effect of heat treatment on α-tocopherol content and...

Effect of Heat Treatment on α-Tocopherol Content and Antioxidant

Activity of Vegetable Oils

By

Hasan Al-attar

Department of Food Science and Agriculture Chemistry

Macdonald Campus, McGill University,

Montreal, Quebec

A thesis submitted to McGill University in partial fulfillment of the

requirements of the degree of Master of Science

June, 2013

©Hasan Al-attar, 2013

ii

Suggested short Title:

α-Tocopherol content and antioxidant activity of oils

iii

ABSTRACT

The objective of this research was to investigate the effect of heating on α-

Tocopherol content and antioxidant activity of different vegetable oils (EVOO, canola

and palm oil). The highest α-Tocopherol content was found in EVOO followed by canola

oil and palm oil (323 ±5, 271 ±2 and 174 ±2 µg/ml) respectively. The effect of heat was

done at 70, 100 and 130 oC, for time intervals of 0.5, 1, 1.5 and 2 h. Thermal degradation

of α-Tocopherol in the oils was minimal at 70 oC and increased at 100

oC and 130

oC.

Heating at 130 oC for 2 h resulted in 100, 24 and 44 % degradation of α-Tocopherol in

EVOO, canola oil and palm oil respectively; EVOO was completely degraded after 1.5 h

heating at 130 oC. Use of 2 cooking methods, pan-frying (250

oC, 5 min) and oven

cooking (130 oC, 30 min) resulted in the degradation of α-Tocopherol in the oils. In the

pan-frying method, both EVOO and palm oil were completely degraded and canola oil

showed 42 % degradation. .With the oven cooking method the degradation for EVOO,

canola oil and palm oil were 18, 13 and 10 %, respectively. The antioxidant activity was

highest with canola oil followed by palm oil and EVOO (59 ±1.72, 51 ±0.84 and 46 ±0.91

%), respectively. At 70 oC there was no significant decrease in the antioxidant activity of

the heated oils. At 100 oC, EVOO showed highest reduction in antioxidant activity

followed by canola oil and palm oil. At 130 oC, the antioxidant activity decreased

gradually in the oil samples. The highest decrease was observed with EVOO followed by

canola oil and palm oil. The decrease of antioxidant activity in oil samples was also

observed with both pan-frying and oven cooking methods, with greater reduction in

antioxidant activity using the pan-frying method.

iv

RESUME

L’objectif de cette recherche a été d’étudier l’effet de la chaleur sur la quantité

d’α-tocophérol et sur l’activité antioxydant de différentes huiles végétales (l’huile d’olive

extra vierge, l’huile de canola, et l’huile de palme). La quantité la plus élevée d’α-

Tocophérol a été trouvée dans l’huile d’olive extra vierge, suivie par l’huile de canola et

l’huile de palme (323 ±5, 271 ±2 and 174 ±2 µg/ml) respectivement. L’effet de la chaleur

a été étudié à 70, à 100 et à 130 o

C durant 0.5, 1, 1.5 et 2 h. La dégradation thermale d’α-

Tocophérol dans les huiles a été minimale à 70 °C et a augmenté à 100 et 130 °C.

Chauffer à 130 o

C durant 2 h a mené à la dégradations de 100, 24 et 44 % d’α-Tocophérol

dans l’huile d’olive extra vierge, dans l’huile de canola, et dans l’huile de palme,

respectivement; l’huile d’olive extra vierge a été complètement dégradée après 1.5 h de

chauffage à 130 o

C. L’utilisation de deux différents façons de cuire, l’utilisation de la

poêle (250 o

C, 5 min) et l’utilisation du four (130 oC, 30 min), a mené à la dégradation

d’α-Tocophérol dans les huiles. En utilisant la poêle, l’huile d’olive extra vierge et l’huile

de palme ont été complètement dégradées et l’huile de canola a démontré une dégradation

de 42 %. En utilisant le four, la dégradation de l’huile d’olive extra vierge, de l’huile de

canola, et de l’huile de palme a été de 18, 13 et 10 % respectivement. L’activité

antioxydant des échantillons a été le plus élevé avec l’huile de canola, suivi par l’huile de

palme et par l’huile d’olive extra vierge (59 ±1.72, 51 ±0.84 et 46 ±0.91 %),

respectivement. À 70 o

C, il n’y avait pas de réduction significative dans l’activité

antioxydant des huiles chauffées. À 100 o

C, l’huile d’olive extra vierge a démontré une

réduction maximale en activité antioxydant suivi par l’huile de canola et par l’huile de

palme. À 130 o

C, l’activité antioxydant des huiles a baissé graduellement. La réduction la

plus élevée a été observée avec de l’huile d’olive extra vierge, suivie par l’huile de canola

et par l’huile de palme. La réduction en activité antioxydant dans les échantillons d’huile

a été aussi observée avec les deux façons de cuire, d’où une réduction plus importante en

activité antioxydant a été observée en utilisant la poêle.

v

ACKNOWLEDGMENT

This thesis would not have been possible to complete without the support of my

supervisor Dr. Inteaz Alli. I would like to thank him for his excellent guidance, patience

at all times, encouragement as well as for his academic advice, and friendship. Also, I

would like to thank his family for their support too.

I take this opportunity to thank Dr. Selim Kermasha for providing me with the

necessary resources to accomplish my work and finish my experiments, Sarya Aziz for

her help and support in mastering HPLC use, Dr. Salwa Karboune for allowing accessing

her laboratory facilities during my research, and also her students Amanda Waglay and

Sooyoun Seo for their positive collaboration, Dr. Varoujan Yaylayan and Dr. Hosahalli

S. Ramaswamy for permitting me to use their equipments. Finally I like to thank Dr.

Jasim Ahmed for his help.

Sincere thanks to my laboratory colleagues, Mohammed Hassan, Amal

Mohammed and Abdulaziz Gassas for their support, help and friendship.

I would also like to thank Kuwait institute for Scientific Research for allowing me

pursue my Master’s degree by granting me a scholarship Lastly I like to thank my family

for their support and encouragement.

vi

TABLE OF CONTENT

ABSTRACT…................................................................................................iii

RESUME……… ............................................................................................ iv

ACKNOWLEDGMENT ................................................................................. v

TABLE OF CONTENT .................................................................................. vi

LIST OF TABLES ........................................................................................... x

LIST OF FIGURES ....................................................................................... xii

CHAPTER 1….. .............................................................................................. 1

1. INTRODUCTION .................................................................................... 1

1.1 General Introduction ............................................................................ 1

1.2 Research Objectives ............................................................................ 1

CHAPTER 2….. .............................................................................................. 2

2. LITERATURE REVIEW ......................................................................... 2

2.1 Vitamin E components ........................................................................ 2

2.2 Vitamin E sources ............................................................................... 3

2.3 Vitamin E and human health ............................................................... 5

2.3.1 Vitamin E and enzyme inhibition and activation .......................... 5

2.3.2 Other functions of vitamin E ......................................................... 6

2.4 Vitamin E and free radical ................................................................... 6

2.4.1 Free radical chain reaction ............................................................ 8

2.5 Vitamin E antioxidant function ........................................................... 8

2.6 Vitamin E effect on diseases ............................................................... 9

vii

2.6.1 Vitamin E and Cardiovascular disease .......................................... 9

2.6.2 Vitamin E and hypertension ........................................................ 11

2.6.3 Vitamin E and diabetes ............................................................... 12

2.7 Antioxidant content in food ............................................................... 12

2.8 Antioxidants and free radicals ........................................................... 13

2.8.1 Antioxidant and Cardiovascular disease ..................................... 15

2.8.2 Antioxidant and hypertension ..................................................... 15

2.8.3 Antioxidant and cancer ............................................................... 15

2.9 Vegetable oils - Olive oil................................................................... 16

2.9.1 Types of olive oil......................................................................... 16

2.9.2 Olive oil vitamin E and phenolic content .................................... 16

2.10 Olive oil and heart disease risk factors............................................ 17

2.11 Vitamins with antioxidant properties status among the Kuwaiti

population ................................................................................................ 17

2.11.1 Vitamin E status ..................................................................... 17

2.11.2 Vitamin E sources .................................................................. 19

CHAPTER 3….. ............................................................................................ 21

MATERIALS AND METHODS ............................................................... 21

3.1 Materials ............................................................................................ 21

3.2 Preparation of standard solutions and sample solutions ................... 21

3.3 Preparation of standard curve ............................................................ 23

3.4 Effect of heating on standard and oils ............................................... 23

3.5 Cooking of the food samples in oils .................................................. 23

3.6 High Performance Liquid Chromatography (HPLC) analysis .......... 23

viii

3.7 DPPH assay ....................................................................................... 24

3.8 Thermal degradation kinetics ............................................................ 26

3.9 Statistical analysis ............................................................................. 26

CHAPTER 4….. ............................................................................................ 27

RESULTS AND DISCUSSION................................................................. 27

4.1 Standard α-Tocopherol concentration curve ..................................... 27

4.2 Thermal degradation of standard α-Tocopherol and α-Tocopherol in

vegetable oils ........................................................................................... 29

i) Thermal degradation of standard α-Tocopherol .............................. 29

ii) Thermal degradation of α-Tocopherol in extra virgin olive oil

(EVOO) ................................................................................................. 33

iii) Thermal degradation of α-Tocopherol in Canola oil ..................... 37

iv) Thermal degradation of α-Tocopherol in Palm oil ........................ 41

4.3 Comparison of α-Tocopherol degradation in vegetable oils at the

same thermal treatments .......................................................................... 45

4.4 Thermal degradation of α-Tocopherol in vegetable oils during

cooking .................................................................................................... 48

4.5 Antioxidant activity: Standard curve for α-Tocopherol antioxidant

activity ..................................................................................................... 53

4.6 Antioxidant activity of standard α-Tocopherol and heated oils ........ 55

i) Antioxidant activity of standard α-Tocopherol ................................. 55

ii) Antioxidant activity in extra virgin olive oil (EVOO) ..................... 57

iii) Antioxidant activity in canola oil ................................................... 59

iv) Antioxidant activity in palm oil ....................................................... 61

4.7 Comparison of antioxidant activity of heated vegetable oils at the

same thermal treatments .......................................................................... 63

ix

4.8 Effect of cooking on antioxidant activity of oils ............................... 66

GENERAL CONCLUSION ......................................................................... 69

Appendix A…… ............................................................................................ 70

REFERENCES…. ......................................................................................... 72

x

LIST OF TABLES

Table 2.1 Selected food items and α-Tocopherol content ..................... 4

Table 2.2 Free radical species and formation ........................................ 7

Table 2.3 Mechanisms by which vitamin E inhibits atherosclerosis .. 11

Table 2.4 Antioxidants in low density lipoprotein (LDL) .................. 13

Table 2.5 Daily intake of vitamin E by gender and age ..................... 18

Table 2.6 Percentage of participants not meeting the estimated

average requirement (EAR) of vitamin E (mg) by gender

and age ................................................................................ 18

Table 2.7 Percentage contribution of foods to average daily vitamin E

intake by sex and age group ............................................... 20

Table 4.1 Effect of heating time and temperature on standard α-

Tocopherol concentration .................................................... 32

Table 4.2 Effect of heating time and temperature on α-Tocopherol

concentration in extra virgin olive oil ................................. 36

Table 4.3 Effect of heating time and temperature on α-Tocopherol

concentration in canola oil .................................................. 40

Table 4.4 Effect of heating time and temperature on α-Tocopherol in

palm oil ................................................................................ 44

Table 4.5 Effect of heating time and temperature on degradation of α-

Tocopherol in Standard α-Tocopherol and vegetable oils

(µg/ml) ................................................................................. 46

Table 4.6 Effect of cooking method on degradation of α-Tocopherolin

vegetable oils ....................................................................... 51

xi

Table 4.7 Antioxidant activity of standard α-Tocopherol at different

concentration ....................................................................... 53

Table 4.8 Effect of heating time and temperature on antioxidant

activity of standard α-Tocopherol (%) ................................ 56


activity of extra virgin olive oil (%) .................................... 58


activity of canola oil (%) ..................................................... 60


activity of palm oil (%) ....................................................... 62


activity of standard α-Tocopherol and vegetable oils (%) .. 64

Table 4.13 Effect of cooking method on antioxidant activity of

vegetable oils (%) ................................................................ 67

xii

LIST OF FIGURES

Figure 2.1 Forms of vitamin E ................................................................ 2

Figure 2.2 Various oils and Tocophenol content .................................. 3

Figure 2.3 2R and 2S stereoisomers of α-Tocopherol ............................ 5

Figure 2.4 Vitamin E regeneration cycle ................................................ 9

Figure 2.5 Formation of foam cell ........................................................ 10

Figure 2.6 Potential sources of reactive oxygen species (ROS) .......... 11

Figure 3.1 Diagram of procedure used for measuring α-Tocopherol in

oil samples ........................................................................... 22

Figure 3.2 Diagram of procedure utilized to measure the antioxidant

activity of α-Tocopherol and vegetable oils ........................ 25

Figure 4.1 HPLC chromatogram of standard α-Tocopherol (A) 20

µg/ml , (B) 30 µg/ml and (C) 40 µg/ml ............................ 28

Figure 4.2 α-Tocopherol standard concentration curve ....................... 28

Figure 4.3 HPLC chromatogram for (A) standard α-Tocopherol (B) α-

Tocopherol in EVOO (C) α-Tocopherol in palm oil and (D)

α-Tocopherol in canola oil .................................................. 30

Figure 4.4 Degradation of standard α-Tocopherol at (I) 70 oC, (II) 100

oC and (III) 130

oC (A) 0 time (B) 0.5 h (C) 1 h (D) 1.5 h

and (E) 2 h ........................................................................... 31

Figure 4.5 Degradation kinetics of standard α-Tocopherol (A) 70 o

C,

(B) 100 oC and (C) 130

oC ................................................. 32

xiii

Figure 4.6 Degradation of α-Tocopherol in EVOO at (I) 70 oC, (II) 100

oC and (III) 130

oC (A) 0 time (B) 0.5 h (C) 1 h (D) 1.5 h

and (E) 2 h ........................................................................... 35

Figure 4.7 Degradation kinetics of α-Tocopherol in extra virgin olive

oil (A) 70 oC (B) 100

oC and (C) 130

oC ............................. 36

Figure 4.8 Degradation of α-Tocopherol in canola oil at (I) 70 oC, (II)

100 oC and (III) 130

oC (A) 0 time (B) 0.5 h (C) 1 h (D) 1.5

h and (E) 2 h ........................................................................ 39

Figure 4.9 Degradation kinetics of α-Tocopherol in canola oil (A) 70 oC (B) 100

oC and (C) 130

oC ............................................. 40

Figure 4.10 Degradation of α-Tocopherol in palm oil at (I) 70 oC, (II)

100 oC and (III) 130

oC (A) 0 time (B) 0.5 h (C) 1 h (D) 1.5

h and (E) 2 h ........................................................................ 43

Figure 4.11 Degradation kinetics of α-Tocopherol in palm oil (A) 70 oC

(B) 100 oC and (C) 130

oC .................................................. 44

Figure 4.12 Degradation kinetics of α-Tocopherol in ( ) standard α-

Tocopherol, ( ) EVOO, ( ) canola oil and ( ) palm

oil at (A) 70 oC (B) 100

oC and (C) 130

oC ........................ 47

Figure 4.13 Degradation of α-Tocopherol in (I) EVOO, (II) canola oil

and (III) palm oil during cooking, (A) 0 time (B) oven

cooking and (C) pan-frying ................................................. 50

Figure 4.14 Effect of cooking method on degradation of α-Tocopherol in

extra virgin olive oil, canola oil and palm oil ..................... 51

Figure 4.15 Degradation % of α-Tocopherol in ( ) extra virgin olive

oil, ( ) canola oil and ( ) palm oil in relation to

cooking method ................................................................... 52

Figure 4.14 Standard α-Tocopherol calibration curve ........................... 53

xiv

Figure 4.15 Antioxidant activity of standard α-Tocopherol in 3 different

concentrations ...................................................................... 54

Figure 4.16 Effect of heating on standard α-Tocopherol antioxidant

activity at (A) 70 oC, (B) 100

oC and (C) 130

oC ................ 56

Figure 4.17 Effect of heating on antioxidant activity of extra virgin olive

oil at (A) 70 oC, (B) 100

oC and (C) 130

oC ........................ 58

Figure 4.18 Effect of heating on antioxidant activity of canola oil at (A)

70 oC, (B) 100

oC and (C) 130

oC ....................................... 60

Figure 4.19 Effect of heating on antioxidant activity of palm oil at (A)

70 oC, (B) 100

oC and (C) 130

oC ....................................... 62

Figure 4.20 Effect of heating on antioxidant activity of ( ) standard

α-Tocopherol, ( ) EVOO, ( ) canola oil and ( ) palm

oil at (A) 70 oC, (B) 100

oC and (C) 130

oC ........................ 65

Figure 4.21 Effect of cooking method on antioxidant activity on

vegetable oils ....................................................................... 67

Figure 4.22 Antioxidant activity loss during cooking ( ) extra virgin

olive oil, ( ) canola oil and ( ) palm oil ...................... 68

1

CHAPTER 1

1. INTRODUCTION

1.1 General Introduction

The minimal requirements of nutritional “macro and micro nutrients” ensures a

healthy human being and failure to meet the minimal intake can lead to malnutrition (Al-

hooti et al., 2009). In a recent study (Al-hooti et al., 2009) on Kuwait population it was

found that there was an inadequate consumption of vitamin E in all age groups and the

beneficial effect of vitamin E could enhance the health status of the Kuwaiti population.

Micronutrients such as vitamins, play a crucial role in the growth and

development of the human body; some micronutrients also have a health and disease

protective role in the human body by acting as antioxidants against free radicals.

Antioxidants are known to help the body to fight diseases such as cardiovascular,

diabetes, atherosclerosis and cancer (Meydani et al., 2005; Salonen et al., 1995; Knekt et

al., 1994). In this study the vitamin E α-Tocopherol was studied for its thermal stability

and antioxidant activity in edible oils as extra virgin olive oil, canola and palm oil.

1.2 Research Objectives

The overall objective of this research is to study the effect of heat treatment on α-

Tocopherol content and antioxidant activity in edible oils; the two specific objectives are

to investigate:

i. The effects of different thermal treatments on α-Tocopherol content in extra virgin

olive oil, canola oil and palm oil.

ii. The antioxidant activity of α-Tocopherol and the 3 edible oils and the effect of

thermal treatment on antioxidant activity.

2

CHAPTER 2

2. LITERATURE REVIEW

2.1 Vitamin E components

Vitamin E includes 8 different components or vitamins. Each compound has a

side chain referred to as phytyl tail, and a chromane ring which contains a phenolic

functional group. The 8 compounds are separated into 2 groups, Tocopherols and

Tocotrienols. Tocopherols have a saturated side chain with 16 carbons and Tocotrienol

have an unsaturated side chain with 16 carbons also. According to the position and

number of methyl group (CH3) attached to the aromatic ring, the 2 groups are classified as

α, β, γ and δ (Colombo, 2010). Figure 2.1 illustrates the different Tocopherols and

Tocotrienol classification.

Figure 2.1 Forms of vitamin E (Bender, 2009)

3

2.2 Vitamin E sources

Vitamin E can be found in various types of foods, in both animal and plant sources.

The animal sources of vitamin E are the fatty tissues of animal. The amount that is found

in animal sources is much less than in plant sources. Plant sources and especially

vegetable oils are considered the richest source of vitamin E. Olive oil, canola, sunflower,

and cottonseed are high in α-Tocopherol. On the other hand, corn oil and soybean have

higher amounts of γ-Tocopherol in comparison to the amount of α-Tocopherol. Figure 2.2

shows the different Tocopherol content in various oils. Other plant sources of α-

Tocopherol are whole-grain, cereals, legumes and some fruits such as kiwi and mango

and vegetables such as spinach and broccoli (Table 2.1; USDA, 2000). Gropper et al.

(2005) showed that the green leaves provide mostly α-Tocopherol and some γ-Tocopherol

which are in the nonchloroplast region of the plant and are the main source of γ, β, and δ

Tocopherols. Wheat, barley, rice and oats are some examples of Tocotreinols containing

cereals. Other sources of vitamin E are foods made from vegetable oils such as salad

dressing, mayonnaise, and also margarine also foods made from nuts such as peanut

butter (Gropper et al., 2005).

Figure 2.2 Various oils and Tocophenol content (USDA, 2000)

4

Table 2.1 Selected food items and α-Tocopherol content (USDA, 2000)

Food mg/100 g

Oil

Wheat – germ 149.4

Sunflower 41.08

Cottonseed 35.3

Safflower 34.1

Canola 14.84

Olive oil 14.35

Corn 14.3

Soybean 8.1

Nuts

Almond 26.22

Peanut 8.33

Spinach, raw 2.03

Egg 1.05

Other sources of vitamin E are supplements or by food fortification. These are

synthetic sources of vitamin E which are either all-racemic-α-Tocopherol acetate or all-

racemic-α-Tocopherol succinate; they are a mixture of equal proportion of all 8 possible

stereoisomers and referred as all-rac-α-Tocopherol and are not active as the natural

Tocopherol. Four of these sterioisomers are in the 2R-sterioisomeric form (RRR, RSR,

RRS, and RSS) and the other four are in the 2S-sterioisomric form (SRR, SSR, SRS, and

SSS) (Figure 2.3; USDA, 2000).

5

Figure 2.3 2R and 2S stereoisomers of α-Tocopherol (USDA, 2000)

2.3 Vitamin E and human health

2.3.1 Vitamin E and enzyme inhibition and activation

Vitamin E plays an important role in maintaining human health, the major function of

vitamin E is as an antioxidant. Other functions of vitamin E are inhibition of protein

kinase C (PKC), a family of enzymes that control various cellular processes such as

differentiation, immune response, transcriptional regulation, proliferation, synaptic

transmission, learning and memory (Win, 2008). Freedman et al. (1996) showed that α-

Tocopherol inhibits the aggregation of platelet through a PKC – dependent mechanism;

the incorporation of α-Tocopherol with the platelets lowered their sensitivity to

aggregation by adenosine 5'-diphosphate, arachidonic acid, and phorbol 12-myristate 13-

acetate (PMA) which gave the highest sensitivity reduction of 100 fold in comparison to

the other compounds. This could explain the beneficial effect of vitamin E on coronary

artery disease and increase in cerebral hemorrhagic risk (Freedman et al., 1996), and

inhibiting the superoxide anion (O2-) produced by monocytes by impairing the assembly

of the NADPH-oxidase (Cachia et al., 1998). Other enzymes that are inhibited by vitamin

6

E are phospholipase A2, protein kinase B (PKB/Akt), 5-lipoxygenase (5-LO) and

cyclooxygenase-2 (COX-2) (Kempna et al., 2004; Jiang et al., 2000; Douglas et al.,

1986). Protein phosphatase 2A (PP2A), diacylglycerol kinase (DAG) and HMG-CoA

reductase are enzymes activated by vitamin E (Khor and Ng, 2000; Ricciarelli et al.,

1998; Tran et al., 1994).

2.3.2 Other functions of vitamin E

Immune response can be enhanced by increasing vitamin E intake. Immune cells

membrane could be damaged by free radicals causing an impaired ability to respond to

pathogenic challenges. Studies showed that increasing the level of vitamin E consumption

improved T cell-mediated function in the aged (5, 7 – 9) (Meydani et al., 2005) and also a

significant increase in delayed-type hypersensitivity skin response (DTH) was evident in

healthy elderly (> 60 years) when given a dose of 800 mg vitamin E per day (Meydani et

al., 1997).

Vitamin E has been associated with enhanced cognition and short term memory along

with other vitamins such as folate, vitamin B6, B12 and minerals such as iron. A study

done on 2889 patients aging 65 to 102 years, after their eating pattern through modified

food frequency questionnaire, showed an association of low cognitive decline with age

and vitamin E intake. Another function of vitamin E is the protection of vitamin A from

oxidation (Whitney and Sharon, 2009a; Morris et al., 2002).

2.4 Vitamin E and free radical

Free radicals are highly reactive and unstable ions. They are atoms or molecules with

unpaired electrons and play an important role in various biological processes such as

metabolic pathways, cell signaling, immune response and a different number of

pathophysiological conditions (Vikram et al., 2010). Free radicals are classified as

oxygen or nitrogen species. The formation of free radical can be generated from

mitochondria, iron overload or lipids, protein, sugar, DNA during oxidation damage,

photosensitization, and atmospheric pollution. Other sources are redox cycling of

xenobiotics, exposure to physiochemical agents like ionizing radiations such as X – ray

7

and γ – ray, drugs that act as photosensitizer or endogenous compound (Devasagayam et

al., 2004). Table 2.2 illustrates the various types of free radicals and formation.

Table 2.2 Free radical species and formation (Devasagayam et al., 2004)

Reactive

Species

Symbol Half-life (in sec) Reactivity / Remarks

Reactive Oxygen Species

(ROS)

Superoxide O2•- 10

-6 s

generated in mitochondria,

in cardiovascular system and others

Hydroxyl

Radical •OH 10

-9 s

very highly reactive, generated during

iron overload and such conditions in

our body

Hydrogen

Peroxide H2O2 stable

formed in our body by large number

of reaction and yields potent species

like •OH

Peroxyl

Radical ROO

• s

reactive and formed from lipids,

proteins, DNA,

sugar etc. during oxidative damage

Organic

Hydroperoxide ROOH stable

react with transient metal ions to yield

reactive species

Singlet

Oxygen ׀O2 10

-6 s

highly reactive, formed during

photosensitization and chemical

reactions

Ozone O3 s

present as an atmospheric pollutant,

can react with various molecules,

yielding ɪO2

Reactive Nitrogen

Species (RNS)

Nitric Oxide NO• s

neurotransmitter and blood pressure

regulator, can yield potent oxidants

during pathological states

Peroxynitrite ONOO- 10

-3 s

formed from NO• and superoxide,

highly reactive

Peroxynitrous

acid ONOOH fairly stable

protonated from ONOO-

Nitrogen

Dioxide NO2 s

formed during atmospheric pollution

Free radical leads to cell damage if left uncontrolled; they attack proteins, nucleic

acids in DNA, polyunsaturated fatty acids (PUFAs) found in cell membrane or

8

intracellular organelles such as the nucleus, mitochondria or endoplasmic reticulum

(Gropper et al., 2005). The damage to these molecules leads to mutation resulting in

cancer; disrupt the protein structure leading to premature degradation of the protein

through amino acid cross-linking and degradation of lipids (Gropper et al., 2005).

2.4.1 Free radical chain reaction

The generation of free radicals through numerous processes such as the exposure

to ultraviolet light, trace metals or enzymatic reaction in the body leads to a series of

sequential reaction which eventually cause damage to the cell. For example the •OH

(hydroxyl radical) often take electron from a nearby organic molecule such as

polyunsaturated fatty acid (PUFA) located in the cell phospholipid protein membrane.

This reaction leads to the formation of lipid-carbon-center radical (L•) and H2O or

reaction with O2 to generate lipid-carbon-center radical (L•) and hydroperoxyl radical

HO2•; this allows for additional radicals to be formed. The spread of L

• leads to the

formation of peroxyl radical (LO2•) by reacting

with O2 and this can abstract a hydrogen

atom from another organic compound (other PUFA) in the membrane or in lipoprotein

(LH) to generate lipid peroxides (Gropper et al., 2005). The chain reaction is presented as

follows;

1. LH + •OH L

• + H2O OR LH + O2 L

• + HO2

•

2. L• + O2 LO2

•

3. LO2• + LH L

• + LOOH

2.5 Vitamin E antioxidant function

Free radicals go through 3 phases; initiation, propagation and termination. The last

phase involves vitamin E (EH). Before the interaction of peroxyl radicals (LO2•) or lipid-

carbon-centered radical (L•) with fatty acids, vitamin E terminates the chain propagation.

This interaction yields a reduced peroxyl radical (LOOH) and a oxidized state of vitamin

E (E•) as shown below;

OR 1. LO2

• + EH LOOH + E

•

2. L•

+ EH LH + E•

The combined ability of the chromanol ring to stabilize an unpaired electron and

the reactivity of the phenolic hydrogen located on its 6 hydroxyl group, allows vitamin E

9

to provide the hydrogen needed for the reduction process thus leaving an oxidized

vitamin E and in order to regain its ability to terminate free radical, it must be

regenerated. The agents that are involved in this regeneration are vitamin C (ascorbic

acid), reduced glutathione (GSH), NADPH, ubiquinol, and dihydrolipoic acid (Figure 2.4;

Gropper et al., 2005).

Figure 2.4 Vitamin E regeneration cycle (Gropper et al., 2005)

2.6 Vitamin E effect on diseases

2.6.1 Vitamin E and Cardiovascular disease

A study done in Finland showed a positive effect of vitamins E and C on coronary

heart disease; 5,133 healthy men and women aged 30 – 69 years showed an inverse

association in both men and women between vitamin E intake and coronary heart disease

(Knekt et al., 1994). Other studies focus only on beneficial effect of vitamin E

supplementation; for instance, a study done on approximately 90,000 nurses showed a

decreased risk of coronary heart disease in women who took vitamin E supplements

(Stampfer et al., 1993). Another study with men showed similar results (Rimm et al.,

1993). A study done to examine the effect of α-Tocopherol doses on myocardial

infarction (MI) “heart attack” showed a reduction in the rate of non-fatal MI (Stephens et

al., 1996). One of the major causes of cardiovascular disease is atherosclerosis, an

inflammatory disease that is targeted by low density lipoprotein (LDL) cholesterol which

accumulates on arterial wall (Ross, 1999). The oxidized LDL (oxLDL) is absorbed by

the macrophages forming lipid-laden foam cells in the fatty streak lesion (Meydani,

10

2001); Vitamin E prevents oxidative modification of LDL (Li et al., 1996; Jialal et al.,

1995; Reaven et al., 1993). Other mechanism can be by down-regulation of the receptor

involved in uptaking oxLDL and forming the foam-cell; the CD36 scavenger receptor in

the aortic smooth muscle cells (SMCs) when treated with α-Tocopherol showed a

decrease in its promoter activity and leads to the reduction of uptake of oxLDL into the

cytosol (Ricciarelli et al., 2000). Other mechanisms on reducing atherosclerosis are by

reducing the formation of cholesteryl ester in macrophages and uptake (Suzukawa et al.,

1994), inhibiting the protein kinase (PKC) pathway decreasing SMC proliferation (Azzi

et al., 1996) and preventing inflammation and monocyte/ macrophage adhesion to the

endothelium. Figure 2.5 illustrates the formation of foam cells (Chan, 1998); other

mechanisms are summarized in Table 2.3; (Meydani, 2001).

Figure 2.5 Formation of foam cell (Chan, 1998)

11

Table 2.3 Mechanisms by which vitamin E inhibits atherosclerosis (Meydani, 2001)

↓ LDL oxidation, ↓ macrophage uptake of oxLDL

↓ Endothelial cell injury

↓ Adhesion molecule expression

↓ Immune/endothelial cell adhesion

↓ Inflammatory cytokines and chemokines

↓ Smooth muscle cell proliferation

↓ Platelet aggregation

↑ NO production, ↑ arterial dilation

↑ Prostacyclin (PGI2), ↓ Thromboxane A2 (TXA2)

2.6.2 Vitamin E and hypertension

The overproduction of reactive oxygen species (ROS) appeared to be the central

common pathway by which different influences may induce and intensify hypertension.

NADPH oxidase, mitochondria, xanthine oxidase, endothelium-derived NO synthase

(eNOS), cyclooxygenase 1 and 2, cytochrome P450 epoxygenase and transition metals

are potential sources of reactive oxygen species (ROS) (Figure 2.6; Harrison et al., 2007).

Figure 2.6 Potential sources of reactive oxygen species (ROS) (Harrison et al., 2007)

As discussed previously, one of vitamin E functions is acting as an enzyme

inhibitor. This inhibition of NADPH oxidase, lipoxygenase, and cyclooxygenase can

12

lower oxidative stress (Kizhakekuttu and Michael, 2010). One study showed little but

significant reduction in blood pressure when treated with vitamin E along with other

compound; zinc sulphate, ascorbic acid and beta-carotene (Galley et al., 1997).

2.6.3 Vitamin E and diabetes

Type 2 diabetes mellitus formally known as non-insulin dependent diabetes

mellitus (NIDDM) is defined by the decreased uptake of glucose by human cells. In most

patients, the insulin molecules and receptors are normal but several intracellular signaling

pathways defect effects are responsible for insulin resistance. (Nolan, 2006).

The extracellular hyperglycemia leads to tissue damage and pathophysiological

complications such as heart disease, atherosclerosis, cataract formation and other

damages. The hyperglycemia stimulates the formation of reactive oxygen species (ROS)

from oxidative phosphorylation, glucose autooxidation, NADPH oxidase, lipooxygenase,

cytochrome P450, monooxygenases, and nitric oxide synthase (NOS) (Valko et al.,

2007).

Some antioxidants have a significant effect on type 2 diabetes, including β-

cryptoxanthin, vitamin E (α-Tocophenol, β-Tocophenol, ϒ-Tocophenol, δ-Tocophenol

and β-Tocotrienol, but not others such as vitamin C (Montonen et al., 2004). A four year

study of 944 men aged 42 – 60 years showed that 45 participants developed diabetes due

to low concentration of vitamin E, and 22 % were at risk of diabetes (Salonen et al.,

1995). Vitamin E plays an important role in improving glycemic control by possibly

reducing pancreatic β-cells damage caused by free radicals (Ruhe and MaDonald, 2001).

2.7 Antioxidant content in food

Vitamin E is not the only antioxidant present in food. Other vitamins as vitamin C

and carotenoids including β-carotene, γ-carotene and lycopene act as antioxidants along

with other functions. Vitamin C is a water soluble vitamin and carotenoids are lipid

soluble and therefor carried within lipoprotein particles with different concentrations

Table 2.4; Esterbauer et al., 1993. Antioxidant function is not limited to vitamins alone; it

includes enzymes and coenzymes such as ubiquinol (CoQH) catalase and copper and

zinc-dependent superoxide dismutase, peptides such as glutathione (GSH) and transition

13

metal-binding proteins such as transferrin and ceruloplasmin (Basu, 1999). Other natural

antioxidants are phenolic compounds including phenolic acids, flavonoids, and phenolic

polymer (tannins) (Fuhrman and Aviram, 2002).

A variety of food items are sources of antioxidant; for example vitamin C is found in

fruits such as oranges and strawberries, and vegetables such as broccoli and bell pepper

(Whitney and Sharon, 2009b). The phenolic compounds are found in all berries, tea,

oranges, tofu, red wine, olive oil and other sources of foods (Servili et al., 2009; Manach

et al., 2004). Glutathione (GSH) on the other hand is synthesized in the cytosol cells (Lu,

2009).

Table 2.4 Antioxidants in low density lipoprotein (LDL) (Esterbauer et al., 1993)

Individual antioxidants mol/mol LDL

α-Tocopherol 7.26 ± 2.52

γ-Tocopherol 0.56 ± 0.24

β-carotene 0.29 ± 0.26

-carotene 0.12 ±0.14

lycopene 0.16 ±0.11

cryptoxanthin 0.14 ±0.13

cantaxanthin 0.02 ± 0.04

Lutein + zeaxanthin 0.04 ± 0.03

phytofluene 0.05 ± 0.03

ubiquinol-10 0.10 ±0.10

2.8 Antioxidants and free radicals

All antioxidants have the same function; they reduce oxidized compounds to

stabilize it and protect the cell from damage. Vitamin C (AH2) protects the body form

several radicals including superoxide radicals, hydrogen peroxide, hydrogen radicals,

singlet molecular oxygen, carbon-centered peroxide and hydroperoxyl radicals. The

interaction between the radicals and vitamin C results in the formation of H2O and

dehydroascorbate (DHAA) or semidehydroascorbate radical (AH-); the regeneration of

vitamin C requires niacin, dihydrolipic acid (DHLA), glutathione and thioredoxin

(Gropper et al., 2005).

14

AH2 + O-

2 H2O + DHAA

AH2 + H2O2 2 H2O + DHAA

AH2 + OH• H2O + AH

-

AH2 + LO• LOH + AH

-

Superoxide dismutase (SOD) is dependent on certain minerals to function and

depending on the location of the SOD in the body these minerals changes; for instance, if

the SOD is in the mitochondria, manganese is the activation mineral whereas in the

extracellular or the intracellular, zinc and copper are the activation minerals (Gropper et

al., 2005). SOD acts on superoxide radicals and form hydrogen peroxide and O2 as shown

below;

SOD + 2O-

2 2 H2O + O2

Glutathione peroxidase (GPx) with the help of glutathione (GSH) eliminates

hydrogen peroxide, carbon-centered peroxide and hydroperoxyl radicals; catalase (an iron

dependent enzyme) on the other hand, eliminates hydrogen peroxide only. The activation

of GPx, requires selenium as a cofactor. Carotenoids such as (β-carotene and lycopene)

and ubiquinol (CoQH) eliminate singlet molecular oxygen, carbon-centered peroxide and

hydroperoxyl radicals. Oxidized glutathion (GSSG) is regenerated via reacting with

DHLA or glutathione reductase with niacin as NADPH. Also DHLA helps in

regenerating CoQH or by the thirodoxin-thirodoxin reductase system (Gropper et al.,

2005).

2GSH + GPx-Se + H2O2 2 H2O + GSSG (oxidized glutathione)

β-carotene + 1O2

3O2 + excited β-carotene β-carotene + heat

CoQH + LOO• CoQH

• + LOOH

Antioxidant activity of phenolic compounds comes from the ability to donate a

hydrogen atom to the peroxyl radical to form an alkyl hydroperoxide. The phenolic

radical can be stabilized by donating another hydrogen atom or by reacting with another

radical and they eliminate hydroxyl and peroxyl radicals, and superoxide anion (Fuhrman

and Aviram, 2002). Another pathway of phenolic compound is by chelation achieved

15

either binding of the ion (such as iron) to the chelating agents preventing their

involvement in generating hydroxyl radicals, or by binding the transition metal ion to an

antioxidant; the redox reaction may not be prevented but the formed radicals are directed

into the antioxidant path (Halliwell, 2002).

ROO· + PPH ROOH + PP·

2.8.1 Antioxidant and Cardiovascular disease

Singh et al. (1992) reported a positive association between a diet with increased

fruits, vegetables, fiber and mineral and the reduction of blood lipoproteins; this study

was done on 505 patients with and acute myocardial infarction, they were divided into 2

groups, both had the same diet but group A had more fruits, vegetables and nuts in their

diet and the results showed a reduction in weight and lipids in group A in comparison

with group B.

Another study used the same strategy and divided participants into three groups;

group A the controlled group, group B similar to A but added more fruit and vegetables to

their diet and the last group C with a low fat diet and more fruit and vegetables and

showed that having a low fat and high fruit and vegetable diet lower the rate of lipid

peroxidation (Miller et al., 1998).

2.8.2 Antioxidant and hypertension

Hypertension increases the risk of atherosclerosis and free radicals are associated

with atherogenic process; this means hypertensive patients with low levels of antioxidant

are at greater risk of developing atherosclerosis (Redon et al., 2003). A study showed that

Vitamin C and thiols levels were significantly lower in hypertensive patients thus free

radicals would be at a higher level (Tse et al., 1994). Hypertension patients showed not

only low levels of vitamin C, but also vitamin E as well (Wen et al., 1996).

2.8.3 Antioxidant and cancer

A study was done in Sweden on the effect of vegetable and fruit as antioxidants

on the risk of having cancer and showed that having more fruits and vegetables rich in

vitamin C, β-carotene and vitamin E can reduce the risk of Cardia cancer (Ekstrom et al.,

2000) and distal cancer (Serafini et al., 2002).

16

2.9 Vegetable oils - Olive oil

2.9.1 Types of olive oil

Olive oil is available in the following 3 main categories: virgin olive oil, olive oil

and refined olive oil;virgin olive oil is furthered differentiated into extra virgin olive oil,

virgin olive oil, lampante virgin olive oil and ordinary virgin olive oil (USDA, 2010).

This classification is based on certain quality criteria such as color, odor and flavor.

Another type of olive oil is olive pomace oil which is obtained from the residue

remaining after extracting the olive oil. It is of lower quality and is separated into olive

pomace oil, refined olive pomace oil and crude olive pomace oil (Appendix A; USDA,

2010).

2.9.2 Olive oil vitamin E and phenolic content

Olive oil contains 14 mg/100 g of vitamin E with mainly α-Tocopherol and high

content of antioxidant phenolic compounds (Owen et al., 2004) and especially in extra

virgin olive oil which has higher levels of phenolic compounds (Owen et al., 2000).

These phenolic compounds are present only in virgin olive oil and not in any other

vegetable oil and are classified as follows: mainly tyrosol, hydroxytyrosol, and their

derivatives, derivatives of 4-hydroxybenzoic,4-hydroxyphenylacetic, and 4-

hydroxycinnamic acids, lignans and flavonoids (Ramirez-Tortosa et al., 2006).

The polarity of the phenolic compounds vary; the more polar phenolic compounds

are 4-acetoxy-ethyl-1, 2-dihydroxybenzene, 1-acetoxypinoresinol, apigenin, caffeic acid,

o- and p-coumaric acids, ferulic acid, gallic acid, homovanillic acid, p-hydroxybenzoic

acid, hydroxytyrosol, luteolin, oleuropein, pinoresinol, protocatechuic acid, sinapic acid,

syringic acid, tyrosol, vanillic acid, and vanillin while the less polar phenolic compounds

are aglycones of oleuropein and ligstroside (the hydroxytyrosol and tyrosol esters of

elenolic acid), deacetoxy and di- aldehydic forms of these aglycones the flavones luteolin

and apigenin, the lignans 1-acetoxypinoresinol and pinoresinol and also elenolic acid and

cinnamic acid (Boskou et al., 2006). Olive variety, degree of ripeness, soil composition,

17

climate, processing techniques and storage are factors effecting the quantity (150 – 700

mg/l) and quality of phenolic compounds in olive oil (Corona et al., 2009).

Olive oil has been reported to have positive effect on certain diseases including

cancer, cardio vascular disease, hypertension, hypercholesterolemia and overall health

status (Covas et al., 2006; Marrugat et al., 2004; Weinbrenner et al., 2004; Madigan et

al., 2000; Owen et al., 2000; Visioli and Galli, 1998).

2.10 Olive oil and heart disease risk factors

A European study involving 192 men aged 20 – 60 years (using high, medium and

low polyphenol content olive oils) showed an increase in HDL-cholesterol levels. Also

the oxidation damage of LDL was lowered depending on polyphenol content of olive oils

(Covas et al., 2006).

The Mediterranean diet is characterized by high consumption of fruit, vegetables

and olive oil. For instance, in Crete, the largest island in Greece, the consumption of fats

reaches up to 40 % of the total caloric intake most of it comes from olive oil (Visioli and

Galli 1998).

Weinbrenner et al. (2004) studied the effect of olive oil on 12 healthy men aged

20 – 22 years who were asked to consume olive oil with different phenolic content; high,

moderate and low; 486, 133 and 10 mg/kg, respectively; the results showed a reduction in

oxLDL, 8-oxo-dG in mitochondrial DNA and urine and an increase of HDL and

glutathione peroxidase.

2.11 Vitamins with antioxidant properties status among the Kuwaiti

population

2.11.1 Vitamin E status

In a 2009 National Nutrition Survey in Kuwait, the mean daily intake of vitamin E

was highest among males aging 6 – 9 and 10 – 19 years 6.4 mg. Females aging 20 – 49

had the highest daily intake 5.3 mg. the mean intake between both genders and age group

did not differ (Table 2.5; Al-hooti et al., 2009).

18

Table 2.5 Daily intake of vitamin E by gender and age (Al-hooti et al., 2009)

Average daily intake of vitamin E - alpha equivalents (mg)

Age Group

(years)

Males Females

Median Mean S.E. Median Mean S.E.

Weighted

3-5 2.7 3.5 0.30 3.2 4.2 0.44

6-9 4.3 6.4 1.08 4.0 5.2 0.45

10-19 5.0 6.4 0.40 4.5 5.2 0.27

20-49 4.9 5.7 0.28 4.3 5.3 0.19

50+ 4.8 5.6 0.36 3.4 4.4 0.46

Total 4.5 5.8 0.26 4.0 5.0 0.14

Unweighted

3-5 3.3 4.0 0.34 3.2 4.0 0.44

6-9 4.3 5.7 0.53 4.0 4.9 0.45

10-19 5.2 6.4 0.39 4.5 5.2 0.28

20-49 5.0 6.2 0.31 4.3 5.2 0.21

50+ 5.2 6.0 0.31 3.4 4.5 0.3

Total 4.84 5.92 0.17 3.99 4.95 0.13

The percentage of individuals not meeting the estimated average requirements

(EAR) was highest among females age ≥ 50 (96 %) and 20 – 49 years of age for males

with 94 %. In total, 86 % and 91 % of males and females respectively did not meet the

EAR (Table 2.6; Al-hooti et al., 2009).

Table 2.6 Percentage of participants not meeting the estimated average requirement

(EAR) of vitamin E (mg) by gender and age (Al-hooti et al., 2009)

Gender Age Group (Years)

3-5 6-9 10-19 20-49 ≥ 50 Total

Weighted

Males 85 68 84 94 92 86

Females 75 78 95 95 96 91

Unweighted

Males 80 73 85 91 91 87

Females 97 82 94 95 95 92

19

2.11.2 Vitamin E sources

In the Kuwait dietary survey, olive oil was the main contributor of the average

daily intake of vitamin E. the overall percent was 9.1 %. French fried potatoes was second

with an overall contribution of 6.5 %, followed by corn oil 5.7 %, sunflower seeds 4.8 %,

and mashkoul (rice with onions) 4.5 % (Table 2.7; Al-hooti et al., 2009).

20

Table 2.7 Percentage contribution of foods to average daily vitamin E intake by sex and age group (Al-hooti et al., 2009)

Age Group (Years)

Food items Men Women

3-5 6-9 10-19 20-49 ≥50 All men 3-5 6-9 10-19 20-49 ≥50 All women All

% % % % % % % % % % % % %

Oil, olive, salad or cooking 5.8 3.9 7.6 9.9 14.1 8.5 6.5 7.7 6.9 11.5 11.1 9.8 9.1

French Fries 10.6 6.2 8.5 5.3 0.8 6.3 13.0 4.6 11.6 5.9 0.5 6.8 6.5

Oil, corn, salad or cooking 5.3 4.6 7.6 4.5 7.2 5.8 5.0 7.0 5.8 5.2 4.8 5.5 5.7

Seeds, sunflower, kernels, dry roasted,

salted 0.0 27.1 5.9 0.7 0.0 5.9 8.8 0.0 3.5 2.9 6.6 3.5 4.8

Mashkoul (Rice with Onion) 6.0 2.9 4.3 4.7 4.8 4.4 7.1 6.2 4.3 4.4 4.0 4.7 4.5

Green salad 2.7 1.2 2.1 4.5 5.6 3.3 1.0 1.1 2.8 4.3 6.8 3.8 3.5

Macbous dajaj (Chicken & Rice) 3.9 2.1 2.5 4.1 2.1 3.1 2.6 3.3 3.0 1.3 2.4 2.1 2.6

Bread, pita, whole wheat 1.6 0.8 0.9 2.7 5.8 2.1 1.0 1.6 1.5 2.2 4.8 2.2 2.1

Juice drink, mango nectar, canned 3.3 3.0 2.3 0.3 0.1 1.5 2.0 1.7 3.0 0.7 0.1 1.3 1.4

Nuts, mixed, with peanuts, dry

roasted, salted 0.0 1.9 0.0 1.8 0.0 1.0 0.0 4.9 0.5 0.1 0.0 0.6 0.8

21

CHAPTER 3

MATERIALS AND METHODS

3.1 Materials

Extra virgin olive oil, canola oil samples were purchased from a local supermarket

Montreal, Canada and palm oil sample was purchased from a local supermarket Kuwait,

Kuwait City. Meat and salmon fish samples were purchased from a local supermarket in

Montreal, Canada. Vitamin E, DL-all-rac-α-Tocopherol (≥ 95 %) and 2,2-Diphenyl-1-

picryl-hydrazl (DPPH) were purchased from Sigma/Aldrich. Ethyl acetate, methanol,

ethanol and n-hexane were purchased from Fisher Scientific.

3.2 Preparation of standard solutions and sample solutions

Standard solutions were prepared according to Gimeno et el., 2000 with

modifications. Standard α-Tocopherol was weighed (0.2, 0.3 and 0.4 g) in a 10 ml

volumetric flask then diluted with 10 ml ethanol. The solution was mixed using a vortex-

mixer for 10 s; 1 ml was transferred to a 10 ml volumetric flask, 9 ml ethanol added and

mixed for 10 s. This procedure was repeated 3 times to obtain standard solutions of (20,

30 and 40 µg/ml) of the prepared solutions which were kept in a dark volumetric flask at -

20 oC for up to 2 weeks.

Solutions of the oil samples were prepared based on the method of Gimeno et al.,

(2000) with modifications (Figure 3.1). The oil samples were diluted in n-hexane (1:1),

the solution was mixed using vortex-mixer for 10 s then 200 µl was transferred to a

centrifuge tube, and 600 µl methanol and 200 µl ethanol were added. The solution was

mixed using a vortex-mixer and centrifuged at 3000 RPM for 5 min, then filtered through

a 0.45 µm pore size filter and 50 µl was injected directly into the chromatograph (refer to

Section 3.6 for details). The prepared oil solutions were kept in dark at -20 oC for up to 1

week.

22

3 ml oil sample + 3 ml n-hexane

Mixed using vortex-mixer for 10 s

200 µl transferred to Eppendorf centrifuge tube (1.5 ml)

Added 600 µl methanol and 200 µl ethanol

Mixed using vortex-mixer for 10 s and centrifuged at 3000 RPM for 5 min using

(Eppendorf Centrifuge, Model minispin plus, Canada)

Precipitate Supernatant filtered with

(AcetatePlus, supported, plain, 0.45 µ, 13 mm,

USA) filter

50 µl injected into the HPLC

Figure 3.1 Diagram of procedure used for extracting α-Tocopherol in oil samples

23

3.3 Preparation of standard curve

Standard solutions were prepared as described in Section 3.2. The solutions were

allowed to warm-up to room temperature before injecting. 50 µl was injected into the

chromatograph and the area under the curve was calculated using the calibration curve

obtained from standard with dilution factors; the analysis was done in triplicate.

3.4 Effect of heating on standard and oils

Heating of the α-Tocopherol and the oils was done according to the method of

Kalantzakis et al., (2006) with some modifications; an oil bath (IKA-HEIZBAD HB-250)

with grape-seed oil was used as heat transfer medium because of its high smoke point

(190 – 250 oC) (Bail et al., 2008) . The effect of heating was carried out using the method

of Pellegrini et al., 2001 with modifications. Four tubes of 10 ml Pyrex test tubes were

filled with 2 ml standard α-Tocopherol and 9 ml oil samples. The tubes were placed in the

oil bath heated at 3 different temperatures (70, 100, and 130 oC). Four heating time

intervals (0.5, 1, 1.5 and 2 h) were chosen, and after each time period the tube was

covered with aluminum foil and stored at -20 oC until analysis.

3.5 Cooking of the food samples in oils

Cooking was done according to the method of Andrikopoulos et al., (2002) with

modifications. The pan-frying was performed in an uncovered stainless steel (35 mm

high, diameter 220 mm). 3 pieces of meat were fried each time in 15 ml oil, for 5 min at

250 (±2) oC, the oil samples were placed in a 50 ml centrifuge tube, sealed and stored at -

20 oC immediately to prevent any further oxidation until analysis. The oven cooking was

performed in an aluminum foilware (146 mm x 121 mm x 35 mm) covered with

aluminum foil. 3 pieces of salmon were oven cooked using 15 ml oil, for 30 min at 130

(±3) oC. The oil samples were placed in a 50 ml centrifuge tube, sealed and stored at -20

oC immediately to prevent any further oxidation until analysis.

3.6 High Performance Liquid Chromatography (HPLC) analysis

HPLC analysis was carried out with Beckman liquid chromatographic system

equipped with a binary high-pressure delivery system (Model 126), a manual injector

(Rheodyne Model 7125i) with a 50 µl final loop and a UV detector (Model 166). The data

24

were stored and analysed with the Beckman Coulter chromatographic software (23 Karat

8.0). 1 ml of solution was centrifuged (3000 RPM, 5 min, Eppendorf Centrifuge, Model

minispin plus, Canada ), the supernatant was filtered with (AcetatePlus, supported, plain

,0.45 µ, 13 mm, USA) filter and injected into a Phenyl column CSC-Inertsil

150A/Phenyl, 5 µm, 150 x 4.6 mm and pre-column Eclipse XDB-C18, 3.5 µm, 4.6 x 56

mm operated at room temperature. The following conditions were used to elute the

sample at flow-rate of 1 ml min-1

from column: solvent A, methanol in water (96:4, v/v)

over 10 min.

3.7 DPPH assay

A DU800 UV/visible spectrophotometer (version 3.0 build 5, 2001, Beckman

Coulter) was used to measure the antioxidant activity for both the standard and the oil

samples. The method of Kalantzakis et al., (2006) was used with modifications. The

unheated and heated samples at 70, 100 and 130 oC for 2 h, were taken every 30 min.

Samples (2.5 ml) were dissolved in 5 ml n-hexane and extracted with 5 ml of a

methanol/water mixture (60:40, v/v). The resulting mixture was shaken vigorously by

means of a mechanical shaker (Vortex) and centrifuged at 8000 RPM for 10 min. The

methanol/water insoluble fraction in the n-hexane was evaporated using nitrogen gas for

30 min (Figure 3.2).

A 1 ml sample of the oil solution in ethyl acetate (7.5 %, v/v), 1 ml was added to 4

ml of a freshly prepared DPPH• Solution (10

-4 M in ethyl acetate) in a screw-capped 10

ml test tube. The reaction mixture was shaken vigorously for 10 s in a Vortex apparatus

and the tube was maintained in the dark for 30 min, after which a steady state was

reached. The absorbance of the mixture was measured at 515 nm against a blank solution

(without radical) water was used. A control sample (without oil) was prepared and

measured daily. The radical scavenging activity (RSA) toward DPPH0 was expressed as

the % reduction in DPPH• concentration by the constituents of the oils:

% [DPPH•]red = 100 X (1–[DPPH

•]30/[DPPH

•]0)

where [DPPH•]0 and [DPPH

•]30 were the concentrations of DPPH

• in the control sample (t

= 0) and in the test mixture after the 30 min reaction, respectively.

25

2.5 ml oil sample + 5 ml n-hexane + 5 ml methanol/water mixture (60:40, v/v)

Mixed using vortex-mixer for 10 s

Centrifuge at 8000 RPM for 10 min

Precipitate Supernatant transferred to

50 ml centrifuge tube

n-hexane evaporated using nitrogen gas for 30 min

0.75 ml dissolved in 9.25 ml ethyl acetate

1 ml added to 4 ml DPPH• Solution

(10-4

M in ethyl acetate) and maintained in

the dark for 30 min

Antioxidant activity was measured

Figure 3.2 Diagram of procedure utilized to measure the antioxidant activity of α-

Tocopherol and vegetable oils

26

3.8 Thermal degradation kinetics

Using different order kinetics (zero, 1st and 2

nd) the results showed that the 1

st

order kinetics best fit the α-Tocopherol degradation results. The 1st order kinetic equation

is;

(1)

where C is the quantity of α-Tocopherol in µg/ml at any time, T is the time in hour and k

is the reaction rate constant in hour

Integrating Eq. (1) and letting C = Co at T = 0 gives:

(2)

The first order equation was obtained from (Ahmad et al., 2012)

3.9 Statistical analysis

All results presented as means (± standard deviation) of triplicate determinations.

ANOVA, Two-Factor without replication was used to analyze the data for significance. A

value of (P < 0.05) was considered as significant. All statistical analyses were done using

Excel 2007.

27

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Standard α-Tocopherol concentration curve

Figure 4.1 shows the HPLC chromatograms of the standard α-Tocopherol

solutions at 3 different concentrations; Figure 4.2 shows the plot of peak area vs. α-

Tocopherol at the three different concentrations for triplicate injections. The equation

obtained was:

y = 26682x -100355 (R2

= 0.9619)

Gimeno et al. (2000) reported an equation of y = 1.048x -0.011 for HPLC

analysis of α-Tocopherol standard using a concentration range of 1 – 25 µg/ml of α-

Tocopherol standard.

28

Figure 4.1 HPLC chromatogram of standard α-Tocopherol (A) 20 µg/ml , (B) 30

µg/ml and (C) 40 µg/ml

Figure 4.2 α-Tocopherol standard concentration curve

29

4.2 Thermal degradation of standard α-Tocopherol and α-Tocopherol in

vegetable oils

Figure 4.3 shows the HPLC chromatogram of unheated (room temperature, 25 oC)

(A) standard α-Tocopherol, (B) α-Tocopherol in EVOO, (C) α-Tocopherol in palm oil

and (D) α-Tocopherol in canola oil. Average α-Tocopherol concentration in the 3 oils

were 323 (±5), 271 (±2) and 174 (±2) µg/ml, respectively.

i) Thermal degradation of standard α-Tocopherol: Standard α-Tocopherol was heated at

70, 100 and 130 oC for 2 h and HPLC was used to determine the degradation of α-

Tocopherol in relation to time and temperature. Figures 4.4i), 4.4ii) and 4.4iii) show the

HPLC chromatogram of standard α-Tocopherol during the thermal treatment, at half-hour

heating time intervals over 2 h at 70, 100 and 130 oC, respectively. Table 4.1 shows the

α-Tocopherol concentration calculated from the HPLC chromatograms and Figure 4.5

shows the first order degradation kinetics curve for standard α-Tocopherol during the heat

treatment. There was no significant degradation (P > 0.05) of α-Tocopherol at 70 oC; at 0

heating time α-Tocopherol concentration was 41.15 (±0.3) µg/ml compared to 40.18

(±1.6), 39.18 (±1.5), 37.17 (±0.4) and 37.8 (±0.9) µg/ml after 0.5, 1, 1.5 and 2 h heating

time, respectively. At 100 oC also, there was no significant decrease (P > 0.05) in α-

Tocopherol during the 2 h heating period; the α-Tocopherol concentration were 39.67

(±0.3), 37.41 (±2.1), 37.37 (±2.7) and 37.29 (±1.2) µg/ml, after 0.5 , 1, 1.5 and 2 h

heating time, respectively. Similarly at 130 oC there was non-significant decrease (P >

0.05) of α-Tocopherol during the 2 h heating period; the α-Tocopherol concentration were

39.12 (±1.6), 37.57 (±1.7), 36.24 (±0.6) and 36.19 (±2) µg/ml after 0.5, 1, 1.5 and 2 h

heating time, respectively. In general, the results show that there was a slight but

statistically non-significant (P > 0.05) decrease of α-Tocopherol concentration with

increasing time during the 2 h heating period at 70, 100 and 130 oC; there was no effect of

the temperature treatment on α-Tocopherol concentration.

Sabliov et al. (2009) reported the degradation of α-Tocopherol at 40, 60, 120,

and 180 oC; their results showed no significant effect of heat on α-Tocopherol

concentration during the 6 h heating period at 40, 60 and 120 oC, but at 180

oC there was

a significant effect of temperature on the decrease in α-Tocopherol concentration during

30

Time (m)

the 6 h heating period. Siro et al. (2006) also reported α-Tocopherol stability with heating

to temperatures up to 190 oC.

Figure 4.3 HPLC chromatogram for (A) standard α-Tocopherol (B) α-Tocopherol in

EVOO (C) α-Tocopherol in palm oil and (D) α-Tocopherol in canola oil

B

A

B

C

D

α-Tocopherol

31

I II III

Figure 4.4 Degradation of standard α-Tocopherol at (I) 70 o

C, (II) 100 oC and (III) 130

oC (A) 0 time (B) 0.5 h (C) 1 h

(D) 1.5 h and (E) 2 h

Time (m) Time (m) Time (m)

32

Table 4.1 Effect of heating time and temperature on standard α-Tocopherol

concentration

Time

α-Tocopherol concentration (µg/ml)

70 °C 100 °C 130 °C

0 h 41.15 (±0.3) 41.15 (±0.3) 41.15 (±0.3)

0.5 h 40.18 (±1.6) 39.67 (±0.3) 39.12 (±1.6)

1 h 39.18 (±1.5) 37.41 (±2.1) 37.57 (±1.7)

1.5 h 37.17 (±0.4) 37.37 (±2.7) 36.24 (±0.6)

2 h 37.80 (±0.9) 37.29 (±1.2) 36.19 (±2.0)

Figure 4.5 Degradation kinetics of standard α-Tocopherol (A) 70 o

C, (B) 100 oC and

(C) 130 oC

33

ii) Thermal degradation of α-Tocopherol in extra virgin olive oil (EVOO): Figures 4.6i),

4.6ii) and 4.6iii) show the HPLC chromatogram of α-Tocopherol during the thermal

treatment of EVOO, at half-hour heating time intervals over 2 h at 70, 100 and 130 oC,

respectively. Table 4.2 shows the α-Tocopherol concentration calculated from the HPLC

chromatograms, and Figure 4.7 shows the first order degradation kinetics curve of α-

Tocopherol in EVOO during the heat treatment. There was a gradual but non-significant

degradation (P > 0.05) of α-Tocopherol at 70 oC; at 0 heating time α-Tocopherol

concentration was 323 (±5.3) µg/ml compared to 283 (±9.4), 273 (±4.6), 289 (±10.7) and

304 (±2.6) µg/ml after 0.5, 1, 1.5 and 2 h heating time, respectively. At 100 oC also, there

was no significant decrease (P > 0.05) of α-Tocopherol in EVOO during the 2 h heating

period; the α-Tocopherol concentration were 291 (±6.2), 282 (±9.0), 279 (±5.8) and 269

(±7.7) µg/ml, after 0.5, 1, 1.5 and 2 h heating time, respectively. On the other hand, at

130 oC there was a significant decrease (P < 0.05) of α-Tocopherol during the 2 h heating

period; the α-Tocopherol concentration were 160 (±2.0), 133 (±0.8) µg/ml after 0.5 h and

1 h heating time, respectively and complete destruction of α-Tocopherol after 1.5 and 2 h

of heating. In general the results show that there was increasing destruction of α-

Tocopherol concentration in EVOO with increasing time at 100 oC and complete

destruction of α-Tocopherol after 1.5 h and 2 h at 130 oC.

The concentration α-Tocopherol in extra virgin olive oil was similar to those

reported by other researches. Boskou et al. (2006); Psomiadou (2000) reported that the

level of α-Tocopherol in extra virgin olive oil range were 55 – 370 µg/ml.

Previous studies have reported on the effect of heat treatment of olive oil at

temperatures above 160 oC (Allouch et al., 2007; Pellegrini et al., 2001); these researches

studied the effect of heat treatment at 160, 175,180,185 and 190 oC on extra virgin olive

oil and olive oil and reported a significant reduction of α-Tocopherol during the heat

treatment, and the results showed a significant effect in relation to heat time but not

temperature. Brenes et al. (2002) studied the effect of microwave heating and boiling of

water on α-Tocopherol concentration in virgin olive oil and reported a significant

reduction of α-Tocopherol concentration after 10 min during the microwave heating,

while for water boiling, no significant change was shown after 30 min. Nissiotis and

34

Tasioula-Margari (2002) reported the effect of heat treatment at 60 and 100 oC over an

extended period of time (3 – 30 days and 9 – 100 hours, respectively) on extra virgin

olive oil and fine virgin olive oil; the results showed no significant decrease after 3 d and

9 h of heat treatment, while a significant reduction after 15 d and 20 h of heat treatment.

Bester et al. (2008) also reported a significant reduction in α-Tocopherol concentration

with heating after 142 h of heat treatment.

35

I II III

Figure 4.6 Degradation of α-Tocopherol in EVOO at (I) 70 o

C, (II) 100 oC and (III) 130

oC (A) 0 time (B) 0.5 h (C) 1 h

(D) 1.5 h and (E) 2 h

36

Table 4.2 Effect of heating time and temperature on α-Tocopherol concentration in

extra virgin olive oil

Time α-Tocopherol concentration in Extra virgin olive oil (EVOO) (µg/ml)

70 °C 100 °C 130 °C

0 h 323 (±5.3) 323 (±5.3) 323 (±5.3)

0.5 h 283 (±9.4) 291 (±6.2) 160 (±2.0)

1 h 273 (±4.6) 282 (±9.0) 133 (±0.8)

1.5 h 289 (±10.7) 279 (±5.8) 0

2 h 304 (±2.6) 269 (±7.7) 0

Figure 4.7 Degradation kinetics of α-Tocopherol in extra virgin olive oil (A) 70 oC

(B) 100 oC and (C) 130

oC

37

iii) Thermal degradation of α-Tocopherol in Canola oil: Figures 4.8i), 4.8ii) and 4.8iii)

show the HPLC chromatogram of α-Tocopherol during the thermal treatment of canola

oil, at half-hour heating time intervals over 2 h at 70, 100 and 130 oC, respectively. Table

4.3 shows the α-Tocopherol concentration calculated from the HPLC chromatogram and

Figure 4.9 shows the first order degradation kinetics curve of α-Tocopherol during the

heat treatment of canola oil. There was a gradual but non-significant degradation (P >

0.05) of α-Tocopherol at 70 oC; at 0 heating time α-Tocopherol concentration was 271

(±2.2) µg/ml compared to 268 (±6.1), 256 (±8.6), 250 (±1.8) and 249 (±5.6) µg/ml after

0.5, 1, 1.5 and 2 h heating time, respectively. At 100 oC also, there was non-significant

decrease (P > 0.05) of α-Tocopherol during the 2 h heating period; the α-Tocopherol

concentration were 245 (±8.8), 222 (±5.5), 216 (±4.7) and 208 (±2.8) µg/ml, after 0.5, 1,

1.5 and 2 h heating time, respectively. At 130 oC there was also a gradual but non-

significant decrease (P > 0.05) of α-Tocopherol during the 2 h heating period; the α-

Tocopherol concentration were 231 (±8.0), 228 (±11.9), 225 (±8.1) and 205 (±10.7)

µg/ml, after 0.5, 1, 1.5 and 2 h heating time , respectively. In general, the results show

that α-Tocopherol in canola oil was stable at 70 oC during the 2-hour heating period; at

100 and 130 oC the degradation of α-Tocopherol increased with time but this increase in

degradation was statistically not significant (P > 0.05).

The concentration of α-Tocopherol in canola oil was similar to those reported by

other researchers. Normand et al. (2001) reported that the levels of α-Tocopherol for

regular canola oil, high oleic canola oil, high oleic-low linolenic canola oil and low

linolenic acid canola oil were 197 (±10), 180 (±2), 290 (±2) and 152 (±13) µg/g,

respectively. Other researches (Przybylski et al., 2005; Bramley et al., 2000) reported 270

and 210 µg/g α-Tocopherol in canola oil.

Previous studies have reported the effect of heat treatment on canola oil at

temperature above 160 oC (Aladedunye and Roman, 2011; Romero et al., 2007); these

researches reported that the effect of temperature of 185 and 180 oC on canola oil showed

a significant reduction of α-Tocopherol during the heat treatment with a significant effect

in relation to heat time but not temperature. Sharayei et al. (2011) reported the effect of

heat treatment on the total Tocopherol content in canola oil at a temperature of 180 oC;

38

the results showed a significant decrease of total Tocopherol during heating treatment

with a significant effect in relation to heat time but not temperature.

39

I II III

Figure 4.8 Degradation of α-Tocopherol in canola oil at (I) 70 o

C, (II) 100 oC and (III) 130

oC (A) 0 time (B) 0.5 h (C) 1 h

(D) 1.5 h and (E) 2 h


40

Table 4.3 Effect of heating time and temperature on α-Tocopherol concentration in

canola oil

Time α-Tocopherol concentration in Canola oil (µg/ml)

70 °C 100 °C 130 °C

0 h 271 (±2.2) 271 (±2.2) 271 (±2.2)

0.5 h 268 (±6.1) 245 (±8.8) 231 (±8.0)

1 h 256 (±8.6) 222 (±5.5) 228 (±11.9)

1.5 h 250 (±1.8) 216 (±4.7) 225 (±8.1)

2 h 249 (±5.6) 208 (±2.8) 205 (±10.7)

Figure 4.9 Degradation kinetics of α-Tocopherol in canola oil (A) 70 oC (B) 100

oC

and (C) 130 oC

41

iv) Thermal degradation of α-Tocopherol in Palm oil: Figures 4.10i), 4.10ii) and 4.10iii)

show the HPLC chromatogram of α-Tocopherol during the thermal treatment, at half-

hour heating time intervals over 2 h at 70, 100 and 130 oC, respectively. Table 4.4 shows

the α-Tocopherol concentration calculation from the HPLC chromatogram and Figure

4.11 shows the first order degradation kinetics curve for α-Tocopherol during the heat

treatment. There was a gradual but non-significant degradation (P > 0.05) of α-

Tocopherol at 70 oC; at 0 heating time α-Tocopherol concentration was 174 (±1.7) µg/ml

compared to 174 (±0.7), 166 (±0.9), 166 (±6.1) and 159 (±2.7) µg/ml after 0.5, 1, 1.5 and

2 h heating time, respectively. At 100 oC also, there was a decrease (non-significant; P >

0.05) of α-Tocopherol during the 2 h heating period; the α-Tocopherol concentration were

158 (±3.6), 156 (±2.2), 152 (±8.7) and 150 (±5.7) µg/ml, after 0.5, 1, 1.5 and 2 h heating

time, respectively. At 130 oC there was also a gradual but non-significant (P > 0.05) of α-

Tocopherol in palm oil during the first 1-hour heating period. During the 2 h heating

period; the α-Tocopherol concentration was 156 (±6.1) and 154 (±6.7) after 0.5 and 1 h

heating time, respectively. There was a significant (P < 0.05) degradation of α-

Tocopherol after the 1.5 and 2 h heating period; the α-Tocopherol concentration was 127

(±1.9) and 97 (±0.9) µg/ml, after 1.5 and 2 h heating time, respectively. In general, the

results show that the α-Tocopherol in palm oil was stable at 70 oC during the 2-hour

heating period; at 100 oC the degradation of α-Tocopherol increased with time but this

increase in degradation was statistically not significant (P > 0.05) while at 130 oC the

degradation of α-Tocopherol increased and this was statistically significant (P < 0.05) at

the 1.5 and 2 h heating times.

The concentration of α-Tocopherol in palm oil was similar to those reported by

other researchers. Simonne and Eitenmiller (1998) reported that the level of α-Tocopherol

for palm oil was 155 (±5) µg/g. Other researches (Marco et al., 2007) reported 185 ppm

of α-Tocopherol in palm oil and Schroeder et al. (2006) reported 193 (±8) and 288 (±9) α-

Tocopherol in yellow palm oil and red palm oil, respectively.

Previous studies have reported on the effect of heat treatment of palm oil and palm

olein (Adam et al., 2007; Barrera-Arellano et al., 2002; Simonne and Eitenmiller, 1998);

these researchers studied the effect of heat treatment at 180 and 185 oC, on palm oil and

42

reported a significant reduction of α-Tocopherol with a significant effect in relation to

heat time but not temperature. Corsini et al. (2009) reported no significant decrease of α-

Tocopherol under the similar heating conditions. Schroeder et al. (2006) studied the

effect of repeated heat treatment at 160 oC on yellow and red palm oil and reported a

significant reduction of α-Tocopherol with repeated heat treatment.

43

I II III

Figure 4.10 Degradation of α-Tocopherol in palm oil at (I) 70 o

C, (II) 100 oC and (III) 130

oC (A) 0 time (B) 0.5 h (C) 1 h

(D) 1.5 h and (E) 2 h


α-Tocopherol

E

D

C

A

B

α-Tocopherol

E

C

α-Tocopherol

E

D

C

B

A

D

B

A

44

Table 4.4 Effect of heating time and temperature on α-Tocopherol in palm oil

Time α-Tocopherol concentration in Palm oil (µg/ml)

70 °C 100 °C 130 °C

0 h 174 (±1.7) 174 (±1.7) 174 (±1.7)

0.5 h 174 (±0.7) 158 (±3.6) 156 (±6.1)

1 h 166 (±0.9) 156 (±2.2) 154 (±6.7)

1.5 h 166 (±6.1) 152 (±8.7) 127 (±1.9)

2 h 159 (±2.7) 150 (±5.7) 97 (±0.9)

Figure 4.11 Degradation kinetics of α-Tocopherol in palm oil (A) 70 oC (B) 100

oC

and (C) 130 oC

45

4.3 Comparison of α-Tocopherol degradation in vegetable oils at the same

thermal treatments

Table 4.5 shows thermal degradation of α-Tocopherol concentration in standard α-

Tocopherol and vegetable oils and Figure 4.12 shows the first order degradation kinetics

of α-Tocopherol in standard α-Tocopherol and vegetable oils during heat treatment. For

all 3 oils and α-Tocopherol standard, the degradation at 70 oC was minimal; the

degradation were 8 % for α-Tocopherol standard, 6 % for EVOO, 8 % for canola oil and

9 % for palm oil after 2 hours heating at 70 oC. Heating at 100

oC for 2 hours results in

higher degradation of α-Tocopherol; the degradation were 9 % for α-Tocopherol standard,

17 % for EVOO, 23 % for canola oil and 14 % for palm oil after 2 hours heating at 100

oC. Heating at 130

oC for 2 hours results in higher degradation of α-Tocopherol; the

degradation were 12 % for α-Tocopherol standard, 100 % for EVOO, 24 % for canola oil

and 44 % for palm oil after 2 hours heating at 130 oC. In general, the results show that the

most stable oil was canola oil followed by palm oil and EVOO.

46

Table 4.5 Effect of heating time and temperature on degradation of α-Tocopherol in Standard α-Tocopherol and vegetable oils (µg/ml)

Time 70

oC α-Tocopherol degradation (%)

Standard α-Tocopherol EVOO Canola Palm Standard α-Tocopherol EVOO Canola Palm

0 h 41.15 (±0.3) 323 (±5.3) 271 (±2.2) 174 (±1.7) 0 0 0 0

0.5 h 40.18 (±1.6) 283 (±9.4) 268 (±6.1) 174 (±0.7) 2 12 1 0

1 h 39.18 (±1.5) 273 (±4.6) 256 (±8.6) 166 (±0.9) 5 15 6 5

1.5 h 37.17 (±0.4) 289 (±10.7) 250 (±1.8) 166 (±6.1) 10 11 8 5

2 h 37.80 (±0.9) 304 (±2.6) 249 (±5.6) 159 (±2.7) 8 6 8 9

Time 100



0 h 41.15 (±0.3) 323 (±5.3) 271 (±2.2) 174 (±1.7) 0 0 0 0

0.5 h 39.67 (±0.3) 291 (±6.2) 245 (±8.8) 158 (±3.6) 4 10 10 9

1 h 37.41 (±2.1) 282 (±9.0) 222 (±5.5) 156 (±2.2) 9 13 18 10

1.5 h 37.37 (±2.7) 279 (±5.8) 216 (±4.7) 152 (±8.7) 9 14 20 13

2 h 37.29 (±1.2) 269 (±7.7) 208 (±2.8) 150 (±5.7) 9 17 23 14

Time 130



0 h 41.15 (±0.3) 323 (±5.3) 271 (±2.2) 174 (±1.7) 0 0 0 0

0.5 h 39.12 (±1.6) 160 (±2.0) 231 (±8.0) 156 (±6.1) 5 50 15 10

1 h 37.57 (±1.7) 133 (±0.8) 228 (±11.9) 154 (±6.7) 9 59 16 11

1.5 h 36.24 (±0.6) 0 225 (±8.1) 127 (±1.9) 12 100 17 27

2 h 36.19 (±2.0) 0 205 (±10.7) 97 (±0.9) 12 100 24 44

47

Figure 4.12 Degradation kinetics of α-Tocopherol in ( ) standard α-Tocopherol, ( )

EVOO, ( ) canola oil and ( ) palm oil at (A) 70 oC (B) 100

oC and (C) 130

oC

48

4.4 Thermal degradation of α-Tocopherol in vegetable oils during cooking

Figure 4.13 shows the HPLC chromatograms for EVOO, canola and palm oil

during cooking, (A) no cooking (B) oven cooking and (C) pan-frying. Table 4.6 shows

the α-Tocopherol concentration calculation from the HPLC chromatograms while Figure

4.14 shows the degradation of α-Tocopherol during cooking and Figure 4.15 shows the

degradation % of α-Tocopherol during cooking .The results suggest that the loss of α-

Tocopherol depended on the type of oil and cooking method. With the oven cooking

method (0.5 h cooking at 130 ±3 oC) extra virgin olive oil showed an α-Tocopherol

concentration of 264 (±8.0) µg/ml; with the frying method (5 min at 250 ± 3 oC) there

was complete degradation of α-Tocopherol. Palm oil showed similar behavior to extra

virgin olive oil with α-Tocopherol concentration of 156 (±2.9) µg/ml; with the oven

cooking method and a complete degradation of α-Tocopherol with the frying method.

With the same oven cooking method, canola oil showed a α-Tocopherol concentration of

236 (±2.3) µg/ml and with the same frying method α-Tocopherol concentration was 156

(±12.0) µg/ml. Overall, the results suggest that α-Tocopherol in canola oil was the most

stable of the three oils when the oils were subjected to the same cooking conditions; α-

Tocopherol in extra virgin olive oil and palm was completely degraded by the higher

temperature of the frying method while α-Tocopherol in canola oil showed a 42 %

degradation.

Andrikopoulos et al. (2002) reported that pan-frying of potatoes in virgin olive oil

at 180 oC 10 times for 6 min, showed a 92 % loss of α-Tocopherol. Brenes et al. (2002)

used microwave cooking (0.5 kW power at 2450 MHz) and reported that the degradation

of α-Tocopherol of extra virgin olive oil decreased with time during microwave heating at

maximum power for 5 and 10 min; for the Picual cultivar of olive oil the α-Tocopherol

degradation was 26 and 62 %, respectively and for Arbequina cultivar the degradation

was 35 and 81 %, respectively. Allouche et al. (2007) reported that the degradation of α-

Tocopherol of extra virgin olive oil decreased with time during heating over 36 hours at

180 oC; for the Arbequina cultivar the α-Tocopherol degradation ranged from 10 – 90%

and for the Picual cultivar the degradation ranged from 8 – 80 %. Normand et al. (2001)

reported that the level of degradation of α-Tocopherol varied depending on canola oil

49

type; canola oils were subjected to a temperature of 175 oC of 72 h. The results showed

the degradation of α-Tocopherol depended on the type of oil. Adam et al. (2007) showed

that heating palm oil for 10 min at 180 oC resulted in α-Tocopherol degradation ranging

from 98.13 and 55.70 % depending on the heating conditions during cooking; similar

results were reported by Bansal et al. (2010) and Schroeder et al. (2006).

50

I II III

Figure 4.13 Degradation of α-Tocopherol in (I) EVOO, (II) canola oil and (III) palm oil during cooking, (A) 0 time (B)

oven cooking and (C) pan-frying

α-Tocopherol α-Tocopherol α-Tocopherol

C

C

B

A

B

A

C

B

A


51

Table 4.6 Effect of cooking method on degradation of α-Tocopherol in vegetable oils

Oil type α-Tocopherol concentration (µg/ml) α-Tocopherol degradation (%)

No cooking Oven cooking (130 oC) Pan-Frying (250

oC) Oven cooking (130

oC) Pan-Frying (250

oC)

Extra virgin olive oil 323 (±5.3) 264 (±8.0) 0 18 100

Canola oil 271 (±2.2) 236 (±2.3) 156 (±12.0) 13 42

Palm oil 174 (±1.7) 156 (±2.9) 0 10 100

Figure 4.14 Effect of cooking method on degradation of α-Tocopherol in extra virgin olive oil, canola oil and palm oil

52

Figure 4.15 Degradation % of α-Tocopherol in ( ) extra virgin olive oil, ( )

canola oil and ( ) palm oil in relation to cooking method

53

4.5 Antioxidant activity: Standard curve for α-Tocopherol antioxidant

activity

Table 4.7 shows the antioxidant activity of α-Tocopherol standard at 3 different

concentrations; Figures 4.14 and 4.15 shows the standard curve for α-Tocopherol

antioxidant activity and the antioxidant activity of α-Tocopherol at 3 different

concentrations; from the standard curve, a concentration of 7.5 % was selected for

determination of the antioxidant activity in the oil samples.

Table 4.7 Antioxidant activity of standard α-Tocopherol at different concentration

Standard α-Tocopherol concentration DPPH reduction (%)

2.5% 99 (±0.05)

5.0% 99 (±0.54)

7.5% 98 (±0.45)

Figure 4.14 Standard α-Tocopherol calibration curve

0

0.004

0.008

0.012

0.016

0.02

0.024

2.5 5 7.5

Abso

rbance

(nm

)

Standard α-Tocopherol concentrations (%)

54

Figure 4.15 Antioxidant activity of standard α-Tocopherol in 3 different

concentrations

0

20

40

60

80

100

120

2.5 5 7.5

DP

PH

red

uct

ion

%

Standard α-Tocopherol concentrations (%)

55

4.6 Antioxidant activity of standard α-Tocopherol and heated oils

i) Antioxidant activity of standard α-Tocopherol: Table 4.8 and Figure 4.16 show the

antioxidant activity of standard α-Tocopherol, at a half –hour heating time over 2 h at 70,

100 and 130 oC. For the standard α-Tocopherol there was no significant effect of heating

at 70, 100 and 130 oC over the 2 hour heating period; this suggests that the antioxidant

activity of α-Tocopherol was not affected by the heat treatment.

A previous study reported the effect of heat treatment on the antioxidant activity

of standard α-Tocopherol (Larrauri et al., 1998); this research studied the effect of heat

treatment at 20, 80, 100 and 120 oC on standard α-Tocopherol and reported no significant

decrease; the results showed no significant effect in relation to temperature or heating

time.

56

Table 4.8 Effect of heating time and temperature on antioxidant activity of standard

α-Tocopherol (%)

Time Temperature

70 oC 100

oC 130

oC

0 h 99 (±0.05) 99 (±0.05) 99 (±0.05)

0.5 h 99 (±0.19) 99 (±0.06) 99 (±0.08)

1 h 99 (±0.05) 99 (±0.10) 99 (±0.49)

1.5 h 99 (±0.10) 99 (±0.08) 99 (±0.36)

2 h 98 (±0.32) 99 (±0.08) 98 (±0.59)

Figure 4.16 Effect of heating on standard α-Tocopherol antioxidant activity at (A) 70 oC, (B) 100

oC and (C) 130

oC

0

20

40

60

80

100

120

0 0.5 1 1.5 2

DPP

H re

duct

ion

%

Time (h)

A

0

20

40

60

80

100

120

DPP

H re

duct

ion

%

B

0

20

40

60

80

100

120

DPP

H re

duct

ion

%

C

57

ii) Antioxidant activity in extra virgin olive oil (EVOO): Table 4.9 and Figure 4.17 show

the effect of heat on antioxidant activity of extra virgin olive oil, at a half –hour heating

time over 2 h at 70, 100 and 130 oC. There was no significant effect of the 70

oC

treatment. With the 100 oC, the antioxidant activity decreased gradually (statistically not

significant) during the 2 h heating period; the antioxidant activity was 45 (±0.49), 42

(±1.51), 41 (±2.09) and 40 (±1.61) %, after 0.5, 1, 1.5 and 2 h heating time, respectively.

With the 130 oC treatment the antioxidant activity decreased during the 4 time intervals

with values of 33 (±0.84), 31 (±0.43), 26 (±3.55) and 24 (±0.72) %, after 0.5, 1, 1.5 and 2

h heating time, respectively; the decrease in antioxidant activity was statistically

significant (P < 0.05).

Previous studies have shown the effect of heat treatment on the antioxidant

activity of olive oil at temperatures 160, 175, 180, 185 and 190 oC (Kalantzakis et al.,

2006; Quiles et al., 2002; Pellegrini et al., 2001; Espin et al., 2000); the research reported

a significant decrease in total antioxidant activity (TAA) during heat treatment and

showed significant effect in relation to heat time but not temperature. Valavanidis et al.

(2004) also studied effect of different thermal treatment on the total antioxidant capacity

(IC50) of extra virgin olive oil and olive oil at 160 and 190 oC; the research results showed

a lower IC50 with heat treatment.

58

Table 4.9 Effect of heating time and temperature on antioxidant activity of extra

virgin olive oil (%)

Time Temperature

70 oC 100

oC 130

oC

0 h 46 (±0.91) 46 (±0.91) 46 (±0.91)

0.5 h 44 (±0.62) 45 (±0.49) 33 (±0.84)

1 h 44 (±1.23) 42 (±1.51) 31 (±0.43)

h 44 (±0.75) 41 (±2.09) 26 (±3.55)

2 h 44 (±1.24) 40 (±1.61) 24 (±0.72)

Figure 4.17 Effect of heating on antioxidant activity of extra virgin olive oil at (A) 70 oC, (B) 100

oC and (C) 130

oC

0

20

40

60

80

100

0 0.5 1 1.5 2

DPP

H re

duct

ion

%

Time (h)

A

0

20

40

60

80

100

DPP

H re

duct

ion

%

B

0

20

40

60

80

100

DPP

H re

duct

ion

%

C

59

iii) Antioxidant activity in canola oil: Table 4.10 and Figure 4.18 show the effect of heat

on antioxidant activity of canola oil, at a half-hour heating time intervals over 2 h at 70,

100 and 130 oC. There was little effect of the 70

oC treatment. With 100

oC treatment, the

antioxidant activity of canola oil decreased gradually (statistically non-significant) during

the 4 time intervals (58 ±1.64, 57 ±0.96, 55 ±1.76 and 53 ±1.48 %, after 0 .5, 1, 1.5 and 2

h heating time, respectively). With 130 oC treatment, the antioxidant activity decreased

(statistically non-significant) during the 4 time intervals with values of 54 (±1.04), 54

(±1.08), 50 (±0.90) and 48 (±1.57) %, after 0.5, 1, 1.5 and 2 h heating time, respectively.

Previous studies have shown the effect of heat treatment on canola oil antioxidant

activity at temperatures of 55 and 95 oC (Cheung at al., 2007; Su et al., 2004) the research

reported a significant decrease in total antioxidant activity (TAA) during heat treatment

with significant effect in relation with heat time but not temperature. Chiavaro et al.

(2010) reported a slight but significant increase of trolox equivalent antioxidant capacity

(TEAC) during microwave heating for up to 6 min, followed by a subsequent decrease

reaching values similar to the initial value and a significant decrease of oxidative stability

index (OSI).

60

Table 4.10 Effect of heating time and temperature on antioxidant activity of canola

oil (%)

Time Temperature

70 oC 100

oC 130

oC

0 h 59 (±1.72) 59 (±1.72) 59 (±1.72)

0.5 h 59 (±0.66) 58 (±1.64) 54 (±1.04)

1 h 58 (±1.80) 57 (±0.96) 54 (±1.08)

1.5 h 58 (±2.54) 55 (±1.76) 50 (±0.90)

2 h 59 (±2.22) 53 (±1.48) 48 (±1.75)

Figure 4.18 Effect of heating on antioxidant activity of canola oil at (A) 70 oC, (B)

100 oC and (C) 130

oC

0

20

40

60

80

100

0 0.5 1 1.5 2

DPP

H re

duct

ion

%

Time (h)

A

0

20

40

60

80

100

DPP

H re

duct

ion

% B

0

20

40

60

80

100

DPP

H re

duct

ion

% C

61

iv) Antioxidant activity in palm oil: Table 4.11 and Figure 4.19 show the effect of heat on

the antioxidant activity of palm oil, at a half-hour heating time intervals over 2 h at 70,

100 and 130 oC and. There was little effect of 70

oC and 100

oC treatments. With 130

oC

treatment the antioxidant activity decreased gradually (statistically non-significant) during

the 4 time intervals with values of 33 (±0.84), 31 (±0.43), 26 (±3.55) and 24 (±0.72) %

after 0.5, 1.0, 1.5, 2 h heating time, respectively.

Previous studies have shown the effect of heat treatment on palm oil antioxidant

activity at temperatures of 170, 180, 210, 250 oC (Valantina et al., 2010; Andrikopoulos

et al., 2002); the research reported that antioxidants were lost during frying. Schroeder et

al. (2006) reported a decrease in antioxidant activity in yellow palm olein and red palm

olein, during repeated deep-fat frying at 163 oC.

62

Table 4.11 Effect of heating time and temperature on antioxidant activity of palm oil

(%)

Time Temperature

70 oC 100

oC 130

oC

0 h 51 (±0.84) 51 (±0.84) 51 (±0.84)

0.5 h 51 (±0.98) 50 (±1.00) 49 (±0.83)

1 h 50 (±0.40) 50 (±0.23) 47 (±0.77)

1.5 h 51 (±1.21) 50 (±0.28) 45 (±0.77)

2 h 50 (±0.49) 49 (±1.14) 39 (±0.30)

Figure 4.19 Effect of heating on antioxidant activity of palm oil at (A) 70 oC,

(B) 100 oC and (C) 130

oC

0

20

40

60

80

100

0 0.5 1 1.5 2

DPP

H re

duct

ion

%

Time (h)

A

0

20

40

60

80

100

DPP

H re

duct

ion

%

B

0

20

40

60

80

100

DPP

H re

duct

ion

%

C

63

4.7 Comparison of antioxidant activity of heated vegetable oils at the same

thermal treatments

Table 4.12 and Figure 4.20 show antioxidant activity of standard α-Tocopherol

and the 3 oils during the thermal treatments at 3 temperatures. The results showed no

significant antioxidant activity reduction as a result of the treatment at 70 oC. At 100

oC,

of the 3 oils, the highest stability of antioxidant activity to the heat treatment was with

palm oil followed by canola oil and EVOO. At 130 oC there was a significant decrease in

antioxidant activity for all oils; the highest stability of antioxidant activity was with

canola oil followed by palm and finally by EVOO (19, 24 and 48 % decrease in

antioxidant activity, respectively).

64

Table 4.12 Effect of heating time and temperature on antioxidant activity of standard α-Tocopherol and vegetable oils (%)

Time 70

oC Antioxidant activity loss (%)


0 h 99 (±0.05) 46 (±0.91) 59 (±1.72) 51 (±0.84) 0 0 0 0

0.5 h 99 (±0.19) 44 (±0.62) 59 (±0.66) 51 (±0.98) 0.1 4.3 0 0

1 h 99 (±0.05) 44 (±1.23) 58 (±1.80) 50 (±0.40) 0.1 4.3 1.7 2.0

1.5 h 99 (±0.10) 44 (±0.75) 58 (±2.54) 51 (±1.21) 0.1 4.3 1.7 0

2 h 98 (±0.32) 44 (±1.24) 59 (±2.22) 50 (±0.49) 0.7 4.3 0 2.0

Time 100

oC Antioxidant activity loss (%)


0 h 99 (±0.05) 46 (±0.91) 59 (±1.72) 51 (±0.84) 0 0 0 0

0.5 h 99 (±0.06) 45 (±0.49) 58 (±1.64) 50 (±1.00) 0.2 2.2 1.7 2.0

1 h 99 (±0.10) 42 (±1.51) 57 (±0.96) 50 (±0.23) 0.2 8.7 3.4 2.0

1.5 h 99 (±0.08) 41 (±2.09) 55 (±1.76) 50 (±0.28) 0 10.9 6.8 2.0

2 h 99 (±0.08) 40 (±1.61) 53 (±1.48) 49 (±1.14) 0.1 13.0 10.2 3.9

Time 130

oC Antioxidant activity (%)


0 h 99 (±0.05) 46 (±0.91) 59 (±1.72) 51 (±0.84) 0 0 0 0

0.5 h 99 (±0.08) 33 (±0.84) 54 (±1.04) 49 (±0.83) 0.2 28.3 8.5 3.9

1 h 99 (±0.49) 31 (±0.43) 54 (±1.08) 47 (±0.77) 0.1 32.6 8.5 7.8

1.5 h 99 (±0.36) 26 (±3.55) 50 (±0.90) 45 (±0.77) 0.1 43.5 15.3 11.8

2 h 98 (±0.59) 24 (±0.72) 48 (±1.57) 39 (±0.30) 0.5 47.8 18.6 23.5

65

Figure 4.20 Effect of heating on antioxidant activity of ( ) standard α-Tocopherol, ( )

EVOO, ( ) canola and ( ) palm oil at (A) 70 oC, (B) 100

oC and (C) 130

oC

66

4.8 Effect of cooking on antioxidant activity of oils

Table 4.13 and Figure 4.21 show the effect of cooking on the antioxidant activity

of the 3 oils while Figure 4.22 shows the antioxidant activity loss during cooking. Canola

oil showed the highest antioxidant activity followed by EVOO and palm oil; antioxidant

reduction percentages were 24, 57 and 57 % for canola oil, EVOO and palm oil

respectively. With canola oil, the antioxidant activity was not affected by the oven

cooking method, while in the frying method there was a significant reduction (P < 0.05)

in the antioxidant activity from 59 (±1.98) to 45 (±1.55) %. With EVOO the antioxidant

activity decreased slightly during the oven cooking method, while in the frying method

produced a significant decrease (P < 0.05) in the antioxidant activity from 46 (±0.91) to

24 (±0.63) %. Palm oil showed similar behavior to canola oil and EVOO; the cooking

oven method produced small decrease in antioxidant activity while frying method showed

a significant decrease (P < 0.05) in the antioxidant activity from 51 (±0.84) to 24 (±0.74)

%.

Gomez-Alonso et al. (2003) showed that the antioxidant activity of virgin olive

oil decreased as the number of frying operations increased. Similar results were obtained

by Quiles et al., (2002) using electrospin resonance as an indication of antioxidant

activity.

67

Table 4.13 Effect of cooking method on antioxidant activity of vegetable oils (%)

Oil type Cooking method Antioxidant activity loss (%)

No cooking Oven cooking (130 oC) Pan-Frying (250

oC) Oven cooking Pan-Frying

Extra virgin olive oil 46 (±0.91) 42 (±1.77) 24 (±0.63) 9 48

Canola oil 59 (±1.72) 59 (±1.98) 45 (±1.55) 0 24

Palm oil 51(±0.84) 49 (±1.60) 24 (±0.74) 4 53

Figure 4.21 Effect of cooking method on antioxidant activity on vegetable oils

0

20

40

60

80

100

No

co

ok

ing

Ov

en c

oo

kin

g

Pan

-fry

ing

No

co

ok

ing

Oven

cookin

g

Pan

-fry

ing

No

coo

kin

g

Oven

cookin

g

Pan

-fry

ing

EVOO Canola Palm

DP

PH

re

du

ctio

n %

Different oils and cooking methods

68

Figure 4.22 Antioxidant activity loss during cooking ( ) extra virgin olive oil, ( )

canola oil and ( ) palm oil

0

20

40

60

80

100

No cooking Oven cooking Pan-Frying

An

tio

xid

an

t a

ctiv

ity

lo

ss %

Cooking method

69

GENERAL CONCLUSION

The effect of heating on α-Tocopherol content and antioxidant activity in

different vegetable oils was investigated; the samples selected were EVOO, canola and

palm oil. These oils were chosen due to their use in the Kuwaiti cuisine, amount of α-

Tocopherol and use in Kuwaiti bakery products. α-Tocopherol content varied depending

on the oil samples; the highest content was found in EVOO followed by canola oil and

finally palm oil (323 ±5, 271 ±2 and 174 ±2 µg/ml) respectively. The effect of heat was

done at 70, 100 and 130 oC, for the following 4 time intervals: 0.5, 1, 1.5 and 2 h.

Thermal degradation of α-Tocopherol in the oils was minimal at 70 oC and increased at

100 oC and 130

oC. Heating at 130

oC for 2 h resulted in 100, 24 and 44 % degradation of

α-Tocopherol in EVOO, canola oil and palm oil respectively; EVOO was completely

degraded after 1.5 h heating at 130 oC. Use of 2 cooking methods, pan-frying (250

oC, 5

min) and oven cooking (130 oC, 30 min) resulted in the degradation of α-Tocopherol in

the oils. In the pan-frying method, both EVOO and palm oil were completely degraded

and canola oil showed 42 % degradation. With the oven cooking method the degradation

for EVOO, canola oil and palm oil were 18, 13 and 10 %, respectively.

DPPH method was used for examining the antioxidant activity of the oil

samples; it was found that highest antioxidant activity was observed with canola oil

followed by palm oil and EVOO (59 ±1.72, 51 ±0.84 and 46 ±0.91 %) respectively. At

70 oC there was no significant decrease in the antioxidant activity of the heated oils. At

100 oC, EVOO showed highest reduction in antioxidant activity followed by canola oil

and palm oil. At 130 oC, the antioxidant activity decreased gradually in the oil samples.

The highest decrease was observed with EVOO followed by canola oil and palm oil. The

decrease of antioxidant activity in oil samples was also observed with both pan-frying and

oven cooking methods, with greater reduction in antioxidant activity using the pan-frying

method.

70

Appendix A

72

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