25

2. REVIEW OF LITERATURE

2.1. PREAMBLE

Antioxidants have possibly been used to preserve fats long before recorded

history. In prehistoric times, it is conceivable that aromatic plants were used to give

fats, to be used for medicinal purposes and foods, pleasant odors and flavors.

Retardation of oxidative reactions by certain compounds was first recorded by

Berthollet in 1926. Early workers explain the phenomenon as “catalysts poisoning”.

In the mid-nineteenth century, Chevreul observed that linseed oil dried much more

slowly in oak wood than on glass or natural surfacing; other woods had the same

effect but not to the same extent and observed that oak wood was an anti-drying agent

for linseed oil. The course of rancidification of fats remained unknown until Duclaux

demonstrated that atmospheric oxygen was the major causative agent of free fatty acid

oxidation. Several years later, perhaps, the first report of antioxidants used in fat was

by Deschamps observed that gum benzoin and poplar tree extracts could retard

rancidity of ointments made with lard. Wright observed that American Indians in the

Ohio Valley preserved bear fat using bark of the slippery elm and found that elm was

effective to preserve lard and butter fat. Elm bark was patented as an antioxidant 30

years later. Present knowledge of the properties of various chemicals to prevent

oxidative breakdown of fats and fatty foods began with the classic studies by Moureu

and Dufraise. During World War I and shortly thereafter these workers tested more

than 500 compounds for AA1. This basic research, combined with the vast importance

of oxidation in practically all manufacturing operations, triggered a search for

chemical additives to regulate oxidation.

According to The Mitochondrial Free Radical Theory of Aging2, free radicals

are a class of molecule with a very simple definition. The nature of atomic structure

26

and of the covalent chemical bond (the features that give an atom its valency) are

fixed by the rule that electrons occupy orbitals of atoms, such that an orbital can

contain zero, one or two electrons, and that electrons carry less energy when they are

one of a pair in an orbital than when they are unpaired. A molecule is only a free

radical if it possesses one or more unpaired electrons (Fig. 2.1).

Fig.2.1. Free Radical Molecule

AA is a fundamental property important for life. Many of the biological

functions, such as antimutagencity, anticarcinogencity, and antiaging, among others,

originate from this property3,4. Many of the natural antioxidants, especially

flavonoids, exhibit a wide range of biological effects, including antibacterial,

antiviral, anti-inflammatory, antiallergic, antithrombotic, and vasodilatory actions.

The AA of several plant materials has recently been reported5,6,7,8,9 & 10 however,

information on the relationship between AA and phenolic content and composition of

many food plants is not available.

2.2. ANTIOXIDANTS AND FREE RADICALS

Numerous antioxidants are plant-based and play a fundamental role in

protecting plants that are open to the elements such as sunlight and severe oxygen

stress. It has been suggested that antioxidants may amend cellular oxidative status and

prevent biologically significant molecules such as DNA, proteins, and membrane

27

lipids from oxidative damage and as a result lessen the risk of several chronic diseases

including cancer and cardiovascular disease11. A sufficient ingestion of natural

antioxidants in food is, therefore, of great consequence for the defense of

macromolecules against oxidative damage 12, 13. According to Stratil14 (2006), the

cells most frequently damaged by oxidative stress are unsaturated fatty acids in lipids,

cholesterol, different functional polypeptides, proteins and nucleic acids.

Some antioxidants provide increased protection with increasing concentration,

while others have optimal levels after which higher levels exert prooxidant effects15.

Moure16(2001) reported that there is a general trend for AA to increase with the

concentration of the antioxidant up to a certain limit, depending on the antioxidant

and the test used, most natural extracts and tests have shown a maximum AA at a

0.05% concentration. Various phenolic extracts from plant sources have been shown

to increase in AA with increasing concentration of the extract17, 18. Gordon19 (1992)

showed that IP of lard samples increased as concentration of hexane extracts of

powdered tanshen (Salvia miltiorrhiza Bunge) increases. Onyeneho17 (1992) also

found that increasing the concentration of freeze dried extracts from the bran of

durum wheat led to increase AA showed by a decrease in PVs of soy oil. Similarly,

ethanolic extracts from red grape marc, peels and seed showed increased AA with

increased extract concentration18. Gamez-Meza20 (1999) showed that AA of extracts

of Thompson grape Bagasse increased as the concentration increased which was

shown by the increased stability of soybean oil. The increase in AA of phenolic

compounds due to increase in their concentration could be due to an increase in the

concentration of potential sites for radical scavenging. On the other hand, at high

concentrations, prooxidant effects could arise due to the involvement of the phenolic

compounds in initiation reactions (i.e., formation of radicals) 21.

28

2.3. EXTRACTION METHODS

Plant-derived antioxidants, especially, the phenolics have gained considerable

importance due to their potential health benefits. Epidemiological studies have shown

that consumption of plant foods containing antioxidants is beneficial to health because

it down-regulates many degenerative processes and can effectively lower the

incidence of cancer and cardio-vascular diseases22. Recovery of antioxidant

compounds from plant materials is typically accomplished through different

extraction techniques taking into account their chemistry and uneven distribution in

the plant matrix. For example, soluble phenolics are present in higher concentrations

in the outer tissues (epidermal and sub-epidermal layers) of fruits and grains than in

the inner tissues (mesocarp and pulp).

Solvent extraction is more frequently used for isolation of antioxidants; both

extraction yield and AA of extracts are strongly dependent on the solvent due to

different antioxidant potential of compounds with different polarity. Julkunen-Tiitto23

(1985) found a different behaviour in the extraction of different compounds and total

extractable polyphenols (TEP). Maximum total phenolic extraction yields were

attained with methanol, where as 50% acetone extracted more selectively

leucoanthocyanins and no significant effects were observed in the extraction of

glycosides. Also for extracts from burdock roots, water yielded the greatest amount of

extract and exhibited the strongest AA24. Aziza25 (1999) reported maximum AA from

cocoa by products in the methanol followed by mixtures of chloroform, ether and

dichloroethane or chloroform, methanol and dichloroethane. Also Przybylski26 (1998)

reported that the AA of buckwheat extracts varied with the polarity of the solvent,

those extracted with methanol being the most active.

29

Literature suggests limitations on reporting antioxidant capacity in cereals

based on the various extraction methods. First, the procedure used to extract

antioxidants may be incomplete. However, the extract yields and resulting antioxidant

activities of the plant materials are strongly dependent on the nature of extracting

solvent, due to the presence of different antioxidant compounds of varied chemical

characteristics and polarities that may or may not be soluble in a particular solvent23.

Polar solvents are frequently employed for the recovery of polyphenols from a

plant matrix. The most suitable of these solvents are (hot or cold) aqueous mixtures

containing methanol, ethanol, acetone, and ethyl acetate. Methanol and ethanol have

been extensively used to extract antioxidant compounds from various plants and

plant-based foods (fruits, vegetables etc.) such as plum, strawberry, pomegranate,

broccoli, rosemary, sage, sumac, rice bran, wheat grain and bran, mango kernel,

mango, citrus and many other fruit peels. In most of these experiments, the most

commonly employed solvent is absolute ethanol27, 28 or ethanol: water in different

proportions29, 30, 31&32 although the extraction of phenolic compounds could be

improved by using more polar solvents such as methanol33.

Some authors use methanol: water as an extraction solvent34, but the solvent

dilution is not regularly acidified, which has shown to improve the extraction35. The

solid-liquid extraction is a heterogeneous, multi component operation involving the

non-steady transfer of solutes from a solid to a fluid. Vegetable materials include

different solutes that can be extracted simultaneously at different rates depending on

the location (outer surface, pores, vacuoles, etc.) and their partition coefficients36.

30

Stability of different extracts from same material was dependent on extracting

solvent used for solubilization and removal of polyphenolic compounds. Methanol

extracts from cocoa by-products were stable up to 500C where as other extracts were

less stable25. Bonoli37 (2004) reported that maximum phenolic compounds were

obtained from barley flour with mixtures of ethanol and acetone. Similarly, aqueous

methanol was found to be more effective in recovering highest amounts of phenolic

compounds from rice bran and Moringa oleifera leaves38. Anwar39 (2003) extracted

antioxidant compounds from various plant materials including rice bran, wheat bran,

oat grouts and hull, coffee beans, citrus peel and guava leaves using aqueous 80%

methanol (methanol: water, 80:20 v/v). A study conducted by Peschel40 (2006) on

some fruits and vegetable by-products showed that apple, strawberry, cucumber,

chicory showed highest phenolic content in methanol extract than water, ethanol,

acetone and hexane. It was found that methanol extract was also having high AA

when tested by superoxide anion scavenging activity, FTC, DPPH and Rancimat

methods40.

The AA also depends on the type and polarity of the extracting solvent, the

isolation procedures, purity of active compounds, as well as the test system and

substrate to be protected by the antioxidant41. It has been suggested that the

determining factor for the AA is the lipophilic nature of the molecules and the affinity

of the antioxidant for the lipid42, 43. A close dependency on the AA of phenolic

acids44 has been reported for phenolic compounds, and even the recommended

concentration of synthetic antioxidants has been indicated for some tests45. The

antioxidant potential of a compound is different according to different antioxidant

assays or, for the same assay when the polarity of the medium differs, since the

interaction of the antioxidant with other compounds play an important role in the

31

activity, dramatic difference in the relative antioxidant potential of model compounds

were observed when one model compound is strongly antioxidant with one method

and prooxidant with another44. A phenomenon known as “polar paradox’ has been

repeatedly reported; hydrophilic antioxidant are more effective than lipophilic

antioxidants in bulk oil, whereas lipophilic antioxidants present greater activity in

emulsions.

2.4. TOTAL PHENOLIC CONTENT (TPC)

It is highly impractical to quantify all of the compounds in plants that exhibit

antioxidant activities; a variety of methods have been developed to quantify total

antioxidant capacity of plant extracts46.

Most natural antioxidants are multifunctional in complex heterogeneous foods;

their activity cannot be assessed by any one method47, 48. Measurement of TPC

quantifies the total concentration of phenolic hydroxyl groups present in an extract

being assayed, regardless of the specific molecules in which the hydroxyl groups

occur49. The assays used to measure total phenols do not, therefore, provide

information on the particular phenolic compounds present in a sample. Some common

methods for the measurement of total phenols included the colorimetric Prussian

Blue, Folin-Denis ferric ammonium citrate or FC methods.

The FC method, however, appears to be the popular method widely used for

the determination of total phenols. The method is based on the reducing power of

hydroxyl groups, which react with the FC phenol reagent (an oxidizing agent

comprised of heteropolyphosphotungstate-molybdate) under basic conditions to form

chromogens that can be detected spectrophotometrically at 760 nm. The reagent is

32

more sensitive to reduction by phenolics, and the addition of lithium sulphate to the

reagent makes it less prone to precipitation and interference by non-phenolics.

The FC method has been used for many years as a means to determine total

phenolics in natural products50. Many studies show the recommended reference

standard (gallic acid) being replaced with tannic acid equivalents, caffeic equivalents,

vanillic acid equivalents and catechin equivalents, among others. Phenolic compounds

can be found in flavonoids, phenolic acids, hydroxycinnamic acid derivatives and

lignans.

2.5. ANTIOXIDANT CAPACITY ASSAYS

It is of great interest to the general public, medical and nutritional experts, and

health and food science researchers to know the antioxidant capacity and constituents

in the foods we consume. Due to the complexity of the composition of foods,

separating each antioxidant compound and studying it individually is costly and

inefficient, notwithstanding the possible synergistic interactions among the

antioxidant compounds in a food mixture. A total antioxidant capacity assay using

only one chemical reaction seems to be rather un-realistic and not easy to rely, yet,

there are numerous published methods claiming to measure total antioxidant capacity

in vitro. In fact, phenols are presumed to be responsible for the beneficial effects

derived from the consumption of whole grains, fruits, and vegetables. Phenolic

compounds have strong in vitro and in vivo AAs associated with their ability to

scavenge free radicals, break radical chain reactions, and chelate metals. Moreover,

high phenol consumption has been correlated with a reduced risk of cardiovascular

diseases and certain cancers37.

33

Several methods have been described to assess AA of compounds both in vitro

and in vivo, such as the measurement of prevention of oxidative damage to

biomolecules such as lipids or DNA and methods assessing radical scavenging

ability51. The following in vitro methods have been described: Ferricthiocyanate

(FTC) method52, β-carotene-linoleate model system 53,19 and free radical scavenging

using 2,2-diphenyl-1 picrylhydrazyl (DPPH)42.

No single assay will accurately reflect all of the radical foundations or all

antioxidants in a mixed or complex system and it must be appreciated at the outset

that there are no simple universal methods by which antioxidant capacity can be

measured accurately and quantitatively50; also too many analytical methods result in

inconsistent results, inappropriate application and interpretation of assays and

improper specifications of antioxidant capacities. There are two reaction mechanisms

in which antioxidants can deactivate radicals. The first of these methods is the single

electron transfer (SET) assay which detects the ability of a potential antioxidant to

transfer one electron to reduce any compound, including metals, carbonyls and

radicals54.

According to Prior50 (2005), SET reactions are usually slow and can require a

lengthy time to reach completion, so the antioxidant capacity calculations are based

on per cent decrease in product rather than kinetics. The second method is the

hydrogen atom transfer (HAT), which measures the antioxidant’s ability to quench

free radicals by hydrogen donation. HAT reactions are solvent and pH independent

and are usually quite rapid. The presence of reducing agents, including metals, is a

complication in HAT assays and can lead to erroneously high apparent reactivity50.

There are methods utilizing both HAT and SET mechanisms. The TEAC and DPPH

34

assays are usually classified as SET reactions, however, these two indicator radicals

in fact may be neutralized either by direct reduction via electron transfers or by

radical quenching via HAT55.

2.5.1. 2, 2,-Diphenyl-1-picrylhydrazyl Activity

DPPH• is one of a few stable and commercially available organic nitrogen

radicals bearing no similarity to the highly reactive and transient peroxyl radicals

involved in various oxidative reactions in vivo56,57. This assay is based on the

measurement of the reducing ability of antioxidants towards DPPH•. The ability can

be evaluated by electron spin resonance or by measuring the decrease of its

absorbance. The measurement of the loss of DPPH color at 515 nm following the

reaction with test compounds is what the antioxidant assays are based on Prior50

(2005). DPPH radical, with a deep violet color, receives a hydrogen atom from the

antioxidant and is converted to a colorless molecule. Using this reagent, the free

radical scavenging ability of the antioxidant can be determined by spectrophotometric

methods. Yen and Duh(1994)58, Chen and Ho (1995)59 have reported that inhibition of

free radical formation by different antioxidants can be measured using very stable free

radicals such as 2,2-diphenyl-1-picrylhydrazyl. Using the DPPH assay has its

advantages. It is simple, rapid and needs only a UV vis spectrophotometer to carry out

the experiment.

2.5.2. Reducing Power (RP)

RP increases according to the increase in absorbance. As more Fe3+ are

reduced to the ferrous form or when more electrons are donated by antioxidant

components; the colour of Perl’s Prussian blue will be darker, resulting in an

increased absorbance reading60. Reductants also react with certain precursors of

35

peroxides, thus preventing the formation of peroxide61. The RP is found to be

dependent on extract concentration. Yen (1995) also reported that RP of tea extracts

increased markedly with increasing extract concentration62.

2.5.3. Ferric Thiocyanate (FTC) Capacity

Real food Systems generally consists of multiple phases in which lipid and

water co exists with some emulsifier. Hence an antioxidant assay using a

heterogenous systems such as oil-in water emulsion is one of the model systems for

such evaluation, satisfying above conditions. The linoleic acid emulsion

system/thiocyanate method has been used here for evaluation under above

conditions63. During peroxidation in an incubator the absorbance values increased

owing to oxidation products, which react to form ferric thiocyanate the colour of red

blood64. Antioxidants can hinder the oxidation and consequently, the increase in

absorbance will be less.

2.5.4. β-carotene bleaching assay

Mechanism of bleaching of β-carotene is a free radical mediated phenomenon

resulting from hydroperoxides formed from linoleic acid. β-carotene, in this model

system undergoes rapid discolouration in the absence of an antioxidant. The linoleic

acid free radical formed upon the abstraction of a hydrogen atom from one of its

diallylic methylene groups attacks the highly unsaturated β-carotene molecule and

looses its chromophore and characteristic orange colour62.

2.6. LIPID OXIDATION

Lipid oxidation is a general term used to describe a complex sequence of

chemical interactions between unsaturated fatty acyl groups in lipids with active

36

oxygen species65. The unsaturated fatty acids on triacylglycerols and phospholipids

have low volatility and do not directly contribute to the aroma of foods. However,

these fatty acids will decompose during lipid oxidation to form small, volatile

molecules that produce the off-aromas associated with oxidative rancidity. These

volatile compounds are detrimental to food quality except in the case of food products

such as fried foods, dried cereal, and cheeses, where small amounts of these volatile

compounds are important in their flavour profile.

The oxidative deterioration of fats and oils in foods is responsible for rancid

odours and flavours, with a consequent decrease in nutritional quality and safety

caused by the formation of secondary, potentially toxic, compounds. The addition of

antioxidants is required to preserve flavor and colour and to avoid vitamin

destruction. The mechanisms of lipid oxidation in a particular food depend on the

nature of the reactive species present and their physico-chemical environment66.

Lipid oxidation reactions are dependent on the chemical reactivity of

numerous components including reactive oxygen species, prooxidants, and

antioxidants. However, research over the past few decades has shown that the

physical properties of food systems are extremely important to the chemistry of lipid

oxidation67, 68.

Understanding how the physical nature of bulk oils impacts lipid oxidation

reactions could lead to the development of new antioxidant technologies and to the

more efficient use of existing antioxidant ingredients. For example, if association

colloids are found to be the major site of oxidation reactions in bulk oils, then altering

these structures or more efficiently delivering antioxidants to the site of oxidation

37

could significantly improve the oxidative stability of oils. Thus, increasing the

understanding of oxidative mechanisms in oils could increase the use of nutritionally

beneficial polyunsaturated lipids in foods.

2.6.1. Mechanism of Lipid Oxidation

Theoretically, lipid oxidation is a free radical chain reaction between

unsaturated fats and oxygen that can occur in an autocatalytic manner. However, lipid

oxidation in many food systems is accelerated by prooxidants such as transition metal

ions, photosensitizers, UV light, and certain enzymes69. The overall mechanism of

lipid oxidation involves three stages:

1. initiation - the formation of free radicals

2. propagation - the free-radical chain reactions and

3. termination - the formation of nonradical products 69,65

A simple scheme for the free radical mechanism can be summarized as

follows65

In the initiation step, a fatty acid radical known as the alkyl radical (L•) is

formed by abstraction of a hydrogen from a fatty acid in the presence of an initiator

(In•). Once the alkyl radical forms, the free radical can delocalize over the double

bond system resulting in double bond shifting and in the case of polyunsaturated fatty

acids, formation of conjugated double bonds. In bulk oils, the ease of formation of

alkyl radicals in fatty acids increases with increasing unsaturation.

The first step of propagation involves the addition of oxygen to the alkyl

radical resulting in the formation of peroxyl radical (LOO•), which has a higher

38

energy than the alkyl radical. Thus, the peroxyl radical can abstract hydrogen from

another unsaturated fatty acid and produce lipid hydro peroxide (LOOH) and a new

alkyl radical. The interaction of two free radicals to form non-radical species will

terminate the process. This step is sometimes not very important in many foods as

initiation and propagation since the food is already rancid before significant

termination reactions take place. An exception is in the low oxygen environment of

frying oils where the termination reaction can occur between alkyl radicals to form

fatty acid dimers65.

2.6.2. Lipid Oxidation Decomposition Products

Rancidity in food occurs when unsaturated fatty acids decompose into volatile

compounds. These volatile oxidation products are produced from the decomposition

of fatty acid hydroperoxides41. The homolytic cleavage of hydroperoxides (LOOH)

between these two oxygen molecules is the most likely hydroperoxide decomposition

pathway. This reaction yields an alkoxyl (LO•) and a hydroxyl radical (•OH). The

alkoxyl radical (LO•), which is more energetic than either the alkyl (L•) or peroxyl

radical (LOO•), can enter into a number of different reaction pathways. Alkoxyl

radicals can attack another unsaturated fatty acid, a pentadiene group within the same

fatty acid or the covalent bonds adjacent to the alkoxyl radical. This last reaction is

known as β-scission reaction and is important to food quality as it can cause fatty

acids to decompose into low molecular weight, volatile compounds that cause

rancidity.

2.6.3. Antioxidants (oxidation inhibitors)

Incorporation of antioxidants into foods is one of the most effective methods

of retarding lipid oxidation. However, many factors can impact the activity of

39

antioxidants with some antioxidants retarding lipid oxidation under certain conditions

but promoting lipid oxidation under other conditions70. Several degradation reactions,

which may occur on heating or during long term storage, deteriorate fats and oils and

the lipid constituents of foods.

Oxidation reactions and the decomposition of oxidation products are the main

processes which result in decreased nutritional value and sensory quality71. Research

has implicated oxidative and free-radical-mediated reactions in degenerative

processes related to ageing and diseases such as cancer, coronary heart disease and

neurodegenerative disorders such as Alzheimer's disease72.

The prevention or retardation of these oxidation processes is essential for the

food producer and almost everyone involved in the entire food chain from “farm to

fork”. Various methodologies may be employed to inhibit oxidization, including

prevention of oxygen access, use of lower temperature, inactivation of enzymes

catalyzing oxidation, reduction of oxygen pressure and the use of suitable

packaging73. Another common method of protection against oxidation is to use

specific additives which inhibit or retard oxidation. These oxidation inhibitors are

generally known as antioxidants.

Antioxidants can be classified according to their mechanisms of action as

either primary or secondary antioxidants. However, some substances have more than

one mechanism of antioxidant activity and are referred to as multiple-function

antioxidants.

40

2.6.4. Primary Antioxidants

Primary or chain-breaking antioxidants retard lipid oxidation by interfering

with chain propagation, initiation, reactions by accepting free radicals and forming

stable free radicals that do not further promote initiation or propagation reactions66.

For a primary antioxidant to be effective it must inactivate free radicals before they

can attack unsaturated fatty acids. It is believed that free radical scavenging

antioxidants interact mainly with peroxyl radicals (LOO•).

Although, there are many synthetic free radical scavenging antioxidants

(BHA, BHT, PG and TBHQ), that are efficient and relatively cheap these synthetic

compounds are “label unfriendly” additives. Special attention has been given to the

use of natural free radical scavengers due to the worldwide trend to avoid or minimize

the use of synthetic food additives. Many natural free radical scavengers such as

catechins (catechin, epicatechin, epigallocatechin, epicatechin gallate,

epigallocatechin gallate) also have health benefits as inhibitors of biologically harmful

oxidation reactions in the body75, 76, &77. Therefore, a better understanding of the

properties of natural free radical scavengers could lead to the development of food

additives that could both prevent oxidative deterioration of foods while also providing

health benefits. Taking advantage of rancidity prevention and health promoting

properties of natural free radical scavengers could help overcome their limitation in

effectiveness, price, associated flavours and colours.

2.6.5. Secondary Antioxidants

Secondary antioxidants prevent the formation of free radicals in the initiation

step by scavenging oxygen, e.g. ascorbic acid or chelating metal ions, e.g. citric acid

and flavonoids78. Some phenolic antioxidants have synergistic effects with ascorbic

41

and citric acids61. Ascorbic acid is primarily an effective oxygen scavenger21, but

when used in combination with tocopherol gives rise to a stronger synergistic

antioxidant effect. The synergistic effect of ascorbic acid with tocopherol is believed

to be due to the ability of ascorbic acid to regenerate tocopherol by reducing the

tocopheroxyl radical resulting from the primary antioxidant effect of the tocopherol78.

2.6.6. Chelators

Transition metals are prooxidants capable of accelerating lipid oxidation

reactions. Chelators are a group of secondary antioxidants that can bind and thus

inactivate or reduce the activity of prooxidant metals. The most common food

chelators are citric acid (and its lipophilic, mono glyceride ester), phosphoric acid

(and its polyphosphate derivatives), and ethylene diamine tetra acetic acid (EDTA). It

should be noted that some chelators can be ineffective when used alone but can

greatly enhance the action of phenolic free radical scavengers when used in

combination. In addition, some chelators may increase oxidative reaction under

certain conditions by increasing metal solubility or altering the redox potential of the

metal 79, 80 For instance, the anti oxidative properties of EDTA is determined by its

concentration in relation to prooxidant metals, and can act as a prooxidant when the

ratio of EDTA to iron is less than 1, and an antioxidant when the ratio is equal or

greater than 180.

2.6.7. Oxygen Scavengers and Reducing Agents

Oxygen is a substrate in lipid oxidation reactions. Ascorbic acid, ascorbyl

palmitate, erythorbic acid, sodium erythorbate, and sulfites are able to scavenge

oxygen and act as reductants. Ascorbic acid and sulfites retard lipid oxidation by

reacting directly with oxygen to eliminate it from food. In recent years, oxygen

42

scavengers have been directly incorporated into packaging materials, however, these

technologies are still too expensive for most food packaging applications.

2.6.8. Singlet Oxygen Quenchers

Singlet oxygen (1O2) is an excited state of oxygen that can be formed by

enzymatic reactions in biological systems or in the presence of a photosensitizer, light

and triplet oxygen. Singlet oxygen can be inactivated through both chemical and

physical quenching pathways by compounds such as tocopherols, carotenoids

(including β-carotene, lycopene, and lutein), polyphenols (including catechins, and

flavonoids), amino acids, peptides, proteins, urate, and ascorbate79.

2.6.9. Interactions between Antioxidants and Multiple Antioxidant Functions

It has been reported that when two antioxidants are used together, a synergistic

or additive effect can be observed. Vitamin E and vitamin C are a well-known

example of a synergistic system. Vitamin E acts as a lipid soluble chain-breaking

antioxidant and vitamin C reduces the tocopherol radical back to its original state so

that it can continue to inactivate free radicals in the lipid phase. If a chain-breaking

and a secondary antioxidant are mixed, both initiation and propagation are

suppressed. Thus, a combination of chelators and free radical scavenging antioxidants

are often very effective. For instance, chelating antioxidants such as citric or other

acids can enhance the activity of phenolic antioxidants. A synergistic relationship

between ascorbic acid and phosphates in retarding lipid oxidation was also observed

in meat.

Antioxidants can also have multiple functions such as free radical scavenging

and metal chelation. Examples include propyl gallate (PG), proteins81 and

43

proanthocyanidins82. Ascorbic acid is also a multifunctional antioxidant in food

systems. It can quench singlet oxygen, reduce free radicals and antioxidant radicals,

and remove molecular oxygen in the presence of metal ions 83.

The antioxidant principles were characterized as phenolic compounds and

phospholipids84. The phenolics were assumed to be mainly gallic acids and gallates.

In another study, gallotannins and condensed tannin-related polyphenols were

reported in mango kernels85. Ethanolic extracts of mango kernels displayed a broad

antimicrobial spectrum and were more effective against Gram-positive than against

Gram-negative bacteria. Their active component was shown to be a polyphenolic-type

structure; however, its exact nature still remains to be elucidated86.

2.7. NATURAL ANTIOXIDANTS

2.7.1. Natural versus synthetic antioxidants

Plants contain a variety of substances called “Phytochemicals” that owe to

naturally occurring components present in plants. The phytochemical preparations in

preventing lipid oxidation have tremendous potential for extending shelf life of food

products. Several research groups around the world have succeeded in finding and

identifying natural antioxidants from herbs and spices using different model

systems87.

Increased intake of dietary antioxidants may help to maintain an adequate

antioxidant status, defined as the balance between antioxidants and oxidants in living

organisms88. Antioxidants are widely used in the manufacture, packaging and storage

of lipid-containing foods. Much interest has developed during the last few decades in

naturally occurring antioxidants because of the adverse attention received by synthetic

44

antioxidants, and also the worldwide trend to avoid or minimize the use of artificial

food additives89. Halliwell90 (1989) defined an antioxidant as any substance that when

present at low concentrations compared to those of an oxidizable substrate

significantly delays or prevents oxidation of that substrate.

The primary purpose of adding antioxidants to lipids is to delay the onset of

oxidation and accumulation of oxidative products91. Antioxidants may delay or inhibit

oxidation, but cannot improve the quality of an already oxidized product92.

Antioxidants may act either as primary (chain breaking) or as secondary (preventive)

antioxidants. Some phenolic antioxidants are classified as synergists due to their

prominent effectiveness in the presence of other non-phenolic substances such as

citric acid.

Hundreds of materials, both synthetic and of natural origin, have been

developed as antioxidants for food preservation, but only BHA and BHT as synthetic

antioxidants and tocopherols as natural ones are practically used. Synthetic

antioxidants such as BHT, BHA, TBHQ and PG are currently widely used in the food

industry to increase the oxidative stability of fats and oils 93, 94. However, the most

commonly used synthetic antioxidants, BHA, BHT, PG and TBHQ are faced

resistance in recent times due to accumulating evidence that they could be toxic,

carcinogenic or mutagenic96, 97, 98, 99 and also suspected of causing liver damage.

There has been a growing interest in the exploration of natural antioxidants as food

additives due to these safety concerns.

On the other hand, tocopherols are widely used as safe natural antioxidants,

but they are not as effective as synthetic antioxidants and the manufacturing cost is

45

high. These circumstances stimulated the isolation of new antioxidants from natural

sources, especially from plant materials. They are added to an extensive variety of

foods in order to prevent or retard oxidation. Since continued use of chemical

antioxidants such as BHA and BHT has been reported to cause teratogenic and

carcinogenic effects in small animals and primates, commonly used vegetable leaves

which are rich in antioxidants would probably prove safe to the health of the

consumers.

Shi99 (2001) stated that natural antioxidants are safer, more potent and more

efficient than synthetic antioxidants. For example, ∝-tocopherol (the most active form

of vitamin E) is more effective than synthetic racemic ∝-tocopherol, primarily

because ∝-tocopherol transfer protein selectively recognizes natural ∝- tocopherol. In

addition, the possible activity of synthetic antioxidants as promoters of carcinogenesis

has become a concern. Therefore, replacing synthetic antioxidants with natural

alternatives, or simply replacing all synthetic food additives with natural choices, has

attracted great interest over the past two decades100.

2.7.2. Plants as sources of antioxidants

Antioxidant properties of common spices and herbs were systematically

investigated in the 1950s. Since then, numerous studies have been devoted to

vegetable sources, not only for their possible utilization as antioxidative extracts but

also to look for new, naturally efficient antioxidants. Several antioxidants have been

characterized from plant leaves and some products or extracts proved to be as good as

the synthetic BHA and BHT101.

46

Natural antioxidants may be found in any plant part. Fruits, vegetables, spices,

nuts, seeds, leaves, roots and barks have been considered as potential sources of

natural antioxidants102. Antioxidants in flaxseed, sunflower, soybean, cottonseed and

canola typify those found in oilseeds. The majority of natural antioxidants are

phenolic compounds, and the most important groups of natural antioxidants are the

tocopherols, flavonoids and phenolic acids that are common to all plant sources103.

Naturally occurring antioxidants are presumed safe since they are found in

food that has been used by man for centuries. The natural antioxidants in food may

also be beneficial because there is evidence that they prevent cancer development and

that they inhibit oxidative reactions in the body system.

Phenolic compounds have both primary and secondary antioxidant activities

by acting both as chain breaking antioxidants by donating hydrogen atoms to lipid

radicals (radical scavenging) or by preventing initiation reactions by such means as

chelating metals104,105,106. Both the chain breaking (radical scavenging) and metal

chelating properties are known to be possible due to the structural features of the

phenolic compounds.

Phenolic extracts from several plant sources have been shown to have

antioxidant activities. These include potato peels107, tomatoes 108, garlic and ginger109,

sunflower seed, wheat germ, fruits, vegetables and medicinal plants110, rape seed111,

tea91, sage and thyme67, plums112, grape marc26, red clove113 and tree spinach46.

Thompson grape Bagasse is byproduct with high phenolic compounds and is a source

of natural antioxidant20. Phenolic compounds from sorghum such as condensed

47

tannin114 flavones (such as naringen) 115 and anthocyanins116 have also been reported

to possess AA.

2.7.3. Factors affecting AA of phenolic compounds

Polymeric polyphenols are more potent antioxidants than simple monomeric

phenolics; Hagerman114 (1998) demonstrated the higher antioxidant ability of

condensed and hydrolysable tannins at quenching peroxyl radicals over simple

phenols; Yamaguchi82 (1999) observed that the higher the polymerization degree of

flavanols, the stronger the superoxide-scavenging activity. A similar effect was

reported for the capacity to inhibit the O2 radical, which increased with the degree of

procycanidin polymerization117 or for the stronger inhibition of lipid peroxidation by

dimers of ferulic than by ferulic acid118. As a general trend, improved stabilization of

the phenoxyl radical is desirable, but the lipohilic nature of the molecules and the

affinity of the antioxidant for the lipids could be determinant43. Also the anti

lipoperoxidant effect depends on the number and position of hydroxyl and methoxyl

groups in the benzene ring and on the possibility of electron delocalization in the

double bonds118. The presence of sugar substituent of flavonols from different

vegetables has been demonstrated to significantly affect AA of flavonols119, 120.

Methoxylation of hydroxyl groups (introduction of a –OCH3 group into the

phenolic structure, replacing the OH group) decreases AA. This reduction in the AA

may be attributed to differences in redox potential of the OH groups and the

substituted groups (such as the –OCH3 group) on the phenyl ring. Hydroxyl groups

have lower redox potential, which makes them good electron-donating groups (i.e.

good reducing agents), hence increasing the efficacy of phenolic antioxidants.

Substituted groups such as-OCH3, however, increase the redox potential of phenols,

48

hence reducing their antioxidant potential due to a reduction in their ability to donate

electrons121.

Phenolic compounds, flavonoids in particular, are able to chelate metals due to

the presence of hydroxyl groups attached to their ring structures104, 105 & 106. Phenolic

compounds are compartmentalized in the cell matrix, but once the cell matrix is

broken during the extraction of phenolic compounds, the compounds become prone to

degradation. It has been shown that temperature123, light124, oxygen125, enzymes126

and pH127 affect stability of phenolic compounds. The changes that occur may in turn

affect the antioxidant properties of the phenolic compounds.

Phenolic compounds can be degraded through enzymatic or chemical

oxidation that may occur during the extraction process. Plant tissues contain the

enzyme polyphenol oxidase, which when in contact with phenolic compounds in the

presence of oxygen as the plant tissues disintegrate, may catalyze the oxidation of the

phenols to quinines126. The phenolic compounds can also be oxidized non-

enzymatically in which the phenol group can be transformed into a quinine upon

exposure to air. The quinones formed can subsequently undergo rapid non-enzymatic

polymerization that would change the original nature of the phenolic compounds,

hence causing changes in its chemical properties. For instance, the disintegration of

plant tissues in potatoes releases polyphenol oxidase, which reacts with phenolic

compounds in the presence of oxygen resulting in the formation of polymerized

brown compounds (enzymatic browning).

Several isolated plant constituents as well as crude extracts of vegetables and

fruits have been recognized to possess beneficial effects against free radicals in

49

biological systems as antioxidants. The beneficial effect of fruits and vegetables can

be attributed to the antioxidant capacity of polyphenols present in them. Green leafy

vegetables form a rich source of dietary fibres, nutraceuticals and vitamins, eg

carotene (a precursor of vitamin A), vitamin C, riboflavin, folic acid and minerals, eg

calcium, iron and phosphorous128, 129. Epidemiological studies have shown that many

phytonutrients of fruits and vegetables might protect the human body against damage

by reactive oxygen species. The consumption of natural antioxidant phytochemicals

was reported to have potential health benefits130. In recent years, there has been a

considerable interest in finding natural antioxidants from plant materials. The

antioxidant phytochemicals from plants, particularly flavonoids and other

polyphenols, have been reported to inhibit the propagation of free radical reactions, to

protect the human body from disease and to retard lipid oxidative rancidity. In

addition, the use of synthetic antioxidants has been questioned because of their

toxicity131. Therefore, there have been numerous researches on these bio resources to

seek for potential natural and possibly economic and effective antioxidants to replace

the synthetic ones132.

Since fruits and leafy vegetables are a rich source of a number of

micronutrients and phytochemicals having antioxidant properties, these are protective

against chronic degenerative health disorders. The levels of antioxidants and

micronutrients may vary depending upon the type of fruit or vegetable, the variety and

agro-climatic conditions. Micronutrient content and antioxidant capacity are affected

by food processing and cooking, indicating the need for data on locally consumed

cooked preparations133.

50

The consumption of fruits and vegetables40 containing antioxidants has been

found to offer protection against diseases. Dietary antioxidants can augment cellular

defenses and help to prevent oxidative damage to cellular components90. Besides

playing an important role in physiological systems, antioxidants have been used in

food industry to prolong the shelf life of foods, especially those rich in

polyunsaturated fats. These components in food are readily oxidized by molecular

oxygen and are major cause of oxidative deterioration, nutritional losses, off flavour

development and discoloration. The search for antioxidants from natural sources has

received much attention and efforts have been put into identify compounds that can

act as suitable antioxidants to replace synthetic ones. In addition, these naturally

occurring antioxidants can be formulated as functional foods and nutraceuticals that

can help to prevent from oxidative damage occurring in the body.

Dietary antioxidants protect against free radicals such as reactive oxygen

species in the human body. Free radicals can cause damage to cellular bio-molecules

such as nucleic acids, enzymes, proteins, lipids and carbohydrates and consequently

may adversely affect immune functions. Fruits and vegetables are low in calories but

they provide significant contributions in the protection of health from harmful effects

of reactive oxygen species. They are good sources of natural antioxidants such as

vitamins, carotenoids and phenols. Several epidemiological studies have shown a

negative association between the intake of fruits, vegetables and certain diseases such

as cancer, cardiovascular disease, cataracts and so on134.

The key structural features that have been shown to enable phenolic

compounds exert RSA are as follows:

51

(i) The number of phenolic hydroxyl groups on the phenolic compound

molecule. Studies have shown that generally, the more the number of

hydroxyl groups on a phenolic structure, the higher the radical scavenging

capacity135, 136,137. More hydroxyl groups on a phenolic compound provide

more sites for radical scavenging, hence the increased AA of compounds with

multiple hydroxyl groups.

(ii) Presence of hydroxyl groups on carbon number 3 and 4 in the B ring (3’, 4’-

dihydroxyl catechol configuration) 138, 122. The strong RSA by the 3, 4’-

dihydroxyl group in the B ring is due to its ability to provide highly stable

phenoxyl radicals after the donation of a hydrogen atom139.

Tocopherols are considered the best known and most widely used

antioxidants140. They are classified into two groups, namely tocopherols and

tocotrienols. There are four isomers (∝-, β-, γ - and δ-) in each group, for a total of

eight tocopherol isomers. Tocopherols exist in nearly all parts of plants. Flavonoids

represent a large group of phenolics that occur naturally in plants and are found in

fruits, vegetables, grains, barks, roots, stems, flowers, tea and wine141. They are

characterized by the carbon skeleton C6–C3–C6. The basic structure of these

compounds consists of two aromatic rings linked by a three carbon aliphatic chain.

Several classes of flavonoids are delineated on the basis of their molecular structure,

but the four main groups that occur in plant tissues are flavones, flavanones, catechins

and anthocyanins141.

Phenolic acids and their derivatives occur widely in the plant kingdom, e.g.,

legumes, cereals, fruits and plant products such as tea, cider, oil, wine, beverages and

medicinal plants142. Phenolic acids can be found in free and conjugated forms in

52

cereals103. They are present in highest concentration in the aleurone layer of grains,

but are also found in the embryo and seed coat. The level of phenolics in plant sources

also depends on such factors as cultivation techniques, cultivar, growing conditions,

ripening process, processing and storage conditions, as well as stress conditions such

as UV radiation, infection by pathogens and parasites, wounding, air pollution and

exposure to extreme temperatures103.

2.7.4. Recent advances in research on natural antioxidants

Recently, research on phytochemicals and their effects on human health have

been intensively studied. In particular, research has focused on a search for

antioxidants, from vegetables, fruits, leaves, tea, spices and medicinal herbs. Since

fruits and leafy vegetables are a rich source of a number of micronutrients and

phytochemicals having antioxidant properties, these are protective against chronic

degenerative health disorders. Several studies have analysed the antioxidant potential

of a wide variety of vegetables and particularly of cocoa beans143, potato144, tomato8,

spinach145, legumes such as Phaseolus vulgaris146 or vegetable such as paprika147. The

levels of antioxidants and micronutrients may vary depending upon the type of fruit or

vegetable, the variety and agro-climatic conditions. Both natural extracts and

commercial products from garlic and ginger148, rosemary149, dietary supplements150,

drinks151, seashore plants152 and seaweeds153 were evaluated for AA.

A number of studies deal with the AA of extracts from herbs, medicinal plants

and spices. Other potential vegetable sources, such as trees, have been evaluated for

antioxidant compounds154. Among the different parts of the plants, leaves deserve

special attention e.g., those from green barley155 or avocado156. Seeds are another

source of antioxidants as reported for tamarind 157, canola158, sesame159, evening

53

prime rose160, buckwheat, sunflower161. Also in cereals162 as in recent study on corn

kernel163, AA was detected.

Hulls contain compounds with antioxidant activity 164 .Active compounds were

detected in hulls from peanut165, mung bean166 and buck wheat167. During the

extraction of oil from oilseeds, the antioxidant compounds present in the hulls could

be incorporated in the oil, as reported for peanut oil extracted from coated seeds,

which contained higher oxidative stability than the oil from dehulled seeds. Baublis162

(2000) found higher inhibition of iron accelerated oxidation of phosphotidylcholine

liposomes for water soluble from high bran wheat varieties than from refined wheat.

The outer layers usually contain a greater amount of polyphenolic compounds, as

expected from their protective function in the plants. Agricultural and industrial

residues are attractive sources of natural antioxidants. Potato peel waste101, skin of

olive168 fruits, olive mill waste waters169, grape seeds170 and grape pomace171 have

been studied as cheap sources of antioxidants and recently increased AA in rat

plasma after oral administration of grape seed extracts was reported. Identification of

polyphenolic compounds from apple pomace172, grape pomace171, citrus seeds and

peels173, carrot pulp waste174, old tea leaves 175, cocoa byproducts25, non volatile

residue from orange essential oil176 and soybean molasses177 has also been reported.

Spent ground coffee oil from the residue from the production of instant coffee was

used to obtain an antioxidant product useful for food preservation and for aroma

stabilization, the AA being due to the 5-hydroxytryptamide carboxylic acids178.

Maillard reaction products were also reported as antioxidant agents. The derivation of

natural products with AA from brewing seeds, grains and or germs has been claimed

by Niwa179 (1991).

54

Among all, mango (Mangifera indica L.) is one of the most important tropical

fruits worldwide in terms of production and consumer acceptance180. It is a rich

source of antioxidants181, 182 including ascorbic acid183, carotenoids184 and phenolic

compounds185, 186, 187. Flavonol and xanthone glycosides as well as galactonnins and

benzophenone derivatives, have been demonstrated to be present mainly in the peels

and seeds and pronounced inter-varietal differences have been observed in terms of

the quantitative composition of these compounds 185,186,188. Mango peels were also

reported to be a good source of dietary fiber containing high amounts of extractable

polyphenolics189. This appreciable content was reflected by high in vitro AA190.

However, since polyphenolics were determined by the FC assay, no conclusions could

be drawn as to their chemical structures. Furthermore, owing to their lack of

selectivity, spectrophotometric methods tend to over-estimate the phenolic content191.

Raw mango has highest phenols compared to ripe whereas carotenoid content was

high in ripe mangoes192. The presence of a broad pattern of phenolic compounds

especially of flavonol glycoside, in mango puree concentrate was demonstrated by

HPLC with diode array and mass spectrometric detection187. It was hypothesized that

these phenolics may partly originate from the peel since mango puree is prepared both

from peeled and unpeeled fruits. Preliminary investigations on peel phenolics of

different mango cultivars confirmed this assumption. Antioxidant compounds

associated with some types of dietary fibers may be responsible in part for the

beneficial effects on health of high dietary fiber diets. AA of a high dietary fibre

mango peel product and of some commercial samples was determined by the FTC

colorimetric method. All Bran, Quaker Oats, lemon and apple fiber did not exhibit

any antioxidant capacity. The production of high dietary fibre products with bioactive

55

compounds could be useful for the food industry and AA may be a new property to

consider in the quality evaluation of these ingredients.

The presence of phenolic compounds in the human diet is associated with

protective effects against some chronic-degenerative diseases related to oxidative

stress193. Flavonols have potent antioxidant194, anticarcinogenic195 and antiatherogenic

activities196. Mangiferin, a xanthone-C-glycoside, has attracted intense interest for its

variety of pharmacological properties, including antioxidant197, antitumor and

antiviral198.

A severe problem associated with the mango processing industry is the

production of large quantities of waste and by-products, in particular peels and seeds.

The main reason to exploit the potential use of the mango peel and kernel are the

environmental consequence caused by the generation of agro-industrial residue by

factories making the juice. These waste and by-products have been shown to

constitute a rich source of polyphenolics which could be used as neutraceuticals and

/or natural antioxidants to replace some synthetic food additive199 & 200 and as cancer-

preventing agents.

Mango kernels were extracted with different organic solvents and phenolics

and phospholipids were studied for AA separately in buffalo ghee in comparison with

BHA at 0.02%. Phenolics and phospholipids showed better activity when added

jointly. PV was tested for stored oil which showed low PV in joint addition84. The

antioxidant effect of mango peel and kernel was due to their high content of

polyphenols, sesquiterpenoids, phytosterols and microelements like selenium, copper

56

and zinc 200,201, however little is known about AA and phenolic profile of mango peel

and kernel.

Pomegranate juice has proved to be a very strong anti-oxidant; however, the

rind/peel is also in possession of life saving chemical properties202. The pomegranate

peel extract (PPE), when introduced into juice, improves the process intensity due to

acceleration of deposit precipitation of the haze-forming substance203. The presence of

antioxidants has been reported from pomegranate juice202. The much less acclaimed

pomegranate rind contains certain compounds that assist in AA204, gastroprotective

effects, antifungal activities205, antimicrobial action206, antiviral effects and

hypoglycemic effects207.

Some tannins are also thought to have strong antioxidant effects as well.

Pomegranates contain particularly a beneficial type of tannin known as an

ellagitannin, which are water-soluble combinations of ellagic acid and glucose that are

more digestible for animals. Ellagic acid is perhaps the most important compound

found in the pomegranate. Recent studies have exposed many of its advantageous

effects. It also acts as a scavenger, binding free-radicals and preventing oxidative

damage. In addition, ellagic acid derived from elagitannins hinders DNA mutation by

masking mutagen binding sites. The other compounds found in the rind of the

pomegranate, such as the flavanols contribute greatly to its many activities as well.

Flavanols have proved to promote cardiovascular health and reduce the severity of

thrombosis. A number of other activities are attributed to them as well. Improvements

in antioxidant defense systems, blood pressure, insulin resistance and glucose

tolerance are only some of the contributions of flavanols.

57

The combined mechanism of the many compounds in pomegranate rind has

manifested itself into an anti-diabetic effect. The National Organization for Drug

Control and Research discovered that pomegranate rind extract can lower blood

glucose levels and increase insulin levels in diabetic rats. The extract showed

significant effects on rat beta cells, the main cells destroyed in diabetes. The strong

evidence determined that "PPE can reduce blood sugar through regeneration of B cells

in the pancreas". This is just one mechanism amongst numerous others of

pomegranate rind that benefits the body. Pomegranate rind and its properties can

support a healthier body and lifestyle.

Areca nut extract suppresses T-cell activation and interferon-gamma

production via the induction of oxidative stress208. Methanolic extract of Areca

catechu showed anti-aging and strong scavenging activity against super-oxide anion

radical209. The areca nut contains tannin, gallic acid, a fixed oil gum, a little terpineol,

lignin, various saline substances and three main alkaloids: Arecoline, Arecaidine and

Guvacine which have vasoconstricting properties210, however little is known about

AA and phenolic profile of areca nut.

In the studies, the anti-diarrhoeal, antipyretic, antimicrobial and bio-

antimutagenic guava leaves have been used as health tea. Its leaf contains copious

amounts of phenolic phytochemicals which inhibit peroxidation reaction in the living

body, and therefore, can be expected to prevent various chronic diseases such as

diabetes, cancer and heart-disease. Furthermore, decreasing of free-radicals has

antioxidizing effect in the body, meaning these guava leaf polyphenols can prevent

arterial sclerosis, thrombosis, cataract and inhibit senescence of the body and skin211.

Many people habitually take medicinal decoction of guava leaf for long treatment of

58

diarrhea, and therefore, the safety of guava leaves have empirically been confirmed.

People in China use guava leaf as anti-inflammatory and haemostatic agent. It was

reported that the leaves of guava contain an essential oil rich in cineol, tannins and

triterpenes. In addition, three flavonoids (quercetin, avicularin, and guaijaverin) have

been isolated from the leaves. Lee212 (2003) have screened out methanolic extracts of

nine medicinal plants traditionally used in Chinese medicine for AA versus

resveratrol which has been shown to protect cells from oxidative damage.

Work showed that curry leaf has unusually high antioxidant activity compared

to other leafy vegetables. Antioxidant role of curry and beetle leaves in ghee by direct

extraction has been examined213. The antioxidant potential of carbozole alkaloids

from oleoresin of curry leaves procured from southern states of India has been

reported by Rao214 (2000). Their properties include much value as an antidiabetic215,

antioxidant216, antimicrobial, anti-inflammatory, hepatoprotective, anti-

hypercholesterolemic etc.

Betel leaves are traditionally added to butter while converting into ghee and in

some of the oily preparations in some parts of our country. These treatments are

reported to enhance the shelf life of the products. It is reported that the AA of Piper

betel has more potential to prevent lipid peroxidation than tea. The alkaloid arakene in

it has properties resembling cocaine in some respects.

2.8. FACTORS INFLUENCING THE RATE OF OXIDATION OF LIPID

FOODS

Oils that are more unsaturated are oxidized quickly than less unsaturated217. As

the degree of unsaturation increases, both the rate of formation and the amount of

59

primary oxidation compounds accumulated at the end of the IP increases218. Crude oil

contains free fatty acids, and oil processing, such as refining, decreases the free fatty

acid content219. But, processing of crude oils to refined oils is generally performed at

high temperature, and it can produce oxidized compounds such as cyclic and

noncyclic carbon-to-carbon-linked dimers and trimers, hydroxy dimers, and dimers

and trimers joined through carbon-to-oxygen linkage.

The oxidation of oil is influenced by the fatty acid composition of the oil, oil

processing, energy of heat or light, the concentration and type of oxygen, and free

fatty acids, mono- and diacylglycerols, transition metals, peroxides, thermally

oxidized compounds, pigments, and antioxidants. These factors interactively affect

the oxidation of oil and it is not easy to differentiate the individual effect of the

factors.

Edible fats and oils deteriorate faster when exposed to light due to its catalytic

effect on lipid oxidation220. Suzuki221 (1996) reported that lipid peroxides

decomposed faster at higher temperatures(370C) than at lower temperature(40C),

which was confirmed by a corresponding increase in levels of carbonyls formed at

higher temperature. Naz222 (2004) reported higher oxidative rates of soybean, corn and

olive oil used for deep-frying at 1800C than oils exposed to air and light. Lowering the

temperature could, therefore, reduce oxidation by slowing down oxidative reactions;

hence, fats and oils kept at low temperatures would be less prone to oxidation.

The study of Azizah25 (1999) also proved that temperature and light are major

factors influencing antioxidant activity during storage. Various studies have shown

this catalytic effect of light on lipid oxidation223. The catalytic effect is due to the

60

presence of natural pigments such as chlorophyll in a fat or oil, which act as

photosensitizers and transfer energy (hv) from light to ground state triplet

oxygen(3O2) to form singlet oxygen,(1O2) 220.

Fats and oils deteriorate in quality with time. Changes that occur during

deterioration include absorption of odours from surroundings (tainting), lipolysis and

oxidation224. Lipolysis is the enzymatic hydrolysis of ester linkages of lipids to release

free fatty acids, resulting in the development of lipolytic or hydrolytic rancidity of the

fats15. Oxidative rancidity occurs when fats containing PUFA are oxidized, resulting

in the breaking of the double bond of the unsaturated fatty acid molecules225 and

subsequent formation of various carbonyl compounds, hence a reduction in shelf life

of oil. Oxidative rancidity is, therefore, one of the major causes of lipid deterioration

leading to the development of off-flavours and off-odours in edible oils and other

lipid containing foods, which may also reduce the nutritional quality of foods226.

Fats and oils with a high degree of unsaturation are more susceptible to lipid

oxidation69. According to the degree of unsaturataion of fatty acids, the relative rates

of oxidation for oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and arachidonic

acids (C18:4) are 1, 12, 25 and 50, respectively227. Chu228 (1998) found the oxidative

stability of various oils (soybean, high oleic safflower, corn, olive and peanut) to be

inversely related to the PUFA content, with linolenic acid (C18:3) having the most

influence on the oxidative stability index, followed by linoleic acid (C18:2) and oleic

acid (C18:1).

61

2.9. OXYGEN CONCENTRATION AND EXPOSED SURFACE AREA OF

THE LIPID

Availability of oxygen determines the rate of oxidation. This is true at low

oxygen concentrations where the rate of oxidation increases with an increase in

oxygen concentration, unlike when oxygen is abundant where the oxidation becomes

independent of oxygen concentration69. Koelsch229 (1991) showed that during the

oxidation of soybean oil at 2300C, the formation of hexanal (an important volatile

product of oxidation commonly used to determine the extent of lipid oxidation), as a

function of time, increased with increasing oxygen concentrations. The rate of

oxidation also increases with the surface area of the lipid that is exposed to air. Tan230

(2002) observed that oils with a high surface -to-volume ratio had lower oxidative

induction times than oils with a lower surface-to-volume ratio because of the fact that

a larger surface area of the oil is more exposed to oxygen.

The storage stability of edible vegetable oil has important bearing on the

safety aspects of human health as because the oxidation products are well known to

cause damage to our body cells resulting in large numbers of diseases. Hence,

enrichment of edible oils with natural antioxidants and not synthetic antioxidants will

ensure nutritional benefit to the people.

2.10. ASSESSMENT OF QUALITY OF OILS

One of the most important indicators of the quality of oils is their oxidative

stability230 which is the resistance of oil to oxidation231. Methods used to determine

the extent of oxidation of oils are based on the measurement of the concentration of

the primary or secondary products of oxidation. Various methods such as OSI232,

Differential Scanning Calorimetry (DSC)233, 234, Rancimat method229, the

62

measurement of conjugated Diene hydroperoxides, volatile compounds such as

aldehydes, anisidine values(AV) 67, 233 and the measurement of PVs 67, 233 have been

used for assessing the extent of oxidation of edible oils. These methods employ

accelerated oxidation conditions such as exposing the oil at a determined constant

temperature, flow of air or the presence or absence of light and catalysts231 and

assessing the extent of oxidation. The measurement of PV and AV can be used to

monitor the formation of primary and secondary oxidation products respectively, as a

means of determining the quality of oils.

No off flavours were detected in sunflower oil stored for two years at room

temperature even when the PV increases from 8.5 to 22.6. Yousuf Ali Khan235 (1979)

noted development of detectable off-odours in sunflower oil stored at ambient

temperature when PV reached 25meq/kg oil. On the other hand, at higher storage

temperatures rancidity is detected at lower PVs. The average PV of soybean, low

erucic acid rapeseed and sunflower oil after eight days of storage at 600C when they

were perceived rancid were 8.3, 11.0 and 13.6 respectively236. In general, it appears

that at a higher storage temperature rate of decomposition of hydroperoxides is much

faster, which could lead to early development of rancidity than at lower

temperatures236.

Another consideration worth noting when interpreting PVs and development

of rancidity in oil samples is that a low PV may not always be an indication of good

quality oil. It is well known that during oxidation of fats and oils, the initial rate of

formation of hydroperoxides exceeds the rate of their decomposition and the relative

rates of these reactions are reversed during the later stages of oxidation237. According

to Dugan15 (1976) and O’Brien238 (2004), measurement of PV may be useful during

the initial stages of oxidation before extensive decomposition of hydroperoxides

begins.

63

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