labibah bt abdaullah 09 24
TRANSCRIPT
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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
4.6 Comparison of free radical scavenging activities of both
water and ethanol Carica papaya peels extract at 500 ppm 27
4.7 Comparison of free radical scavenging activities of both
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.