be subdivided into hydrolysable and condensed tannins. Hydrolysable tannins are esters of Gallic acid while condensed tannins are polymers of polyhydroxyflavan-3-ol monomers (Bravo, 1998; Porter, 1989).

2.3.4 Benefits of phenolic compounds

Phenolic compounds play some important functions in plants which help the plant to adapt to changing environment and give plant its colour, taste, technological properties and health promoting benefits. Besides that, phenolic compounds are also plant secondary metabolites, which play important roles in resisting disease (Servili and Montedoro, 2002), protection against pests and species continuation. The activity of this secondary metabolic is reported due to the presence of polyphenols which exhibit antibacterial, anti-inflammatory and vasodilatory actions that might be contributed by the antioxidant activity (Bocco et al., 1998).

Phenolic compounds are also well known for their antioxidant activity. The antioxidant activity of phenolic compounds is mainly due to their ability to scavenge free radicals, donate hydrogen atoms or electron or chelate metal cations (Afanas, 1989). Currently the interest in these compounds in foods has increased greatly due to this antioxidant activity and its potential beneficial roles in human health in reducing the risk of cancer, cardiovascular and other diseases. Phenolic compounds that can be commonly found in fruits and vegetables include hydroxybenzoic acids, hydroxycinnamic acids, hydrolysable tannins, flavonols, proanthocyanidins, and anthocyanins (Vasco et al., 2009). Fruits and vegetables that are rich in antioxidant phenolic compounds include most of the berry type fruit, fruit trees and onions.

2.4 Extraction methods

Extraction method is the important step in quantification of phenolic compound from plant based material. The extraction process should be, of course aim to provide for the maximum yield of substances and of the highest quality in terms of the concentration of target compound and antioxidant power of the extracts. Extraction is being influenced by the chemical nature of the compound to be extracted, extraction method used, the storage time and conditions and also the presence of interfering substances (Robbins & Agric, 2003).

Solvent extraction is the most widely used extraction method where this method is an inexpensive since it involves the usage of organic solvents. Other extraction methods that are suitable to be used include ultrasound-assisted extraction (UAE), supercritical fluid extraction (SFE), microwave-assisted extraction (MAE) and pressurized liquid extraction (PLE).

2.4.1 Solvent Extraction

Generally, solvent extraction method involves the usage of solvent to separate the soluble phenolic compound from a solid matrix which is the plant tissue. Many factors are identified to affect the efficiency of solvent extraction process and these variables have been investigated up to now which include type of solvent used, method used in pre-treatment of the sample, solvent/sample ratio, time of extraction, pH and temperature of extraction. A study by Rosana et al. (2006) on the optimization of extraction conditions of antioxidant phenolic compounds from mashua tubers are done where five factors that affect the efficiency of extraction process are studied. The factors studied including type of extraction solvent, pH, solvent/water ratio, extraction time and different acidified solvents used. The best condition for extraction process is determined by the total phenolic content that able to be extracted. In this study, several types of solvent that can be used in the extraction process were tried out which include water, ethanol, methanol, acetone and hexane. In this study, it is being proved that methanol is able to extract the highest phenolic content followed by water, acetone, ethanol and lastly hexane. Methanol is a solvent with very high polarity give the best extraction result while hexane exhibit low extracting ability due to its low solvent strength.

The extraction process is also best performed at pH range of 1.45 to 2.11 where HCl is added into the solvent to reduce the pH value. In low pH, the extracting ability of the solvent will apparently increase as the acid has increased the stability of the phenolic compound such as anthocyanins. Secondly, the acid addition to the extraction solvent appears to favour the dissolution of phenolic compounds such as hydroxycinnamic acid through hydrolysis mechanism. The presence of acid is also believed to improve the disintegration of cell wall where it will facilitate the solubilisation and diffusion of phenolic compounds from plant material.

The recovery of phenolic compound as a function of the solvent-water ratio is also evaluated. Solvent-water ratio is one of the factors that affect the efficiency of extracting ability. Solvent-water ratio will determine the degree of polarity of the solvent used. It is being observed that when the water proportion is more than 50%, the yield of extraction of phenolic compound will not be higher. On the other hand, methanol proportion higher than 70% is needed to inactivate the polyphenol oxidase widely distributed in plant to achieve a maximum recovery of phenolic compounds.

2.5 Quantification of phenolic compounds

After the extraction process, the extracted phenolic compounds need to be analyzed for the concentration and total phenolic acid content. Both can be determined through Follin-Ciocalteu reagent method in comparison with standard Gallic acid where a calibration curve of standard Gallic acid concentration (g/mL) against the absorbance (nm) is first constructed. The concentration of phenolic acid of A.muricata samples are expressed in term of Gallic acid equivalent g GAE/ml. On the other hand, total phenol contents in the A.muricata samples can be calculated from the following expression: C = c where C is the total phenolic content of A.muricata extracts (g GAE/g dry sample)c is the concentration of Gallic acid established from calibration curve (g GAE/mL)V is the volume of the extract (mL)m is the weight of the A.muricata extract (g)

2.6 Antioxidant activity

Phenolic compound can be characterized by its antioxidant activities and several methods are used to quantify the antioxidant capacity of phenolic compounds which include the usage of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay, Ferrous Ion Chelating (FIC) assay and -Carotene bleaching method. DPPH and FIC method are the most commonly used method in quantifying antioxidant activities of a plant extract. In this research DPPH assay method is used as it is suitable method for phenolic compound and it has high reliability in determining the antioxidant activity.

DPPH assay is free-radical scavenging assay in which this method is based on the quantification of free-radical scavenging by the sample extract. The samples extract are mixed with DPPH reagent and being incubated at room temperature in the dark. After 30 minutes of incubation, the absorbance of the mixture is measured at 517 nm using spectrophotometer. The free-radical scavenging activities in terms of % inhibition can be calculated by the following equation:% inhibition = [(A0 Ae) / A0] x 100%where A0 is the absorbance value of the blank while Ae is the absorbance of the extract.

Theoretically, as antioxidant interacts with DPPH, it will donate an electron or a hydrogen atom to this radical, thus neutralising its free radical character. The transfer of electron cause the bleaching of a purple coloured of DPPH solution to yellow and the absorbance value will gradually decreased. The antioxidant capacity is also expressed as the concentration of sample extract required to scavenge 50% of free radical, IC50. IC50 values can be calculated through % inhibition of different concentration of sample extract to identify at which concentration of sample extract to reach 50% inhibition of free radical (Norshazila et al., 2010).

2.7 Summary

The previous researches have shown that different part of A.muricata contain valuable bioactive compounds that have potential to be commercialize as food supplements. Therefore this research aims to quantify the phenolic compounds that can be extracted from parts of A.muricata that usually being discarded which are the seeds and peels. Their concentration and total phenolic contents will be compared with the phenolic compounds from the edible part which is the pulp. Solvent extraction with methanol as the solvent was chosen and the extraction process will be carried out with studied parameters to ensure a maximum recovery of phenolic compounds. This research is basically a preliminary stage where conventional method is preferred prior to the investigation by using modern alternative methods.

CHAPTER 3

RESEARCH METHODOLOGY

3.1 Overview

The main objectives of this research project were to quantify and compare the total phenolic content and antioxidant activity of phenolic acid extracted from the peel, seeds and pulp of A.muricata. In order to achieve these objectives, several steps were performed which include preparation of raw material, the extraction process and the quantification of extracted phenolic compounds and its antioxidant activities. The preparation of the raw material is basically to make the peel, seeds and pulp of A.muricata in a powder form that will ease the extraction process.

Next, the most crucial step in this research was during the extraction process where the aim of the extraction process was of course to extract a maximum yield and highest quality of phenolic compounds from the A.muricata. Next, the last step was the analysis of extracted samples where the extract phenolic compounds were quantified by using Follin-Ciocalteu reagent and the antioxidant activity of extracted phenolic compounds was being analyzed by using DPPH assay. The flow of overall processes is shown in Figure 3.1 below.

Figure 3.1: Flow chart of overall research methodology

3.2 Materials and Chemicals

Ripe A.muricata originates from Kelantan; eastern part of Malaysia was being used in this experiment. The A.muricata used was from the species of Annona muricata Linn. The peel, seeds and pulp of A.muricata were being used in this research and being pre-treated to form sample powder prior to extraction process. The methanol used for the extraction process is being acidified with HCI acid until the pH change to 2. Methanol and HCI acid were purchased from Fisher Scientific (M) Sdn Bhd. Other chemical used which are Follin-Ciocalteu reagent, Gallic acid () and Sodium Carbonate () were purchased from Merck Schuchardt OHG, Germany. DPPH powder was purchased from Sigma-Aldrich (M) Sdn Bhd. All these chemical were of standard analytical grade.

Figure 3.2: Annona muricata Linn species

3.3 Sample preparation of A.muricata

The preparation of A.muricata samples is to convert the original form of the peel, seeds and pulp of A.muricata into powder form that will ease the extraction process. A medium size of ripe A.muricata was cut into two and then being separated into peel, seeds and pulp. Peel, seeds and the pulp of A.muricata were frozen for a day in the refrigerator prior to freeze drying. The freeze drying processed took three days to complete. The dried samples were grinded to obtain the powder. The powder obtained was stored in a freezer at -20C prior to extraction process.

3.4 Extraction of phenolic compounds

As for the extraction process, solvent extraction method was performed by using Soxhlet extractor where methanol was used as the extraction solvent. The samples powder was first weighted. In the extraction process, the sample powder was placed inside the extractor thimble about three-third full. The round bottom flask was filled with 200 ml of pH 2 methanol-water solvent (v/v 90:10). The thimble was placed inside the extractor fitting and then being connected with the top of the round bottom flask. After that, the condenser was connected on the top of the extractor fitting. The heater was turned on to heat up the methanol for extraction period of 8 hours.

After the extraction process completed, the round bottom flask containing the solution was then transferred into the evaporator for further oils separation. The oil separation process is carried out until no more methanol was vaporised out from the round bottom flask. The oil was kept inside the bottle sample at -20C prior to further analysis. The above extraction procedure was according to the method described by Rosana et al. (2006).

3.5 Phenolic compounds analysis

The analysis on phenolic compounds from extracted A.muricata samples were carried out by using Follin method. Before the analyst, several steps were carried out which include preparation of stock solution and construction of standard curve.

3.5.1 Preparation of stock solution and standard curve

There are two types of stock solutions that need to be prepared which are Gallic acid solution and sodium carbonate solution. Both stock solutions are needed for the standard curve of total phenolic contents using Follin method. For preparation of Gallic acid solution, 0.008g of Gallic acid powder is dissolved in 100 ml of methanol to produce 80 of Gallic acid solution. Next, serial dilution is performed to produce 40, 20, 10 and 5 of Gallic acid solution. On the other hand, the 20% sodium carbonate solution is prepared by mixing 20g of sodium carbonate powder with 100mL distilled water. The mixture is stirred to make sure all the sodium carbonate is dissolved.

These five different concentrations of Gallic acid were used for the standard curve of Follin analysis method. 1mL of Gallic acid solution was mixed with 0.5 mL of the Follin-Ciocalteu reagent, 3mL of 20% sodium carbonate and 10mL of distilled water. This mixture is left in room at ambient temperature for 2 hours of reaction. After 2 hours, the absorbance was measured at 765 nm by using methanol which replacing Gallic acid as the blank. A curve of absorbance against Gallic acid concentration was then constructed.

3.5.2 Concentration and total phenolic acid content analysis

The oil extracted will be analyzed for its concentration and total phenolic acid content by using Follin-Ciocalteu reagent method. This method was according to Taghreed et al. (2010). 1mL of sample extract is mixed with 0.5 mL of the Follin-Ciocalteu reagent, 3mL of 20% sodium carbonate and 10mL of distilled water. This mixture is left in room at ambient temperature for 2 hours of reaction. After 2 hours, the absorbance was measured at 765 nm by using methanol which replacing the sample extract as the blank. The concentration of phenolic acid is determined in comparison with the standard curve constructed. On the other hand, the total phenolic content in the A.muricata extract was calculated according to the equation below:C = c x whereC is the total phenolic content of sample extract (g GAE/g)c is the concentration of gallic acid established from standard curve ( g GAE/ml)V is the volume of the sample extract (ml)m is the dry weight of sample (g)

3.6 Antioxidant activity analysis

The antioxidant activity of the extracts, on the basis of the scavenging activity of the stable DPPH free radical was determined by the method described by Braca et al. (2002) with some modifications. 1ml of 0.002% DPPH in methanol was added into 1ml of phenolic extract. The mixture was kept in dark at room temperature for 30 minutes. The absorbance of the mixture was measured at 517nm by using methanol as the blank. The mixture of 1 ml of methanol and 1ml of 0.002% DPPH were used as the control. The % of inhibition activity can be calculated by the following equation:% inhibition = [(A0 Ae) / A0] x 100%where A0 is the absorbance value of the control while Ae is the absorbance of the extract.

Theoretically, as antioxidant interacts with DPPH, it will donate an electron or a hydrogen atom to this radical, thus neutralising its free radical character. The transfer of electron cause the bleaching of a purple coloured of DPPH solution to yellow and the absorbance value will gradually decreased. 5 different concentrations of sample extracts were being test for the % inhibition. The antioxidants activity of the phenolic content was being compared by using the value of IC50 which is the concentration of sample extract needed to inhibit 50% of the free radical of DPPH solution.

Figure 3.3: A.muricata fruit was cut into two.Figure 3.4: A.muricata fruit was separated into pulp, seeds and peel.

Figure 3.5: The drying process of A.muricata samples using freeze dryerFigure 3.6: The grinding process of dried samples.

Figure 3.7: The Soxhlet apparatus for extraction process using acidified methanol as solvent.Figure 3.8: The oil separation process using rotary evaporator.

CHAPTER 4

RESULTS AND DISCUSSIONS

4.1 Introduction

This research aimed to quantify and compare the phenolic compounds and its antioxidant activities extracted from the peel, seeds and pulp of A.muricata. Throughout this research, steps which include the preparation of samples, solvent extraction process by using Soxhlet extractor and quantification of phenolic contents and its antioxidants activities were performed. The extraction process was performed by using Soxhlet extractor with acidified methanol as the solvent for 8 hours. The concentration and total phenolic acid content were determined by using Follin-Ciocalteu reagent method. The antioxidant activities of the extracted phenolic compounds were quantified using DPPH assay method. The findings of this research will be discussed in this chapter and then comparison was made with the previous studies done by other researchers.

4.2 Concentration and total phenolic acid content

After the extraction process, the analysis on the concentration and total phenolic acid content were performed. In order to determine the concentration of phenolic acid in peel, pulp and seeds of A.muricata, a standard curve of Gallic acid was first constructed. Table B.1 and Figure B.1 in Appendix B shows the raw data and the standard curve for Gallic acid respectively. Phenolic compounds are plant secondary metabolites, which play important roles in resisting disease (Servili and Montedoro, 2002), protection against pests and species continuation. The activity of this secondary metabolic is reported due to the presence of polyphenols which exhibit antibacterial, anti-inflammatory and vasodilatory actions that might be contributed by the antioxidant activity (Bocco et al., 1998).

The concentration of phenolic acid in every samples was determined by using Follin-Ciocalteu reagent in terms of Gallic acid equivalent (GAE) with standard curve equation of y = 0.014x + 0.0152. There are three samples which each extracted from the peel, seeds and pulp of A.muricata. All samples were being diluted with methanol with the dilution factor of 10 before being analysed for its phenolic acid concentration and total phenolic content. The absorbance value obtained will determine the concentration of phenolic content through the standard curve. The concentration obtained will be multiplied with the dilution factor to get the exact concentration of phenolic content. Figure 4.1 and Figure 4.2 showed the result of both concentration and total phenolic acid content found in peel, seeds and pulp of A.muricata based on the standard curve of Gallic acid.

As being shown by Figure 4.1, it can be seen that, peel had the highest concentration of phenolic acid compared to the pulp and the seed extract of the A.muricata with the concentration of 1895.60 g GAE/ml followed by 1191.30 g GAE/ml and 967.00 g GAE/ml respectively. Quantitative analysis of total phenolic content showed by Figure 4.2 also revealed the highest value in peel extract with 3791.20 g GAE g-1 dry sample compared to pulp and seeds extract at 2382.60 g GAE g-1 dry sample and 773.60 g GAE g-1 dry sample respectively.

A similar trend was also observed in a study done by Monica et al. (2012) where the peel of A.cherimola, another species which belongs to genus Annona contains 14.6 mg of chlorogenic acid equivalent/100 g fresh weight compare than the pulp with 12.6 mg of chlorogenic acid equivalent/100 g fresh weight where chlorogenic acid is also one of the standard used to quantify bioactive compound in plant based material. A higher phenolic content of peel compare to pulp was also observed in A.squamosa with 12.1 and 1.5 g Gallic acid equivalent/100g dry weight respectively (Huang et al., 2010). On top of that, none of study has reported the concentration and total phenolic acid content in seed of A.muricata. In this research, seed of A.muricata showed the lowest concentration of phenolic acid and total phenolic content among other samples.

Figure 4.1: The concentration of phenolic acid of peel, pulp and seeds of A.muricata.

Figure 4.2: The total phenolic content of peel, pulp and seeds of A.muricata.4.3 Antioxidant activity

The antioxidant activities of the samples were determined by using DPPH assay. DPPH assay is a stable free radical that being used to test the ability of samples to acts as free radical scavengers or hydrogen donor. The antioxidant activity was determined by the value of IC50; concentration of sample extract needed to inhibit 50% of the free radical activity. Before that, different concentrations of sample extract were prepared and % inhibition of each concentration towards free radical was calculated as being shown in Table 4.1. The calculated % inhibition of DPPH were showed at Appendix A. Theoretically, when the sample extract interacts with DPPH, it will donate an electron or a hydrogen atom to this radical, thus neutralising its free radical character. The transfer of electron cause the bleaching of a purple coloured of DPPH solution to yellow and the absorbance value will gradually decreased.

Peel extract of A.muricata demonstrated the highest DPPH activity with IC50 value of 0.0409 g/mL followed by pulp and seeds with IC value of 5.0162 g/mL and 0.037 g/mL respectively. This finding was in agreement with Monica et al. (2012) who found the highest antioxidant activity in the peel extract of A.cherimola with an IC50 value of 57.7 g/mL compare to the pulp extract of A.cherimola which showed a lower antioxidant activity with an IC50 value of 72.2 g/mL. There were no study has reported the antioxidant activity in seed of A.muricata. In this research, seed of A.muricata showed the lowest antioxidant activity among other samples. The results showed that the peel of A.muricata exhibit highest antioxidant activities compare than the pulp and the seeds itself and has the potential as the sources of food supplements.

Table 4.1: % inhibition of A.muricata sample extract at different concentrationSeedsPulpPeel

Concentration (g/ml)% inhibitionConcentration (g/ml)% inhibitionConcentration (g/ml)% inhibition

0.3112.990.382.50.015246.24

1.5517.231.9135.280.07655.12

7.7465.549.5371.390.3866.34

38.6875.4247.6578.891.9177.56

193.4089.55238.2692.509.5383.41

Table 4.2: IC50 value for A.muricata sample extractSample extractIC50 (g/ml)

Seeds5.745

Pulp5.0162

Peels0.0409

CHAPTER 5

CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

This study was performed to determine the phenolic content and its potential from peel, seeds and pulp of A.muricata. Several researches were done investigating the cancer curing potential in A.muricata fruits which focused more on the edible part of the A.muricata. But none of them had determined the phenolic content, another useful and therapeutic substances with high antioxidant activity from various part of A.muricata fruits. In this research, the total phenolic content of peel, seeds and pulp of A.muricata were investigated and this phenolic content was determined by using Follin-Ciocalteu reagent method. The total phenolic content in peel of A.muricata was the highest compare to the seeds and the peel of A.muricata and it also showed the highest antioxidant activity towards free radical. It showed the potential of the peel of A.muricata as food supplement which usually being discarded during fruit processing. As phenolic acid is being characterised by its antioxidants activity, sample with high phenolic content also contributed to high antioxidant activity with low value of IC50.

Countries where A.muricata fruit are grown and processed commercially should endeavour to collect the peel and seeds of A.muricata and process them further to be used as a source of healthy compounds for the development of dietary supplements and to protect against oxidative stress. The seeds of A.muricata also contains an acceptable amount of phenolic compounds and can be further process to obtain the oil cake and the vegetable oil. The former may be suitable for use as a food supplement for cattle feed. The oil should have several commercial applications such as in the soap industry and for making certain pharmaceutical preparations. Since the oil has been reported to be toxic, further studies, especially processing to remove the toxic constituents, are essential in order to exploit it for culinary purposes.

5.2 Recommendations

After experiment had been performed, there are some recommendations that have the potential to be proposed for future study. The extraction method used in this study is a conventional method which uses Soxhlet extractor. For future study, other modern technique of extraction process may be employed for a high efficiency of extraction process for example microwaves extraction, supercritical carbon dioxide extraction and many other modern technique of extraction. Secondly, as seeds, pulp and peel of A.muricata contain high phenolic content and have high potential as food supplement, a safe and consumable solvent should be used for the extraction process. Thus for future study, extraction process may use consumable solvent or extractant such as water compare than using chemical solvents or a safer extraction method can be used such as supercritical carbon dioxide which these methods do not use any chemical solvent.