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ANTIOXIDANT ACTIVITY OF THE PEELS OF GUAVA, PAPAYA AND

PINEAPPLE

LABIBAH BT ABDULLAH

Final Year Project Report Submitted in

Partial Fulfilment of the Requirements for the

Degree of Bachelor of Science (Hons.)Chemistry

in the Faculty of Applied Sciences

Universiti Teknologi MARA

MAY 2009


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ACKNOWLEDGEMENT

In the name of ALLAH, my greatest thanks are to Him for giving me good health and

fully patient. I am very grateful to Him for letting me complete my final project report.

Here, I want to express my thanks and gratitude to all people that involve and give all

their contribution to my final project report to make it better.

Special thank to Dr. Norizan Ahmat, for her guidance, advise, fully support and high

ancouragement until this final report is completed. Her contributions in the completion

of this report were very meaningful to me.

I also would like to thank to all stuff from Chemistry Laboratory at Faculty Applied

Science for giving me the permission to use their facilities.

A special thank to En. Adnan, En. Khairul and En. Kadim for their helping especially in

the laboratory works. Not forgotten the lecturers of B.Sc (Hons) Chemistry for their

cooperation during for my final project.

Last but not least, I would like to express my gratitude to my family and friends who

has supported me through the ups and downs.

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TABLE OF CONTENT

Page

ACKNOWLEDGEMENT iii

TABLE OF CONTENTS iv

LIST OF TABLES vi

LIST OF FIGURE vii

LIST OF SCHEME viii

LIST OF ABBREVIATIONS ix

ABSTRACT x

ABSTRAK xi

CHAPTER 1 INTRODUCTION

1.1 Background 1

1.2 Antioxidant in plan 2

1.3 Significant of study 4

1.4 Objectives 4

CHAPTER 2 LITERATURE REVIEW

2.1 Guava (Psidium guajava L.) 5

2.2 Papaya (Carica papaya) 7

2.3 Pineapple (Ananas Comosus) 7

2.4 Antioxidant activity 8

2.5 Categories of antioxidant 9

2.6 Antioxidant activity of guava, papaya and pineapple 10

CHAPTER3 METHODOLOGY

3.1 Instruments 13

3.2 Materials chemical 13

3.3 Plant material 14

3.4 Extraction of papaya(carica papaya), guava(guajavapsidium),

Pineapple (ananas comosus) 14

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3.5 Antioxidant method 16

3.5.1 Total phenolic content 16

3.5.1.1 preparation and measurement of standard

Gallic acid 16

3.5.1.2 Determination of total phenolic content 18

3.5.2 2,2-Diphenyl -1-picrylhydrazyl (DPPH)

method-scavenging assay 20

CHAPTER 4 RESULT AND DISCUSSION

4.1 Total phenolic content 20

4.2 2,2 Diphenyl 1 picrylhydrazhyl (DPPH) 24

CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 30

CITED REFERENCES 33

APPENDICES 37

CURRICULUM VITAE 39

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LIST OF TABLES

Table Page

3.1 Guidelines for the preparation of standard Gallic Acid 16

4.1 total phenolic content ofguajava psidium 21

4.2 total phenolic content ofcarica papaya 21

4.3 total phenolic content ofAnanas comosus 21

LIST OF SCHEMES

Scheme Page

3.1 The extraction method 15

3.2 Guidelines for the preparation of standard Gallic Acid 17

3.3 Preparation of sample for the determination of Total

PhenolicContent (TPC) 18

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LIST OF FIGURES

Figure Page

2.1 'Thai Maroon' guavas, a red Apple Guava cultivar

extremely rich ia ntioxidants 6

2.2 Green apple guavas are less rich in antioxidants 6

4.1 Standard calibration curve of gallic acid at 760nm 20

4.2 Free radical scavenging activities of water and methanol

guajava psidium peels extract on different concentration 24

4.3 Free radical scavenging activities of water and methanol 25Carica papaya peels extract on different concentration

4.4 Free radical scavenging activities of water and methanol

ananas comosus peels extract on different concentration 25

4.5 Comparison of free radical scavenging activities of both

water and ethanol guajava psidium peels extract at 500 ppm 27


water and ethanol Carica papaya peels extract at 500 ppm 27


water and ethanolAnanas comosus peels extract at 500 ppm 28

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LIST OF ABBREVIATIONS

GAE/g : Gallic Acid Equivalent per gram

Mg : milligram

% : percent

GLE : Guava leaf extract

DPPH : 2, 2 diphenyl -1- picrylhydrazhyl

TPC : total phenolic content

G : gram

L : liter

Ml : mililiter

(DNA) : deoxyribonucleic acid

FRAP : ferric reduction antioxidant power

UV-Visible spectrophotometer : ultra violet visible

(Na2CO3) : Sodium carbonate

Ppm : part permillion

FA : Follin Ciocalteu reagent

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ABSTRACT

ANTIOXIDANT ACTIVITY OF THE PEELS OF GUAVA, PAPAYA AND

PINEAPPLE

The antioxidant activity of the peels of guava, papaya and pineapple was evaluated by 2,

2 diphenyl -1- picrylhydrazhyl (DPPH) method. This study was carried out to

determine the total phenolic content (TPC) of these peels of fruits. For this testing,

ethanol and water was used as extracting solvent. The total phenolic content of the

crude extract of peels were determined from the standard calibration curve of gallic

acid. From the results, it showed that the ethanol extract had higher total phenoliccontent than the water extract for all of fruits peel extract. In guajava psidium, value of

ethanol extract is (1210 mg GAE/g ) compared water extract (324 mg GAE/g), carica

papaya (353.2 mg GAE/g) for ethanol extract compared to water extract is (102.4 mg

GAE/g) and also forananas comosus with ethanol extract (236.4 mg GAE/g) compared

to water extract (80 mg GAE/g). Ethanol extract also showed the higher radical

scavenging for all of fruits peel. Forguajava psidium, ethanol extract showed (98.4 %)

compared to water (76.9%). While for radical scavenging activities ofcarica papaya in

ethanol extract was (83%) compared water extract was (82.7%). Ethanol extract of

ananas comosus peel also showed higher scavenging activities with (76.1%) compared

to water extract was (73.3%). From the testing on 2, 2 diphenyl- 1- hydrazhyl (DPPH)

method, it showed that the extract of guajava psidium had high radical scavengingacivities and also had high total phenolic content .

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ABSTRAK

AKTIVITI ANTIOKSIDA DARIPADA KULIT JAMBU, BETIK DAN NENAS

Aktiviti antioksida dalam jambu batu, betik dan nenas ditentukan dengan kaedah DPPH

(2, 2 diphenyl-1- picrylhydrazhyl). Kajian ini juga dijalankan untuk menentukan

kandungan fenolik bagi semua kulit buah. Untuk ujian ini, etanol dan air telah

digunakan sebagai pelarut. kandungan fenolik daripada ekstrak kulit ditentukan

daripada graf bacaan penyerapan larutan piawai asid galik. Keputusan yang diperolehi

menunjukkan ekstrak etanol adalah lebih tinggi kandungan fenolik berbanding denganekstrak air untuk ketiga-tiga ekstrak kulit buah tersebut. Nilai ekstrak etanol untuk

guajava psidium adalah (1210 mg GAE /g) berbanding dengan ekstrak air (324 mg

GAE/g). Untukcarica papaya, nilai ekstrak etanol adalah lebih tinggi berbanding air

ekstrak iaitu (353.2 mg GAE/g) dan (102.4 mg GAE/g). Manakala untuk ananas

comosus pula, nilai etanol ekstrak juga adalah lebih tinggi dibandingkan dengan nilai air

ekstrak iaitu (236.4 mg GAE/g) dan (80 mg GAE/g). Ekstrak etanol juga menunjukkan

aktiviti memerangkap radikal yang tinggi untuk semua ekstrak kulit buah. Bagi guajava

psidium, etanol esktrak menunjukkan dibangdingkan dengan air iaitu (98.4 %) dan

(76.9%). Manakala untuk aktiviti memerangkap radikal daripada carica papaya dalam

etanol ekstrak (83%) dan air ekstrak (82.7%). Ekstrak daripada kulit ananas comosus

dalam etanol juga menunjukkan ativiti memerangkap radikal yang tinggi dengan

(76.1%) dibandingkan dengan ekstrak daripada air (73.3%). Daripada ujian ke atas

kaedah 2, 2 diphenyl- 1- hydrazhyl (DPPH), menunjukkan bahawasanya ekstrak

daripada guajava psidium mempunyai aktiviti memerangkap radikal yang paling tinggi

juga dan mempunyai kuantti kandungan fenolik yang paling tinggi.

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CHAPTER 1

INTRODUCTION

1.1 Background

Nowadays, many of the plants around the world are used for medicine. These plants

actually have healing properties which are known as medicinal plants. Most of plants

are usually related to natural products, which are known or actually refer to herbs and

dietary supplement. Natural products are generally either of prebiotic originates from

microbes, plants, or animal sources (Nakanishi, 1999). Natural antioxidants, particularly

in fruits and vegetables have gained increasing interest among consumers and the

scientific community because epidemiological studies have indicated that frequent

consumption of natural antioxidants is associated with a lower risk of cardiovascular

disease and cancer (Renaudet al., 1998; Temple,2000). The defensive effects of natural

antioxidants in fruits and vegetables are related to three major groups: vitamins,

phenolics, and carotenoids. Ascorbic acid and phenolics are known as hydrophilic

antioxidants, while carotenoids are known as lipophilic antioxidants (Halliwell, 1996)


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Recent studies on cultured mammalian cells and animals indicate that polyphenolic

compounds from numerous fruits and vegetables exert several health-promoting

functions, including reducing the risks of cancer and heart and neurodegenerative

diseases (Joseph et al., 2005, Vita 2005). Besides that, epidemiological studies also

show positive associations between intake of fruits and vegetables and reduced

mortality rate from heart diseases, common cancers, and other degenerative diseases

(Kaur and Kapoor, 2001; Art and Hollman, 2005; Scalbert et al., 2005). The free-radical

scavenging capability and consequent antioxidant properties of the phenolics play an

important role in protecting the cells and tissues from oxidative stress and other

biological effects associated with these chronic diseases (Rimbach et al., 2005).

1.2 Antioxidant in plant

Substances that delay the onset of oxidative rancidity in fats have been sought

because of the unpalatability of rancid fats. These substances are known as

antioxidants. Most of the antioxidants in use today are phenolic antioxidants

approved for use in fats is butylated hydroxyanisole (BHA), butylated

hydroxytoluene (BHT), and propyl gallate. Labels on containers for margarine

and many rich crackers indicate the presence of one or more of these

antioxidants. (Helen 1982.; John Willey and n suns, inc.)


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Convincing epidemiological evidence shows that the phytonutrients of fruits and

vegetables play an important preventive role in the development of some diseases, such

as cancer, heart and neurodegenerative diseases (Diplock et al,. 1998). Consequently,

there has been an increased interest in understanding the reason for the protective effect

of fruits and vegetables. It is now widely accepted that the antioxidant nutrients

vitamins C and E, carotenoids and other phenolic compounds may help to protect the

human body against damage by reactive oxygen and nitrogen species (Halliwell, 1997).

Consumption of fruits and vegetables has been highly associated with the reduced risk

of cancer (Doll et al., 1990, Ames et al., 1979). In the status of normal metabolism, the

levels of oxidants and antioxidants in humans are maintained in balance, which is

important for sustaining optimal physiological conditions (Temple et al., 2000,

Thompson et al., 1994).

Besides that, other epidemiological studies show that consumption of fruits and

vegetables with high phenolic content correlate with reduced cardio- and

cerebrovascular diseases and cancer mortality (Hertog a,b et al., 1997).

There is evidence for beneficial roles of fruit and vegetables in the human diet

providing protection against cellular damage caused by exposure to high levels of free

radicals. Radical scavengers have attracted special interest because they can protect the

human body from free radicals that may cause many diseases, including cancer, and

contribute to the aging process (Ames et al., 1993). Fruits contain many different

antioxidant compounds (i.e. vitamin C and E, carotenoids and phenolic compounds),


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that serve as radical scavengers and it is relatively difficult to measure each antioxidant

component separately.

1.3 Significance of study

The function of natural antioxidants and dietary fiber in foods and biological systems

has received much attention. Fruits and vegetables play a significant role in the human

diet providing protection against cellular damage caused by exposure to high levels of

free radicals. (Ames et el., 1993). From the most evidence and the previous research, it

proves that the fruits and vegetables contain the antioxidant activities. Thus, the aim of

this study is to investigate the antioxidant activity of the peels of papaya, guava and

pineapple.

1.4 ObjectivesThe objectives of this study are:

1. To extract the chemical components from the peels of papaya,guava and pineapple using ethanol and water as solvents.

2. To determined the Total Phenolic Content (TPC) in the extractsfrom different peels of fruit.

3. To evaluate the antioxidant activity of the peels of papaya, guavaand pineapple by using 2, 2-diphenyl-2-picrylhydrazyl (DPPH) test.


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CHAPTER 2

LITERATURE REVIEW

Free radicals are molecules that are present in the environment and also occur in the body as a

natural part of physiological functions. They can cause cell damage, however, and when present

in high levels, may contribute to chronic conditions such as cancer, diabetes, and heart disease.

It's impossible to avoid free radicals, but antioxidants can minimize their effect. One of the best

sources of antioxidants is a diet rich in deeply colored fruits and vegetables; the pigment in

these foods is responsible for the antioxidant activity. In a recent study analyzing the antioxidant

capacity of tropical fruits, red guava came out ahead of other exotic fruits, including white

guava, mango, and green papaya.

2.1 Guava (Psidium guajava L.)

Guava (Psidium guajava) is an important tropical fruit, mostly consumed fresh. The

fruit is a berry, which consists of a fleshy pericarp and seed cavity with fleshy pulp and

numerous small seeds. World production of guava was estimated at about 500,000

metric tons. Of the South American countries, Brazil, Colombia, Mexico, and

Venezuela produce significant quantities of guava. ( Adsule et al,. 1995)

Guavas are plants in the myrtle family (Myrtaceae) genus Psidium, which contains

about 100 species of tropical shrubs and small trees which originating from Central

America. It is a food plant that contains yellow carotenoids. This fruit also had

nutritional values, whereas contain is about 5% sugar. But the vitamin C content is


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between 23 and 468 mg/100g (Harbone et al., 1928) and also has good levels of the

dietary minerals.

Guava fruit, usually 4 to 12 cm long, are round or oval depending on the species. Its

outer skin is rough, with sweet taste. Usually this fruit are canned or made into jelly,

paste or juice.Guava wood is used for meat smoking such as in barbecues, providing a

nice smoked flavor.

Figure 2.1: 'Thai Maroon' guavas, a red Apple Guava cultivar extremely rich in

antioxidants

Figure 2.2: Green apple guavas are less rich in antioxidants


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2.2. Papaya (Carica papaya)

Papaya contains 'papain'which helps to digest food. The papaya is the fruit of the plant

Carica papaya, in the genus Carica. Papaya is plants in the myrtle family (Caricaceae).

It has nutritional value where this fruits contain about 9% sugars and with only a low

organic acid content about 0.2%. Its also have the vitamin C leve is about 60mg/100g.

Their flesh is a rich orange color due to carotenoids contain. (Harbone etal., 1928).

These nutrients help prevent the oxidation of cholesterol. The fruits of papaya have the

shape like sphericals which can be as long as 20 inches. The seeds of the fruit are black

and round shape. It has a large tree-like plant, growing about 5 to 10 meters tall, with

spirally arranged leaves confined to the top of the trunk. The leaves are large with 7

lobes. The tree is usually unbranched if unlopped.

2.3 Pineapple (ananas comosus)

Bromeliaceae (the bromeliads) is a family of monocot flowering plants. The family

includes terrestrial species, such as the pineapple (Ananas comosus). Pineapple (Ananas

comosus) is the common name for an edible tropical plant and also its fruit. A.comosus

have leaves to 1.5 long, 1.5 cm wide and sometimes with short spines. Its scape is about

30-60 cm. It also have floral bract small, not covering the ovaries, toothed or entire. Its

flower with violet petals and fruits held erect on the stout scape, large and succulent.

(Spencer et al.,). The shapes of pineapples are cylindrical shape, a scaly green, brown or

yellow skin and have a crown on top with blue-green leaves. It has nutritional value

where pineapple contains a proteolytic enzyme bromelain, which breaks down protein.

Pineapple juice also used as a marinade and tenderizer for meat. Pineapple can get with


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fresh or canned and it is also available as a juice. It's also used in desserts and salads, as

an added value to meat dishes and in fruit cocktail.

2.4 Antioxidant activity

Related to the important role of antioxidant in our daily life, many studies on the

antioxidant activities have been done by researcher. There are chemical that a major

contributor for antioxidant activity, such classes of compound of terpenoids,

polyketides, amino acids, peptides, proteins, carbohydrates, lipids, nucleic acids bases

deoxyribonucleic acid (DNA) an so on.

Epidemiological studies have shown that dietary patterns were significantly associated

with the prevention of chronic diseases such as heart disease, cancer, diabetes, and

Alzheimerss disease (Temple et al., 2000, Willet et al., 2002).

Overproduction of oxidants in certain conditions can cause an imbalance, leading to

oxidative damage to large biomolecules such as lipids, DNA, and proteins (Liu et al.,

2002). More and more evidence suggests that this potentially cancer-inducing oxidative

damage might be prevented or limited by dietary antioxidants found in fruits and

vegetables. Phytochemicals in fruits and vegetables can have complementary and

overlapping mechanisms of oxidative agents, stimulation of the immune system,

regulation of gene expression in cell proliferation and apoptosis, hormone metabolism,

and antibacterial and antiviral effects (Liu et al., 2002).


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2.5 Categories of antioxidant

In general, there are two basic categories of antioxidants, natural and synthetic.

Recently, interest has increased considerably in finding naturally occurring antioxidants

for use in foods or medicinal materials to replace synthetic antioxidants, which are

being restricted due to their carcinogenicity (Zheng et al., 2001).

Natural antioxidant is due to free radical. Free radicals are everywhere can

found. Radical process which proceeds via a chain reaction including induction,

propagation and termination steps. During the induction period, alkyl radicals are

formed which undergo reaction with oxygen molecules to form hydroperoxides and

peroxide radicals during the propagation phase. Termination proceeds via association of

two radicals to form a stable adduct. In its radical form, DPPH absorbs at 515 nm, but

upon reduction by an antioxidant (AH) or a radical species (Re), the absorption

disappears. (Brand et al., 1994)

DPPH + AH DPPH-H + A

DPPH + R DPPH-R

Synthetic antioxidant butylated hydroxytoluenpropyle (BHT), butylated hydroxyanisole

(BHA) and propyl gallate. Both types of these antioxidant are added to food to prevent

from oxidize.


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2.6 Antioxidant activity of guava, papaya and pineapple

Cells in humans and other organisms are constantly exposed to a variety of oxidizing

agents, some of which are necessary for life. Overproduction of oxidants can cause

oxidative stress, which is associated with chronic diseases. Therefore, increased

consumption of fruits and vegetables containing high levels of antioxidants (mainly

phytochemicals) has been recommended to prevent or slow the oxidative stress caused

by free radicals. However, total phenolic contents of fruits in the literature were

underestimated because the bound phenolics were not included. It is important to know

the profiles of total phenolics, including both soluble free and bound forms in fruits and

vegetables, and their potential to improve human nutrition and health.

(Sun et al,.2002)

Guava (Psidium guajava ) fruit is considered a highly nutritious fruit because it contains

a high level of ascorbic acid (50300 mg/100 g fresh weight), which is three to six

times higher than oranges. Beside that, red-fleshed Brazilian guava has several

carotenoids such as phytofluene, b-carotene, bcryptoxanthin, g-carotene, lycopene,

rubixanthin, cryptoflavin, lutein, and neochrome (Mercadante et al., 1999). In other

report, Indonesian guava is an excellent source of provitamin A carotenoids. According

to Miean and Mohamed, (2001) and Misra and Seshadri, (1968) Phenolic compounds

such as myricetin and apigenin, ellagic acid, and anthocyanins are also at high levels in

guava fruits. Turns out guava is tops among tropical fruits when it comes to disease-

fighting antioxidants.


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Consumption of fruits and vegetables has been associated with reduced risk of chronic

diseases such as cardiovascular disease and cancer. Phytochemicals, especially

phenolics, in fruits and vegetables are suggested to be the major bioactive compounds

for the health benefits. (Sun et al., 2002)

The previous result showed that the pineapple is the among some fruit that has an

antioxidant activity of free phytocmical with 100mg/g total phenolic content. (Sun et

al., 2002).

The significance of bound phytochemicals in fruits to human health is highly possible

that different fruits with different amounts of bound phytochemicals can be digested and

absorbed at different sites of the gastrointestinal tract and plays their unique health

benefits. Bound phytochemicals, mainly in -glycosides, cannot be digested by human

enzymes and could survive stomach and small intestine digestion to reach the colon,

providing site specific health benefits (Sosulski et al.,). For example, banana and

pineapple, with a high percentage of bound phytochemicals, maybe able to survive the

stomach and small intestine digestion to reach the colon and be digested by bacteria

flora to release phytochemicals locally to have health benefits. It is interesting to note

that the fruit has been commonly used in traditional Chinese medicine for treating

constipation in humans (Jiangsu, 1986, Zhang, 1990). The phytochemical extracts of

fruits showed potent antioxidant activities.

(Sun et al., 2002)


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Vitamin C has been considered as the major antioxidant in fruits. Here showed that the

contributions of vitamin C to the total antioxidant activities in pineapple, where the

majority of the total antioxidant activity was from other phytochemicals in fruits.

Pineapple had contained vitamin C contribution to the total antioxidant activity with

(5.20%) (Sun et al., 2002).

Ripe papaya exhibited higher antioxidant activity by DPPH testing. According to

Jimenez-Escrig et al., (2001) showed that the DPPH radical can be scavenged by

carotenoids. Besides that the differences in phenolic composition may be more

associated with the higher level of DPPH in papaya than in other fruit, regardless of the

fact that total phenolic concentrations in papaya were similar to the levels in other fruit.


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CHAPTER 3

METHODOLOGY

3.1 Instruments

Rotary evaporator

UV-Visible spectrophotometer (Perkin Elmer)

3.2 Materials Chemical

Methanol

Ethanol

1,1-Diphenyl-2-picrylhydrazyl (DPPH)

Folin-Ciocalteu reagent

Sodium carbonate powder (Na2CO3)

Gallic Acid


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3.3 Plant material

The raw material of fresh papaya (Carica papaya), guava (Psidium guajava) and

pineapple an (Ananas comosus) peel was used in this study were obtained from

University Technology Mara Caf Shah Alam, Selangor. These samples were air- dried

for a few days until a constant weight was obtained. Then, the size of the raw material is

reduced by using a grinder

3.4 Extractionofpapaya(Caricapapaya),guava(Psidiumguajava)andpineapple(Ananascomosus)peels.

According to the method of extraction, the extracts of sample peels are carried out. The

size of sample that reduced to finely condition by grinder is soaked by ethanol and

water respectively for 48 hour. The solvents were removed by filtration to evaporate

under reduced pressure to get the crude extracts. This process are repeated for a few

time by adding the fresh solvent to the plant material and again evaporated under

reduced pressure to get a dark viscous mass. Then, the weight of crude was recorded.

These crude extract, was stored at 40

C until tested.

labibah bt abdaullah 09 24

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