POLYPHENOL extraction.pdf

DETERMINATION OF TOTAL FLAVONOIDS, TOTAL PHENOLIC AND ASCORBIC ACID CONTENT IN FRESH AND COOKED LONGAN (Dimocarpus longan Lour.), NUTMEG (Myristica

fragrans) AND SNAKE FRUIT (Salacca edulis Reins.) SEEDS.

FARHANA BINTI MOHAMED WAZIR

BACHELOR OF SCIENCE (Hons.) FOOD SCIENCE AND TECHNOLOGY FACULTY OF APPLIED SCIENCES

UNIVERSITI TEKNOLOGI MARA

JANUARY 2012

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ACKNOWLEDGEMENTS

In preparing this project, I was in contact with many people, academicians and

practitioners. They have contributed times and knowledge towards my

understanding and thoughts. In particular, I would like to express my sincere

appreciation to my main project supervisor, Madam Suzaira bt Bakar for her

encouragement, support, guidance, critics and friendship. Without her continued

support and interest, this project would not have been the same as presented here.

Not forget to my programme mates who support, help and encourage me to finish

my project on time. Not forgetting to my lovely parents who are supporting me in

mental and financial while doing this project. Finally, thanks to all my lectures of

Food Science and Technology in Faculty of Applied Sciences and to anyone who

was not mention above, whom indirectly gave me an advice, support, ideas and all

sorts of things that help me finish this project.

Farhana binti Mohamed Wazir

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS iii

TABLE OF CONTENT iv

LIST OF TABLES vi

LIST OF FIGURES vii

LIST OF ABBREVIATIONS viii

ABSTRACT ix

ABSTRAK x

CHAPTER 1 INTRODUCTION

1.1 Background and problem statement 1

1.2 Significance of study 3

1.3 Objectives of study 4

CHAPTER 2 LITERATURE REVIEW

2.1 Nutmeg (Myristica fragrans)

2.1.1 Illustration taxanomy 5

2.1.2 General information 6

2.1.3 Pharmacologically active parts of the plant 6

2.1.4 Chemical composition 7

2.1.5 Antioxidant properties of nutmeg 7

2.2 Longan (Dimocarpus longan)



2.2.3 Antioxidant properties of longan 9

2.2.4 Food exploitation 10

2.3 Snake fruit (Salacca edulis Reinw.)



2.3.3 Antioxidant properties of snake fruit 12

2.3.4 Food exploitation 12

2.4 Antioxidants 13

2.4.1 Classification of antioxidant based on mechanism of action 14

2.4.2 Free Radical 15

2.4.3 Flavonoid Compound 16

2.4.4 Ascorbic acid 17

2.4.5 Phenolic compound 17

2.4.6 Synthetic antioxidant 18

CHAPTER 3 METHODOLOGY

3.1 Materials

3.1.2 Samples 19

3.1.3 Chemicals 19

3.14 Equipment 19

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3.2 Methods

3.2.1 Preparation of fresh sample 20

3.2.2.1 Longan (Dimocarpus longan) 20

3.2.2.2 Snake fruit (Salacca edulis Reinw) 20

3.2.2.3 Nutmeg (Myristica fragnans) 20

3.2.3 Preparation of cooked sample 20

3.2.3.1 Longan (Dimocarpus longan) 20

3.2.3.2 Snake fruit (Salacca edulis Reinw) 20

3.2.3.3 Nutmeg (Myristica fragnans) 21

3.2.4 Extraction of plant sample 22

3.2.2 Antioxidants test

3.2.5.1 Total flavonoid content 22

3.2.5.2 Total phenolic content 22

3.2.5.3 Total ascorbic acid 22

3.2.6 Statistical analysis 25

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Total flavonoid content 26

4.2 Total phenolic content 28

4.3 Total ascorbic acid content 30

4.4 Correlation between total phenolic content with

total flavonoid content 33

CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 5.1 Conclusion 34

5.2 Recommendations 35

CITED REFERENCES 36

APPENDICES 43

CURICULUM VITAE 47

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LIST OF TABLES

Table Caption Page

4.1 Total flavonoid content in fresh and cooked longan, 26

nutmeg and snake fruit

4.2 Total phenolic content in fresh and cooked longan, 28


4.3 Ascorbic acid content in fresh and cooked longan, 30


4.4 Correlation coefficients between total flavonoid content 33

and total phenolic content in fresh and cooked longan,

nutmeg and snake fruit.

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LIST OF FIGURES

Figure Caption Page

2.1 Nutmeg seeds 5

2.2 Seeds of longan 8

2.3 Snake fruit pulp and seeds 11

2.4 Basic chemical structure of antioxidants 13

2.5 Cause and effect of free radicals 15

2.6 Basic flavanoid structure 16

3.1 Flowchart of preparation of seed extracts 21

4.1 Graph of flavonoid content 27

4.2 Graph of phenolic content 29

4.3 Graph of ascorbic acid content 31

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LIST OF ABBREVIATIONS

AOAC : Association of Official Analytical Chemists

ANOVA : Analysis of Variance

AlCl3 : Aluminium Chloride

CH3COOH : Acetic Acid

DCIP : 2, 6-dichloroindophenol

GAE : Gallic Acid Equivalent

HPO3 : Metaphosphoric Acid

NaNO2 : Sodium Nitrate

Na2CO3 : Sodium Bicarbonate

RE : Rutin Equivalent

mL : miliLitre

cm : centimeter

°C : degree Celcius

% : percent

g : gram

wt : weight

M : Molarity

mg/L : milligram per Litre

rpm : rotation per minutes

SPSS : Statistical Package for Social Sciences

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ABSTRACT

DETERMINATION OF TOTAL FLAVONOIDS, TOTAL PHENOLIC AND

ASCORBIC ACID CONTENT IN FRESH AND COOKED LONGAN

(Dimocarpus longan Lour.), NUTMEG (Myristica fragrans) AND SNAKE

FRUIT (Salacca edulis Reins.) SEEDS.

The aim of this study is to determine the content of total flavonoid, phenolic and

ascorbic acid content in six samples which are fresh and cooked longan, nutmeg,

and snake fruit seeds. This study was carried out to observe the effects of cooking

on total flavonoid, phenolic content and ascorbic acid in six samples. Before the

analysis, all fresh and cooked samples were extracted by using ethanol to obtain

extracts of sample for total flavonoid and phenolic content. For ascorbic acid test, all

samples were homogenized with metaphophoric acid-acetic acid solution. The total

flavonoid content were determined by using total flavonoid content method at 510

nm against a standard curve with rutin as a standard. The highest amount of total

flavonoid content was in fresh longan seeds with 715.19 ± 3.56 mg RE/100g and the

lowest amount of flavonoid content was in cooked snake fruit seeds with 163.99 ±

6.58 mg RE/100g. The total phenolic content were determined by using total

phenolic content method through direct spectrophotometric absorption at 750 nm

against a standard curve with gallic acid as a standard. The highest amount of total

phenolic content was in fresh longan seeds with 952.60 ± 5.84 mg GAE/100g and

the lowest amount of phenolic content was in cooked snake fruit seeds with 277.24

± 2.68 mg GAE/100g. Ascorbic acid content was determined by using AOAC

method. The highest amount of ascorbic acid content was fresh longan seeds with

118.375 ± 5.92 mg ascorbic acid/100g and the lowest amount of ascorbic acid was

cooked snake fruit seeds with 44.686 ± 2.25 mg ascorbic acid/100g. All samples are

significant differences between fresh and cooked samples at (p < 0.05). Cooked

samples shown lower amount of total flavonoid, phenolic and ascorbic acid

compared to the fresh samples because nutrients may lost through oxidation,

especially during cooking or heating. This is more easily happened to ascorbic acid

because it very sensitive to heat. It is also because decreases in synthesis or

increases in degradation.

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ABSTRAK

PENENTUAN JUMLAH KANDUNGAN FLAVONOID, FENOLIK DAN

ASID ASKORBIK PADA BIJI LONGAN (Dimocarpus longan Lour.), PALA

(Myristica fragrans) DAN SALAK (Salacca edulis Reins.) SEGAR DAN

TELAH DI MASAK.

Kajian ini dijalankan adalah untuk menentukan kandungan flavonoid, fenolik dan

asid askorbik di dalam enam sampel iaitu biji longan, pala, salak segar dan yang

telah di masak. Kesemua sampel telah diekstrak dengan menggunakan ethanol untuk

mendapatkan ekstrak fenolik dan flavonoid sebelum analisis dijalankan. Bagi

kandungan asid askorbik, kesemua sampel telah dicampurkan dengan menggunakan

asid metafosforik-asid asetik. Kandungan flavonoid ditentukan dengan

menggunakan kaedah flavonoid yang menggunakan rutin sebagai piawaian. Jumlah

flavonoid paling banyak adalah pada biji longan segar iaitu sebanyak 715.19 ± 3.56

mg RE/100 g dan jumlah flavonoid yang paling sedikit iaitu biji salak yang dimasak

sebanyak 163.99 ± 6.58 mg RE/100 g sampel. Kandungan fenolik ditentukan

dengan menggunakan kaedah fenolik yang menggunakan asid galik sebagai

piawaian. Jumlah fenolik paling banyak adalah biji longan segar iaitu sebanyak

952.60 ± 5.84 mg GAE/100 g sampel dan jumlah fenolik paling sedikit adalah biji

salak yang dimasak iaitu sebanyak 277.24 ± 2.68 mg GAE/100 g sampel.

Kandungan asid askorbik ditentukan dengan menggunakan kaedah AOAC. Jumlah

asid askorbik paling banyak ditunjukkan pada biji longan segar dengan 118.375 ±

5.92 mg askorbic asid/100 g sampel untuk biji longan segar dan jumlah asid

askorbik paling sedikit adalah biji salak yang dimasak iaitu sebanyak 44.686 ± 2.25

mg ascorbic acid/100 g sampel. Kesemua sampel mempunyai perbezaan signifikan

di antara sampel segar dan yang di masak pada (p < 0.05). Sampel yang telah di

masak menunjukkan jumlah keseluruhan flavonoid, fenolik dan askorbik asid lebih

rendah berbanding sampel segar kerana nutrien boleh hilang melalui osidasi

terutamanya sewaktu masak atau di panaskan. Ini lebih mudah berlaku kepada

askorbik asid kerana ia lebih sensitif kepada haba. Ia juga kerana penurunan dalam

sintesis atau peningkatan dalam degradasi.

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CHAPTER 1

INTRODUCTION

1.1 Background and problem statement

Antioxidant components are microconstituents present in the diet. It can

delay or inhibit lipid peroxidation by inhibiting the initiation or propagation

of oxidising chain reactions. It also involved in scavenging free radicals.

Food such as honey, fruits, vegetables and grains are reported to contain a

wide variety of antioxidant components, including some vitamins, as well as

L-ascorbic acid, phenolic compounds and others. These compounds are

found to be well correlated with antioxidant activity (Katalinic et al., 2004).

According to Michels et al., (2000), incidence of degenerative diseases

including cancer, heart disease, inflammation, arthritis, immune system

decline, brain dysfunction and cataracts can be lowered by associated with a

high consumption of fruits and vegetables. Many researchers believe that

vitamin supplements can reduce free radical damage. It also prevents and

delays the chronic degenerative diseases, and also possibly extends lifespan

(Jacob and Sotoudeh, 2002). Besides that, the synergistic effect which could

exist between different antioxidants means that the total antioxidant effect

may be greater than the sum of the individual antioxidant activities and the

isolation of one compound will not exactly reflect the overall action (Jia et

al., 1998).

Antioxidants can act as a protection agent which is able to terminate the

initiation of oxidizing chain reactions. It also can inhibit or delay oxidation

by other molecules (Suchandra et al., 2007). Oxidation processes are

important because it can control the production of free radicals and the

unbalanced mechanism of antioxidant protection that can cause diseases and

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accelerated ageing (Dawidowicz et al., 2006). Free radicals can also initiate

the oxidation of biomolecules which will lead into cell injury and death

(Freidovich, 1999).

The dietary intake of antioxidants is an important role in the protection of the

human organism against free radicals. Many studies show the connection

between the antioxidant activity of the substances present in the diet and the

prevention from diseases such as cardiovascular diseases or carcinogenesis

(Kris-Etherton et al. 2002). Free radicals play an important role in affecting

human health by causing several diseases including cancer, hypertension,

heart attack and diabetes. There are generated during body metabolism.

Exogenous intake of antioxidants can help the body scavenge free radicals

effectively. These effects have been attributed to antioxidant components

such as plant phenolics, flavonoids and phenylpropanoids (Rice-Evans et al.,

1996). Antioxidants also can neutralise chemically active products of

metabolism. Sources of natural antioxidants are primarily phenolics that may

occur in all products and parts of a plant such as fruits, vegetables, nuts,

seeds, leaves, roots, and bark.

Many studies have shown that the increased dietary intake of natural

phenolics can reduced coronary heart disease and cancer mortality with

longer life expectancy (Halliwell, 2007). Moreover, these polyphenolic

compounds have been found effective in many health-related properties,

such as antioxidant, anticancer, antiviral and anti-inflammatory activities

(Amin et al., 2006). On the other hand, concern about safety of the common

used of synthetic antioxidants such as butylated hydroxyanisole (BHA) and

tertiary butylhydroquinone (TBHQ) have led to increase the interest on

consumption of natural antioxidants which occur in plants as secondary

metabolites. The synthetic antioxidant which is commonly used in processed

foods has some unwanted side effects and some also are carcinogenic to

human being.

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1.2 Significance of study

This study was proposed to investigate the natural activities of longan,

nutmeg and snake fruit seeds by methanol and water extraction. It would

give an opportunity on ways to replace the synthetic antioxidant in the

processed foods, since synthetic antioxidant is not healthy.

Fruits and vegetables consumption have been attributed in many

epidemiological studies to reduce cancer and cardiovascular disease. Recent

cancer literatures have focused on antioxidant supplementation during

chemotherapy and found that with supplementation, there was a significant

in higher survival rate, higher tumour response, fewer toxicities and

increased chemotherapy efficacy (Block et al., 2007).

Malaysia has many types of fruits and vegetables. Malaysians are not

interested in trying a new fruits and vegetables that are not being

commercialised yet or called as indigenous fruits and vegetables. Most of

these fruit and vegetable are usually found in the rural area. Because of this,

there are not expose to the outsider. But for the people that live in the rural

area, they are familiar with some of the fruits or vegetables that are not in the

market yet because of their daily consumption of that food.

There is a growing interest in the use of natural antioxidants for expanding

the shelf life of food without the need of synthetic antioxidants. Examples of

synthetic antioxidants are butylated hydroxyanisole (BHA), butylated

hydroxytoluene (BHT) and tertiary butyhydroquinone (TBHQ). These foods

additive have been used to prevent lipid peroxidation that may possess

possible toxic and carcinogenic effect on health (Ito et al., 1985). Thus,

efforts have been made to search for novel natural antioxidants from tea,

fruits, vegetables, herb and spices and by product such as skin and seeds.

The replacement of synthetic antioxidants by natural antioxidant may have

benefits due to health implications and functionality. However, some of them

such as those from spices and herbs have limited applications in spite of their

high antioxidant activity. Naturally occurring antioxidant substances also

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need safety testing. Caution regarding an assumption of safety of natural

antioxidants has been repeatedly advised, since the fact than an antioxidant

comes from a natural source does not prove its safety.

1.3 Objectives

The objectives of this study are as follows:

i. to determine the content of total flavonoid, phenolic and ascorbic

acid of fresh and cooked nutmeg, snake fruit and longan seeds.

ii. to observe the effects of cooking on total flavonoid, phenolic and

ascorbic acid content of nutmeg, snake fruit and longan seeds

compare to fresh.

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CHAPTER 2

LITERATURE REVIEW

2.1 Nutmeg (Myristica fragrans)

Figure 2.1 Nutmeg seeds

Source: Rudgley, (1998)

2.1.1 Illustration taxanomy

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Magnoliales

Species : Myristica

Family : Myristicaceae

Genus : Myristica

Source: Anon, (1995)

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2.1.2 General information

Nutmeg seed is grayish-brown colour. It is a wrinkled kernel of the fruit of

the Myristica fragrens Houtt tree. It can be found in spherical and oval

shape. Both of them appear hard, but are easily grated. When the kernel is

cut transversely, many dark brown veins, containing the volatile oils, become

visible. Nutmeg is in the category of tree. This tree is indigenous to the

Banda islands in the Moluccas. Nutmeg is the species of the genus Myristica.

It have distributed from India and South-East Asia to North Australia and the

Pacific Islands.

Nutmeg tree can reach a height of 4 to 10 metres. This tree is dioecious with

male and female flowers occurring on different trees. It is obligatory cross

pollinated an ant mimicking flower beetle and effective pollinator in South

India (Armstrong and Drummond, 1986). The fruits of this tree are

pendulous, broadly pyriform, yellow and smooth. The fruits can be 7–10 cm

long. Its flesh is split open into two halves when ripe, showing the ovoid 2–3

cm long dark brown shining seed with hard seed coat, surrounded by a

lanciate red aril attached to the base of the seed. The seed of nutmeg is large

with ruminate endosperm and is considered as the most primitive among the

flowering plants (Corner, 1976).

2.1.3 Pharmacologically active parts of the plant

The most important part of the plant in terms of its pharmacological activity

and commerce is of course the dried kernel which is seed. Intoxication from

the use of the aril of the fruit, generally known as mace, has also been

reported, but rare. The oil of nutmeg has also been used for medicinal

purposes. This is because nutmeg contains the pharmacologically active

components. It is also used as a spice in various dishes, as components of tea

and soft drinks or mixed in milk and alcohol. Sometimes nutmeg is used as a

stomachic. It is used in traditional medicine. It is also used as a stimulant,

carminative as well as for intestinal catarrh and colic, to stimulate appetite,

to control flatulence and it has a reputation as an emmenagogue and

abortifacient (Nadkarni, 1988).

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2.1.4 Chemical composition

The main constituents of M. fragrans have been found to be alkyl benzene

derivatives such as myristicin, elemicin and safrole, terpenes, alpha-pinene,

beta-pinene, myristic acid and trimyristin (Yang et al., 2008). Nutmeg

contains about 10% essential oil. It is mostly composed of terpene

hydrocarb, terpene derivatives and phenylpropanoids. Of the latter group,

myristicin is responsible for the hallucinogenic effect of nutmeg. Oil of mace

contains the same aroma components but the total fraction of terpenoids is

increased to almost 90%. Nutmeg contains about 2% of lignans, which are

non volatile dimers of phenylpropanoid constituents of the essential oil for

example is dehydrodiisoeugenol (Anonymous, 1995). The main glycoside is

trimyristin having anxiogenic activity (Sonavane et al., 2002).

2.1.5 Antioxidant properties of nutmeg

Murcia et al., (2004) has carried out a study on the antioxidant properties of

some spices and compared with those of the common food antioxidants

butylated hydroxyanisole (BHA) (E-320), butylated hydroxytoluene (BHT)

(E-321) and propylgallate (E-310). From the results, nutmeg showed the

strongest protection in the deoxyribose assay. Nutmeg improved the stability

of oils which are sunflower, corn, olive and fats which are butter and

margarine against oxidation at 110°C. Trolox Equivalent Antioxidant

Capacity (TEAC) assay is used to provide a ranking order of antioxidant

activity. When used this assay, the antioxidant capacity of nutmeg was found

to be higher than BHT. According to Murcia et al. (2004), they reported that

phenylpropanoid compound extracts from nutmeg possessed antioxidant

activity.

Recently Checker et al., (2008) observed that lignans present in aqueous

extract of fresh nutmeg mace possess antioxidant, radioprotective and

immunomodulatory effects in mammalian cells. High antioxidant activity

has been reported in monoterpenoid rich extracts such as terpinene-4-ol,

alpha-terpineol and 4- allyl-2,6-dimethoxyphenol in nutmeg seed (Maeda et

al., 2008). According to Yadav and Bhatnagar (2007) , they reported that aril

part of M. fragrans have significant antioxidant activity due to its ability to

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inhibit lipid peroxidation and superoxide radical scavenging activity in rat.

Pretreatment with M. fragrans effectively protects the mice against

radiation-induced biochemical alterations as evident by decrease in lipid

peroxidation level and acid phosphatase activity and simultaneous increase

in hepatic glutathione and alkaline phosphatase activity (Sharma and Kumar,

2007).

2.2 Longan (Dimocarpus longan)

Figure 2.2 Seeds of Dimocarpus longan

Source : Crane et al., (2005)

2.2.1 Illustrated taxanomy

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Sapindales

Family : Sapindaceae

Genus : Dimorcarpus Lour.

Synonym names : Nephelium longan (Lam.) Carm.;

Euphoria longana Steud.

Scienctific names : Dimocarpus longan Lour.

Source: Crane et al., (2005)

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Longan, Dimocarpus longan Lour., the most popular members in

Sapindacea family. This fruit is a close cousin with lychee. This fruit is a

highly attractive subtropical fruit widely distributed in the south of China. It

is a tree fruit that grows in clusters. The individual fruits are round with a

diameter of about 1 inch and are covered with a brown skin that has a

smooth texture. Inside the fruit is a single, round seed. It produces fruit with

sweet, translucent and juicy flesh. The flesh of the longan is translucent,

white and crisp. It can be eaten fresh, frozen, canned, dried, or processed

into juice, wine, pickles, preserves, ice-cream and yoghurt. Longan is a

small, round, undistinguished looking fruit. The brittle light brown skin

encloses delicious translucent, juicy soft flesh around a single large, black

inedible pit. The Chinese name for this fruit is long yan rou, which literally

means “dragon eye flesh”.

Longans are adapted to tropical and warm subtropical areas with high

rainfall. They grow and crop best in areas with short cool frost-free winters

and long hot humid and wet summers. The temperature regime for fruit set

and development is similar to that for lychee, but, the minimum temperature

required inducing panicle and flower initiation appears to be less.

According to Morton (1987) longan seeds are traditional used as a folklore

medicine, which are administered to counteract heavy sweating and the

pulverized kernel serves as a styptic. Longan seeds have previously been

shown to possess potent antioxidant activities which could be ascribed to

their phenolic contents (Soong and Barlow, 2005). However, there is a little

study on longan peel which usually regards as a waste material. No previous

study on the antioxidant property of longan seed so far as we know.

2.2.3 Antioxidant properties of longan

Longans have higher in sugar and contain several vitamins and minerals.

Longan seeds have been found to be a rich source of antioxidant phenolic

compounds which are promising as functional food ingredients or natural

preservatives. According to Soong and Barlow, (2005) reported longan seeds

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contained high levels of gallic acid, corilagin and ellagic acid. It has been

proven to possess strong free radical scavenging activity (Rangkadilok et al.,

2005). However, Rangkadilok et al., (2005) reported that the afore

mentioned three characterised polyphenols might not be the only

contributors for the high antioxidant activity of longan seeds. Instead, other

phenolic constituents might also play important roles. On the other hand,

besides gallic acid and ellagic acid, many other phenolic glycosides such as

monogalloyl-glucose, monogalloyl-diglucose, digalloyl-diglucose, penta - to

heptagalloyl-glucose, ellagic acid-pentose conjugate, galloyl-

hexahydroxydiphenoyl (HHDP)-glucopyranose, pentagalloyl-HHDP-

glucopyranose, etc., were found in longan seeds by HPLC–ESIMS analysis

(Soong and Barlow, 2005). However, neither the complete structures of

these polyphenols nor their individual antioxidant activities have been

determined.

2.2.4 Food exploitation

Longans can be dried. For drying, this is the oldest processing method

known. It was developed in China before other technologies for preserving

the fruit became available (Chen and Huang, 2001). For this processing, the

fruits are first heated to shrink the flesh and facilitate peeling of the rind.

Then the seeds are removed and the flesh dried over a slow fire. The dried

product is black, leathery and smoky in flavor and is mainly used to prepare

an infusion drunk for refreshment. A liqueur is made by macerating the

longan flesh in alcohol. Longans also can be canned, juiced and frozen

(Subhadrabandhu and Yapwattanaphun, 2001).

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2.3 Snake fruit (Salacca edulis Reins.)

Figure 2.3 Snake fruit pulp and seeds.


2.3.1 Illustrate taxanomy

Kingdom : Plantae

(unranked) : Angiosperms

(unranked) : Monocots

(unranked) : Commelinids

Order : Arecales

Family : Areacaccae

Genus : Salacca

Species : S. zalacca

Binomial name : Salacca zalacca

Synonym names : Calamus zalacca, Salacca edulis



This fruits have been called “The Future of Our Health” and “The

Superheroes of Functionality” (Starling and Shane, 2007). Snake fruit or

salak (Salacca edulis Reinw) belongs to the class of Salacca originated from

South East Asia. This fruit is egglike in shape. The skin of the fruit is brown

and looks like a snake skin. It contains three pieces of seeds covered with

white flesh. In Indonesia there are many snake fruit cultivars; however, most

of them have an astringent taste and are not sweet.

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Snake fruit is a spiny palm. It is not a form of trunk but rather sprouts the

leaves from the ground level. Fruits are in tight, globose bunches, and round.

The fruit skin is covered with regularly arranged scales, giving the

appearance of a reptile skin. The edible part is the aromatic and translucent

whitish pulp, resembling in taste a mixture of pineapple and banana. Each

fruit contains 1 to 3 dark brown seeds. The pulp is edible and consists of

three lobes. The lobes have the consistency of three large peeled garlic

cloves, yet the taste is sweet and acidic with an apple-like texture.

In Indonesia, snake fruit is widely cultivated in the lowlands throughout the

islands. There are many different snake fruit cultivars; each of those has its

particular taste and fruit characteristics. Regardless, the present major

problems in the development of snake fruit production are quality, quantity

and continuity of supply. Currently, there is an increasing interest in

investigating snake fruit production techniques and postharvest properties in

Indonesia.

2.3.3 Antioxidant properties of snake fruit

In a study published by European Food Research and Technology

(Leontowicz et al., 2006), snake fruit was found to contain a high

concentration of bioactive compounds, high antioxidant potentials and to

positively affect plasma lipid profiles and plasma antioxidant activity in rats

fed with cholesterol-containing diets. It can be identified as chlorogenic acid,

epicatechin, singly-linked proanthocyanidins that mainly existed as dimers

through hexamers of catechin or epicatechin (Shui, 2004).

2.3.4 Food exploitation

Snake fruit usually consumed fresh as well as made in the form of candy

using sugar. Malaysia mostly consumed this fruit as in original form. This is

because to avoid any nutrient damage if this fruit going under treatment.

There is no information about the uses of the snake fruit seed. Usually the

seed is non-edible portion of the fruit and will be thrown away after finish

consuming the pulp (Leontowicz et al., 2006).

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2.4 Antioxidant

Antioxidants may be defined as a substance, when present at low

concentrations compared to oxidizable substrates, significantly delay or

inhibit oxidization of those substrates. Antioxidants, as a tradition, are

divided into two groups, chain breaking as primary group and preventing as

secondary group (Antolovich et al., 2002). In more detailed way,

antioxidants can be grouped as inhibitors of free-radical oxidation reactions,

inhibitors interrupting the propagation step of autoxidation, singlet oxygen

quenchers, synergist antioxidants, reducing agents and metal chalators

(Pokorny, 2007).

Figure 2.4 Basic chemical structure of antioxidants

Source: Pokorny, (2007)

Oxidation process occurs naturally in human body and defined as electron

transfer from one atom to another. Since oxygen is the ultimate electron

acceptor in the electron flow system that produces energy in the form of

ATP, oxidation is an essential part of aerobic life and human metabolism.

But the problem may arise when electrons flow from oxidation process

become unpaired and then subsequently generates free radicals, known as

Reactive Oxygen Species (ROS), such as superoxide (O2•-), peroxyl

(ROO•), alkoxyl (RO•), hydroxyl (HO) and nitric oxide (NO•). Free radicals

are very reactive and rapidly attack molecules in nearby cells (Pietta, 2000).

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2.4.1 Classification of antioxidant based on mechanism of action

Very common way to classify antioxidants is to divide them into two

mechanistically distinct groups: primary and secondary (Dapkevicius, 2002).

A similar classification of antioxidants is to divide them as chain breaking,

preventive and complementary (Williams and Elliot, 1997).

Primary antioxidants delay or inhibit the initiation step and interrupt the

propagation step of the radical chain reaction. Antioxidants act by

transferring a hydrogen atom to the peroxy radical. The resulting radicals

from the oxidised antioxidant are stabilised by resonance and are relatively

unreactive and therefore are not capable of initiating or propagating the

oxidative reaction.

Most of the antioxidants used in food protection are primary antioxidants.

Basically they are different phenolic compounds with various ring

substitutions: phenolic acids, catechins, flavonoids, anthocyanidins, lignans,

tannins and coumarins. Synthetic antioxidants, like butylated hydroxyanisole

(BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone

(TBHQ), propyl gallate (PG) also have a phenolic structure.

The effectiveness of the phenolic antioxidant depends on the resonance

stabilization of the phenoxy radical. Substitution at ortho and para position

increases the reactivity and formed radicals are more stable. Bulky

substituents such as the tertiary alkyl groups of BHA and BHT create steric

hindrance and provide stability to the phenoxy radical, however they also

lower the reaction rate with peroxy radicals.

Several antioxidants are used in combinations because of synergistic effects.

For instance, because of the earlier described steric hindrance of BHA and

BHT, they are often used in combination with other antioxidants such as

propyl gallate and TBHQ. Another class of antioxidants is the secondary or

preventive antioxidants. They include metal chelating agents, singlet oxygen

quenchers, peroxide destructors and some others (Wong, 1989).

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2.4.2 Free radical

Oxidation metabolism is an essential process for survival of living things

that can causes formation of free radicals (Pourmorad et al., 2006). Although

they are unwanted metabolic by-products, they are continuously released by

aerobic metabolisms (Mantle et al., 2000). Free radicals can also be

produced by light energy, photochemical smog, tobacco products,

polyunsaturated fats, alcohol, radiation, physical stress that leads depletion

of immune system antioxidants and modification of proteins caused by gene

expression changes (Pourmorad et al., 2006).

Figure 2.5 Cause and effect of free radicals

Source: Pourmorad et al., (2006)

Free radicals are unstable. It has a tendency of being stabilized in a way of

reducing their energy level by transferring their excess electron to nearby

substances. As an example, when they are formed within body, they attack

nearby tissues by oxidizing membrane lipids, cellular proteins, DNA that

causes complete shutdown of cellular activities such as respiration and

terminates the cell. Furthermore, the interaction of oxygen free radicals with

members of lipidic portion of body leads to formation of new radicals such

as hydroperoxides, superoxide, lipid oxides and hydroxyl radical whose type

may interact with biological systems in a citotoxic manner (Benavente-

Garcia et al., 2000).

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