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 TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURE LIST OF SCHEME LIST OF ABBREVIATIONS ABSTRACT ABSTRAK CHAPTER 1 INTRODUCTION 1.1 1.2 1.3 1.4 Background Antioxidant in plan Significant of study Objectives 1 2 4 4 iii iv vi vii viii ix x xi

CHAPTER 2 LITERATURE REVIEW 2.1 2.2 2.3 2.4 2.5 2.6 Guava (Psidium guajava L.) Papaya (Carica papaya) Pineapple (Ananas Comosus) Antioxidant activity Categories of antioxidant Antioxidant activity of guava, papaya and pineapple 5 7 7 8 9 10

CHAPTER 3 METHODOLOGY 3.1 3.2 3.3 Instruments Materials chemical Plant material 13 13 14

3.4 Extraction of papaya(carica papaya), guava(guajavapsidium), Pineapple (ananas comosus) 14

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3.5

Antioxidant method 3.5.1 Total phenolic content 3.5.1.1 preparation and measurement of standard Gallic acid 3.5.1.2 Determination of total phenolic content 3.5.2 2,2-Diphenyl -1-picrylhydrazyl (DPPH) method-scavenging assay

16 16 16 18

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CHAPTER 4 RESULT AND DISCUSSION 4.1 4.2 Total phenolic content 2,2 Diphenyl 1 picrylhydrazhyl (DPPH) 20 24 30 33 37 39

CHAPTER 5 CONCLUSION AND RECOMMENDATIONS CITED REFERENCES APPENDICES CURRICULUM VITAE

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LIST OF TABLES Table 3.1 4.1 4.2 Guidelines for the preparation of standard Gallic Acid total phenolic content of guajava psidium total phenolic content of carica papaya Page 16 21 21

4.3

total phenolic content of Ananas comosus

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LIST OF SCHEMES Scheme 3.1 3.2 3.3 The extraction method Guidelines for the preparation of standard Gallic Acid Preparation of sample for the determination of Total PhenolicContent (TPC) 18 Page 15 17

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LIST OF FIGURES Figure 2.1 'Thai Maroon' guavas, a red Apple Guava cultivar extremely rich ia ntioxidants 2.2 4.1 4.2 Green apple guavas are less rich in antioxidants Standard calibration curve of gallic acid at 760nm Free radical scavenging activities of water and methanol guajava psidium peels extract on different concentration 4.3 Free radical scavenging activities of water and methanol Carica papaya peels extract on different concentration 24 25 6 6 20 Page

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 Comparison of free radical scavenging activities of both water and ethanol Carica papaya peels extract at 500 ppm Comparison of free radical scavenging activities of both water and ethanol Ananas comosus peels extract at 500 ppm

4.6

27

4.7

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LIST OF ABBREVIATIONS

GAE/g : Gallic Acid Equivalent per gram Mg % GLE : milligram : percent : Guava leaf extract

DPPH : 2, 2 diphenyl -1- picrylhydrazhyl TPC : total phenolic content G L Ml : gram : liter : 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 phenolic content 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 for ananas 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. For guajava psidium, ethanol extract showed (98.4 %) compared to water (76.9%). While for radical scavenging activities of carica 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 scavenging acivities 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 dengan ekstrak 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). Untuk carica 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 (Renaud et 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

Objectives The 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 extracts from different peels of fruit. 3. To evaluate the antioxidant activity of the peels of papaya, guava and 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 diseasefighting 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

Extraction of papaya(Carica papaya), guava(Psidium guajava) and pineapple(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 4 0C until tested.

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