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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).

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 byproducts 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

1.4

To quantify the antioxidant activity from the extracted phenolic compounds

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 muricata Linn or soursop is a small tree from the Annonaceae family. 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 the A.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.muricata was 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 antiHSV-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 classes


Class

Structure

Acethophenones, phenylacetic acids


Biflavonoids

)2

Condensed tannins (proanthocyanidins or

)n

flavolans
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, phydroxybenzoic, 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).

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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,

anthocyanidins (Hollman & Katan, 1999).

isoflavones,

flavanonols

and

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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).

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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).

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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.

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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)

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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).

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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.

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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.

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Pre-treatment of peel, seeds and pulp of A.muricata

Extraction of phenolic compounds using solvent extraction by Soxhlet extractor

Quantification of total phenolic contents and its antioxidants activities

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 (
Carbonate (

) and Sodium

) 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.

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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

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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.

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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=cx
where
C 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)

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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.

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Figure 3.3: A.muricata fruit was cut into Figure


two.

3.4:

A.muricata

fruit

was

separated into pulp, seeds and peel.

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

samples.

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Figure 3.7: The Soxhlet apparatus for Figure 3.8: The oil separation process
extraction

process

methanol as solvent.

using

acidified using rotary evaporator.

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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 FollinCiocalteu 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

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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

27

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.

Concentration of phenolic acid from standard


curve (mg GAE/ml)

2500

2000

1500

Peel
Pulp

1000

Seed

500

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

Total phenolic content, C (mg GAE/g dry


sample)

4000
3500
3000
2500
2000
1500

Peel
Pulp
Seed

1000
500
0

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

28

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.

29

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


Seeds

Pulp

Peel

Concentration

Concentration

Concentration

(g/ml)

inhibition

(g/ml)

inhibition

(g/ml)

inhibition

0.31

12.99

0.38

2.5

0.0152

46.24

1.55

17.23

1.91

35.28

0.076

55.12

7.74

65.54

9.53

71.39

0.38

66.34

38.68

75.42

47.65

78.89

1.91

77.56

193.40

89.55

238.26

92.50

9.53

83.41

Table 4.2: IC50 value for A.muricata sample extract


Sample extract

IC50 (g/ml)

Seeds

5.745

Pulp

5.0162

Peels

0.0409

30

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

31

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.